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[Cancer Research 62, 4054-4060, July 15, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

The Fragile Histidine Triad/Common Chromosome Fragile Site 3B Locus and Repair-deficient Cancers1

Bruce C. Turner, Michelle Ottey, Drazen B. Zimonjic, Magdalena Potoczek, Walter W. Hauck, Edward Pequignot, Catherine L. Keck-Waggoner, Cinzia Sevignani, C. Marcelo Aldaz, Peter A. McCue, Juan Palazzo, Kay Huebner2 and Nicholas C. Popescu

Departments of Radiation Oncology [B. C. T., M. P.], Microbiology and Immunology [M. O., C. S., K. H.], Medicine [W. W. H., E. P.], and Pathology [P. A. M., J. P.], Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892 [D. B. Z., C. L. K- W., N. C. P.]; and Department of Carcinogenesis, University of Texas, M. D. Anderson Cancer Center, Science Park Research Division, Smithville, Texas 77030 [C. M. A.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In various studies of sporadic breast cancers, 40–70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several earlier studies suggested to us that activity of repair proteins was important in maintaining integrity of common fragile regions (1, 2, 3) . Alteration of the FHIT3 gene in breast carcinomas has been reported (4, 5, 6) in concordance with LOH at chromosome region 3p14.2 in breast cancer (7 , 8) and benign proliferative breast disease (9) . A significantly higher frequency of LOH at the FHIT locus was observed for breast cancers with deleterious mutations of BRCA2 (10) . In a study of breast tumors that were either sporadic or exhibited founder Icelandic BRCA2 999del5 mutations, there was a significant association of LOH at 3p14.2 with reduced expression of Fhit, and the BRCA2 999del5 tumors demonstrated significantly lower levels of Fhit protein compared with sporadic breast cancers (3) . Thus, alteration at the FHIT locus led to loss of Fhit protein in a significant fraction of sporadic breast cancers and a larger fraction of familial breast cancers with an inherited BRCA2 mutation, consistent with the idea that loss of Brca2 function affects stability of the FHIT/FRA3B locus at the common fragile region in chromosome band 3p14.2.

Recent studies of Fhit knockout mice suggested a connection between Fhit inactivation and MTS, defined as the coexistence of one or more sebaceous tumors with one or more visceral carcinomas (11) ; the syndrome shares clinical and pathological characteristics with HNPCC (12) and is, in fact, allelic to HNPCC. A large subgroup of MTS cases exhibit microsatellite instability and germ-line mutations in MSH2 or MLH1 genes (11) . Mice heterozygous for a mutant Fhit allele (Fhit+/- mice) exhibited an N-nitrosomethylbenzylamine (NMBA)-induced MTS-like syndrome with sebaceous and visceral tumors (13) but no involvement of mismatch repair deficiency. If human and mouse MTS cases arise through similar mechanisms, then the FHIT gene may be a target of damage in some mismatch repair-deficient tumors, leading to Fhit protein loss and clonal expansion of Fhit-negative cells. In fact, it was previously observed that two of three human pancreatic cancer cell lines with high microsatellite instability, diagnostic of mismatch repair deficiency, had homozygous deletions within FHIT (2) , and more recently it has been shown that a large fraction of Msh2-deficient colon tumors are Fhit negative (3) . To further examine the connection between loss of DNA repair function and expression of common fragile sites, we assessed expression of Fhit in breast cancers with inactivated BRCA1, a gene involved in repair of DNA double-strand breaks (14, 15, 16, 17) , and we have assessed the level of induction of fragile sites in repair-deficient cell lines.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells.
Onco-98 and Onco-71 human lymphoblastoid lines with a mutant PMS2 and a mutant APC allele, respectively, were obtained from Bert Vogelstein (Johns Hopkins University). Onco-98 cells express no mismatch repair activity because of a possibly dominant negative PMS2 mutation (18 , 19) . Cancer cell lines were obtained from American Type Culture Collection (Capan-1, MDA-MB-468, MCF-7, SKBR3, and SW626).

Mouse cell long-term primary cultures were established by us (Msh2-/- kidney cells, passage 5) from Msh2-deficient mice; p53-/- fibroblasts (passage 12) from deficient mice, Msh2-/- p53-/- fibroblasts (passage 15) from deficient mice (20) ; Fhit-/- kidney cells from deficient mice; or obtained from other labs (wild-type mouse kidney cells from Albert Wong, established from normal kidney of an NIH mouse, passages 6–12; Mlh1-/- mouse embryo fibroblasts, passage 3, from Winfried Edelmann; Brca1+/-, passage 13, and Brca1-/- mouse embryo fibroblasts, passage 6, on p53-/- background were from Beverly Koller, Ref. 21 ). All cells were established and maintained in DMEM with 10–15% fetal bovine serum and were used at tissue culture passages <15.

Fragile Site Induction.
For expression of fragile sites, cells were treated 1 day after subculturing with aphidicolin (0.2–0.4 µM; Sigma, St. Louis, MO) in DMSO or in ethanol (0.2%) for 26 h. To enhance the expression of fragile sites, cultures were exposed to caffeine (2.2 µM; Sigma) for 6 h before chromosome preparation (22 , 23) . Metaphase spreads were prepared after 3–4 h Colcemid (50 µg/ml) treatment by standard KCl hypotonic treatment, acetic acid-methanol fixation, and air-dried slide preparation. All cell line identifications were coded so that these experiment were carried out blind. A minimum of 25 DAPI- or Giemsa-stained metaphases was scored for each sample.

FISH.
DAPI-stained chromosome preparations from human cell lines used for scoring the incidence of gaps and breaks were destained, washed, rehydrated in a series of alcohols and hybridized with YAC probes for regions 3p14.2,7q32 and 16q23.3. To visualize the signal for all three probes at the same time, one probe was labeled with biotin, the second with digoxigenin, and the third with a combination of both fluorochromes. The combined fluorochromes generated a yellow color distinctive from red and green. The conditions for FISH, detection of the signal, and digital imaging were carried out as described previously (24 , 25) . The metaphases examined for aberrations were relocated, and position of fluorescent signal for the YAC probes was evaluated relative to chromosome integrity.

The YAC clones used were: 933H-2 for FRA16D at 16q23.2 (26) ; 850A6 encompassing FRA3B (25) ; and YAC 746A5 encompassing FRA7G (27) . 0.5–1 µg of each YAC DNA was labeled for hybridization to metaphases.

Tissues.
Paraffin-embedded breast tumor samples from 44 BRCA1/BRCA2 wild-type and 22 BRCA1 mutant breast cancer patients were obtained for expression analysis from the Breast Tumor Data Banks at Thomas Jefferson University and Yale University. All patients underwent comprehensive sequencing of the BRCA1 and BRCA2 genes to identify any deleterious mutations of these genes. The control patients were individually matched to the BRCA1 mutant breast cancer patients with respect to age (within 5 years), stage, tumor grade, and disease-free survival.

Immunohistochemistry.
Fhit expression was analyzed by standard immunostaining protocols, which included deparaffinization from xylene to 95% alcohol and rehydration (28) . The deparaffinization process included a 30-min methanol peroxide block for endogenous peroxidase activity. The primary antibody was a polyclonal rabbit immunoglobulin raised against gstFhit protein at a 1:3000 dilution incubated for 60 min at room temperature in a humidified chamber. After incubation, slides were washed and incubated for 30 min with a biotinylated IgG secondary antibody (Dako, LSAB kit) and exposed to streptavidin-biotin complex for 30 min. After a wash, slides were subjected to two consecutive 4-min applications of 3,3'-diaminobenzidine/peroxide solution (Dako). Treated slides were counterstained with hematoxylin and cover slipped before evaluation. Fhit expression in tumor cells was evaluated for intensity of Fhit staining and rated on a 4-point scale: 0, none; 1+, light; 2+, moderate; 3+, heavy; and 4+, intense, as described previously (29 , 30) . Distribution of staining within the invasive component was scored as 0–100% staining, and the H-score, a total Fhit staining score for each tumor, was defined as the product of intensity and distribution scores. To set cutoffs for dichotomizations of data into high (positive) and low (negative) expression groups, the mean H-score results for the entire data set were displayed as bar-histograms, and an H-score <100 was determined to represent background staining, and an H-score of 100 was considered the cutoff for Fhit positivity. All slides were evaluated by a single pathologist (J. P.) who was blinded to the clinical and genetic histories of the patients.

Statistical Analysis.
Odds ratios and related Ps, for analysis of Fhit expression in Brca1-deficient breast cancers, were calculated using exact multivariate logistic regression (LogXact 2.0; Cytel Software Corporation). The significance of differences in median number of aberrations/chromosome between cell lines was tested using a Wilcoxon test (Ref. 31 ; comparing all cell lines to wild type). For cell lines Msh2-/-, (Msh2-/-,p53-/-), Mlh1-/-, (Brca1-/-,p53-/-), and Fhit-/- where the number of aberrations was sometimes recorded as a lower bound, a Wilcoxon test modified to handle such data was used (32) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fhit Expression in BRCA1-deficient Cancers.
Expression of Fhit protein was determined in 22 breast cancers with definitive deleterious mutations in the BRCA1 gene (Table 1)Citation . For each BRCA1 mutant breast cancer, there were two control breast tumors that contained wild-type BRCA1 and BRCA2 genes that were matched for both clinical and pathological variables, including age, stage, tumor grade, and disease-free survival. Examples of immunostaining are shown in Fig. 1Citation , and results are summarized in Table 1Citation . As demonstrated in Fig. 1, D–FCitation , there was absence of Fhit immunoreactivity in BRCA1 mutant tumors compared with BRCA wild-type control breast tumors (Fig. 1, A–C)Citation that demonstrated Fhit immunoreactivity localized to the cytoplasm of the cells. In contrast to previous reports that suggest that 40–70% of breast tumors express the Fhit protein, only 2 of 22 (9%) BRCA1 mutant breast tumors were found to have Fhit expression. Of the double individually matched pairs of sporadic breast tumors, both control pairs were positive in 8 cases, and for all other pairs, at least 1 breast tumor was Fhit immunoreactive. The marked decrease in Fhit expression in BRCA1 mutant breast cancers compared with the matched controls with wild-type BRCA1 and BRCA2 genes was statistically significant (P < 0.001; odds ratio: 0.09; 95% confidence interval: 1%, 38%). There was not a significant association between Fhit expression and specific BRCA1 mutations, age at diagnosis, stage, tumor grade, or disease-free survival.


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  		Table 1 Immunohistochemical analysis of Fhit expression in Brca1-deficient breast cancers

Fhit expression in tumor cells was evaluated for intensity and distribution. The product of the intensity and distribution scores, the H score, was determined for each tumor, and bar-histograms of the scores determined that scores <100 represented background. Thus, a H score of <100 was considered negative for Fhit expression; a score of >=100 was considered Fhit positive.

 


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  		Fig. 1. Immunohistochemical detection of Fhit expression in Brca1 mutant breast cancers. A–C, BRCA1 wild-type (WT) breast tumors stained with Fhit antibody and expressing varying levels of Fhit protein (A and B, x250; C, x400). D–F, BRCA1 mutant breast tumors lacking Fhit protein expression (x400).

 
Aphidicolin Induction of Chromosome Aberrations in Human Cells.
If breast cancers with inactivated BRCA1 and BRCA2 genes show enhanced LOH at 3p14.2 and increased frequency of Fhit protein loss, it seemed possible that cancer cell lines derived from such BRCA1- or BRCA2-deficient cancers might show a higher level of activation of common fragile sites; i.e., perhaps these sites are more fragile in BRCA1 or BRCA2 cancers. Thus, we tested a panel of cancer cell lines for appearance of fragile site gaps and breaks after aphidicolin treatment. Aphidicolin is an inhibitor of DNA polymerases {alpha} and {delta} and inhibits progression of the DNA replication fork. Capan-1, a pancreatic cancer cell line with inactivated BRCA2 (33) , showed more gaps and breaks/chromosome than did the three breast cancer cell lines included in the study (Table 2)Citation , and only the BRCA2-deficient cell line, Capan-1, showed some metaphases with 100 or more gaps and breaks. Because cancer cell lines have been through long selection histories to become cancers and then to be established in cell culture, there could be numerous alterations that could affect responses to aphidicolin, so the differences between Capan-1 and the other cells need not reflect the differences at the BRCA2 locus. Thus, we were interested in testing more nearly normal human cells with repair deficiencies for the effect of aphidicolin on gaps and breaks at fragile sites. Normal human cell lines lacking BRCA1 or BRCA2 do not exist, but the Brca1 protein is known to be part of a protein complex that includes DNA damage repair proteins Msh2, Msh6, and Mlh1 (34) , so nearly normal human cells missing mismatch repair functions might also exhibit higher fragility as seen in Capan-1 cells. Human lymphoblastoid cell lines derived from individuals carrying a germ-line mutation in the APC gene (Onco-71) or PMS2 (Onco-98; Refs. 18 , 19 ) were treated with 0.4 µM aphidicolin, the concentration used to induce common fragile sites, and metaphase chromosomes were prepared and assessed for expression of the gaps and breaks typical of common fragile sites. Results are summarized in Table 3Citation . Interestingly, the Onco-98 metaphases showed more than twice as many aberrations/chromosome as the Onco-71 metaphases, a highly significant difference (P < 0.001) as shown in Table 3Citation . The Onco-98 cells are completely lacking mismatch repair activity (18 , 19) . To illustrate that many of the gaps and breaks were in fragile sites, fluorescent YAC probes for three common fragile regions were hybridized to the same metaphase chromosomes, and the number of fluorescent signals was tallied at the individual fragile regions, FRA3B at 3p14.2, FRA7G at 7q32, and FRA16D at 16q23 (Fig. 2)Citation . The data are summarized in Table 3Citation and shows that in both cells lines, a higher incidence of gaps and breaks occurs at FRA3B and FRA16D than at FRA7G as reported previously (22) .


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  		Table 2 Aphidicolin induction of gaps and breaks in human tumor cell linesa

 

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  		Table 3 Aphidicolin induction of chromosome aberrations in human lymphoblastoid cells

Cells were treated with 0.4 M aphidicolin and 1% ethanol for 26 h; caffeine (2.2 M) and Colcemid were added 6 and 4 h, respectively, before chromosome fixation. Fifty metaphases were scored on DAPI-stained preparation and used for FISH with YAC probes.

 


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  		Fig. 2. Breaks at fragile regions in human lymphoblastoid cells. The Onco-71 and Onco-98 lymphoblast cell lines were treated with aphidicolin as described in Table 3Citation , and the metaphases were processed for counting of chromosome gaps and breaks. The slides were then treated to denaturing conditions and hybridized to fluorescent YACs for FRA3B (3p14.2), FRA7G (7q32), and FRA16D (16q23). A shows an Onco-71 metaphase with aberrations indicated by arrows. B shows the same metaphase after FISH in which it can be observed that the fragile region probes mapped at the sites of breaks. C shows an Onco-98 metaphase with D illustrating fluorescent probes at chromosome breaks.

 
We expected to see about a 2-fold increase in the number of FISH signals encompassing gaps and breaks because we hypothesize that certain repair deficiencies would lead to an increase in aphidicolin-induced gaps and breaks in common fragile sites. Between 60 and 80% of gaps and breaks induced by 0.2–0.4 µM aphidicolin were reported to occur in common fragile sites with the remaining aberrations apparently occurring randomly (22 , 23) . There is no reason to suppose that the aberrations would be skewed toward random breaks in these repair-deficient cells. It is possible that the PMS2-dominant negative mutant in the Onco-98 cells, which abrogates mismatch repair (18 , 19) , might cause increased fragility at common fragile sites not assayed in our FISH experiment. The distribution of common fragile sites activated in specific repair-deficient cell types will require extensive future investigations.

Aphidicolin Sensitivity in Repair-deficient Mouse Cells.
Elevated induction of gaps and breaks in the nearly normal mismatch repair deficient human cell line Onco-98 was consistent with the hypothesis that aphidicolin sensitivity is elevated in mismatch repair-deficient lymphoblastoid cells. To study this in more detail in cells with different repair genes mutated, it was necessary to use repair-deficient mouse cell lines established from recombinant mice or mouse embryos as listed in Table 4Citation . Wild type, Msh2-/-, and Fhit-/- cells were from kidneys of young wild type and Msh2 or Fhit knockout mice. Msh2-/-p53-/- cells were colonic fibroblasts from double knockout mice. Other lines were from 11–13 d mouse embryos of the designated genotype (Table 4)Citation . Cell lines of each genotype were treated with aphidicolin to induce gaps and breaks and metaphase chromosomes were examined. By inspection of the columns in Table 4Citation , it can be seen that repair-deficient cells (Msh2, Mlh1, Brca1 negative cells) show 2- to >3-fold increases in numbers of aberrations/chromosome, and cells completely missing repair function show metaphases with multiple aberrations (see last column of Table 4Citation ). Thus, the first three cells in the table, wild type, p53-/-, and Brca1+/-p53-/-, show a low level of aberrations/chromosome, similar to the level seen in Onco-71 cells (compare to Table 3Citation ). All of the other cell lines, including Fhit-/- cells, show elevated numbers of aberrations (P < 0.001 to P = 0.007). Examples of the chromosome gaps and breaks are shown in Fig. 3, A and BCitation . Of the cell lines listed in Table 4Citation , only the wild-type and Fhit-/- cells were mostly diploid after aphidicolin treatment (Fig. 3C)Citation . The majority of metaphases of the other cell lines was tetraploid (Fig. 3C)Citation . Tetraploidy may be contributed by absence of p53 in the three p53-deficient cell lines. Because these are early passage mouse cell cultures, it is unlikely that the increase in chromosome aberrations in these cells is attributable to downstream mutations resulting from mutant phenotypes because chromosome aberrations are increased in most cells in the population; but the increase need not be a direct effect of absence of the individual genes. That absence of Fhit also increased gaps and breaks was unexpected; it seemed unlikely that a gene that can be damaged by alteration to a fragile site would be involved in protection from DNA damage. This will require additional investigation.


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  		Table 4 Aphidicolin induction of gaps and breaks in repair-deficient mouse cells

Cells were treated with 0.4 µM aphidicolin and 1% ethanol for 26 h. Caffeine (2.2 M) and Colcemid were added at 6 and 4 h, respectively, before chromosome fixation.

 


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  		Fig. 3. Chromosome aberrations in repair-deficient mouse cells. Mouse cell lines were treated with aphidicolin and assessed for chromosome aberrations as summarized in Table 4Citation . A, wild-type mouse kidney cells. B, Msh2-/- mouse kidney cells. Arrows indicate gaps, breaks. C, ploidy of the aphidicolin-treated murine cell lines.

 
The p53-/- cells showed the fewest aberrations of any of the cell lines, including wild type. It is likely that the apparent low number of gaps and breaks observed was influenced by the polyploidy level with 80–160 chromosomes in most of the metaphases (Fig. 3C)Citation and the occurrence of a small fraction of the spreads with very few aberrations.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A number of familial cancer syndromes, especially those resulting in HNPCC, breast/pancreatic cancers, and breast/ovarian cancers, are because of germ-line mutations in genes required for repair of various forms of DNA damage, such as base mismatches and double-strand break repair. MSH2 and MLH1 are the genes most frequently mutated in HNPCC, although PMS2 can also be involved (12) . The BRCA1 and BRCA2 genes are both involved in repair complexes that deal with double-strand break repair (14, 15, 16 , 35) . Disruption of the murine Brca1 gene causes {gamma}-irradiation hypersensitivity and genetic instability. Although both Brca1 and Brca2 are very large proteins and are likely to be involved in a number of functions, it has been thought that loss of the DNA repair function, leading to an increased incidence of mutations, underlies the tumor suppressor activity of the two genes (17) . We had speculated (1) that the loss of repair function of the BRCA2 gene might contribute to damage at common fragile sites. Thus, we wanted to observe aphidicolin-induced fragility in BRCA2-deficient cells, which were not available. We also wished to determine whether breast cancers with inactivated BRCA1 genes would show reduced Fhit protein expression, suggesting that BRCA1, like BRCA2, was important in protecting FRA3B from damage. Indeed, we have now observed that BRCA1-associated breast cancers are mostly negative for Fhit expression, consistent with the idea that Brca1 is also important for maintaining intact FRA3B, and by extension of other fragile sites, possibly including FRA16D (36, 37, 38) . This fragile region encodes the WWOX gene, a gene that is involved in LOH in breast, prostate, and other cancers and is altered or homozygously deleted in some cancer cell lines and primary tumors. WWOX/FRA16D, like FHIT/FRA3B, exhibits hallmarks of a tumor suppressor gene for a fraction of breast and other cancers (36 , 37 , 39) . FRA3B and FRA16D, harboring tumor suppressor genes, were considerably more susceptible to aphidicolin induction than FRA7G, confirming earlier observations (22) . Genes targeted for damage in cancers with inactivated BRCA1/2 are thus far unknown, but genes at fragile sites seemed possible targets because of their apparent susceptibility to double-strand breaks and misrepair. Thus, we proceeded to test a Brca1-deficient mouse cell line for increased susceptibility to induction of gaps and breaks typical of common fragile sites and did observe such an increase.

While these studies were in progress, several reports suggested an increased frequency of Fhit inactivation in mismatch repair-deficient cancer cells. Thus, we also studied Mlh1- and Msh2-deficient mouse and PMS2-deficient human cells and observed increased gaps and breaks similar to those induced at common fragile sites by aphidicolin. This was somewhat unexpected because mismatches were not the type of damage incurred at common fragile sites. Fhit is absent in a large fraction of MSH2-deficient cancers, possibly because of misrepaired damage at FRA3B. Thus, it could be that genes at fragile sites are frequently directly involved in tumorigenesis of repair-deficient cancers, and by cloning and studying genes at common fragile sites, we may find important tumor suppressor genes for familial and sporadic breast cancers.

Brca1 has been shown to associate physically with a group of proteins that are important for DNA repair. The protein complex, called BASC for Brca1-associated genome surveillance complex, includes Brca1, Msh2, Mlh1, Atm, Blm, and the Rad50-Mrell-Nbs1 protein complex (34) . All members of this complex have roles in recognition of abnormal DNA structures or damaged DNA, suggesting that it may serve as a DNA damage sensor. Thus, it is thought that Brca1 (or other proteins in the complex) functions as coordinator of activities required for maintenance of genomic integrity during DNA replication. It is not surprising then that inactivation of specific protein members of the complex such as Brca1, Msh2, and Mlh1 could affect the frequency of common fragile site induction. These genetic alterations may be critical events in the development of hereditary cancers and suggest that both hereditary and sporadic tumorigenesis may have common distal pathways.


   FOOTNOTES
 
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.

1 Supported by P01CA77738, Cancer Center Support Grant CA56036 and Training Grant 5T32-CA09678 (to M. O.) from the National Cancer Institute, National Institutes of Health, and the Susan G. Komen Breast Cancer Foundation, respectively. Back

2 To whom requests for reprints should be addressed, at Kimmel Cancer Center, BLSB, Room 1008, 233 South 10th Street, Philadelphia, PA 19107. Phone: (215) 503-4656; Fax: (215) 923-3528; E-mail: K_Huebner{at}springfield.jci.tju.edu Back

3 The abbreviations used are: FHIT, fragile histidine triad; LOH, loss of heterozygosity; MTS, Muir-Torre syndrome; HNPCC, hereditary nonpolyposis colorectal cancer; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; YAC, yeast artificial chromosome; FRA3B, common chromosome fragile site 3B. Back

Received 1/31/02. Accepted 5/ 8/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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