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PubMed2006

Cellular response to etoposide treatment.

Etoposide is a potent anti-tumor drug that belongs to the class of topoisomerase poisons. Although its molecular target, i.e. DNA topoisomerase II, has been identified more than 20 years ago, the cellular response to etoposide is still poorly understood. The cytotoxicity of the drug stems from its ability to stabilize a covalent complex between DNA topoisomerase II and DNA that results in a high level of DNA damage. Here, we review the present knowledge about the strategy used by the cells to deal with the etoposide-induced DNA damage. New and unanticipated effects of topoisomerase II poisoning on cell metabolism are recently emerging, among which the ability to activate cell cycle checkpoint pathways and to affect gene expression at different levels, including chromatin remodeling and alternative splicing of gene transcripts. The elucidation of the effects of etoposide on cell metabolism will increase our ability to exploit this drug in cancer therapy and will expand our comprehension of the cancerous cell.

Animals↗
PubMed1999

The human G2 checkpoint control protein hRAD9 is a nuclear phosphoprotein that forms complexes with hRAD1 and hHUS1.

Eukaryotic cells actively block entry into mitosis in the presence of DNA damage or incompletely replicated DNA. This response is mediated by signal transduction cascades called cell cycle checkpoints. We show here that the human checkpoint control protein hRAD9 physically associates with two other checkpoint control proteins, hRAD1 and hHUS1. Furthermore, hRAD1 and hHUS1 themselves interact, analogously to their fission yeast homologues Rad1 and Hus1. We also show that hRAD9 is present in multiple phosphorylation forms in vivo. These phosphorylated forms are present in tissue culture cells that have not been exposed to exogenous sources of DNA damage, but it remains possible that endogenous damage or naturally occurring replication intermediates cause the observed phosphorylation. Finally, we show that hRAD9 is a nuclear protein, indicating that in this signal transduction pathway, hRAD9 is physically proximal to the upstream (DNA damage) signal rather than to the downstream, cytoplasmic, cell cycle machinery.

Animals↗
PubMed2004

A genomic approach reveals a novel mitotic pathway in papillomavirus carcinogenesis.

More than 90% of cervical carcinomas are associated with human papillomavirus (HPV) infection. The two viral oncogenes E6 and E7 play a major role in transforming the cells by disrupting p53- and pRb-dependent cell cycle checkpoints. A hallmark of HPV-associated cervical carcinoma is loss of the expression of the viral E2 protein, often by disruption of E2-encoding gene. We showed previously that reintroduction of E2 in HPV18-associated cervical carcinoma cells induces cell cycle arrest in G(1) because of the transcriptional repression of the viral oncogenes E6 and E7 and concomitant reactivation of the p53 and pRb pathways. Here we describe global gene profiling of HeLa cells expressing different HPV18 E2 mutants to study the effects of repression of the viral oncogenes. We identified 128 genes transcriptionally regulated by the viral oncogenes in cervical carcinoma. Surprisingly, E2 repressed a subset of E2F-regulated mitotic genes in an E6/E7-dependent pathway. This was corroborated by the observation that E2 delayed mitotic progression, suggesting the involvement of a mitotic pathway in HPV carcinogenesis. These mitotic genes constitute an as yet unrecognized set of genes, which were also found deregulated in other HPV-associated cervical carcinoma cell lines and therefore represent new targets for both diagnosis and therapeutic approaches in cervical cancer.

Adenoviridae↗
PubMed2002

c-Abl tyrosine kinase regulates the human Rad9 checkpoint protein in response to DNA damage.

The ubiquitously expressed c-Abl tyrosine kinase is activated in the apoptotic response of cells to DNA damage. The mechanisms by which c-Abl signals the induction of apoptosis are not understood. Here we show that c-Abl binds constitutively to the mammalian homolog of the Schizosaccharomyces pombe Rad9 cell cycle checkpoint protein. The SH3 domain of c-Abl interacts directly with the C-terminal region of Rad9. c-Abl phosphorylates the Rad9 Bcl-2 homology 3 domain (Tyr-28) in vitro and in cells exposed to DNA-damaging agents. The results also demonstrate that c-Abl-mediated phosphorylation of Rad9 induces binding of Rad9 to the antiapototic Bcl-x(L) protein. The regulation of Rad9 by c-Abl in the DNA damage response is further supported by the demonstration that the interaction between c-Abl and Rad9 contributes to DNA damage-induced apoptosis. These findings indicate that Rad9 is regulated by a c-Abl-dependent mechanism in the apoptotic response to genotoxic stress.

Apoptosis↗
PubMed2002

The human papillomavirus type 16 E6 and E7 oncoproteins independently induce numerical and structural chromosome instability.

The development of genomic instability is a hallmark of high-risk human papillomavirus (HPV) associated cervical carcinogenesis. We have previously shown that the HPV-16 E7 oncoprotein rapidly subverts mitotic fidelity by inducing abnormal centrosome numbers and multipolar mitotic spindles. Here we report that expression of HPV-16 E6 and E7 independently results in various mitotic abnormalities. HPV-16 E6 and E7 were each associated with unaligned or lagging chromosomal material, indicating relaxation of spindle checkpoint control. Moreover, by overwhelming checkpoint control mechanisms that may prevent cells with multiple spindle poles to enter anaphase, expression of HPV-16 E6 and E7 leads to a small but significant number of cells with altered polarity at later stages of the cell division process. In addition to changes that have the potential to give rise to numerical chromosome imbalances, we discovered that expression of HPV-16 E7 could trigger anaphase bridge formation to an extent similar to that of high-risk HPV E6. Anaphase bridges typically develop after chromosomal breaks and alterations of chromosomal structure. Further investigation of mechanisms by which HPV-16 E6 and E7 contribute to the destabilization of the host cell genome revealed that both high-risk HPV oncoproteins induce DNA damage. Moreover, expression of HPV-16 E7 was associated with an increased number of cells exhibiting nuclear foci of phosphorylated histone H2AX as well as activation of cell cycle checkpoints triggered by DNA repair. Our results therefore suggest that HPV oncoproteins are a source for both numerical and structural chromosome instability during HPV-associated carcinogenesis.

Anaphase↗
PubMed2004

Telomeres are double-strand DNA breaks hidden from DNA damage responses.

A network of ATM/ATR-mediated events regulates cell cycle checkpoints and genomic integrity and contributes to the processing of DNA double-strand breaks in both genomic DNA and at telomeres. In yeast and in human cells, investigators, including, and Herbig et al., published in this issue of Molecular Cell, are beginning to decipher the signaling pathways involved at the telomeres.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

Protection against chemotherapy-induced cytotoxicity by cyclin-dependent kinase inhibitors (CKI) in CKI-responsive cells compared with CKI-unresponsive cells.

Inactivation of the retinoblastoma (Rb) protein caused by gene mutation, association with oncoproteins from small DNA viruses, mutational inactivation of p16(Ink4a), or overexpression of cyclin D is a common feature of many human cancer cells and is causally associated with the aberrant proliferation control of cancer cells; whereas normal cells maintain an integrated cell cycle machinery and are subject to cell cycle checkpoint control by cyclin-dependent kinase (CDK) inhibitors (CKIs). To determine whether this difference can be translated into a therapeutic advantage to protect normal cells from adverse cytotoxicity caused by chemotherapy, we established cell model systems for ecdysone-inducible expression of p16(Ink4a), p21(Waf1), and p27(Kip1) in one CKI-responsive cell line (A431 human vulvar epidermoid carcinoma cells with functional Rb) and one CKI-unresponsive cell line (SiHa human cervical cancer cells with nonfunctional Rb). Expression of p16(Ink4a), p21(Waf1), or p27(Kip1) in both SiHa and A431 cells strongly inhibited CDK2 activity, indicating functional expression of the CDK inhibitors in both cell lines. However, only in A431 cells did expression of p16(Ink4a), p21(Waf1), or p27(Kip1) cause Rb dephosphorylation, arrest cell cycle traversal, and potently inhibit cell proliferation. Induction of p16(Ink4a), p21(Waf1), or p27(Kip1) in SiHa cells failed to cause Rb dephosphorylation or to arrest cell cycle traversal, and such induction only minimally inhibited cell proliferation. We then compared the chemosensitivity of clones derived from these two cell lines when the CKIs were and were not induced. Induction of p16(Ink4a), p21(Waf1), or p27(Kip1) conferred strong resistance to paclitaxel- or cisplatin-mediated cytotoxicity on the CKI-responsive A431 cells but not on the CKI-unresponsive SiHa cells. Our results support a novel chemotherapy strategy for treating patients with Rb pathway-impaired cancers by concurrent administration of chemotherapy with CKIs as chemoprotective agents for normal cells.

Cell Cycle Proteins↗
PubMed2004

Interactions between BRCT repeats and phosphoproteins: tangled up in two.

The C-terminal region of the breast-cancer-associated protein BRCA1 contains a pair of tandem BRCA1 C-terminal (BRCT) repeats that are essential for the tumour suppressor function of the protein. Similar repeat sequences have been identified in many proteins that seem to mediate cellular mechanisms for dealing with DNA damage. The BRCT domain in BRCA1 has been recently shown to constitute a module for recognizing phosphorylated (phospho-) peptides, with a recognition groove that spans both BRCT repeats. The fact that many other BRCT-containing proteins have phospho-peptide binding activity suggests that BRCT repeats might mediate phosphorylation-dependent protein-protein interactions in processes that are central to cell-cycle checkpoint and DNA repair functions.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

The contribution of homologous recombination in preserving genome integrity in mammalian cells.

Although it is clear that mammalian somatic cells possess the enzymatic machinery to perform homologous recombination of DNA molecules, the importance of this process in mitigating DNA damage has been uncertain. An initial genetic framework for studying homologous recombinational repair (HRR) has come from identifying relevant genes by homology or by their ability to correct mutants whose phenotypes are suggestive of recombinational defects. While yeast has been an invaluable guide, higher eukaryotes diverge in the details and complexity of HRR. For eliminating DSBs, HRR and end-joining pathways share the burden, with HRR contributing critically during S and G2 phases. It is likely that the removal of interstrand cross-links is absolutely dependent on efficient HRR, as suggested by the extraordinary sensitivity of the ercc1, xpf/ercc4, xrcc2, and xrcc3 mutants to cross-linking chemicals. Similarly, chromosome stability in untreated cells requires intact HRR, which may eliminate DSBs arising during DNA replication and thereby prevent chromosome aberrations. Complex regulation of HRR by cell cycle checkpoint and surveillance functions is suggested not only by direct interactions between human Rad51 and p53, c-Abl, and BRCA2, but also by very high recombination rates in p53-deficient cells.

Amino Acid Sequence↗
PubMed2001

Cellular responses to ionizing radiation damage.

PURPOSE: The purpose of this report is to provide current perspectives on studies of DNA damage and cell cycle response after ionizing radiation, and their applications in radiation oncology. METHODS AND MATERIALS: Presentations at the Seventh Annual Radiation Oncology Workshop, held at the International Festival Institute at Round Top, TX, were summarized. RESULTS: Eighteen speakers presented their current work covering a wide range of studies on cellular responses to ionizing radiation. These presentations and discussions form the framework of our report. CONCLUSION: In response to ionizing radiation, cells immediately activate a series of biochemical pathways that promote cell survival while maintaining genetic integrity. The main cellular defense system against ionizing radiation exposure is composed of two distinct types of biochemical pathways, that is, the DNA damage cell cycle checkpoint pathways and the DNA repair pathways. The DNA damage checkpoint pathways are activated directly by DNA damage, while the repair pathways are constitutively active and are likely modulated by checkpoint signals. Discussions here emphasize that the ATM protein is a central component of the ionizing radiation-responsive pyramid and is essential for activating divergent molecular responses that involve transcriptional regulation, cell cycle arrest, and modulation of DNA repair. The relationship between homologous recombinational repair and nonhomologous end joining of double-strand breaks is also discussed.

Animals↗
PubMed2004

Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number.

Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number.

Amino Acid Sequence↗
PubMed2000

Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype.

Immortal epithelial cell lines were previously established after transduction of the HPV16-E6E7 genes into primary cultures of normal pancreatic duct epithelial cells. Single clones were isolated that demonstrated near normal genotype and phenotype. The proliferation of HPDE6-E6E7c7 and c11 cells is anchorage-dependent, and they were nontumorigenic in SCID mice. The cell lines demonstrated many phenotypes of normal pancreatic duct epithelium, including mRNA expression of carbonic anhydrase II, MUC-1, and cytokeratins 7, 8, 18, and 19. These cells have normal Ki-ras, p53, c-myc, and p16(INK4A) genotypes. Cytogenetic studies demonstrated losses of 3p, 10p12, and 13q14, the latter included the Rb1 gene. The wild-type p53 protein was detectable at very low levels consistent with the presence of E6 gene product, and the lack of functional p53 pathway was confirmed by the inability for gamma-irradiation to up-regulate p53 and p21waf1/cip1 protein. The p110/Rb protein level was also not detectable consistent with the expression of E7 protein and haploid loss of Rb1 gene. Despite this, the proliferation of both c7 and c11 cells were markedly inhibited by transforming growth factor-beta1. This was associated with up-regulation of p21cip1/waf1 but not p27kip1. Further studies showed that p130/Rb2 and cyclin D3 were expressed, suggesting that p130/Rb2 may have partially assumed the maintenance of G(1) cell cycle checkpoint regulation. These results indicate that except for the loss of p53 functional pathway, the two clones of HPDE6-E6E7 cells demonstrated a near normal genotype and phenotype of pancreatic duct epithelial cells. These cell lines will be useful for future studies on the molecular basis of pancreatic duct cell carcinogenesis and islet cell differentiation.

Animals↗
PubMed2003

G2M arrest, blocked apoptosis, and low growth fraction may explain indolent behavior of head and neck paragangliomas.

Head and neck paragangliomas are characterized by unusually slow growth and a strong hereditary component, which is associated with inactivating mutations in subunits of complex II of the mitochondrial respiratory chain. It is unclear how mutations induce tumorigenesis and lead to the indolent clinical behavior that often plays a prominent role in treatment strategies. To better understand the natural course of the tumors, we studied a number of growth-related parameters in 42 hereditary and sporadic paragangliomas. Computerized image analysis showed that the fraction of Ki-67-positive cells was generally below 1%, in accordance with the slow growth. Weak or negative immunohistochemical staining indicated wild-type TP53 status, whereas p-21(waf) expression was heterogeneous. Most tumors showed strong expression of Bcl-x(L), and no apoptotic cells could be detected with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Flow cytometry showed abnormal DNA content profiles in 52% of the tumors, including overt aneuploidy as well as G(2)/M arrest or tetraploidization. These results fit into a model in which a stress-activated cell cycle checkpoint at the G(2) to M transition and inhibition of apoptosis permit the expansion of only a minor fraction of cycling cells with high likelihood of polyploidization.

Adult↗
PubMed2005

Caffeine sensitizes nondividing human fibroblasts to x rays by inducing a high frequency of misrepair.

Caffeine sensitizes cells to ionizing radiation, and this effect is believed to be associated with the disruption of DNA damage-responsive cell cycle checkpoints, which is controlled by ATM. Recent studies suggest that misrejoining of DSBs is one of the underlying mechanisms of AT cell hyper-radiosensitivity. In this study, we investigated the effects of caffeine and radiation on nongrowing G0 normal human fibroblast cells by determining cell survival and scoring aberrations in calyculin A-induced G2 chromosomes. Results from the cell survival study indicate that after X-ray exposure G0 cells were sensitized by 24 h treatment with caffeine. Analysis of chromosome aberrations using FISH (fluorescence in situ hybridization) revealed a high frequency of aberrant cells and color junctions in the caffeine-treated cells. Since most DNA repair in nongrowing G0 cells is believed to result from nonhomologous end joining (NHEJ), caffeine may influence the fidelity of the NHEJ pathway in irradiated G0 cells.

Caffeine↗
PubMed1998

Cell cycle transition in early embryonic development of Xenopus laevis.

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.

Animals↗
PubMed1997

atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity.

Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia and Li-Fraumeni syndrome, respectively. The two genes are believed to interact in a number of pathways, including regulation of DNA damage-induced cell-cycle checkpoints, apoptosis and radiation sensitivity, and cellular proliferation. Atm-null mice, as well as those null for p53, develop mainly T-cell lymphomas, supporting the view that these genes have similar roles in thymocyte development. To study the interactions of these two genes on an organismal level, we bred mice heterozygous for null alleles of both atm and p53 to produce all genotypic combinations. Mice doubly null for atm and p53 exhibited a dramatic acceleration of tumour formation relative to singly null mice, indicating that both genes collaborate in a significant manner to prevent tumorigenesis. With respect to their roles in apoptosis, loss of atm rendered thymocytes only partly resistant to irradiation-induced apoptosis, whereas additional loss of p53 engendered complete resistance. This implies that the irradiation-induced atm and p53 apoptotic pathways are not completely congruent. Finally-and in contrast to prior predictions-atm and p53 do not appear to interact in acute radiation toxicity, suggesting a separate atm effector pathway for this DNA damage response and having implications for the prognosis and treatment of human tumours.

Acute Disease↗
PubMed2003

A multifaceted role for ATM in genome maintenance.

The pleiotropic nature of the clinical phenotypes of patients with ataxia-telangiectasia (A-T)--which encompass cerebellar degeneration (leading to ataxia), gonadal atrophy, and cancer predisposition--suggests multiple functions of the gene responsible for the disease. The ataxia-telangiectasia mutated gene product (ATM), whose loss of function is responsible for ataxia-telangiectasia, is a protein kinase that interacts with several substrates and is implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control and telomere maintenance. This review focuses on the critical roles that ATM appears to play in cell-cycle checkpoints, DNA repair, telomere metabolism and oxidative stress, indicating how defects in these processes might lead to ataxia-telangiectasia.

Ataxia Telangiectasia↗
PubMed2000

The in vitro phosphorylation of the co-chaperone mSTI1 by cell cycle kinases substantiates a predicted casein kinase II-p34cdc2-NLS (CcN) motif.

The co-chaperone murine stress-inducible protein 1 (mSTI1), a Hsp70/Hsp90 organizing protein (Hop) homolog, functions as a physical link between Hsp70 and Hsp90 by mediating the formation of the mSTI1/ Hsp70/Hsp90 chaperone heterocomplex. We show here that mSTI1 is an in vitro substrate of cell cycle kinases. Casein kinase II (CKII) phosphorylates mSTI1 at S189, and cdc2 kinase (p34cdc2) at T198, substantiating a predicted CKII-p34cdc2-NLS (CcN) motif. The possible implications of this phosphorylation as a cell cycle checkpoint are discussed.

Amino Acid Motifs↗