Steven P. Linke

Centrosome Amplification and a Defective G2–M Cell Cycle Checkpoint Induce Genetic Instability in BRCA1 Exon 11 Isoform–Deficient Cells

Germline mutations of the Brca1 tumor suppressor gene predispose women to breast and ovarian cancers. To study mechanisms underlying BRCA1-related tumorigenesis, we derived mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of the Brca1 gene. We show that the mutant cells maintain an intact G1-S cell cycle checkpoint and proliferate poorly. However, a defective G2-M checkpoint in these cells is accompanied by extensive chromosomal abnormalities. Mutant fibroblasts contain multiple, functional centrosomes, which lead to unequal chromosome segregation, abnormal nuclear division, and aneuploidy. These data uncover an essential role of BRCA1 in maintaining genetic stability through the regulation of centrosome duplication and the G2-M checkpoint and provide a molecular basis for the role of BRCA1 in tumorigenesis.

Nitric oxide-induced cellular stress and p53 activation in chronic inflammation

Free radical-induced cellular stress contributes to cancer during chronic inflammation. Here, we investigated mechanisms of p53 activation by the free radical, NO. NO from donor drugs induced both ataxia-telangiectasia mutated (ATM)- and ataxia-telangiectasia mutated and Rad3-related-dependent p53 posttranslational modifications, leading to an increase in p53 transcriptional targets and a G2/M cell cycle checkpoint. Such modifications were also identified in cells cocultured with NO-releasing macrophages. In noncancerous colon tissues from patients with ulcerative colitis (a cancer-prone chronic inflammatory disease), inducible NO synthase protein levels were positively correlated with p53 serine 15 phosphorylation levels. Immunostaining of HDM-2 and p21WAF1 was consistent with transcriptionally active p53. Our study highlights a pivotal role of NO in the induction of cellular stress and the activation of a p53 response pathway during chronic inflammation.

Genetic interactions between tumor suppressors Brca1 and p53 in apoptosis, cell cycle and tumorigenesis

Breast cancer is a chief cause of cancer-related mortality that affects women worldwide. About 8% of cases are hereditary, and approximately half of these are associated with germline mutations of the breast tumor suppressor gene BRCA1 (refs. 1,2). We have previously reported a mouse model in which Brca1 exon 11 is eliminated in mammary epithelial cells through Cre-mediated excision. This mutation is often accompanied by alterations in transformation-related protein 53 (Trp53, encoding p53), which substantially accelerates mammary tumor formation. Here, we sought to elucidate the underlying mechanism(s) using mice deficient in the Brca1 exon 11 isoform (Brca1Δ11/Δ11). Brca1Δ11/Δ11 embryos died late in gestation because of widespread apoptosis. Unexpectedly, elimination of one Trp53 allele completely rescues this embryonic lethality and restores normal mammary gland development. However, most female Brca1Δ11/Δ11 Trp53+/− mice develop mammary tumors with loss of the remaining Trp53 allele within 6–12 months. Lymphoma and ovarian tumors also occurr at lower frequencies. Heterozygous mutation of Trp53 decreases p53 and results in attenuated apoptosis and G1–S checkpoint control, allowing Brca1Δ11/Δ11 cells to proliferate. The p53 protein regulates Brca1 transcription both in vitro and in vivo, and Brca1 participates in p53 accumulation after γ-irradiation through regulation of its phosphorylation and Mdm2 expression. These findings provide a mechanism for BRCA1-associated breast carcinogenesis.

BLM helicase‐dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co‐localized with each other and with RAD51 at sites of stalled DNA replication forks. HU‐induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild‐type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM‐corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.

DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2/ING1L. Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G1-phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

The p53 network in lung carcinogenesis

The p53 tumor suppressor gene lies at the crossroads of multiple cellular response pathways that control a cells fate in response to endogenous or exogenous stresses. Positive and negative regulatory loops both upstream and downstream of p53 cooperate to finely tune its functions as a transcription factor, a DNA damage sensor, and possibly, a protein-assembly scaffold. Through this plethora of activities, p53 is a major determinant of cell survival and a safeguard against genetic instability. Functional inactivation of p53 pathways through genetic and epigenetic events affecting the p53 gene itself and/or its interacting partners occur with a high frequency in lung cancer. The p53 mutational spectrum provides molecular evidence of the etiology of lung cancer and supports abundant epidemiological data indicating the role of tobacco smoke in the causation of this disease.

Involvement of Crm1 in hepatitis B virus X protein-induced aberrant centriole replication and abnormal mitotic spindles.

ABSTRACT Hepatitis B virus (HBV) includes an X gene (HBx gene) that plays a critical role in liver carcinogenesis. Because centrosome abnormalities are associated with genomic instability in most human cancer cells, we examined the effect of HBx on centrosomes. We found that HBx induced supernumerary centrosomes and multipolar spindles. This effect was independent of mutations in the p21 gene. Furthermore, the ability of HBV to induce supernumerary centrosomes was dependent on the presence of physiological HBx expression. We recently showed that HBx induces cytoplasmic sequestration of Crm1, a nuclear export receptor that binds to Ran GTPase, thereby inducing nuclear localization of NF-κB. Consistently, supernumerary centrosomes were observed in cells treated with a Crm1-specific inhibitor but not with an HBx mutant that lacked the ability to sequester Crm1 in the cytoplasm. Moreover, a fraction of Crm1 was found to be localized at the centrosomes. Immunocytochemical and ultrastructural examination of these supernumerary centrosomes revealed that inactivation of Crm1 was associated with abnormal centrioles. The presence of more than two centrosomes led to an increased frequency of defective mitoses and chromosome transmission errors. Based on this evidence, we suggest that Crm1 is actively involved in maintaining centrosome integrity and that HBx disrupts this process by inactivating Crm1 and thus contributes to HBV-mediated carcinogenesis.

Functional interaction between BLM helicase and 53BP1 in a Chk1-mediated pathway during S-phase arrest

Blooms syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of γ-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.

Identification of a Functional Domain in a GADD45-mediated G2/M Checkpoint

Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G2/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivowith the G2 cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA), and the cell cycle kinase inhibitor p21 waf1 . The ability of GADD45 to induce a G2/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G2/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G2/M arrest. A mutant with a deletion of the central region (residues 50–76), which is conserved in the family members GADD45β and GADD45γ, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21 waf1 in vivo. Consistently, either GADD45β or GADD45γ bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45β nor GADD45γ inhibited the Cdc2 kinase or induced G2/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR residues. Alanine substitutions in the region abolished GADD45 induction of a G2/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21 waf1 . Therefore, the binding of GADD45 to Cdc2 was insufficient to induce a G2/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G2/M checkpoint.

Genetic instability as a consequence of inappropriate entry into and Progression through S-phase

SummaryThe stability of the mammalian genome depends on the proper function of G1 and G2 cell cycle control mechanisms. Two tumor suppressors, p53 and retinoblastoma (Rb), play key roles in progression from G1 into S-phase. We address the mechanisms by which these proteins mediate a G1 arrest in response to DNA damage and limiting metabolic conditions. Gamma-irradiation induced a prolonged, p53-dependent G1 arrest associated with a long-term increase in the levels of the cdk-inhibitor p21WAF1/Cip1 (p21). Microinjection of linear plasmid DNA also caused a G1 arrest. The p53-dependent arrest induced by inhibitors of UMP biosynthesis was reversible and occurred in the absence of detectable DNA damage. Both arrest mechanisms contribute to limiting the formation and propagation of damaged genomes. Cells containing mutant p53 but wild-type Rb do not generate methotrexate (Mtx) resistant variants. However, pre-treatment with DNA damaging agents prior to drug selection resulted in resistant clones containing amplified dihydrofolate reductase (DHFR) genes, suggesting that DNA breakage is a rate limiting step for gene amplification. The Mtx-induced arrest did not occur in cells with non-functional Rb. Rb acts as a negative regulator of the E2F transcription factors, and Rb-deficient primary mouse embryo fibroblasts (MEFs) produced elevated levels of mRNA and protein for key E2F target genes. Failure to prevent entry into S-phase in Rb−/- MEFs exposed to DNA-damaging or nutrient limiting conditions caused apoptosis and correlated with p53 induction. Taken together, these findings indicate a link between p53 and Rb function and suggest that their coordination insures correct entry into S-phase, minimizing the emergence of genetic variants.