CHROMIUM(VI) TREATMENT OF NORMAL HUMAN LUNG-CELLS RESULTS IN GUANINE-SPECIFIC DNA-POLYMERASE ARREST, DNA-DNA CROSS-LINKS AND S-PHASE BLOCKADE OF CELL-CYCLE

Previous studies have shown that in vitro treatment of a synthetic dou ble-stranded DNA template with chromium(III), or chromium(VI) in the p resence of ascorbate, resulted in guanine-specific DNA polymerase arre sts that correlated strongly with DNA-DNA cross-linking, In vivo chrom ium(VI) undergoes a more complicated intracellular cascade of reductiv e metabolism than is achievable in an in vitro model, Moreover, in liv ing cells, DNA is highly packaged in the form of chromatin which may a lter the accessibility of DNA to chromium. A repetitive primer-extensi on assay was employed to determine whether chromium forms polymerase-a rresting lesions in vivo, Normal human lung fibroblasts treated with c hromium(VI) exhibited adduct levels of 0.13-0.92 mmol Cr/mol DNA-nucle otides in the total genome (0.26-1.84 Cr adducts/Kbp DNA) and DNA inte rstrand cross-links, Genomic DNA was isolated and alphoid sequences (1 -5% of the genome) were used as a substrate for repetitive primer exte nsion using Tag polymerase. The results showed a dose-dependent, guani ne-specific, replication termination, even at low doses resulting in g reater than 90% survival, The same treatment resulted in a dose-depend ent suppression of thymidine incorporation into DNA immediately after treatment, Thymidine incorporation increased during the first 6 h afte r the 2-h exposure, probably related to the repair of single strand br eaks, but then returned to high suppression levels at 24 h, The chroma te treatments inhibited cell growth by specific blocking of the progre ssion of cells through S-phase of the cell cycle, The results confirme d our studies in cell-free systems and taken together they strongly in dicate that guanine-guanine DNA interstrand cross-links induced by chr omate in living cells is the lesion responsible for blocking DNA repli cation processivity.