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 oligonucleotide probes for each of the six non-neuronal human MT (hMT) isoforms and used them to assay hMT isoform expression in three pairs of CDDP-resistant and -sensitive human carcinoma cell lines, i.e., SCC25/CP versus SCC25 cells, H69/CP versus H69 cells, and SW2/CP versus SW2 cells. We found a 9-fold increase in basal hMT-IIa mRNA levels and a 5-fold increase in hMT-le mRNA levels in SCC25/CP cells, compared with 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 their parental cells. No significant basal expression of hMT-Ia, -Ib, -If, or Ig was detected in any cells, suggesting that overexpression of these isoforms was not commonly associated with the CDDP-resistant phenotype. Levels of constitutively expressed hMT isoforms, as well as hMT-If, could be elevated by treatment of all cells with 100 microM zinc. The universal overexpression of hMT-IIa suggests a role of this particular isoform in CDDP resistance. Using our isoform-specific hMT-IIa probe and the demethylating agent 5'-azacytidine (AZC), we found that AZC pretreatment increased basal hMT-IIa mRNA levels in SCC25 but not SCC25/CP cells, suggesting that DNA hypomethylation was responsible for higher 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 methylation-sensitive enzymes, however, revealed no obvious differences in the methylation status of the hMT-IIa promoter in either SCC25 or SCC25/CP cells.
Yang
Y Y
YY
Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Woo
E S
ES
Reese
C E
CE
Bahnson
R R
RR
Saijo
N
N
Lazo
J S
JS
D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology*; D004351:Drug Resistance; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D008670:Metals; D012333:RNA, Messenger; D008668:Metallothionein; D001374:Azacitidine; D002945:Cisplatin