Patients with the rare genetic disease, Fanconi anemia (FA), are highly susceptible to squamous cell carcinomas arising at multiple anatomic sites including the head and neck region. Human papillomaviruses (HPVs), particularly HPV16, are associated with ∼20% of head and neck squamous cell carcinomas (HNSCCs) in the general population. Some but not other investigators have reported that HNSCCs in FA patients are much more frequently positive for HPV. In addition, studies have demonstrated an interaction between the HPV16 E7 oncoprotein and the FA pathway, a DNA damage response pathway deficient in FA patients. On the basis of these studies, it was hypothesized that the FA pathway contributes to repair of DNA damage induced by HPV16 E7, providing one explanation for why FA patients are predisposed to HPV-associated HNSCCs. To determine the importance of the FA pathway in modulating the oncogenic abilities of E7, we crossed K14E7 transgenic (K14E7) and fancD2 knockout mice (FancD2−/−) to establish K14E7/FancD2−/− and K14E7/FancD2+/+ mice and monitored their susceptibility to HNSCC when treated with a chemical carcinogen. K14E7/FancD2−/− mice had a significantly higher incidence of HNSCC compared with K14E7/FancD2+/+ mice. This difference correlated with an increased proliferative index and the increase in expression of biomarkers that are used to assess levels of DNA damage. These animal studies support the hypotheses that FA patients have increased susceptibility to HPV-associated cancer and that the FA DNA damage response pathway normally attenuates the oncogenic potential of HPV16 E7. Cancer Res; 70(23); 9959–68. ©2010 AACR.

Fanconi anemia (FA) is a heterogeneous, recessive, genetic disease characterized by congenital abnormalities, progressive bone marrow failure, and a predisposition to leukemia and solid tumors, particularly squamous cell carcinomas (SCCs) including head and neck SCCs (HNSCCs), at remarkably young ages. The frequency of HNSCCs in FA patients is highly elevated compared with that of the general population (1–4). So far, hematopoietic stem cell transplantation (HSCT) is the only way to treat bone marrow failure, which is the main cause of death in FA patients. Even after HSCT, FA patients suffer from a high incidence of HNSCCs (5–7) most likely due to defects in DNA damage repair, and consequent increases in genomic instability, in epithelial cells of the head and neck region.

Fanconi anemia is caused by biallelic mutations in 1 of at least 13 different genes (FA genes). FA proteins (Fanc proteins; Fanc A, B, C, D1, D2, E, F, G, I, J, L, M, and N) encoded by these genes act together to protect genomic integrity, though the mechanism by which they do this remains poorly understood. The FA pathway is known to be activated by DNA interstrand cross-links (ICLs) induced by chemicals and/or found at the sites of stalled DNA replication forks (8). Eight of the FA gene products interact in a holo-complex that localizes to the sites of damage or stalled replication forks. This, in turn, leads to the monoubiquitination of another FA gene product, FancD2 (9). DNA damage induced by ultraviolet infrared radiation and chemical compounds can also activate the FA pathway through phosphorylation of FancD2 protein by the ATM kinase; however, sensitivity of the FA pathway to these forms of damage is less than that to ICLs (10–12).

High-risk human papillomaviruses (HPVs) are etiologic agents in approximately 20% of HNSCCs in the general population, primarily amongst tumors of the oropharynx. HPV type 16 (HPV16) is present in more than 90% of the HPV-positive HNSCCs (13, 14). HPV16 encodes 3 oncogenes, E5, E6, and E7. A mouse model for HPV-associated HNSCC has been developed in which HPV16 transgenic mice, when treated with a chemical carcinogen that induces DNA adducts akin to those caused by tobacco-associated carcinogens synergizes with HPV16 oncogenes to induce HNSCC. The cancers that arise in these mice share many histopathologic properties with HPV-positive HNSCC in humans. The site at which tumors arise is less restrictive than in humans, presumably reflective of the pattern of HPV transgene expression throughout the mouse oral/upper gastrointestinal epithelia. Between E6 and E7, HPV16 E7 has greater potential to cause head and neck cancer in animal models (15), with E6 contributing to increased incidence in combination with E7 (16). The contribution of HPV16 E5 to HNSCC in this mouse model has not been fully evaluated but appears to be much less than that of E7 (Strati and Lambert, unpublished data).

In FA patients from North America, a high percentage (84%) of HNSCC arising at various sites in the head and neck region (i.e., not restricted to the oropharynx) were found to be positive for high-risk HPV DNA. Consistent with a role of HPV in these cancers, mutations in p53, a tumor suppressor that is inactivated by HPV16 E6, were not found in HNSCCs from these patients. However, in patients from European countries, HNSCCs were negative for HPV DNA and more than 50% of these tumors had p53 mutations (17, 18). Given these conflicting clinical data, it remains unclear whether HPVs play a major role in HNSCCs arising in FA patients.

Several studies point to a contribution of FA deficiency in HPV-associated disease at the molecular level. In tissue culture, expression of the HPV16 E7 oncogene was shown to stimulate transcription of fancD2 (19) and activate the FA pathway (20). In another study making the use of organotypic cultures of human keratinocytes to recapitulate the HPV life cycle, HPV16- and HPV18-induced epithelial hyperplasia was increased in fancA-deficient cells or in cells knocked down in its expression whereas restoration of fancA expression attenuated this effect (21). These studies indicate that there is an interplay between HPV and the FA pathway. The implications of this interplay on HPV-associated carcinogenesis remain unclear.

Given these findings, we performed experiments with mice to ask whether deficiency in the FA pathway leads to an increase in HPV16 E7–mediated HNSCC. We crossed K14E7 transgenic mice, which display heightened susceptibility to head and neck cancer (15, 22), to fancD2 gene knockout mice (23) and the incidence of head and neck cancers in HPV transgenic and nontransgenic mice was scored in fancD2-sufficient and fancD2 -deficient backgrounds. We found a significantly increased incidence of head and neck tumors in K14E7/FancD2−/− mice compared with that in K14E7/FancD2+/+ mice. No tumors arose in nontransgenic mice regardless of fancD2status. Increased tumor incidence in the FA-deficient, E7transgenic mice correlated with increases in cell proliferation and DNA damage foci. In addition, we found that 2 biomarkers, p16 and MCM7 (22, 24), used in distinguishing between HPV-positive and HPV-negative HNSCC arising in humans are upregulated in the tumors arising in E7 transgenic mice regardless of FA-status. Our findings support the hypotheses that FA patients have greater susceptibility to HPV-associated HNSCC and that the oncogenic properties of E7 are attenuated by a functional FA pathway. On the basis of these conclusions, HPV-specific prophylactic vaccines could be useful in reducing SCC burden in FA patients.

Mice

K14E7 transgenic mice (25), on the FVB genetic background, were crossed with fancD2 gene knockout (FancD2−/−) mice (23), on the 129 genetic background, to generate F1 mice, K14E7/FancD2+/− and NTG/FancD2+/− (FVB/129 mixed background). All experimental mice(NTG/FancD2+/+, NTG/FancD2−/−, K14E7/FancD2+/+, and K14E7/FancD2−/− mice) were males generated by intercrossing F1 mice. All mice were genotyped by PCR. Mice were injected intraperitoneally with 0.3 mL of bromodeoxyuridine (BrdUrd; 12.5 mg/mL) 1 hour before being sacrificed to measure cell proliferation. Tongues and esophagi were harvested and processed as described previously (22). Mice were housed in the Association for Assessment of Laboratory Animal Care–approved McArdle Laboratory Animal Care Unit. All procedures were carried out in accordance with an animal protocol approved by the University of Wisconsin Institutional Animal Care and Use Committee.

4-Nitroquinoline-1-oxide–induced head and neck carcinogenesis study and histologic analysis

The treatment and guidelines for histologic analysis of tumors were described previously (22). Briefly, mice were treated with 4-nitroquinoline-1-oxide (4-NQO; 10 μg/mL) in their drinking water for 8 weeks and then held off treatment for an additional 16 weeks. At the end point or when mice became moribund, they were sacrificed, tumors in the tongue and the esophagus were scored, and tissues were collected for histopathologic analyses.

Immunofluorescence

Immunofluorescence was performed as described previously (26, 27). Antibodies used included anti-p16 (1:50 in 5% nonfat milk/5% horse serum; M156, Santa Cruz Biotech), anti-Mcm7 (1:200 in 5% horse serum; Neomarkers), anti-cytokeratin 14 conjugated to FITC (CK14; 1:100 in 5% horse serum; CBL 197F, Millipore), and anti-BrdUrd (1:50 in 5% horse serum; Calbiochem).

Quantifying BrdUrd-positive nuclei and γH2AX- and 53BP1-positive foci cells

At least 3 mice of each genotype, NTG/FancD2+/+, NTG/FancD2−/−, K14E7/FancD2+/+, and K14E7/FancD2−/− mice, were selected and ∼8 to 10 frames (×400) of cells within the suprabasal (CK14 negative) and basal (CK14 positive) layers of tongue and esophagus epithelia were quantified for each mouse.

Statistical analysis

The MSTAT software program4 was used for determining statistical significance. Two-sided Fisher's exact test was used to determine the significance of differences in tumor incidence between each group of mice (Table 1). To determine the significance for BrdUrd, γH2AX foci, and 53BP1-positive foci cells between each group of mice, a 1-sided Wilcoxon rank-sum test was used (Figs. 2 and 3).

Table 1.

Comparison of 4-NQO induced over tumor incidences in animal tissues (tongue and esophagus)

GenotypeGroup size, nAnimal tissues with overt tumor, n (%)

Tongue and esophagusTongueEsophagus

 
NTG/FancD2+/+ 24 0 (0) 0 (0) 0 (0) 
NTG/FancD2−/− 26 0 (0) 0 (0) 0 (0) 
K14E7/FancD2+/+ 20 4 (20) 2a(10) 3a (15) 
K14E7/FancD2−/− 21 11 (52) 5 (24) 6 (29) 

 
GenotypeGroup size, nAnimal tissues with overt tumor, n (%)

Tongue and esophagusTongueEsophagus

 
NTG/FancD2+/+ 24 0 (0) 0 (0) 0 (0) 
NTG/FancD2−/− 26 0 (0) 0 (0) 0 (0) 
K14E7/FancD2+/+ 20 4 (20) 2a(10) 3a (15) 
K14E7/FancD2−/− 21 11 (52) 5 (24) 6 (29) 

 

NOTE: The difference in tumor incidence between K14E7/FancD2+/+ and K14E7/FancD2−/ groups was statistically significant(P = 0.05). Also, the differences in the tumor incidence between K14E7/FancD2+/+ and NTG/FancD2+/+ (P = 0.04), and NTG/FancD2−/− groups (P = 0.03) are statistical significant. All statistical comparisons were performed using a 2-sided Fisher's exact test.

aOne K14E7/FancD2+/+ mouse had tumors in both the tongue and the esophagus.

fancD2deficiency sensitizes mice to HPV-associated tumorigenesis

To test the hypothesis that FA deficiency leads to increased susceptibility to head and neck cancers caused by HPV, we made use of a mouse model for HPV-associated head and neck cancer (15, 22) in which HPV transgenic mice develop head and neck cancers when exposed to a chemical carcinogen, 4-NQO. This chemical carcinogen is absorbed by the epithelia lining the oral cavity and esophagus where a metabolite of 4-NQO causes DNA mutations. Prolonged treatment (16 weeks) with 4-NQO at a high dose (×10 that used in our own studies) is sufficient to efficiently induce head and neck cancers in nontransgenic mice (28). At the 10-fold lower dose used in our studies, 4-NQO synergizes with HPV16oncogenes to induce head and neck cancers with few if any cancers arising in nontransgenic mice (22). In the current study, we treated mice at the dose used in our past studies but for a shorter duration (8 weeks treatment compared with 16 weeks). This shorter treatment regimen was chosen because it confers lower incidence of tumors in E7 transgenic mice (Jabbar et al., manuscript in preparation), thereby providing a better context in which to monitor any contribution of FA deficiency in increasing the incidence of head and neck cancer. Differences have been observed in the tumor susceptibility of male versus female fancD2−/−/p53+/− mice (29), with higher incidence of spontaneous tumors observed in female mice owing largely to tumors arising in the mammary glands and ovaries. In the present study, only male mice were used.

As stated earlier, of the HPV16 oncogenes, E7 contributes greatest to head and neck cancer in mice (15). Because of this, and the fact that tissue culture studies indicated that the FA pathway promotes repair of E7-induced damage (21), we crossed K14E7 mice to fancD2-null mice (23) to generate 4 different genotypes: NTG/FancD2+/+, NTG/FancD2−/−, K14E7/FancD2+/+, and K14E7/FancD2−/−. Male mice were treated with 4-NQO for 8 weeks and then held for an additional 16 weeks before being sacrificed. Visible tumors on the tongues and esophagi of mice were counted, and tissues were collected for histopathologic analyses. No tumors were detected in either the NTG/FancD2+/+ or NTG/FancD2−/− mice, indicating that FA deficiency alone was not sufficient to induce head and neck cancer under this carcinogen treatment regimen (Table 1). Only 4 of 20 K14E7/FancD2+/+ mice (20%) had overt tumors (Table 1), consistent with prior observations that tumors arise in only a small fraction of E7 mice treated for only 8 weeks with 4-NQO (Jabbar et al., manuscript in preparation). However, a much higher fraction (11 of 21; 52%) of K14E7/FancD2−/− mice developed overt tumors (Table 1). This incidence of tumors in the K14E7/FancD2−/− mice was significantly higher than in the K14E7/FancD2+/+ mice (P = 0.05; 2-sided Fisher's exact test). These data indicate that FA deficiency increases the susceptibility of mice to E7-induced head and neck tumors.

The majority of overt tumors arising in K14E7/FancD2−/− mice are carcinomas

Tissues from mice with overt tumors were harvested, fixed, sectioned, and subjected to detailed histopathologic analysis. Specifically, every tenth 5-μm section of the tongue and esophagus was stained with hematoxylin and eosin (H&E) and analyzed histopathologically to determine the worst stage of neoplastic disease in each animal (Table 2). As described previously (22), a progressive disease was observed in the E7 transgenic mice in this study (see Supplementary Fig. S1), ranging from normal epithelium to benign papillomas to invasive cancer (i.e., carcinomas). Carcinomas were subdivided into grades I, II, and III, based on the degree of differentiation (keratinization) within the cancer. While 1 of the tumors arising in the K14E7/FancD2+/+ mice was a carcinoma, 8 of 11 tumors arising in the K14E7/FancD2−/− mice were carcinomas (Table 2). These histopathologic findings demonstrate that FA deficiency increases the severity of neoplastic head and neck disease in E7 transgenic mice.

Table 2.

Histopathologic analysis of sample with overt lesions

GenotypeGrade of overt lesions, n (%)

PapillomaCarcinoma

Grade IGrade IIGrade III

 
K14E7/FancD2+/+ 3 (75)   1 (25) 
K14E7/FancD2−/− 3 (27) 3 (27) 2 (18) 3 (27) 

 
GenotypeGrade of overt lesions, n (%)

PapillomaCarcinoma

Grade IGrade IIGrade III

 
K14E7/FancD2+/+ 3 (75)   1 (25) 
K14E7/FancD2−/− 3 (27) 3 (27) 2 (18) 3 (27) 

 

Expression of 2 biomarkers in carcinomas arising in K14E7/FancD2+/+ and K14E7/FancD2−/− mice

Two biomarkers, p16 and MCM7, have proven useful in distinguishing HPV-positive from HPV-negative HNSCCs both in mice (22) and in humans (26, 30–32). HPV16 E7 is thought to play a dominant role in the induction of MCM7 and p16 in both head and neck (22) and cervical (26) cancers. To determine whether these proteins were also upregulated by E7 in head and neck cancers arising on a FA-deficient background, sections from cancers arising in K14E7/FancD2+/+ and K14E7/FancD2−/− mice were subjected to p16- and MCM7-specific immunofluorescence. Both proteins were highly expressed in cancers arising in E7 transgenic mice regardless of fancD2 status (Fig. 1). Furthermore, the expression patterns of MCM7 and p16 in normal epithelium of the tongue and esophagus correlated with E7 expression and not fancD2 status (Supplementary Fig. S2). Consistent with these findings, we previously observed MCM7 and p16 staining exclusively in HPV-positive head and neck tumors in our mouse model (22). These findings, when taken together, suggest that these biomarkers will retain their usefulness in identifying HPV-positive disease in the FA patients with HNSCCs.

Figure 1.

Examination of 2 biomarkers, p16 and MCM7, in carcinomas arising on the tongue and esophagus in K14E7/FancD2+/+ and K14E7/FancD2−/− mice treated with 4-NQO, a carcinogen. Shown are representative images from sections stained with anti-p16 (green) and anti-MCM7 (red) antibodies and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Magnified images of areas highlighted in the dashed boxes are shown to the right. Scale bar, 50 μm.

Figure 1.

Examination of 2 biomarkers, p16 and MCM7, in carcinomas arising on the tongue and esophagus in K14E7/FancD2+/+ and K14E7/FancD2−/− mice treated with 4-NQO, a carcinogen. Shown are representative images from sections stained with anti-p16 (green) and anti-MCM7 (red) antibodies and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Magnified images of areas highlighted in the dashed boxes are shown to the right. Scale bar, 50 μm.

Close modal

Deficiency in fancD2 induces DNA synthesis in the basal epithelial compartment but not in the suprabasal epithelial compartment, and this activity is independent of E7

A prior study showed that FA deficiency could lead to hyperplasia in the context of organotypic raft cultures of HPVoncogene–immortalized human keratinocytes (21). This raised the possibility to us that increases in cell proliferation could contribute to the increase in head and neck cancers in the fancD2-deficient, E7 transgenic mice. We, therefore, monitored cell proliferation in the epithelia lining the tongue and esophagus of the different mouse genotypes used in this study. For this experiment, groups of mice (n = 3) of each genotype were injected intraperitoneally with BrdUrd 1 hour before being sacrificed, and BrdUrd-specific immunofluorescence was performed to detect its incorporation into newly synthesized DNA (Fig. 2A). The frequency of BrdUrd-positive cells was then quantified in each tissue (Fig. 2B). On the fancD2-sufficient background, E7 caused significant increases in the frequency of BrdUrd-positive cells in both the basal and suprabasal compartments of the tongue and esophagus epithelia (K14E7/FancD2+/+ vs. NTG/FancD2+/+ mice; P < 0.05), consistent with prior observations (15). In the absence of E7, deficiency in fancD2 alone caused a significant increase in the proliferative index in the basal but not in the surpabasal compartment of the epithelia in both the tongue and the esophagus (NTG/FancD2−/− vs. NTG/FancD2+/+ mice; P < 0.05 for basal only). In the presence of E7, deficiency in fancD2 also caused a significant increase in the proliferative index in the basal but not in the surpabasal compartment of the epithelia in both the tongue and the esophagus (K14E7/FancD2−/− vs. K14E7/FancD2+/+ mice; P < 0.05 for basal only). In addition, we scored cells progressing through G2/M by using immunofluorescence of histone 3 phosphorylated at serine-10 (H3S10), which is a well-known marker for mitosis (33). We found that fancD2 deficiency or expression of E7 led to increased numbers of H3S10-positive cells (Supplementary Fig. S3). This correlated with the increase in DNA synthesis in these same mouse strains (Fig. 2). fancD2 deficiency, however, led to only subtle increases in the percentage of H3S10-positive cells in the E7 transgenic tissue (Supplementary Fig. S3). This correlates with the observation that the induction of DNA synthesis of E7 is only slightly enhanced on a fancD2-deficient background (Fig. 2). Taken together, these findings indicate that both HPV16 E7 oncoprotein and fancD2 deficiency act can independently and additively induce cell-cycle progression specifically in the basal epithelia of the animal tissues.

Figure 2.

Examination of abilities of HPV16 E7 on DNA synthesis under the deficiency of fancD2 gene. A, to assess the newly synthesized DNA at the suprabasal and basal epithelial layers of the tongue and esophagus tissues, mice were injected with BrdUrd before sacrifice and their tissues were stained for anti-BrdUrd (red) and anti-cytokeratin 14 (CK14) protein (green) antibodies. DAPI (blue) is used for a nuclear counterstaining. Scale bar, 20 μm. B, at least 3 mice of each genotype, NTG/FancD2+/+, NTG/FancD2−/−, K14E7/FancD2+/+, and K14E7/FancD2−/− mice, were selected and ∼8 to 10 frames of cells at the suprabasal (CK14 negative) and basal (CK14 positive) layers of the tongue and esophagus epithelia were quantified for each mouse. The amount of BrdUrd-positive nuclei over the number of total cells was plotted in each case (columns); bars, SD. Asterisk (*) means that in the basal epithelial layer of the tongue and esophagus tissues, both the deficiency of fancD2 gene and the expression of HPV16 E7 oncogene were showing significantly increased DNA synthesis between genotypes but not in the suprabasal epithelial layer. All statistical comparisons were performed using a 1-sided Wilcoxon rank-sum test (*, P = 0.02).

Figure 2.

Examination of abilities of HPV16 E7 on DNA synthesis under the deficiency of fancD2 gene. A, to assess the newly synthesized DNA at the suprabasal and basal epithelial layers of the tongue and esophagus tissues, mice were injected with BrdUrd before sacrifice and their tissues were stained for anti-BrdUrd (red) and anti-cytokeratin 14 (CK14) protein (green) antibodies. DAPI (blue) is used for a nuclear counterstaining. Scale bar, 20 μm. B, at least 3 mice of each genotype, NTG/FancD2+/+, NTG/FancD2−/−, K14E7/FancD2+/+, and K14E7/FancD2−/− mice, were selected and ∼8 to 10 frames of cells at the suprabasal (CK14 negative) and basal (CK14 positive) layers of the tongue and esophagus epithelia were quantified for each mouse. The amount of BrdUrd-positive nuclei over the number of total cells was plotted in each case (columns); bars, SD. Asterisk (*) means that in the basal epithelial layer of the tongue and esophagus tissues, both the deficiency of fancD2 gene and the expression of HPV16 E7 oncogene were showing significantly increased DNA synthesis between genotypes but not in the suprabasal epithelial layer. All statistical comparisons were performed using a 1-sided Wilcoxon rank-sum test (*, P = 0.02).

Close modal
Figure 3.

DNA damage response induced by HPV16 E7 via γH2AX and 53BP1 under the deficiency of fancD2 gene. A and C, to assess induced DNA damage response by HPV16 E7 in the deficiency of fancD2 gene, tissues from each group were stained for anti-γH2AX (red), anti-53BP1 (red), and anti-cytokeratin 14 (CK14) protein (green) antibodies. DAPI is used for a nuclear counterstaining. Scale bar, 20 μm. 53BP1-positive nuclear foci cells are highlighted by yellow arrows in the images of panel C. Insets in panel C provide magnified views of cells with 53BP1-positive nuclear foci. B and D, at least 3 mice of each genotype, NTG/FancD2+/+, NTG/FancD2−/−, K14E7/FancD2+/+, and K14E7/FancD2−/− mice, were selected and ∼8 to 10 frames of cells at the basal (CK14 positive) and suprabasal (CK14 negative) layers of the tongue and esophagus epithelia were quantified for each mouse. The amount of cells with γH2AX-positive foci over the number of total cells was plotted in each case (columns); bars, SD. The differences in the number of cells with γH2AX foci between the groups were statistically compared (K14E7/FancD2+/+ vs. K14E7/FancD2−/− suprabasal P = 0.06/P = 0.02, basal P = 0.02/P = 0.02 at tongue/esophagus; B). The differences in the number of cells with 53BP1 foci (yellow arrow) between the groups were statistically compared (NTG/FancD2+/+ vs. K14E7/FancD2+/+ suprabasal P < 0.05/P < 0.05, basal P = 0.06/P = 0.02 at tongue/esophagus; NTG/FancD2+/+ vs. NTG/FancD2−/− suprabasal P = 0.02/P = 0.02 at tongue/esophagus; K14E7/FancD2+/+ vs. K14E7/FancD2−/− suprabasal P = 0.06/P = 0.02, basal P = 0.02/P = 0.02 at tongue/esophagus; D). All statistical comparisons were performed using a 1-sided Wilcoxon rank-sum test (*, P = 0.02 and **, P = 0.06). Note: Exposure times used were the same for all samples analyzed. Some cells in tissues of K14E7/FancD2+/+ and K14E7/FancD2−/− mice had very bright γH2AX-specific nuclear staining at the exposure used. Shorter exposure confirmed that these cells retained punctuate nuclear staining reflective of DNA damage foci.

Figure 3.

DNA damage response induced by HPV16 E7 via γH2AX and 53BP1 under the deficiency of fancD2 gene. A and C, to assess induced DNA damage response by HPV16 E7 in the deficiency of fancD2 gene, tissues from each group were stained for anti-γH2AX (red), anti-53BP1 (red), and anti-cytokeratin 14 (CK14) protein (green) antibodies. DAPI is used for a nuclear counterstaining. Scale bar, 20 μm. 53BP1-positive nuclear foci cells are highlighted by yellow arrows in the images of panel C. Insets in panel C provide magnified views of cells with 53BP1-positive nuclear foci. B and D, at least 3 mice of each genotype, NTG/FancD2+/+, NTG/FancD2−/−, K14E7/FancD2+/+, and K14E7/FancD2−/− mice, were selected and ∼8 to 10 frames of cells at the basal (CK14 positive) and suprabasal (CK14 negative) layers of the tongue and esophagus epithelia were quantified for each mouse. The amount of cells with γH2AX-positive foci over the number of total cells was plotted in each case (columns); bars, SD. The differences in the number of cells with γH2AX foci between the groups were statistically compared (K14E7/FancD2+/+ vs. K14E7/FancD2−/− suprabasal P = 0.06/P = 0.02, basal P = 0.02/P = 0.02 at tongue/esophagus; B). The differences in the number of cells with 53BP1 foci (yellow arrow) between the groups were statistically compared (NTG/FancD2+/+ vs. K14E7/FancD2+/+ suprabasal P < 0.05/P < 0.05, basal P = 0.06/P = 0.02 at tongue/esophagus; NTG/FancD2+/+ vs. NTG/FancD2−/− suprabasal P = 0.02/P = 0.02 at tongue/esophagus; K14E7/FancD2+/+ vs. K14E7/FancD2−/− suprabasal P = 0.06/P = 0.02, basal P = 0.02/P = 0.02 at tongue/esophagus; D). All statistical comparisons were performed using a 1-sided Wilcoxon rank-sum test (*, P = 0.02 and **, P = 0.06). Note: Exposure times used were the same for all samples analyzed. Some cells in tissues of K14E7/FancD2+/+ and K14E7/FancD2−/− mice had very bright γH2AX-specific nuclear staining at the exposure used. Shorter exposure confirmed that these cells retained punctuate nuclear staining reflective of DNA damage foci.

Close modal

DNA damage positive foci cells accumulate in head and neck epithelia in E7-expressing mice: influence of FA pathway

A number of observations support the hypothesis that HPV16 E7induces genomic instability. Expression of E7 in cells leads to an accumulation of DNA breaks on the basis of the use of the comet assay, which scores for the presence of single- and/or double-strand DNA breaks (DSBs), as well as DNA ICLs damage (34, 35). Expression of E7 in cells also causes an accumulation of chromosomal abnormalities as evidenced by losses or gains of specific alleles (34, 36) or gross changes scored by karyotypic analyses for chromatid breaks, chromatid fusions, or polyploidy (36). That E7 causes an accumulation of damaged DNA is further supported by the observation that in the nuclei of cells expressing E7, there is an increased frequency of DNA damage foci (34), as detected by using antibodies specific to histone 2AX phosphorylated at serine-139 (γH2AX), which is a marker for activated DNA damage responses triggered by the presence of damaged DNA in the nuclei of cells (37, 38).

All of these studies were conducted in tissue culture. We wanted to determine whether E7 is capable of inducing DNA damage in the context of our mouse model for head and neck cancer and, if so, learn whether this property of E7 is affected by the status of the FA pathway. Specifically, we wanted to test the hypothesis that the FA pathway suppresses the capacity of E7to induce DNA damage. It is not possible to directly quantify DNA damage in situ in the context of tissue samples; therefore, we monitored for the presence of DNA damage indirectly by quantifying the abundance of cells with DNA damage foci. γH2AX-specific immunofluorescence was performed on sections of the tongue and esophagus from E7 transgenic and nontransgenic mice on the fancD2-sufficient and fancD2-deficient backgrounds (Fig. 3A). E7 led to a large and statistically significant increase in cells with γH2AX-positive nuclear foci in both the basal and suprabasal compartments of the epithelia lining the tongue and the esophagus (Fig. 3B), consistent with prior findings in tissue culture (34). This E7-dependent induction in the number of cells with γH2AX-positive foci was further increased on the fancD2-deficient background (Fig. 3B). This latter increase was statistically significant in the basal and suprabasal compartments of the esophagus and the suprabasal compartment of the tongue and marginally significant in the basal compartment of the tongue (Fig. 3B). The fact that there was greater frequency of cells with DNA damage foci in FA-deficient, E7 transgenic epithelia than in FA-sufficient, E7 transgenic epithelia is consistent with the hypothesis that the FA pathway suppresses the capacity of E7 to induce DNA damage. Similar results were observed in the epidermis lining the K14E7 mouse torso, another stratified epithelia in which E7 is expressed. In this tissue, we again observed E7-dependent induction of γH2AX-positive nuclear foci in both the basal and suprabasal compartments, and this induction was further heightened on the fancD2-deficient background (Supplementary Fig. S4).

A recent study carried out in tissue culture also supports the hypothesis that the FA pathway promotes repair of E7-induced damage (21). Specifically, in HPV18 E6/E7-immortalized, fancA-deficient keratinocytes grown in organotypic cultures to produce stratified epithelium, there was observed an increased frequency of cells with high numbers of 53BP1-positive nuclear foci compared with a derivative cell line that had been complemented for fancA. 53BP1, like γH2AX, is a marker for DNA damage foci and provides an indirect measure of the level of DNA damage in a cell (39). We repeated our own analysis of mouse tissues by using 53BP1 as an alternative marker for DNA damage foci (Fig. 3C). As observed with γH2AX-specific immunofluorescence (Fig. 3B), E7 caused a large and statistically significant induction in the frequency of cells with 53BP1-positive nuclear foci in both the basal and suprabasal compartments of the tongue and esophagus epithelia (Fig. 3D). In fancD2-deficient tissue, E7 caused a further increase in the frequency of cells with 53BP1-positive nuclear foci in both the basal and suprabasal compartments of the tongue and esophagus. This further increase was again statistically significant in both the compartments of the esophagus and the suprabasal compartment of the tongue and marginally significant in the basal compartment of the tongue (Fig. 3D). Thus, using 2 different markers for DNA damage foci, we find evidence in support of the hypothesis that the FA pathway suppresses E7-dependent induction of DNA damage.

In the absence of E7, we found no evidence for fancD2 deficiency leading to an increase in the frequency of cells with γH2AX-positive nuclear foci, but there was a modest yet significant increase in the frequency of cells with 53BP1-positive nuclear foci specifically in the suprabasal compartment of the tongue and esophagus epithelia (Fig. 3D). It is unclear what this modest increase reflects, as in nontransgenic mice, the compartment of the epithelia is postmitotic; therefore, it is not likely to reflect DNA damage arising from normal DNA replicative processes.

FA pathway suppresses HPV-associated carcinogenesis in the tongue and the esophagus

A link between HPVs and SCCs from FA patients is of interest to both the clinical and basic research fields. In prior studies of FA patients, geographic and technical differences between study groups make establishing such a link unclear (17, 18). Some researchers, however, have demonstrated clear correlations between the oncogenic activity of HPVs and deficiency in the FA pathway in vitro (20, 21). Our in vivo findings that HPV-associated cancer incidence is significantly induced in the tongue and the esophagus when the FA pathway is compromised by the lack of fancD2 gene (Tables 1 and 2) supports the conclusions of prior in vitro studies. Our findings might explain why HPV16 E7 activates the FA pathway in human cells (20). Such an activation could counteract the oncogenic activities of E7. Our results are also consistent with the observation in vitro that rescue of the FA pathway inhibits HPV-associated hyperplasia (21). On the basis of the cumulative studies, we hypothesize that the FA pathway suppresses the oncogenic activities of HPV16 E7. Moreover, these data provide further argument for the value of early vaccination of FA patients against HPVs to prevent the onset of HPV-associated neoplasia.

FancD2 controls cell proliferation in basal epithelial cells

We observed fancD2 deficiency increased the frequency of cells supporting DNA replication in the basal epithelium, even in the absence of HPV16 E7 expression (Fig. 2). This supports the hypothesis that increases in cell proliferation contribute to epithelial cancers in FA patients. This finding also correlates with prior findings that posttranslational modification of FancD2 protein by the ATM and ATR pathways plays a role in the S-phase checkpoint in cells in vitro (10, 12, 40). In the absence of exposure to exogenous DNA-damaging agents, ubiquitinated FancD2 protein is observed in the S phase of cells (10, 12, 41). Furthermore, lymphoblastoid cell lines deficient in the FA pathway lack the ability to delay S-phase progression in response to DNA damage from DNA cross-linking agents (42, 43). In the latter cell type, a corresponding induction of programmed cell death is thought to contribute ultimately to bone marrow failure in FA patients (44).

Deregulation of cell proliferation by HPV16 E7is induced by deficiency in the FA pathway

Unscheduled entry into S phase and the deregulation of cyclin expression in epithelium are the well-known properties of HPV16 E7 (45). fancD2 gene knockdown increases the proliferation in organic raft culture system by using keratinocytes immortalized by HPV16 and HPV18 oncogenes in vitro (21). Our finding that deficiency in the FA pathway increases cell proliferation in epithelium in vivo induced by E7 expression (Fig. 2) is consistent with the previous observation. We did not observe an effect of fancD2 deficiency on proliferation within the suprabasal compartment of epithelia in the presence of E7 expression. This would suggest that the ability of E7to deregulate the cell in the suprabasal compartment is not modulated by the FA pathway. However, deficiency in the FA pathway did result in increased proliferation in the basal compartment of epithelium expressing the E7 oncoprotein (Fig. 2B). But because FA deficiency, in the absence of E7 expression, can induce basal cell proliferation (see above), it remains unclear whether the increase in basal cell proliferation in fancD2-null, E7 transgenic epithelia reflects an ability of the FA pathway to partially suppress E7-induced proliferation or an ability of FA deficiency to act independently to cause cell proliferation. Regardless, the predisposition of FA-deficient, E7 transgenic mice to develop cancer correlated with an overall heightened rate of basal cell proliferation.

FA pathway modulates DNA damage induced by HPV16 E7

Whether the FA pathway acts directly to suppress DNA damage induced by HPV16 E7 is an important question in light of the fact that E7 is more oncogenic on an FA-deficient background in mice. E7 activates FA pathway as shown by the accumulation of monoubiquitinated FancD2 protein in vitro(20). E7 induces DNA damage, as detected by the comet assay (34), and DNA damage response, as measured by the induction of γH2AX-positive nuclear foci in vitro (34). Our findings that E7 -dependent DNA damage responses were increased on the FA-deficient background in vivo, as evidenced by the increase in numbers of cells with γH2AX- and 53BP1-positive nuclear foci (Fig. 3), is consistent with recent in vitro results showing that fancA gene complementation significantly reduced the number of 53BP1 foci per nucleus in fancA gene–deficient keratinocytes immortalized by HPV18 (21). Together, these results support the hypothesis that the FA pathway suppresses E7 -induced DNA damage and raise the interesting corollary that this property of the FA pathway contributes to its ability to suppress HPV-induced neoplasia.

One of the possible forms of DNA damage induced by E7 is DSBs, given that γH2AX- and 53BP1-positive nuclear foci are commonly considered to be readouts for DSBs and DNA damage responses to infrared radiation (46, 47). γH2AX/53BP1 double-positive nuclear foci were observed in the epithelium expressing HPV16 E7 (data not shown). In addition, anaphase bridges, which represent chromosomal fusion caused by DSBs, have been observed in cells expressing E7 (35). These data are consistent with the hypothesis that E7 induces DSBs and that E7-induced DSBs accumulate to a greater extent on an FA-deficient background. Further studies are needed to clearly define the nature of the DNA damage arising in the context of our mouse model for HPV-associated HNSCC.

HPV and HNSCCs in FA patients

Taken together, our studies and that of others in the field point to a model in which cell proliferation and DNA damage induced by the dominant HPV oncogene, E7, arise to a greater extent in an FA-deficient cellular context. Consistent with this model, recent data indicate a role of the FancD2 protein in preventing chromosome instability and aneuploidy (48). Accumulated, unrepaired DNA damage is likely to lead to increased genomic instability. This instability of the human genome may, in turn, contribute to increases in the incidence of SCCs in HPV-positive FA patients. In addition, the increased epithelial proliferation seen in FA-deficient cells/tissues may increase the infectivity of HPVs, thereby increasing the chance for persistent infections arising in FA patients (21). Together, these properties conferred by FA deficiency are likely to contribute to increased susceptibility of FA patients to HPV-associated cancers.

No potential conflict of interest were disclosed.

We thank Dr. Doug Lowy (NCI, Bethesda, MD) for first proposing to us to make use of our mouse models to investigate the relationship between HPV oncogenesis and the FA pathway.

Grant SupportThis research was supported by grants from the NIH (DE017315) to P.F. Lambert and (CA112598-) to S. Duensing.

1.
Kutler
DI
,
Auerbach
AD
,
Satagopan
J
,
Giampietro
PF
,
Batish
SD
,
Huvos
AG
, et al
High incidence of head and neck squamous cell carcinoma in patients with Fanconi anemia
.
Arch Otolaryngol Head Neck Surg
2003
;
129
:
106
12
.
2.
Kutler
DI
,
Singh
B
,
Satagopan
J
,
Batish
SD
,
Berwick
M
,
Giampietro
PF
, et al
A 20-year perspective on the International Fanconi Anemia Registry (IFAR)
.
Blood
2003
;
101
:
1249
56
.
3.
Lowy
DR
,
Gillison
ML
. 
A new link between Fanconi anemia and human papillomavirus-associated malignancies
.
J Natl Cancer Inst
2003
;
95
:
1648
50
.
4.
Rosenberg
PS
,
Greene
MH
,
Alter
BP
. 
Cancer incidence in persons with Fanconi anemia
.
Blood
2003
;
101
:
822
6
.
5.
Masserot
C
,
de Latour
R Peffault
,
Rocha
V
,
Leblanc
T
,
Rigolet
A
,
Pascal
F
, et al
Head and neck squamous cell carcinoma in 13 patients with Fanconi anemia after hematopoietic stem cell transplantation
.
Cancer
2008
;
113
:
3315
22
.
6.
Rosenberg
PS
,
Socie
G
,
Alter
BP
,
Gluckman
E
. 
Risk of head and neck squamous cell cancer and death in patients with Fanconi anemia who did and did not receive transplants
.
Blood
2005
;
105
:
67
73
.
7.
Salum
FG
,
Martins
GB
,
de Figueiredo
MA
,
Cherubini
K
,
Yurgel
LS
, et al
Squamous cell carcinoma of the tongue after bone marrow transplantation in a patient with Fanconi anemia
.
Braz Dent J
2006
;
17
:
161
5
.
8.
Moldovan
GL
,
D'Andrea
AD
. 
How the Fanconi anemia pathway guards the genome
.
Annu Rev Genet
2009
;
43
:
223
49
.
9.
de Winter
JP
,
Joenje
H
. 
The genetic and molecular basis of Fanconi anemia
.
Mutat Res
2009
;
668
:
11
9
.
10.
Taniguchi
T
,
Garcia-Higuera
I
,
Andreassen
PR
,
Gregory
RC
,
Grompe
M
,
D'Andrea
AD
. 
S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51
.
Blood
2002
;
100
:
2414
20
.
11.
Wang
X
,
D'Andrea
AD
. 
The interplay of Fanconi anemia proteins in the DNA damage response
.
DNA repair
2004
;
3
:
1063
9
.
12.
Taniguchi
T
,
Garcia-Higuera
I
,
Xu
B
,
Andreassen
PR
,
Gregory
RC
,
Kim
ST
, et al
Convergence of the Fanconi anemia and ataxia telangiectasia signaling pathways
.
Cell
2002
;
109
:
459
72
.
13.
Gillison
ML
,
Koch
WM
,
Capone
RB
,
Spafford
M
,
Westra
WH
,
Wu
L
, et al
Evidence for a causal association between human papillomavirus and a subset of head and neck cancers
.
J Natl Cancer Inst
2000
;
92
:
709
20
.
14.
Ragin
CC
,
Modugno
F
,
Gollin
SM
. 
The epidemiology and risk factors of head and neck cancer: a focus on human papillomavirus
.
J Dent Res
2007
;
86
:
104
14
.
15.
Strati
K
,
Lambert
P.F.
Role of Rb-dependent and Rb-independent functions of papillomavirus E7 oncogene in head and neck cancer
.
Cancer Res
2007
;
67
:
11585
93
.
16.
Jabbar
S
,
Strati
K
,
Pitot
HC
,
Lambert
PF
. 
Human papillomavirus type 16 E6 and E7 oncoproteins act synergistically to cause head and neck cancer in mice
.
Virology
2010
;
407
:
60
7
.
17.
Kutler
DI
,
Wreesmann
VB
,
Goberdhan
A
,
Ben-Porat
L
,
Satagopan
J
,
Ngai
I
, et al
Human papillomavirus DNA and p53 polymorphisms in squamous cell carcinomas from Fanconi anemia patients
.
J Natl Cancer Inst
2003
;
95
:
1718
21
.
18.
van Zeeburg
HJ
,
Snijders
PJ
,
Wu
T
,
Gluckman
E
,
Soulier
J
,
Surralles
J
, et al
Clinical and molecular characteristics of squamous cell carcinomas from Fanconi anemia patients
.
J Natl Cancer Inst
2008
;
100
:
1649
53
.
19.
Hoskins
EE
,
Gunawardena
RW
,
Habash
KB
,
Wise-Draper
TM
,
Jansen
M
,
Knudsen
ES
, et al
Coordinate regulation of Fanconi anemia gene expression occurs through the Rb/E2F pathway
.
Oncogene
2008
;
27
:
4798
808
.
20.
Spardy
N
,
Duensing
A
,
Charles
D
,
Haines
N
,
Nakahara
T
,
Lambert
PF
, et al
The human papillomavirus type 16 E7 oncoprotein activates the Fanconi anemia (FA) pathway and causes accelerated chromosomal instability in FA cells
.
J Virol
2007
;
81
:
13265
70
.
21.
Hoskins
EE
,
Morris
TA
,
Higginbotham
JM
,
Spardy
N
,
Cha
E
,
Kelly
P
, et al
Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture
.
Oncogene
2009
;
28
:
674
85
.
22.
Strati
K
,
Pitot
HC
,
Lambert
PF
. 
Identification of biomarkers that distinguish human papillomavirus (HPV)-positive versus HPV-negative head and neck cancers in a mouse model
.
Proc Natl Acad Sci USA
2006
;
103
:
14152
7
.
23.
Houghtaling
S
,
Timmers
C
,
Noll
M
,
Finegold
MJ
,
Jones
SN
,
Meyn
MS
, et al
Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice
.
Genes Dev
2003
;
17
:
2021
35
.
24.
Klussmann
JP
,
Gultekin
E
,
Weissenborn
SJ
,
Wieland
U
,
Dries
V
,
Dienes
HP
, et al
Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus
.
Am J Pathol
2003
;
162
:
747
53
.
25.
Herber
R
,
Liem
A
,
Pitot
H
,
Lambert
PF
. 
Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene
.
J Virol
1996
;
70
:
1873
81
.
26.
Brake
T
,
Connor
JP
,
Petereit
DG
,
Lambert
PF
. 
Comparative analysis of cervical cancer in women and in a human papillomavirus-transgenic mouse model: identification of minichromosome maintenance protein 7 as an informative biomarker for human cervical cancer
.
Cancer Res
2003
;
63
:
8173
80
.
27.
Riley
RR
,
Duensing
S
,
Brake
T
,
Munger
K
,
Lambert
PF
,
Arbeit
JM
. 
Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis
.
Cancer Res
2003
;
63
:
4862
71
.
28.
Tang
XH
,
Knudsen
B
,
Bemis
D
,
Tickoo
S
,
Gudas
LJ
. 
Oral cavity and esophageal carcinogenesis modeled in carcinogen-treated mice
.
Clin Cancer Res
2004
;
10
:
301
13
.
29.
Houghtaling
S
,
Granville
L
,
Akkari
Y
,
Torimaru
Y
,
Olson
S
,
Finegold
M
, et al
Heterozygosity for p53 (Trp53+/−) accelerates epithelial tumor formation in Fanconi anemia complementation group D2 (Fancd2) knockout mice
.
Cancer Res
2005
;
65
:
85
91
.
30.
Pyeon
D
,
Newton
MA
,
Lambert
PF
,
den Boon
JA
,
Sengupta
S
,
Marsit
CJ
, et al
Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers
.
Cancer Res
2007
;
67
:
4605
19
.
31.
Begum
S
,
Cao
D
,
Gillison
M
,
Zahurak
M
,
Westra
WH
. 
Tissue distribution of human papillomavirus 16 DNA integration in patients with tonsillar carcinoma
.
Clin Cancer Res
2005
;
11
:
5694
9
.
32.
Gillison
ML
,
Lowy
DR
. 
A causal role for human papillomavirus in head and neck cancer
.
Lancet
2004
;
363
:
1488
9
.
33.
Hendzel
MJ
,
Wei
Y
,
Mancini
MA
,
Van Hooser
A
,
Ranalli
T
,
Brinkley
BR
, et al
Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation
.
Chromosoma
1997
;
106
:
348
60
.
34.
Duensing
S
,
Munger
K
. 
The human papillomavirus type 16 E6 and E7 oncoproteins independently induce numerical and structural chromosome instability
.
Cancer Res
2002
;
62
:
7075
82
.
35.
Duensing
S
,
Munger
K
. 
Human papillomaviruses and centrosome duplication errors: modeling the origins of genomic instability
.
Oncogene
2002
;
21
:
6241
8
.
36.
White
A.E.
,
Livanos
E.M.
,
Tlsty
T.D
. 
Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins
.
Genes Dev
1994
;
8
:
666
77
.
37.
Fernandez-Capetillo
O
,
Celeste
A
,
Nussenzweig
A
. 
Focusing on foci: H2AX and the recruitment of DNA-damage response factors
.
Cell Cycle
2003
;
2
:
426
7
.
38.
Fernandez-Capetillo
O
,
Lee
A
,
Nussenzweig
M
,
Nussenzweig
A
. 
H2AX: the histone guardian of the genome
.
DNA repair
2004
;
3
:
959
67
.
39.
Rappold
I
,
Iwabuchi
K
,
Date
T
,
Chen
J
. 
Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways
.
J Cell Biol
2001
;
153
:
613
20
.
40.
Pichierri
P
,
Rosselli
F
. 
The DNA crosslink-induced S-phase checkpoint depends on ATR-CHK1 and ATR-NBS1-FANCD2 pathways
.
EMBO J
2004
;
23
:
1178
87
.
41.
Pichierri
P
,
Rosselli
F
. 
Fanconi anemia proteins and the s phase checkpoint
.
Cell Cycle
2004
;
3
:
698
700
.
42.
Sala-Trepat
M
,
Rouillard
D
,
Escarceller
M
,
Laquerbe
A
,
Moustacchi
E
,
Papadopoulo
D
. 
Arrest of S-phase progression is impaired in Fanconi anemia cells
.
Exp Cell Res
2000
;
260
:
208
15
.
43.
Centurion
SA
,
Kuo
HR
,
Lambert
WC
. 
Damage-resistant DNA synthesis in Fanconi anemia cells treated with a DNA cross-linking agent
.
Exp Cell Res
2000
;
260
:
216
21
.
44.
Bagby
GC
 Jr
. 
Genetic basis of Fanconi anemia
.
Curr Opin Hematol
2003
;
10
:
68
76
.
45.
Song
S
,
Gulliver
GA
,
Lambert
PF
. 
Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways
.
Proc Natl Acad Sci USA
1998
;
95
:
2290
5
.
46.
Podhorecka
M.
[gamma H2AX in the recognition of DNA double-strand breaks]
(
online
).
Postepy Hig Med Dosw
2009
;
63
:
92
8
.
47.
Zgheib
O
,
Huyen
Y
,
DiTullio
RA
 Jr.
,
Snyder
A
,
Venere
M
,
Stavridi
ES
, et al
ATM signaling and 53BP1
.
Radiother Oncol
2005
;
76
:
119
22
.
48.
Naim
V
,
Rosselli
F
. 
The FANC pathway and mitosis: a replication legacy
.
Cell Cycle
2009
;
8
:
2907
11
.

Supplementary data