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PubMed2003

[Histone deacetylase inhibitor blocks proliferation of cells transformed with oncogenes E1A and cHa-ras].

Rat embryonic fibroblasts, transformed with E1A and cHa-ras oncogenes, are unable to stop in the cell cycle checkpoints under growth factor withdrawal and genotoxic stresses (Bulavin et al., 1999). In the present paper, we showed that sodium butyrate, an inhibitor of histone deacetyase activity, decreased the share of cells being in S-phase, and caused G1/S and G2/M blocks of the cell cycle in the transformants. By means of RT-PCR and immunoblotting, we found that NaB significantly changed the expression of genes involved in proliferation: cyclins D1, A, E and cyclin-dependent kinases Cdk2 and Cdk4, whereas the amount of p21Waf1 and p27Kip1 inhibitors greatly increased. Along with accumulation of p21Waf1 protein content, that of Cdk2-bound p21 increases. Taken together, these data allow to suggest that NaB treatment does evidently restore the capability of p21Waf1 to inhibit cyclin-kinase activity. One may suppose that inhibition of HDAC activity by sodium butyrate leads to activation of yet unknown HDAC-dependent genes, which is followed by restoration of p21Waf1 function in spite of the E1A oncogene expression.

Adenovirus E1A Proteins↗
PubMed2003

Repression of mRNA for the PLK cell cycle gene after DNA damage requires BRCA1.

DNA damage activates the G2 cell cycle checkpoint to allow time for DNA repair before mitotic entry. The mechanism involves inhibition of the enzymatic activity for polo-like kinase 1 (Plk1), rendering Cdc25C with a basal phosphatase activity that is insufficient for converting Cdc2 to the fully active G2/M transition kinase. We found that cell cycle arrest at the G2/M boundary after ionizing radiation (IR) of breast carcinoma cells may involve repression of the gene for Plk1, PLK, mediated by the tumor-suppressor protein BRCA1. The p53-defective MT-1 cell line had an apparent accumulation of G2/M phase cells 12 h after irradiation. This response was preceded by a transient downregulation of PLK mRNA expression with a barely detectable level 6 h after exposure to IR but recovered after 12 h. A significantly lower fraction of irradiated BRCA1(-/-) HCC1937 cells arrested in the G2/M phase after 12 h, and the transient response of PLK mRNA was also considerably impaired. After reconstitution of wild-type BRCA1 in the HCC1937 cells however, downregulation of PLK mRNA as well as Plk1 protein expression after IR was restored. Moreover, the suppression of PLK mRNA expression 6 h after irradiation was completely abolished by the specific CHEK1 kinase inhibitor UCN-01, further indicating that the effector mechanism of DNA damage on PLK signals through BRCA1 and its downstream CHEK1. Our observations provide new information about the diversity of regulatory mechanisms governed by BRCA1 in DNA damage checkpoint control.

BRCA1 Protein↗
PubMed2006

Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells.

High-fidelity maintenance of genomic integrity in eukaryotes is ensured by cell cycle checkpoints and DNA repair. The checkpoint kinase, Chk2, has been implicated in both of these responses. In response to DNA damage, Chk2 is initially phosphorylated at Thr-68, which leads to its full activation. The fully activated Chk2 then phosphorylates downstream substrates of cell cycle control. However, the mechanism of inactivation of Chk2 is still unknown. Protein phosphatase type 2A (PP2A) plays an essential role in cell cycle regulation and induction of G2 arrest by a mechanism of phosphorylation/dephosphorylation with a variety of protein kinases. Data from our investigation provide evidence that, in response to cisplatin exposure, PP2A associates with Chk2 as a complex in cells and functions as a negative regulator of Chk2 activation by dephosphorylating p-Chk2. Results from immunostaining and coimmunoprecipitation demonstrate that Chk2 and PP2A can colocalize in cells, and the holoenzyme of PP2A (subunits A, B and C) coimmunoprecipitates with p-Chk2. Further, inhibition of PP2A by okadaic acid, an inhibitor of PP2A, and by small interfering RNA (siRNA) to PP2A results in enhanced Chk2 phosphorylation, implicating a direct enzyme-substrate relationship. An in vitro PP2A dephosphorylation assay shows that PP2A dephosphorylates p-Chk2 in a cell-free system. These findings suggest that the protein serine/threonine kinase, Chk2, is activated after cisplatin exposure and negatively regulated by a tightly associated protein serine/threonine phosphatase, PP2A.

Antineoplastic Agents↗
PubMed2000

DNA damage-related gene expression as biomarkers to assess cellular response after gamma irradiation of a human lymphoblastoid cell line.

Since defects in molecular mechanisms controlling DNA repair, cell cycle checkpoint and apoptosis could modify cellular sensitivity to DNA damaging agents, we have conducted a multiparametric molecular analysis for better understanding the regulation pathways leading to cell survival or cell death after irradiation. Using a human lymphoblastoid cell line, we have analysed, following gamma irradiation (0.5, 1, 2, 4, 8, 16 and 32 Gy, at 0.5, 24, 48 and 72 h after treatment), the correlation between proliferation, cell cycle analysis, apoptosis and micronuclei frequency with the expression of TP53, WAF1, DNA LIGASE 1, PCNA, BAX, BLC-2, BAK, DAD1, ICH1-Long and -Short forms mRNAs. We have found that whereas TP53, BAK, ICH1-Short form, and DAD1 were expressed at constant levels, WAF1, PCNA, BAX were up-regulated, ICH1-Long form, DNA LIGASE 1, and BCL-2 were down-regulated. These modifications of expression were significantly correlated with doses, survival, proliferation, cell cycle delays, and apoptosis. A positive correlation of WAF1 and BAX, and a borderline negative correlation with BCL-2 expressions were observed with micronuclei frequency for doses ranging from 0.5 to 4 Gy. In conclusion, our data clearly demonstrate that gene expression profiling, which is easier and more rapid to conduct than the assessments of classical phenotypic responses, could be useful to improve knowledge concerning pathways involved in cellular response to irradiation, knowing that such biomarkers could constitute tools to assess radio-sensitivity/radio-resistance. Oncogene (2000) 19, 916 - 923.

Apoptosis↗
PubMed2025

The genome of the polyextremophilic yeast, Naganishia friedmannii, reveals adaptations involved in stress response pathways, carbohydrate metabolism expansion, and a limited DNA repair repertoire.

Here we report the draft genome sequence of Naganishia friedmannii (formerly Cryptococcus friedmannii) isolate, a Basidiomycota yeast commonly found in some of the most extreme environments of the Earth's cryosphere. We isolated N. friedmannii strain Llullensis from soils at 6000 m above sea level on Volcán Llullaillaco, Argentina. The genome was 22.2 Mb with 6251 identified protein coding genes. Proteins known to be associated with thermal, osmotic, and radiation stress were identified in the genome. Comparative analysis with seven other Naganishia genomes revealed unique features underlying its polyextremophilic lifestyle. Naganishia friedmannii showed an expansion of genes involved in breaking down plant-derived carbohydrates, supporting the hypothesis that it survives at high elevations by metabolizing wind-deposited organic matter. Surprisingly, many genes involved in cell-cycle checkpoints and DNA repair were missing, as in several other Naganishia species. This extensive loss may be adaptive in extreme environments prone to abiotic stress, where a high mutation rate could generate advantageous traits, and reduced cell-cycle control may allow for faster reproduction that would be advantageous for rapid growth during brief periods of soil wetting following rare snow events.

Carbohydrate Metabolism↗
PubMed1996

The Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the human ATM gene product, is required for G1 arrest following radiation treatment.

The Saccharomyces cerevisiae gene MEC1 represents a structural homolog of the human gene ATM mutated in ataxia telangiectasia patients. Like human ataxia telangiectasia cell lines, mec1 mutants are defective in G2 and S-phase cell cycle checkpoints in response to radiation treatment. Here we show an additional defect in G1 arrest following treatment with UV light or gamma rays and map a defective arrest stage at or upstream of START in the yeast cell cycle.

Ataxia Telangiectasia↗
PubMedDate not supplied

Differential p53 protein expression level in human cancer-derived cell lines after estradiol treatment.

BACKGROUND: p53 has a remarkable number of biological activities, including a central role in cell cycle checkpoints, apoptosis, senescence, and maintenance of genomic integrity. Its expression is modified by estradiol in some epithelial cancer-derived cell lines from the reproductive tract. The aim of this study was to evaluate the effect of low and high doses of estradiol in p53 gene expression in epithelial cancer-derived cell lines from the reproductive tract. METHODS: p53 gene expression was assessed by Northern and Western blot methods in three human epithelial cancer-derived cell lines after estradiol treatment. RESULTS: These indicated that no changes in p53 mRNA content occurred after estradiol treatment at both low (10 nM) and high (1 micro M) doses of estradiol in HeLa, CaLo, and C-33 cell lines. p53 protein content was nearly constant in HeLa and C-33 cell lines at administration of 10 nM of estradiol. However, when estradiol was administered at a higher dose (1 micro M), an increase in p53 protein was observed over time in HeLa and CaLo cell lines. In contrast, estradiol was without variations in C-33. CONCLUSIONS: Overall results indicate that estradiol induces variations of p53 protein levels in epithelial cancer-derived cell lines from the reproductive tract in vitro and that this effect may be related with status p53 and/or presence of E6/E7 from human papillomavirus.

Blotting, Northern↗
PubMed2000

ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway.

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.

Ataxia Telangiectasia↗
PubMed2003

P53 is a regulator of the metastasis suppressor gene Nm23-H1.

p53, a tumor suppressor gene involved in the G1 cell cycle checkpoint, is also the most frequently mutated gene in human cancer. In addition, p53 modifies the ability of tumor cells to metastasize. The metastasis-associated gene Nm23-H1, which encodes an 18-kDa nucleoside diphosphate kinase, was previously identified in cells with low metastatic potential. Although p53 and Nm23-H1 proteins play an important part in regulating the progression of cancer, any functional relationship between these two proteins is currently unknown. Here we report an association between p53 levels and expression of the Nm23-H1 gene. Our data indicate that wild-type (wt) p53 upregulated the expression of Nm23-H1 at protein and mRNA levels in MCF-7 and J7B cells. This capacity of wt p53 to regulate expression of Nm23-H1 was not only dependent on the endogenous but also the exogenous origin of p53, and could not be reproduced with mutant p53. Subsequently, the invasive ability of MCF-7 and J7B cells was suppressed upon induction of the Nm23-H1 protein by p53. In contrast, increased levels of p53 downregulated the expression of Nm23-H1 at the protein and mRNA levels in RKO and H1299 cells and, as a consequence, increased the invasive ability of both cell types. Thus, our results implicated the differential regulation of Nm23-H1 by p53 in different cell types as an important component in the molecular mechanisms of tumor metastasis.

Antigens, Neoplasm↗
PubMed2005

[Direct role of p53 on homologous recombination].

The tumor suppressor gene p53, which is the most frequently mutated gene in human tumors, controls cell cycle checkpoint and apoptosis via the transactivation of the transcription of a collection of genes. These activities avoid proliferation of cell bearing alteration of genetic material. However, like a two-edged sword, p53 can also directly participate to genome stability maintenance by repressing homologous recombination (HR), independently of the transactivation activity. This parallel activity allows to limit the deleterious consequences on an excess of HR. Beside genetic interactions, p53 protein physically interacts with both HR proteins and HR intermediates (heteroduplex and Holliday junctions). The core domain of p53 is required for interaction with Rad51 at an early step and the carboxy-terminal domain of p53 is involved in the interaction with Rad54 and HR intermediates, at a late step. We discuss here the putative consequences of this parallel activity of p53 on genome stability, speciation and tumor protection.

DNA-Binding Proteins↗
PubMed2001

Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation?

Cytolethal distending toxins (CDT) constitute a family of genetically related bacterial protein toxins able to stop the proliferation of numerous cell lines. This effect is due to their ability to trigger in target cells a signaling pathway that normally prevents the transition between the G2 and the M phase of the cell cycle. Produced by several unrelated Gram-negative mucosa-associated bacterial species, CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, cdtC) encoding proteins whose respective role is not yet fully elucidated. The CDT-B protein presents sequence homology to several mammalian and bacterial phosphodiesterases, such as DNase I. The putative nuclease activity of CDT-B, together with the activation by CDT of a G2 cell cycle checkpoint, strongly suggests that CDT induces an as yet uncharacterized DNA alteration. However, the effective entry of CDT into cells and subsequent translocation into the nucleus have not yet been demonstrated by direct methods. The relationship between the potential DNA-damaging properties of this original family of toxins and their role as putative virulence factors is discussed.

Animals↗
PubMed2005

Chromosomal instability in oral cancer cells.

Chromosomal instability is a common feature of human tumors, including oral cancer. Although a tumor karyotype may remain quite stable over time, chromosomal instability can lead to 'variations on a theme' of a clonal cell population, often with each cell within a tumor possessing a different karyotype. Thus, chromosomal instability appears to be an important acquired feature of tumor cells, since propagation of such a diverse cell population may facilitate evasion of standard therapies. There are several sources of chromosomal instability, although the primary causes appear to be defects in chromosomal segregation, telomere stability, cell-cycle checkpoint regulation, and the repair of DNA damage. Our understanding of the biological basis of chromosomal instability in cancer cells is increasing rapidly, and we are finding that the seemingly unrelated origins of this phenomenon may actually be related through the complex network of cellular signaling pathways. Here, we review the general causes of chromosomal instability in human tumors. Specifically, we address the state of our knowledge regarding chromosomal instability in oral cancer, and discuss various mechanisms that enhance the ability of cancer cells within a tumor to express heterogeneous karyotypes. In addition, we discuss the clinical relevance of factors associated with chromosomal instability as they relate to tumor prognosis and therapy.

Carcinoma, Squamous Cell↗
PubMed1996

Drug Resistance.

Chemotherapy fails to cure most cancer patients with advanced disease, particularly patients with the most common forms of solid tumors. The presence or development of resistance to anticancer agents is the major cause of this failure. Several of the mechanisms underlying drug resistance to cytotoxic drugs have been elucidated in the last two decades, largely by employing in vitro drug-selected cancer cell lines. In unselected cell lines and probably also in human cancer, multiple mechanisms are redundantly present to defend the organism from the insults of drugs. Mechanisms have been unraveled by which cross-resistance ensues to multiple drugs (multidrug resistance), similar to what is commonly seen in patients. More recently, the identification of downstream genes, intimately involved in cell-cycle checkpoints, appears also to directly contribute to determining the sensitivity to cytotoxic drugs by regulating the response of the cell to the drug damage. The identification of mechanisms of drug resistance has provided ways of attempting to revert the drug resistance. Although, so far, attempts to revert P-glycoprotein-mediated multidrug resistance have only sorted out limited efficacy, new drugs and new strategies are being devised and implemented, such as high-dose chemotherapy and gene transfer.

Journal Article↗
PubMedDate not supplied

Carboplatin-induced cell death in model prostate cancer systems.

BACKGROUND: Many chemotherapeutic agents are believed to kill cancer cells by inflicting cellular damage which triggers the cell to enter apoptosis (programmed cell death). We investigated the means by which carboplatin induces cell death in three model cancer systems: the human prostate carcinoma cell lines PC-3 and LNCaP and the human cervical carcinoma cell line HeLa. MATERIALS AND METHODS: Drug cytotoxicity, cell cycle effects, bcl-2 deactivation, and multiple markers for apoptosis were utilized to examine carboplatin activity within these cell lines. RESULTS: In HeLa cells, carboplatin appears to induce an S-phase block followed by apoptosis. In contrast, PC-3 and LNCaP cells show no cell cycle phase block and die from necrosis rather than apoptosis. The effects of carboplatin contrast sharply with the effects of paclitaxel, which induces an M-phase block and apoptosis in all three cell lines. CONCLUSIONS: These results show that PC-3 and LNCaP cells are relatively resistant to carboplatin and suggest two causes of resistance: bypassing the cell cycle checkpoints which serve as points of entry into apoptosis, and incomplete execution of the effector mechanisms of apoptosis. Carboplatin resistance in the prostate cancer cell lines fits into the developing scheme of apoptosis-necrosis and raises valuable questions about the root causes of cancer resistance to chemotherapeutic agents.

Apoptosis↗
PubMed2006

Mechanisms of induction of cell cycle arrest and cell death by cryptolepine in human lung adenocarcinoma a549 cells.

We investigated p53-dependent and -independent molecular events associated with cell cycle alteration and cell death in human lung adenocarcinoma A549 cells using cryptolepine, a DNA-damaging agent. After a 24-h treatment, cryptolepine caused an accumulation of p53 at concentrations of 1.25-10 microM and induction of p21(Cip1/WAF1) but only at concentrations up to 5muM. p21(Cip1/WAF1) was also strongly induced by cryptolepine (2.5-5 microM) in cells with p53 largely ablated via small interfering RNA-mediated gene silencing. Cryptolepine induced G1-phase block at 1.25-2.5 microM, S-phase and G2/M-phase block at 2.5-5 microM, and cell death at 10 microM. The dead cells displayed condensed and fragmented nuclei, features of apoptosis. Wortmannin, an inhibitor of ataxia telangiectasia-mutated and DNA-dependent protein kinase (DNA-PK), caused cell cycle arrest at G1 phase without inducing p53 and p21(Cip1/WAF1) expression and cell death. The addition of wortmannin partially prevented cryptolepine-induced expression of p53 and p21(Cip1/WAF1) together with the S-phase block and sensitized cells to induction of cell death. NU7026, a DNA-PK-specific inhibitor, showed neither induction of cell cycle arrest and apoptosis nor the expression of p53 and p21(Cip1/WAF1). The presence of NU7026 caused further reduction of cells in G1 phase induced by cryptolepine at 5 microM without affecting the induction of p53 and p21(Cip1/WAF1) and cell death. This study using the A549 cell as a model demonstrated that cryptolepine selects different molecular pathways to cell cycle checkpoint activation in a dose-specific manner and evokes a wortmannin-sensitive antiapoptosis response.

Adenocarcinoma↗
PubMed1996

Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin.

Loss of p53 function in cancer cells commonly results in a condition of genomic instability. This is believed to emanate from a loss of the G1 checkpoint response to DNA damage. While the role of p53 in the induction of a G1 arrest is well-accepted, additional p53 functions are being discovered. Cell cycle checkpoints presumably function to allow additional time for DNA repair after damage is incurred, however, genetic studies in yeast suggest that components of the checkpoint pathway may also be involved in DNA lesion processing (Lydall and Weinert, 1995). Recent evidence suggests that this may also be the case for p53, as suggested by numerous reports linking p53 function to DNA repair. Thus, loss of p53 function might contribute to genomic instability independent of G1-arrest. In the present study, we explored the effect of p53 disruption and consequences of antisense GADD45 expression on the DNA repair capacity of human colon carcinoma RKO cells. DNA repair was assayed using host-cell reactivation of u.v.-damaged reporter plasmids and unscheduled DNA synthesis experiments in transiently-transfected cells. We show that a number of transfected genes that suppress p53 function reduce the ability of cells to repair u.v.-induced DNA damage. Moreover, cells in which expression of the p53-regulated gene GADD45 was blocked by antisense vectors, also showed altered levels of DNA repair. Blocking Gadd45 expression by constitutive antisense expression sensitized cells to killing by u.v.-radiation or by cis-platinum (II) diamine-dichloride (CDDP, or cisplatin), a cancer chemotherapy drug which produces DNA cross-links. These findings suggest the involvement of downstream effectors of the p53 pathway in the coordination of cell cycle arrest and DNA repair.

Antineoplastic Agents↗
PubMed2002

Characterization and changes of a chromosomal-scaffolding protein in human epithelia.

Chromosomal-scaffolding proteins exert DNA structural functions during mitosis, and gene regulatory functions such as RNA splicing/polymerization and DNA replication in interphase, allowing the progression of the cell cycle. Recently, it has been reported that topoisomerases play a key role in DNA repair, suggesting an additional regulatory mechanism of the chromosome structure on DNA metabolism and cell cycle checkpoints. Despite the progress made toward the understanding of the genome organization and expression, few changes have been reported in the chromosome scaffold of malignant cells associated with the cancer phenotype. In a previous work, we reported LFM-1 protein (Licensing Factor Model-1) as a chromosomal-scaffold component transiently associated with mitotic chromosomes in MDCK (Madin Darby canine kidney) epithelial cells (Vega-Salas and Salas 1996). In this work, we explore LFM-1 expression in human epithelia with contrasting tumorigenicity during the progression of the cell cycle. Although cell metabolic labeling shows synthesis of a common 87-kDa LFM-1 precursor during G(2)-phase in both non-tumorigenic and cancer cells, surprisingly, the post-translational LFM-1 chromosome-bound polypeptide displays a different apparent molecular weight and binding to chromosomes in the cancer phenotype. The finding of a highly phosphorylated LFM-1 60-kDa form with abnormal binding to chromosomes in human carcinoma cells suggests a structural/regulatory role(s) of the chromosome-scaffold/matrix in DNA metabolism in cancer-related events of cell proliferation.

Animals↗
PubMed2004

[Mitotic catastrophe: a special case of apoptosis].

Mitotic catastrophe is a poorly defined type of cell death linked to the abnormal activation of cyclin B/Cdk1. Here we propose that a conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe, provided that cell cycle checkpoints are inhibited, in particular the DNA structure checkpoints and the spindle assembly checkpoint. Two subtypes of mitotic catastrophe can be distinguished. First, mitotic catastrophe can kill the cell during or close to the metaphase, in a p53-independent fashion, as this occurs in Chk2-inhibited heterokarya generated by fusion. Second, mitotic catastrophe can occur after failed mitosis, during the activation of the polyploidy checkpoint, in a partially p53-dependent fashion. In these conditions, cells die as a result of caspase activation and mitochondrial membrane permeabilization that constitute hallmarks of apoptosis. Prevention of caspase activation and/or mitochondrial damage avoids mitotic catastrophe, indicating that this form of cell death indeed constitutes a special case of apoptosis. Importantly, the suppression of mitotic catastrophe can favor asymmetric division and the generation of aneuploid cells. This delineates a molecular pathway through which failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities which are likely to participate in oncogenesis.

Animals↗