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Results for “Checkpoint Kinase 1”

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PubMed2006

Mammary tumorigenesis following transgenic expression of a dominant negative CHK2 mutant.

A truncating allele of the cell cycle checkpoint kinase CHK2 is present in 1% of the population, conferring a moderate increase in breast cancer risk, and inactivation of chk2 enhances mammary tumorigenesis in mice with targeted inactivation of brca1. We used the mouse mammary tumor virus (MMTV) promoter to target expression of a kinase-dead CHK2 allele (D347A). Mammary tumors, of predominantly micropapillary histology, developed in 40% of MMTV-CHK2-D347A transgenic mice with an average latency of 20 months. Tumors metastasized to lung and spleen; tumor-derived cell lines were frequently aneuploid and showed suppression of irradiation-induced p53 function. Primary hematopoietic malignancies were also observed in the spleen, another site of MMTV expression. The increased rate of tumor formation in MMTV-CHK2-D347A mice, compared with the relatively low incidence in chk2-null mice, provides a model to study modifiers of CHK2-dependent transformation.

Alleles↗

PubMed2001

Up-regulation of cyclin-dependent kinase 4/cyclin D2 expression but down-regulation of cyclin-dependent kinase 2/cyclin E in testicular germ cell tumors.

Testicular germ cell tumors (GCT) characteristically display two chromosome 12 abnormalities: the isochromosome i(12p) and concomitant deletions of the long arm. Some genes important in the control of the G(1)-S cell cycle checkpoint G(1)-S, i.e., cyclin-dependent kinases 2 and 4, cyclin D2 are located on this chromosomal region. Therefore, testicular GCTs were analyzed as to the expression of CDK2, CDK4, CDK6, and the expression of their catalytic partners cyclins D1, D2 and E by semiquantitative reverse transcription-PCR. Cyclin D2, located on 12p, was overexpressed in 69% (31 of 45) of the tumors by a mean factor of 8, including all histological subtypes. In addition, the cyclin D2 partner CDK4 was increased in 41% (21 of 51) of all tumors by a factor of 6, most strongly in embryonal carcinomas. Sixty-four percent of the seminomas and 23% of the non-seminomas had decreased expression of CDK6 by a mean factor of 5 (P = 0.009). Statistical analysis using configural frequency analysis and regression analysis revealed that cyclin D2 and CDK4 expression were strongly correlated (r(2) = 0.682; P = 0.000052), whereas expression of CDK6 did not correlate with either of them (r(2) = 0.382; P = 0.00085). CDK2 and its catalytic partner cyclin E were down-regulated in 40% (19 of 47) and 42% (19 of 45) of the tumors, respectively, by a factor of 7 each. Western blots and immunohistochemical experiments confirmed cyclin D2 and CDK4 overrepresentation and reduced expression of cyclin E and CDK2 tumors in the few tumors under protein study. Despite its localization on 12q13, a hot spot for loss of heterozygosity in testicular GCTs (>40%), Southern blotting revealed no gross DNA alteration of the CDK2 gene. Because up-regulation of the cyclin D2/CDK4 complex and down-regulation of cyclin E/CDK2 complex were found in seminomas as well as in non-seminomas and in all tumor stages, these findings seem to be early events during tumorigenesis of testicular GCTS: Together with previous findings that retinoblastoma mRNA and protein expression is strongly decreased in these tumors, these data suggest an unusual deregulated G(1)-S checkpoint as a decisive event for germ cell tumors.

CDC2-CDC28 Kinases↗

PubMed2005

Protein kinase C delta stimulates apoptosis by initiating G1 phase cell cycle progression and S phase arrest.

Overexpression of protein kinase C delta (PKCdelta) stimulates apoptosis in a wide variety of cell types through a mechanism that is incompletely understood. PKCdelta-deficient cells are impaired in their response to DNA damage-induced apoptosis, suggesting that PKCdelta is required to mount an appropriate apoptotic response under conditions of stress. The mechanism through which it does so remains elusive. In addition to effects on cell survival, PKCdelta elicits pleiotropic effects on cellular proliferation. We now provide the first evidence that the ability of PKCdelta to stimulate apoptosis is intimately linked to its ability to stimulate G(1) phase cell cycle progression. Using an adenoviral-based expression system to express PKCalpha,-delta, and -epsilon in epithelial cells, we demonstrate that a modest increase in PKCdelta activity selectively stimulates quiescent cells to initiate G(1) phase cell cycle progression. Rather than completing the cell cycle, PKCdelta-infected cells arrest in S phase, an event that triggers caspase-dependent apoptotic cell death. Apoptosis was preceded by the activation of cell cycle checkpoints, culminating in the phosphorylation of Chk-1 and p53. Strikingly, blockade of S phase entry using the phosphatidylinositol 3-kinase inhibitor LY294002 prevented checkpoint activation and apoptosis. In contrast, inhibitors of mitogen-activated protein kinase cascades failed to prevent apoptosis. These findings demonstrate that the biological effects of PKCdelta can be extended to include positive regulation of G(1) phase cell cycle progression. Importantly, they reveal the existence of a novel, cell cycle-dependent mechanism through which PKCdelta stimulates cell death.

Adenoviridae↗

PubMed2006

Beta-lapachone activates a Mre11p-Tel1p G1/S checkpoint in budding yeast.

Beta-lapachone is an anticancer agent that selectively induces cell death in several human cancer cells. The mechanism of beta-lapachone cytotoxicity is not yet fully understood. Here we report that beta-lapachone treatment delayed cell cycle progression at the G(1)/S transition, incremented phosphorylation of the Rad53p checkpoint kinase and decreased cell survival in the budding yeast Saccharomyces cerevisiae. Furthermore, beta-lapachone induced phosphorylation of histone H2A at serine 129. These checkpoint responses were regulated by Mec1p and Tel1p kinases. Mec1p was required for Rad53p/histone H2A phosphorylation and cell survival following beta-lapachone treatment in asynchronous cultures, but not for the G(1) delay. The tel1Delta mutation increased sensitivity to beta-lapachone in a mec1 defective strain and compromised checkpoint responses in G(1). Both Rad53p phosphorylation and G(1) delay were fully dependent on a functional Mre11p-Rad50p Xrs2p (XMR) complex, and mutants in the XMR complex were hypersensitive to beta-lapachone treatment. Finally, XRS2 and TEL1 worked epistatically regarding beta-lapachone sensitivity and Xrs2p was phosphorylated in a Tel1p-dependent manner after beta-lapachone treatment. Taken together, these findings indicate that beta-lapachone activates a Mre11p-Tel1p checkpoint pathway in budding yeast. Given the conserved nature of the Mre11p-Tel1p pathway, these results suggest that activation of the Mre11-Tel1p checkpoint could be of significance for beta-lapachone anti-tumour activity.

Anti-Infective Agents↗

PubMed2005

BRCA1-mediated G2/M cell cycle arrest requires ERK1/2 kinase activation.

Germline mutations in the BRCA1 gene are associated with an increased susceptibility to the development of breast and ovarian cancers. Evidence suggests that BRCA1 protein plays a key role in mediating DNA damage-induced checkpoint responses. Several studies have shown that ectopic expression of BRCA1 in human cells can trigger cellular responses similar to those induced by DNA damage, including G2/M cell cycle arrest and apoptosis. While the effects of ectopic BRCA1 expression on the G2/M transition and apoptosis have been extensively studied, the factors that dictate the balance between these two responses remain poorly understood. We have recently shown that ectopic expression of BRCA1 in MCF-7 human breast cancer cells resulted in activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and G2/M cell cycle arrest. Furthermore, inhibition of BRCA1-induced ERK1/2 activation using mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-specific inhibitors resulted in increased apoptosis, suggesting a potential role of ERK1/2 kinases in BRCA1-mediated G2/M checkpoint response. In this study, we assessed the role of ERK1/2 kinases in the regulation of BRCA1-mediated G2/M cell cycle arrest. Results indicate that BRCA1-induced G2/M cell cycle arrest and ERK1/2 activation correlate with changes in the level and/or activity of several key regulators of the G2/M checkpoint, including activation of Chk1 and Wee1 kinases, induction of 14-3-3, and down-regulation of Cdc25C. Furthermore, inhibition of ERK1/2 kinases using MEK1/2-specific inhibitors results in a marked attenuation of the BRCA1-induced G2/M arrest. Biochemical studies established that ERK1/2 inhibition abolished the effects of BRCA1 on components of the G2/M checkpoint, including regulation of Cdc25C expression and activation of Wee1 and Chk1 kinases. These results implicate a critical role of ERK1/2 signaling in the regulation of BRCA1 function on controlling the G2/M checkpoint responses.

14-3-3 Proteins↗

PubMed2003

Cell cycle alterations in the blastoid variant of mantle cell lymphoma (MCL-BV) as detected by gene expression profiling of mantle cell lymphoma (MCL) and MCL-BV.

Overexpression of cyclin D1 is necessary but by itself insufficient for the development of mantle cell lymphoma (MCL). To identify pathways in the pathogenesis of MCL and the blastoid variant (MLC-BV), we compared the gene-expression profiles of microdissected normal mantle cells, MCL, and MCL-BV by oligonucleotide microarrays and quantitative reverse transcriptase PCR (QRT-PCR). We identified and confirmed the overexpression of several genes in MCL-BV that are involved in the cell cycle control at the G1/S and G2/M checkpoints or inhibit apoptotic cell death. The highly expressed cyclin dependent kinase 4 (CDK4) is a cell cycle kinase that associates with cyclin D1 for the progression through the G1/S checkpoint, whereas overexpression of cdc28 protein kinase 1 (CKS1) blocks the inhibition of the cyclin D1/CDK4 complex by the CDK inhibitor p27/Kip1. Other highly expressed genes in MCL-BV that promote the cells through the G1/S-checkpoint include the oncogenes B-Myb, PIM1, and PIM2, and passage through the G2/M-checkpoint is enhanced by high levels of cdc25B. Furthermore, two highly expressed genes that inhibit apoptosis are defender against cell death (DAD1) and RSK1. In summary, our microarray and QRT-PCR analyses identified several candidate genes whose expression increased when comparing normal follicular mantles with MCL and MCLBV, suggesting a potential pathogenic role in the evolution of MCL-BV.

Cell Cycle↗

PubMed2004

CHK1 kinase activity assay.

Mammalian CHK1 is a Ser/Thr effector kinase that plays critical roles in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3-related protein kinase). This chapter is focused on describing an assay to measure CHK1 activity in vitro. The basic mechanism of this assay is to observe the phosphorylated levels of a fragment of CDC25C containing the site that can be phosphorylated by CHK1 in vitro. This assay includes five major steps: (1) preparing extracts from the control or treated cells; (2) preparing substrate; (3) immunoprecipitating CHK1 protein from the cells; (4) assembling the kinase assay; and (5) analyzing the phosphorylated level of the substrates by CHK1. Besides CHK1, CHK2 is another important checkpoint regulator that responds to DNA damage. Because CHK1 and CHK2 share some substrates such as CDC25C in vitro, this assay could also be used for CHK2 activity assay, except that the CHK2 antibody will replace the CHK1 antibody.

Antibodies, Monoclonal↗

PubMed2002

Development of a highly specialized cDNA array for the study and diagnosis of epithelial ovarian cancer.

Ovarian cancer is a major cause of cancer death in women. Unfortunately, the molecular pathways underlying ovarian cancer progression are poorly understood, making the development of novel diagnostic and therapeutic strategies difficult. On the basis of our previous observations obtained from serial analysis of gene expression, we have constructed a specialized cDNA array for the study of ovarian cancer. Small, specialized arrays have several practical advantages and can reveal information that is lost in the "noise" generated by irrelevant genes present in larger arrays. The array, which we named Ovachip, contains 516 cDNAs chosen from our serial analysis of gene expression and cDNA array studies for their relevance to ovarian cancer. The gene expression patterns revealed with the Ovachip are highly reproducible and extremely consistent among the different ovarian specimens tested. This array was extremely sensitive at differentiating ovarian cancer from colon cancer based on expression profiles. The Ovachip revealed clusters of coordinately expressed genes in ovarian cancer. One such cluster, the IGF2 cluster, is particularly striking and includes the insulin-like growth factor II, the cisplatin resistance-associated protein, the checkpoint suppressor 1, the cyclin-dependent kinase 6, and a protein tyrosine phosphatase receptor. We also identified a cluster of down-regulated genes that included the cyclin-dependent kinase 7 and cyclin H. Thus, the Ovachip allowed us to identify previously unidentified clusters of differentially expressed genes that may provide new paradigms for molecular pathways important in ovarian malignancies. Because of the relevance of the arrayed genes, the Ovachip may become a powerful tool for investigators in the field of ovarian cancer and may facilitate progress in understanding the etiology of this disease and in its clinical management.

Carcinoma, Papillary↗

PubMed2026

Age-related genomic characterization and therapeutic targets in Chinese breast cancer: insights from prospective targeted sequencing and clinical data analysis.

BACKGROUND: In China, breast cancer occurs at a much younger age and has a higher recurrence and mortality rate. However, with changes in lifestyle, there has been a trend towards an older age of breast cancer incidence in Chinese women. There is a paucity of large-scale next-generation sequencing cohorts for the analysis of genomic characterization in these populations and the identification of potential therapeutic targets. METHODS: To address this gap, we performed prospective targeted sequencing of tumor and blood samples from Chinese patients and collected detailed clinical information. We then categorized patients into two groups based on age (< 40 years, n = 637; ≥ 40 years, n = 3442) and proceeded to provide comprehensive descriptions of somatic and germline mutations in both groups. RESULTS: The somatic mutation analysis revealed that PIK3CA, FOXA1, and TBX3 mutations were more prevalent in elderly patients. By leveraging the aforementioned mutational characteristics, we employed our institution's FUTURE-SUPER clinical trial, an umbrella study targeting metastatic breast cancer, to confirm the potential benefits of PI3K-AKT-mTOR pathway inhibitors among elderly patients with breast cancer. Furthermore, TP53 and ERBB2 were more likely to be co-mutated in young women. Patients with TP53 and ERBB2 co-mutation tend to have a poorer prognosis, but through investigation of the SPARK cohort, patients carrying the TP53 and ERBB2 co-mutation are more likely to benefit from immune checkpoint inhibitor combination with tyrosine kinase inhibitor therapy. In our study, we observed a higher frequency of mutations in the DNA homology-dependent recombination pathway in young patients with breast cancer, which was associated with an elevated Ki67 index. Additionally, we confirmed a significant prevalence of germline breast cancer susceptibility gene 1 (gBRCA1) mutations in young patients, whereas germline checkpoint kinase 2 (gCHEK2) mutations are more common in elderly patients. CONCLUSIONS: Our study, which makes use of the largest Chinese breast cancer sequencing cohort, sought to characterize the age-related genomic profile of breast cancer patients and identify novel therapeutic opportunities for individuals with breast cancer.

Adult↗

PubMed2004

Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G1 tetraploidy checkpoint function.

Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G(1) cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G(1) tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine-threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Rat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G(1) tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G(1) tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G(1) tetraploidy checkpoint.

Actins↗

PubMed2002

Inhibition of cyclin-dependent kinase 2 by the Chk1-Cdc25A pathway during the S-phase checkpoint activated by fludarabine: dysregulation by 7-hydroxystaurosporine.

Human myeloid leukemia ML-1 cells responded to cytostatic concentrations of fludarabine nucleoside (F-ara-A) by instituting an arrest in S-phase that involved the inhibition of cyclin-dependent kinase 2 (Cdk2). This seemed to be mediated by 1) persistent phosphorylation on the Tyr(15) residue of Cdk2 and 2) an increased association of Cdk2 with p21. S-phase arrest was also associated with an increase in Chk1 kinase activity. Concomitantly, the activity of Cdc25A phosphatase was decreased. Immunoprecipitation studies demonstrated complexes of Cdk2, Cdc25A, and Chk1. The addition of the Chk1 kinase inhibitor 7-hydroxystaurosporine (UCN-01) to F-ara-A-arrested S-phase cells resulted in a rapid decrease in the fraction of cells with an S-phase DNA content and a corresponding increase in the fraction of apoptotic cells. Under these conditions, the kinase activity of Chk1 was reduced, Cdc25A phosphatase activity was increased, the level of Tyr(15) phosphorylation of Cdk2 was reduced, and the kinase activity associated with immunoprecipitates of Cdk2 and cyclin A was reactivated. UCN-01 also had no effect on the association of p21 with Cdk2. Lastly, cells incubated with UCN-01 before F-ara-A addition did not arrest in S-phase. Thus, the DNA damage induced by F-ara-A initiated a hierarchical regulatory cascade through Chk1 and Cdc25A that resulted in Cdk2 inhibition, affecting an S-phase checkpoint that was dysregulated by UCN-01. These results suggest a mechanism by which UCN-01 enhances the cytotoxicity of agents that cause an S-phase arrest.

Alkaloids↗

PubMed2004

RACK1 regulates G1/S progression by suppressing Src kinase activity.

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.

Animals↗

PubMed2006

Induction of centrosome amplification during arsenite-induced mitotic arrest in CGL-2 cells.

Arsenite-induced mitotic abnormalities result in mitotic death in several cancer cell lines. However, how arsenite induces these effects is not known. We have previously shown that arsenite induces mitotic arrest, mitotic abnormalities, and mitotic death in CGL-2 cells. To further delineate the mechanism of action of arsenite, we examined its effect on centrosome duplication and the possible link between centrosome dysregulation and arsenite-induced mitotic death. Immunofluorescence staining of gamma-tubulin revealed that centrosome amplification was induced in arsenite-arrested mitotic cells but not in nocodazole-arrested cells. When S phase-enriched cells were treated with arsenite, they progressed into and arrested at mitosis and then formed supernumerary centrosomes. A further increase in arsenite-induced centrosome amplification was seen during the prolonged mitotic arrest. The arsenite-induced supernumerary centrosomes might result from uneven fragmentation of centrosome, overexpression of pericentriolar materials, and inhibition of centrosomal coalescence during mitosis. Furthermore, termination of mitotic arrest by treatment of arsenite-arrested mitotic cells with cyclin-dependent kinase 1 inhibitors or by suppression of spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 or Mad2 markedly reduced the induction of centrosome amplification and mitotic death in arsenite-treated cells. These results indicate that centrosome amplification is induced in arsenite-arrested mitotic CGL-2 cells in a spindle checkpoint-dependent manner and is involved in the induction of arsenite-induced mitotic death.

Apoptosis↗

PubMed2004

Altered expression of Chk1 disrupts cell cycle remodeling at the midblastula transition in Xenopus laevis embryos.

Studies in several model systems, including Xenopus laevis oocytes and embryos, have indicated that the checkpoint kinase, Chk1, is required for early development, even in the absence of damaged or unreplicated DNA. Chk1 is transiently activated at the midblastula transition (MBT) in Xenopus, a time when the cell cycle remodels from rapid embryonic cleavage cycles to longer, more regulated somatic cell cycles. To better understand the role of Chk1 in cell cycle remodeling, mRNA encoding Chk1 was microinjected into 1-cell stage embryos, and the effects on both the MBT and on the expression of several cell cycle regulators were examined. Zygotic transcription, a hallmark of the MBT that depends upon the nucleocytoplasmic (N/C) ratio, was blocked, as was degradation of maternal cyclin E, an event of the MBT that occurs independent of the N/C ratio. Levels of mitotic cyclins were elevated throughout early development, consistent with cell cycle arrest at G2/M. In these embryos, Cdc25A level was low, whereas Cdc25C level was not affected. Furthermore, the level of Wee1 increased at 6 hrs post-fertilization (pf), the time at which the MBT normally occurs, even though these embryos did not demonstrate any known markers of the MBT. These studies suggest that in addition to targeting Cdc25A for degradation, Chk1 may also function in cell cycle remodeling at the MBT by stabilizing Wee1 until it is replaced by the somatic Wee2 protein during gastrulation.

Animals↗

PubMed2002

Polo-like kinase 1 and Chk2 interact and co-localize to centrosomes and the midbody.

Chk2 is a protein kinase intermediary in DNA damage checkpoint pathways. DNA damage induces phosphorylation of Chk2 at multiple sites concomitant with activation. Chk2 phosphorylated at Thr-68 is found in nuclear foci at sites of DNA damage (1). We report here that Chk2 phosphorylated at Thr-68 and Thr-26 or Ser-28 is localized to centrosomes and midbodies in the absence of DNA damage. In a search for interactions between Chk2 and proteins with similar subcellular localization patterns, we found that Chk2 coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome segregation, mitotic entry, and mitotic exit. Plk1 overexpression enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates recombinant Chk2 in vitro. Indirect immunofluorescence (IF) microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes in early mitosis and to the midbody in late mitosis. These findings suggest lateral communication between the DNA damage and mitotic checkpoints.

Cell Cycle Proteins↗

PubMed2004

Potent inhibition of checkpoint kinase activity by a hymenialdisine-derived indoloazepine.

The marine sponge metabolite hymenialdisine is a potent inhibitor of a variety of kinases including MEK-1, GSK-3 beta, and CK1. In addition, hymenialdisine and debromohymenialdisine exhibit inhibition of the G(2) cell cycle checkpoint at micromolar concentrations. We report herein the potent inhibition of cell cycle kinase Chk2 by the indolic-hymenialdisine indoloazepine 1 (IC(50)=8 nM).

Azepines↗

PubMed2004

A novel pathological role of p53 in kidney development revealed by gene-environment interactions.

Gene-environment interactions are implicated in congenital human disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Previously, our laboratory demonstrated that gestational salt stress of bradykinin B(2) receptor (B(2)R)-null mice induces renal dysgenesis and early death of the offspring. In contrast, salt-stressed B(2)R +/+ or +/- littermates have normal development. The present study investigates the mechanisms underlying the susceptibility of B(2)R-null mice to renal dysgenesis. Proteomic and conventional Western blot screens identified E-cadherin among the differentially repressed proteins in B(2)R-/- kidneys, whereas the checkpoint kinase Chk1 and its substrate P-Ser(20) p53 were induced. We tested the hypothesis that p53 mediates repression of E-cadherin gene expression and is causally linked to the renal dysgenesis. Genetic crosses between B(2)R -/- and p53+/- mice revealed that germline reduction of p53 gene dosage rescues B(2)R-/- mice from renal dysgenesis and restores kidney E-cadherin gene expression. Furthermore, gamma-irradiation induces repression of E-cadherin gene expression in p53+/+ but not -/- cells. In transient transfection assays, p53 repressed human E-cadherin promoter-driven reporter activity, whereas a mutant p53, which cannot bind DNA, did not. Functional promoter analysis indicated the presence of a p53-responsive element in exon 1, which partially mediates p53-induced repression. Chromatin immunoprecipitation assays revealed that p53 inhibits histone acetylation of the E-cadherin promoter. Treatment with a histone deacetylase inhibitor reversed both p53-mediated promoter repression and deacetylation. In conclusion, this study demonstrates that gene-environment interactions cooperate to induce congenital defects through p53 activation.

Animals↗

PubMed2001

Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G(1)/S checkpoint activation in response to DNA damage.

ATR [ataxia-telangiectasia-mutated (ATM)- and Rad3-related] is a protein kinase required for both DNA damage-induced cell cycle checkpoint responses and the DNA replication checkpoint that prevents mitosis before the completion of DNA synthesis. Although ATM and ATR kinases share many substrates, the different phenotypes of ATM- and ATR-deficient mice indicate that these kinases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on Ser(635) and Ser(645) in vitro. In undamaged synchronized human cells, these two sites were phosphorylated in late G(1), S, and G(2)/M, but not in early-mid G(1). Treatment of cells with genotoxic stress induced phosphorylation of hRad17 in cells in early-mid G(1). Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues were phosphorylated in ionizing radiation (IR)-treated ATM-deficient human cell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-deficient tissues, but induction of Ser(645) was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR-induced activation of the G(1)/S checkpoint in MCF-7 cells. These results suggest ATR and hRad17 are essential components of a DNA damage response pathway in mammalian cells.

Amino Acid Sequence↗