Ultraviolet light exposure triggers nuclear accumulation of p21^WAF1 and accelerated senescence in human normal and nucleotide excision repair‐deficient fibroblast strains

Abstract

Induction of the p21(WAF1) protein (hereafter called p21) following genotoxic stress is known to inhibit proliferating cell nuclear antigen (PCNA)-dependent DNA repair, downregulate apoptosis, and trigger a sustained growth-arrested phenotype called accelerated senescence. Studies with immortalized human and murine cell lines have revealed that exposure to ultraviolet light (UVC; 254 nm) results in the degradation of p21 to facilitate DNA repair and promote cell survival, or may lead to apoptotic cell death. The objective of the present study was to determine whether exposure of non-transformed human fibroblast strains to relatively low fluences of UVC (i.e., fluences typically used in the clonogenic survival assay) might induce sustained nuclear accumulation of p21, leading to accelerated senescence. We have evaluated the responses of normal human fibroblast (NHF) strains and nucleotide excision repair (NER)-deficient fibroblast strains representing xeroderma pigmentosum (XP) complementation groups A and G and Cockayne syndrome (CS) complementation groups A and B. We report that exposure of NHFs to < or =15 J/m(2) of UVC, and NER-deficient fibroblasts to < or =5 J/m(2) of UVC, results in sustained nuclear accumulation of p21 and growth arrest through accelerated senescence. With each fibroblast strain examined, exposure to UVC fluences that resulted in approximately 90% loss of clonogenic potential triggered significant (>60%) accelerated senescence, but only marginal (<5%) apoptosis. We conclude that nuclear accumulation of p21 accompanied by accelerated senescence may be an integral component of the response of human fibroblasts to UVC-induced DNA damage, irrespective of their DNA repair capabilities.

Human Genetic Disorders Associated with Genome Instability, Premature Aging and Cancer Predisposition

“…Thus, normal fibroblast cultures contained a significant proportion of apoptotic cells after exposure to supralethal fluences of UVC (e.g., 30 J/m 2 , resulting in >99% loss of clonogenic potential), but not after exposure to 15 J/m 2 or lower fluences [87]. In addition, we have recently demonstrated the existence of a threshold for UVC-induced apoptosis in NER-deficient fibroblast strains representing the XP-A, XP-G, CS-A and CS-B complementation groups, albeit shifted to lower fluences as compared to the threshold seen with normal fibroblasts [113]. We observed little, if any, induction of apoptosis in normal human fibroblasts exposed to 15 J/m 2 , in XP-A and XP-G fibroblasts exposed to 2 J/m 2 , and in CS-A and CS-B fibroblasts exposed to 4 J/m 2 .…”

“…We observed little, if any, induction of apoptosis in normal human fibroblasts exposed to 15 J/m 2 , in XP-A and XP-G fibroblasts exposed to 2 J/m 2 , and in CS-A and CS-B fibroblasts exposed to 4 J/m 2 . These fluences of UVC cause more than 90% overall cell killing in the clonogenic assay [87,113]. On the other hand, exposure to these fluences triggered accelerated senescence, as evident from the sustained nuclear accumulation of p21 protein accompanied by the development of cells that exhibit flattened and enlarged morphology, cease to divide, retain viability and remain adherent for prolonged times (e.g., 7 days) after UVC exposure, and express high levels of the senescence marker senescence-associated--galactosidase [113].…”

“…These fluences of UVC cause more than 90% overall cell killing in the clonogenic assay [87,113]. On the other hand, exposure to these fluences triggered accelerated senescence, as evident from the sustained nuclear accumulation of p21 protein accompanied by the development of cells that exhibit flattened and enlarged morphology, cease to divide, retain viability and remain adherent for prolonged times (e.g., 7 days) after UVC exposure, and express high levels of the senescence marker senescence-associated--galactosidase [113]. Collectively, these results demonstrated that: (i) sustained nuclear accumulation of p21 associated with a proliferative block through the process of accelerated senescence is an integral component of the response of NER-proficient and -deficient human fibroblast cultures to relatively low fluences of UVC that are typically used in the clonogenic assay; and (ii) apoptosis does not appear to contribute significantly to the loss of clonogenic potential of non-transformed human fibroblasts exposed to such fluences of UVC.…”

Molecular bio-dosimetry for carcinogenic risk assessment in survivors of Bhopal gas tragedy

“…Metaphase spreads obtained from treated cells showed an array of structural chromosomal aberrations in the form of chromatid fragmentation, p-p arm Robertsonian fusions, premature centromeric separation, dicentrics, chromatid breaks, ring, end-to end fusion, endo-reduplication, markers, double minutes, tri-or quadric-radials. Simultaneously, evaluation of the numerical cytogenetic index revealed a significant percent gain and loss of whole chromosomal number indicating propensity towards aneuploidy and IJOMEH 2015;28(6) 931 changes, thereby compromising the tissue architecture and function [60]. Consistent with this, our investigation in B/CMBA.Ov cells showed an increase in the cell size, a characteristic feature of senescence with early induction of positive staining for β-galactosidase in MIC exposed cells in-advance with large granular nucleus and focal enrichment of lysosome-related β-galactosidase activity at vacuoles which persisted progressively along the incremental time course [47].…”

“…Toxic oxygen metabolites such as hydroxyl radical can covalently attack nucleophilic sites of nucleic acids causing base hydroxylation, cross-linking or scission of DNA strands and wreak havoc in cells by causing single-or double-strand DNA breakage which could lead to mutagenic effects [42]. Due to their electron loving chemistry, isocyanates have been known to react with exocyclic amino group of deoxynucleotides (dNTP's) to yield carbamoylated DNA cross links/adducts and thus attribute to the biotransformation reactions [43,44].…”