Acquisition of mitochondrial dysregulation and resistance to mitochondrial-mediated apoptosis after genotoxic insult in normal human fibroblasts: A possible model for early stage carcinogenesis

AbstractAcquisition of death-resistance is critical in the evolution of neoplasia. Our aim was to model the early stages of carcinogenesis by examining intracellular alterations in cells that have acquired apoptosis-resistance after exposure to a complex genotoxin. We previously generated sub-populations of BJ-hTERT human diploid fibroblasts, which have acquired death-resistance following exposure to hexavalent chromium [Cr(VI)], a broad-spectrum genotoxicant. Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis. Here, we report on the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with 5μM Cr(VI) (DR1, DR2), or selected by dilution-based cloning without treatment (CC1). Following Cr(VI) treatment, CC1 cells downregulated expression of the anti-apoptotic protein Bcl-2 and exhibited extensive expression of cleaved caspase 3. In contrast, the DR cells exhibited no cleaved caspase 3 expression and maintained expression of Bcl-2 following recovery from 24h Cr(VI) exposure. The DR cells also exhibited attenuated mitochondrial-membrane depolarization and mitochondrial retention of cytochrome c and SMAC/DIABLO following Cr(VI) exposure. The DR cells exhibited less basal mtDNA damage, as compared to CC1 cells, which correlates with intrinsic (non-induced) death-resistance. Notably, there was no difference in p53 protein expression before or after treatment among all cell lines. Taken together, our data suggest the presence of more resilient mitochondria in death-resistant cells, and that death-resistance can be acquired in normal human cells early after genotoxin exposure. We postulate that resistance to mitochondrial-mediated cell death and mitochondrial dysregulation may be an initial phenotypic alteration observed in early stage carcinogenesis

Androgen Receptor-Target Genes in African American Prostate Cancer Disparities

The incidence and mortality rates of prostate cancer (PCa) are higher in African American (AA) compared to Caucasian American (CA) men. To elucidate the molecular mechanisms underlying PCa disparities, we employed an integrative approach combining gene expression profiling and pathway and promoter analyses to investigate differential transcriptomes and deregulated signaling pathways in AA versus CA cancers. A comparison of AA and CA PCa specimens identified 1,188 differentially expressed genes. Interestingly, these transcriptional differences were overrepresented in signaling pathways that converged on the androgen receptor (AR), suggesting that the AR may be a unifying oncogenic theme in AA PCa. Gene promoter analysis revealed that 382 out of 1,188 genes contained cis-acting AR-binding sequences. Chromatin immunoprecipitation confirmed STAT1, RHOA, ITGB5, MAPKAPK2, CSNK2A,1 and PIK3CB genes as novel AR targets in PCa disparities. Moreover, functional screens revealed that androgen-stimulated AR binding and upregulation of RHOA, ITGB5, and PIK3CB genes were associated with increased invasive activity of AA PCa cells, as siRNA-mediated knockdown of each gene caused a loss of androgen-stimulated invasion. In summation, our findings demonstrate that transcriptional changes have preferentially occurred in multiple signaling pathways converging (“transcriptional convergence”) on AR signaling, thereby contributing to AR-target gene activation and PCa aggressiveness in AAs

Alternative Splicing Promotes Tumour Aggressiveness and Drug Resistance in African American Prostate Cancer.

linical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 (idelalisib), in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa

Alternative Splicing Promotes Tumour Aggressiveness and Drug Resistance in African American Prostate Cancer.

linical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 (idelalisib), in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa

Alternative Splicing Promotes Tumour Aggressiveness and Drug Resistance in African American Prostate Cancer.

linical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 (idelalisib), in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa

Lung Inflammation, Injury, and Proliferative Response after Repetitive Particulate Hexavalent Chromium Exposure

BACKGROUND: Chronic inflammation is implicated in the development of several human cancers, including lung cancer. Certain particulate hexavalent chromium [Cr(VI)] compounds are well-documented human respiratory carcinogens that release genotoxic soluble chromate and are associated with fibrosis, fibrosarcomas, adenocarcinomas, and squamous cell carcinomas of the lung. Despite this, little is known about the pathologic injury and immune responses after repetitive exposure to particulate chromates. OBJECTIVES: In this study we investigated the lung injury, inflammation, proliferation, and survival signaling responses after repetitive exposure to particulate chromate. METHODS: BALB/c mice were repetitively treated with particulate basic zinc chromate or saline using an intranasal exposure regimen. We assessed lungs for Cr(VI)-induced changes by bronchoalveolar lavage, histologic examination, and immunohistochemistry. RESULTS: Single exposure to Cr(VI) resulted in inflammation of lung tissue that persists for up to 21 days. Repetitive Cr(VI) exposure induced a neutrophilic inflammatory airway response 24 hr after each treatment. Neutrophils were subsequently replaced by increasing numbers of macrophages by 5 days after treatment. Repetitive Cr(VI) exposure induced chronic peribronchial inflammation with alveolar and interstitial pneumonitis dominated by lymphocytes and macrophages. Moreover, chronic toxic mucosal injury was observed and accompanied by increased airway pro-matrix metalloprotease-9. Injury and inflammation correlated with airways becoming immuno reactive for phosphorylation of the survival signaling protein Akt and the proliferation marker Ki-67. We observed a reactive proliferative response in epithelial cells lining airways of chromate-exposed animals. CONCLUSIONS: These data illustrate that repetitive exposure to particulate chromate induces chronic injury and an inflammatory microenvironment that may promote Cr(VI) carcinogenesis

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

Generation of S phase-dependent DNA double-strand breaks by Cr(VI) exposure: Involvement of ATM in Cr(VI) induction of γ-H2AX

Certain hexavalent chromium [Cr(VI)] compounds are implicated as occupational respiratory carcinogens. Cr(VI) induces a broad spectrum of DNA damage, but Cr(VI)-induced DNA double-strand breaks (DSBs) have not been reported. Previously we found that Cr(VI) activates the ataxia telangiectasia mutated (ATM) kinase. ATM is activated specifically in response to DSBs. Therefore, the objective of this study was to investigate DSB induction by Cr(VI) exposure with the overarching hypothesis that S phase-dependent DSBs are produced by Cr(VI) exposure. To test this hypothesis, normal human fibroblasts were treated with either Cr(VI) or neocarzinostatin (NCS). DSBs were analyzed by both comet assay under neutral conditions, which detects primarily DNA DSBs, and phosphorylation of histone H2AX (γ-H2AX) and the resultant formation of nuclear foci, which are considered to be indicative of DSBs. Induction of DSBs was observed after Cr(VI) exposure, however, the Cr(VI)-induced DSBs were abrogated by G1 synchronization. Furthermore, our data showed that Cr(VI)-induced DSBs were only observed in the S phase population, whereas no significant DSBs were observed in Cr(VI)-treated G1 synchronized cells. In contrast, NCS-induced DSBs were equally distributed in all cell cycle phases in both asynchronous and G1 synchronized cells. Moreover, Cr(VI -induced γ-H2AX foci formation was restricted to PCNA-positive cells, whereas NCS-induced γ-H2AX foci formed in both PCNA-positive and PCNA-negative cells. These results indicate that Cr(VI -induced DSBs are S phase-dependent. Finally, our data showed that Cr(VI -induced γ-H2AX production was significantly decreased in ATM-/- cells compared with ATM+/+ cells. Taken together, these results suggest that Cr(VI)-induced activation of ATM involves the formation of S phase-dependent DSBs. Examining the mechanism of Cr(VI)-induced DSBs will aid in understanding the inter-related mechanisms of Cr(VI) toxicity and carcinogenesis. © Oxford University Press 2004; all rights reserved

Chromium genotoxicity: A double-edged sword

Certain forms of hexavalent chromium [Cr(VI)] are known respiratory carcinogens that induce a broad spectrum of DNA damage. Cr(VI)-carcinogenesis may be initiated or promoted through several mechanistic processes including, the intracellular metabolic reduction of Cr(VI) producing chromium species capable of interacting with DNA to yield genotoxic and mutagenic effects, Cr(VI)-induced inflammatory/immunological responses, and alteration of survival signaling pathways. Cr(VI) enters the cell through non-specific anion channels, and is metabolically reduced by agents including ascorbate, glutathione, and cysteine to Cr(V), Cr(IV), and Cr(III). Cr(III) has a weak membrane permeability capacity and is unable to cross the cell membrane, thereby trapping it within the cell where it can bind to DNA and produce genetic damage leading to genomic instability. Structural genetic lesions produced by the intracellular reduction of Cr(VI) include DNA adducts, DNA-strand breaks, DNA-protein crosslinks, oxidized bases, abasic sites, and DNA inter- and intrastrand crosslinks. The damage induced by Cr(VI) can lead to dysfunctional DNA replication and transcription, aberrant cell cycle checkpoints, dysregulated DNA repair mechanisms, microsatelite instability, inflammatory responses, and the disruption of key regulatory gene networks responsible for the balance of cell survival and cell death, which may all play an important role in Cr(VI) carcinogenesis. Several lines of evidence have indicated that neoplastic progression is a result of consecutive genetic/epigenetic changes that provide cellular survival advantages, and ultimately lead to the conversion of normal human cells to malignant cancer cells. This review is based on studies that provide a glimpse into Cr(VI) carcinogenicity via mechanisms including Cr(VI)-induced death-resistance, the involvement of DNA repair mechanisms in survival after chromium exposure, and the activation of survival signaling cascades in response to Cr(VI) genotoxicity. © 2010 Elsevier Ireland Ltd