AKT1 mediates bypass of the G1/S checkpoint after genotoxic stress in normal human cells

Certain forms of hexavalent chromium [Cr(VI)] are human carcinogens. Our recent work has shown that a broad range protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SOV), abrogated both Cr(VI)-induced growth arrest and clonogenic lethality. Notably, SOV enhanced Cr(VI) mutation frequency, ostensibly through forced survival of genetically damaged cells. In the present study, co-treatment with this PTP inhibitor bypassed the Cr(VI)-induced G1/S checkpoint arrest in diploid human lung fibroblasts (HLF). Moreover, the PTP inhibitor abrogated the Cr(VI)-induced decrease in the expression of key effectors of the G1/S checkpoint [Cyclin D1, phospho Ser 807/811 Rb (pRB), p27]. Cr(VI)-induced G1 arrest was associated with the cytoplasmic appearance of pRb and the nuclear localization of p27, both of which were reversed by the PTP inhibitor. The PTP inhibitor’s reversal of G1/S checkpoint effector localization after Cr exposure was found to be Akt1-dependent, as this was abrogated by transfection with either akt1 siRNA or an Akt1-kinase dead plasmid. Furthermore, Akt1 activation alone was sufficient to induce G1/S checkpoint bypass and to prevent Cr(VI)-induced changes in pRb and p27 localization. In conclusion, this work establishes Akt1 activation to be both sufficient to bypass the Cr(VI)-induced G1/S checkpoint, as well as necessary for the observed PTP inhibitor effects on key mediators of the G1/S transition. The potential for Akt to bypass G1/S checkpoint arrest in the face of genotoxic damage could increase genomic instability, which is a hallmark of neoplastic progression.     

Protein tyrosine phosphatase (PTP) inhibition enhances chromosomal stability after genotoxic stress: Decreased chromosomal instability (CIN) at the expense of enhanced genomic instability (GIN)?

Inappropriate survival signaling after DNA damage may facilitate clonal expansion of genetically compromised cells, and it is known that protein tyrosine phosphatase (PTP) inhibitors activate key survival pathways. In this study we employed the genotoxicant, hexavalent chromium [Cr(VI)], which is a well-documented carcinogen of occupational and environmental concern. Cr(VI) induces a complex array of DNA damage, including DNA double strand breaks (DSBs). We recently reported that PTP inhibition bypassed cell cycle arrest and abrogated Cr(VI)-induced clonogenic lethality. Notably, PTP inhibition resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of DNA damage may lead to genomic instability (GIN), via cell cycle checkpoint bypass. The aim of the present study was to determine the effect of PTP inhibition on DNA DSB formation and chromosomal integrity after Cr(VI) exposure. Diploid human lung fibroblasts were treated with Cr(VI) in the presence or absence of the PTP inhibitor, sodium orthovanadate, for up to 24h, and cells were analyzed for DNA DSBs and chromosomal damage. Cr(VI) treatment induced a rapid increase in DNA DSBs, and a significant increase in total chromosomal damage (chromatid breaks and gaps) after 24h. In sharp contrast, PTP inhibition abrogated both DNA DSBs and chromosomal damage after Cr(VI) treatment. In summary, PTP inhibition in the face of Cr(VI) genotoxic stress decreases chromosomal instability (CIN) but increases mutagenesis, which we postulate to be a result of error-prone DNA repair.     

Mechanism of apoptosis and determination of cellular fate in chromium(VI)-exposed populations of telomerase-immortalized human fibroblasts.

The cellular responses to carcinogen exposure influence cellular fate, which in turn modulates the neoplastic response. Certain hexavalent chromium [Cr(VI)] compounds are implicated as occupational respiratory carcinogens at doses that are both genotoxic and cytotoxic. We examined the mechanism of Cr(VI)-induced apoptosis in normal human fibroblasts (BJ) immortalized by human telomerase gene transfection (BJ-hTERT), and we assessed the spectrum of cumulative cellular fates [(a) regaining of replicative potential; (b) terminal growth arrest; or (c) apoptosis] for a narrow range of increasingly genotoxic doses of Cr(VI). Exposure of BJ-hTERT cells to Cr(VI) resulted in a dose-dependent increase in apoptosis that involved mitochondrial disruption as evidenced by mitochondrial membrane depolarization and cytochrome c release. The initial response to Cr(VI) exposure was inhibition of cell cycle progression. At the lowest dose tested (1 microM; 32% clonogenic survival), the cell cycle inhibition led to terminal growth arrest but no apoptosis. The fraction of terminally growth arrested cells increased as the dose was increased to 3 microM but then decreased at 4, 5, and 6 microM as apoptosis became the predominant cell fate. Our results suggest that cell populations exposed to Cr(VI) have a different spectrum of responses, depending on the extent of DNA damage, and that the regaining of replicative potential after relatively higher genotoxic exposures may be attributable to either escape from, or resistance to, terminal growth arrest or apoptosis.     

Chromium (VI) activates ataxia telangiectasia mutated (ATM) protein. Requirement of ATM for both apoptosis and recovery from terminal growth arrest

The ataxia telangiectasia mutated (ATM) protein plays a central role in early stages of DNA double strand break (DSB) detection and controls cellular responses to this damage. Although hypersensitive to ionizing radiation-induced clonogenic lethality, ataxia telangiectasia cells are paradoxically deficient in their ability to undergo ionizing radiation-induced apoptosis. This contradiction illustrates the complexity of the central role of ATM in DNA damage response and the need for further understanding. Certain hexavalent chromium (Cr(VI)) compounds are implicated as occupational respiratory carcinogens at doses that are both genotoxic and cytotoxic. Cr(VI) induces a broad spectrum of DNA damage, but Cr(VI)-induced DSBs have not been reported. Here, we examined the role of ATM in the cellular response to Cr(VI) and found that Cr(VI) activates ATM. We also show that physiological targets of ATM, p53 Ser-15 and Chk2 Thr-68, were phosphorylated by Cr(VI) exposure in an ATM-dependent fashion. We found that ATM-/- cells were markedly resistant to Cr(VI)-induced apoptosis but considerably more sensitive to Cr(VI)-induced clonogenic lethality than wild type cells, indicating that resistance to Cr(VI)-induced apoptosis did not confer a selective survival advantage. However, analysis of long term growth arrest revealed a striking difference: ATM-/- cells were markedly less able to recover from Cr(VI)-induced growth arrest. This indicates that terminal growth arrest is the fate of these apoptosis-resistant cells. In summary, ATM is involved in cellular response to a complex genotoxin that may not directly induce DSBs. Our data suggest that ATM is a major signal initiator for genotoxin-induced apoptosis but, paradoxically, also contributes to maintenance of cell survival by facilitating recovery/escape from terminal growth arrest. The results also strongly suggest that terminal growth arrest is not merely an extended or even irreversible form of checkpoint arrest, but instead an independent and unique cell fate pathway.     

Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae

A broad spectrum of genetic damage results from exposure to hexavalent chromium. These lesions can result in DNA and RNA polymerase arrest, chromosomal aberrations, point mutations and deletions. Because of the complexity of Cr genotoxicity, the repair of Cr(VI)-induced DNA damage is poorly understood. Therefore, our aim was to investigate the sensitivities of DNA repair-deficient Saccharomyces cerevisiae strains to Cr(VI)-induced growth inhibition and lethality. Wild-type, translesion synthesis (rev3) and excision repair (apn1, ntg1, ntg2, rad1) mutants exhibited similar survival following Cr(VI) treatment (0-50mM) and underwent at least one population doubling within 2-4h post-treatment. The simultaneous loss of several excision repair genes (apn1 rad1 ntg1 ntg2) led to slower growth after Cr(VI) exposure (10mM) manifested as an initial delay in S phase progression. Higher concentrations of Cr(VI) (25mM) resulted in a prolonged transit through S phase in every strain tested. A G(2)/M arrest was evident within 1-2h after Cr(VI) treatment (10mM) in all strains and cells subsequently divided after this transient delay. In contrast to all other strains, only recombination-deficient (rad52, rad52 rev3) yeast were markedly hypersensitive towards Cr(VI) lethality. RAD52 mutant strains (rad52, rad52 rev3) also exhibited a significant delay (>6h) in the resumption of replication after Cr(VI) exposure which was related to the immediate and apparently terminal arrest of these yeast in G(2)/M after Cr(VI) treatment. These results, taken together with the recombinogenic effects of Cr(VI) in yeast containing a functional RAD52 gene, suggest that RAD52-mediated recombination is critical for the normal processing of lethal Cr-induced genetic lesions and exit from G(2) arrest. Furthermore, only the combined inactivation of multiple excision repair genes affects cell growth after Cr(VI) treatment.     

Enhanced clonogenic survival induced by protein tyrosine phosphatase (PTP) inhibition after Cr(VI) exposure is mediated by c-Raf and Ras activity

Our recent studies showed that maintenance of protein tyrosine phosphorylation by PTP inhibition enhanced cell growth, clonogenic survival, and mutagenesis after a single low-level Cr(VI) exposure, thereby suggesting that tyrosine phosphorylation-dependent signaling may govern inappropriate survival in human lung fibroblasts (HLFs). Our goal is to identify specific phospho-tyrosine regulator(s)/ downstream effectors involved in enhanced survival after Cr(VI) exposure and PTP inhibition. Phosphotyrosine profiling array showed that PTP inhibition following Cr(VI) exposure increased tyrosine phosphorylation of specific proteins, such as FGR and ABL, which are upstream regulators of both Erk and Akt pathways. To explore the roles of these pathways in the PTP-induced increase in clonogenic survival after Cr(VI) exposure, we examined the effect of combined Akt1 and Erk1/2 knockdown via siRNA technology. Akt1 and/or Erk1/2 silencing had no effect on the PTP inhibitor-induced increase in survival following Cr(VI) exposure, suggesting the presence of non-Akt/non-Erk-mediated survival signaling. Interestingly, geldanamycin, an HSP90 inhibitor and non-specific Raf inhibitor, abrogated the PTP inhibitor-mediated increase in survival following Cr(VI) exposure and abolished the expression/activity of c-Raf and activity of Mek. These findings prompted us to explore upstream regulators of Erk, i.e., Ras, c-Raf and Mek for their potential roles in clonogenic survival. GW5074, a specific c-Raf kinase inhibitor did not alter the effect of the PTP inhibitor but decreased Cr(VI)-mediated clonogenic lethality, potentially though Mek hyperactivation. A genetic approach with a c/a Mek1 mutant also showed that Mek activity was not directly associated with the PTP inhibitor effect. Finally, a genetic approach with d/n or c/a Ras and c-Raf mutants, showed that Ras and c-Raf activities play a substantive role in enhancing clonogenic survival by PTP inhibition following Cr(VI) insult. In conclusion, these studies highlight a novel pro-survival mechanism for clonogenic survival in the face of genotoxic stress in the presence of PTP inhibition via an Erk/Mek-independent and Ras/c-Raf-dependent regulation in normal human lung fibroblasts.     

Role of reactive oxygen species and p53 in chromium(VI)-induced apoptosis

Apoptosis is a programmed cell death mechanism to control cell number in tissues and to eliminate individual cells that may lead to disease states. The present study investigates chromium(VI) (Cr(VI))-induced apoptosis and the role of reactive oxygen species (ROS) and p53 in this response. Treatment of human lung epithelial cells (A549) with Cr(VI) caused apoptosis as measured by DNA fragmentation, mitochondria damage, and cell morphology. Cr(VI)-induced apoptosis is contributed to ROS generation, resulting from cellular reduction of Cr(VI) as measured by flow cytometric analysis of the stained cells, oxygen consumption, and electron spin resonance spin trapping. Scavengers of ROS, such as catalase, aspirin, and N-acetyl-L-cysteine, decreased Cr(VI)-induced apoptosis, whereas NADPH and glutathione reductase, enhancers of Cr(VI)-induced ROS generation, increased it. p53 is activated by Cr(VI), mostly by ROS-mediated free radical reactions. Cr(VI)-induced ROS generation occurred within a few minutes after Cr(VI) treatment of the cells, whereas p53 induction took at least 5 h. The level of Cr(VI)-induced apoptosis was similar in both p53-positive cells and p53-negative cells independent of p53 status in the early stage (0-3 h) of Cr(VI) treatment. However, at the later stage (3-24 h), the level of the apoptosis is higher in p53-positive cells than in p53-negative cells. These results suggest that ROS generated through Cr(VI) reduction is responsible to the early stage of apoptosis, whereas p53 contributes to the late stage of apoptosis and is responsible for the enhancement of Cr(VI)-induced apoptosis at this stage.     

Critical role of chromium (Cr)-DNA interactions in the formation of Cr-induced polymerase arresting lesions

The genotoxicity associated with the metabolic reduction of hexavalent chromium [Cr(VI)] is complex and can impede DNA polymerase-mediated replication in vitro. The exact biochemical nature of Cr-induced polymerase arresting lesions (PALs) is not understood, but is believed to involve the formation of Cr-DNA interstrand cross-links (ICLs). The aim of this investigation was to determine the dependence of direct Cr-DNA interactions on the development of PALs in DNA treated with trivalent Cr [Cr(III)] or with Cr(VI) in the presence of ascorbic acid (Asc), a major intracellular reductant, using an in vitro, acellular system. The formation of Cr-DNA adducts, ICLs, and PALs was maximal at Asc:Cr(VI) molar ratios of 0.5-2, but gradually decreased at higher ratios. EDTA, a Cr(III) chelator, significantly decreased Cr-DNA binding and ICL and PAL formation. Co-treatment of DNA with Cr(VI)/Asc and mannitol, a Cr(V) chelator, selectively inhibited the formation of mono/bifunctional DNA adducts and PALs produced by Cr(VI) reduction, but had no effect on Cr(III)-DNA binding or Cr(III)-induced polymerase arrest. Blocking Cr-DNA phosphate interaction by preincubation of DNA with MgCl(2) abrogated DNA binding and ICL and PAL production. DNA strand breaks and abasic sites may lead to the in vitro arrest of DNA polymerases; however, we failed to detect significant increases in the frequency of these lesions following Cr(VI)/Asc treatment. These data indicate that the bifunctional adduction of Cr to DNA phosphates (ICLs) constitutes a major PAL. Furthermore, the generation of DNA strand breaks and abasic sites by Cr(VI) reduction is insufficient to explain PALs observed in vitro.     

The ATM-SMC1 pathway is essential for activation of the chromium[VI]-induced S-phase checkpoint

Hexavalent chromium (Cr[VI]) is a common industrial waste product, an environmental pollutant, and a recognized human carcinogen. Following cellular uptake, Cr[VI] can cause DNA damage, however, the mechanisms by which mammalian cells respond to Cr-induced DNA damage remain to be elucidated. Using single cell gel electrophoresis (e.g., Comet Assay) and immunofluoresence microscopy to detect the presence of gamma-H2AX foci, we find that Cr[VI] induces DNA double-strand breaks similar to ionizing radiation (IR). We also demonstrated that ataxia telangiectasia mutated (ATM) is activated in response to Cr[VI] and exposure to Cr[VI] triggers a dose and ATM-dependent S-phase arrest. Further, we document that ATM is required for phosphorylation of the structural maintenance of chromosome protein 1 (SMC1). Finally, we find that ATM-dependent phosphorylation of SMC1 is required to facilitate S-phase cell-cycle arrest in response to Cr[VI] exposure. Collectively, these results indicate that the ATM-SMC1 pathway plays a critical role in cellular response to Cr[VI].     

A role for Mus81 in the repair of chromium-induced DNA damage

Hexavalent chromium (Cr[VI]) is a toxic environmental contaminant that is capable of producing a broad spectrum of DNA damage. The ability of Cr[VI] to induce mutagenesis and neoplastic transformation has been attributed to its genotoxic action, however our understanding of molecular mechanisms involved in the repair of Cr[VI]-induced DNA damage remains incomplete. Here, we report that Mus81, an enzyme that participates with Eme1 in the resolution of replication fork damage caused by certain lesions, is involved in the repair of Cr[VI]-induced DNA damage. Mus81-deficient cells were found to be more susceptible to Cr[VI]-induced proliferation arrest and more sensitive to the long-term cytotoxic effects of Cr[VI] than isogenic wild-type cells. Following Cr[VI] exposure, Mus81-deficient cells displayed a lag in the disappearance of Rad51 foci, exhibited elevated replication-associated γ-H2AX and showed an increased incidence of chromosomal instability compared to wild-type cells. Our findings support a role for Mus81 in the resolution of replication-associated DNA damage associated with this genotoxic agent, by converting Cr[VI]-DNA lesions into a form more amenable for homologous recombination.     

Response of antioxidant defense system to chromium (VI)-induced cytotoxicity in human diploid cells

The aim of this study is to establish antioxidant indicators of chromium toxicity in fetal human lung fibroblasts (HLF). The results obtained corroborate and develop our earlier observation of low-dose and long-term action of Cr(VI) on human cells in culture. In the case of a nontoxic chromium dose, temporary oxidative stress is overcome by increased activity of the antioxidant system with correlation to cell cycle re-entry. The toxic concentrations misbalance the cell antioxidant defense systems and cause irreversible growth arrest and massive cell death by apoptosis. Sub-toxicity is defined as toxicity stretched in time. The activity of GPx (glutathione peroxidase) is proposed as a biomarker of oxidative stress caused by Cr(VI), and the GR (glutathione reductase) inhibition is considered as a marker of the toxicity developed under the complex Cr(VI) action. In HLF cells the glutathione dependent defense system is the first system destroyed in response to toxic chromium action. Only the balance between SOD (superoxide dismutase) and H₂O₂ degrading enzymes (catalase and GPx), should play an important role in the fate of a cell, not individual enzymes.     

Involvement of the p38 MAP kinase in Cr(VI)-induced growth arrest and apoptosis

Hexavalent chromium [Cr(VI)] is a carcinogenic genotoxin commonly found in industry and the environment. DNA damage resulting from Cr(VI) exposure triggers numerous stress responses, including activation of cell cycle checkpoints and initiation of apoptosis. Mechanisms controlling these responses, while extensively studied, have yet to be fully elucidated. Here, we demonstrate that the p38 mitogen-activated protein kinase (MAPK) is activated by Cr(VI) exposure and that inhibition of p38 function using the selective inhibitor SB203580 results in abrogation of S-phase and G2 cell cycle checkpoints in response to Cr(VI). Also, we observe that inhibition of p38 results in decreased cell survival and increased percentage of apoptotic cells following Cr(VI) treatment. Taken together, these results indicate that p38 function is critical for optimal stress response induced by Cr(VI) exposure.     

Gene expression profile in response to chromium-induced cell stress in A549 cells

Chromium (Cr) is a trace element required for life. Biological activities of Cr are complicated and remain to be fully investigated. It is known that the valence of Cr plays an important role in the biological activities of Cr. For example, Cr (VI) is classified as a metal carcinogen, but Cr (III) is widely used as a nutritional supplement. Establishment of a gene expression profile for Cr-induced cellular response is necessary to facilitate investigation of the biological activities of Cr. In the present study, a large-scale gene expression analysis was conducted using RNA of human lung epithelial cells after in vitro exposure to Cr (VI). Utilizing high-density oligonucleotide arrays representing 2400 genes, we observed that expression of 150 genes was up-regulated, and that of 70 genes were down-regulated by Cr (VI). Functional analysis of these responsive genes led to an outline of potential biological activities of Cr in six aspects. The gene expression profile reveals that Cr may involves in redox stress, calcium mobilization, energy metabolism, protein synthesis, cell cycle regulation and carcinogenesis in the cell. The results provide a critical clue for understanding molecular mechanisms of the biological activities of Cr.     

Chromium induces chromosomal instability,which is partly due to deregulation of BubR1 and Emi1, two APC/C inhibitors

Disruption of cell cycle checkpoints and interference with the normal cell cycle progression frequently result in cell death or malignant transformation. Hexavalent chromium [Cr(VI)] is a well-known carcinogen that has been implicated in the occurrence of many types of human malignancies, including lung cancer. However, the exact mechanism by which Cr(VI) causes malignant transformation in the lung remains unknown. We have demonstrated that chronic exposure to a noncytotoxic concentration of Cr(VI) induced a variety of chromosomal abnormalities, including premature sister chromatid separation, chromosomal breakage and the presence of lagging/misaligned chromosomes. After treatment with nocodazole, both HeLa and normal lung bronchial epithelial cells were arrested at mitosis. However, Cr(VI) significantly compromised M-phase arrest induced by nocodazole. Cr(VI) suppressed BubR1 activation and reduced expression of Emi1, leading to an unscheduled activation of APC/C. Consistent with this observation, Cr(VI) treatment caused enhanced polyubiquitination of geminin during mitotic release, while it deregulated the activity of Cdt1, a DNA replication licensing factor. Combined, these results suggest that Cr(VI)-induced chromosomal instability is partly due to a perturbation of APC/C activities, leading to chromosomal instability.Key words: chromium, checkpoint, chromosome instability, APC/C, BubR1, Emi1