Extracellular matrix regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes.
Previous studies reported that, in the absence of drug exposure, multidrug resistance, including resistance to Adriamycin (ADR), could develop in primary rat hepatocyte cultures (B. Carr, Proc. Am. Assoc. Cancer Res., 29:1158, 1988). However, the hepatocytes in that report were cultured on plastic without the benefit of an extracellular matrix (ECM). Because the ECM regulates hepatic gene expression, we have critically evaluated in primary cultures of rat hepatocytes how the ECM affects hepatic ADR resistance, the level of the drug efflux transporter associated with MDR, P-glycoprotein (pgp), and transport of a prototypical pgp substrate, vincristine. Hepatocytes cultured on type I collagen (Vitrogen) had greater resistance to ADR toxicity accompanied by parallel increases in the level of pgp mRNA, decreased drug accumulation, and enhanced drug efflux when compared with the hepatocytes maintained on the basement membrane matrix Matrigel. The development of ADR resistance coincided with the time course of increased pgp mRNA but was not coincident with the time course of expression of either the placental isozyme of glutathione S-transferase or P-450 reductase, proteins associated with MDR in some resistance models. Southern blot analysis revealed neither gross changes in pgp gene structure or gene copy number to account for the increase in pgp RNA levels for hepatocytes cultured on Vitrogen. ECM also regulated xenobiotic-inducible expression of hepatic pgp, since chemotherapeutic agents, including vincristine and colchicine, induced pgp mRNA exclusively in hepatocytes cultured on Vitrogen. The critical matrix proteins in Matrigel responsible for regulation of pgp were determined by the selective addition of its components to the culture environment. The presentation of the individual matrix elements as a rigid substratum to the hepatocyte did not decrease pgp mRNA. In contrast, the presentation to the same hepatocytes of either laminin or type IV collagen in a nonrigid state (solubly in the medium) selectively decreased hepatocellular pgp mRNA. We conclude that primary rat hepatocytes develop ADR resistance with time in culture due to increased expression of pgp and that ECM proteins represent endogenous physiological modulators of both basal and chemotherapeutically inducible expression of hepatic P-glycoprotein.
Schuetz
J D
JD
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Schuetz
E G
EG
D000818:Animals; D002478:Cells, Cultured; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance / Q000502:physiology; D005109:Extracellular Matrix / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D010641:Phenotype; D051381:Rats
D004317:Doxorubicin