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PubMed1997

Inhibition of DNA topoisomerase II alpha gene expression by the p53 tumor suppressor.

DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II alpha is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II alpha gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II alpha mRNA and protein following incubation for 24 h at 32 degrees C (p53 with wt conformation) than at 39 degrees C (p53 with mutant conformation). The effect of p53 on the human topo II alpha gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II alpha promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp -557 to +90) topo II alpha promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II alpha promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (-32 to +90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (-68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II alpha expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II alpha gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II alpha and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic stability associated with neoplasia.

Animals↗
PubMed2006

[Effects of genistein on the expressions of cell cycle proteins in salivary adenoid cystic carcinoma cell line SACC-83].

PURPOSE: To investigate the molecular mechanism of cell cycle arrest induced by tyrosine protein kinase inhibitor, genistein, in human salivary adenoid cystic carcinoma cell line SACC-83. METHODS: SACC-83 cells cultured in vitro were treated with genistein, the expressions of CyclinB1, Cdk1, CyclinD1 and Cdk4 proteins were detected with Western blotting, and the results were quantitatively analyzed by FluorChem V2.0 software, statistical analysis was performed with analysis of variance using SPSS11.5 software. RESULTS: With the increase of concentration of genistein and the elongation of time, the expression of CyclinB1, Cdk1, CyclinD1 and Cdk4 proteins was significantly decreased. Treated with 220 micromol/L of genistein for 3 days, the expression level of CyclinB1, Cdk1, CyclinD1 and Cdk4 proteins was 58%, 64%, 46% and 43% of the control group, respectively (P<0.01). CONCLUSION: The cell cycle G2/M arrest induced by genistein in human salivary adenoid cystic carcinoma cell line SACC-83 may be associated with the downregulations of CyclinB1, Cdk1, CyclinD1 and Cdk4 protein expressions.

Carcinoma, Adenoid Cystic↗
PubMed1995

Ataxia-telangiectasia and cellular responses to DNA damage.

Ataxia-telangiectasia (A-T) is a human disease characterized by high cancer risk, immune defects, radiation sensitivity, and genetic instability. Although A-T homozygotes are rare, the A-T gene may play a role in sporadic breast cancer and other common cancers. Abnormalities of DNA repair, genetic recombination, chromatin structure, and cell cycle checkpoint control have been proposed as the underlying defect in A-T; however, previous models cannot satisfactorily explain the pleiotropic A-T phenotype. Two recent observations help clarify the molecular pathology of A-T: (a) inappropriate p53-mediated apoptosis is the major cause of death in A-T cells irradiated in culture; and (b) ATM, the putative gene for A-T, has extensive homology to several cell cycle checkpoint genes from other organisms. Building on these new observations, a comprehensive model is presented in which the ATM gene plays a crucial role in a signal transduction network that activates multiple cellular functions in response to DNA damage. In this Damage Surveillance Network model, there is no intrinsic defect in the machinery of DNA repair in A-T homozygotes, but their lack of a functional ATM gene results in an inability to: (a) halt at multiple cell cycle checkpoints in response to DNA damage; (b) activate damage-inducible DNA repair; and (c) prevent the triggering of programmed cell death by spontaneous and induced DNA damage. Absence of damage-sensitive cell cycle checkpoints and damage-induced repair disrupts immune gene rearrangements and leads to genetic instability and cancer. Triggering of apoptosis by otherwise nonlethal DNA damage is primarily responsible for the radiation sensitivity of A-T homozygotes and results in an ongoing loss of cells, leading to cerebellar ataxia and neurological deterioration, as well as thymic atrophy, lymphocytopenia, and a paucity of germ cells. Experimental evidence supporting the Damage Surveillance Network model is summarized, followed by a discussion of how defects in the ATM-dependent signal transduction network might account for the A-T phenotype and what insights this new understanding of A-T can offer regarding DNA damage response networks, genomic instability, and cancer.

Apoptosis↗
PubMed2005

RNA silencing of checkpoint regulators sensitizes p53-defective prostate cancer cells to chemotherapy while sparing normal cells.

p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G(2) checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.

Antibiotics, Antineoplastic↗
PubMed2004

Cell death by mitotic catastrophe: a molecular definition.

The current literature is devoid of a clearcut definition of mitotic catastrophe, a type of cell death that occurs during mitosis. Here, we propose that mitotic catastrophe results from a combination of deficient cell-cycle checkpoints (in particular the DNA structure checkpoints and the spindle assembly checkpoint) and cellular damage. Failure to arrest the cell cycle before or at mitosis triggers an attempt of aberrant chromosome segregation, which culminates in the activation of the apoptotic default pathway and cellular demise. Cell death occurring during the metaphase/anaphase transition is characterized by the activation of caspase-2 (which can be activated in response to DNA damage) and/or mitochondrial membrane permeabilization with the release of cell death effectors such as apoptosis-inducing factor and the caspase-9 and-3 activator cytochrome c. Although the morphological aspect of apoptosis may be incomplete, these alterations constitute the biochemical hallmarks of apoptosis. Cells that fail to execute an apoptotic program in response to mitotic failure are likely to divide asymmetrically in the next round of cell division, with the consequent generation of aneuploid cells. This implies that disabling of the apoptotic program may actually favor chromosomal instability, through the suppression of mitotic catastrophe. Mitotic catastrophe thus may be conceived as a molecular device that prevents aneuploidization, which may participate in oncogenesis. Mitotic catastrophe is controlled by numerous molecular players, in particular, cell-cycle-specific kinases (such as the cyclin B1-dependent kinase Cdk1, polo-like kinases and Aurora kinases), cell-cycle checkpoint proteins, survivin, p53, caspases and members of the Bcl-2 family.

Animals↗
PubMed1998

Induction of apoptosis in MCF-7:WS8 breast cancer cells by beta-lapachone.

Beta-lapachone (beta-lap) affects a number of enzymes in vitro, including type I topoisomerase (Topo I); however, its exact intracellular target(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells after 4-h pulses of beta-lap or camptothecin (CPT), a known Topo I poison. A direct correlation between loss of survival and apoptosis was seen after beta-lap treatment (LD50 = 2.5 microM). A concentration-dependent, transient sub-2 N preapoptotic cell population was observed at 4-8 h. Estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies revealed an apoptotic exit point near the G1-S border. Apoptosis activated by beta-lap was closely correlated with cleavage of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphorylated forms of the retinoblastoma protein was observed within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 microM beta-lap. Topo I and Topo IIalpha levels decreased at > 24 h. Logarithmic-phase MCF-7 cells were not affected by < or = 1 microM beta-lap. In contrast, dramatic and irreversible G2-M arrest with no apoptosis was observed in MCF-7 cells treated with 1 microM CPT, monitored for 6-10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 microM) resulted in only approximately 20% apoptosis. No correlation between apoptosis and loss of survival was observed. MCF-7 cells exposed to > 5 microM CPT arrested at key cell cycle checkpoints (i.e., G1, S, and G2-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2-5-fold decreases in bcl-2, Topo I, Topo IIalpha, and cyclins A and B1, with no change in cyclin E, were observed. Temporal decreases in bcl-2 and cleavage of lamin B corresponded to the minimal apoptotic response observed. Beta-lap activated apoptosis without inducing p53/p21 or cell cycle arrest responses and killed MCF-7 cells solely by apoptosis. In contrast, concentration-dependent increases in nuclear p53/p21 and various cell cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despite dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cells showed low levels of apoptosis, possibly due to protective cell cycle checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.

Antineoplastic Agents, Phytogenic↗
PubMed2004

Epothilone induced cytotoxicity is dependent on p53 status in prostate cells.

BACKGROUND: Epothilones are a promising class of drugs in clinical trials of prostate cancer that target the microtubules, similar to taxanes, and induce apoptosis in taxane resistant tumors. The tumor suppressor p53 is one important molecular mechanism of chemotherapy resistance that in some studies predicted tumor sensitivity to paclitaxel. We hypothesized that epothilone induced cytotoxicity would be influenced by the status of p53 in prostate cells. METHODS: LNCaP, DU145, and a transformed rat prostate (RP) epithelial cell line with a temperature sensitive mutant p53 (val 135) were studied for the effect of epothilone on cell viability, cell cycle, and cell cycle checkpoint proteins. RESULTS: Epothilone had greater cytotoxicity in p53 mutant cancer cells compared to wild type cells. We confirmed our findings by creating a transformed RP epithelial cell line with a temperature sensitive mutant p53 (val 135). Using a tetrazolium (MTT) assay we found that epothilone (100 nM) decreased cell viability in RP cells by 90% with mutant p53 compared to 45% with wild type p53 (P < 0.01). Epothilone induced G2/M arrest in 50% of cells with mutant p53 compared to 25% with wild type p53 (P < 0.01). To begin to understand mechanism of epothilone induced G2/M arrest, we assessed cell cycle checkpoint proteins. We found that the effect to enhance G2/M cell cycle arrest was associated with dephosphorylation of cdc2 in both p53 wild type and p53 mutant RP cells. CONCLUSIONS: These results demonstrate that epothilone is more active against transformed prostate epithelial cells with mutant compared to wild type p53. Epothilone is capable of dephosphorylation of cdc2 in both p53 wild type and mutant cells, which is associated with G2/M cell cycle arrest. These data provide a basis for further study of p53, and the phosphorylation status of cdc-2, as markers for epothilone sensitivity in clinical studies.

Animals↗
PubMed2005

Novel indication for cancer therapy: Chk1 inhibition sensitizes tumor cells to antimitotics.

Paclitaxel (Taxol) is the most-prescribed anti-mitotic agent for a variety of advanced metastatic cancers. It induces mitotic arrest leading to apoptosis through microtubule stabilization. Chk1 is the major cell-cycle checkpoint kinase mediating S- and G2-arrests in response to various DNA-damages. Chk1 inhibitor is anticipated and has been demonstrated to potentiate the cytotoxicity of DNA-damaging agents through abrogation of cell-cycle checkpoints. Paclitaxel does not, however, induce Chk1 activation, and Chk1 has not been shown to function in mitotic checkpoint. Thus, Chk1 inhibitor is not expected to enhance the toxicity of paclitaxel. Here we show that downregulation of Chk1 sensitizes tumor cells to the toxicity of paclitaxel in cell proliferation assay. Fluorescence microscopy showed that Chk1 knockdown augments mitotic catastrophe and apoptosis in paclitaxel-treated cancer cells. Further, we elucidated the mechanism of this sensitization. Chk1 inhibition facilitates paclitaxel-induced M-phase entry by activation of Cdc2 kinase and accumulation of cyclin B1, the required cofactor for Cdc2 kinase activity. Moreover, Chk1 downregulation inhibits M phase exit through induction of the anaphase inhibitor, securin/PDS1. Collectively, Chk1 elimination sustains a more effective mitotic arrest as demonstrated by the more efficient accumulation of M-phase marker phospho-histone H3. We show that Chk1 elimination attenuates the paclitaxel-induced activation of the anti-apoptotic p42/p44 (ERK1/2) MAP kinase pathway, additionally contributing to the sensitization. Our results suggest that in addition to its well-established role as an enforcer of S and G2-checkpoints in response to genotoxic stress, Chk1 also plays a protective role in mitotic checkpoint to lessen mitotic catastrophe and thereby limits cell-death. Therefore Chk1 downregulation can not only potentiate DNA-damaging agents, but also enhance the toxicity of anti-microtubule agents, which significantly broadens its therapeutic applications.

Antineoplastic Agents↗
PubMed1996

Chemical induction of mitotic checkpoint override in mammalian cells results in aneuploidy following a transient tetraploid state.

Populations of tetraploid cells are found in a variety of human tumours where they may act as precursors of aneuploidy and tumorigenesis. Here we demonstrate the drug induction of tetraploid cells at mitosis by interference with cell cycle checkpoints and the coordination of mitotic events. Tetraploid cells result from mitotic exit in the absence of either chromosome segregation or cytokinesis. One class of agents that induces tetraploidy causes override of cell cycle checkpoints that require metaphase chromosome alignment as a pre-condition for initiating exit from mitosis. As a result cells exposed to such drugs progress partially through mitosis, but exit without chromosome segregation or cytokinesis. Inhibitors of microtubule assembly comprise a second class of agents that induce tetraploidy. Many cell types are incapable of maintaining indefinite mitotic arrest in the presence of microtubule inhibitors and finally exit mitosis without microtubule dependent chromosome segregation. Inhibitors of topoisomerase II represent a third class of drugs that induce tetraploidy at mitosis. By inhibiting DNA decatenation required for sister chromatid separation at the onset of anaphase such drugs block chromosome segregation. When topoisomerase II activity is inhibited, cells nonetheless reform nuclei and exit from mitosis without chromosome segregation. Finally, inhibition of cleavage furrow formation by agents such as cytochalasins represents a fourth mechanism of tetraploidization at mitosis. We find that when Chinese Hamster Ovary cells become tetraploid, regardless of which mechanism induces this state, they are genetically unstable and become aneuploid at the subsequent mitosis. In conclusion, the failure of mitotic checkpoint function can generate gross aneuploidy from which cells with an advantage for tumor growth may be selected.

2-Aminopurine↗
PubMed2003

Characterisation of telomerase immortalised normal human oesophageal squamous cells.

BACKGROUND AND AIMS: Oesophageal cell lines derived from malignancies have numerous genetic abnormalities and therefore are of limited value for studying the early events in carcinogenesis. Reported attempts to establish normal human oesophageal cell lines either have failed to achieve immortalisation or have achieved it by disrupting important cell functions. We have used telomerase technology to establish normal human oesophageal cell lines. METHODS: Endoscopic biopsy specimens of normal oesophageal squamous epithelium were trypsinised, dispersed into single cell suspensions, and cocultivated with ATCC Swiss 3T3 cells. Oesophageal cells were infected with the catalytic subunit of human telomerase (hTERT) using a defective retroviral vector. The integrity of cell cycle checkpoints was tested by measuring p53 response to UV irradiation, and p16 response to infection with H-RasGV12. Expression of a differentiation marker was tested by measuring involucrin response to calcium exposure. RESULTS: Cultures of uninfected oesophageal cells had weak telomerase activity at baseline but exhibited loss of telomerase activity and progressive telomere shortening before undergoing senescence between population doublings (PD) 40-45. In contrast, hTERT infected cells exhibited sustained telomerase activity and stabilisation of telomere length. These cells have reached PD 100 with no diminution in growth rate, while cell cycle checkpoint integrity and involucrin response to calcium exposure have remained intact. CONCLUSIONS: By introducing telomerase into normal human oesophageal squamous cells cocultivated with feeder layers, we have established a cell line that retains normal cell cycle checkpoints and normal differentiation markers. This cell line may be useful for studying the early events in oesophageal carcinogenesis.

3T3 Cells↗
PubMed2005

Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway.

When exposed to DNA-damaging insults such as ionizing radiation (IR) or ultraviolet light (UV), mammalian cells activate checkpoint pathways to halt cell cycle progression or induce cell death. Here we examined the ability of five commonly used anticancer drugs with different mechanisms of action to activate the Chk1/Chk2-Cdc25A-CDK2/cyclin E cell cycle checkpoint pathway, previously shown to be induced by IR or UV. Whereas exposure of human cells to topoisomerase inhibitors camptothecin, etoposide, or adriamycin resulted in rapid (within 1 h) activation of the pathway including degradation of the Cdc25A phosphatase and inhibition of cyclin E/CDK2 kinase activity, taxol failed to activate this checkpoint even after a prolonged treatment. Unexpectedly, although the alkylating agent cisplatin also induced degradation of Cdc25A (albeit delayed, after 8-12 h), cyclin E/CDK2 activity was elevated and DNA synthesis continued, a phenomena that correlated with increased E2F1 protein levels and consequently enhanced expression of cyclin E. These results reveal a differential impact of various classes of anticancer chemotherapeutics on the Cdc25A-degradation pathway, and indicate that the kinetics of checkpoint induction, and the relative balance of key components within the DNA damage response network may dictate whether the treated cells arrest their cell cycle progression.

Antibiotics, Antineoplastic↗
PubMed2003

14-3-3 sigma positively regulates p53 and suppresses tumor growth.

The 14-3-3 sigma (sigma) protein, a negative regulator of the cell cycle, is a human mammary epithelium-specific marker that is downregulated in transformed mammary carcinoma cells. It has also been identified as a p53-inducible gene product involved in cell cycle checkpoint control after DNA damage. Although 14-3-3 sigma is linked to p53-regulated cell cycle checkpoint control, detailed mechanisms of how cell cycle regulation occurs remain unclear. Decreased expression of 14-3-3 sigma was recently reported in several types of carcinomas, further suggesting that the negative regulatory role of 14-3-3 sigma in the cell cycle is compromised during tumorigenesis. However, this possible tumor-suppressive role of 14-3-3 sigma has not yet been characterized. Here, we studied the link between 14-3-3 sigma activities and p53 regulation. We found that 14-3-3 sigma interacted with p53 in response to the DNA-damaging agent adriamycin. Importantly, 14-3-3 sigma expression led to stabilized expression of p53. In studying the molecular mechanism of this increased stabilization of p53, we found that 14-3-3 sigma antagonized the biological functions of Mdm2 by blocking Mdm2-mediated p53 ubiquitination and nuclear export. In addition, we found that 14-3-3 sigma facilitated the oligomerization of p53 and enhanced p53's transcriptional activity. As a target gene of p53, 14-3-3 sigma appears to have a positive feedback effect on p53 activity. Significantly, we also showed that overexpression of 14-3-3 sigma inhibited oncogene-activated tumorigenicity in a tetracycline-regulated 14-3-3 sigma system. These results defined an important p53 regulatory loop and suggested that 14-3-3 sigma expression can be considered for therapeutic intervention in cancers.

14-3-3 Proteins↗
PubMed1998

Mathematical model of the fission yeast cell cycle with checkpoint controls at the G1/S, G2/M and metaphase/anaphase transitions.

All events of the fission yeast cell cycle can be orchestrated by fluctuations of a single cyclin-dependent protein kinase, the Cdc13/Cdc2 heterodimer. The G1/S transition is controlled by interactions of Cdc13/Cdc2 and its stoichiometric inhibitor, Rum1. The G2/M transition is regulated by a kinase-phosphatase pair, Wee1 and Cdc25, which determine the phosphorylation state of the Tyr-15 residue of Cdc2. The meta/anaphase transition is controlled by interactions between Cdc13/Cdc2 and the anaphase promoting complex, which labels Cdc13 subunits for proteolysis. We construct a mathematical model of fission yeast growth and division that encompasses all three crucial checkpoint controls. By numerical simulations we show that the model is consistent with a broad selection of cell cycle mutants, and we predict the phenotypes of several multiple-mutant strains that have not yet been constructed.

Anaphase↗
PubMed2002

Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest.

In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.

Amino Acid Sequence↗
PubMed2001

Checking out the G(2)/M transition.

Tight regulation of cell cycle progression is essential for the maintenance of genomic integrity. The orderly progression from one cell cycle phase to the other is mediated by timed activation of distinct cyclin/cdk complexes. For example, onset of mitosis is regulated by the activation of cyclin B/cdc2 and this event is controlled by several cell cycle checkpoints. Such checkpoints ensure that chromosome segregation does not occur in the case of unreplicated or damaged DNA, or misaligned chromosomes. Recently, new insights into the targets of the DNA damage checkpoint help to unravel more of the complex mechanisms of cell cycle checkpoints. This review focuses on the factors controlling the transition from G(2) phase to mitosis. Also, the pathways contributing to the DNA damage checkpoints in these phases of the cell cycle will be discussed.

Animals↗
PubMed1994

Role of retinoblastoma gene product in p53-mediated DNA damage response.

The cellular response to DNA-damaging agents involves the activation of cell cycle checkpoints. Checkpoints provide a transient delay in cell cycle progression, presumably to allow time for the cell to repair the damage. A most important checkpoint, active in the G1 phase of the cell cycle, is mediated by the p53 tumor suppressor gene product. To investigate the role of downstream components of the cell cycle machinery in p53-mediated G1 arrest, the possible involvement of the RB gene product was examined. Rb and p53 proteins were studied by immunoprecipitation and Western blotting experiments in the presence and absence of DNA-damaging treatment. The phosphorylation status of Rb was altered following DNA damage in p53 wild-type cell lines, but was not altered in p53 mutant cell lines, nor in cell lines where p53 function was abrogated by viral gene products. These findings indicate that Rb probably plays a role in the activation of the p53-mediated checkpoint.

Cell Cycle↗
PubMed2004

C/EBPalpha is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes.

The basic leucine zipper transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha), is abundantly expressed in keratinocytes of the skin; however, its function in skin is poorly characterized. UVB radiation is responsible for the majority of human skin cancers. In response to UVB-induced DNA damage, keratinocytes activate cell cycle checkpoints that arrest cell cycle progression and prevent replication of damaged DNA, allowing time for DNA repair. We report here that UVB radiation is a potent inducer of C/EBPalpha in human and mouse keratinocytes, as well as in mouse skin in vivo. UVB irradiation of keratinocytes resulted in the transcriptional up-regulation of C/EBPalpha mRNA, producing a >70-fold increase in C/EBPalpha protein levels. N-Methyl-N'-nitro-N-nitrosoguanidine, etoposide, and bleomycin also induced C/EBPalpha. UVB-induced C/EBPalpha was accompanied by an increase in p53 protein and caffeine, an inhibitor of ataxia-telangiectasia-mutated kinase, and ataxia-telangiectasia-mutated and Rad3-related kinase inhibited UVB-induced increases in both C/EBPalpha and p53. UVB irradiation of p53-null or mutant p53-containing keratinocytes failed to induce C/EBPalpha. UVB irradiation of C/EBPalpha knockdown keratinocytes displayed a greatly diminished DNA damage G(1) checkpoint, and this was associated with increased sensitivity to UVB-induced apoptosis. Our results uncover a novel role for C/EBPalpha as a p53-regulated DNA damage-inducible gene that has a critical function in the DNA damage G(1) checkpoint response in keratinocytes.

Animals↗
PubMed1996

Genes involved in cell cycle G1 checkpoint control are frequently mutated in human melanoma metastases.

A common characteristic of cancer cells is unrestrained cell division. This may be caused by mutational changes in genes coding for components of cell cycle-controlling networks. Alterations in genes involved in G1 checkpoint control have been registered in many human tumours, and investigations from several laboratories show that such alterations, taken together, are the most frequent changes detected in cancer cells. The present paper describes mutational analysis by polymerase chain reaction-single-strand conformation polymorphism (PCR/SSCP) and nucleotide sequence analysis of the genes coding for the p15, p53 and N-ras proteins in 26 metastases from 25 melanoma patients. The registered mutation frequencies add together with previously registered mutations in p16 in the same patient samples to a substantial total frequency of 44% of patients with mutation in at least one of the investigated genes. These results show the occurrence of heterogeneous defects among components of the cell cycle controlling machinery in a human melanoma tumour sample collection and demonstrate that the total frequency of detected alterations increases with the number of cell cycle controlling genes included in the screening panel.

Carrier Proteins↗