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.     

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr–DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA–Cr–protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr–DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr–DNA adducts processed by NER, the incision of CrCl₃ [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl₃) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 μM we observed 2 Cr(III)–DNA adducts per plasmid. At this same concentration of Cr(III) we found that 17% of the plasmid DNA contained ICLs (0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 μM) was incubated with Bca UvrABC we observed 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)–DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.     

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

Acquisition 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 5 μM 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 24 h 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.     

Soluble vs insoluble hexavalent chromate

Soluble CaCrO₄ and insoluble PbCrO₄ were tested for induction of mutation to 6-thioguanine (base-substitution, deletion, addition, and frameshift mutations) or ouabain (base-substitution mutations) resistance in Chinese hamster ovary cells and morphological transformation in C3H/101/2 mouse embryo cells. CaCrO₄ induced dose-dependent cytotoxicity and mutation to 6-thioguanine resistance, but did not induce mutation to ouabain resistance or morphological transformation. Highly cytotoxic amounts of CaCrO₄ induced conversion of 10T1/2 cells to adipocytes, but cell lines derived from such cells were not transformed. PbCrO₄ was not mutagenic in either mutation assay but induced a dose-dependent, low frequency of focus formation. Cell lines established from these foci had a 3–5-fold increased saturation density, grew in soft agarose, and were tumorigenic in nude mice. Chronic exposure to CaCrO₄ or PbCl₂ did not induce transformation, PbCl₂ was inactive even at acutely cytotoxic concentrations, and sequential treatments with CaCrO₄ and PbCl₂ did not induce transformation. Light and scanning electron microscopy showed progressive cytoplasmic engulfment of PbCrO₄ particles and extensive vacuolization of cells in contact with the particles. No particles were observed inside of vacuoles. We suggest that internalization of PbCrO₄ and the associated cellular stress response may be related to PbCrO₄-induced neoplastic transformation of 10T1/2 cells.     

FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure: Activation is not required for repair of Cr(VI)-induced DSBs

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by γ-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult.     

Ca(2+)-regulated serine protease associated with the nuclear scaffold.

The nuclear scaffold (NS) is a proteinaceous network of orthogonally arrayed intermediate filament proteins, termed lamins, which is responsible for nuclear structure. Recent work has demonstrated that a subset of lamins A/C is proteolytically cleaved to produce an ATP-binding protein. This proteolytic cleavage is accomplished by a NS protease activity, which shows a considerable selectivity for lamins A/C and is stringently regulated by Ca2+ in vitro, suggesting that it might also participate in control of NS breakdown in various scenarios. Here, we identify the major NS protease as a novel serine protease with a predominantly chymotryptic-like substrate preference, and we show that even transient perturbations in cytosolic Ca2+ have significant effects on the NS protease activity. This NS protease activity shows extensive similarities to the multicatalytic proteinase complex. In addition to a potential role in control of NS breakdown at mitosis and/or under pathological conditions, this NS protease is also strategically located for other functions, such as inactivation of various oncogenic proteins or maturation-promoting factor.     

FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure: activation is not required for repair of Cr(VI)-induced DSBs

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by gamma-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.