Constitutive Activation of Epidermal Growth Factor Receptor Promotes Tumorigenesis of Cr(VI)-transformed Cells through Decreased Reactive Oxygen Species and Apoptosis Resistance Development

Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing to Cr(VI)-induced tumorigenesis.     

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 non-cytotoxic 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.     

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     

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.     

Ascorbate acts as a highly potent inducer of chromate mutagenesis and clastogenesis: linkage to DNA breaks in G2 phase by mismatch repair

Here we examined the role of cellular vitamin C in genotoxicity of carcinogenic chromium(VI) that requires reduction to induce DNA damage. In the presence of ascorbate (Asc), low 0.2–2 μM doses of Cr(VI) caused 10–15 times more chromosomal breakage in primary human bronchial epithelial cells or lung fibroblasts. DNA double-strand breaks (DSB) were preferentially generated in G₂ phase as detected by colocalization of γH2AX and 53BP1 foci in cyclin B1-expressing cells. Asc dramatically increased the formation of centromere-negative micronuclei, demonstrating that induced DSB were inefficiently repaired. DSB in G₂ cells were caused by aberrant mismatch repair of Cr damage in replicated DNA, as DNA polymerase inhibitor aphidicolin and silencing of MSH2 or MLH1 by shRNA suppressed induction of γH2AX and micronuclei. Cr(VI) was also up to 10 times more mutagenic in cells containing Asc. Increasing Asc concentrations generated progressively more mutations and DSB, revealing the genotoxic potential of otherwise nontoxic Cr(VI) doses. Asc amplified genotoxicity of Cr(VI) by altering the spectrum of DNA damage, as total Cr-DNA binding was unchanged and post-Cr loading of Asc exhibited no effects. Collectively, these studies demonstrated that Asc-dependent metabolism is the main source of genotoxic and mutagenic damage in Cr(VI)-exposed cells.     

Metabolism of Cr(VI) by ascorbate but not glutathione is a low oxidant-generating process

Genotoxic activity of hexavalent chromium (chromate) results from its reductive activation inside the cell. Cr(VI) metabolism in vivo is primarily driven by ascorbate (Asc) but in cultured cells by glutathione (GSH). Given the common use of cultured cells for mechanistic studies, it is important to establish whether Cr(VI) activated by Asc and GSH displays the same genotoxic properties. Using 2',7' dichlorofluorescin (DCFH) as a redox sensitive probe, we found that Asc-dependent reduction of Cr(VI) in vitro under physiological conditions generated 25-80 times lower yields of oxidants compared to GSH. When both reducers were present, Asc dominated Cr(VI) metabolism and inhibited DCFH oxidation. Consistent with the findings in defined chemical reactions, restoration of physiological levels of Asc in human lung H460 cells led to the loss of their hypersensitivity to clonogenic killing by Cr(VI) in the presence of methoxyamine, which inhibits base excision repair of oxidative DNA damage. Despite suppressed oxidative damage, Asc-containing cells formed a large number of DNA double-strand breaks after exposure to a dose of Cr(VI) corresponding to the drinking water standard of 100 ppb. Our results indicate that Asc-driven metabolism of Cr(VI) shifts its genotoxicity toward nonoxidative mechanisms.     

Hexavalent chromium,a lung carcinogen,confers resistance to thermal stress and interferes with heat shock protein expression in human bronchial epithelial cells

Exposure to hexavalent chromium [Cr(VI)], a lung carcinogen, triggers several types of cellular stresses, namely oxidative, genotoxic and proteotoxic stresses. Given the evolutionary character of carcinogenesis, it is tempting to speculate that cells that survive the stresses produced by this carcinogen become more resistant to subsequent stresses, namely those encountered during neoplastic transformation. To test this hypothesis, we determined whether pre-incubation with Cr(VI) increased the resistance of human bronchial epithelial cells (BEAS-2B cells) to the antiproliferative action of acute thermal shock, used here as a model for stress. In line with the proposed hypothesis, it was observed that, at mildly cytotoxic concentrations, Cr(VI) attenuated the antiproliferative effects of both cold and heat shock. Mechanistically, Cr(VI) interfered with the expression of two components of the stress response pathway: heat shock proteins Hsp72 and Hsp90α. Specifically, Cr(VI) significantly depleted the mRNA levels of the former and the protein levels of the latter. Significantly, these two proteins are members of heat shock protein (Hsp) families (Hsp70 and Hsp90, respectively) that have been implicated in carcinogenesis. Thus, our results confirm and extend previous studies showing the capacity of Cr(VI) to interfere with the expression of stress response components.     

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.     

Appearance of receptors for Macaca speciosa red blood cells on human fibroblasts transformed by simian virus 40

Receptors for monkey red blood cells (MRBC) that are expressed on subpopulations of human lymphoid cells are coded by genes linked to the major histocompatibility complex (MHC) region. Since malignant transformation of cells is associated with changes in structures coded by the MHC region, 10 cultured human melanoma and sarcoma cells and autologous SV40-transformed fibroblasts were tested for expression of MRBC receptors and compared with normal autologous fibroblasts. Only 2 of the tumor cell lines and normal fibroblasts from the same individual formed rosettes with MRBC. On the other hand, SV40 transformation induced in all the fibroblasts expression of receptors for MRBC. MRBC receptors on SV40-transformed fibroblasts show properties similar to those on B lymphoid cells.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line

We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, down-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.     

Proteomic identification of betaig-h3 as a lysophosphatidic acid-induced secreted protein of human mesenchymal stem cells: paracrine activation of A549 lung adenocarcinoma cells by betaig-h3

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.     

ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.     

Deficient repair of particulate hexavalent chromium-induced DNA double strand breaks leads to neoplastic transformation

Hexavalent chromium (Cr(VI)) is a potent respiratory toxicant and carcinogen. The most carcinogenic forms of Cr(VI) are the particulate salts such as lead chromate, which deposit and persist in the respiratory tract after inhalation. We demonstrate here that particulate chromate induces DNA double strand breaks in human lung cells with 0.1, 0.5, and 1 microg/cm(2) lead chromate inducing 1.5, 2, and 5 relative increases in the percent of DNA in the comet tail, respectively. These lesions are repaired within 24 h and require Mre11 expression for their repair. Particulate chromate also caused Mre11 to co-localize with gamma-H2A.X and ATM. Failure to repair these breaks with Mre11-induced neoplastic transformation including loss of cell contact inhibition and anchorage-independent growth. A 5-day exposure to lead chromate induced loss of cell contact inhibition in a concentration-dependent manner with 0, 0.1, 0.5, and 1 microg/cm(2) lead chromate inducing 1, 78, and 103 foci in 20 dishes, respectively. These data indicate that Mre11 is critical to repairing particulate Cr(VI)-induced double strand breaks and preventing Cr(VI)-induced neoplastic transformation.