Gene Therapy and Molecular Biology Vol 12, page 229 clastogenic effects of both water-soluble and insoluble Cr(VI) compounds (Sen and Costa 1986; Wise et al, 1994, 2002, 2006; Seoane et al, 2002; O’Brien et al, 2003; Glaviano et al, 2005; Holmes et al, 2006;). However, the exposure consequences are different, since particulate Cr(VI) was reported to induce an increase in both the number of metaphases with too few chromosomes (hypodiploidy) and the number of metaphases with twice the number of chromosomes (tetraploidy) in human lung fibroblasts (Holmes et al, 2006), whereas soluble Cr(VI) [sodium dichromate] induced only hypodiploidy (Seoane et al, 2002). These different clastogenic outcomes appear to be dependent not only on the solubility characteristics of the Cr(VI) compound but also on the cell line and exposure regimens used. In the case of particulate Cr(VI), exposures to very low and low to moderate concentrations lead, in human lung fibroblasts, to a long-term increase in tetraploid cells, while short-term exposures to low and moderate concentrations induced an increase in hypodiploid cells (Holmes et al, 2006; Xie et al, 2007). In addition, short-term exposures to very low concentrations did not appear to have any effect on centrosome amplification. In contrast, low to moderate doses induced, both in human lung fibroblasts and human bronchial epithelial cells, centrosome amplification (Holmes et al, 2006; Xie et al, 2007). These findings lead to the hypothesis that, in the aforementioned cell lines, centrosome amplification was inducing hypodiploidy, whereas tetraploidy was induced by a different mechanism possibly by effects on the spindle assembly checkpoint (Holmes et al, 2006). It is important to note that hypodiploidy was reported to occur before tetraploidy in human lung cells exposed to low to moderate particulate Cr(VI) concentrations (Holmes et al, 2006). In contrast, in cells exposed to soluble Cr(VI), tetraploidy was the first outcome of centrosome amplification (Güerci et al, 2000; Seoane et al, 2002). Thus, there are apparently different mechanisms for Cr(VI)-induced hypodiploidy and tetraploidy (Holmes et al, 2006) which cannot be only ascribed to the solubility and the exposure regimen (concentration and exposure time) to the Cr(VI) compound. The cell line used appears to be the key feature. In fact, the same exposure regime to particulate Cr(VI) induced only hypodiploidy in hTERTimmortalised human lung epithelial cells, while in human lung fibroblasts tetraploid cells exceeded by far the number of hypodiploid cells (Holmes et al, 2006). It is possible that these dissimilar results may be explained by Cr(VI) effects on hTERT expression, since recent studies revealed that although hTERT protected cells against many features of Cr(VI)-induced genomic instability, it allowed Cr(VI) to induce tetraploidy rather than aneuploidy (Glaviano et al, 2005). Aberrant mitotic figures (lagging metaphase, cmetaphase and ball metaphase, as well as lagging and disorganized anaphase and mitotic catastrophe) (Holmes et al, 2006) may explain Cr(VI) effects on chromosome instability, particularly the tetraploid phenotype, one of the hallmarks of lung cancer (Masuda and Takahashi, 2002). Both soluble and particulate Cr(VI) compounds were also reported to induce structural chromosome
modifications (chromatid lesions, isochromatid lesions, dicentric chromosomes and centromere spreading) in both human lung epithelial cells and human lung fibroblasts (Manning et al, 1994; Wise et al, 2002, 2003, 2004b, 2006; Xie et al, 2004; Xie et al, 2007). All these findings are in line with the epidemiological evidence suggesting that chromosomal abnormalities and genomic instability may be involved in the induction of human lung cancer by Cr(VI) (Kondo et al, 1997; Hirose et al, 2002; Takahashi et al, 2005). Therefore, determining how Cr(VI) causes genomic instability will be a significant step forward in the prevention of Cr(VI)-induced cancers and events that are important to lung cancer progression in general and, ultimately, in the design of new treatment approaches.
C. Genomic instability: the success of proliferating pathways and the failure of apoptotic pathways Although undoubtedly relevant, the establishment of a whole spectrum of Cr(VI)-induced DNA lesions cannot, by itself, explain Cr(VI)-induced toxicity and carcinogenicity and a lot more effort will have to be put into the elucidation of the signalling pathways mediating the cellular responses to Cr(VI) exposure. In fact, it must be acknowledge that, in terms of signalling pathways, many of the results obtained to this day are probably seriously compromised by the use of inadequate systems and/or of exposure regimens that are not representative of any toxicologically relevant human exposures. In particular, which signalling pathways are activated and their role on genomic instability and consequently in lung cancer onset and progression needs to be established. In vitro studies revealed that extensive Cr-DNA interactions play a critical role in DNA polymerase arrest (O’Brien et al, 2002) and on the processivity of both prokaryotic and mammalian DNA and RNA polymerases (Bridgewater et al, 1994a,b, 1998; Xu et al, 1996; O’Brien et al, 2001, 2002), leading generally to apoptosis. Instead, low levels of DNA damage can induce the activation and/or the inactivation of signalling pathways which may contribute to Cr(VI)-induced genomic instability because they allow cells with unrepaired DNA damage to progress through the cell cycle. One such a signalling pathway is mediated by the ataxia telangiectasia mutated kinase (ATM), a serine/threonine kinase that belongs to a family of large proteins that contain the phosphatidylinositol 3-kinaserelated domain (Beamish et al, 1996). This protein is a key element in multiple biochemical pathways linking, through phosphorylation of various substrates, the recognition and repair of chromatin structure lesions (ICLs and doublestrand breaks) to downstream cellular processes, such as activation of cell cycle checkpoints, DNA repair, apoptosis and also cell proliferation (Kastan and Lim, 2000) (Figure 7). As revealed by in vitro studies, in normal human dermal fibroblasts and human bronchial cells, Cr(VI)induced double-strand breaks formation activates ATM which was shown to be a major signal initiator for Cr(VI)induced apoptosis that also contributes to cell survival by facilitating recovery/escape from terminal growth arrest 229