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

Abstract

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.

A bioenergetic profile of non-transformed fibroblasts uncovers a link between death-resistance and enhanced spare respiratory capacity

“…Two death-resistant cell lines (DR1, DR2), as well as a death-susceptible clonogenic control line (CC1) were the focus of a recently published report from our laboratory (Nickens et al, 2012). Our data showed that, as compared to the death-susceptible cells, the DR cells were resistant to apoptosis as indicated by lack of cleaved caspase 3 expression following exposure to both Cr(VI) and cisplatin.…”

“…Subclonal populations were derived as previously described (Nickens et al, 2012). The cell lines used in the present study include untreated clonogenic control cell lines, CC1 and CC2, as well as clones derived from clonogenic survivors of Cr(VI) exposure, DR1, DR2, DR3, and DR4, which display an apoptosis resistant phenotype.…”

“…The cell lines used in the present study include untreated clonogenic control cell lines, CC1 and CC2, as well as clones derived from clonogenic survivors of Cr(VI) exposure, DR1, DR2, DR3, and DR4, which display an apoptosis resistant phenotype. As previously reported, all CC and DR cell lines were derived from human foreskin fibroblasts transfected with the hTERT gene (BJ-hTERT; Geron Corp.), and further subcloned (at passage 138) into both CC and DR cell lines (Nickens et al, 2012; Pritchard et al, 2005). The original hTERT-immortalized foreskin fibroblast cell line, BJ-5ta, was derived by transfecting the BJ foreskin fibroblast cell line with the pGRN145 hTERT-expressing plasmid (ATCC MBA-141) (Bodnar et al, 1998).…”

“…This system is unique in that it models initial molecular events that occur in a normal cell that survived a single, acute, initiating genotoxic challenge. While the selection model in this study was generated by Cr(VI) treatment, it also exhibits a cross-resistance to the well-known chemotherapeutic agent, cisplatin, as well as to H 2 O 2 (Nickens et al, 2012). Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis (IARC, 1990).…”

“…Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis (IARC, 1990). Our recent report investigated the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with Cr(VI) (DR), or selected by dilution-based cloning without treatment (CC) (Nickens et al, 2012). Notably, our data suggested the presence of more resilient mitochondria in DR cells, and that death resistance can be acquired in normal human cells early after genotoxin exposure.…”