{
    "v1_Abstract": "The end replication problem, which occurs in normal somatic cells inducing replicative senescence, is solved in most of cancer cells by activating telomerase. The activity of telomerase is highly associated with carcinogenesis which makes the enzyme an attractive biomarker in cancer diagnosis and treatment. The indole alkaloid harmine has multiple pharmacological properties including DNA intercalation which can lead to frame shift mutations. In this study, harmine was applied to human breast cancer MCF-7 cells. Its activity towards telomerase was analyzed by utilizing the telomeric repeat amplification protocol (TRAP). Our data indicate that harmine exhibits a pronounced cytotoxicity and induces an anti-proliferation state in MCF-7 cells which is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by overexpressing elements of the p53/p21 pathway.",
    "v1_col_introduction": "introduction  : The end replication problem results in a continuous shortening of each end of a chromosome. In most somatic cells the shortened fragments cannot be compensated. Cells stop dividing when telomeres reach a critical length and replicative senescence is initiated consequently. However, most cancer cells can conquer this obstacle, because their telomerase, a ribonucleoprotein that replicates telomere sequences at each cell division, remains active. Telomerase is highly associated with carcinogenesis. It is detectable in 85\u201390% of human cancers and over 70% of immortalized human cell lines (Kim et al., 1994; Shay & Wright, 1996a,b), whereas it is undetectable in non-transformed somatic cells. Therefore, telomerase is an attractive target for the development of anti-cancer drugs.\nTelomerase is a cellular reverse transcriptase containing two components: A protein\nelement, telomerase reverse transcriptase (in human, hTERT) serving as catalytic subunit and an RNA element, hTR, providing a template for telomere synthesis (Nakamura et al., 1997).\nRecent evidence suggests that increased telomere dysfunction leads to a loss of\nchromosome end protection and induces the senescence state. But senescence can also be induced without continuous telomere shortening suggesting that telomere integrity is critical regardless of telomere length. Tumour suppressor proteins such as p53 are required for the senescence arrest (Liu & Kulesz-Martin, 2001; Gorbunova et al., 2002; Gewirtz et al., 2008).\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\n28\n29\nPeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nHarmine, a naturally occurring \u03b2-carboline alkaloid, has long been used in folk medicine\nin the Middle East and in Asia (Sourkes, 1999) and as a hallucinogenic drug (Wink & van Wyk, 2008). It was first isolated from the seeds of Peganum harmala L. in 1874 (Budavari, 1989; Roberts & Wink, 1998; Wink & van Wyk, 2008; Wink et al., 1998b). Harmine has multiple pharmacological properties including antiplasmodial activity (Astulla et al., 2008), antimutagenic and antiplatelet properties (Im et al., 2009). In vitro studies demonstrate that the planar structure of harmine leads to DNA intercalation. Since DNA intercalation causes frame shift mutations, these alkaloids are known to be mutagenic, cytotoxic and antimicrobial (Roberts & Wink, 1998; Wink, 2007). Burger and the colleagues had observed about decades ago that harmine could inhibit monoamine oxidase (Burger and Nara, 1965), through which way to regulate the metabolism of neurotransmitters (Kim et al. 1997; Wink et al., 1998). Recent data indicate that harmine and related alkaloids act as agonists at serotonin receptors (Wink et al., 1998; Glennon et al., 2000; Song et al., 2004). Harmine and other \u03b2-carboline alkaloids therefore exhibit hallucinogenic properties (Wink & van Wyk, 2008).\nData obtained from cell viability assays indicate that harmine is a promising inhibitor of\ncell proliferation in a variety of cancer cell lines (Song et al., 2004). It blocks the cell cycle at G0/G1 phase (Hamsa and Kuttan, 2011) accompanied with a decrease of cyclin-dependent kinase activity (Song et al. 2002, 2004). DNA intercalation is also involved in the inhibition of cell division as it prevents the transcription of several genes and causes frame shift mutations. Previous findings indicate that telomeres are a also target of intercalating drugs (Shammas et al., 2003, 2004); they can induce very stable G-quadruplex structures which cannot be replicated by telomerase (Burge et al., 2006). Some known anticancer drugs exhibit DNA intercalation, such as isoquinoline, quinoline, and indole alkaloids (Wink et al., 1998; Wink, 2007).\nAmong these alkaloids, the simple indole alkaloid harmine was identified in our\nlaboratory as a potent DNA intercalating and cytotoxic natural product (Rosenkranz et al., 2007). It has been reported that a few DNA-intercalating alkaloids, including berbamine (Ji et al., 2002), chelidonine (Noureini & Wink, 2009) and 9-hydroxyellipticine (Sasaki et al., 1992) are inhibitors of telomerase activity. Because harmine is an intercalating and cytotoxic alkaloid a possible telomerase inhibition was evaluated in this research. The aim of this research was to examine the effects of harmine in human breast cancer MCF-7 cells and its possible interaction with telomerase. Anti-telomerase activity was analyzed using the telomeric repeat amplification protocol (TRAP). Harmine causes a pronounced cytotoxicity and induces an anti-proliferation\n30\n31\n32\n33\n34\n35\n36\n37\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n49\n50\n51\n52\n53\n54\n55\n56\n57\n58\n59\n60\n61\nPeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013)\nR ev ie w in g M an\nus cr ip t\nstate in MCF-7 cells. This process is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by over-expressing p53/p21.",
    "v2_Abstract": "The end replication problem, which occurs in normal somatic cells inducing replicative senescence, is solved in most of cancer cells by activating telomerase. The activity of telomerase is highly associated with carcinogenesis which makes the enzyme an attractive biomarker in cancer diagnosis and treatment. The indole alkaloid harmine has multiple pharmacological properties including DNA intercalation which can lead to frame shift mutations. In this study, harmine was applied to human breast cancer MCF-7 cells. Its activity towards telomerase was analyzed by utilizing the telomeric repeat amplification protocol (TRAP). Our data indicate that harmine exhibits a pronounced cytotoxicity and induces an anti-proliferation state in MCF-7 cells which is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by over-expressing elements of the p53/p21 pathway.",
    "v2_col_introduction": "introduction  : The end replication problem results in a continuous shortening of each end of a chromosome. In most somatic cells the shortened fragments cannot be compensated. Cells stop dividing when telomeres reach a critical length and replicative senescence is initiated consequently. However, most cancer cells can conquer this obstacle, because their telomerase, a ribonucleoprotein that replicates telomere sequences at each cell division, remains active. Telomerase is highly associated with carcinogenesis. It is detectable in 85\u201390% of human cancers and over 70% of immortalized human cell lines (Kim et al., 1994; Shay & Wright, 1996a,b), whereas it is undetectable in non-transformed somatic cells. Therefore, telomerase is an attractive target for the development of anti-cancer drugs. Telomerase is a cellular reverse transcriptase containing two components: A protein element, telomerase reverse transcriptase (in human, hTERT) serving as catalytic subunit and an RNA element, hTR, providing a template for telomere synthesis (Nakamura et al., 1997). Recent evidence suggests that increased telomere dysfunction leads to a loss of chromosome end protection and induces the senescence state. But senescence can also be induced without continuous telomere shortening suggesting that telomere integrity is critical regardless of telomere length. Tumour suppressor proteins such as p53 are required for the senescence arrest (Liu & Kulesz-Martin, 2001; Gorbunova et al., 2002; Gewirtz et al., 2008). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t\n3 Harmine, a naturally occurring \u03b2-carboline alkaloid, has long been used in folk medicine in the Middle East and in Asia (Sourkes, 1999) and as a hallucinogenic drug (Wink & van Wyk, 2008). It was first isolated from the seeds of Peganum harmala L. in 1874 (Budavari, 1989; Roberts & Wink, 1998; Wink & van Wyk, 2008; Wink et al., 1998b). Harmine has multiple pharmacological properties including antiplasmodial activity (Astulla et al., 2008), antimutagenic and antiplatelet properties (Im et al., 2009). In vitro studies demonstrate that the planar structure of harmine leads to DNA intercalation. Since DNA intercalation causes frame shift mutations, these alkaloids are known to be mutagenic, cytotoxic and antimicrobial (Roberts & Wink, 1998; Wink, 2007). Burger and the colleagues had observed about decades ago that harmine could inhibit monoamine oxidase (Burger and Nara, 1965), through which way to regulate the metabolism of neurotransmitters (Kim et al. 1997; Wink et al., 1998). Recent data indicate that harmine and related alkaloids act as agonists at serotonin receptors (Wink et al., 1998; Glennon et al., 2000; Song et al., 2004). Harmine and other \u03b2-carboline alkaloids therefore exhibit hallucinogenic properties (Wink & van Wyk, 2008). Data obtained from cell viability assays indicate that harmine is a promising inhibitor of cell proliferation in a variety of cancer cell lines (Song et al., 2004). It blocks the cell cycle at G0/G1 phase (Hamsa and Kuttan, 2011) accompanied with a decrease of cyclin-dependent kinase activity (Song et al. 2002, 2004). DNA intercalation is also involved in the inhibition of cell division as it prevents the transcription of several genes and causes frame shift mutations. Previous findings indicate that also telomeres are a target of intercalating drugs (Shammas et al., 2003, 2004); they can induce very stable G-quadruplex structures which cannot be replicated by telomerase (Burge et al., 2006). Some known anticancer drugs exhibit DNA intercalation, such as isoquinoline, quinoline, and indole alkaloids (Wink et al., 1998; Wink, 2007). Among these alkaloids, the simple indole alkaloid harmine was identified as a potent DNA intercalating and cytotoxic natural product (Rosenkranz et al., 2007). Whether human telomeres and telomerase can be affected by harmine has not been reported. The aim of this research was to examine the effects of harmine in human breast cancer MCF-7 cells and its possible interaction with telomerase. Anti-telomerase activity was analyzed using the telomeric repeat amplification protocol (TRAP). Harmine causes a pronounced cytotoxicity and induces an anti-proliferation state in MCF-7 cells. This process is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by over-expressing p53/p21. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t\n4",
    "v3_Abstract": "The end replication problem, which occurs in normal somatic cells inducing replicative senescence, is solved in most of cancer cells by activating telomerase. The activity of telomerase is highly associated with carcinogenesis which makes the enzyme an attractive biomarker in cancer diagnosis and treatment. The indole alkaloid h armine has multiple pharmacological properties including DNA intercalation which can lead to frame shift mutations. In this study, harmine was applied to human breast cancer MCF-7 cells. Its activity towards telomerase was analyzed by utilizing the telomeric repeat amplification protocol (TRAP). Our data indicate that harmine exhibits a pronounced cytotoxicity and induces an anti-proliferation state in MCF-7 cells which is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by over-expressing elements of the p53/p21 pathway.",
    "v3_col_introduction": "introduction  : The end replication problem results in a continuous shortening of each end of a chromosome. In most somatic cells the shortened fragments cannot be compensated. Cells stop dividing when telomeres reach a critical length and replicative senescence is initiated consequently. However, most cancer cells can conquer this obstacle, because their telomerase, a ribonucleoprotein that replicates telomere sequences at each cell division, remains active. Telomerase is highly associated with carcinogenesis. It is detectable in 85\u201390% of human cancers and over 70% of immortalized human cell lines (Kim et al., 1994; Shay & Wright, 1996a,b), whereas it is undetectable in non-transformed somatic cells. Therefore, telomerase is an attractive target for the development of anti-cancer drugs.\nTelomerase is a cellular reverse transcriptase containing two components: A protein\nelement, telomerase reverse transcriptase (in human, hTERT) serving as catalytic subunit and an RNA element, hTR, providing a template for telomere synthesis (Nakamura et al., 1997).\nRecent evidence suggests that increased telomere dysfunction leads to a loss of\nchromosome end protection and induces the senescence state. But senescence can also be induced without continuous telomere shortening suggesting that telomere integrity is critical\n1\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\n11\n12\n13 14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\n28\n29\n30\n31\nPeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013)\nR ev ie w in g M an\nus cr ip t\nregardless of telomere length. Tumour suppressor proteins such as p53 are required for the senescence arrest (Liu & Kulesz-Martin, 2001; Gorbunova et al., 2002; Gewirtz et al., 2008).\nHarmine, a naturally occurring \u03b2-carboline alkaloid, has long been used in folk medicine\nin the Middle East and in Asia (Sourkes, 1999) and as a hallucinogenic drug (Wink & van Wyk, 2008). It was first isolated from the seeds of Peganum harmala L in 1874 (Budavari, 1989; Roberts & Wink, 1998; Wink & van Wyk, 2008; Wink et al., 1998b). Harmine has multiple pharmacological properties including antiplasmodial activity (Astulla et al., 2008), antimutagenic and antiplatelet properties (Im et al., 2009). In vitro studies demonstrate that the planar structure of harmine leads to DNA intercalation. Since DNA intercalation causes frame shift mutations, these alkaloids are known to be mutagenic, cytotoxic and antimicrobial (Roberts & Wink, 1998; Wink, 2007). Burger and the colleagues had observed about decades ago that harmine could inhibit monoamine oxidase (Burger and Nara, 1965), through which way to regulate the metabolism of neurotransmitters (Kim et al. 1997; Wink et al., 1998). Recent data indicate that harmine and related alkaloids act as agonists at serotonin receptors ( Wink et al., 1998; Glennon et al., 2000; Song et al., 2004). Harmine and other \u03b2-carboline alkaloids therefore exhibit hallucinogenic properties (Wink & van Wyk, 2008).\nData obtained from cell viability assays indicate that harmine is a promising inhibitor of\ncell proliferation in a variety of cancer cell lines (Song et al., 2004). It blocks the cell cycle at G0/G1 phase (Hamsa and Kuttan, 2011). Such effect may be strongly associated with the decrease of cyclin-dependent kinase activity, such as Cdk1/cyclin B, Cdk2/cyclin A, and Cdk5/p25 (Song et al. 2002, 2004). DNA intercalation is also involved in the inhibition of cell division as it prevents the transcription of several genes and causes frame shift mutations. Telomeres are affected by intercalating drugs; whether human telomeres and telomerase can be affected by harmine has not been reported.\nThe aim of this research was to examine the effects of harmine in human breast cancer\nMCF-7 cells and its possible interaction with telomerase. Anti-telomerase activity was analyzed using the telomeric repeat amplification protocol (TRAP). Harmine causes a pronounced cytotoxicity and induces an anti-proliferation state in MCF-7 cells. This process is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by over-expressing p53/p21.\n2\n32\n33\n34\n35\n36\n37\n38\n39\n40\n41\n42\n43\n44\n45\n46\n47\n48\n49\n50\n51\n52\n53\n54\n55\n56\n57\n58\n59\n60\n61 62\nPeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013)\nR ev ie w in g M an\nus cr ip t",
    "v1_text": "materials & methods : results : Harmine is cytotoxic to MCF-7 cells in a dose- and time-dependent manner and induces accelerated senescence The cytotoxicity of harmine in MCF-7 cells is shown in Fig. 1. Cell viability at various time points was determined by MTT assay. The results indicate that harmine arrests cell growth in a dose- and time-dependent manner. Concentrations of 20 and 30 \u00b5M harmine significantly 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t reduced cell growth after 48 to 96 h. Concentrations between 10 and 20 \u00b5M did not influence viability of MCF-7 cells within the first 24 h, and were therefore used in the subsequent experiments. In the next step we tried to study whether senescent cells could be identified in response to harmine treatment. In MCF-7 cells, harmine arrests cell proliferation and induces a senescence morphology. \u03b2-Galactosidase activity, as a senescence marker, was detectable as early as 2 d after treatment with harmine, and became intense and expressed in virtually every cell of the culture at day 4 (Fig. 2). Cells, which were \u03b2-galactosidase positive, were larger in size or multinucleated (indicated with arrows), both of which are morphological features indicative of a senescent state. The SA-\u03b2-gal staining was not detected or barely detected in untreated control cells. Telomerase activity Telomerase activity of MCF-7 cells, treated with or without harmine, was evaluated as evidenced by the TRAP assay. A decreased telomerase activity (Fig. 3A) was detected after the incubation of the cells with 20 \u00b5M harmine. Telomerase activity was inhibited by 81.87% after 96 h treatment as compared to the untreated control (Fig. 3B). Treatment at a lower concentration, e.g., 10 \u00b5M, did not show a significant reduction of telomerase activity. Expression analysis of human TERT splicing variants by RT-PCR. RT-PCR analysis was performed with a pair of primers which covers all hTERT transcripts. In theory, four hTERT variants should be expected under the identical PCR conditions at the same time (full length hTERT with 457 bp; \u03b1 variant with 421 bp; \u03b2 variant with 275 bp and \u03b1+ \u03b2 variant with 239 bp). However, in our investigation the \u03b1+ \u03b2 variant could not be detected (Fig. 4A). Treatment of the cells with 20 \u00b5M harmine significantly down-regulated all hTERT subunits in a time-dependent manner (Fig. 4B). Expression analysis of human TERT Human hTERT, p21, and CDK2 mRNA transcripts were examined by real time PCR. Data were analyzed by Relative Expression Software Tool (REST2008). PCR efficiency was set as 2 as indicated by the software and the housekeeping gene \u03b2-actin was regarded as a control. A significant up-regulation of p21 mRNA was detected as early as 12 h after harmine treatment. The mRNA concentration was 3.9 fold higher than that of the untreated control, and the upregulation became 6.5 fold with respect to the control after 96 h (Fig. 5). Within the first 24 h of treatment, no alteration on of hTERT and CDK2 mRNA expression was detected, while an extended treatment up to 48 h showed that a significant down-regulation was observed for these two genes. 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t figure captions : Figure 1 Cytotoxicity of harmine in MCF-7 cells. MCF-7 cells were incubated with harmine at different concentrations (0 \u00b5M, 10 \u00b5M, 20 \u00b5M and 30 \u00b5M) and multiple time periods (24 h, 48 h, 72 h and 96 h). Cell metabolic activity was determined by MTT assay. Viability of vehicle-treated samples was set 100%: 24 h, white bars; 48 h, black bars; 72 h, hatched bars; 96 h, grey bars. Results are derived from two independent experiments performed in quadruplicate (mean \u00b1 SD). Figure 2 Harmine-induced senescence: SA- \u03b2-gal staining image of MCF-7 cells after harmine treatment. MCF-7 cells were firstly treated with 20 \u00b5M harmine for 48 h and 96 h, respectively. At the end of treatment, SA- \u03b2-gal staining was investigated following a standard protocol. All images were taken at 10 x magnification. Percentage of \u03b2-gal positive cells were quantified by ImageJ software. Graph established from two independent areas (mean \u00b1 S.D). p values indicate the significant difference in positive \u03b2-gal staining for the sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p \u2264 0.05; ***p \u2264 0.001. Figure 3 Effect of harmine on telomerase activity in MCF-7 cells. (A) MCF-7 cells were incubated with harmine at two concentrations (10 \u00b5M and 20 \u00b5M) for 24 h, 48 h, 72 h and 96 h. At the end of incubation, telomerase activity was evaluated by applying TRAP assay; the TRAP products were then separated on a 12% PAGE gel and their intensity (all bands) was quantified by using ImageJ software and values were plotted in (B): Ctr, vehicle control, black bar; cells treated with 10 \u00b5M of harmine, white bars; cells treated with 20 \u00b5M of harmine, dotted bars. Results derived from two independent experiments (mean \u00b1 S.D). p values indicate the significant changes in relative telomerase activity for the sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p <0.05; **p \u2264 0.01. Figure 4 Harmine inhibits telomerase expression in MCF-7 cells in a dose- and timedependent manner. (A) MCF-7 cells were incubated with harmine at final concentration of 10 \u00b5M and 20 \u00b5M, respectively, then total RNA was isolated and analyzed by using RT-PCR; (B) 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t MCF-7 cells were incubated with harmine at a final concentration of 20 \u00b5M for 48 h or 96 h, respectively, then total RNA was isolated and analyzed by RT-PCR. Figure 5 mRNA levels of hTERT, p21, and CDK2 in response to harmine treatment. MCF7 cells were exposed to harmine at a final concentration of 20 \u00b5M for 12 h, 24 h, 48 h and 96 h, then mRNA expression of each target gene was analyzed by real time PCR: hTERT, white bars; p21, black bars; CDK2, hatched bars. Results are representative of two independent experiments in triplicate (mean \u00b1 SD). p values measure significant changes in mRNA expression for the target gene treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p > 0.05; **p \u2264 0.01; ***p \u2264 0.001. Figure 6 Harmine induces a general DNA damage response by over-expressing p53/p21 and \u03b3H2AX. (A) MCF-7 cells were incubated with harmine at 20 \u00b5M for 24 h, 48 h and 96 h, then 25 \u00b5g of total protein extracted from cells after treatment of harmine or vehicle only was separated by PAGE and analyzed by western blotting; (B) the changes in protein level after the treatment were calculated with respect to vehicle controls (100%): p21, black bars; p53, white bars; \u03b3H2AX, grey bars; c-Myc, hatched bars. 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Table 1 Primers for RT-PCR PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Table 1 Primers for RT-PCR Gene primer sequence (5\u2019-3\u2019) \u03b2 \u2013 actin-f \u03b2 \u2013 actin-r CCTGGCACCCAGCACAAT GGGCCGGACTCGTCATAC 2007hTERT-f 2007hTERT-r ACGGCGACATGGAGAACAA CACTGTCTTCCGCAAGTTCAC p21-f p21-r TTTCTCTCGGCTCCCCATGT GCTGTATATTCAGCATTGTGGG Cdk2-f Cdk2-r CCTCCTGGGCTGCAAATA CAGAATCTCCAGGGAATAGGG p53-f p53-r TGCGTGTGGAGTATTTGGATG TGGTACAGTCAGAGCCAACCAG 1 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Figure 1 Cytotoxicity of harmine in MCF-7 cells MCF-7 cells were incubated with harmine at different concentrations (0 \u00b5M, 10 \u00b5M, 20 \u00b5M and 30 \u00b5M) and multiple time periods (24 h, 48 h, 72 h and 96 h). Cell metabolic activity was determined by MTT assay. Viability of vehicle-treated samples was set 100%: 24 h, white bars; 48 h, black bars; 72 h, hatched bars; 96 h, grey bars. Results are derived from two independent experiments performed in quadruplicate (mean \u00b1 SD). PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Figure 2 Harmine-induced senescence: SA- - gal staining image of MCF-7 cells after harmine \u03b2 treatment. MCF-7 cells were firstly treated with 20 \u00b5M harmine for 48 h and 96 h, respectively. At the end of treatment, SA- - gal staining was investigated following a standard protocol. All \u03b2 images were taken at 10 x magnification. Percentage of - gal positive cells were quantified \u03b2 by ImageJ software. Graph established from two independent areas (mean \u00b1 S.D). p values indicate the significant difference in positive - gal staining for the sample treated with \u03b2 harmine with respect to the vehicle treated controls. Unpaired t test: *p 0.05; ***p 0.001.\u2264 \u2264 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Figure 3 Effect of harmine on telomerase activity in MCF-7 cells (A) MCF-7 cells were incubated with harmine at two concentrations (10 \u00b5M and 20 \u00b5M) for 24 h, 48 h, 72 h and 96 h. At the end of incubation, telomerase activity was evaluated by applying TRAP assay; the TRAP products were then separated on a 12% PAGE gel and their intensity (all bands) was quantified by using ImageJ software and values were plotted in (B): Ctr, vehicle control, black bar; cells treated with 10 \u00b5M of harmine, white bars; cells treated with 20 \u00b5M of harmine, dotted bars. Results derived from two independent experiments (mean \u00b1 S.D). p values indicate the significant changes in relative telomerase activity for the sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p <0.05; **p 0.01.\u2264 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Figure 4 Harmine inhibits telomerase expression in MCF-7 cells in a dose- and time-dependent manner. (A) MCF-7 cells were incubated with harmine at final concentration of 10 \u00b5M and 20 \u00b5M, respectively, then total RNA was isolated and analyzed by using RT-PCR; (B) MCF-7 cells were incubated with harmine at a final concentration of 20 \u00b5M for 48 h or 96 h, respectively, then total RNA was isolated and analyzed by RT-PCR. PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Figure 5 mRNA levels of hTERT, p21, and CDK2 in response to harmine treatment. MCF-7 cells were exposed to harmine at a final concentration of 20 \u00b5M for 12 h, 24 h, 48 h and 96 h, then mRNA expression of each target gene was analyzed by real time PCR: hTERT, white bars; p21, black bars; CDK2, hatched bars. Results are representative of two independent experiments in triplicate (mean \u00b1 SD). p values measure significant changes in mRNA expression for the target gene treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p > 0.05; **p 0.01; ***p 0.001.\u2264 \u2264 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t Figure 6 Harmine induces a general DNA damage response byover-expressing p53/p21 and H \u03b3 acknowledgements : We thank Holger Sch\u00e4fer for helpful discussions. discussion : The indole alkaloid harmine exhibits multiple pharmacological properties in vivo and in vitro (Wink & van Wyk, 2008; Wink & Schimmer, 2010). Among other effects, harmine significantly arrests cell proliferation and induces cell death in a number of tumour cell lines. A dose- and time- dependent cytotoxicity of harmine could be confirmed in our experiments with MCF-7 cells (Fig. 1). Cytotoxicity can result from an adverse interaction of harmine and other alkaloids with one or more important targets present in a cell including DNA, RNA, or associated enzymes (Roberts & Wink, 1998; Wink & van Wyk, 2008; Wink, 2007). Harmine is known to intercalate DNA and through this it can cause mutations and DNA damage (Wink et al., 1998a; Wink & Schimmer, 2010). Through these interactions cell proliferation can be interrupted or cell death induced (Lansiaux et al., 2002; M\u00f6ller et al., 2007; Wink, 2007). In addition, the inhibition of cycline-dependent kinases such as CDK2 and CDK5 (Song et al., 2002) might also contribute to the cytotoxicity of harmine. Furthermore, it has been shown that harmine can repress cytochrome P450 activity (Tweedie et al., 1988) and selectively inhibit DNA topoisomerase (Funayama et al., 1996). Another mechanism for cytotoxicity of alkaloids might involve the intercalation of telomeres and the inhibition of telomerase. Several DNA-intercalating alkaloids, including berbamine (Ji et al., 2002), chelidonine (Noureini & Wink, 2009) and 9-hydroxyellipticine (Sasaki et al., 1992) could significantly inhibit telomerase activity which could lead to the 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t interruption of the genomic stability as well as cell growth arrest (Shay & Bacchetti, 1997). Because harmine is an intercalating alkaloid a possible telomerase inhibition was evaluated in this research. Indeed, as shown in our investigation, harmine induces a remarkable reduction of telomerase activity in MCF-7 cells as measured by TRAP (a PCR-based assay to detect telomerase activity) (Fig. 3). Harmine also triggers a significant inhibition of telomerase activity in Hela cells (unpublished result), the concentrations applied in both cell lines were very similar (20 \u00b5M in MCF-7 cells, 30 \u00b5M in HeLa cells). Under our experimental condition, we did not find the same senescent phenotype with HeLa cells. Also no down-regulation could be detected on hTERT mRNA expression although telomerase activity was significantly inhibited after harmine treatment. The mTOR pathway might be involved which needs to be further investigated. The regulation of telomerase activity involves various signalling pathways (Shay and Wright, 1996a,b). It is commonly accepted that the expression of hTERT is critical for telomerase activity (Meyerson et al., 1997; Nakamura et al., 1997; Bodnar et al., 1998). The transcription of hTERT mRNA was significantly down-regulated in response to harmine treatment (Fig. 4 & 5). The down-regulation became apparent about 24 h earlier than the reduction of telomerase activity (TRAP assay) whereas no decrease in telomerase activity could be seen at the same condition. Such an observation coincides with the report that telomerase activity has a half-life longer than 24 h in almost all cell lines (Holt et al., 1997) whereas the half-life of the hTERT mRNA is about 2 h (Xu et al. 1999). Our hypothesis is harmine does not induce a direct inhibition on telomerase activity in MCF-7 cells but through down-regulating hTERT at transcriptional level. Another factor could be c-Myc which plays an important role in the transcriptional regulation of hTERT (Wu et al., 1999; Kyo et al., 2000). Overexpressed c-Myc protein leads to a remarkable E-box dependent increase in the hTERT promoter activity. Moreover, c-Myc could induce the expression of endogenous hTERT mRNA and telomerase activity in normal human cells (Wang et al. 1998; Greenberg et al. 1999). In our experiments, a time-dependent downregulation of cMyc was observed (Fig. 6) which might be correlated with the down-regulation of hTERT (Fig. 4). The tumor suppressor protein p53 is a nuclear transcription factor that accumulates in response to cellular stress, including DNA damage and oncogene activation (Wink, 2007). P53 protein is a critical determinant of the cell fate following certain types of DNA damage (Clarke et al., 1993; Liu & Kulesz-Martin, 2001). DNA damage triggers transcriptional transactivation of p53 target genes such as p21, leading to cell cycle arrest, senescence and/or apoptosis (Levine, 1997; Farnebo et al., 2010). P53 is essential for both senescence and apoptosis pathways, 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t specifically, in cell cycle arrest at G1 phase; p53 enhances p21 transcription, which in turn inhibits CDK activity. As reported, overexpressed wild-type p53 could inhibit telomerase activity via down-regulating hTERT transcription (Gollahon et al., 1998; Kusumoto et al., 1999). However, such a reduction cannot be directly achieved by p53 because the binding site between p53 and the hTERT promoter is missing (Gualberto and Baldwin, 1995; Bargonetti et al., 1997; Kyo et al., 2000). In our study, p53 was overexpressed after harmine exposure (Fig. 6); an induction could be detected as early as 12 h after treatment. This enhancement was accompanied by an increase of mRNA level as well as on protein level of p21 (Fig. 5 & 6). The question arises as to whether the inhibition of hTERT is a consequence of overexpressed p53 or harmine-induced cell cycle arrest. Harmine is able to interrupt DNA replication in vivo (Boeira et al., 2001; Moura et al., 2007; Sasaki et al., 1992) and in vitro (Wink, 2007). Other studies have found that harmine induces chromosome aberrations and produces DNA breakage in cultured mammalian cells (Boeira et al., 2001). Moreover, harmine can inhibit topoisomerase I (Cao et al., 2005; Wink et al., 1998), therefore blocking an important enzyme which can repair DNA damage and fix mutations (Sasaki et al., 1992; Wang, 1998). The accumulation of phosphorylated H2AX (\u03b3H2AX) is an early sign of genomic events reflecting induction of double strands breaks (Tanaka et al. , 2007; Albino et al., 2004). In this study, an increase of \u03b3H2AX was detected at 24 h after the treatment of harmine. Our hypothesis is that intercalating harmine induces a general timedependent DNA damage response. Instead of triggering apoptosis, such damage apparently initiates an accelerated senescence in MCF cells (Fig. 2). Similar results were obtained in other studies, in which MCF-7 cells failed to undergo apoptotic cell death but underwent accelerated senescence after the exposure of ionizing radiation and adriamycin (Elmore et al., 2002a; Jones et al., 2005). On the other hand, when p53 protein was diminished by infection with HPV-E6 oncogene, MCF-7-E6 cells entered delayed programmed cell death (Elmore et al., 2002b). A number of studies have demonstrated that replicative senescence induced by telomere shortening and DNA damage-induced senescence leads to a very similar cell morphology (Oh et al., 2001; Gorbunova et al., 2002; Gewirtz et al., 2008). Both events involve the participation of p53, the mechanisms however, remain unclear. In conclusion, the treatment of MCF-7 cells with the DNA intercalator harmine induces a time-dependent general DNA damage response. P53 senses the damage and stops cell cycle progression by transactivating p21. Alternatively, the overexpressed p53 could directly inhibit 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t hTERT transcription. The inhibited telomerase could then facilitate cell growth arrest in MCF-7 cells, and directs damaged cells into accelerated senescence and not into apoptotic pathway. 2 ax. : (A) MCF-7 cells were incubated with harmine at 20 \u00b5 M for 24 h, 48 h and 96 h, then 25 \u00b5g of total protein extracted from cells after treatment of harmine or vehicle only was separated by PAGE and analyzed by western blotting; (B) changes in protein level after the treatment were calculated with respect to vehicle controls (100%): p21, black bars; p53, white bars; H 2 AX,\u03b3 grey bars; c-Myc, hatched bars. PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t chemicals : Harmine (C13H12N2O; MW 212.25) was purchased from Sigma-Aldrich. The stock solution was prepared in dimethyl sulfoxide (DMSO) with a concentration of 100 \u00b5M, which was stored at -20 \u00b0C. Cell culture and harmine treatment Human breast cancer cell line MCF-7 was kindly provided by Prof. Dr. S. W\u00f6lfl (IPMB, Heidelberg University). Cells were routinely cultured in Dulbeccos's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 \u00b5g/ml streptomycin (Invitrogen, USA), and 10% heat-inactivated fetal bovine serum. Cells were incubated at 37 \u00b0C in 5% CO2 and 100% humidity. Twenty-four h after plating, cells were treated with harmine and incubated up to different time points depending on the experimental design. A DMSO control was included in each analysis. Metabolic cell activity assay Ten microliters of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/ml) prepared in phosphate-buffered saline buffer were added to each well after given time intervals; all plates were gently shaken by hands for several times and incubated at 37 \u00b0C for 3\u20134 h. At the end of incubation, the solution with MTT was carefully removed and 100 \u03bcl of lysis buffer (20% SDS in 1:1 N,N\u2032-dimethylformamide : water/ 2% acetic acid/ 2.5% HCl 1 M) was added per well. Then the plates were placed on a shaker at low speed for 30 min at room temperature to ensure that the formazan formed was completely solubilized; it was quantified by measuring the OD value at 570 nm in a 96-well plate reader (Spectramax 384 plus, Molecular Devices). telomerase activity assay : Proteins were isolated from MCF-7 cells with CHAPS lysis buffer (10 mM Tris-HCl, pH 7.5; 1 mM MgCl2, 1 mM EDTA, 0.5% CHAPS, and 10% glycerol). All buffers and solutions were prepared with RNase-free water. Telomerase activities was determined with 0.5 \u00b5g protein extract using TRAP as described previously (Kim et al., 1994b). Briefly, the protein extract was firstly incubated with TS primer (5\u2019AATCCGTCGAGCAGAGTT 3\u2019) for 30 min at 30 \u00b0C, after addition 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t of CX primer (5\u2019 AATCCCATTCCCATTCCCATTCCC 3\u2019) the products were then subjected to PCR-amplification at 94 \u00b0C for 30 sec, and at 60 \u00b0C 30 sec for 29 cycles. The PCR products were separated on a 12.5% polyacrylamide gel by PAGE. The gel was stained with SYBR green (Amersham Biosciences) and directly visualized under a UV-transilluminator. A 36-bp internal control was amplified to serve as a standard for the normalization of telomerase expression. The intensity of all bands were photo scanned using ImageJ software (National Institutes of Health, America), the relative telomerase activity (RTA) was determined by the formula (Betts and King 1999). (s-b)/ ics RTA = \u00d7100 (pc-b)/icpc Values are expressed as % of the control sample. Reverse transcription PCR of endogenous hTERT expression Total RNA was isolated from MCF-7 cells using RNeasy kit (Qiagen, Germany). One \u00b5g of total RNA was reverse transcribed in a 20 \u00b5L reaction volume using ImProm-IITM Reverse Transcription System (Promega, Germany). A 1 \u00b5L aliquot of cDNA was analyzed by PCR amplifications. Global hTERT was amplified using the primer 5\u2019- CGGAAGAGTGTCTGGAGCAA-3\u2019 paired with 5\u2019-GGATGAAGCGGAGTCTGGA-3\u2019; variant-hTERT was amplified with the primer 5\u2019-GCCTCAGCTGTACTTTGTCAA-3\u2019 paired with 5\u2019-CGCAAACAGCTTGTTCTCCATGTC-3'. The thermocycling conditions for global hTERT amplification were: 94 \u00b0C 2 min followed by 33 cycles of 94 \u00b0C for 45 sec, 60 \u00b0C for 45 sec, and 72 \u00b0C for 90 sec; for variant hTERT amplification, the thermocycling conditions were: 94 \u00b0C for 2 min followed by 35 cycles of 94 \u00b0C for 15 sec, 60 \u00b0C for 15 sec, and 72 \u00b0C for 30 sec. The housekeeping gene \u03b2-actin was amplified with the primer 5'-CCTGGCACCCAGCACAAT-3' paired with 5'-GGGCCGGACTCGTCATAC-3' under the same thermocycling conditions described above with only 20 cycles. Amplified products (global hTERT: 145-bp; variant hTERT: full length variant, 457-bp; \u03b1 variant, 421-bp; and \u03b2 variant 275-bp; \u03b2-actin: 143-bp) were separated by gel electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. Semi-quantitative PCR analysis s: Intensity of sample pc: Intensity of positive control b: Intensity of background ic: Intensity of internal control 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 PeerJ reviewing PDF | (v2013:05:541:2:0:NEW 7 Sep 2013) R ev ie w in g M an us cr ip t One microliter of cDNA was applied in 10 \u00b5L PCR reaction in capillaries containing 1 \u00d7 SYBR Green Master Mix (ABgene), 0.3 \u00b5M of each primer. A non-template control was included as the negative control. The PCR reaction was performed in LightCycler3 (Roche, Germany) with initial10 min denaturation at 95 \u00baC, then followed with 45 cycles: 95 \u00b0C 10 sec; 60 \u00b0C 10 sec. All crossing point (cp) values were assessed by using REST software relative to the expression of \u03b2actin. Primers which were used in Real-Time PCR are listed in Table 1. \u03b2-Galactosidase staining MCF-7 cells were incubated with harmine for 48 h or 96 h before \u03b2-galactosidase activity was determined. Then cells were washed twice in PBS and fixed in fixation solution containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min. The fixation solution was removed by washing the cells twice in PBS, and then the staining solution was added. Cells were then incubated at 37 \u00b0C in a CO2 free environment for 8 h. The percentage of positively stained cells was determined after counting three random fields of 100 cells each. Representative microscopic fields were photographed under a 20x objective. Western blot analysis for p53 and p21waf-1 proteins MCF-7 cells were treated with 20 \u00b5M harmine for multiple time points (12, 24, 48, and 96 h) prior to lysing the cells in Nonidet-P40 (NP40) lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP40, and 10% glycerol). The constitutive levels of p53 and p21waf1 were assessed with respect to isogenic untreated MCF-7 cultures. Protein concentration was firstly determined with standard Bradford assay (Bradford 1976), then a 25 \u00b5g aliquot of the protein extract was separated on a 12% of SDS-PAGE and transferred onto a PVDF membrane (Millipore, Germany) by electroblotting. A standard blotting protocol was then performed using p53 (DO1, Santa Cruz Biotech, Germany) and a p21waf-1 monoclonal (BD Biosciences, Germany) antibody followed by horseradish peroxidase-conjugated anti-mouse IgG (Dianova GmbH, Germany). An enhanced chemiluminescent reaction (ECL Reagent, Amersham) was applied for the detection. harmine induces an over-expression of p53 and of of p21 : We had shown before that harmine arrested MCF-7 cell growth and induced senescence (Fig. 1 & 2). In order to define the mechanism of harmine-induced cell arrest a serial of immunoblot analyses were performed (Fig. 6A). MCF-7 cells were cultured with harmine in a final concentration of 20 \u00b5M and cell samples were collected at different time points (24, 48, and 96 h). An enhanced expression of the phosphorylated H2AX (\u03b3H2AX) protein was detected after harmine treatment (Fig 6B). An overexpressed p53 protein was identified by immunoblot analysis as early as 24 h accompanied by an increased p21 protein. c-Myc is a known transcriptional enhancer of hTERT expression. In our investigation, c-Myc was apparently down-regulated (Fig. 6B). Compared with the changes in other genes, the decrease of c-Myc was more moderate in response to the treatment with harmine.",
    "v2_text": "materials & methods : results : Harmine is cytotoxic to MCF-7 cells in a dose- and time-dependent manner and induces accelerated senescence The cytotoxicity of harmine in MCF-7 cells is shown in Fig. 1. Cell viability at various time points was determined by MTT assay. The results indicate that harmine arrests cell growth in a dose- and time-dependent manner. Concentrations of 20 and 30 \u00b5M harmine significantly reduced cell growth after 48 to 96 h. Concentrations between 10 and 20 \u00b5M did not influence viability of MCF-7 cells within the first 24 h, and were therefore used in the subsequent experiments. 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 7 In the next step we tried to study whether senescent cells could be identified in response to harmine treatment. In MCF-7 cells, harmine arrests cell proliferation and induces a senescence morphology. \u03b2-Galactosidase activity, as a senescence marker, was detectable as early as 2 d after treatment with harmine, and became intense and expressed in virtually every cell of the culture at day 4 (Fig. 2). Cells, which were \u03b2-galactosidase positive, were larger in size or multinucleated (indicated with arrows), both of which are morphological features indicative of a senescent state. The SA-\u03b2-gal staining was not detected or barely detected in untreated control cells. figure captions : Figure 1 Cytotoxicity of harmine in MCF-7 cells. MCF-7 cells were incubated with harmine at different concentrations (0 \u00b5M, 10 \u00b5M, 20 \u00b5M and 30 \u00b5M) and multiple time periods (24 h, 48 h, 72 h and 96 h). Cell metabolic activity was determined by MTT assay. Viability of vehicle-treated samples was set 100%: 24 h, white bars; 48 h, black bars; 72 h, hatched bars; 96 h, grey bars. Results are derived from two independent experiments performed in quadruplicate (mean \u00b1 SD). Figure 2 Harmine-induced senescence: SA- \u03b2-gal staining image of MCF-7 cells after harmine treatment. MCF-7 cells were firstly treated with 20 \u00b5M harmine for 48 h and 96 h, respectively. At the end of treatment, SA- \u03b2-gal staining was investigated following a standard protocol. All images were taken at 10 x magnification. Percentage of \u03b2-gal positive cells were quantified by ImageJ software. Graph established from two independent areas (mean \u00b1 S.D). p values indicate the significant difference in positive \u03b2-gal staining for the sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p \u2264 0.05; ***p \u2264 0.001. Figure 3 Effect of harmine on telomerase activity in MCF-7 cells. (A) MCF-7 cells were incubated with harmine at two concentrations (10 \u00b5M and 20 \u00b5M) for 24 h, 48 h, 72 h and 96 h. At the end of incubation, telomerase activity was evaluated by applying TRAP assay; the TRAP products were then separated on a 12% PAGE gel and their intensity (all bands) was quantified by using ImageJ software and values were plotted in (B): Ctr, vehicle control, black bar; cells treated with 10 \u00b5M of harmine, white bars; cells treated with 20 \u00b5M of harmine, dotted bars. Results derived from two independent experiments (mean \u00b1 S.D). p values indicate the significant changes in relative telomerase activity for the sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p <0.05; **p \u2264 0.01. 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 R e v ie w in g M a n u s c ri p t 18 MCF-7 cells were incubated with harmine at a final concentration of 20 \u00b5M for 48 h or 96 h, respectively, then total RNA was isolated and analyzed by RT-PCR. Figure 5 mRNA levels of hTERT, p21, and CDK2 in response to harmine treatment. MCF7 cells were exposed to harmine at a final concentration of 20 \u00b5M for 12 h, 24 h, 48 h and 96 h, then mRNA expression of each target gene was analyzed by real time PCR: hTERT, white bars; p21, black bars; CDK2, hatched bars. Results are representative of two independent experiments in triplicate (mean \u00b1 SD). p values measure significant changes in mRNA expression for the target gene treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p > 0.05; **p \u2264 0.01; ***p \u2264 0.001. Figure 6 Harmine induces a general DNA damage response by over-expressing p53/p21 and \u03b3H2AX. (A) MCF-7 cells were incubated with harmine at 20 \u00b5M for 24 h, 48 h and 96 h, then 25 \u00b5g of total protein extracted from cells after treatment of harmine or vehicle only was separated by PAGE and analyzed by western blotting; (B) the changes in protein level after the treatment were calculated with respect to vehicle controls (100%): p21, black bars; p53, white bars; \u03b3H2AX, grey bars; c-Myc, hatched bars. 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 19 Table 1(on next page) Table 1 Primers for RT-PCR PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 20 Table 1 Primers for RT-PCR Gene primer sequence (5\u2019-3\u2019) \u03b2 \u2013 actin-f \u03b2 \u2013 actin-r CCTGGCACCCAGCACAAT GGGCCGGACTCGTCATAC 2007hTERT-f 2007hTERT-r ACGGCGACATGGAGAACAA CACTGTCTTCCGCAAGTTCAC p21-f p21-r TTTCTCTCGGCTCCCCATGT GCTGTATATTCAGCATTGTGGG Cdk2-f Cdk2-r CCTCCTGGGCTGCAAATA CAGAATCTCCAGGGAATAGGG p53-f p53-r TGCGTGTGGAGTATTTGGATG TGGTACAGTCAGAGCCAACCAG 1 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 21 Figure 1 Cytotoxicity of harmine in MCF-7 cells MCF-7 cells were incubated with harmine at different concentrations (0 \u00b5M, 10 \u00b5M, 20 \u00b5M and 30 \u00b5M) and multiple time periods (24 h, 48 h, 72 h and 96 h). Cell metabolic activity was determined by MTT assay. Viability of vehicle-treated samples was set 100%: 24 h, white bars; 48 h, black bars; 72 h, hatched bars; 96 h, grey bars. Results are derived from two independent experiments performed in quadruplicate (mean \u00b1 SD). PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 22 Figure 2 Harmine-induced senescence: SA- \u03b2-gal staining image of MCF-7 cells after harmine treatment. MCF-7 cells were firstly treated with 20 \u00b5M harmine for 48 h and 96 h, respectively. At the end of treatment, SA- \u03b2-gal staining was investigated following a standard protocol. All images were taken at acknowledgements : We thank Holger Sch\u00e4fer for helpful discussions. discussion : The indole alkaloid harmine exhibits multiple pharmacological properties in vivo and in vitro (Wink & van Wyk, 2008; Wink & Schimmer, 2010). Among other effects, harmine significantly arrests cell proliferation and induces cell death in a number of tumour cell lines. A dose- and time- dependent cytotoxicity of harmine could be confirmed in our experiments with MCF-7 cells (Fig. 1). Cytotoxicity can result from an adverse interaction of harmine and other alkaloids with one or more important targets present in a cell including DNA, RNA, or associated enzymes (Roberts & Wink, 1998; Wink & van Wyk, 2008; Wink, 2007). Harmine is known to intercalate DNA and through this it can cause mutations and DNA damage (Wink et al., 1998a; Wink & Schimmer, 2010). Through these interactions cell proliferation can be interrupted or cell death induced (Lansiaux et al., 2002; M\u00f6ller et al., 2007; Wink, 2007). In addition, the inhibition of cycline-dependent kinases such as CDK2 and CDK5 (Song et al., 2002) might also contribute to the cytotoxicity of harmine. Furthermore, it has been shown that harmine can repress cytochrome P450 activity (Tweedie et al., 1988) and selectively inhibit DNA topoisomerase (Funayama et al., 1996). Another mechanism for cytotoxicity of alkaloids might involve the intercalation of telomeres and the inhibition of telomerase. Several DNA-intercalating alkaloids, including berbamine (Ji et al., 2002), chelidonine (Noureini & Wink, 2009) and 9-hydroxyellipticine (Sasaki et al., 1992) could significantly inhibit telomerase activity which could lead to the interruption of the genomic stability as well as cell growth arrest (Shay & Bacchetti, 1997). 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 9 Because harmine is an intercalating alkaloid a possible telomerase inhibition was evaluated in this research. Indeed, as shown in our investigation, harmine induces a remarkable reduction of telomerase activity in MCF-7 cells as measured by TRAP (a PCR-based assay to detect telomerase activity) (Fig. 3). Harmine also triggers a significant inhibition of telomerase activity in Hela cells (unpublished result), the concentrations applied in both cell lines were very similar (20 \u00b5M in MCF-7 cells, 30 \u00b5M in HeLa cells). Under our experimental condition, we did not find the same senescent phenotype with HeLa cells. Also no down-regulation could be detected on hTERT mRNA expression although telomerase activity was significantly inhibited after harmine treatment. The mTOR pathway might be involved which needs to be further investigated. The regulation of telomerase activity involves various signalling pathways (Shay and Wright, 1996a,b). It is commonly accepted that the expression of hTERT is critical for telomerase activity (Meyerson et al., 1997; Nakamura et al., 1997; Bodnar et al., 1998). The transcription of hTERT mRNA was significantly down-regulated in response to harmine treatment (Fig. 4 & 5). The down-regulation became apparent about 24 h earlier than the reduction of telomerase activity (TRAP assay) whereas no decrease in telomerase activity could be seen at the same condition. Such an observation coincides with the report that telomerase activity has a half-life longer than 24 h in almost all cell lines (Holt et al., 1997) whereas the half-life of the hTERT mRNA is about 2 h (Xu et al. 1999). Our hypothesis is harmine does not induce a direct inhibition on telomerase activity in MCF-7 cells but through down-regulating hTERT at transcriptional level. Another factor could be c-Myc which plays an important role in the transcriptional regulation of hTERT (Wu et al., 1999; Kyo et al., 2000). Overexpressed c-Myc protein leads to a remarkable E-box dependent increase in the hTERT promoter activity. Moreover, c-Myc could induce the expression of endogenous hTERT mRNA and telomerase activity in normal human cells (Wang et al. 1998; Greenberg et al. 1999). In our experiments, a time-dependent down-regulation of cMyc was observed (Fig. 6) which might be correlated with the down-regulation of hTERT (Fig. 4). The tumor suppressor protein p53 is a nuclear transcription factor that accumulates in response to cellular stress, including DNA damage and oncogene activation (Wink, 2007). P53 protein is a critical determinant of the cell fate following certain types of DNA damage (Clarke et al., 1993; Liu & Kulesz-Martin, 2001). DNA damage triggers transcriptional transactivation of p53 target genes such as p21, leading to cell cycle arrest, senescence and/or apoptosis (Levine, 1997; Farnebo et al., 2010). P53 is essential for both senescence and apoptosis pathways, specifically, in cell cycle arrest at G1 phase; p53 enhances p21 transcription, which in turn inhibits 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 10 CDK activity. As reported, overexpressed wild-type p53 could inhibit telomerase activity via down-regulating hTERT transcription (Gollahon et al., 1998; Kusumoto et al., 1999). However, such a reduction cannot be directly achieved by p53 because the binding site between p53 and the hTERT promoter is missing (Gualberto and Baldwin, 1995; Bargonetti et al., 1997; Kyo et al., 2000). In our study, p53 was overexpressed after harmine exposure (Fig. 6); an induction could be detected as early as 12 h after treatment. This enhancement was accompanied by an increase of mRNA level as well as on protein level of p21 (Fig. 5 & 6). The question arises as to whether the inhibition of hTERT is a consequence of overexpressed p53 or harmine-induced cell cycle arrest. Harmine is able to interrupt DNA replication in vivo (Boeira et al., 2001; Moura et al., 2007; Sasaki et al., 1992) and in vitro (Wink, 2007). Other studies have found that harmine induces chromosome aberrations and produces DNA breakage in cultured mammalian cells (Boeira et al., 2001). Moreover, harmine can inhibit topoisomerase I (Cao et al., 2005; Wink et al., 1998), therefore blocking an important enzyme which can repair DNA damage and fix mutations (Sasaki et al., 1992; Wang, 1998). The accumulation of phosphorylated H2AX (\u03b3H2AX) is an early sign of genomic events reflecting induction of double strands breaks (Tanaka et al. , 2007; Albino et al., 2004). In this study, an increase of \u03b3H2AX was detected at 24 h after the treatment of harmine. Our hypothesis is that intercalating harmine induces a general timedependent DNA damage response. Instead of triggering apoptosis, such damage apparently initiates an accelerated senescence in MCF cells (Fig. 2). Similar results were obtained in other studies, in which MCF-7 cells failed to undergo apoptotic cell death but underwent accelerated senescence after the exposure of ionizing radiation and adriamycin (Elmore et al., 2002a; Jones et al., 2005). On the other hand, when p53 protein was diminished by infection with HPV-E6 oncogene, MCF-7-E6 cells entered delayed programmed cell death (Elmore et al., 2002b). A number of studies have demonstrated that replicative senescence induced by telomere shortening and DNA damage-induced senescence leads to a very similar cell morphology (Oh et al., 2001; Gorbunova et al., 2002; Gewirtz et al., 2008). Both events involve the participation of p53, the mechanisms however, remain unclear. In conclusion, the treatment of MCF-7 cells with the DNA intercalator harmine induces a time-dependent general DNA damage response. P53 senses the damage and stops cell cycle progression by transactivating p21. Alternatively, the overexpressed p53 could directly inhibit hTERT transcription. The inhibited telomerase could then facilitate cell growth arrest in MCF-7 cells, and directs damaged cells into accelerated senescence and not into apoptotic pathway. 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 11 rna was isolated and analyzed by rt-pcr. : PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 27 Figure 5 mRNA levels of hTERT, p21, and CDK2 in response to harmine treatment. MCF-7 cells were exposed to harmine at a final concentration of 20 \u00b5M for 12 h, 24 h, 48 h and 96 h, then mRNA expression of each target gene was analyzed by real time PCR: hTERT, white bars; p21, black bars; CDK2, hatched bars. Results are representative of two independent experiments in triplicate (mean \u00b1 SD). p values measure significant changes in mRNA expression for the target gene treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p > 0.05; **p \u2264 0.01; ***p \u2264 0.001. PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 28 Figure 6 Harmine induces a general DNA damage response byover-expressing p53/p21 and \u03b3H 2 AX. (A) MCF-7 cells were incubated with harmine at 20 \u00b5 M for 24 h, 48 h and 96 h, then 25 \u00b5g of total protein extracted from cells after treatment of harmine or vehicle only was separated by PAGE and analyzed by western blotting; (B) changes in protein level after the treatment were calculated with respect to vehicle controls (100%): p21, black bars; p53, white bars; \u03b3H 2 AX, grey bars; c-Myc, hatched bars. PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 29 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 10 x magnification. percentage of \u03b2-gal positive cells were quantified by imagej software. graph : established from two independent areas (mean \u00b1 S.D). p values indicate the significant difference in positive \u03b2-gal staining for the sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p \u2264 0.05; ***p \u2264 0.001. PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 23 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 24 Figure 3 Effect of harmine on telomerase activity in MCF-7 cells (A) MCF-7 cells were incubated with harmine at two concentrations (10 \u00b5M and 20 \u00b5M) for 24 h, 48 h, 72 h and 96 h. At the end of incubation, telomerase activity was evaluated by applying TRAP assay; the TRAP products were then separated on a 12% PAGE gel and their intensity (all bands) was quantified by using ImageJ software and values were plotted in (B): Ctr, vehicle control, black bar; cells treated with 10 \u00b5M of harmine, white bars; cells treated with 20 \u00b5M of harmine, dotted bars. Results derived from two independent experiments (mean \u00b1 S.D). p values indicate the significant changes in relative telomerase activity for the sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p <0.05; **p \u2264 0.01. PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 25 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 26 Figure 1 Harmine inhibits telomerase expression in MCF-7 cells in a dose- and time-dependent manner. (A) MCF-7 cells were incubated with harmine at final concentration of 10 \u00b5M and 20 \u00b5M, respectively, then total RNA was isolated and analyzed by using RT-PCR; (B) MCF-7 cells were incubated with harmine at a final concentration of 20 \u00b5M for 48 h or 96 h, respectively, then total chemicals : Harmine (C13H12N2O; MW 212.25) was purchased from Sigma-Aldrich. The stock solution was prepared in dimethyl sulfoxide (DMSO) with a concentration of 100 \u00b5M, which was stored at -20 \u00b0C. Cell culture and harmine treatment Human breast cancer cell line MCF-7 was kindly provided by Prof. Dr. S. W\u00f6lfl (IPMB, Heidelberg University). Cells were routinely cultured in Dulbeccos's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 \u00b5g/ml streptomycin (Invitrogen, USA), and 10% heat-inactivated fetal bovine serum. Cells were incubated at 37 \u00b0C in 5% CO2 and 100% humidity. Twenty-four h after plating, cells were treated with harmine and incubated up to different time points depending on the experimental design. A DMSO control was included in each analysis. Metabolic cell activity assay Ten microliters of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/ml) prepared in phosphate-buffered saline buffer were added to each well after given time intervals; all plates were gently shaken by hands for several times and incubated at 37 \u00b0C for 3\u20134 h. At the end of incubation, the solution with MTT was carefully removed and 100 \u03bcl of lysis buffer (20% SDS in 1:1 N,N\u2032-dimethylformamide : water/ 2% acetic acid/ 2.5% HCl 1 M) was added per well. Then the plates were placed on a shaker at low speed for 30 min at room temperature to ensure that the formazan formed was completely solubilized; it was quantified by measuring the OD value at 570 nm in a 96-well plate reader (Spectramax 384 plus, Molecular Devices). telomerase activity assay : Proteins were isolated from MCF-7 cells with CHAPS lysis buffer (10 mM Tris-HCl, pH 7.5; 1 mM MgCl2, 1 mM EDTA, 0.5% CHAPS, and 10% glycerol). All buffers and solutions were prepared with RNase-free water. Telomerase activities was determined with 0.5 \u00b5g protein extract using TRAP as described previously (Kim et al., 1994b). Briefly, the protein extract was firstly incubated with TS primer (5\u2019AATCCGTCGAGCAGAGTT 3\u2019) for 30 min at 30 \u00b0C, after addition of CX primer (5\u2019 AATCCCATTCCCATTCCCATTCCC 3\u2019) the products were then subjected to PCR-amplification at 94 \u00b0C for 30 sec, and at 60 \u00b0C 30 sec for 29 cycles. The PCR products were separated on a 12.5% polyacrylamide gel by PAGE. The gel was stained with SYBR green 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 5 (Amersham Biosciences) and directly visualized under a UV-transilluminator. A 36-bp internal control was amplified to serve as a standard for the normalization of telomerase expression. The intensity of all bands were photo scanned using ImageJ software (National Institutes of Health, America), the relative telomerase activity (RTA) was determined by the formula (Betts and King 1999). (s-b)/ ics RTA = \u00d7100 (pc-b)/icpc Values are expressed as % of the control sample. Reverse transcription PCR of endogenous hTERT expression Total RNA was isolated from MCF-7 cells using RNeasy kit (Qiagen, Germany). One \u00b5g of total RNA was reverse transcribed in a 20 \u00b5L reaction volume using ImProm-IITM Reverse Transcription System (Promega, Germany). A 1 \u00b5L aliquot of cDNA was analyzed by PCR amplifications. Global hTERT was amplified using the primer 5\u2019- CGGAAGAGTGTCTGGAGCAA-3\u2019 paired with 5\u2019-GGATGAAGCGGAGTCTGGA-3\u2019; variant-hTERT was amplified with the primer 5\u2019-GCCTCAGCTGTACTTTGTCAA-3\u2019 paired with 5\u2019-CGCAAACAGCTTGTTCTCCATGTC-3'. The thermocycling conditions for global hTERT amplification were: 94 \u00b0C 2 min followed by 33 cycles of 94 \u00b0C for 45 sec, 60 \u00b0C for 45 sec, and 72 \u00b0C for 90 sec; for variant hTERT amplification, the thermocycling conditions were: 94 \u00b0C for 2 min followed by 35 cycles of 94 \u00b0C for 15 sec, 60 \u00b0C for 15 sec, and 72 \u00b0C for 30 sec. The housekeeping gene \u03b2-actin was amplified with the primer 5'-CCTGGCACCCAGCACAAT-3' paired with 5'-GGGCCGGACTCGTCATAC-3' under the same thermocycling conditions described above with only 20 cycles. Amplified products (global hTERT: 145-bp; variant hTERT: full length variant, 457-bp; \u03b1 variant, 421-bp; and \u03b2 variant 275-bp; \u03b2-actin: 143-bp) were separated by gel electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. Telomerase activity of MCF-7 cells, treated with or without harmine, was evaluated as evidenced by the TRAP assay. A decreased telomerase activity (Fig. 3A) was detected after the incubation of the cells with 20 \u00b5M harmine. Telomerase activity was inhibited by 81.87% after 96 h treatment as compared to the untreated control (Fig. 3B). Treatment at a lower concentration, e.g., 10 \u00b5M, did not show a significant reduction of telomerase activity. Expression analysis of human TERT splicing variants by RT-PCR. RT-PCR analysis was performed with a pair of primers which covers all hTERT transcripts. In theory, four hTERT variants should be expected under the identical PCR conditions at the same time (full length hTERT with 457 bp; \u03b1 variant with 421 bp; \u03b2 variant with 275 bp and \u03b1+ \u03b2 variant with 239 bp). However, in our investigation the \u03b1+ \u03b2 variant could not be detected (Fig. 4A). Treatment of the cells with 20 \u00b5M harmine significantly down-regulated all hTERT subunits in a time-dependent manner (Fig. 4B). Expression analysis of human TERT Human hTERT, p21, and CDK2 mRNA transcripts were examined by real time PCR. Data were analyzed by Relative Expression Software Tool (REST2008). PCR efficiency was set as 2 as indicated by the software and the housekeeping gene \u03b2-actin was regarded as a control. A significant up-regulation of p21 mRNA was detected as early as 12 h after harmine treatment. The mRNA concentration was 3.9 fold higher than that of the untreated control, and the upregulation became 6.5 fold with respect to the control after 96 h (Fig. 5). Within the first 24 h of treatment, no alteration on of hTERT and CDK2 mRNA expression was detected, while an extended treatment up to 48 h showed that a significant down-regulation was observed for these two genes. Harmine induces an over-expression of wt p53, independent of p21 induction 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 8 We had shown before that harmine arrested MCF-7 cell growth and induced senescence (Fig. 1 & 2). In order to define the mechanism of harmine-induced cell arrest a serial of immunoblot analyses were performed (Fig. 6A). MCF-7 cells were cultured with harmine in a final concentration of 20 \u00b5M and cell samples were collected at different time points (24, 48, and 96 h). An enhanced expression of the phosphorylated H2AX (\u03b3H2AX) protein was detected after harmine treatment (Fig 6B). An overexpressed p53 protein was identified by immunoblot analysis as early as 24 h accompanied by an increased p21 protein. c-Myc is a known transcriptional enhancer of hTERT expression. In our investigation, c-Myc was apparently down-regulated (Fig. 6B). Compared with the changes in other genes, the decrease of c-Myc was more moderate in response to the treatment with harmine. semi-quantitative pcr analysis : One microliter of cDNA was applied in 10 \u00b5L PCR reaction in capillaries containing 1 \u00d7 SYBR Green Master Mix (ABgene), 0.3 \u00b5M of each primer. A non-template control was included as the negative control. The PCR reaction was performed in LightCycler3 (Roche, Germany) with initial10 min denaturation at 95 \u00baC, then followed with 45 cycles: 95 \u00b0C 10 sec; 60 \u00b0C 10 sec. All s: Intensity of sample pc: Intensity of positive control b: Intensity of background ic: Intensity of internal control 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 PeerJ reviewing PDF | (v2013:05:541:1:1:REVIEW 15 Aug 2013) R e v ie w in g M a n u s c ri p t 6 crossing point (cp) values were assessed by using REST software relative to the expression of \u03b2actin. Primers which were used in Real-Time PCR are listed in Table 1. \u03b2-Galactosidase staining MCF-7 cells were incubated with harmine for 48 h or 96 h before \u03b2-galactosidase activity was determined. Then cells were washed twice in PBS and fixed in fixation solution containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min. The fixation solution was removed by washing the cells twice in PBS, and then the staining solution was added. Cells were then incubated at 37 \u00b0C in a CO2 free environment for 8 h. The percentage of positively stained cells was determined after counting three random fields of 100 cells each. Representative microscopic fields were photographed under a 20x objective. Western blot analysis for p53 and p21waf-1 proteins MCF-7 cells were treated with 20 \u00b5M harmine for multiple time points (12, 24, 48, and 96 h) prior to lysing the cells in Nonidet-P40 (NP40) lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP40, and 10% glycerol). The constitutive levels of p53 and p21waf1 were assessed with respect to isogenic untreated MCF-7 cultures. Protein concentration was firstly determined with standard Bradford assay (Bradford 1976), then a 25 \u00b5g aliquot of the protein extract was separated on a 12% of SDS-PAGE and transferred onto a PVDF membrane (Millipore, Germany) by electroblotting. A standard blotting protocol was then performed using p53 (DO1, Santa Cruz Biotech, Germany) and a p21waf-1 monoclonal (BD Biosciences, Germany) antibody followed by horseradish peroxidase-conjugated anti-mouse IgG (Dianova GmbH, Germany). An enhanced chemiluminescent reaction (ECL Reagent, Amersham) was applied for the detection.",
    "v3_text": "results : Harmine is cytotoxic to MCF-7 cells in a dose- and time-dependent manner and induces accelerated senescence 5 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t The cytotoxicity of harmine in MCF-7 cells is shown in Fig. 1. Cell viability at various time points was determined by MTT assay. The results indicate that harmine arrests cell growth in a dose- and time-dependent manner. Concentrations of 20 and 30 \u00b5M harmine significantly reduced cell growth after 48 to 96 h. Concentrations between 10 and 20 \u00b5M did not influence viability of MCF-7 cells within the first 24 h, and were therefore used in the subsequent experiments. In the next step we tried to study whether senescent cells could be identified in response to harmine treatment. In MCF-7 cells, harmine arrests cell proliferation and induces a senescence morphology. \u03b2-Galactosidase activity, as a senescence marker, was detectable as early as 2 d after treatment with harmine, and became intense and expressed in virtually every cell of the culture at day 4 (Fig. 2). Cells, which were \u03b2-galactosidase positive, were larger in size or multinucleated (indicated with arrows), both of which are morphological features indicative of a senescent state. The SA-\u03b2-gal staining was not detected or barely detected in untreated control cells. Telomerase activity Telomerase activity of MCF-7 cells, treated with or without harmine, was evaluated as evidenced by the TRAP assay. A decreased telomerase activity (Fig. 3A) was detected after the incubation of the cells with 20 \u00b5M harmine. Telomerase activity was inhibited by 81.87% after 96 h treatment as compared to the untreated control (Fig. 3B). Treatment at a lower concentration, e.g., 10 \u00b5M, did not show a significant reduction of telomerase activity. Expression analysis of human TERT splicing variants by RT-PCR. RT-PCR analysis was performed with a pair of primers which covers all hTERT transcripts. In theory, four hTERT variants should be expected under the identical PCR conditions at the same time (full length hTERT with 457 bp; \u03b1 variant with 421 bp; \u03b2 variant with 275 bp and \u03b1+ \u03b2 variant with 239 bp). However, in our investigation the \u03b1+ \u03b2 variant could not be detected (Fig. 4A). Treatment of the cells with 20 \u00b5M harmine significantly down-regulated all hTERT subunits in a time-dependent manner (Fig. 4B). Expression analysis of human TERT Human hTERT, p21, and CDK2 mRNA transcripts were examined by real time PCR. Data were analyzed by Relative Expression Software Tool (REST2008). PCR efficiency was set as 2 as indicated by the software and the housekeeping gene \u03b2-actin was regarded as a control. A 6 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t significant up-regulation of p21 mRNA was detected as early as 12 h after harmine treatment. The mRNA concentration was 3.9 fold higher than that of the untreated control, and the up-regulation became 6.5 fold with respect to the control after 96 h (Fig. 5). Within the first 24 h of treatment, no alteration on of hTERT and CDK2 mRNA expression was detected, while an extended treatment up to 48 h showed that a significant down-regulation was observed for these two genes. Down-regulation of hTERT mRNA, mediated by wt p53, is independent of p21 induction We had shown before that harmine arrested MCF-7 cell growth and induced senescence (Fig. 1 & 2). In order to define the mechanism of harmine-induced cell arrest a serial of immunoblot analyses were performed (Fig. 6A). MCF-7 cells were cultured with harmine in a final concentration of 20 \u00b5M and cell samples were collected at different time points (24, 48, and 96 h). An enhanced expression of the phosphorylated H2AX (\u03b3H2AX) protein was detected after harmine treatment (Fig 6B). An overexpressed p53 protein was identified by immunoblot analysis as early as 24 h accompanied by an increased p21 protein. c-Myc is a known transcriptional enhancer of hTERT expression. In our investigation, c-Myc was apparently down-regulated (Fig. 6B). Compared with the changes in other genes, the decrease of c-Myc was more moderate in response to the treatment with harmine. discussion : The indole alkaloid harmine exhibits multiple pharmacological properties in vivo and in vitro (Wink & van Wyk, 2008; Wink & Schimmer, 2010). Among other effects, harmine significantly arrests cell proliferation and induces cell death in a number of tumour cell lines. A dose- and time- dependent cytotoxicity of harmine could be confirmed in our experiments with MCF-7 cells (Fig. 1). Cytotoxicity can result from an adverse interaction of harmine and other alkaloids with one or more important targets present in a cell including DNA, RNA, or associated enzymes (Roberts & Wink, 1998; Wink & van Wyk, 2008; Wink, 2007). Harmine is known to intercalate DNA and through this it can cause mutations and DNA damage (Wink et al., 1998a; Wink & Schimmer, 2010). Through these interactions cell proliferation can be interrupted or cell death induced (Lansiaux et al., 2002; M\u00f6ller et al., 2007; Wink, 2007). In addition, the inhibition of 7 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t cycline-dependent kinases such as CDK2 and CDK5 (Song et al., 2002) might also contribute to the cytotoxicity of harmine. Furthermore, it has been shown that harmine can repress cytochrome P450 activity (Tweedie et al., 1988) and selectively inhibit DNA topoisomerase (Funayama et al., 1996). Another mechanism for cytotoxicity of alkaloids might involve the intercalation of telomeres and the inhibition of telomerase. Several DNA-intercalating alkaloids, including berbamine (Ji et al., 2002), chelidonine (Noureini & Wink, 2009) and 9-hydroxyellipticine (Sasaki et al., 1992) could significantly inhibit telomerase activity which could lead to the interruption of the genomic stability as well as cell growth arrest (Shay & Bacchetti, 1997). Because harmine is an intercalating alkaloid a possible telomerase inhibition was evaluated in this research. Indeed, as shown in our investigation, harmine induces a remarkable reduction of telomerase activity in MCF-7 cells as measured by TRAP (a PCR-based assay to detect telomerase activity) (Fig. 3). Harmine also triggers a significant inhibition of telomerase activity in Hela cells (unpublished result), the concentrations applied in both cell lines were very similar (20 \u00b5M in MCF-7 cells, 30 \u00b5M in HeLa cells). The regulation of telomerase activity involves various signalling pathways (Shay and Wright 1996a,b). It is commonly accepted that the expression of hTERT is critical for telomerase activity (Meyerson et al., 1997; Nakamura et al., 1997; Bodnar et al., 1998). The transcription of hTERT mRNA was significantly down-regulated in response to harmine treatment (Fig. 4 & 5). The down-regulation became apparent about 24 h earlier than the reduction of telomerase activity (TRAP assay). This observation strongly indicates that the harmine-induced inhibition of telomerase activity is initiated by the suppression of hTERT transcription. Another factor could be c-Myc which plays an important role in the transcriptional regulation of hTERT (Wu et al., 1999; Kyo et al., 2000). Overexpressed c-Myc protein leads to a remarkable E-box dependent increase in the hTERT promoter activity. Moreover, c-Myc could induce the expression of endogenous hTERT mRNA and telomerase activity in normal human cells (Wang et al. 1998; Greenberg et al. 1999). In our experiments, a time-dependent down-regulation of cMyc was observed (Fig. 6) which might be correlated with the down-regulation of hTERT (Fig. 4). The tumor suppressor protein P53 is a nuclear transcription factor that accumulates in response to cellular stress, including DNA damage and oncogene activation (Wink, 2007). p53 8 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t protein is a critical determinant of the cell fate following certain types of DNA damage (Clarke et al., 1993; Liu & Kulesz-Martin, 2001). DNA damage triggers transcriptional transactivation of p53 target genes such as p21, leading to cell cycle arrest, senescence and/or apoptosis (Levine, 1997; Farnebo et al., 2010). P53 is essential for both senescence and apoptosis pathways, specifically, in cell cycle arrest at G1 phase; p53 enhances p21 transcription, which in turn inhibits CDK activity. As reported, overexpressed wild-type p53 could inhibit telomerase activity via down-regulating hTERT transcription (Gollahon et al., 1998; Kusumoto et al., 1999). However, such a reduction cannot be directly achieved by p53 because the binding site between p53 and the hTERT promoter is missing (Gualberto and Baldwin, 1995; Bargonetti et al., 1997; Kyo et al., 2000). In our study, p53 was overexpressed after harmine exposure (Fig. 6); an induction could be detected as early as 12 h after treatment. This enhancement was accompanied by an increase of mRNA level as well as on protein level of p21 (Fig. 5 & 6). The question arises as to whether the inhibition of hTERT is a consequence of overexpressed p53 or harmine-induced cell cycle arrest. Harmine is able to interrupt DNA replication in vivo (Boeira et al., 2001; Moura et al., 2007; Sasaki et al., 1992) and in vitro (Wink, 2007). Other studies have found that harmine induces chromosome aberrations and produces DNA breakage in cultured mammalian cells (Boeira et al., 2001). Moreover, harmine can inhibit topoisomerase I (Cao et al., 2005; Wink et al., 1998), therefore blocking an important enzyme which can repair DNA damage and fix mutations (Sasaki et al., 1992; Wang, 1998). The accumulation of phosphorylated H2AX (\u03b3H2AX) is an early sign of genomic events reflecting induction of double strands breaks (Tanaka et al. , 2007; Albino et al., 2004). In this study, an increase of \u03b3H2AX was detected at 24 h after the treatment of harmine. Our hypothesis is that intercalating harmine induces a general time-dependent DNA damage response. Instead of triggering apoptosis, such damage apparently initiates an accelerated senescence in MCF cells (Fig. 2). Similar results were obtained in other studies, in which MCF-7 cells failed to undergo apoptotic cell death but underwent accelerated senescence after the exposure of ionizing radiation and adriamycin (Elmore et al., 2002a; Jones et al., 2005). On the other hand, when p53 protein was diminished by infection with HPV-E6 oncogene, MCF-7-E6 cells entered delayed programmed cell death (Elmore et al., 2002b). A number of studies have demonstrated that replicative senescence induced by telomere shortening and DNA damage-induced senescence leads to a very similar cell morphology (Oh et al., 2001; 9 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t Gorbunova et al., 2002; Gewirtz et al., 2008). Both events involve the participation of p53, the mechanisms however, remain unclear. In conclusion, the treatment of MCF-7 cells with the DNA intercalator harmine induces a time-dependent general DNA damage response. p53 senses the damage and stops cell cycle progression by transactivating p21. Alternatively, the overexpressed p53 could directly inhibit hTERT transcription. The inhibited telomerase could then facilitate cell growth arrest in MCF-7 cells, and directs damaged cells into accelerated senescence and not into apoptotic pathway. acknowledgements : We thank Holger Sch\u00e4fer for helpful discussions. materials & methods : figure captions : Figure 1 Cytotoxicity of harmine in MCF-7 cells. MCF-7 cells were incubated with harmine at different concentrations (0 \u00b5M, 10 \u00b5M, 20 \u00b5M and 30 \u00b5M) and multiple time periods (24 h, 48 h, 72 h and 96 h). Cell viability was determined by MTT assay. Viability of vehicle-treated samples was set 100%: 24 h, white bars; 48 h, black bars; 72 h, hatched bars; 96 h, grey bars. Results are derived from two independent experiments performed in quadruplicate (mean \u00b1 SD). Figure 2 Harmine-induced senescence: SA- \u03b2-gal staining image of MCF-7 cells after harmine treatment. MCF-7 cells were firstly treated with 20 \u00b5M harmine for 48 h and 96 h, respectively. At the end of treatment, SA- \u03b2-gal staining was investigated following a standard protocol. All images were taken at 10 x magnification. Figure 3 Effect of harmine on telomerase activity in MCF-7 cells. (A) MCF-7 cells were incubated with harmine at two concentrations (10 \u00b5M and 20 \u00b5M) for 24 h, 48 h, 72 h and 96 h. At the end of incubation, telomerase activity was evaluated by applying TRAP assay; the TRAP products were then separated on a 12% PAGE gel and their intensity (all bands) was quantified by using ImageJ software and values were plotted in (B): Ctr, vehicle control, black bar; cells treated with 10 \u00b5M of harmine, white bars; cells treated with 20 \u00b5M of harmine, dotted bars. Results derived from two independent experiments (mean \u00b1 S.D). p values indicate the significant changes in relative telomerase activity for the 15 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t sample treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p > 0.05; **p \u2264 0.01. Figure 4 Harmine inhibits telomerase expression in MCF-7 cells in a dose- and time-dependent manner. (A) MCF-7 cells were incubated with harmine at final concentration of 10 \u00b5M and 20 \u00b5M, respectively, then total RNA was isolated and analyzed by using RT-PCR; (B) MCF-7 cells were incubated with harmine at a final concentration of 20 \u00b5M for 48 h or 96 h, respectively, then total RNA was isolated and analyzed by RT-PCR. Figure 5 mRNA levels of hTERT, p21, and CDK2 in response to harmine treatment. MCF-7 cells were exposed to harmine at a final concentration of 20 \u00b5M for 12 h, 24 h, 48 h and 96 h, then mRNA expression of each target gene was analyzed by real time PCR: hTERT, white bars; p21, black bars; CDK2, hatched bars. Results are representative of two independent experiments in triplicate (mean \u00b1 SD). p values measure significant changes in mRNA expression for the target gene treated with harmine with respect to the vehicle treated controls. Unpaired t test: *p > 0.05; **p \u2264 0.01; ***p \u2264 0.001. Figure 6 Harmine induces a general DNA damage response by over-expressing p53/p21 and \u03b3H2AX. (A) MCF-7 cells were incubated with harmine at 20 \u00b5M for 24 h, 48 h and 96 h, then 25 \u00b5g of total protein extracted from cells after treatment of harmine or vehicle only was separated by PAGE and analyzed by western blotting; (B) the changes in protein level after the treatment were calculated with respect to vehicle controls (100%): p21, black bars; p53, white bars; \u03b3H2AX, grey bars; c-Myc, hatched bars. FIGURES 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 R ev ie w in g M an us cr ip t 17 488 489 490 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t Figure 2 18 491 492 493 494 495 496 497 498 499 500 501 502 503 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t Figure 3 (A) (B) 19 504 505 506 507 508 509 510 511 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t Figure 4 20 A B 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 R ev ie w in g M an us cr ip t 22 A B 545 546 547 548 549 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Table 1 Primers for RT-PCR PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t Table 1 Primers for RT-PCR Gene primer sequence (5\u2019-3\u2019) \u03b2 \u2013 actin-f \u03b2 \u2013 actin-r CCTGGCACCCAGCACAAT GGGCCGGACTCGTCATAC 2007hTERT-f 2007hTERT-r ACGGCGACATGGAGAACAA CACTGTCTTCCGCAAGTTCAC p21-f p21-r TTTCTCTCGGCTCCCCATGT GCTGTATATTCAGCATTGTGGG Cdk2-f Cdk2-r CCTCCTGGGCTGCAAATA CAGAATCTCCAGGGAATAGGG p53-f p53-r TGCGTGTGGAGTATTTGGATG TGGTACAGTCAGAGCCAACCAG PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t cell viability assay : Ten microliters of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/ml) prepared in phosphate-buffered saline buffer were added to each well after given time intervals; all plates were gently shaken by hands for several times and incubated at 37 \u00b0C for 3\u20134 h. At the end of incubation, the solution with MTT was carefully removed and 100 \u03bcl of lysis buffer (20% SDS in 1:1 N,N\u2032-dimethylformamide : water/ 2% acetic acid/ 2.5% HCl 1 M) was added per well. Then the plates were placed on a shaker at low speed for 30 min at room temperature to ensure that the formazan formed was completely solubilized; it was quantified by measuring the OD value at 570 nm in a 96-well plate reader (Spectramax 384 plus, Molecular Devices). chemicals : Harmine (C13H12N2O; MW 212.25) was purchased from Sigma-Aldrich. The stock solution was prepared in dimethyl sulfoxide (DMSO) with a concentration of 100 \u00b5M, which was stored at -20 \u00b0C. Cell culture and harmine treatment Human breast cancer cell line MCF-7 was kindly provided by Prof. Dr. S. W\u00f6lfl (IPMB, Heidelberg University). Cells were routinely cultured in Dulbeccos's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 \u00b5g/ml streptomycin (Invitrogen, USA), and 10% heat-inactivated fetal bovine serum. Cells were incubated at 37 \u00b0C in 5% CO2 and 100% humidity. Twenty-four h after plating, cells were treated with harmine and incubated up to different time points depending on the experimental design. A DMSO control was included in each analysis. telomerase activity assay : Proteins were isolated from MCF-7 cells with CHAPS lysis buffer (10 mM Tris-HCl, pH 7.5; 1 mM MgCl2, 1 mM EDTA, 0.5% CHAPS, and 10% glycerol). All buffers and solutions were prepared with RNase-free water. Telomerase activities was determined with 0.5 \u00b5g protein extract using TRAP as described previously (Kim et al., 1994b). Briefly, the protein extract was firstly incubated with TS primer (5\u2019AATCCGTCGAGCAGAGTT 3\u2019) for 30 min at 30 \u00b0C, after addition of CX primer (5\u2019 AATCCCATTCCCATTCCCATTCCC 3\u2019) the products were then subjected to PCR-amplification at 94 \u00b0C for 30 sec, and at 60 \u00b0C 30 sec for 29 cycles. The PCR 3 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t products were separated on a 12.5% polyacrylamide gel by PAGE. The gel was stained with SYBR green (Amersham Biosciences) and directly visualized under a UV-transilluminator. A 36-bp internal control was amplified to serve as a standard for the normalization of telomerase expression. The intensity of all bands were photo scanned using ImageJ software (National Institutes of Health, America), the relative telomerase activity (RTA) was determined by the formula (Betts and King 1999). (s-b)/ ics RTA = \u00d7100 (pc-b)/icpc Values are expressed as % of the control sample. Reverse transcription PCR of endogenous hTERT expression Total RNA was isolated from MCF-7 cells using RNeasy kit (Qiagen, Germany). One \u00b5g of total RNA was reverse transcribed in a 20 \u00b5L reaction volume using ImProm-IITM Reverse Transcription System (Promega, Germany). A 1 \u00b5L aliquot of cDNA was analyzed by PCR amplifications. Global hTERT was amplified using the primer 5\u2019- CGGAAGAGTGTCTGGAGCAA-3\u2019 paired with 5\u2019-GGATGAAGCGGAGTCTGGA-3\u2019; variant-hTERT was amplified with the primer 5\u2019-GCCTCAGCTGTACTTTGTCAA-3\u2019 paired with 5\u2019-CGCAAACAGCTTGTTCTCCATGTC-3'. The thermocycling conditions for global hTERT amplification were: 94 \u00b0C 2 min followed by 33 cycles of 94 \u00b0C for 45 sec, 60 \u00b0C for 45 sec, and 72 \u00b0C for 90 sec; for variant hTERT amplification, the thermocycling conditions were: 94 \u00b0C for 2 min followed by 35 cycles of 94 \u00b0C for 15 sec, 60 \u00b0C for 15 sec, and 72 \u00b0C for 30 sec. The housekeeping gene \u03b2-actin was amplified with the primer 5'-CCTGGCACCCAGCACAAT-3' paired with 5'-GGGCCGGACTCGTCATAC-3' under the same thermocycling conditions described above with only 20 cycles. Amplified products (global hTERT: 145-bp; variant hTERT: full length variant, 457-bp; \u03b1 variant, 421-bp; and \u03b2 variant 275-bp; \u03b2-actin: 143-bp) were separated by gel electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. Semi-quantitative PCR analysis 4 s: Intensity of sample pc: Intensity of positive control b: Intensity of background ic: Intensity of internal control 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 PeerJ reviewing PDF | (v2013:05:541:0:2:NEW 31 May 2013) R ev ie w in g M an us cr ip t One microliter of cDNA was applied in 10 \u00b5L PCR reaction in capillaries containing 1 \u00d7 SYBR Green Master Mix (ABgene), 0.3 \u00b5M of each primer. A non-template control was included as the negative control. The PCR reaction was performed in LightCycler3 (Roche, Germany) with initial10 min denaturation at 95 \u00baC, then followed with 45 cycles: 95 \u00b0C 10 sec; 60 \u00b0C 10 sec. All crossing point (cp) values were assessed by using REST software relative to the expression of \u03b2-actin. Primers which were used in Real-Time PCR are listed in table 1. \u03b2-Galactosidase staining MCF-7 cells were incubated with harmine for 48 h or 96 h before \u03b2-galactosidase activity was determined. Then cells were washed twice in PBS and fixed in fixation solution containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min. The fixation solution was removed by washing the cells twice in PBS, and then the staining solution was added. Cells were then incubated at 37 \u00b0C in a CO2 free environment for 8 h. The percentage of positively stained cells was determined after counting three random fields of 100 cells each. Representative microscopic fields were photographed under a 20x objective. Western blot analysis for p53 and p21waf-1 proteins MCF-7 cells were treated with 20 \u00b5M harmine for multiple time points (12, 24, 48, and 96 h) prior to lysing the cells in Nonidet-P40 (NP40) lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP40, and 10% glycerol). The constitutive levels of p53 and p21waf1 were assessed with respect to isogenic untreated MCF-7 cultures. Protein concentration was firstly determined with standard Bradford assay (Bradford 1976), then a 25 \u00b5g aliquot of the protein extract was separated on a 12% of SDS-PAGE and transferred onto a PVDF membrane (Millipore, Germany) by electroblotting. A standard blotting protocol was then performed using p53 (DO1, Santa Cruz Biotech, Germany) and a p21waf-1 monoclonal (BD Biosciences, Germany) antibody followed by horseradish peroxidase-conjugated anti-mouse IgG (Dianova GmbH, Germany). An enhanced chemiluminescent reaction (ECL Reagent, Amersham) was applied for the detection.",
    "url": "https://peerj.com/articles/175/reviews/",
    "review_1": "Xiaolei Huang \u00b7 Sep 13, 2013 \u00b7 Academic Editor\nACCEPT\nI appreciate the detailed responses from the authors. The issues raised by reviewers have been appropriately addressed. I am happy to accept the manuscript.",
    "review_2": "Xiaolei Huang \u00b7 May 14, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nBoth reviewers think the paper is worthy of publication but needs some revisions. One reviewer suggests the second part (patterns in data reuse) of the paper could be shortened and more information on this aspect may need to be provided in the Introduction. Please try to address the reviewers' concerns about \"open access citation benefit\" and the usage of some terms including the maybe too positive \"boost\". Both reviewers raise questions about tables and figures used in the manuscript. I suggest the authors fully address the questions from the reviewers, and think about their thoughtful comments, some of which may be beyond the current manuscript.",
    "review_3": "Reviewer 1 \u00b7 May 13, 2013\nBasic reporting\nThe aims of the paper are to : a) assess whether making datasets public and available at time of article publication increases citations to the article and (b) describe the pattern of data reuse once data are made public. It does so using studies based on gene expression microarray data. The objectives of the paper are highly relevant since a clear demonstration that making data widely available provides authors with a citation benefit would be a strong incentive for scientists to do so.\nThe paper is well organized and clear, but the second objective was less convincingly introduced than the first one, although it makes an important part of the manuscript (especially in terms of number of figures). I found this second part a bit lengthy, and I suggest that the authors shorten it by removing data and/or figures (for example, Fig. 5 and 6) give the same basic information, and one of the two could be deleted while giving only results for the second one in the text.\nIsn\u2019t the term \u201cboost\u201d a bit too strong for a 9-10% higher citation rate? Wouldn\u2019t \u201cbenefit\u201d be more appropriate?\nSome minor points to modify in the text and/or figures:\n- p. 2, line 11: delete one of the \u201ccharacterized\u201d\n- p. 2, line 42: this repeats the sentence of p. 2 lines 13-17\n- p. 3, line 2: Table 1 does not show the content discussed in the text (attributes that correlate with citation rates);\n- p. 3, line 14: give a reference for MeSH terms\n- p. 3, line 37: add \"they\" between \"because\" and \"included\"\n- Fig. 2 and Table 3 give the same information: remove Table 3\n- p 7, line 16: chage \"two\" by \"to\"\nExperimental design\nThe first aim is addressed using a data set of 10,555 papers screened using full-text query method, but I could not find the number of papers in each category: with or without publicly available data. This information should be provided.\n- p. 4, line 7 : why switch to a different source of data here (use of ISI web of knowledge instead of Scopus: p. 2, lines 17-21)\nThe second aim (data reuse) is addressed using direct mentions to the data sets in papers where these are mentioned.\nOverall, the methods of data collection from the various sources and the treatment of data appear to have been correctly conducted.\nValidity of the findings\nThe analyses show that papers making microarray data sets publicly available are, on average, cited 9% more than those which do not. This impact is apparent only at least three years after publication. This finding is interesting, although the effect is weaker than previously shown with a more restricted data set. The trend found for data reuse shows that original authors tend to use their own data sets until approximately two years after these are published, while the use by third party authors tend to increase after these two years. The study also show that the number of data sets reused in any one paper tends to increase with time: most papers reused one or two data sets until 2005, and this number increases substantially thereafter.\nI found the last part of the result section (p. 8, lines 1-18) and figures 8 and 9 a bit overdone, all the more that this aspect of the paper is not properly introduced ealier, especially in the introduction.\nAdditional comments\nI would recommend shortening the second part of the result section pertaining to data reuse, which increases substantially the amount fo data presented in the paper and tends to dilute its main messages. Given that this aspect is only poorly introduced, shortening this part would certainly strengthen the paper.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Data reuse and the open data citation advantage (v0.1)\". PeerJ https://doi.org/10.7287/peerj.175v0.1/reviews/1",
    "review_4": "Iain Hrynaszkiewicz \u00b7 Apr 28, 2013\nBasic reporting\nI am not qualified to comprehensively review the methods and statistical analysis but my reading raised the following:\n\nIn the \u2018Primary analysis\u2019 section, is a table mislabelled or missing? The authors state:\n\u201cWe used a subset of the 124 attributes from (Piwowar, 2011d) previously shown or suspected to correlate with citation rate (Table 1).\u201d\nI expected to see the list of attributes but Table one is headed: \u201cProportion of sample published in most common journals\u201d\nIf so, this needs to be resolved before publication. I am also interested in the evidence on which the correlates are based. The authors mention the open access citation advantage a few times in their paper, for example. Have they assumed that open access papers receive more citations in the present analysis? The open access citation benefit is much debated. Furthermore, my understanding of the cited paper by Craig et al 2007 is that it documents *decreasing* evidence of a citation benefit for open access articles. I am not aware of a systematic review of the impact of open access on citations, but a bibliography of studies, which the authors may find relevant, is here http://opcit.eprints.org/oacitation-biblio.html\nExperimental design\nSee above.\nValidity of the findings\nThe results of this paper are welcome evidence of the benefits to individual researchers for sharing their research data. They should and will likely be used to develop further support and policy development for data sharing and will be much discussed on social media. However, I feel the authors should tone down the positivity of the language used to describe the citation increase in their conclusions (\u201crobust\u201d; a \u201cboost\u201d, etc), given the citation benefit is just 9%.\n\nI wondered if the authors could comment on if and how much their results could be generalised to other areas of research. Also, where should the research community\u2019s priorities be for establishing citation or other benefits to individuals from sharing their research? Which fields? Which benefits? We know, for example, that clinical trialists are willing to share their data but practical issues and fears over inappropriate reanalysis are more important barriers to sharing \u2013than lack of individual incentives/citations (Rathi et al. http://dx.doi.org/10.1136/bmj.e7570).\nAdditional comments\nI assume the opening quote is not intended to add colour to paper but to provide the opening part of the introduction. As such, I found the author quoting themselves at this length from a 6 year old study was not the best way to open a paper.\n\nThroughout the manuscript the authors refer to \u201copen data\u201d meaning, I assume in this context, data which are freely accessible on the web. However, there is growing recognition that \u201copen\u201d data means data which are available under legal terms \u2013 a license or waiver \u2013 which permit sharing and reuse with the minimum of barriers. Open data is about more than just accessibility. \u201cOpen\u201d data must be free to \u201cdownload, copy, analyse, re-process, pass them to software or use them for any other purpose without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself\u201d [http://pantonprinciples.org/] . To my knowledge the authors are aware of the appropriate use of \u201copen\u201d in open data and open access and I recommend they use an alternate term to \u201copen data\u201d in the present manuscript to avoid engendering confusion about the term \u201copen data\u201d.\n\nI\u2019m not convinced the account of the authors\u2019 problems in obtaining access to Scopus should be included in the current manuscript. It seems tangential to thrust of the manuscript and its analysis and would be more appropriate for a blog (I believe some of this was described on Piwowar\u2019s blog) or commentary, and would make the manuscript more focused. Also, elsewhere in the paper the authors note that Scopus now has an API for programmatic access. The authors have previously written about the problems faced by researchers in gaining access to the literature for research \u2013 text mining, citation analysis \u2013 elsewhere.\n\nI noticed a few typos (this is not an exhaustive list) e.g. \u201cThe lack of conventions and tool support for data Attribution\u201d; \u201cWhich types of data reuse are facilitates by robust data standards and which types are unaffected?\u201d\n\nI am aware one of the authors has previously discussed publicly the present study\u2019s data and preliminary results (http://researchremix.wordpress.com/2012/07/16/many-datasets-are-reused-not-just-an-elite-few/). Open science should not preclude papers being published, and the fact the authors discussed the data from this study before submission of their paper should have no influence on the paper\u2019s acceptance or rejection. However, best practice regarding potentially duplicate or overlapping publications would be to state in the paper and cite the previous publications, including blogs, posters etc, in the submitted manuscript.\n\nRecommendation to PeerJ staff regarding competing interests statements from authors. Please include these in the review version of the manuscript.\nCite this review as\nHrynaszkiewicz I (2013) Peer Review #2 of \"Data reuse and the open data citation advantage (v0.1)\". PeerJ https://doi.org/10.7287/peerj.175v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/175v0.2/submission",
    "pdf_2": "https://peerj.com/articles/175v0.1/submission",
    "all_reviews": "Review 1: Xiaolei Huang \u00b7 Sep 13, 2013 \u00b7 Academic Editor\nACCEPT\nI appreciate the detailed responses from the authors. The issues raised by reviewers have been appropriately addressed. I am happy to accept the manuscript.\nReview 2: Xiaolei Huang \u00b7 May 14, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nBoth reviewers think the paper is worthy of publication but needs some revisions. One reviewer suggests the second part (patterns in data reuse) of the paper could be shortened and more information on this aspect may need to be provided in the Introduction. Please try to address the reviewers' concerns about \"open access citation benefit\" and the usage of some terms including the maybe too positive \"boost\". Both reviewers raise questions about tables and figures used in the manuscript. I suggest the authors fully address the questions from the reviewers, and think about their thoughtful comments, some of which may be beyond the current manuscript.\nReview 3: Reviewer 1 \u00b7 May 13, 2013\nBasic reporting\nThe aims of the paper are to : a) assess whether making datasets public and available at time of article publication increases citations to the article and (b) describe the pattern of data reuse once data are made public. It does so using studies based on gene expression microarray data. The objectives of the paper are highly relevant since a clear demonstration that making data widely available provides authors with a citation benefit would be a strong incentive for scientists to do so.\nThe paper is well organized and clear, but the second objective was less convincingly introduced than the first one, although it makes an important part of the manuscript (especially in terms of number of figures). I found this second part a bit lengthy, and I suggest that the authors shorten it by removing data and/or figures (for example, Fig. 5 and 6) give the same basic information, and one of the two could be deleted while giving only results for the second one in the text.\nIsn\u2019t the term \u201cboost\u201d a bit too strong for a 9-10% higher citation rate? Wouldn\u2019t \u201cbenefit\u201d be more appropriate?\nSome minor points to modify in the text and/or figures:\n- p. 2, line 11: delete one of the \u201ccharacterized\u201d\n- p. 2, line 42: this repeats the sentence of p. 2 lines 13-17\n- p. 3, line 2: Table 1 does not show the content discussed in the text (attributes that correlate with citation rates);\n- p. 3, line 14: give a reference for MeSH terms\n- p. 3, line 37: add \"they\" between \"because\" and \"included\"\n- Fig. 2 and Table 3 give the same information: remove Table 3\n- p 7, line 16: chage \"two\" by \"to\"\nExperimental design\nThe first aim is addressed using a data set of 10,555 papers screened using full-text query method, but I could not find the number of papers in each category: with or without publicly available data. This information should be provided.\n- p. 4, line 7 : why switch to a different source of data here (use of ISI web of knowledge instead of Scopus: p. 2, lines 17-21)\nThe second aim (data reuse) is addressed using direct mentions to the data sets in papers where these are mentioned.\nOverall, the methods of data collection from the various sources and the treatment of data appear to have been correctly conducted.\nValidity of the findings\nThe analyses show that papers making microarray data sets publicly available are, on average, cited 9% more than those which do not. This impact is apparent only at least three years after publication. This finding is interesting, although the effect is weaker than previously shown with a more restricted data set. The trend found for data reuse shows that original authors tend to use their own data sets until approximately two years after these are published, while the use by third party authors tend to increase after these two years. The study also show that the number of data sets reused in any one paper tends to increase with time: most papers reused one or two data sets until 2005, and this number increases substantially thereafter.\nI found the last part of the result section (p. 8, lines 1-18) and figures 8 and 9 a bit overdone, all the more that this aspect of the paper is not properly introduced ealier, especially in the introduction.\nAdditional comments\nI would recommend shortening the second part of the result section pertaining to data reuse, which increases substantially the amount fo data presented in the paper and tends to dilute its main messages. Given that this aspect is only poorly introduced, shortening this part would certainly strengthen the paper.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Data reuse and the open data citation advantage (v0.1)\". PeerJ https://doi.org/10.7287/peerj.175v0.1/reviews/1\nReview 4: Iain Hrynaszkiewicz \u00b7 Apr 28, 2013\nBasic reporting\nI am not qualified to comprehensively review the methods and statistical analysis but my reading raised the following:\n\nIn the \u2018Primary analysis\u2019 section, is a table mislabelled or missing? The authors state:\n\u201cWe used a subset of the 124 attributes from (Piwowar, 2011d) previously shown or suspected to correlate with citation rate (Table 1).\u201d\nI expected to see the list of attributes but Table one is headed: \u201cProportion of sample published in most common journals\u201d\nIf so, this needs to be resolved before publication. I am also interested in the evidence on which the correlates are based. The authors mention the open access citation advantage a few times in their paper, for example. Have they assumed that open access papers receive more citations in the present analysis? The open access citation benefit is much debated. Furthermore, my understanding of the cited paper by Craig et al 2007 is that it documents *decreasing* evidence of a citation benefit for open access articles. I am not aware of a systematic review of the impact of open access on citations, but a bibliography of studies, which the authors may find relevant, is here http://opcit.eprints.org/oacitation-biblio.html\nExperimental design\nSee above.\nValidity of the findings\nThe results of this paper are welcome evidence of the benefits to individual researchers for sharing their research data. They should and will likely be used to develop further support and policy development for data sharing and will be much discussed on social media. However, I feel the authors should tone down the positivity of the language used to describe the citation increase in their conclusions (\u201crobust\u201d; a \u201cboost\u201d, etc), given the citation benefit is just 9%.\n\nI wondered if the authors could comment on if and how much their results could be generalised to other areas of research. Also, where should the research community\u2019s priorities be for establishing citation or other benefits to individuals from sharing their research? Which fields? Which benefits? We know, for example, that clinical trialists are willing to share their data but practical issues and fears over inappropriate reanalysis are more important barriers to sharing \u2013than lack of individual incentives/citations (Rathi et al. http://dx.doi.org/10.1136/bmj.e7570).\nAdditional comments\nI assume the opening quote is not intended to add colour to paper but to provide the opening part of the introduction. As such, I found the author quoting themselves at this length from a 6 year old study was not the best way to open a paper.\n\nThroughout the manuscript the authors refer to \u201copen data\u201d meaning, I assume in this context, data which are freely accessible on the web. However, there is growing recognition that \u201copen\u201d data means data which are available under legal terms \u2013 a license or waiver \u2013 which permit sharing and reuse with the minimum of barriers. Open data is about more than just accessibility. \u201cOpen\u201d data must be free to \u201cdownload, copy, analyse, re-process, pass them to software or use them for any other purpose without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself\u201d [http://pantonprinciples.org/] . To my knowledge the authors are aware of the appropriate use of \u201copen\u201d in open data and open access and I recommend they use an alternate term to \u201copen data\u201d in the present manuscript to avoid engendering confusion about the term \u201copen data\u201d.\n\nI\u2019m not convinced the account of the authors\u2019 problems in obtaining access to Scopus should be included in the current manuscript. It seems tangential to thrust of the manuscript and its analysis and would be more appropriate for a blog (I believe some of this was described on Piwowar\u2019s blog) or commentary, and would make the manuscript more focused. Also, elsewhere in the paper the authors note that Scopus now has an API for programmatic access. The authors have previously written about the problems faced by researchers in gaining access to the literature for research \u2013 text mining, citation analysis \u2013 elsewhere.\n\nI noticed a few typos (this is not an exhaustive list) e.g. \u201cThe lack of conventions and tool support for data Attribution\u201d; \u201cWhich types of data reuse are facilitates by robust data standards and which types are unaffected?\u201d\n\nI am aware one of the authors has previously discussed publicly the present study\u2019s data and preliminary results (http://researchremix.wordpress.com/2012/07/16/many-datasets-are-reused-not-just-an-elite-few/). Open science should not preclude papers being published, and the fact the authors discussed the data from this study before submission of their paper should have no influence on the paper\u2019s acceptance or rejection. However, best practice regarding potentially duplicate or overlapping publications would be to state in the paper and cite the previous publications, including blogs, posters etc, in the submitted manuscript.\n\nRecommendation to PeerJ staff regarding competing interests statements from authors. Please include these in the review version of the manuscript.\nCite this review as\nHrynaszkiewicz I (2013) Peer Review #2 of \"Data reuse and the open data citation advantage (v0.1)\". PeerJ https://doi.org/10.7287/peerj.175v0.1/reviews/2\nReview 5: nan\nReview 6: \n",
    "LLM_extracted_review": "['Review 1: I appreciate the detailed responses from the authors. The issues raised by reviewers have been appropriately addressed. I am happy to accept the manuscript.\\n\\nReview 2: Both reviewers think the paper is worthy of publication but needs some revisions. One reviewer suggests the second part (patterns in data reuse) of the paper could be shortened and more information on this aspect may need to be provided in the Introduction. Please try to address the reviewers\\' concerns about \"open access citation benefit\" and the usage of some terms including the maybe too positive \"boost\". Both reviewers raise questions about tables and figures used in the manuscript. I suggest the authors fully address the questions from the reviewers, and think about their thoughtful comments, some of which may be beyond the current manuscript.\\n\\nReview 3: The aims of the paper are to: a) assess whether making datasets public and available at the time of article publication increases citations to the article and (b) describe the pattern of data reuse once data are made public. The objectives of the paper are highly relevant since a clear demonstration that making data widely available provides authors with a citation benefit would be a strong incentive for scientists to do so. The paper is well organized and clear, but the second objective was less convincingly introduced than the first one, although it makes an important part of the manuscript (especially in terms of number of figures). I found this second part a bit lengthy, and I suggest that the authors shorten it by removing data and/or figures (for example, Fig. 5 and 6) give the same basic information, and one of the two could be deleted while giving only results for the second one in the text. Isn\u2019t the term \u201cboost\u201d a bit too strong for a 9-10% higher citation rate? Wouldn\u2019t \u201cbenefit\u201d be more appropriate? Some minor points to modify in the text and/or figures: - p. 2, line 11: delete one of the \u201ccharacterized\u201d - p. 2, line 42: this repeats the sentence of p. 2 lines 13-17 - p. 3, line 2: Table 1 does not show the content discussed in the text (attributes that correlate with citation rates); - p. 3, line 14: give a reference for MeSH terms - p. 3, line 37: add \"they\" between \"because\" and \"included\" - Fig. 2 and Table 3 give the same information: remove Table 3 - p 7, line 16: change \"two\" by \"to\" The first aim is addressed using a data set of 10,555 papers screened using full-text query method, but I could not find the number of papers in each category: with or without publicly available data. This information should be provided. - p. 4, line 7: why switch to a different source of data here (use of ISI web of knowledge instead of Scopus: p. 2, lines 17-21) The second aim (data reuse) is addressed using direct mentions to the data sets in papers where these are mentioned. Overall, the methods of data collection from the various sources and the treatment of data appear to have been correctly conducted. The analyses show that papers making microarray data sets publicly available are, on average, cited 9% more than those which do not. This impact is apparent only at least three years after publication. This finding is interesting, although the effect is weaker than previously shown with a more restricted data set. The trend found for data reuse shows that original authors tend to use their own data sets until approximately two years after these are published, while the use by third party authors tends to increase after these two years. The study also shows that the number of data sets reused in any one paper tends to increase with time: most papers reused one or two data sets until 2005, and this number increases substantially thereafter. I found the last part of the result section (p. 8, lines 1-18) and figures 8 and 9 a bit overdone, all the more that this aspect of the paper is not properly introduced earlier, especially in the introduction. I would recommend shortening the second part of the result section pertaining to data reuse, which increases substantially the amount of data presented in the paper and tends to dilute its main messages. Given that this aspect is only poorly introduced, shortening this part would certainly strengthen the paper.\\n\\nReview 4: In the \u2018Primary analysis\u2019 section, is a table mislabelled or missing? The authors state: \u201cWe used a subset of the 124 attributes from (Piwowar, 2011d) previously shown or suspected to correlate with citation rate (Table 1).\u201d I expected to see the list of attributes but Table one is headed: \u201cProportion of sample published in most common journals.\u201d If so, this needs to be resolved before publication. I am also interested in the evidence on which the correlates are based. The authors mention the open access citation advantage a few times in their paper, for example. Have they assumed that open access papers receive more citations in the present analysis? The open access citation benefit is much debated. Furthermore, my understanding of the cited paper by Craig et al 2007 is that it documents *decreasing* evidence of a citation benefit for open access articles. I am not aware of a systematic review of the impact of open access on citations, but a bibliography of studies, which the authors may find relevant, is here http://opcit.eprints.org/oacitation-biblio.html. The results of this paper are welcome evidence of the benefits to individual researchers for sharing their research data. They should and will likely be used to develop further support and policy development for data sharing and will be much discussed on social media. However, I feel the authors should tone down the positivity of the language used to describe the citation increase in their conclusions (\u201crobust\u201d; a \u201cboost\u201d, etc), given the citation benefit is just 9%. I wondered if the authors could comment on if and how much their results could be generalized to other areas of research. Also, where should the research community\u2019s priorities be for establishing citation or other benefits to individuals from sharing their research? Which fields? Which benefits? We know, for example, that clinical trialists are willing to share their data but practical issues and fears over inappropriate reanalysis are more important barriers to sharing than lack of individual incentives/citations (Rathi et al. http://dx.doi.org/10.1136/bmj.e7570). I assume the opening quote is not intended to add color to the paper but to provide the opening part of the introduction. As such, I found the author quoting themselves at this length from a 6-year-old study was not the best way to open a paper. Throughout the manuscript, the authors refer to \u201copen data\u201d meaning, I assume in this context, data which are freely accessible on the web. However, there is growing recognition that \u201copen\u201d data means data which are available under legal terms \u2013 a license or waiver \u2013 which permit sharing and reuse with the minimum of barriers. Open data is about more than just accessibility. \u201cOpen\u201d data must be free to \u201cdownload, copy, analyze, re-process, pass them to software or use them for any other purpose without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself\u201d [http://pantonprinciples.org/]. To my knowledge, the authors are aware of the appropriate use of \u201copen\u201d in open data and open access and I recommend they use an alternate term to \u201copen data\u201d in the present manuscript to avoid engendering confusion about the term \u201copen data.\u201d I\u2019m not convinced the account of the authors\u2019 problems in obtaining access to Scopus should be included in the current manuscript. It seems tangential to the thrust of the manuscript and its analysis and would be more appropriate for a blog (I believe some of this was described on Piwowar\u2019s blog) or commentary, and would make the manuscript more focused. Also, elsewhere in the paper, the authors note that Scopus now has an API for programmatic access. The authors have previously written about the problems faced by researchers in gaining access to the literature for research \u2013 text mining, citation analysis \u2013 elsewhere. I noticed a few typos (this is not an exhaustive list) e.g. \u201cThe lack of conventions and tool support for data Attribution\u201d; \u201cWhich types of data reuse are facilitated by robust data standards and which types are unaffected?\u201d I am aware one of the authors has previously discussed publicly the present study\u2019s data and preliminary results (http://researchremix.wordpress.com/2012/07/16/many-datasets-are-reused-not-just-an-elite-few/). Open science should not preclude papers from being published, and the fact the authors discussed the data from this study before submission of their paper should have no influence on the paper\u2019s acceptance or rejection. However, best practice regarding potentially duplicate or overlapping publications would be to state in the paper and cite the previous publications, including blogs, posters, etc., in the submitted manuscript. \\n\\nReview 5: nan\\n\\nReview 6: ']"
}