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PubMed1998

Commitment and differentiation of lung cell lineages.

To form a large diffusible interface capable of conducting respiratory gases to and from the circulation, the lung must undergo extensive cell proliferation, branching morphogenesis, and alveolar saccule formation, to generate sufficient surface area. In addition, the cells must differentiate into at least 40 distinct lung cell lineages. Specific transcriptional factors, peptide growth factor receptor-mediated signaling pathways, extracellular matrix components, and integrin-signaling pathways interact to direct lung morphogenesis and lung cell lineage differentiation. Branching mutants of the respiratory tracheae in Drosophila have identified several functionally conserved genes in the fibroblast growth factor signaling pathway that also regulate pulmonary organogenesis in mice and probably also in man. Key transcriptional factors including Nkx2.1, hepatocyte nuclear factor family forkhead homologues, GATA family zinc finger factors, pou and homeodomain proteins, as well as basic helix-loop-helix factors, serve as master genes to integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Lung mesenchyme serves as a 'compleat' inducer of lung morphogenesis by secreting soluble peptide growth factors. In general, peptide growth factors signaling through cognate receptors with tyrosine kinase intracellular signaling domains such as epidermal growth factor receptor, fibroblast growth factor receptors, hepatocyte growth factor/scatter factor receptor, c-met, insulin-like growth factor receptor, and platelet-derived growth factor receptor, stimulate lung morphogenesis, while the cognate receptors with serine/threonine kinase intracellular signaling domains, such as the transforming growth factor-beta receptor family are inhibitory. The extracellular matrix also plays a key role in determining branching morphogenesis. Pulmonary neuroendocrine (PNE) cells differentiate earliest in gestation among lung epithelial cells. PNE cells are principally derived from endoderm and not neural crest. PNE cells have been proposed to function as airway chemoreceptors, while PNE cell secretory granules contain many bioactive substances such as GRP which may direct proliferation of adjacent epithelial cells. Mammalian achaete-schute homolog-1 null mutant mice do not develop PNE cells. Candidate molecular switches in the transition from a quiescent to a proliferative alveolar epithelial cell (AEC) phenotype and back again following acute hyperoxia, include autocrine peptide growth factor signaling pathways and cell cycle regulatory elements. AEC type 2 also appear capable of reversible transdifferentiation into AEC type 1 and intermediate phenotypes in response to cues from extracellular matrix and cell shape, as well as soluble factors. Evidence for expression of telomerase by alveolar epithelial stem cells, which correlates with self-renewal potential, is now beginning to emerge. Lung regeneration following lobectomy in juvenile rodents is associated with co-ordinated cell proliferation, re-expression of elastin and formation of alveoli. Retinoic acid has recently shown promise as a stimulator of alveolization in juvenile rats. Our future goal is to devise new rational and gene therapeutic strategies to stimulating lung growth and maturation, ameliorating lung injury, augmenting lung repair, and inducing lung regeneration. The ideal agent or agents would therefore mimic the instructive role of lung mesenchyme, correctly inducing the temporospatial pattern of lung cell lineages necessary to restore pulmonary gas diffusing capacity.

Animals↗
PubMed2006

Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin-dependent kinase 1 (Cdk1).

Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.

Amino Acid Sequence↗
PubMed2005

Lack of PTEN sequesters CHK1 and initiates genetic instability.

Pten-/- cells display a partially defective checkpoint in response to ionizing radiation (IR). The checkpoint defect was traced to the ability of AKT to phosphorylate CHK1 at serine 280, since a nonphosphorylated mutant of CHK1 (S280A) complemented the checkpoint defect and restored CDC25A degradation. CHK1 phosphorylation at serine 280 led to covalent binding of 1 to 2 molecules of ubiquitin and cytoplasmic CHK1 localization. Primary breast carcinomas lacking PTEN expression and having elevated AKT phosphorylation had increased cytoplasmic CHK1 and displayed aneuploidy (p <0.005). We conclude that loss of PTEN and subsequent activation of AKT impair CHK1 through phosphorylation, ubiquitination, and reduced nuclear localization to promote genomic instability in tumor cells.

Animals↗
PubMed1999

CHK1 frameshift mutations in genetically unstable colorectal and endometrial cancers.

The protein encoded by the CHK1 gene plays an important role in the G2 checkpoint in mammalian cells. In its coding region it presents a sequence of nine consecutive adenines that are a potential site of mutations in tumors with microsatellite instability (MSI). We analyzed the presence of frameshift mutations in the CHK1 gene in human colon and endometrial cancer samples. In the same cancer samples genes known to be altered in these tumors (BAX, TGFBRII, and IGFIIR) were also analyzed. CHK1 frameshfit mutations were found in 1 out 10 colon cancers and 2 out of 7 endometrial cancers showing MSI. CHK1 alterations were associated with the presence of a high degree of MSI. No alterations were found in patients with tumors showing low frequency or lacking instability (microsatellite stable). The same was true for the other four genes analyzed. The insertion or deletion of one A in the poly A tract resulted in a truncated protein. Alterations of the CHK1 gene could represent an alternative way of cancer cells to escape from cell cycle control. Genes Chromosomes Cancer 26:176-180, 1999.

Biomarkers, Tumor↗
PubMed2002

A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle.

The role of the PI 3-kinase cascade in regulation of cell growth is well established [1]. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects [2,3] and the role it plays in initiation of S phase [4]. Here, we show that PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas caPKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery.

Animals↗
PubMed1998

Destabilization of yeast micro- and minisatellite DNA sequences by mutations affecting a nuclease involved in Okazaki fragment processing (rad27) and DNA polymerase delta (pol3-t).

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase delta) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the "proofreading" exonuclease domain of DNA polymerase delta) mutations were synthetically lethal.

Checkpoint Kinase 1↗
PubMed2006

Development of 6-substituted indolylquinolinones as potent Chek1 kinase inhibitors.

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.

Animals↗
PubMed1995

Mutations in RAD27 define a potential link between G1 cyclins and DNA replication.

The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions. To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2. We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2. rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to a DNA-damaging agent, increased mitotic recombination rates, and increased spontaneous mutation rates. Unlike the block in cell cycle progression, these phenotypes are not suppressed by CLN1 or CLN2. CLN1 and CLN2 may activate an RAD27/ERC11-independent pathway specific for DNA synthesis that CLN3 is incapable of activating. Alternatively, CLN1 and CLN2 may be capable of overriding a checkpoint response which otherwise causes cln1 cln2 rad27/erc11 cells to arrest. These results imply that CLN1 and CLN2 have a role in the regulation of DNA replication. Consistent with this, GAL-CLN1 expression in checkpoint-deficient, mec1-1 mutant cells results in both cell death and increased chromosome loss among survivors, suggesting that CLN1 overexpression either activates defective DNA replication or leads to insensitivity to DNA damage.

Checkpoint Kinase 1↗
PubMed2006

Oxidative stress induces a prolonged but reversible arrest in p53-null cancer cells, involving a Chk1-dependent G2 checkpoint.

Reactive oxygen species (ROS), the principal mediators of oxidative stress, induce responses such as apoptosis or permanent growth arrest/senescence in normal cells. Moreover, p53 activation itself contributes to ROS accumulation. Here we show that treatment of p53-null cancer cells with sublethal concentrations of ROS triggered an arrest with some morphological similarities to cellular senescence. Different from a classical senescent arrest in G(1), the ROS-induced arrest was predominantly in the G(2) phase of the cell cycle, and its establishment depended at least in part on an intact Chk1-dependent checkpoint. Chk1 remained phosphorylated only during the repair of double strand DNA breaks, after which Chk1 was inactivated, the G(2) arrest was suppressed, and some cells recovered their ability to proliferate. Inhibition of Chk1 by an RNAi approach resulted in an increase in cell death in p53-null cells, showing that the Chk1-dependent G(2) checkpoint protected cells that lacked a functional p53 pathway from oxidative stress. It has been proposed that the induction of a senescent-like phenotype by antineoplastic agents can contribute therapeutic efficacy. Our results indicate that oxidative stress-induced growth arrest of p53-null tumor cells cannot be equated with effective therapy owing to its reversibility and supports the concept that targeting Chk1 may enhance the effects of DNA-damaging agents on cancer progression in such tumors.

Animals↗
PubMed1999

The COP9/signalosome complex is conserved in fission yeast and has a role in S phase.

The COP9/signalosome complex is conserved from plant to mammalian cells. In Arabidopsis, it regulates the nuclear abundance of COP1, a transcriptional repressor of photomorphogenic development [1] [2]. All COP (constitutive photomorphogenesis) mutants inappropriately express genes that are normally repressed in the dark. Eight subunits (Sgn1-Sgn8) of the homologous mammalian complex have been purified [3] [4]. Several of these have been previously identified through genetic or protein interaction screens. No coherent model for COP9/signalosome function has yet emerged, but a relationship with cell-cycle progression by transcriptional regulation, protein localisation or protein stability is possible. Interestingly, the COP9/signalosome subunits possess domain homology to subunits of the proteasome regulatory lid complex [5] [6]. Database searches indicate that only Sgn5/JAB1 is present in Saccharomyces cerevisiae, precluding genetic analysis of the complex in cell-cycle regulation. Here we identify a subunit of the signalosome in the fission yeast Schizosaccharomyces pombe through an analysis of the DNA-integrity checkpoint. We provide evidence for the conservation of the COP9/signalosome complex in fission yeast and demonstrate that it functions during S-phase progression.

COP9 Signalosome Complex↗
PubMed2004

chk-1 is an essential gene and is required for an S-M checkpoint during early embryogenesis.

The chk1 gene was first discovered in screens for radiation sensitive mutants in S. pombe.(1) Genetic analysis revealed that chk1 is involved in a DNA damage G(2)-M checkpoint. Chk1 becomes activated in response to DNA damage and prevents entry into mitosis by inhibiting the cell cycle machinery. This checkpoint decreases the risk of defective DNA being inherited by daughter cells, therefore reducing the risk of genetic instability. In higher eukaryotes, chk1 homologues have similar checkpoint functions. For example, an avian B-lymphoma cell line that is defective for Chk1 fails to arrest in G(2)-M after DNA damage. Nonetheless, these Chk1 defective cells are viable indicating that Chk1 is not essential for normal somatic cells to divide.(2) In spite of this, mouse and Drosophila homozygous Chk1 mutants die during embryogenesis suggesting that this is an essential gene for embryonic cell cycles.(3,4) What particular role does Chk1 have in directing embryonic cell divisions? Here we used the model organism, C. elegans, to address the role of chk-1 during development. As expected, disruption of chk-1 by RNAi eliminated the DNA damage checkpoint response in C. elegans. In addition, we revealed that chk-1 was predominantly expressed during embryogenesis and in the postembryonic germline. Indeed, we found that chk-1 had an essential role in embryo and germline development. More specifically, disruption of chk-1 expression resulted in embryo lethality, which was attributed to a defect in an intrinsic S-M checkpoint hence causing premature entry into M-phase.

Animals↗
PubMed2003

Suppression of Tousled-like kinase activity after DNA damage or replication block requires ATM, NBS1 and Chk1.

The human Tousled-like kinases 1 and 2 (TLK) have been shown to be active during S phase of the cell cycle. TLK activity is rapidly suppressed by DNA damage and by inhibitors of replication. Here we report that the signal transduction pathway, which leads to transient suppression of TLK activity after the induction of double-strand breaks (DSBs) in the DNA, is dependent on the presence of a functional ataxia-telangiectasia-mutated kinase (ATM). Interestingly, we have discovered that rapid suppression of TLK activity after low doses of ultraviolet (UV) irradiation or aphidicolin-induced replication block is also ATM-dependent. The nature of the signal that triggers ATM-dependent downregulation of TLK activity after UVC and replication block remains unknown, but it is not due exclusively to DSBs in the DNA. We also demonstrate that TLK suppression is dependent on the presence of a functional Nijmegan Breakage Syndrome protein (NBS1). ATM-dependent phosphorylation of NBS1 is required for the suppression of TLK activity, indicating a role for NBS1 as an adaptor or scaffold in the ATM/TLK pathway. ATM does not phosphorylate TLK directly to regulate its activity, but Chk1 does phosphorylate TLK1 GST-fusion proteins in vitro. Using Chk1 siRNAs, we show that Chk1 is essential for the suppression of TLK activity after replication block, but that ATR, Chk2 and BRCA1 are dispensable for TLK suppression. Overall, we propose that ATM activation is not linked solely to DSBs and that ATM participates in initiating signaling pathways in response to replication block and UV-induced DNA damage.

Aphidicolin↗
PubMed1999

The topoisomerase I poison camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission yeast.

The protein kinase Chk1 is essential for the DNA damage checkpoint. Cells lacking Chk1 are hypersensitive to DNA-damaging agents such as UV light and gamma-irradiation because they fail to arrest the cell cycle when DNA damage is generated. Phosphorylation of Chk1 occurs after DNA damage and is dependent on the integrity of the DNA damage checkpoint pathway. We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA damage in the fission yeast Schizosaccharomyces pombe that results in Chk1 phosphorylation. We demonstrate that Chk1 is phosphorylated in response to CPT treatment in a time- and dose-dependent manner and that phosphorylation is dependent on an intact DNA damage checkpoint pathway. Furthermore, we show that cells must be actively dividing in order for CPT to generate a Chk1-responsive DNA damage signal. This observation is consistent with a model whereby the cytotoxic event caused by CPT treatment is the production of a DNA double-strand break resulting from the collision of a DNA replication fork with a trapped CPT-topoisomerase I cleavable complex. Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpoint pathway can be an important determinant for CPT sensitivity or resistance. Finally, as a well-characterized, soluble agent that specifically causes DNA damage, CPT will allow a biochemical analysis of the checkpoint pathway that responds to DNA damage.

Camptothecin↗
PubMed2002

Analysis of 11q21-24 loss of heterozygosity candidate target genes in breast cancer: indications of TSLC1 promoter hypermethylation.

Loss of heterozygosity (LOH) at the distal half of chromosome arm 11q is frequent in a variety of human tumors, including breast cancer, and is often associated with poor prognosis. In an ongoing attempt to locate and characterize the main target genes within this chromosome region, we first looked for aberrations in known genes either suggested to be involved in tumorigenesis or shown to suppress tumor formation. We examined 31 primary breast tumors showing LOH in 11q21-24 for mutations in the MRE11A, CHK1, PPP2R1B, and TSLC1 genes. The absence of intragenic alterations related to cancer led us next to evaluate possible gene silencing resulting from promoter region CpG hypermethylation, using the bisulfite sequencing technique. In addition to the four genes mentioned above, we also analyzed the ATM gene, which had been investigated for certain germline mutations in an earlier study. Only the TSLC1 promoter region exhibited aberrant methylation patterns, and altogether 33% (10/30) of the successfully analyzed tumors showed evidence of elevated levels of TSLC1 CpG methylation. Ten percent (3/30) of the tumors showed significantly increased methylation. Thus, as has been shown in lung and some other forms of cancer, hypermethylation of the TSLC1 promoter region is also frequently a second hit along with LOH in breast cancer.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2002

Poly(ADP-ribose) polymerase inhibitor increases growth inhibition and reduces G(2)/M cell accumulation induced by temozolomide in malignant glioma cells.

Temozolomide (TZM) is a novel methylating agent currently under investigation for treatment of recurrent high-grade gliomas. Although TZM generates a wide spectrum of methyl adducts, its cytotoxicity has been attributed to mismatch repair (MR)-mediated processing of O(6)-methylguanine:T mispairs. N3-methyladenine and N7-methylguanine adducts are promptly repaired by the base excision repair system, unless a poly(ADP-ribose) polymerase (PARP) inhibitor is combined to TZM. In this case, the repair process of N-methylpurines cannot be completed and the deriving DNA strand breaks contribute to cytotoxicity. In this study, we investigated the influence on cell growth and cell cycle of treatment with TZM + PARP inhibitor in glioma cells characterized by different susceptibility to TZM. The results indicated that PARP inhibitor increases growth inhibition induced by TZM in either p53-wild-type or p53-mutant glioblastoma cells, as early as 24 h after drug exposure. The enhancing effect exerted by PARP inhibitor was particularly evident in glioma cells characterized by a defective expression of MR, since these cells are tolerant to O(6)-methylguanine damage and show low sensitivity to TZM. In O(6)-alkylguanine-DNA alkyltransferase (OGAT)-deficient and MR-proficient tumor cells bearing wild-type p53, the drug combination markedly reduced cell accumulation in the G(2)/M phase of cell cycle and induction of the G(2) checkpoint regulator Chk1 kinase. In short-term cultures of glioma cells derived from surgical specimens, PARP inhibitor enhanced chemosensitivity to TZM and this effect was especially evident in OGAT-proficient tumors. Thus, a pharmacological strategy based on the interruption of N-methylpurine repair might represent a novel strategy to restore or increase glioma sensitivity to TZM.

Antineoplastic Agents, Alkylating↗
PubMed2003

Development of resistance to a trinuclear platinum complex in ovarian carcinoma cells.

BBR3464 is a trinuclear platinum complex that exhibits a potent cytotoxicity and efficacy against cisplatin-resistant tumors. To better understand the determinants of cellular resistance to BBR3464, we selected a resistant ovarian carcinoma cell line after exposure to the complex. The resistant cells (A2780/BBR3464) exhibited a high level of resistance to the selecting agent, but a marginal cross-resistance to cisplatin. Although cellular accumulation of BBR3464 was similar in parental and in resistant cells, DNA platination was decreased in A2780/BBR3464 cells, suggesting a reduced drug accessibility to DNA. This behavior reflected a partial drug inactivation at cytoplasmic level, as a consequence of increased levels of nucleophilic molecules including metallothioneins and human neurofilament low, but not glutathione. A2780/BBR3464 cells also exhibited a reduced susceptibility to apoptosis, which was consistent with reduced expression of Bax, and an alteration of DNA mismatch repair system, as reflected by lack of expression of MLH1 and PMS2, which could impair the recognition/repair of DNA lesions. Whereas both platinum drugs induced G2/M arrest in the parental cells, BBR3464, but not cisplatin, caused a late G1 arrest of resistant cells. Cisplatin induced an appreciable increase of p21(WAF1) levels in both models, in contrast to BBR3464 that produced a substantial upregulation of p21(WAF1) only in parental cells. An inverse relationship with p21(WAF1) modulation was found for CHK1 in parental cells treated with both agents and in resistant cells treated with cisplatin. This pattern of response is consistent with a regulatory loop involving p53 and p21(WAF1) at G2 checkpoint. In contrast, no modulation of CHK1 was found in A2780/BBR3464 treated with the triplatinum compound. These findings, indicating a different activation of regulatory pathways at DNA damage checkpoints in response to cisplatin and BBR3464, support an altered ability of resistant cells to recognize or tolerate sublethal lesions induced by BBR3464.

Antineoplastic Agents↗
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↗
PubMed2005

Involvement of the Fhit gene in the ionizing radiation-activated ATR/CHK1 pathway.

Fragile Histidine Triad (Fhit) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified, although it has been shown that Fhit overexpression leads to apoptosis in many cancer cell lines. We report in this study that Fhit-/- cells derived from gene knockout mice show much stronger S and G2 checkpoint responses than their wild type counterparts. The strong checkpoint responses are regulated by the ATR/CHK1 pathway, which contributes to the radioresistance of Fhit-/- cells. These results indicate an association of Fhit gene inactivation with increased survival after DNA damage, which is related to the over-active checkpoints regulated by the ATR/CHK1 pathway. These results also suggest the potential effects of Fhit-dependent DNA damage response on tumor progression.

Acid Anhydride Hydrolases↗