Abstract
P-glycoprotein gene (mdrl) amplification and expression were examined in murine leukaemia P388/DX and melanoma B16VDXR cell lines, which exhibit a high level of resistance to a selecting agent, doxorubicin, and express a multidrug-resistant phenotype because they are cross-resistant to multiple cytotoxic drugs. The multidrug-resistant phenotype was obtained in different conditions of selection (in vivo and in vitro for P388/DX and B16VDXR, respectively). In both multidrug-resistant cell lines, an increased expression of P-glycoprotein gene (5 kb transcript detected in Northern blots) was observed and the level of P-glycoprotein mRNA correlated with the degree of resistance. In addition, high molecular weight mRNAs homologous to mdrl gene sequence were consistently detected only in P388/DX cells. Overexpression was associated with a high level of gene amplification only in resistant melanoma cells, whereas it occurred in P388/DX cells with a marginal increase in gene copy number. These results, suggesting that different genetic mechanisms could be responsible for P-glycoprotein overexpression, emphasise the complexity of genetic regulation that may affect tumour cell sensitivity to cytotoxic agents.
Highlights
High levels of mdrl gene expression were observed in resistant cells, and a low level of expression in sensitive cells
Under conditions of high hybridisation stringency at least two mRNA of around 5.7 and 9.5 kb were detected in autoradiograms of both total and poly(A)+RNA from P388/DX cells, whereas such/ high molecular weight transcripts were not observed either in P388 cells or in sensitive and resistant B16 cells
Our findings show that mdrl gene is overexpressed in P388/DX and B16VDXR cells, strongly suggesting that P-glycoprotein may contribute to the high degree of resistance of these two resistant cell lines (Table I)
Summary
Anthracyclines were obtained from Farmitalia-Carlo Erba (Milan, Italy); vincristine sulphate from Lilly France SA (Fegersheim, France); cisplatin, VP16 and VM26 from Bristol Italiana (Latina, Italy). Restriction endonuclease EcoRI was purchased from Bethesda Research Laboratories (Eggenstein, West Germany); deoxycytidine-5' [Oa-32P]triphosphate (specific activity 3,000 Ci mmol- 1) and restriction endonucleases Hindlll and SalI from Amersham International (Amersham, United Kingdom). Other chemicals of the highest grade were supplied by Fluka (Switzerland). Plasmid pcDR 1.3, containing a 1.3kb insert. Received 15 April 1988, and in revised form, 21 November 1988
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