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[Cancer Research 65, 85-91, January 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Heterozygosity for p53 (Trp53+/–) Accelerates Epithelial Tumor Formation in Fanconi Anemia Complementation Group D2 (Fancd2) Knockout Mice

Scott Houghtaling1, Laura Granville2, Yassmine Akkari1, Yumi Torimaru1, Susan Olson1, Milton Finegold2 and Markus Grompe1

1 Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon and 2 Department of Pathology, Texas Children's Hospital, Baylor College of Medicine, Houston, Texas

Requests for reprints: Scott Houghtaling, Department of Molecular and Medical Genetics, Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road, Mail Code L103, Portland, OR 97239. Phone: 503-494-6206. E-mail: houghtal{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure and an increased susceptibility to cancer. FA is genetically heterogeneous, consisting of at least 11 complementation groups, FA-A through L, including FA-D1 (BRCA2) and D2. We have previously reported an increased incidence of epithelial tumors in Fancd2 knockout mice. To further investigate the role of the FA pathway in tumor prevention, Fancd2 mutant mice were crossed to mice with a null mutation in the tumor suppressor gene, Trp53. The tumor spectrum in Fancd2–/–/Trp53+/– mice included sarcomas expected in Trp53 heterozygotes, as well as mammary and lung adenocarcinomas that occur rarely in Trp53 heterozygotes. These tumors occurred earlier than in Fancd2–/– control mice. Therefore, the Fancd2–/–/Trp53+/– mice represent an improved model for the study of adenocarcinoma in FA. In addition, it was found that Fancd2–/– mouse embryonic fibroblasts but not Fancd2–/–/Trp53–/– mouse embryonic fibroblasts arrest following DNA damage. Therefore, Trp53 is required for the S phase checkpoint activation observed in Fancd2 mutant cells. Fancd2–/–/Trp53–/– cells showed an increase in aneuploidy and had multiple gross chromosomal rearrangements.

Key Words: Fanconi anemia • Fancd2 • Trp53 • Mammary Adenocarcinoma


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with an increased incidence of cancer (1). Patients often present with multiple abnormalities including short stature, microphthalmia, radial ray defects, infertility, and pigmentation defects. The majority of patients succumb to bone marrow failure. Acute myelogenous leukemia is the most common malignancy, but patients, particularly those surviving following treatment by bone marrow transplantation, also have an increased risk of solid tumors including aerodigestive and gynecologic carcinomas (2, 3). FA is genetically heterogeneous, consisting of at least 11 complementation groups, FA-A, B, C, D1, D2, E, F, G, I, J, and L (4). Eight causative genes have been identified including FANCA, C, FANCD1/BRCA2, D2, E, F, G/XRCC9, and L (PHF9) (5–12). Although patients with mutations in BRCA2 (complementation group D1) have an elevated incidence of brain tumors and may have an increased predisposition to solid tumors early in childhood, patients from all other groups have an equal risk for developing malignancies (13, 14).

The precise function of the FA pathway remains uncertain. However, much evidence points to a role for the proteins in response to DNA damage and in maintenance of genomic stability (15). Following DNA damage and during S phase of the cell cycle, FANCD2 is monoubiquitinated and forms nuclear foci that colocalize with known DNA repair proteins, BRCA1 and RAD51 (16). This posttranslational modification is dependent on a nuclear complex of FANC proteins and is required for the targeting of FANCD2 to chromatin in which it facilitates the loading of BRCA2 into a chromatin complex (17, 18). FANCL has been implicated as the ubiquitin ligase responsible for the modification of FANCD2 (12, 19). BRCA2 and the yet unidentified FANCJ act downstream of other FA proteins because FANCD2 is monoubiquitinated in cell lines from FA-D1 and FA-J patients, whereas FANCI has been placed upstream of FANCD2 based on the lack of monoubiquitinated FANCD2 in FA-I cell lines (4, 7). Recent studies have suggested the FA/BRCA pathway functions in the repair of DNA damage and is particularly important in the repair of DNA interstrand cross-links, which are processed to double-strand breaks during S phase of the cell cycle (20).

Mouse models of FA faithfully represent some aspects of the human condition. Fanca (21–23), Fancc (24, 25), Fancg (26, 27), and Fancd2 (28) mice share many phenotypes including reduced fertility, small size, and a cellular sensitivity to interstrand cross-linking agents. The most striking phenotype of the Fancd2 mice is an elevated incidence of epithelial cancer including hepatocellular, mammary, lung, and ovarian adenocarcinoma that typically develop with a late onset (28).

Trp53 is a tumor suppressor gene coding for a transcription factor that has been implicated in regulating cell cycle control, apoptosis, and repair of damaged DNA. Mice harboring homozygous mutations in Trp53 are viable but develop malignant lymphomas by 6 months of age (29, 30). Trp53 heterozygous mice are also susceptible to tumors, predominantly sarcomas and lymphomas, which develop by 18 months of age. However, mammary adenocarcinoma or other tumors of epithelial origin are rare in Trp53 heterozygous mice of any strain, accounting for <10% of their tumors (31).

There is evidence to suggest that loss of p53 function may facilitate tumor development in patients with FA and mouse models. Recent studies by two groups have identified a higher proportion of human papillomavirus–positive squamous cell carcinomas among patients with FA than among controls, indicating that loss of functional p53 facilitates the development of FA tumors (32, 33). Mice harboring mutations in Brca1 or Brca2 have been intercrossed to Trp53 mutant mice with the general trend of increasing tumor incidence and decreasing tumor latency (34–38). Finally, Fancc mutant mice heterozygous at Trp53 developed blood and solid malignancies that appear earlier than mice with mutations in Trp53 alone (39). Taken together, these reports indicate that the FA/BRCA proteins and p53 cooperate in the prevention of various types of tumors. In this study, we show that Fancd2 and Trp53 cooperate in the prevention of tumors, specifically mammary adenocarcinoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Husbandry. Fancd2 mutant mice, previously described by our laboratory, were maintained on a 129S4 background (28). Trp53 mutant mice, obtained from the laboratory of Dr. Allan Bradley (Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX) were maintained on 129S4 background (29). Fancd2+/–/Trp53+/– breeding pairs were crossed to generate Fancd2–/–/Trp53+/– animals and littermate controls. Animals were examined thrice per week for tumor development and sacrificed if tumors were present or if animal was otherwise unhealthy. Animals were housed at the Oregon Health & Science University Department of Animal Care according to an approved Institutional Animal Care and Use Committee protocol.

Survival Curves. Tumor-free and epithelial cancer–free survival curves were generated using Prism software (Graphpad Software, Inc., San Diego, CA, www.graphpad.com). Statistical significance between genotypes was determined using built-in analysis for survival curves consisting of a log rank test yielding a P value.

Tumor Histology. Tumors fixed in 10% phosphate-buffered formalin for 24 hours (pH 7.4) were dehydrated in 80% ethanol and embedded in paraffin wax at 58°C. Then 4-µm sections were rehydrated and stained with H&E.

Southern Blot Analysis. Genomic DNA was extracted from tumors or tail as previously described (40). Southern blot analysis was done as previously described (29). Briefly, genomic DNA was digested with BamHI, separated on 0.8% agarose gel, and transferred to Hybond N+ (Amersham Biosciences, Piscataway, NJ). The radiolabeled probe consisted of exons 2 to 6 of Trp53. Trp53 mutant and wild-type bands were normalized to the pseudogene and the relative intensity compared to determine if loss of heterozygosity (LOH) had occurred.

Mouse Embryonic Fibroblast Growth Experiments. Primary mouse embryonic fibroblasts (MEF) were generated from pregnant Fancd2+/–/Trp53+/– or Fancd2+/–/Trp53–/– crossed to Fancd2+/–/Trp53+/– males on the 129S4 background at between 12.5 and 14.5 days' gestation. Passage 1 MEFs were seeded in triplicate at a density of 4,200 cells/cm2 on 12-well plates. Cells were grown in DMEM (Mediatech Inc., Herndon, VA) supplemented with 15% fetal bovine serum (HyClone Laboratories, Logan, UT), 1xpenicillin/streptomycin (Mediatech), and 1x L-glutamine (Mediatech). Cells were treated as previously described with slight modification (41). To induce DNA cross-linking, cells were incubated in the dark for 10 minutes in HBSS + 2% fetal bovine serum containing 0.0 or 0.2 ng/mL 4'-hydroxymethyl-4,5', 8-trimethylpsoralen plus UV A (HMT + UVA; Sigma, St. Louis, MO). Cells were irradiated with UV A for 30 minutes and HBSS +HMT was replaced with complete medium. At 3, 5, and 7 days post treatment, cells were washed in PBS, trypsinized, and resuspended in 2.0mLcomplete medium. Cells were counted using a Multisizer II (Beckman-Coulter, Fullerton, CA) according to manufacturer's instructions. Cell number is represented as percentage of cells present on day of treatment (day 0). Error bars indicate ± SE.

Bromodeoxyuridine. Passage 1 MEFs were seeded at a density of 4,200 cells/cm2 on glass coverslips in a 6-well plate and treated as described above to induce DNA cross-links. Bromodeoxyuridine (BrdUrd, Sigma) was added to media at a final concentration of 50 µmol/L for 2 hours. Cells were washed in PBS and fixed for 2 minutes in 60% ethanol/2.5% paraformaldehyde/4% glacial acetic acid. Cells were washed 3 x 2 minutes in PBS and denatured in 0.07 N NaOH for 3 minutes. Cells were washed 3 x 2 minutes in PBS (pH 8.5) and blocked in PBS + 0.5% Tween 20 for 10 minutes. Anti-BrdUrd antibody (Becton Dickinson) was diluted 1:10 in blocking solution and added to cells for 30 minutes. Cells were washed in PBS 3 x 2 minutes and FITC conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, Westgrove, PA) diluted 1:100 in PBS + 0.5% Tween 20 was added for 30 minutes. Cells were washed 3 x 2 minutes in PBS. Cover slips were mounted in SlowFade Light Antifade solution with 4', 6-diamidino-2-phenylindole (DAPI, Molecular Probes) and analyzed by fluorescent microscopy. Images were captured using OpenLAB software at 20x magnification.

Phosphorylated Histone H3 Detection. Passage 1 MEFs were seeded at a density of 4,200 cells/cm2 on glass coverslips in a 6-well plate and treated as described above to induce DNA cross-links. Cells were fixed in 4% paraformaldehyde, washed thrice in PBS for 2 minutes and permeabalized in PBS + 0.1% Triton X-100 for 3 minutes. Cells were blocked in PBS + 1% bovine serum albumin. Cells were washed in PBS for 15 minutes and incubated for 1.5 hours at room temperature in antiphosphorylated histone H3 antibody (Upstate Biotechnology, Lake Placid, NY) diluted 1:100 in blocking solution. Cells were washed 3 x 5 minutes in PBS and incubated for 30 minutes in Cy3 conjugated goat anti-rabbit antibody (Jackson Immunoresearch) diluted 1:100 in blocking solution for 30 minutes. Cells were washed 3 x 5 minutes in PBS. Cover slips were mounted in SlowFade Light Antifade solution with DAPI (Molecular Probes) and analyzed by fluorescent microscopy. Images were captured using OpenLab software at 20x magnification.

Cytogenetics. MEFs were plated on 100-mm dishes in DMEM + 10% fetal bovine serum and treated as described above to induce interstrand cross-links or mock treated. After 5 days, colcemid (Sigma) was added at a final concentration of 150 ng/mL. After 4 to 6 hours, cells were digested with trypsin, placed in hypotonic medium consisting of 5% FCS and 75 mmol/L KCl, and fixed to slides. Slides were G-banded by treatment with 10% trypsin for 45 to 55 seconds followed by Wright's stain (Fisher Scientific, Pittsburgh, PA) for 2 minutes 30 seconds. Cells were observed using a Nikon E800 fluorescence microscope, and captured using CytoVision software (Applied Imaging, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heterozygosity for Trp53 Accelerates Tumor Phenotype of Fancd2 Knockout Mice. To test the hypothesis that Fancd2 and Trp53 cooperate in tumor prevention we intercrossed Fancd2+/– and Trp53+/– mice on the 129S4 strain background. A cohort of 20 Trp53+/– and 22 Fancd2–/–/Trp53+/– mice were monitored for 20 months and compared to Fancd2–/– or control (Fancd2–/+ or wild-type) animals on the 129S4 strain background. Animals were sacrificed if tumors were palpable or if they were otherwise unhealthy.

A total of 15 tumors were identified in 13 (65%) of 20 Trp53+/– control mice (Fig. 1; Table 1). The median age of tumor free survival for these mice was 468 days and the majority of tumors were either lymphoma or sarcoma as previously reported (30)(31). One Fancd2+/–/Trp53+/– mouse developed two independent carcinomas and an adenoma of a pancreatic islet (Table 1).



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  		Figure 1. Heterozygosity for Trp53 accelerates formation of tumors in Fancd2–/– mice. A, tumor-free survival curves include all tumor types observed. B, epithelial cancer–free survival curves show an increased incidence of epithelial tumors in Fancd2–/–/Trp53+/– mice compared to Trp53+/–or control mice. C, epithelial cancer–free survival curves for female mice show a significant increase in tumors in Fancd2–/–/Trp53+/– mice compared to Fancd2–/–, Trp53+/–, or control mice. Fancd2–/– and control genotype curves were adapted from ref. (28). Statistical significance between curves was determined using log rank test. *P < 0.05, **P < 0.01, ***P < 0.001; P > 0.05 (NS).

 

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  		Table 1. Tumor spectrum from Fancd2–/–, Fancd2–/–/Trp53+/– and control mice

 


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  		Figure 3. Mammary adenocarcinomas from Fancd2–/–/Trp53+/– mice undergo LOH for the wild-type allele of Trp53. This representative Southern blot shows near-equal hybridization at wild-type and Trp53 mutant bands in nontumor (NT) genomic DNA (tail) but reduced hybridization for the wild-type band in genomic DNA from mammary adenocarcinomas (Mamm AC). Numbers 1 through 4 refer to mice in Table 1.

 
Heterozygosity for Trp53 had a significant effect on and origin of tumors in the Fancd2–/–/Trp53+/– mice compared to the Fancd2–/– mice. Fifteen (68.2%) of 22 animals developed a total of 23 tumors (Table 1). The mice had a median age of tumor-free survival of 404 days (Fig. 1). Fifteen (65.2%) of 23 tumors involved nonepithelial tissue, whereas the remaining 8 (34.8%) of 23 involved epithelial components of lung, ovary, or mammary glands (Table 1; Fig. 2). Six of the 22 Fancd2–/–/Trp53+/– mice had multiple independent tumors compared to only 1 of 20 controls.



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  		Figure 2. Examples of tumor histology from Fancd2–/–/Trp53+/– mice. A, mammary adenocarcinoma occurred in 10-month-old female mouse. The carcinoma, seen on the right side, had a sheet like growth pattern with some gland formation and compressed the residual normal parenchyma, seen on the left side. Original magnification x100. B, diffusely effacing the ovary, a primary ovarian adenocarcinoma occurred in a 13-month-old mouse. Numerous mitoses were present. Original magnification x200. C, mammary adenocarcinoma, which had obvious gland formation with pushing borders and a lymphocytic response, occurred in a 15-month-old female mouse. Original magnification x100. D, lung tumor embolic to pulmonary arteries occurred in 13-month-old mouse. Original magnification x100. E, Osteosarcoma occurred in 10-month-old Fancd2–/–/Trp53+/– mouse. Malignant osteoid was rimmed by atypical cells with nuclear pleomorphism and hyperchomasia. Original magnification x100. F, malignant fibrous histiocytoma with highly pleomorphic spindled to epithelioid cells had scattered atypical mitoses. This tumor contained no evidence of differentiation and occurred in a 10-month-old mouse. Original magnification x200.

 
The incidence of epithelial cancer was particularly striking when only female Fancd2–/–/Trp53 +/– mice were compared to controls Fig. 1C. Ten of the 22 Fancd2–/–/Trp53+/– mice in the experimental group were female. Six (60%) of these 10 developed at least one adenocarcinoma. Of the 14 tumors that formed in female Fancd2–/–/Trp53+/– mice, 8 (57.1%) involved epithelial tissue. The female Fancd2–/–/Trp53+/– mice began to develop adenocarcinoma at a time when Fancd2–/– females have not yet developed epithelial cancer. This difference was statistically significant and shows that heterozygosity at Trp53 can accelerate the tumor phenotype of Fancd2 mutant mice (Fig. 1C).

LOH at Trp53 Contributes to Formation of Solid Tumors in Fancd2–/–/Trp53 +/– Mice. In cells heterozygous for a mutant allele of a tumor suppressor gene, LOH can result in loss of the wild-type allele and contribute to tumor formation. LOH for the wild-type allele of Trp53 was observed in 55% of tumors from Trp53+/– mice in one study (31). In mouse models with conditional loss of Brca1 or Brca2 and heterozygosity at Trp53, most of the mammary tumors that formed had lost the wild-type allele of Trp53 (37, 38) .

We analyzed the status of the wild-type copy of Trp53 in tumors from Fancd2–/–/Trp53+/– and Trp53+/– mice by Southern blot analysis (Fig. 3). Comparison of intensity between wild-type and mutant Trp53 bands shows that four of four mammary adenocarcinomas from Fancd2–/–/Trp53+/– mice had undergone LOH at Trp53. The absence of complete loss of the wild-type band in genomic DNA from the mammary adenocarcinoma may be due to contaminating normal tissue or to a mix of tumor material in which not all cells have undergone LOH. In addition to the four mammary adenocarcinomas, eight of nine osteosarcomas or soft tissue sarcomas from Fancd2–/–/Trp53+/– mice exhibited loss of the wild-type copy of Trp53. Five of seven osterosarcomas or soft tissue sarcomas examined from control Trp53+/– mice had also undergone LOH at Trp53. Thus, consistent with previous reports (31), LOH may not be necessary for tumor formation in cells heterozygous for Trp53, but may be a necessary step for the development of certain tumor types, such as mammary adenocarcinoma in Fancd2–/–/Trp53+/– animals.

Primary Fancd2–/–/Trp53–/– MEFs Continue to Proliferate following DNA Damage. Both wild-type and FA cells are capable of repairing interstrand cross-links and arrest with near 4N DNA content in late S phase (41, 42). To examine the role of Trp53 in this late S-phase arrest, primary MEFs deficient in Fancd2, Trp53, or both genes and Fancd2+/–/Trp53+/– control MEFs were treated to induce interstrand cross-links. Following a dose of 0.2 ng/mL HMT + UVA, Fancd2+/–/Trp53+/–, Fancd2–/–, and Fancd2–/–/Trp53–/– but not Trp53–/– MEFs stop accumulating in cell number (Fig. 4B)) relative to mock-treated controls (Fig. 4A).



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  		Figure 4. Cell growth kinetics of primary MEFs. Cell number is represented as a percentage of total cells at time of treatment. Cells were mock treated with 0.0 ng/mL (A) or treated with 0.2 ng/mL (B) HMT + UVA. Following treatment with HMT + UVA, Trp53–/– cells continue to increase cell number, whereas Fancd2–/–/Trp53+/–, Fancd2–/–/Trp53–/–, Fancd2+/–/Trp53+/,– and MEFs stop increasing cell number. Points, mean ± SE. At 5 days after induction of DNA cross-links, cells were allowed to incorporate BrdUrd to detect cycling cells (C). BrdUrd-positive cells were scored using an antibody against BrdUrd. Nuclei were counterstained with DAPI. Fancd2–/–/Trp53–/– and Trp53–/– MEFs continue to incorporate BrdUrd following treatment, whereas Fancd2–/–/Trp53+/– and Fancd2+/–/Trp53+/– MEFs do not.

 
To investigate whether Fancd2–/–Trp53–/– MEFs had undergone a cell cycle arrest, cells were allowed to incorporate BrdUrd 5days after treatment and immunocytochemistry was done to detect cells in S phase. Whereas mock-treated Fancd2+/–/Trp53+/– and Fancd2–/– MEFs incorporated BrdUrd, their treated counterparts failed to incorporate BrdUrd (Table 2), consistent with previous results showing that wild-type and FA cells undergo a cell cycle arrest following induction of interstrand cross-links (41, 42). Trp53–/– MEFs continued to incorporate BrdUrd following treatment, demonstrating that they have actively replicating DNA, consistent with their increase in cell number. The percentage of treated Fancd2–/–/Trp53–/– MEFs that were positive for BrdUrd was similar to Trp53–/– MEFs in multiple independent experiments, indicating that these cells are also replicating DNA. We also did immunocytochemistry to identify cells in mitosis. Consistent with the BrdUrd data, both Trp53 mutant and double-mutant cells failed to stop progression through the cell cycle (Table 2). The seemingly paradoxical result of double-mutant cells continuing to progress through the cell cycle but failing to increase in cell number can be explained if these cells die following attempts to divide after induction of DNA damage that cannot be repaired. Thus, the cell cycle arrest observed in both wild-type and Fanconi cells is dependent on Trp53.


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  		Table 2. Measure of primary MEFs in S phase and M phase at day 5 after interstrand cross-link induction

 
To further investigate the effects of continued cycling with unrepaired DNA damage, we did detailed cytogenetic analysis on Trp53 mutant and Fancd2/Trp53 double-mutant MEFs 5 days after mock treatment or treatment with 0.2 ng/mL HMT + UVA. In two independent, blinded experiments the treated double-mutant MEFs were identified as the most genetically abnormal sample. Both the untreated and treated double-mutant samples contained numerous gross chromosomal rearrangements (GCR; (Fig. 5A)). These included deletions, translocations, and chromosomes rearranged beyond recognition (markers). An average of 0.4 GCRs were identified per cell in untreated Trp53 mutant MEFs, and 0.3GCRs were identified per cell in treated Trp53 mutant MEFs. Loss of Fancd2 contributed greatly to the genetic instability observed in double-mutant cells as shown by an average of 1.3 GCRs identified per metaphase in untreated double-mutant MEFs and a striking 2.9 GCRs identified per metaphase from treated double-mutant MEFs. Metaphase spreads were also analyzed for total chromosome number (Fig.5B). All cells examined showed a moderate increase in aneuploidy; however, most of the untreated or treated Trp53 mutant and untreated double-mutant cells contained near-diploid (2N) or tetraploid (4N) genomic content. In contrast, nearly all treated double-mutant MEFs showed marked aneuploidy. Thus, both Fancd2 and Trp53 cooperate in maintaining genomic integrity.



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  		Figure 5. Fancd2–/–/Trp53–/– MEFs show an increase in GCRs and aneuploidy. A, Fancd2–/–/Trp53–/– MEFs show an increase in the total number of GCRs per cell. The GCRs included deletions, translocations, and rearranged marker chromosomes. The mean number of GCRs per cell is shown above the individual scatterplot. P < 0.001 for p53T versus D2p53T. B, the majority of cells from both untreated (UT) and treated (T) Trp53–/– (p53) and untreated Fancd2–/–/Trp53–/– (D2p53) contain a near diploid (2N) or tetraploid (4N) number of chromosomes. However, the majority of cells from treated (T) Fancd2–/–/Trp53–/– cells have a total number of chromosomes that reflects marked aneuploidy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Useful Model of Spontaneous Adenocarcinoma. Here we describe an improved model of epithelial cancer in FA mice. Fancd2–/–/Trp53+/– mice provide a useful model of solid tumor formation in patients with FA. All patients with FA are at an increased lifetime risk of developing a variety of solid tumors (2, 3). Understanding this risk will become increasingly important as treatments for the hematologic complications of FA improve and more patients survive bone marrow failure. Whereas the tumor spectrum in Fancd2–/–/Trp53+/– mice is not identical to that observed in human patients, this is the first animal model of FA in which a significant fraction of the tumors observed originate in epithelial tissues. Thus, Fancd2–/–/Trp53–/+ mice are a useful model for testing the efficacy of potential chemoprevention regimens.

The difference in incidence of adenocarcinoma, particularly mammary adenocarcinoma, between female and male Fancd2–/–/Trp53+/– mice is particularly striking and suggests testable hypotheses for future experiments. Although the most obvious hypothesis involves the larger population of target cells in female versus male mice, other potential explanations exist. Perhaps androgens are protective and prevent the development of adenocarcinoma. If this is correct, castrated male Fancd2–/–/Trp53+/– mice may develop mammary adenocarcinoma at an increased rate. Alternatively, estrogen may promote the development of tumors in the female Fancd2–/–/Trp53+/– mice. If female hormones are tumor-promoting factors, male Fancd2–/–/Trp53+/– mice given estrogen should develop tumors at a similar rate as female Fancd2–/–/Trp53+/– mice. Investigating the cause of the different incidence between male and female Fancd2–/–/Trp53+/–micewill be particularly important given that androgen therapy is often used in the treatment of anemia associated with FA (43).

Many of the pleiotropic phenotypes we have previously described in Fancd2 mutant mice, including small size, microphthalmia, and reduced germ cell number, may be attributed to reduced function of stem cells that give rise to the affected tissues (28). It has been proposed that increased turnover of hematopoietic stem cells in patients with FA results in the shortened telomeres observed in peripheral blood mononuclear cells and correlates with the onset of aplastic anemia (44–46). Other investigators have showed increased breakage at telomeric repeats and an increased frequency of chromosome end fusions in FA lymphocytes (47). Thus, it remains possible that increased telomere shortening and/or breakage predispose patients with FA to myeloid malignancies. The existence of mammary stem cells has been shown by serial transplantation studies in mice (48, 49), and deregulation of normal self-renewal in mammary stem cells has been proposed as a mechanism by which mammary adenocarcinoma forms (50). Thus, it is reasonable to consider that loss of the FA pathway may increase genomic instability in mammary stem cells and predispose to mammary adenocarcinoma. This possibility could be further investigated in the future by serially passaging mammary stem cells from Fancd2–/–/Trp53+/– females and controls. Mammary progenitor cells from Fancd2–/–/Trp53+/– females may form mammary tumors at an increased frequency compared to controls when transferred to recipient animals. Alternatively, mammary tumors from Fancd2–/–/Trp53+/– mice could be analyzed for the presence of particular cell surface markers such as Sca–1, associated with mammary stem cells to further substantiate this theory (51).

Finally, the tumor spectrum of Fancd2–/–/Trp53+/– mice is markedly different from that of Fancc–/–/Trp53+/– mice (39). Whereas approximately a third of Fancd2–/–/Trp53+/– mice developed adenocarcinoma, Fancc–/–/Trp53+/– mice do not develop adenocarcinoma and only 1 ovarian tumor was observed in a population of 22 animals (the representation of males and females was not indicated). This difference in phenotype is important for at least two reasons. First, it shows that a null mutation in Fancd2 causes a more severe phenotype in mice than a null mutation in Fancc. This observation supports a model in which both isoforms of Fancd2 (Fancd2-S and Fancd2-L) perform qualitatively similar functions but that complete loss of both isoforms, as observed in Fancd2 null mice, is required to reach a critical threshold at which certain phenotypes, such as mammary adenocarcinoma, are observed. Second, it suggests that patients with FA, with mutations in genes proposed to function downstream in the FA pathway, may be at an increased risk of developing solid tumors. Increased surveillance for solid tumors may be warranted for patients from complementation group D2 as has been proposed for patients with FA from group D1 (14).

Trp53 Is Required for S Phase Arrest following DNA Damage. We have generated primary MEFs deficient in Fancd2, Trp53, or both genes, and controls and shown that Trp53 is required for the cell cycle arrest following induction of interstrand cross-links. Trp53–/– and double-mutant cells continued replicating DNA following induction of interstrand cross-links, whereas Fancd2 and control cells were capable of arresting their cell cycle following a dose of 0.2 ng/mL HMT + UVA. It is reasonable to assume that cells lacking the appropriate checkpoints to halt cycling may compound the unrepaired DNA damage by dividing before repair has occurred. Cytogenetic analysis of treated double-mutant MEFs revealed many GCRs, including deletions and translocations, as well as marked aneuploidy. It is likely that loss of the FA pathway leads to unrepaired lesions and an inability to arrest and successfully repair contributes to the genomic instability leading to adenocarcinoma observed in Fancd2–/–/Trp53+/– mice.


   Acknowledgments
 
Grant support: NHLBI Program Project grant 1PO1HL48546 (M. Grompe) and a training grant appointment NIH 5 T32 GM008617-08 (S. Houghtaling).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/ 5/04. Revised 10/13/04. Accepted 10/27/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tischkowitz MD, Hodgson SV. Fanconi anaemia.J Med Genet 2003;40:1–10.[Abstract/Free Full Text]
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