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Folate levels modulate oncogene‐induced replication stress and tumorigenicity

2015; Springer Nature; Volume: 7; Issue: 9 Linguagem: Inglês

10.15252/emmm.201404824

1757-4684

Noa Lamm, Karin Maoz, Assaf C. Bester, Michael M. Im, Donna S. Shewach, Rotem Karni, Batsheva Kerem,

RNA modifications and cancer

Research Article21 July 2015Open Access Folate levels modulate oncogene-induced replication stress and tumorigenicity Noa Lamm Noa Lamm Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Karin Maoz Karin Maoz Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Assaf C Bester Assaf C Bester Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Michael M Im Michael M Im Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA Search for more papers by this author Donna S Shewach Donna S Shewach Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA Search for more papers by this author Rotem Karni Rotem Karni Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Search for more papers by this author Batsheva Kerem Corresponding Author Batsheva Kerem Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Noa Lamm Noa Lamm Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Karin Maoz Karin Maoz Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Assaf C Bester Assaf C Bester Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Michael M Im Michael M Im Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA Search for more papers by this author Donna S Shewach Donna S Shewach Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA Search for more papers by this author Rotem Karni Rotem Karni Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Search for more papers by this author Batsheva Kerem Corresponding Author Batsheva Kerem Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Author Information Noa Lamm1, Karin Maoz1, Assaf C Bester1, Michael M Im2, Donna S Shewach2, Rotem Karni3 and Batsheva Kerem 1 1Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem, Israel 2Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA 3Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem, Israel *Corresponding author. Tel: +972 2 6585689; E-mail: [email protected] EMBO Mol Med (2015)7:1138-1152https://doi.org/10.15252/emmm.201404824 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Chromosomal instability in early cancer stages is caused by replication stress. One mechanism by which oncogene expression induces replication stress is to drive cell proliferation with insufficient nucleotide levels. Cancer development is driven by alterations in both genetic and environmental factors. Here, we investigated whether replication stress can be modulated by both genetic and non-genetic factors and whether the extent of replication stress affects the probability of neoplastic transformation. To do so, we studied the effect of folate, a micronutrient that is essential for nucleotide biosynthesis, on oncogene-induced tumorigenicity. We show that folate deficiency by itself leads to replication stress in a concentration-dependent manner. Folate deficiency significantly enhances oncogene-induced replication stress, leading to increased DNA damage and tumorigenicity in vitro. Importantly, oncogene-expressing cells, when grown under folate deficiency, exhibit a significantly increased frequency of tumor development in mice. These findings suggest that replication stress is a quantitative trait affected by both genetic and non-genetic factors and that the extent of replication stress plays an important role in cancer development. Synopsis Oncogene-induced replication stress is shown here as a quantitative trait enhanced by non-genetic factors such as the essential dietary nutrient folate. The combination of oncogene expression and folate deficiency enhances replication-induced genomic instability and cancer development in vivo. Folate deficiency by itself leads to replication stress in a concentration-dependent manner that can be rescued by nucleoside supplementation. The extent of oncogene-induced replication stress can be enhanced by an additional source of stress, resulting in enhanced DNA damage. Activation of the DNA damage response pathways by ATM and ATR is enhanced by the combination of oncogene expression and folate deficiency. Tumorigenicity potential in vitro and tumor development in vivo caused by oncogene expression are significantly enhanced by folate deficiency. Introduction Chromosomal instability is a hallmark of nearly all solid tumors and adult-onset leukemias (Hanahan & Weinberg, 2011). Enormous efforts have been made in the last few decades to understand the cellular and environmental factors leading to genomic instability and cancer development (Lengauer et al, 1998; McGranahan et al, 2012; Ozeri-Galai et al, 2012). In recent years, it has become apparent that in early stages of cancer development, DNA instability is caused by perturbed DNA replication (Ames & Wakimoto, 2002; Gorgoulis et al, 2005; Tsantoulis et al, 2008). This replication stress is defined as perturbations in the dynamics of the replication machinery and is characterized by slow fork progression, and in some cases even fork collapse, activation of additional origins, and asymmetric progression of replication forks emerging from the same origin (Hills & Diffley, 2014). In the early stages of cancer development, oncogene activation leads to replication stress (Bartkova et al, 2005; Di Micco et al, 2006; Tsantoulis et al, 2008; Bester et al, 2011), which underscores the role of DNA replication in cancer development (Halazonetis et al, 2008; Negrini et al, 2010). Several mechanisms by which oncogenes induce replication stress were recently identified, including insufficient nucleotide pools to support the extensive enforced DNA replication (Bester et al, 2011; Mannava et al, 2013), interference with the pre-replication complex assembly (Ekholm-Reed et al, 2004) and the collision between replication and transcription (Jones et al, 2013). However, it remains unclear whether the extent of the replication stress can affect the probability of neoplastic transformation. Moreover, whether enhanced replication stress can be driven by a combination of genetic, cellular, and environmental factors is largely unknown. Micronutrients are important environmental factors for normal cellular proliferation. Suboptimal levels (a deficiency) of micronutrients increase the risk of many types of cancer (reviewed in (Vidal et al, 2011; Ames & Wakimoto, 2002). One classic example of such a micronutrient is folate, a B9 water-soluble vitamin found mainly in green leafy vegetables (Camilo et al, 1996). Folate is the general term for many derivatives found in intracellular equilibrium, which except for de novo synthesis by intestinal microflora cannot be produced by most mammals (Camilo et al, 1996). Folic acid is the fully oxidized monoglutamyl form of folate, which is frequently used as a nutritional supplement. Therefore, folate must be obtained from dietary or supplementary sources (Shane, 1989). Folate is required for one-carbon transfer reactions including the synthesis of thymidine and purines and the methylation of cytosines in DNA (reviewed in (Duthie, 2011; Kim, 1999b; Shane, 1989). It has been shown that folate deficiency caused by the use of antifolate reagents perturbs the size and balance of the nucleotide pool (Shane, 1989). However, the effect of folate deficiency on DNA replication dynamics remains unclear. Many epidemiological studies have shown that suboptimal levels of folate are associated with several types of cancer, including colon (Giovannucci et al, 1995; Zhang et al, 1999; Rohan et al, 2000), cervical (Rampersaud et al, 2002; García-Closas et al, 2005), gastric, and esophageal cancers (Mayne et al, 2001). Studies in human cultured cells and in vivo studies in both animal models and humans have shown that severe folate deficiency is associated with double-strand breaks (DSBs), chromosome instability, and micronuclei formation (Chen et al, 1989; James & Yin, 1989; Duthie & McMillan, 1997; MacGregor et al, 1997; Pogribny et al, 1997; Melnyk et al, 1999; Duthie et al, 2000a,b, 2008; Beetstra et al, 2005). The main mechanism linking folate deficiency to DNA damage is presumed to be the incorporation of dUMP into the DNA, which is thought to culminate in futile cycles of uracil excision, single-strand breaks, and possibly chromosomal breakage (Blount et al, 1997). Importantly, it was shown that folate deficiency enhances the activity of various chemical carcinogens in numerous organs (Eto & Krumdieck, 1986). To date, however, a mechanism that can account for the co-carcinogenic role of folate deficiency has yet to be found. Folate deficiency has a dual effect on the tumorigenic potential of the cells depending on the duration and extent of the folate deficiency and on the cell stage (tumorigenicity). In neoplastic cells, there is extensive DNA replication and cell division. In these cells, folate deficiency causes ineffective DNA synthesis, resulting in inhibition of tumor growth (Kim, 1999a,b; Choi & Mason, 2002). Indeed, this has been the basis for cancer chemotherapy using a number of antifolate agents (e.g., methotrexate and 5-fluorouracil) (Kim, 1999a,b; Choi & Mason, 2002). Like most chemotherapies, antifolate drugs are toxic to both normal and neoplastic cells and prolonged folate deficiency eventually results in growth arrest and cell death regardless of the tumorigenicity of the cells. However, under shorter and milder folate deficiency conditions, neoplastic cells and other extensive proliferating cells will die, whereas normal cells will survive. An accumulating body of epidemiological, clinical, and experimental evidence suggests that normal cells that survived folate deficiency are predisposed to neoplastic transformation (Kim, 1999a,b, 2003). This dual effect of folate deficiency, which is also known as the “double-edged sword” effect, explains why methotrexate therapy is associated with increased risk of secondary malignancy (Schmiegelow et al, 2009). In the current study, we investigated the combined effect of genetic and dietary factors on replication dynamics, genome stability, and cancer development. Our results show that suboptimal levels of folate lead to replication stress and DSBs in a concentration-dependent manner. Importantly, folate deficiency significantly enhances oncogene-induced replication stress, DNA damage, and tumorigenicity in vitro. Furthermore, oncogene-expressing cells grown under folate deficiency show a significant increase in the frequency of tumor development in mice. These findings suggest that replication stress is a quantitative trait that can be affected by both genetic and non-genetic (e.g., dietary) factors. Results Folate deficiency perturbs cellular DNA replication dynamics To investigate the role of folate levels in tumorigenesis, we first analyzed the effects of folate deficiency on DNA replication dynamics. For this purpose, immortalized primary foreskin fibroblasts (BJ-hTert) were grown for 7 days in a folate-free medium (folate-free DMEM). During this time, the folate-deficient cells exhibited a similar growth rate as their counterparts that were cultured in a normal medium (Fig 1A), indicating that differences between the cultures were not a result of impaired growth. To investigate the effect of the folate-free medium on cellular DNA replication, we took advantage of the high-resolution DNA combing approach which enables replication analysis on single DNA molecules. The newly synthesized DNA, labeled with IdU and CldU, can be detected by fluorescent antibodies (green and red, respectively) (Fig 1B). First, we analyzed the effect of folate deficiency on the cellular replication fork rate (Fig 1C and D). The results showed a dramatic decrease in the mean replication rate, from 1.59 Kb/min in cells cultured in a normal medium to 0.78 Kb/min in cells grown in a folate-free medium (P < 1.6 × 10−32). Importantly, a dramatic increase in the percentage of very slow forks (0.75 Kb/min and below) was observed following growth in a folate-free medium (from 3% under normal conditions to 54% under folate deficiency; Fig 1D). Similar results were obtained in three independent experiments (Fig 1E; Appendix Fig S1A). These results indicate that folate deficiency leads to a significant decrease in fork progression rate. Figure 1. Growth rate and replication dynamics in BJ cells grown in a folate-free medium with and without nucleoside supplementation Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days. Example of a single combed DNA molecule labeled with IdU (green) and CldU (red), showing replication from three adjacent origins. Horizontal white arrows indicate fork orientation. Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate-free medium. Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138). Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361). Fork distance (Kb) distribution. The color code is as in (D). Control (n = 72); −folate (n = 71); −folate + AGCT (n = 75). Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 212); −folate (n = 215); −folate + AGCT (n = 209). Data information: (E, G) Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. **P < 0.0001. Download figure Download PowerPoint When DNA replication is perturbed, the number of active origins increases in an attempt to compensate for the slow fork progression (Anglana et al, 2003; Ge et al, 2007; Courbet et al, 2008). For this reason, we studied the effect of growth in a folate-free medium on origin density by measuring the distance between two sister forks, which in unsynchronized cells is approximately half of the replicon length (Maya-Mendoza et al, 2007). The replicon length scales with increasing inter-origin distances and is therefore a readout of the distance between activated origins. The results showed a significant decrease in the mean fork distance from 195 Kb in the control cells to only 107 Kb in the folate-deficient cells (P < 4 × 10−11) (Fig 1C and F). Similar results were obtained in three independent experiments (Fig 1G; Appendix Fig S1B). Altogether, these results indicate that folate deficiency leads to dramatic replication perturbations. We hypothesized that this observed replication stress was due to an insufficient nucleotide pool generated by folate deficiency. For this purpose, BJ cells were grown for 7 days in a folate-free medium and were supplemented with 50 μM of each of the four nucleosides for the last 48 h. Evaluating the replication dynamics using DNA combing revealed that the exogenous supply of nucleosides almost completely restored the average fork rate (Fig 1D and E; Appendix Fig S1A) and the average fork distance (Fig 1F and G; Appendix Fig S1B). Using the high-performance liquid chromatography (HPLC) method, the concentrations of the cellular dNTPs were measured. As expected, the concentration of the cellular dTTP in cells grown under folate deficiency for 15–30 days was significantly reduced compared to the concentration in same cells grown in a normal medium (Appendix Fig S2). The levels of the dATP, dGTP, and dCTP were below detection. Since the level of dTTP in the folate-deficient medium is very low, uracil misincorporation into the DNA in the cells is expected (Duthie & Hawdon, 1998; Fenech, 2012). The extent of replication stress is affected by the levels and duration of folate deficiency In cultured cells, a folate concentration in the 12–120 nM range was shown to be negatively correlated with DNA damage and micronuclei formation (reviewed in Fenech, 2012). Whereas 20 nM is considered a severe folate deficiency in tissue cultured cells and 100 nM is considered to be mild, 500 nM has not, to the best of our knowledge, been reported to induce any DNA damage. Hence, we studied the effect of different folate concentrations on replication dynamics. We grew BJ cells in a folate-free medium and in a medium containing 20, 100, 500, and 9,040 nM folate. The latter is the regular concentration in the commercial DMEM. First, the effect of various folate concentrations on cell growth was studied by analysis of population doublings (PDs). As can be seen in Fig 2A, the effect was concentration dependent. Cells cultured with 500 nM folate showed a similar growth rate as control cells during the 48 days of culturing, whereas cells cultured with 100 nM folate showed a reduced growth rate, but continued to grow during the whole experiment. In contrast, cells cultured with 20 nM folate showed a major decrease in growth rate starting at ~21 days of culturing and stopped growing after ~35 days. The effect of the folate-free medium was even stronger, leading to growth arrest after only 21 days (Fig 2A). Figure 2. Growth rate and replication dynamics in BJ cells grown under various folate concentrations with and without nucleoside supplementation A. Population doublings (PDs) determined in BJ cells cultured at the indicated folate concentrations for 48 days. B, C. The average replication rate ± SEM (B) and the average fork distance ± SEM (C) in the indicated folate concentrations at 14 and 21 days. At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate. At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance. D–G. BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation. (D) Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 115); gray bars: BJ cells that were cultured for 14 days in 100 nM folate (n = 117); blue bars: BJ cells cultured for 14 days in 100 nM folate and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 117). (E) Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 352); 100 nM folate (n = 364); 100 nM folate + AGCT (n = 355). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. (F) Fork distance (Kb) distribution. The color code is as in (D). Control (n = 69); 100 nM folate (n = 74); 100 nM folate + AGCT (n = 72). (G) Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 201); 100 nM folate (n = 220); 100 nM folate + AGCT (n = 228). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. **P < 0.001. Download figure Download PowerPoint Next, we studied the effect of various folate concentrations on the DNA replication dynamic in cells grown for 14 and 21 days (Fig 2B and C). On day 14, cells cultured at 100 nM, 20 nM, or in a folate-free medium exhibited a concentration-dependent decrease in the average fork rate and distance (Fig 2B and C). Consistent with the above, the average fork rate and distance did not significantly differ between cells cultured with 500 nM folate and the control cells (Fig 2B and C). The effect of folate deficiency on the average replication rate and fork distance significantly increased with time (Fig 2B and C). Remarkably, cells grown in a medium with 500 nM folate, which did not affect cell proliferation (Fig 2A), also showed a significant decrease in their average replication rate with time: After 14 days, the replication rate was 1.22 Kb/min (the same rate as in the control cells), whereas after 21 days the rate was significantly lower (Fig 1B). The average fork distance in the 500 nM folate cultures decreased during this period of time from 127 to 97 Kb (Fig 1C). We further analyzed the effect of nucleoside supplementation on replication stress under mild folate deficiency. As can be seen in Fig 2, BJ cells grown for 14 days in 100 nM folate showed a reduced replication rate (from 1.2 to 0.9 Kb/min) (P < 4.1 × 10−10) (Fig 2B and D). Similar results were obtained in three independent experiments (Fig 2E). In accordance with the reduced replication rate, the fork distance reduced from an average of 136 to 85 Kb (P < 2.3 × 10−3) (Fig 2C and F). Similar results were obtained in three independent experiments (Fig 2G). Supplementation of nucleosides for 48 h resulted in almost complete rescue of the average fork rate (P < 3.3 × 10−9) and distance (P < 0.005), (Fig 2D–G). It is worth noting that the replication stress preceded the impaired proliferation, since cell growth for 14 days in 100 nM folate showed perturbed replication dynamics but no effect on cell proliferation. This indicates that the replication stress induced by folate deficiency was not secondary to decreased proliferation. Altogether, our data show that the extent of replication stress is determined by folate deficiency in a concentration-dependent manner. Moreover, the effect of folate deficiency exacerbates with time, and even a mild chronic suboptimal folate level that does not hinder cell proliferation eventually results in stress on DNA replication. Enhanced replication stress and DNA damage in oncogene-expressing cells caused by folate deficiency Next, we studied whether the replication stress conferred by folate deficiency can enhance the replication stress induced by an oncogene. First, we expressed the oncogene cyclin E, which is frequently overexpressed in many types of human precancerous and cancerous lesions (Hwang & Clurman, 2005). Aberrant expression of cyclin E was shown to induce replication stress (Bester et al, 2011; Jones et al, 2013). Using retroviral infection, BJ cells were transfected with a cyclin E construct. Cyclin E expression was verified by Western blot analysis (Appendix Fig S3A). The experiments were performed in newly transformed cells, no later than 6 weeks following cyclin E infection. Cells were cultured for 7 days in a normal or folate-free medium. As can be seen in Fig 3, folate deficiency significantly enhanced the replication stress conferred by cyclin E expression. Whereas cyclin E expression by itself decreased the average replication rate from 1.18 Kb/min in cells expressing an empty vector to 0.79 Kb/min (P < 2.4 × 10−21), folate deficiency further reduced the average replication rate to 0.59 Kb/min (P < 1 × 10−13) (Fig 3A). The fraction of very slow replicating forks found in cyclin E-expressing cells was further increased when cells were cultured in a folate-free medium (Fig 3A). Similarly, the average fork distance was further decreased when cyclin E-expressing cells were cultured in a folate-free medium, from 129 Kb in the control cells to 94 Kb in cyclin E-expressing cells (P < 8.4 × 10−4) and to 70 Kb in cyclin E-expressing cells grown in a folate-deficient medium (P < 1 × 10−3) (Fig 3B). Similar results were obtained in three independent experiments (Appendix Fig S3B and C). Figure 3. The effect of folate deficiency on replication dynamics and DSB formation in cyclin E-expressing cellsCyclin E-expressing BJ cells were grown for 7 days with and without folate. Fork rate (Kb/min) distribution. White bars: BJ cells expressing an empty vector (n = 145); dark gray bars: BJ cells expressing the cyclin E oncogene (n = 147); light gray bars: BJ cells cultured for 7 days in a folate-free medium (n = 135); black bars: BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate-free medium (n = 138). Fork distance distribution (Kb). The color code is as in (A). Empty vector (n = 78); CycE (n = 79); empty vector −folate (n = 71); CycE −folate (n = 80). Percent of origins with the indicated progression ratio between sister forks. Empty vector (n = 158); CycE (n = 155); empty vector −folate (n = 160); CycE −folate (n = 154). *P < 0.05. Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression (CycE) (n = 65), empty vector (n = 65), folate-free medium for 7 days (empty vector −folate) (n = 67) or oncogene expression under folate-free conditions (CycE −folate) (n = 70). Red: γH2AX; green: 53BP1; blue: DAPI staining. Percent of nuclei with the indicated number of γH2AX-53BP1 co-localized foci. **P < 0.01. Data information: Bars represent average values. Download figure Download PowerPoint Two replication forks that emerge from the same origin (sister forks) tend to exhibit the same replication rate (Anglana et al, 2003). However, under replication stress conditions, perturbed fork progression might lead to asymmetric progression of the sister forks (Di Micco et al, 2006). As previously suggested (Anglana et al, 2003), the progression of sister forks is considered symmetric when the ratio between them is > 0.75. Our analysis revealed a significant increase in the asymmetry between sister forks, from 23% in the control cells to 42% in cells grown under folate deficiency and 43% in cyclin E-expressing cells (Fig 3C). Importantly, cyclin E-expressing cells grown under folate deficiency showed a further increase in the fraction of asymmetric forks to 67% (Fig 3C). These results indicate that the replication perturbation induced by aberrant oncogene expression can be enhanced by an additional source of stress such as folate deficiency. Next, we studied the effect of folate deficiency in cells expressing another oncogene, the human papilloma virus 16 (HPV16) E6/E7. In recent years, a correlation between folate deficiency and the development of HPV-induced cervical carcinoma has been reported (Rampersaud et al, 2002; García-Closas et al, 2005). We further investigated the effect of folate deficiency on replication dynamics in primary keratinocytes derived from adult skin biopsies expressing the HPV16 oncogenes E6/E7. This is a highly powerful model system for studying events in early stages of cervical cancer development, as primary keratinocytes are the natural host for HPV infection. All the experiments were performed in newly transformed cells 2–6 weeks following E6/E7 infection and before anaphase bridges and micronuclei were visible. Replication analysis was performed on E6/E7-expressing cells grown in a normal and a folate-free medium for 4 weeks. The average replication rate of the E6/E7-expressing keratinocytes in the normal medium was 0.79 Kb/min, whereas in the folate-free medium the average fork rate was significantly reduced to 0.58 Kb/min (P < 1.5 × 10−5) (Appendix Fig S4A), indicating that folate deficiency significantly enhances the effect of E6/E7 oncogenes on cellular DNA fork progression. We further studied the effect of folate deficiency on fork distance. We found that in E6/E7-expressing cells grown in a folate-free medium, the average fork distance was significantly shorter than in E6/E7-expressing cells grown in a normal medium (P < 5 × 10−3) (Appendix Fig S4B). Overall, our data show that the enhancement of oncogene-induced replication stress by folate deficiency is not oncogene or cell type specific. We further studied the effect of folate deficiency on genome stability by analyzing the formation of DSBs (indicated by the γH2AX-53BP1 foci) in cyclin E-expressing cells grown for 7 days in a folate-free medium. Cyclin E-expressing cells cultured in the folate-free medium showed a significant increase in the number of γH2AX-53BP1 foci per nucleus compared to each treatment by itself (average of 7.8 and 4.6 foci/cell, respectively, Fig 3D and E). In particular, the fraction of cells with a high level of γH2AX-53BP foci increased in cyclin E-expressing cells from 4% in the control cells to 25%