Reduced sister chromatid cohesion acts as a tumor penetrance modifier

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.     

p53 (TRP53) deficiency-mediated antiapoptosis escape after 5 Gy X irradiation still induces stem cell leukemia in C3H/He mice: comparison between whole-body assay and bone marrow transplantation (BMT) assay

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53(+/-) (TRP53(+/-)) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53(+/-) mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.     

Heterozygous inactivation of human Ku70/Ku86 heterodimer does not affect cell growth, double-strand break repair, or genome integrity

Ku, the heterodimer of Ku70 and Ku86, plays crucial roles in non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. It has recently been reported that heterozygous disruption of the human KU86 locus results in haploinsufficient phenotypes, including retarded growth, increased radiosensitivity, elevated p53 levels and shortened telomeres. In this paper, however, we show that heterozygous inactivation of either the KU70 or KU86 gene does not cause any defects in cell proliferation or DSB repair in human somatic cells. Moreover, although these heterozygous cell lines express reduced levels of both Ku70 and Ku86, they appear to maintain overall genome integrity with no elevated p53 levels or telomere shortening. These results clearly indicate that Ku haploinsufficiency is not a commonly observed phenomenon in human cells. Our data also suggest that the impact of KU70/KU86 mutations on telomere metabolism varies between cell types in humans.     

BRCA1 Haploinsufficiency,but not Heterozygosity for a BRCA1-truncating Mutation,Deregulates Homologous Recombination

Humans heterozygous for BRCA1 mutations have a high risk of losing the remaining wild-type BRCA1 allele and developing breast/ovarian cancer, but a molecular basis for this has not yet been determined. It is thought that heterozygosity status — reduced wild-type BRCA1 protein dosage (haploinsufficiency) and/or the presence of a mutant BRCA1 protein — may affect BRCA1 functions and heighten the risk of cancer promoting mutations. BRCA1 maintains genome stability, at least in part, by regulating homologous recombination according to the type of DNA damage. To investigate whether this BRCA1 function is affected by heterozygosity status, we employed, as recombination reporters, human breast cancer MCF-7 cells known to have a single wild-type BRCA1 allele and reduced BRCA1 protein dosage. These cells revealed: 1) a spontaneous hyper-recombination phenotype; 2) reduced efficiency in homologous recombination repair of DNA double-strand breaks (DSBs); and 3) sensitivity to the DSB-inducing chemotherapeutic agent mitomycin C. Correction of BRCA1 protein dosage to the wild-type level reversed all these phenotypes, whereas physiological expression of the cancer-eliciting BRCA1 5382insC mutant allele had no effect on either phenotype. These findings implicate BRCA1 C-terminal domain in recombination control, and indicate that BRCA1 haploinsufficiency alone, which is also a feature of sporadic breast/ovarian cancer, is sufficient to compromise genome stability by triggering spontaneous recombination events that are likely to account for the loss of the remaining wild-type BRCA1 allele and increased cancer risk. Our observations may also have implications for the medical management of cancer patients and cancer prevention.     

Radiation-induced genetic instability in vivo depends on p53 status

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.     

Germline BRCA1 mutations predispose to pancreatic adenocarcinoma

Although the association of germline BRCA2 mutations with pancreatic adenocarcinoma is well established, the role of BRCA1 mutations is less clear. We hypothesized that the loss of heterozygosity at the BRCA1 locus occurs in pancreatic cancers of germline BRCA1 mutation carriers, acting as a “second-hit” event contributing to pancreatic tumorigenesis. Seven germline BRCA1 mutation carriers with pancreatic adenocarcinoma and nine patients with sporadic pancreatic cancer were identified from clinic- and population-based registries. DNA was extracted from paraffin-embedded tumor and nontumor samples. Three polymorphic microsatellite markers for the BRCA1 gene, and an internal control marker on chromosome 16p, were selected to test for the loss of heterozygosity. Tumor DNA demonstrating loss of heterozygosity in BRCA1 mutation carriers was sequenced to identify the retained allele. The loss of heterozygosity rate for the control marker was 20%, an expected baseline frequency. Loss of heterozygosity at the BRCA1 locus was 5/7 (71%) in BRCA1 mutation carriers; tumor DNA was available for sequencing in 4/5 cases, and three demonstrated loss of the wild-type allele. Only 1/9 (11%) sporadic cases demonstrated loss of heterozygosity at the BRCA1 locus. Loss of heterozygosity occurs frequently in pancreatic cancers of germline BRCA1 mutation carriers, with loss of the wild-type allele, and infrequently in sporadic cancer cases. Therefore, BRCA1 germline mutations likely predispose to the development of pancreatic cancer, and individuals with these mutations may be considered for pancreatic cancer-screening programs.     

Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing

Stable expression of Rad51 siRNA was used to generate mouse hybridoma cell lines in which endogenous Rad51 levels were depleted by as much as 60%. Stable Rad51 knockdowns feature reduced homologous recombination responses. The relative ease with which stable Rad51 knockdowns were recovered was surprising, given the embryonic lethality of Rad51 ablation. Interestingly, Rad51-depleted hybridoma cell lines are characterized by reduced levels of p53 protein. Completely unexpected, was the finding that Rad51-depleted hybridoma cell lines are also reduced for the breast cancer susceptibility 2 (Brca2) protein. Additionally, hybridoma cell lines that are siRNA depleted for mouse Brca2 show a corresponding reduction in Rad51 and p53 proteins. Furthermore, cellular levels of Rad51, Brca2 and p53 can be elevated in these cell lines by ectopic expression of wild-type human Rad51 and wild-type human BRCA2. In marked contrast, hybridoma cell lines that are siRNA depleted for mouse p53 feature relatively normal Rad51 and Brca2 levels. These results suggest that cellular levels of Brca2 and Rad51 are mutually dependent on each other, and that low levels of these proteins provide selective pressure for reduction of p53, which permits cell growth.     

Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae.

Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae. The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected. Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone. Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry. p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs. These results suggest that wild-type p53 has a growth-inhibitory activity in S. cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.     

BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

BRCA1 is involved in maintaining genomic integrity and, as a regulator of the G2/M checkpoint, contributes to DNA repair and cell survival. The overexpression of BRCA1 elicits diverse cellular responses including apoptosis due to the stimulation of specific signaling pathways. BRCA1 is normally regulated by protein turnover, but is stabilized by BARD1 which can recruit BRCA1 to the nucleus to form a ubiquitin E3 ligase complex involved in DNA repair or cell survival. Here, we identify BARD1 as a regulator of BRCA1-dependent apoptosis. Using transfected MCF-7 breast cancer cells, we found that BRCA1-induced apoptosis was independent of p53 and was stimulated by BRCA1 nuclear export. Conversely, BARD1 reduced BRCA1-dependent apoptosis by a mechanism involving nuclear sequestration. Regulation of apoptosis by BARD1 was reduced by BRCA1 cancer mutations that disrupt Ub ligase function. Transfection of BRCA1 N-terminal peptides that disrupted the cellular BRCA1-BARD1 interaction caused a loss of nuclear BRCA1 that correlated with increased apoptosis in single cell assays, but did not alter localization or expression of endogenous BARD1. Reducing BARD1 levels by siRNA caused a small increase in apoptosis. Our findings identify a novel apoptosis inhibitory function of BARD1 and suggest that nuclear retention of BRCA1-BARD1 complexes contributes to both DNA repair and cell survival.     

brca2 and tp53 Collaborate in Tumorigenesis in Zebrafish

Germline mutations in the tumor suppressor genes BRCA2 and TP53 significantly influence human cancer risk, and cancers from humans who inherit one mutant allele for BRCA2 or TP53 often display loss of the wildtype allele. In addition, BRCA2-associated cancers often exhibit mutations in TP53. To determine the relationship between germline heterozygous mutation (haploinsufficiency) and somatic loss of heterozygosity (LOH) for BRCA2 and TP53 in carcinogenesis, we analyzed zebrafish with heritable mutations in these two genes. Tumor-bearing zebrafish were examined by histology, and normal and neoplastic tissues were collected by laser-capture microdissection for LOH analyses. Zebrafish on a heterozygous tp53^M214K background had a high incidence of malignant tumors. The brca2^Q658X mutation status determined both the incidence of LOH and the malignant tumor phenotype. LOH for tp53 occurred in the majority of malignant tumors from brca2 wildtype and heterozygous mutant zebrafish, and most of these were malignant peripheral nerve sheath tumors. Malignant tumors in zebrafish with heterozygous mutations in both brca2 and tp53 frequently displayed LOH for both genes. In contrast, LOH for tp53 was uncommon in malignant tumors from brca2 homozygotes, and these tumors were primarily undifferentiated sarcomas. Thus, carcinogenesis in zebrafish with combined mutations in tp53 and brca2 typically requires biallelic mutation or loss of at least one of these genes, and the specific combination of inherited mutations influences the development of LOH and the tumor phenotype. These results provide insight into cancer development associated with heritable BRCA2 and TP53 mutations.     

Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background

muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous for muREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.