HUMAN METALLOTHIONEIN ISOFORM GENE-EXPRESSION IN CISPLATIN-SENSITIVE AND RESISTANT CELLS

Overexpression of metallothioneins (MTs) has been observed in some cis -diamminedichloroplatinum (CDDP)-resistant cells. We have developed ol igonucleotide probes for each of the six non-neuronal human MT (hMT) i soforms and used them to assay hMT isoform expression in three pairs o f CDDP-resistant and -sensitive human carcinoma cell lines, i.e., SCC2 5/CP versus SCC25 cells, H69/CP versus H69 cells, and SW2/CP versus SW 2 cells. We found a 9-fold increase in basal hMT-IIa mRNA levels and a 5-fold increase in hMT-Ie mRNA levels in SCC25/CP cells, compared wit h SCC25 cells. Nuclear run-on studies also revealed a 3-fold increase in hMT-IIa transcription rate. Basal hMT-IIa steady state mRNA levels were 2-3.6-fold greater in H69/CP and SW2/CP cells, compared with thei r parental cells. No significant basal expression of hMT-Ia, -Ib, -If, or -Ig was detected in any cells, suggesting that overexpression of t hese isoforms was not commonly associated with the CDDP-resistant phen otype. Levels of constitutively expressed hMT isoforms, as well as hMT -If, could be elevated by treatment of all cells with 100 muM zinc. Th e universal overexpression of hMT-IIa suggests a role of this particul ar isoform in CDDP resistance. Using our isoform-specific hMT-IIa prob e and the demethylating agent 5'-azacytidine (AZC), we found that AZC pretreatment increased basal hMT-IIa mRNA levels in SCC25 but not SCC2 5/CP cells, suggesting that DNA hypomethylation was responsible for hi gher basal hMT-IIa mRNA levels in SCC25/CP cells. AZC had little or no effect on hMT-If or -Ig expression. Limited restriction analysis by m ethylation-sensitive enzymes, however, revealed no obvious differences in the methylation status of the hMT-IIa promoter in either SCC25 or SCC25/CP cells.