Cyclin A-CDK phosphorylates Sp1 and enhances Sp1-mediated transcription.

Cyclin A-mediated activation of cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. Cyclin A activity is regulated on several levels and cyclin A elevation in a number of cancers suggests a role in tumorigenesis. In the present study, we used a modified DNA binding site selection and PCR amplification procedure to identify DNA binding proteins that are potential substrates of cyclin A-CDK. One of the sequences identified is the Sp1 transcription factor binding site. Co-immunoprecipitation experiments show that cyclin A and Sp1 can interact physically. In vitro and in vivo phosphorylation studies indicate that cyclin A-CDK complexes can phosphorylate Sp1. The phosphorylation site is located in the N-terminal region of the protein. Cells overexpressing cyclin A have elevated levels of Sp1 DNA binding activity, suggesting that cyclin A-CDK-mediated phosphorylation augments Sp1 DNA binding properties. In co-transfection studies, cyclin A expression stimulated transcription from an Sp1-regulated promoter. Mutation of the phosphorylation site abrogated cyclin A-CDK-dependent phosphorylation, augmentation of Sp1 transactivation function and DNA binding activity.

Mammary tumor induction and premature ovarian failure in ApcMin mice are not enhanced by Brca2 deficiency.

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1+/-0.4 and 0.3+/-0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.

BRCA2-null embryonic survival is prolonged on the BALB/c genetic background.

Women who inherit mutations in the BRCA2 cancer susceptibility gene have an 85% chance of developing breast cancer. The function of the BRCA2 gene remains elusive, but there is evidence to support its role in transcriptional transactivation, tumor suppression, and the maintenance of genomic integrity. Individuals with identical BRCA2 mutations display a different distribution of cancers, suggesting that there are low-penetrance genes that can modify disease outcome. We hypothesized that genetic background could influence embryonic survival of a Brca2 mutation in mice. Brca2-null embryos with a 129/SvEv genetic background (129(B2-/-)) died before embryonic day 8. 5. Transfer of this Brca2 mutation onto the BALB/cJ genetic background (BALB/c(B2-/-)) extended survival to embryonic day 10.5. These results indicate that the BALB/c background harbors genetic modifiers that can prolong Brca2-null embryonic survival. The extended survival of BALB/c(B2-/-) embryos enabled us to ask whether transcriptional regulation of the Brca1 and Brca2 genes is interdependent. The interdependence of Brca1 and Brca2 was evaluated by studying Brca2 gene expression in BALB/c(B1-/-) embryos and Brca1 gene expression in BALB/c(B2-/-) embryos. Nonisotopic in situ hybridization demonstrated that Brca2 transcript levels were comparable in BALB/c(B1-/-) embryos and wild-type littermates. Likewise, reverse transcriptase-polymerase chain reactions confirmed Brca1 mRNA expression in embryonic day 8.5 BALB/c(B2-/-) embryos that was comparable to Brca2-heterozygous littermates. Thus, the Brca1 and Brca2 transcripts are expressed independently of one another in Brca1- and Brca2-null embryos. Mol. Carcinog. 28:174-183, 2000.

Mice heterozygous for a Brca1 or Brca2 mutation display distinct mammary gland and ovarian phenotypes in response to diethylstilbestrol.

Women who inherit mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are predisposed to the development of breast and ovarian cancer. We used mice with a Brca1 mutation on a BALB/cJ inbred background (BALB/cB1+/- mice) or a Brca2 genetic alteration on the 129/SvEv genetic background (129B2+/- mice) to investigate potential gene-environment interactions between defects in these genes and treatment with the highly estrogenic compound diethylstilbestrol (DES). Beginning at 3 weeks of age, BALB/cB1+/-, 129B2+/-, and wild-type female mice were fed a control diet or a diet containing 640 ppb DES for 26 weeks. DES treatment caused vaginal epithelial hyperplasia and hyperkeratosis, uterine inflammation, adenomyosis, and fibrosis, as well as oviductal smooth muscle hypertrophy. The severity of the DES response was mouse strain specific. The estrogen-responsive 129/SvEv strain exhibited an extreme response in the reproductive tract, whereas the effect in BALB/cJ and C3H/HeN(MMTV-) mice was less severe. The Brca1 and Brca2 genetic alterations influenced the phenotypic response of BALB/cJ and 129/SvEv inbred strains, respectively, to DES in the mammary gland and ovary. The mammary duct branching morphology was inhibited in DES-treated BALB/cB1+/- mice compared with similarly treated BALB/cB1+/+ littermates. In addition, the majority of BALB/cB1+/- mice had atrophied ovaries, whereas wild-type littermates were largely diagnosed with arrested follicular development. The mammary ductal architecture in untreated 129B2+/- mice revealed a subtle inhibited branching phenotype that was enhanced with DES treatment. However, no significant differences were observed in ovarian pathology between 129B2+/+ and 129B2+/- mice. These data suggest that estrogenic compounds may modulate mammary gland or ovarian morphology in BALB/cB1+/- and 129B2+/- mice. These observations are consistent with the hypothesis that compromised DNA repair processes in cells harboring Brca1 or Brca2 mutations lead to inhibited growth and differentiation compared with the proliferative response of wild-type cells to DES treatment.

Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.

Characterization of the rat and mouse homologues of the BRCA2 breast cancer susceptibility gene.

Inherited BRCA2 mutations confer profound susceptibility to human breast and ovarian cancer. The rat and mouse Brca2 homologues share 58% and 59% identity (72% similarity), respectively, with the human BRCA2 protein. The Brca2 proteins also share a potential nuclear localization signal (human codons 3263-3269) and a highly conserved large carboxyl region (77% identity, 86% similarity between human and rodents) that may represent important functional domains. At least six of eight previously described BRC repeats have been highly conserved in rats and mice. Expression studies demonstrate an 11-12 Kb transcript with rodent tissue-specific patterns of expression consistent with human BRCA2. These results will facilitate studies of Brca2 function during normal and neoplastic development.

Loss of heterozygosity on chromosome 17q11-21 in cancers of women who have both breast and ovarian cancer.

OBJECTIVE: Our purpose was to determine the frequency of allele loss in the region of the BRCA1 gene in cancers of women who have both breast and ovarian cancer. STUDY DESIGN: Four polymorphic microsatellite markers on chromosome 17q11-21 were examined by the polymerase chain reaction in deoxyribonucleic acid from paraffin blocks of normal tissues, breast cancers, and ovarian cancers in 24 women who had primary cancers in both sites. RESULTS: Loss of heterozygosity was seen in one or more markers on chromosome 17q11-21 in 46% of breast cancers and 78% of ovarian cancers. In 38% of cases allele loss was seen in both cancers, and in all these cases the same allele was lost in both cancers. Significantly younger ages at diagnosis of both breast and ovarian cancer were noted among cases with allele loss in both cancers compared with cases in which allele loss was found only in the ovarian cancer (p < 0.05). CONCLUSIONS: Because cases in which 17q11-21 allele loss was seen in both cancers had a young age of onset and the same allele was always deleted in both cancers, hereditary alterations in BRCA1 may play a role in this subset. The older age of onset in cases in which allele loss was seen only in the ovarian cancer suggests that the development of these cancers is not related to an inherited defect in BRCA1.

Allelotype of endometrial carcinoma.

An allelotype analysis of endometrial carcinoma was undertaken to identify chromosomal loci that are relevant to this tumor type. A total of 70 highly polymorphic microsatellite markers, distributed among all nonacrocentric chromosome arms, were examined for evidence of loss of heterozygosity or allelic imbalance in DNA samples from matched normal and tumor tissues. An average of 21 informative tumor cases were obtained for each marker. Allelic deletions or imbalance were observed on 31 of 41 chromosome arms with no marker showing an allelic loss ratio of greater than 33%. Those chromosome arms most frequently involved were 3p, 8p, 9p, 14q, 16q and 18q. There was a strong correlation between loss of heterozygosity on chromosome 14q and death from disease. These data indicate that the molecular genetic character of endometrial carcinoma is complex and that a relatively large number of different chromosomal loci are likely to play a role in the etiology and progression of this tumor type.