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PubMed2006

The 3p21.3 tumor suppressor NPRL2 plays an important role in cisplatin-induced resistance in human non-small-cell lung cancer cells.

NPRL2 is one of the novel candidate tumor suppressor genes identified in the human chromosome 3p21.3 region. The NPRL2 has shown potent tumor suppression activity in vitro and in vivo and has been suggested to be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. In this study, we analyzed the endogenous expression of the NPRL2 protein and the cellular response to cisplatin in 40 non-small-cell lung cancer cell lines and found that expression of NPRL2 was significantly and reciprocally correlated to cisplatin sensitivity, with a Spearman correlation coefficient of -0.677 (P < 0.00001). Exogenously introduced expression of NPRL2 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly resensitized the response to cisplatin, yielding a 40% greater inhibition of tumor cell viability and resulting in a 2- to 3-fold increase in induction of apoptosis by activation of multiple caspases in NPRL2-transfected cells compared with untransfected cells at an equal dose of cisplatin. Furthermore, a systemic treatment with a combination of NPRL2 nanoparticles and cisplatin in a human H322 lung cancer orthotopic mouse model significantly enhanced the therapeutic efficacy of cisplatin and overcame cisplatin-induced resistance (P < 0.005). These findings implicate the potential of NPRL2 as a biomarker for predicting cisplatin response in lung cancer patients and as a molecular therapeutic agent for enhancing response and resensitizing nonresponders to cisplatin treatment.

Animals↗
PubMed1997

Molecular perspectives on cancer, the cell cycle and the inherited disorder ataxia-telangiectasia.

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder which presents a wide array of clinical symptoms including enhanced cancer predisposition and progressive cerebellar degeneration leading to general neuromotor dysfunction. The A-T cellular phenotype consists of higher levels of chromosome breakage, increased sensitivity to ionizing radiation and radiomimetic drugs, and defective cell cycle checkpoints in response to genome damage. Positional-cloning of the gene mutated in A-T, designated ATM, identified a 13 kb transcript encoding a 3056 amino acid protein which possesses a carboxy-terminal domain with distinct homology to phosphatidylinositol-3 kinase. Furthermore, ATM related proteins have been identified in yeast, Drosophila and other mammalian species which are involved in cell cycle control and cellular responses to DNA damage. Development of cellular and animal models for A-T can serve to better dissect the role and involvement of ATM in cell cycle regulation, cancer development, neuronal cell death and other hallmark symptoms of this disorder.

Ataxia Telangiectasia↗
PubMed1998

Monitoring and signaling of radiation-induced damage in mammalian cells.

This paper reviews the functions of and connections between the presumed DNA damage sensors: poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), the protein product of the ataxia telangiectasia mutated (ATM) gene, and the tumor suppressor, p53. Recognition of DNA damage is associated with the generation of alarm signals. The possible alarm signals include synthesis of poly(ADP-ribose) polymers and initiation of phosphorylation cascades by kinases complexed with the DNA damage sensors, DNA-PK and ATM; the role of other factors is discussed, among them BRCA1 and 2, IRF-1 and RB (retinoblastoma). Alarm signal molecules generated in the cytoplasm or plasma membrane are reactive oxygen species and ceramide. Some of the signal pathways are discussed. The p53 protein, which is poised in the central junction of the postirradiation signaling, as well as p53-independent signaling pathways form an intricate network that executes concerted and partly overlapping functions in the cellular response to ionizing radiation. These functions comprise activation of specific groups of genes, control of progression through the cell cycle checkpoints, inhibition of replication and transcription, induction of apoptosis, or an adaptive response; these features of the cellular response to radiation are discussed. They affect the fate of the irradiated mammalian cell as markedly as the DNA repair efficiency. This is shown in examples of the effect of inhibition of signaling on the adaptive response of human lymphocytes and on survival of tumor cells.

Adaptation, Physiological↗
PubMed2003

A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein.

BRCA1 is a central component of the DNA damage response mechanism and defects in BRCA1 confer sensitivity to a broad range of DNA damaging agents. BRCA1 is required for homologous recombination and DNA damage-induced S and G(2)/M phase arrest. We show here that BRCA1 is required for ATM- and ATR-dependent phosphorylation of p53, c-Jun, Nbs1 and Chk2 following exposure to ionizing or ultraviolet radiation, respectively, and is also required for ATM phosphorylation of CtIP. In contrast, DNA damage-induced phosphorylation of the histone variant H2AX is independent of BRCA1. We also show that the presence of BRCA1 is dispensable for DNA damage-induced phosphorylation of Rad9, Hus1 and Rad17, and for the relocalization of Rad9 and Hus1. We propose that BRCA1 facilitates the ability of ATM and ATR to phosphorylate downstream substrates that directly influence cell cycle checkpoint arrest and apoptosis, but that BRCA1 is dispensable for the phosphorylation of DNA-associated ATM and ATR substrates.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

Animals↗
PubMed1999

Hypophosphorylated retinoblastoma protein is associated with G2 arrest in esophageal squamous cell carcinoma.

Hypophosphorylated retinoblastoma (Rb) gene product binds critical transcription factors, leading to G1 arrest in a number of conditions, including following DNA damage. We have previously shown that irradiated esophageal squamous cell carcinoma (ESSC) cells undergo predominantly G2 arrest, with increases in inhibitors of Rb phosphorylation. We thus hypothesized that this G2 arrest would be accompanied by increases in hypophosphorylated Rb protein (pRb). We sequenced the Rb genes of three human ESSC lines (KYSE) following reverse transcription polymerase chain reaction of exons A-E. Western gels were performed on protein extracts for pRb. Cells were irradiated at 6 Gy, and protein was extracted at 6 h. ELISA was used to measure hypophosphorylated pRb in radiated versus control cells. Student's t test was used to compare results. All lines had wild-type Rb genes. Western gels confirmed the presence of pRb. There were significant increases in hypophosphorylated pRb in all three lines following irradiation (no line with less than a 100% increase). We have thus shown that irradiation-induced G2 arrest occurs in association with wild-type Rb genes and that there is associated hypophosphorylation of pRb. This supports our data describing a further role for other G1 mediators, such as p21, in G2 arrest. Further investigations into therapies to expoit this cell cycle checkpoint are warranted and planned.

Blotting, Western↗
PubMed1995

p53 mediated tumor cell response to chemotherapeutic DNA damage: a preliminary study in matched pairs of breast cancer biopsies.

Wild type p53 plays a crucial role in maintaining genomic stability in both normal and tumor cells in vitro. When DNA damage occurs, p53 acts as a cell cycle checkpoint and induces a cellular response that aims at restoring genomic integrity. p53 may either allow the repair of damaged DNA by inducing a transient G1 arrest or may eliminate the damaged cells by triggering apoptosis. Mutant p53 fails to mediate any of these effects. From this, a p53 status-dependent response to therapy might be expected when tumors are treated with DNA-damaging genotoxic agents: Although wild type p53-harboring tumors have an intact checkpoint that might allow them to restore genomic integrity back to a pre-exposure level, mutant p53 tumors have a corrupted checkpoint that could lead to an accelerated loss of genomic stability. Until now, no studies have been described that examine such a p53-mediated effect in vivo. The authors tested this response model in vivo comparing 32 matched biopsy pairs from patients with breast cancer before and after rigorously standardized polychemotherapy. Four of the five drugs specifically induce a wild type p53-mediated checkpoint response. Tumor tissue from matched pairs of untreated and treated biopsies of the same patient were analyzed for treatment-associated changes of p53 protein expression by immunocytochemistry and, in a few available specimens, of p53 genotype changes by polymerase chain reaction-based DNA analysis. Treatment-associated changes of the p53 immunophenotype, which the authors speculate to reflect clonal selection, occurred in 39% (12 of 31) of the specimens. One specimen was not informative. Most tumors undergoing clonal selection originally harbored mutant p53 (nine of 12), and only three of 12 tumors were wild type. This study shows that exposure to genotoxic agents is commonly associated with a change in p53 immunophenotype. Although the limited material in this cohort prevented direct analysis of genetic instability, these results suggest that tumors with altered p53 may be genomically less stable and, therefore, may be more likely to undergo treatment-induced clonal changes than wild type tumors. This study also shows that the rigorous matched sample approach, although difficult to obtain, is an important tool that allows the in vivo assessment of the tumor response to genotoxic therapy in a controlled fashion.

Adult↗
PubMed2001

DNA replication and daughter cell budding are not tightly linked in the protozoan parasite Toxoplasma gondii.

In the protozoan parasite Toxoplasma gondii, cell division occurs by an unusual internal budding process whereby two daughter cells develop within and eventually subsume the mother cell. We have examined this process using inhibitors targeted at specific events in the cell cycle. By adding inhibitors to newly established parasites we were able to examine the effects of the inhibitors on parasites treated at the start of intracellular development and many hours prior to the onset of daughter cell budding. As with other eukaryotes, inhibitors of nuclear DNA synthesis blocked parasite DNA synthesis and prevented cell division. Examination of parasites treated with the nuclear DNA synthesis inhibitor aphidicolin showed that the formation of daughter apical complexes and the initiation of budding occurred as normal and only the inability of the nucleus to become incorporated into the daughter cells prevented successful cell division. Moreover, these inhibitory effects of aphidicolin were not reversible. The initiation of nuclear DNA synthesis and cell division in newly invaded Toxoplasma required both gene transcription and protein synthesis, although inhibitors of mitochondrial DNA synthesis, transcription and protein synthesis did not block parasite division. Thus, unlike most eukaryotes, Toxoplasma tachyzoites have separated nuclear DNA replication and mitosis from the events associated with cell division (daughter cell budding). This implies that Toxoplasma tachyzoites may have dispensed with specific cell cycle checkpoints present in other eukaryotes with, in particular, a DNA-replication checkpoint control either missing, or downregulated in this stage of the parasite life cycle.

Animals↗
PubMed2003

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells.

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

Apoptosis↗
PubMed2006

Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle.

We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and gammaH2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G(1)/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G(2) resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G(1) cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G(1)-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4> or =AT>Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of endogenous damage.

Cell Cycle↗
PubMed1994

A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells/.

The enzyme DNA topoisomerase II, which removes the catenations formed between the DNA molecules of sister chromatids during replication and is a structural component of chromosome cores, is needed for chromosome condensation in yeast and in Xenopus extracts. Inhibitors of topoisomerase II arrest mammalian cells before mitosis in the G2 phase of the cell cycle, but also produce DNA damage, which causes arrest through established checkpoint controls. It is open to question whether cells need topoisomerase II to leave G2, or control late-cycle progression in response to its activity. Bisdioxopiperazines are topoisomerase II inhibitors that act without producing direct DNA damage; the most potent, ICRF-193, blocks mammalian entry into but not exit from mitosis. Here we show that checkpoint-evading agents such as caffeine override this block to produce abortively condensed chromosomes, indicating that topoisomerase II is needed for complete condensation. We find that exit from G2 is regulated by a catenation-sensitive checkpoint mechanism which is distinct from the G2-damage checkpoint.

Animals↗
PubMed2000

A common E2F-1 and p73 pathway mediates cell death induced by TCR activation.

Strong stimulation of the T-cell receptor (TCR) on cycling peripheral T cells causes their apoptosis by a process called TCR-activation-induced cell death (TCR-AICD). TCR-AICD occurs from a late G1 phase cell-cycle check point independently of the 'tumour suppressor' protein p53. Disruption of the gene for the E2F-1 transcription factor, an inducer of apoptosis, causes significant increases in T-cell number and splenomegaly. Here we show that T cells undergoing TCR-AICD induce the p53-related gene p73, another mediator of apoptosis, which is hypermethylated in lymphomas. Introducing a dominant-negative E2F-1 protein or a dominant-negative p73 protein into T cells protects them from TCR-mediated apoptosis, whereas dominant-negative E2F-2, E2F-4 or p53 does not. Furthermore, E2F-1-null or p73-null primary T cells do not undergo TCR-mediated apoptosis either. We conclude that TCR-AICD occurs from a late G1 cell-cycle checkpoint that is dependent on both E2F-1 and p73 activities. These observations indicate that, unlike p53, p73 serves to integrate receptor-mediated apoptotic stimuli.

Animals↗
PubMed2003

Expression of the cell cycle regulatory proteins p34cdc2, p21waf1, and p53 in node negative invasive ductal breast carcinoma.

AIMS: To look for correlations between expression of cell cycle regulatory proteins p34(cdc2), p21(WAF1), and p53 in node negative invasive ductal breast carcinoma, or between these proteins and clinicopathological parameters, and to assess their prognostic value. METHODS: Immunohistochemistry using formalin fixed, paraffin wax embedded sections from 94 breast carcinomas. Adjacent benign epithelial breast tissue was available in 74 cases. Median follow up was 72 months. RESULTS: Nuclear and cytoplasmic p34(cdc2) expression was seen in 80 and 62 tumours, respectively; nuclear expression was seen in adjacent benign epithelium in 12 cases. p21(WAF1) and p53 were positive in 48 and 21 tumours, respectively. High expression of p34(cdc2) in neoplastic nuclei was associated with higher histological grade and p53 expression, but not with tumour size, steroid receptor status, patient age, menopausal status, recurrence, metastasis, disease free survival (DFS), or overall survival (OS). p34(cdc2) in tumour cytoplasm was associated with p34(cdc2) nuclear positivity, high tumour grade, and DFS in univariate but not multivariate analysis. In contrast, p34(cdc2) expression in benign tissue independently predicted DFS and OS in univariate and multivariate analysis. Expression of p53 was associated with high tumour grade and negative steroid receptors, but not with recurrence, metastasis, DFS, or OS. p21(WAF1) expression was not associated with the examined parameters. CONCLUSIONS: p34(cdc2), p21(WAF1), and p53 expression does not predict outcome in node negative breast carcinoma, although p34(cdc2) expression in benign tissue is related to prognosis. The association between p34(cdc2) and p53 implicates p53 in G2-M cell cycle checkpoint control, possibly via mediators unrelated to p21(WAF1).

Adult↗
PubMed2000

[p53 activation by PI-3K family kinases after DNA double-strand breaks].

p53 plays a central role in the cellular response to DNA double-strand breaks (DSBs), and to DNA damage in general. The protein kinases ATM, ATR and DNA-PK detect DSBs and transmit this information to p53 by phosphorylation. This phosphorylation dissociates p53 from its negative regulator, mdm2. p53 then undergoes further modification and activates transcription of the genes responsible for cell cycle arrest. In certain circumstances, p53 also activates transcription of the genes responsible for apoptosis. The dysfunction of this cascade of events is oncogenic, with P53 itself being the most commonly mutated gene in malignant cells, although mutations in both the DNA damage sensors and cell cycle checkpoint and apoptosis effectors are frequent. A more complete understanding of p53 and the proteins it interacts with may allow the development of new cancer treatments.

Acetylation↗
PubMed2007

BRCA1 contributes to cell cycle arrest and chemoresistance in response to the anticancer agent irofulven.

Tumor suppressor gene BRCA1 is frequently mutated in familial breast and ovarian cancer. BRCA1 plays pivotal roles in maintaining genomic stability by interacting with numerous proteins in cell cycle control and DNA repair. Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is one of a new class of anticancer agents that are analogs of mushroom-derived illudin toxins. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. We demonstrated previously that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and that BRCA1 may affect chemosensitivity by controlling cell cycle checkpoints, DNA repair, and genomic stability in response to irofulven treatment. We observed that irofulven induces the formation of chromosome breaks and radials and the activation and foci formation of gamma-H2AX, BRCA1, and RAD51. We also provided evidence that irofulven induces the generation of DNA double-strand breaks. By using BRCA1-deficient or -proficient cells, we demonstrated that in response to irofulven, BRCA1 contributes to the control of S and G(2)/M cell cycle arrest and is critical for repairing DNA double-strand breaks and for RAD51-dependent homologous recombination. Furthermore, we found that BRCA1 deficiency results in increased chromosome damage and chemosensitivity after irofulven treatment.

Antineoplastic Agents↗
PubMed2003

Overexpression of a protein fragment of RNA helicase A causes inhibition of endogenous BRCA1 function and defects in ploidy and cytokinesis in mammary epithelial cells.

The breast- and ovarian-specific tumor suppressor, BRCA1, has been implicated to function in many nuclear processes, including DNA damage repair, recombination, transcription, ubiquitination, cell cycle checkpoint enforcement, and centrosome regulation. Utilizing a previously described interaction between BRCA1 and RNA helicase A (RHA), we have developed a dominant-negative approach to block BRCA1 function in human breast epithelial cells. Overexpression of a truncated RHA peptide that can bind to the BRCA1 carboxy-terminus prevents normal BRCA1 function, such as BRCA1 association with nuclear foci following DNA damage. Overexpression of this dominant-negative protein induces pleomorphic nuclei, aberrant mitoses with extra centrosomes, and tetraploidy. This model system allows us to observe changes to mammary epithelial cells that occur acutely following loss of BRCA1 function. Furthermore, inhibition of BRCA1 via overexpressing the RHA fragment coincides with a reduction in PARP-1 protein expression, suggesting a possible mechanism for BRCA1 in the maintenance of genomic integrity.

Aneuploidy↗
PubMed2005

Reprogramming specific gene expression pathways in B-cell lymphomas.

The BCL6 transcriptional repressor is normally expressed during the germinal center phase of B-cell differentiation. Germinal center B-cells typically undergo rapid proliferation in spite of accumulating DNA damage caused by class switch recombination and somatic hypermutation. BCL6 is required to license B-cells for the germinal center reaction and its down regulation appears to be important for cells to exit this stage and undergo further differentiation. BCL6 appears to mediate these biological effects by recruiting corepressor complexes to silence critical cell cycle checkpoint and differentiation related genes. Based on our data and recent publications, we propose that these gene pathways are regulated through distinct transcriptional mechanisms, which can be specifically targeted to reprogram B-cells preferentially for either growth suppression and apoptosis, or differentiation. As BCL6 plays a central role in the pathogenesis of diffuse large B-cell lymphoma, we predict that targeting BCL6 transcriptional repression complexes in malignant B-cells may constitute a novel form of transcription therapy for lymphomas and possibly other tumors.

Animals↗
PubMed2006

The Rad9 protein enhances survival and promotes DNA repair following exposure to ionizing radiation.

Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.

Cell Cycle Proteins↗