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PubMed1998

The role of DNA mismatch repair in drug resistance.

Loss of DNA mismatch repair (MMR) has been observed in a variety of human cancers. In addition to predisposing to oncogenesis, loss of MMR activity is of concern with respect to the use of chemotherapeutic agents to treat established tumors. Loss of MMR results in drug resistance directly by impairing the ability of the cell to detect DNA damage and activate apoptosis and indirectly by increasing the mutation rate throughout the genome. The MMR proteins are involved in mediating the activation of cell cycle checkpoints and apoptosis in response to DNA damage. MMR-deficient cells have been reported to be resistant to the methylating agents procarbazine and temozolomide, the alkylating agent busulfan, the platinum-containing drugs cisplatin and carboplatin, the antimetabolite 6-thioguanine, and the topoisomerase II inhibitors etoposide and doxorubicin. In the case of cisplatin, busulfan, temozolomide, and procarbazine, the degree of resistance has been shown to be sufficient to produce a large difference in clinical responsiveness in vivo in tumor model systems. The available preclinical data suggest that tumors that contain a significant fraction of cells deficient in MMR will demonstrate reduced responsiveness to specific drugs. The challenge now is to assess the clinical significance of the presence of deficient cells in tumors and to discover drugs that retain activity against MMR-deficient cells.

Alkylating Agents↗
PubMed2006

Interactions between the RNA interference effector protein Ago1 and 14-3-3 proteins: consequences for cell cycle progression.

The Argonaute family member Ago1 is required for formation of pericentric heterochromatin and small interfering RNA (siRNA)-mediated post-transcriptional gene silencing in the fission yeast Schizosaccharomyces pombe. In addition, we have recently demonstrated that Ago1 function is required for enactment of cell cycle checkpoints (Carmichael, J. B., Provost, P., Ekwall, K., and Hobman, T. C. (2004) Mol. Biol. Cell 15, 1425-1435). Here, we provide evidence that the amino terminus of Ago1 binds to proteins that function in cell cycle regulation including 14-3-3 proteins. Interestingly, the amino terminus of human Ago2, the endonuclease that cleaves siRNA-targeted mRNAs, was also demonstrated to bind 14-3-3 proteins. Overexpression of the Ago1 amino terminus in yeast resulted in cell cycle delay at the G(2)/M boundary. Further investigation revealed that nuclear import of the mitosis-inducing phosphatase Cdc25 is inhibited by overexpression of the Ago1 amino terminus. Under these conditions, we found that the cyclin-dependent kinase Cdc2 is constitutively phosphorylated on tyrosine 15, thereby reducing the activity of this kinase, a situation that delays entry into mitosis. We hypothesize that 14-3-3 proteins are required for Argonaute protein functions in cell cycle and/or gene-silencing pathways.

14-3-3 Proteins↗
PubMed1998

Small contribution of G1 checkpoint control manipulation to modulation of p53-mediated apoptosis.

Deregulation of the S-phase promoting E2F-1 transcription factor has been shown to cooperate with p53 to induce apoptosis. BaF3 cells undergo rapid, p53-dependent apoptosis when irradiated in the absence of IL-3. Rapid apoptosis induced by ionizing radiation (IR) coincides with attenuated p21(WAF1/Cip1) induction. Failure to adequately induce p21 could result in inappropriate release of E2F from Rb which may then cooperate with p53 to induce apoptosis in cells deprived of growth factor. We engineered BaF3 cells to express exogenous p21 and tested whether overexpressing p21 in cells irradiated in the absence of IL-3 protects from IR-induced apoptosis. Enforced p21 expression resulted in a consistent, but partial, protection of cells from undergoing IR-induced apoptosis. However, deregulating E2F activity through expression of HPV E7 failed to sensitize cells to IR-induced apoptosis in the presence of IL-3. Together, these data strongly suggest that the IL-3-responsive factors which modulate p53-mediated apoptosis in BaF3 cells are largely independent of G1 cell cycle checkpoint control mediated by p21.

Animals↗
PubMed1999

The FHA domain is a modular phosphopeptide recognition motif.

FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes.

Amino Acid Sequence↗
PubMed2000

Cleavage of ATM during radiation-induced apoptosis: caspase-3-like apoptotic protease as a candidate.

PURPOSE: To study the relationship of ionizing radiation-induced apoptosis with the integrity of ATM (mutated in ataxia telangiectasia) that has a critical role in DNA damage sensing and repair, cell-cycle checkpoint controls and maintenance of genomic stability. MATERIALS AND METHODS: U937 cells were treated with gamma-radiation. Sub-G1 DNA content, DNA fragmentation, cleavage of PARP, active caspase-3, cleavage of ATM in vivo and in vitro were measured by flow cytometry, agarose gel electrophoresis, cleaving of colorimetric caspase-3 substrate and Western blotting. RESULTS: ATM is specifically cleaved in cells during the induction of apoptosis by ionizing radiation exposure. The time-course of cleavage coincided with the appearance of cells with a sub-G1 DNA content and activation of caspase-3. ATM was cleaved with similar kinetics as PARP and DEVD-FMK could abolish the cleavage. In vitro studies showed that ATM was cleaved by caspase-3 or related subfamily members at a DIVD/G site. CONCLUSION: ATM belongs to a group of repair proteins, including PARP, DNA-PK and HsRad51, which are specifically cleaved during apoptosis. These findings support the idea that repair mechanisms need to be inactivated for apoptosis to proceed efficiently.

Apoptosis↗
PubMed2006

ATM regulates ATR chromatin loading in response to DNA double-strand breaks.

DNA double-strand breaks (DSBs) are among the most deleterious lesions that can challenge genomic integrity. Concomitant to the repair of the breaks, a rapid signaling cascade must be coordinated at the lesion site that leads to the activation of cell cycle checkpoints and/or apoptosis. In this context, ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) protein kinases are the earliest signaling molecules that are known to initiate the transduction cascade at damage sites. The current model places ATM and ATR in separate molecular routes that orchestrate distinct pathways of the checkpoint responses. Whereas ATM signals DSBs arising from ionizing radiation (IR) through a Chk2-dependent pathway, ATR is activated in a variety of replication-linked DSBs and leads to activation of the checkpoints in a Chk1 kinase-dependent manner. However, activation of the G2/M checkpoint in response to IR escapes this accepted paradigm because it is dependent on both ATM and ATR but independent of Chk2. Our data provides an explanation for this observation and places ATM activity upstream of ATR recruitment to IR-damaged chromatin. These data provide experimental evidence of an active cross talk between ATM and ATR signaling pathways in response to DNA damage.

Ataxia Telangiectasia↗
PubMed2004

Medaka as a model system for the characterisation of cell cycle regulators: a functional analysis of Ol-Gadd45gamma during early embryogenesis.

Numerous studies, mostly performed on mammalian cell cultures, have implicated the Gadd45 family of small acidic proteins in cell cycle control (arrest and/or engagement in the apoptotic pathway). We report here the cloning, detailled expression pattern and functional characterisation in embryonic development of Ol-Gadd45gamma, the Oryzias latipes ortholog of mammalian Gadd45gamma. Its expression pattern, notably in the developing brain (optic tectum) strongly suggests that it is involved in cell cycle exit. Gain-of-function experiments (through mRNA injection) slowed down early development, and produced embryos clearly reduced in size, while morpholino knockdowns resulted in small embryos over-sensitive to DNA damage (UV irradiation). We further demonstrated that, following Ol-Gadd45gamma overexpression, cells are proliferation-arrested before both G1/S and G2/M cell cycle checkpoints, while in the MO-Ol-Gadd45 loss-of-function experiments cells are engaged in apoptosis rather than prevented from proliferating. These results show that Ol-Gadd45gamma is likely to play an important role in coordinating cell fate decisions during neurogenesis; they also demonstrate that the medakafish is a promising model to analyse in vivo the developmental control of the cell cycle.

Amino Acid Sequence↗
PubMed2005

High levels of Cdc7 and Dbf4 proteins can arrest cell-cycle progression.

Cdc7-Dbf4 serine/threonine kinase is essential for initiation of DNA replication. It was previously found that overexpression of certain replication proteins such as Cdc6 and Cdt1 in fission yeast resulted in multiple rounds of DNA replication in the absence of mitosis. Since this phenomenon is dependent upon the presence of wild-type Cdc7/Hsk1, we hypothesized that high levels of Cdc7 and/or Dbf4 could also cause multiple rounds of DNA replication, or could facilitate entry into S phase. To test this hypothesis, we transiently overexpressed hamster Cdc7, Dbf4 or both in CHO cells. Direct observations of individual cells by fluorescence microscopy and flow cytometric analysis on cell populations suggest that overexpression of Cdc7 and/or Dbf4 does not result in multiple rounds of DNA replication or facilitating entry into S phase. In contrast, moderately increased levels of Dbf4, but not Cdc7, cause cell-cycle arrest in G2/M. This G2/M arrest coincides with hyperphosphorylation of Cdc2/Cdk1 at Tyr-15, raising the possibility that high levels of Dbf4 may activate a G2/M cell-cycle checkpoint. Further increase in Cdc7 and/or Dbf4 by 2-4 fold can arrest cells in G1 and significantly slow down S-phase progression for the cells already in S phase.

Animals↗
PubMed1995

Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast.

The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1-arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule-disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.

Amino Acid Sequence↗
PubMed2002

Caenorhabditis elegans HUS-1 is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis.

BACKGROUND: The inability to efficiently repair DNA damage or remove cells with severely damaged genomes has been linked to several human cancers. Studies in yeasts and mammals have identified several genes that are required for proper activation of cell cycle checkpoints following various types of DNA damage. However, in metazoans, DNA damage can induce apoptosis as well. How DNA damage activates the apoptotic machinery is not fully understood. RESULTS: We demonstrate here that the Caenorhabditis elegans gene hus-1 is required for DNA damage-induced cell cycle arrest and apoptosis. Following DNA damage, HUS-1 relocalizes and forms distinct foci that overlap with chromatin. Relocalization does not require the novel checkpoint protein RAD-5; rather, relocalization appears more frequently in rad-5 mutants, suggesting that RAD-5 plays a role in repair. HUS-1 is required for genome stability, as demonstrated by increased frequency of spontaneous mutations, chromosome nondisjunction, and telomere shortening. Finally, we show that DNA damage increases expression of the proapoptotic gene egl-1, a response that requires hus-1 and the p53 homolog cep-1. CONCLUSIONS: Our findings suggest that the RAD-5 checkpoint protein is not required for HUS-1 to relocalize following DNA damage. Furthermore, our studies reveal a new function of HUS-1 in the prevention of telomere shortening and mortalization of germ cells. DNA damage-induced germ cell death is abrogated in hus-1 mutants, in part, due to the inability of these mutants to activate egl-1 transcription in a cep-1/p53-dependent manner. Thus, HUS-1 is required for p53-dependent activation of a BH3 domain protein in C. elegans.

Amino Acid Sequence↗
PubMed2000

Quiescence in S-phase and G1 arrest induced by irradiation and/or hyperthermia in six human tumour cell lines of different p53 status.

PURPOSE: Quiescent S-phase cells, i.e. cells with a DNA content intermediate between G1 and G2 that nevertheless do not synthesize DNA have been previously observed in human melanoma cells exposed to radiation and/or hyperthermia. This phenomenon has now been studied in more detail comparing six human tumour cell lines of different p53 status and thus different cell-cycle checkpoint control. MATERIALS AND METHODS: Two melanoma (Be11, MeWo), two squamous carcinoma (4197, 4451) and two glioma (EA14, U87) cell lines were used. Changes in the cell-cycle distribution after treatment were studied using two-parameter flow cytometry in order to measure DNA content and BrdU incorporation simultaneously. RESULTS: The fraction of unlabelled cells in the S-phase compartment was determined at daily intervals after treatment. Only background levels of such cells were seen in three of the cell lines (Be11, 4197, EA14). With the other three cell lines (MeWo, 4451, U87) we observed a time- and dose-dependent increase: a few days after treatment up to 20% of all cells did not incorporate BrdU. It is interesting to note that Bell, 4197 and EA14 are p53 wild-types and show a G1 block of several hours after irradiation and/or hyperthermia, while MeWo and 4451 are p53 mutants unable to exhibit such a delay, and U87 in spite of being a p53 wild-type has a reduced ability to do so. CONCLUSIONS: The MeWo, 4451 and U87 cell lines have less time available for the repair of DNA damage before entering into the S-phase, which leads to problems during replication and causes some kind of interphase death. Radiation-induced apoptosis does not seem to be involved here, as it is not unequivocally correlated with the induction of a G1 block or with p53 status.

Dose-Response Relationship, Radiation↗
PubMed2006

Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum.

Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of gamma-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G(2) phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human gamma-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA.

Antineoplastic Agents↗
PubMed2006

NF-kappaB activation by double-strand breaks.

Cellular response to DNA damage is complex and relies on the simultaneous activation of different networks. It involves DNA damage recognition, repair, and induction of signalling cascades leading to cell cycle checkpoint activation, apoptosis, and stress related responses. The fate of damaged cells depends on the balance between pro- and antiapoptotic signals. In this decisive life or death choice, the transcription factor NF-kappaB has emerged as a prosurvival actor in most cell types. As corollary, it appears to be associated with tumorigenic process and resistance to therapeutic strategies as it protects cancerous cells from death. In this review, we will focus on NF-kappaB activation by double-strand breaks inducing agents, such as ionizing radiation and DNA topoisomerase I and II inhibitors routinely used in cancer therapy. Coinciding with the 20th anniversary of the NF-kappaB discovery, major steps of the DSB-triggered cascade have been recently identified. Two parallel cascades are necessary for NF-kappaB activation. The first one depends on ATM (activated by double-strand breaks) and the second on PIDD (activated by an unknown stress signal). The phosphorylation of NEMO by ATM is the point of convergence of these two cascades. The identification of ATM/NEMO complex as the long searched "nuclear to cytoplasm" signal leading to IKK activation is also a major piece of the puzzle. The knowledge of the precise steps leading to DSB-initiated NF-kappaB activation will allow the development of specific blocking compounds reducing its prosurvival function.

Animals↗
PubMed2006

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation.

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

Disruption of cell cycle control by human papillomaviruses with special reference to cervical carcinoma.

Human papillomaviruses (HPVs) play a major role in neoplastic transformation of squamous epithelial cells. The viral genome is small in size and only encodes a limited number of proteins, so one of the major functions of the viral proteins is to modulate the function of key cellular proteins involved in cell cycle control and DNA replication. During this process important host cell cycle checkpoints are lost which may lead to the accumulation of genetic abnormalities and eventual malignant transformation. This review briefly describes the normal cell cycle and also the mechanisms by which HPVs interfere with cell cycle control both as part of their productive life cycle and in the process of neoplastic transformation.

Journal Article↗
PubMed1998

Recovery of the normal p53 response after UV treatment in DNA repair-deficient fibroblasts by retroviral-mediated correction with the XPD gene.

Among the major responses of human cells to DNA damage is accumulation of the p53 tumor suppressor protein, which plays a crucial role as a cell-cycle checkpoint. We have already shown that this response is different in cells from the UV-hypersensitive human syndromes xeroderma pigmentosum (XP) and trichothiodystrophy (TTD), which overlap with each other and arise from mutations in genes involved in nucleotide excision repair. In this paper we report that correction of the repair defect by retroviral-mediated transduction of the wild-type XPD gene in XP-D and TTD/XP-D untransformed primary fibroblasts leads to a normal p53 response in these cells. Thus, the complemented cells, like normal human fibroblasts, require higher UV doses (10 J/m2) for p53 induction than the parental repair-deficient XP-D or TTD/XP-D cells (both mapping at the XPD locus), which accumulate p53 protein at very low UV doses (2.5 and 5 J/m2). The p53 protein levels return to normal 24 h after irradiation when UV-induced lesions have been efficiently repaired by the restored NER activity. These data confirm our earlier results that p53 accumulation following UV treatment is directly related to the presence of unrepaired cyclobutane dimers on the transcribed strand of active genes.

DNA Repair↗
PubMed2001

p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells.

Temozolomide (TMZ) is a DNA-methylating agent that has recently been introduced into Phase II and III trials for the treatment of gliomas. TMZ produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair can lead to a p53-associated apoptotic cell death, although this mechanism has been described mostly in hematopoietic neoplasms. We studied the action of TMZ in gliomas and the role p53 might play by using U87 glioma cells that were either p53-wild-type or p53-deficient (by virtue of expression of the viral oncoprotein E6). LN-Z308 cells, in which p53 gene is deleted, were also used. p53-proficient U87 MG cells underwent a prolonged, p53- and p21(Waf1/Cip1)-associated G2-M arrest beginning 2 days after TMZ treatment. Although very few of these cells underwent apoptosis, most underwent senescence over a 10-day period. p53-deficient (E6-transfected U87 and LN-Z308) cells similarly underwent G2-M arrest in response to TMZ, but this arrest was accompanied by only minor changes in p53 or p21(Waf1/Cip1) and was reversed within 7 days of TMZ treatment in association with the appearance of cells with either 8n or subG1 DNA content. These results suggest that glioma cells respond to TMZ by undergoing G2-M arrest. p53 is not necessary for this G2-M arrest to occur but is important in the duration of G2-M arrest and in the ultimate fate of TMZ-treated cells. Therefore, the integrity of the G2-M cell cycle checkpoint may be important in the cytotoxicity of TMZ in glioma cells.

Antineoplastic Agents, Alkylating↗
PubMed2005

The two edges of the ATM sword: co-operation between repair and checkpoint functions.

ATM is a central component of a signal transduction process that responds to DNA double strand breaks (DSBs) ultimately effecting cell cycle checkpoint arrest and/or apoptosis. Recent studies have shown that ATM also regulates a mechanism of processing a subset of DNA ends that appear to be difficult to ligate, since they are rejoined with slow kinetics in control cells. In the absence of this process, which involves the nuclease, Artemis, the DSBs either remain unrejoined or potentially undergo misrejoining. Thus, ATM's checkpoint function specifically facilitates its repair function. Here, we discuss the contribution of this novel function of ATM to survival after ionising irradiation and to cancer avoidance. We suggest that ATM's strength as a damage response protein lies in the co-ordination of its repair and checkpoint functions making a razor sharp knife out of two blunter edges.

Animals↗