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PubMed2004

Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells.

The 'ataxia telangiectasia mutated' (Atm) gene maintains genomic stability by activating a key cell-cycle checkpoint in response to DNA damage, telomeric instability or oxidative stress. Mutational inactivation of the gene causes an autosomal recessive disorder, ataxia-telangiectasia, characterized by immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasia, defective spermatogenesis, premature ageing and a high incidence of lymphoma. Here we show that ATM has an essential function in the reconstitutive capacity of haematopoietic stem cells (HSCs) but is not as important for the proliferation or differentiation of progenitors, in a telomere-independent manner. Atm-/- mice older than 24 weeks showed progressive bone marrow failure resulting from a defect in HSC function that was associated with elevated reactive oxygen species. Treatment with anti-oxidative agents restored the reconstitutive capacity of Atm-/- HSCs, resulting in the prevention of bone marrow failure. Activation of the p16(INK4a)-retinoblastoma (Rb) gene product pathway in response to elevated reactive oxygen species led to the failure of Atm-/- HSCs. These results show that the self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress.

Acetylcysteine↗
PubMed2000

High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer.

Expression of 14-3-3 final sigma (final sigma) is induced in response to DNA damage, and causes cells to arrest in G(2). By SAGE (serial analysis of gene expression) analysis, we identified final sigma as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, final sigma mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at final sigma such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the final sigma gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of final sigma is functionally important, because treatment of final sigma-non-expressing breast cancer cell lines with the drug 5-aza-2'-deoxycytidine resulted in demethylation of the gene and synthesis of final sigma mRNA. Breast cancer cells lacking final sigma expression showed increased number of chromosomal breaks and gaps when exposed to gamma-irradiation. Therefore, it is possible that loss of final sigma expression contributes to malignant transformation by impairing the G(2) cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of final sigma expression are the most consistent molecular alterations in breast cancer identified so far.

14-3-3 Proteins↗
PubMed2004

Transcription - guarding the genome by sensing DNA damage.

Cells induce the expression of DNA-repair enzymes, activate cell-cycle checkpoints and, under some circumstances, undergo apoptosis in response to DNA-damaging agents. The mechanisms by which these cellular responses are triggered are not well understood, but there is recent evidence that the transcription machinery might be used in DNA-damage surveillance and in triggering DNA-damage responses to suppress mutagenesis. Transcription might also act as a DNA-damage dosimeter where the severity of blockage determines whether or not to induce cell death. Could transcription therefore be a potential therapeutic target for anticancer strategies?

Aging↗
PubMed1999

The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction.

In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [alpha-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [alpha-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [alpha-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth.

Animals↗
PubMed2004

Hallmarks of senescence in carcinogenesis and cancer therapy.

Cellular senescence is a signal transduction program leading to irreversible cell cycle arrest. This growth arrest can be triggered by many different mechanisms including recognition by cellular sensors of DNA double-strand breaks leading to the activation of cell cycle checkpoint responses and recruitment of DNA repair foci. Senescence is initiated by the shortening of telomeres (replicative senescence) or by other endogenous and exogenous acute and chronic stress signals (STASIS: stress or aberrant signaling-induced senescence). The process of carcinogenesis involves a series of changes that allow tumor cells to bypass the senescence program. Nevertheless, tumor cells retain the capacity to undergo senescence. Treatment of tumor cells with many conventional anticancer therapies activates DNA damage signaling pathways, which induce apoptosis in some cells and senescence in others. Overexpression of tumor suppressors or inhibition of oncogenes can also induce rapid senescence in tumor cells. Senescent cells, while not dividing, remain metabolically active and produce many secreted factors, some of which stimulate and others inhibit the growth of tumors. The emerging knowledge about the pathways that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.

Animals↗
PubMed2002

Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses.

Rad50/Mre11/NBS1 (R/M/N) is a multi-functional protein complex involved in DNA repair, cell cycle checkpoint activation, DNA replication and replication block-induced responses. Ionizing radiation (IR) induces the phosphorylation of NBS1 and nuclear foci formation of the complex. Although it has been suggested that the R/M/N complex is associated with DNA damage sites, we present here biochemical evidence for chromatin association of the complex. We show that the chromatin association of R/M/N is independent of IR and ataxia telangiectasia mutated (ATM). We also demonstrate that optimal chromatin association of the Rad50/Mre11/NBS1 proteins requires both the conserved forkhead-associated (FHA) and breast cancer C-terminus (BRCT) domains of NBS1. Moreover, both these domains of NBS1 are required for its phosphorylation on Ser343 but not on Ser278. Importantly, both the FHA and BRCT domains are essential for IR-induced foci (IRIF) formation of R/M/N and S phase checkpoint activation, but only the BRCT domain is needed for cell survival after IR. These data demonstrate that the FHA and BRCT domains of NBS1 are crucial for the functions of the R/M/N complex.

Acid Anhydride Hydrolases↗
PubMed2001

Early development of cyclin dependent kinase modulators.

The protein kinase family presents remarkable opportunities for drug discovery and development targeting mainly to the ATP binding cleft. Cyclin-dependent kinases CDKs control the cell division in by controlling its sub phases. The regulation of CDKs is altered in a number of tumor types, and therefore CDKs are a particularly attractive target group of kinases with reference to proliferative disorders including cancer, but also extending to graft stenosis, and autoimmune disorders. Screening of chemical modulators of CDKs that modulate aberrant CDK activity might be beneficial for cancer therapy by directly inhibiting kinase activity, or influencing cell cycle "checkpoint" function, which is mediated through effects of exogenous cellular regulators of CDK activity. In this regard small molecule modulators such as flavopiridol and UCN-01 are in early clinical trials. Other more selective modulators of CDK function are being actively sought, and initial results with flavopiridol analogs, indirubins, paullones, and purine-based inhibitors will be considered.

Animals↗
PubMed1998

Alterations of the p16-pRb pathway and the chromosome locus 9p21-22 in non-small-cell lung carcinomas: relationship with p53 and MDM2 protein expression.

The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas, respectively. Analysis of the region that encodes for p16 by deletion mapping, a polymerase chain reaction (PCR)-based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16ink4a, p14ARF, and MTS2/p15ink4b. In addition, microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases, respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P = 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb, p53/MDM2, MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P = 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P = 0.013) and p53 (P = 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P = 0.04) and the immunophenotypes p53(P)/MDM2(P) (P = 0.04) and p16(Ab)/pRb(Ab)/p53(P)/MDM2(P) (P = 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.

Adult↗
PubMed2001

DNA strand break-sensing molecule poly(ADP-Ribose) polymerase cooperates with p53 in telomere function, chromosome stability, and tumor suppression.

Genomic instability is often caused by mutations in genes that are involved in DNA repair and/or cell cycle checkpoints, and it plays an important role in tumorigenesis. Poly(ADP-ribose) polymerase (PARP) is a DNA strand break-sensing molecule that is involved in the response to DNA damage and the maintenance of telomere function and genomic stability. We report here that, compared to single-mutant cells, PARP and p53 double-mutant cells exhibit many severe chromosome aberrations, including a high degree of aneuploidy, fragmentations, and end-to-end fusions, which may be attributable to telomere dysfunction. While PARP(-/-) cells showed telomere shortening and p53(-/-) cells showed normal telomere length, inactivation of PARP in p53(-/-) cells surprisingly resulted in very long and heterogeneous telomeres, suggesting a functional interplay between PARP and p53 at the telomeres. Strikingly, PARP deficiency widens the tumor spectrum in mice deficient in p53, resulting in a high frequency of carcinomas in the mammary gland, lung, prostate, and skin, as well as brain tumors, reminiscent of Li-Fraumeni syndrome in humans. The enhanced tumorigenesis is likely to be caused by PARP deficiency, which facilitates the loss of function of tumor suppressor genes as demonstrated by a high rate of loss of heterozygosity at the p53 locus in these tumors. These results indicate that PARP and p53 interact to maintain genome integrity and identify PARP as a cofactor for suppressing tumorigenesis.

Animals↗
PubMed2004

Genetics of breast cancer.

Breast cancer has all the hallmarks of a multistep genetic disease. Somatic and germ-line mutations have been described in several tumor suppressor genes, and oncogenes are found to be amplified. Genes in the ATM-CHK2-TP53 cell-cycle checkpoint pathway are mutated in relation to breast cancer, particularly TP53 at the somatic level. Germ-line mutations in BRCA1 and BRCA2, in which DNA repair function is interrupted, account for the majority of familial breast cancers. The mechanism behind the frequent instability of the genomes of breast cancer cells has been poorly understood, but recent functional findings on oncogenes and tumor suppressor genes have provided substantial information on the matter. Some recent developments in drug therapy are based on molecular and genomic findings about breast cancer pathogenesis.

Animals↗
PubMed2006

RNA interference effector proteins localize to mobile cytoplasmic puncta in Schizosaccharomyces pombe.

Ago1, Dcr1 and Rdp1 are the core components of the RNA interference (RNAi) apparatus in the fission yeast Schizosaccharomyces pombe. They function in distinct gene-silencing pathways that direct homology-dependent degradation of mRNA and modification of chromatin. In addition, Ago1 and Dcr1 regulate enactment of Cdc2-dependent cell cycle checkpoints. The ability of the RNAi apparatus to perform multiple roles in these divergent pathways is sure to require dynamic localization of Ago1, Dcr1 and/or Rdp1. Although limited information is available, comprehensive studies regarding the relative localizations of Ago1, Dcr1 and Rdp1 are lacking. To this end, we employed live-cell imaging and immunoelectron microscopy to study the intracellular localizations of these proteins. In contrast to previous reports, our study results indicate that the bulk of Ago1 and Dcr1 form stable complexes and are associated with large, mobile, highly dynamic cytoplasmic elements. The majority of Rdp1 is localized to the nucleus, but a pool of Rdp1 is associated with the same cytoplasmic structures. The movements of these structures were dependent upon ATP and intact microtubules. Recruitment of the RNAi core proteins to these structures was not dependent upon siRNAs. Together, our data indicate that the enzymes required for the initiation and effector phases of RNA-dependent gene silencing are concentrated in a common intracellular location, an arrangement that would be expected to result in highly efficient post-transcriptional gene silencing.

Base Sequence↗
PubMed2006

The sensitivity of human mesenchymal stem cells to ionizing radiation.

PURPOSE: Recent studies have shown that mesenchymal stem cells (MSCs) obtained from bone marrow transplantation patients originate from the host. This clinical observation suggests that MSCs in their niches could be resistant to irradiation. However, the biologic responses of bone marrow MSCs to irradiation have rarely been described in the literature. METHODS AND MATERIALS: In this study, human bone marrow-derived, clonally expanded MSCs were used to investigate their sensitivity to irradiation in vitro, and the cellular mechanisms that may facilitate resistance to irradiation. The human lung cancer cell line A549 and the breast cancer cell line HCC1937 were used as controls for radiosensitivity; the former line has been shown to be radioresistant and the latter radiosensitive. We then examined their in vitro biologic changes and sensitivities to radiation therapy. RESULTS: Our results suggest that MSCs are characterized as resistant to irradiation. Several cellular mechanisms were demonstrated that may facilitate resistance to irradiation: ATM protein phosphorylation, activation of cell-cycle checkpoints, double-strand break repair by homologous recombination and nonhomologous end joining (NHEJ), and the antioxidant capacity for scavenging reactive oxygen species. CONCLUSIONS: As demonstrated, MSCs possess a better antioxidant reactive oxygen species-scavenging capacity and active double-strand break repair to facilitate their radioresistance. These findings provide a better understanding of radiation-induced biologic responses in MSCs and may lead to the development of better strategies for stem cell treatment and cancer therapy.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2000

Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development.

XRCC4 is a non-homologous end-joining protein employed in DNA double strand break repair and in V(D)J recombination. In mice, XRCC4-deficiency causes a pleiotropic phenotype, which includes embryonic lethality and massive neuronal apoptosis. When DNA damage is not repaired, activation of the cell cycle checkpoint protein p53 can lead to apoptosis. Here we show that p53-deficiency rescues several aspects of the XRCC4-deficient phenotype, including embryonic lethality, neuronal apoptosis, and impaired cellular proliferation. However, there was no significant rescue of impaired V(D)J recombination or lymphocyte development. Although p53-deficiency allowed postnatal survival of XRCC4-deficient mice, they routinely succumbed to pro-B-cell lymphomas which had chromosomal translocations linking amplified c-myc oncogene and IgH locus sequences. Moreover, even XRCC4-deficient embryonic fibroblasts exhibited marked genomic instability including chromosomal translocations. Our findings support a crucial role for the non-homologous end-joining pathway as a caretaker of the mammalian genome, a role required both for normal development and for suppression of tumours.

Animals↗
PubMed2002

Sequence-dependent synergism between the new generation platinum agent ZD0473 and paclitaxel in cisplatin-sensitive and -resistant human ovarian carcinoma cell lines.

ZD0473 is a new generation hindered platinum agent currently undergoing worldwide Phase II clinical studies. The in vitro cytotoxicity of ZD0473 either alone or in combination with the anticancer drugs paclitaxel, gemcitabine, vinorelbine, topotecan and doxorubicin was determined using four human ovarian carcinoma cell lines and by the sulphorhodamine B assay (SRB). The lines included one model of acquired cisplatin resistance and one isogenic pair differing only in their p53 status. Notably, the simultaneous exposure to ZD0473 and paclitaxel for 96 h resulted in synergy (as defined by a median effect analysis) in all four cell lines (i.e. independent of cisplatin resistance and p53 status). In addition, synergy was observed in 3/4 lines and 2/4 lines following concomitant exposure to topotecan or gemcitabine, respectively. Sequencing studies with ZD0473 and paclitaxel revealed that, for three of the four cell lines, the combination of ZD0473 administered for 24 h prior to paclitaxel for 24 h conferred a greater growth inhibitory effect than the reverse sequential combination. This scheduling effect was particularly marked for the acquired cisplatin-resistant A2780CisR cell line; synergy being observed with ZD0473/paclitaxel, but antagonism with paclitaxel/ZD0473. This effect did not appear to be correlated with changes in drug-induced cell cycle checkpoints. These data suggest that ZD0473 may be usefully combined with various cytotoxics in the clinic, including paclitaxel, topotecan and gemcitabine.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

Mutations in hamster single-strand break repair gene XRCC1 causing defective DNA repair.

The molecular basis for the DNA repair dysfunction observed in mutant Chinese hamster ovary cell lines of X-ray repair cross complementing group 1 (XRCC1) is unknown and the exact role of the XRCC1 protein remains unclear. To help clarify the role of the XRCC1 gene we analyzed four mutant cell lines of this complementation group and a revertant cell line for XRCC1 protein content and for sequence alterations in the XRCC1 coding region. Immunoblot analysis of cellular extracts indicated that each of four mutant lines was lacking XRCC1 protein, whereas the repair-proficient revertant line derived from one of these mutants contained a normal level of XRCC1. Although each of these cell lines expressed XRCC1 mRNA, we found in all cases a distinct point mutation resulting in crucial alterations in the encoded XRCC1 protein sequence of 633 amino acids. Two of the mutations cause non-conservative amino acid changes, Glu102-->Lys and Cys390-->Tyr, at positions that are invariant among hamster, mouse and human XRCC1 sequences and are located in putative functional domains. A third debilitating mutation disrupts RNA splicing, generating multiple transcripts of different length that contain deletions spanning a region of >100 amino acids in the midsection of the XRCC1 coding sequence. A fourth mutation results in a termination codon that shortens the open reading frame to 220 amino acids, however, in the revertant cell line a further mutation in the same codon, Stop221-->Leu, permits translation of a full-length functional variant protein. These mutational data indicate the importance of the putative functional regions in XRCC1, such as the BRCA1 C-terminal (BRCT) domain found in common with BRCA1 and other DNA repair and cell cycle checkpoint proteins, and also regions necessary for interaction with DNA polymerase beta and DNA ligase III.

Alternative Splicing↗
PubMed1999

The ataxia-telangiectasia related protein ATR mediates DNA-dependent phosphorylation of p53.

Levels of the tumour suppressor protein p53 are increased in response to a variety of DNA damaging agents. DNA damage-induced phosphorylation of p53 occurs at serine-15 in vivo. Phosphorylation of p53 at serine-15 leads to a stabilization of the polypeptide by inhibiting its interaction with Mdm2, a protein that targets p53 for ubiquitin-dependent degradation. However, the mechanisms by which DNA damage is signalled to p53 remain unclear. Here, we report the identification of a novel DNA-activated protein kinase that phosphorylates p53 on serine-15. Fractionation of HeLa nuclear extracts and biochemical analyses indicate that this kinase is distinct from the DNA-dependent protein kinase (DNA-PK) and corresponds to the human cell cycle checkpoint protein ATR. Immunoprecipitation studies of recombinant ATR reveal that catalytic activity of this polypeptide is required for DNA-stimulated phosphorylation of p53 on serine-15. These data suggest that ATR may function upstream of p53 in a signal transduction cascade initiated upon DNA damage and provide a biochemical assay system for ATR activity.

Ataxia Telangiectasia↗
PubMed2006

Lysine 63-polyubiquitination guards against translesion synthesis-induced mutations.

Eukaryotic cells possess several mechanisms to protect the integrity of their DNA against damage. These include cell-cycle checkpoints, DNA-repair pathways, and also a distinct DNA damage-tolerance system that allows recovery of replication forks blocked at sites of DNA damage. In both humans and yeast, lesion bypass and restart of DNA synthesis can occur through an error-prone pathway activated following mono-ubiquitination of proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, and recruitment of specialized translesion synthesis polymerases. In yeast, there is evidence for a second, error-free, pathway that requires modification of PCNA with non-proteolytic lysine 63-linked polyubiquitin (K63-polyUb) chains. Here we demonstrate that formation of K63-polyUb chains protects human cells against translesion synthesis-induced mutations by promoting recovery of blocked replication forks through an alternative error-free mechanism. Furthermore, we show that polyubiquitination of PCNA occurs in UV-irradiated human cells. Our findings indicate that K63-polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability.

Carcinogens↗
PubMed2003

CyclinB1 expression is elevated and mitosis is delayed in HeLa cells expressing autonomous CaMKII.

Calcium is a second messenger that is implicated in the regulation of cell cycle transitions. Calmodulin is a ubiquitous protein that translates intracellular calcium signals and activates several enzymes including calcium/calmodulin-dependent protein kinase II (CaMKII). Pharmacological inhibitors and constitutively active mutants have implicated CaMKII in cell cycle mediation. Specifically, constitutively active CaMKII impedes mitosis. In order to elucidate the molecular mechanisms underlying this phenomenon, the effect of constitutively active CaMKII gene expression on cdc2/cyclin B1 was investigated. As seen in previous studies with S. pombe, constitutively active CaMKII-hindered mitosis. However, this report shows that CaMKII does not cause permanent cell cycle arrest but delays progression into mitosis. Constitutive CaMKII expression also leads to elevations in cyclin B1 expression and cdc2 tyrosine-15 phosphorylation, analogous to observations in cells treated with hydroxyurea. Taken together, these data suggest that constitutive CaMKII may delay mitosis by activating a cell cycle checkpoint.

Calcium Signaling↗