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Results for “Cell Cycle Checkpoints”

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PubMed1998

cDNA cloning and gene mapping of human homologs for Schizosaccharomyces pombe rad17, rad1, and hus1 and cloning of homologs from mouse, Caenorhabditis elegans, and Drosophila melanogaster.

Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of human homologs of yeast DNA repair/cell cycle checkpoint genes should yield candidates for human tumor suppressor genes as well as identifying potential targets for cancer therapy. The Schizosaccharomyces pombe genes rad17, rad1, and hus1 have been identified as playing roles in DNA repair and cell cycle checkpoint control pathways. We have cloned the cDNA for the human homolog of S. pombe rad17, RAD17, which localizes to chromosomal location 5q13 by fluorescence in situ hybridization and radiation hybrid mapping; the cDNA for the human homolog of S. pombe rad1, RAD1, which maps to 5p14-p13.2; and the cDNA for the human homolog of S. pombe hus1, HUS1, which maps to 7p13-p12. The human gene loci have previously been identified as regions containing tumor suppressor genes. In addition, we report the cloning of the cDNAs for genes related to S. pombe rad17, rad9, rad1, and hus1 from mouse, Caenorhabditis elegans, and Drosophila melanogaster. These include Rad17 and Rad9 from D. melanogaster, hpr-17 and hpr-1 from C. elegans, and RAD1 and HUS1 from mouse. The identification of homologs of the S. pombe rad checkpoint genes from mammals, arthropods, and nematodes indicates that this cell cycle checkpoint pathway is conserved throughout eukaryotes.

Amino Acid Sequence↗

PubMed1996

Expression of HPV16 E6 or E7 increases integration of foreign DNA.

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.

Cinnamates↗

PubMed1998

Splitting the ATM: distinct repair and checkpoint defects in ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is an autosomal recessive human disorder that, because of its multisystem nature, is of interest to scientists and clinicians from many disciplines. A-T patients have defects in the neurological and immune systems, telangiectasia in the eyes and face, and are, in addition, cancer-prone and radiation-sensitive. A-T cell lines have a range of diverse phenotypes including sensitivity to ionizing radiation and defects in cell-cycle checkpoint control. The ATM protein is a member of the PI 3-kinase-like superfamily, and it has been widely accepted that A-T cells represent mammalian cell-cycle checkpoint mutants and that the radiation sensitivity is a consequence of this defect. However, several lines of evidence suggest that A-T cells have distinct repair and checkpoint defects. A-T cells therefore appear to harbour dual checkpoint/repair defects. Here, we review the evidence supporting this contention and consider its implications for an analysis of the A-T phenotype.

Animals↗

PubMed1998

Molecular mechanisms of DNA double strand break repair.

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.

Animals↗

PubMed1996

Molecular and cellular responses to DNA damage in a murine pituitary adenoma cell line.

Loss of cell cycle control and the inability of the cell to repair DNA at cell cycle checkpoints results in the propagation of genetic lesions which ultimately leads to cancer. To further our understanding of these pathways in pituitary tumorigenesis, we have investigated the effects of DNA damage by gamma radiation in a murine pituitary adenoma (AtT20) cell line with attention to cell cycle checkpoint responses, the induction of apoptosis, and the expression of known regulators of these processes. Irradiated cells exhibited characteristic morphologic changes of apoptosis beginning at 24 h, which included cell shrinkage, chromatin condensation, and cytoplasmic vacuolization, yet the ability to exclude trypan blue was retained for several days. DNA fragmentation could be demonstrated by ethidium bromide staining beginning at 24 h post-irradiation. By propidium iodide staining and flow cytometry, irradiated cells demonstrated G1 and G2 arrest at 24 h, followed at 48 h by a shift to a sub-G1 position of the apoptotic cell population. The G1 arrest coincided with an induction of p53 protein by Western blot analysis which peaked at 4 h post-radiation and persisted beyond 48 h. Expression of c-myc in irradiated cells was found to progressively decrease at 12, 24, and 48 h. Basal expression of the bcl-2 gene in AtT20 cells was found to be 15-fold higher than in normal mouse pituitary by RNase protection assay. Bcl-2 mRNA and protein levels, however, remained unchanged at 24 and 48 h following gamma-irradiation, suggesting that apoptosis occurs independently of bcl-2 gene expression in these cells following this stimulus, as reported in other cell types. We conclude that AtT20 cells undergo G1 and G2 arrest following DNA damage and that a significant proportion of cells then undergo apoptosis. The G1 arrest at 24 h is concurrent with a strong induction of p53 protein, while c-myc expression progressively diminishes. Bcl-2 is highly expressed in this cell line. The absence of variation in bcl-2 expression during apoptosis could be related to its high basal level in these cells.

Adenoma↗

PubMed1998

A human homologue of the Schizosaccharomyces pombe rad1+ checkpoint gene encodes an exonuclease.

In the fission yeast Schizosaccharomyces pombe the rad1(+) gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human homologue of the S. pombe rad1(+) gene, designated Hrad1, as well as a mouse homologue: Mrad1. Two Hrad1 alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B. Hrad1A has 60% identity to the S. pombe rad1+ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad1 is located on chromosome 5p13. 2-13.3. This region is subject to loss of heterozygosity in several human cancers. Hrad1 also shares homology with the Saccharomyces cerevisiae RAD17 and Ustilago maydis REC1 proteins. REC1 has previously been characterized as a 3' --> 5' exonuclease with a C-terminal domain essential for cell cycle checkpoint function. We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3' --> 5' exonuclease activity, whereas HisHrad1B lacks such activity. The biological functions of the two proteins remain to be determined.

Alternative Splicing↗

PubMed1998

The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response.

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity. We have isolated the gene encoding p95, a member of the hMre11/hRad50 double-strand break repair complex. The p95 gene mapped to 8q21.3, the region that contains the NBS locus, and p95 was absent from NBS cells established from NBS patients. p95 deficiency in these cells completely abrogates the formation of hMre11/hRad50 ionizing radiation-induced foci. Comparison of the p95 cDNA to the NBS1 cDNA indicated that the p95 gene and NBS1 are identical. The implication of hMre11/hRad50/p95 protein complex in NBS reveals a direct molecular link between DSB repair and cell cycle checkpoint functions.

Acid Anhydride Hydrolases↗

PubMed1999

p21(cip-1/waf-1) deficiency causes deformed nuclear architecture, centriole overduplication, polyploidy, and relaxed microtubule damage checkpoints in human hematopoietic cells.

A recent hypothesis suggests that tumor-specific killing by radiation and chemotherapy agents is due to defects or loss of cell cycle checkpoints. An important component of some checkpoints is p53-dependent induction of p21(cip-1/waf-1). Both p53 and p21 have been shown to be required for microtubule damage checkpoints in mitosis and in G1 phase of the cell cycle and they thus help to maintain genetic stability. We present here evidence that p21(cip-1/waf-1) deficiency relaxes the G1 phase microtubule checkpoint that is activated by microtubule damage induced with nocodazole. Reduced p21(cip-1/waf-1) expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and thus helps to maintain genomic integrity. The inability to efficiently upregulate p21(cip-1/waf-1) in p21(cip-1/waf-1) antisense-expressing cells in response to microtubule damage could uncouple the centrosome cycle from the DNA cycle and lead to nuclear abnormalicies and polyploidy. A centrosome duplication checkpoint could be a new target for novel chemotherapy strategies.

Cell Cycle↗

PubMed1997

Transcriptional analysis of the Caulobacter 4.5 S RNA ffs gene and the physiological basis of an ffs mutant with a Ts phenotype.

A temperature-sensitive (ts) mutation in the ffs gene, encoding 4.5 S RNA, gives rise to cell division and DNA replication defects in Caulobacter crescentus. The ffs gene is transcribed throughout the cell-cycle and is transcribed at similar rates in mutant (ffs36) and wild-type strains, but in the mutant the 4.5 S RNA is unstable leading to lower 4.5 S RNA levels. The ffs36 phenotype results from a single base change in one of the non-conserved stems of the mature RNA, and is completely rescued by a compensating mutation in the opposite strand, providing confirmation of the predicted secondary structure of the 4.5 S RNA. The Caulobacter ffs gene was shown to be functionally comparable to the Escherichia coli ffs gene by complementation. Comparison of the ffs36 strain to a ts secA strain of Caulobacter, also having cell-cycle and DNA replication phenotypes, showed that both exhibit a permanent induction of a heat shock response at the restrictive temperature. To explain the phenotype of both the secA and ffs36 strains, we propose that a cell-cycle checkpoint prevents further progression through the cell-cycle in response to increased intracellular levels of heat shock and misfolded proteins.

Amino Acid Sequence↗

PubMed1998

Manipulation of metallothionein expression in the regenerating rat liver using antisense oligonucleotides.

Metallothioneins (MTs) are low molecular weight, zinc-binding proteins that by activating zinc metalloenzymes participate in the regulation of growth and development. The present study was designed to examine the roles of MTs in cell proliferation using an in vivo model of liver regeneration following partial hepatectomy (PH) in rats. The levels of MT-I and MT-II were studied with respect to regulation of proliferative potential, cell cycle checkpoint activity, and oxidative stress in the rat PH model. We synthesized a 17-mer antisense phosphorothioate oligodeoxynucleotide (S-ODN), named aMT, complimentary to the start site of the MT-I mRNA sequence and an appropriate control. Both S-ODNs were administered intraperitoneally at the dose of 5 mg/kg following 70% PH. MT became induced 57.4 +/- 9.8-fold following PH and the said effect became attenuated dramatically following administration of aMT. In addition, PH rats treated with aMT exhibited decreased rate of liver regeneration as measured by expression of proliferating cell nuclear antigen and elevated cell cycle checkpoint activity as determined by expression of p53. The results of these studies suggest that MT isoforms with their high thiol contents do play an important role in cellular functions and especially during stressful states induced by a broad range of mediators generating free radicals.

Animals↗

PubMed1997

Telomeres, senescence and cellular radiation response.

Telomeres shield the ends of chromosomes from degradation and end-to-end fusions. They shorten at each cell division and when they reach a critically short length, cells arrest in the G1 phase of the cell cycle and undergo senescence. This effectively limits the proliferative potential of cells. Senescence functions as a tumour suppressor mechanism and appears to contribute to the process of ageing. If senescence is circumvented by tumour viruses, proliferation is re-initiated until cells enter crisis. Activation of telomerase prevents telomere attrition and cells become immortal. Cellular response to ionizing radiation involves induction of cell cycle checkpoint arrests and programmed cell death. Because radiation produces double strand breaks in DNA, which cause telomere-less chromosome ends, radiation response appears to be the result of inappropriate induction of cellular senescence mechanisms.

Animals↗

PubMed1998

Induction of cell cycle arrest by the endogenous product of lipid peroxidation, malondialdehyde.

We have investigated the effect of the endogenous genotoxin malondialdehyde (MDA) on cell cycle kinetics and the expression and biochemical activity of several cell cycle regulatory proteins. MDA treatment of two human cell lines (RKO and H1299) resulted in a 3- to 6-fold elevation in the levels of the major detectable MDA-DNA adduct, M1G-dR. The increase in M1G-dR was accompanied by irreversible cell cycle arrest, elevation in p53 and p21 protein levels, and inhibition of cyclin E- and cyclin B-associated kinase activities. The decrease in cyclin E- and cyclin B-dependent kinase activities was caused by increased p21 and decreased cdc2 levels, respectively. Comparable levels of p21 induction were observed in RKO (wild-type p53) and H1299 (p53-null) cells. Thus, MDA was able to engage cell cycle checkpoint function in human cell lines when used at concentrations that produce M1G-dR levels of the same magnitude found in human tissues.

Antibiotics, Antineoplastic↗

PubMed1993

Delays in anaphase initiation occur in individual nuclei of the syncytial Drosophila embryo.

The syncytial divisions of the Drosophila melanogaster embryo lack some of the well established cell-cycle checkpoints. It has been suggested that without these checkpoints the divisions would display a reduced fidelity. To test this idea, we examined division error frequencies in individuals bearing an abnormally long and rearranged second chromosome, designated C(2)EN. Relative to a normal chromosome, this chromosome imposes additional structural demands on the mitotic apparatus in both the early syncytial embryonic divisions and the later somatic divisions. We demonstrate that the C(2)EN chromosome does not increase the error frequency of the late larva neuroblast divisions. However, in the syncytial embryonic nuclear divisions, the C(2)EN chromosome produces a 10-fold increase in division errors relative to embryos with a normal karyotype. During late anaphase of the neuroblast divisions, the sister C(2)EN chromosomes cleanly separate from one another. In contrast, during late anaphase of the syncytial divisions in C(2)EN-bearing nuclei, large amounts of chromatin often lag on the metaphase plate. Live analysis of C(2)EN-bearing embryos demonstrates that individual nuclei in the syncytial population of dividing nuclei often delay in their initiation of anaphase. These delays frequently lead to division errors. Eventually the products of the nuclei delayed in anaphase sink inward and are removed from the dividing population of syncytial nuclei. These results suggest that the Drosophila embryo may be equipped with mechanisms that monitor the fidelity of the syncytial nuclear divisions. Unlike checkpoints that rely on cell cycle delays to identify and correct division errors, these embryonic mechanisms rely on cell cycle delays to identify and discard the products of division errors.

Anaphase↗

PubMed1996

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion.

Alkylating Agents↗

PubMedDate not supplied

[Molecular mechanisms of senescence in cell culture].

Clones of non-transformed human cells are mortal, that is, the cells divide only a limited number of times before they approach a state of proliferative senescence. This state has long been regarded as a cellular model of organismal aging or as aging in vitro because of its close relationship to the aging process of the donor of the cells. The underlying molecular mechanisms of this particular aging process have only been recently understood and are reviewed in the present paper. Cell division is accompanied by progressive telomere shortening, which is due both to properties of the replicative apparatus (the "end-replication problem") and to oxidative damage to telomere DNA under conventional cell culture conditions. If shortening of telomeres reaches a certain critical level, it is recognized as DNA damage by the cell's "guardian of the genome", the tumor suppressor p53. Stabilization of p53 activates the well characterized cell cycle checkpoint at the G1/S phase boundary and blocks the cell cycle irreversibly. Two recent results prove that telomere shortening is in fact the trigger of the checkpoint control in cellular senescence: First, acceleration of telomere shortening by increased oxidative stress results in accelerated proliferative senescence. Second, stabilization of telomere length, typically by activation of telomere, appears to be a necessary prerequisite for the immortalization of cells. Proliferative senescence, therefore, should be understood as an important means to counteract genetic instability and cancer.

Aged↗

PubMed1998

Control of Swe1p degradation by the morphogenesis checkpoint.

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.

Base Sequence↗

PubMed2026

WEE1 kinase in cancer: Molecular mechanisms and inhibitor insights.

WEE1 kinase is a main regulator of the G2/M cell cycle checkpoint. It plays an important role in maintaining genomic stability by inhibiting CDK1 through a phosphorylation process at Tyr15. WEE1 is found to be overexpressed in several cancers and also act as a protective mechanism that allows cancer cells to repair DNA damage and survive under replicative stress. So, pharmacological inhibition of WEE1 has emerged as a promising therapeutic strategy. Many conventional chemotherapeutic agents act by inducing DNA damage, so it enables the activation of WEE1 in cancer cells to arrest the cell cycle and repair this damage by preventing cell death. Inhibition of WEE1 disrupts this protective checkpoint, which ultimately leads to mitotic catastrophe. Therefore, targeting WEE1 represents a promising and rational therapeutic approach, mainly in tumors with TP53 mutations. We have comprehensively discussed the structural features of WEE1, its regulation in DNA damage response, epigenetic control, and its role in cancer progression. We have also summarized the clinical development of major WEE1 inhibitors such as adavosertib, azenosertib (ZN-c3), and Debio 0123. Moreover, recently synthesized small-molecule inhibitors are also discussed with special focus on structure-activity relationship (SAR) insights, dual-target inhibitors, and PROTACs and molecular glue-based degraders. Two compounds, 8 and 11, were found to be the most potent WEE1 inhibitors with excellent enzymatic inhibition. This explains the importance of rational scaffold optimization and electron-withdrawing group insertion for enhanced activity. Overall, this review serves as a valuable reference for medicinal chemists in the development of next-generation WEE1 inhibitors. See also the graphical abstract(Fig. 1).

WEE1 kinase↗

PubMed1996

Molecular biology of head and neck tumorigenesis: the role of p53 expression and genetic instability.

Head and neck cancers progress as multistep tumorigenesis through accumulation of genetic instability. The p53 tumor-suppressor gene encodes a cell-cycle checkpoint protein that functions in the G1 phase of the cell cycle. When DNA damage is incurred, p53 transactivates a number of downstream genes whose products, with diverse biologic activities, contribute to the cellular response to DNA damage. One major p53-mediated function in response to DNA damage is to induce the G1 cell-cycle arrest, or delay, which probably allows time for the cell to repair DNA damage prior to S-phase entry. In cell lacking of p53 function, a condition of genetic instability results from checkpoint loss (Fig. 4.). These events occur early from ANL to SCC and increase gradually through multistep tumorigenesis. Due to the potential role of p53 expression and genetic instability, both might be useful biomarkers in assessing the risk of head and neck tumorigenesis.

Animals↗