Yy. Yang et al., HUMAN METALLOTHIONEIN ISOFORM GENE-EXPRESSION IN CISPLATIN-SENSITIVE AND RESISTANT CELLS, Molecular pharmacology, 45(3), 1994, pp. 453-460
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.