Gene Expression Analysis of Tumor Spheroids Reveals a Role for Suppressed DNA Mismatch Repair in Multicellular Resistance to Alkylating Agents

Abstract

Drug resistance is a major obstacle in the successful treatment of cancer. Thus, elucidation of the mechanisms responsible is a critical first step in trying to prevent or delay such manifestations of resistance. In this regard, three-dimensional multicellular tumor cell spheroids are intrinsically more resistant to virtually all anticancer cytotoxic drugs than conventional monolayer cultures. We have employed the EMT-6 subline PC5T, which forms highly compact spheroids, and differential display to identify candidate genes whose expression differs between monolayer and spheroids. Approximately 5,000 bands were analyzed, revealing 26 to be differentially expressed. Analysis of EMT-6 tumor variants selected in vivo for acquired resistance to alkylating agents identified eight genes whose expression correlated with drug resistance in tumor spheroids. Four genes (encoding Nop56, the NADH SDAP subunit, and two novel sequences) were found to be down-regulated in EMT-6 spheroids and four (encoding 2-oxoglutarate carrier protein, JTV-1, and two novel sequences) were up-regulated. Analysis of the DNA mismatch repair-associated PMS2 gene, which overlaps at the genomic level with the JTV-1 gene, revealed PMS2 mRNA to be down-regulated in tumor spheroids, which was confirmed at the protein level. Analysis of PMS2^−/− mouse embryo fibroblasts confirmed a role for PMS2 in sensitivity to cisplatin, and DNA mismatch repair activity was found to be reduced in EMT-6 spheroids compared to monolayers. Dominant negative PMS2 transfection caused increased resistance to cisplatin in EMT-6 and CHO cells. Our results implicate reduced DNA mismatch repair as a determinant factor of reversible multicellular resistance of tumor cells to alkylating agents.

We are extremely grateful to Michael Liskay for providing PMS2^−/− cells and discussing unpublished results. We are grateful for the excellent secretarial assistance of Cassandra Cheng and Lynda Woodcock. Thanks are extended to Guido Bocci, Ralph Durand, Bert Vogelstein, and Shane Green for review of the manuscript and to the anonymous reviewers for their suggestions.

Giulio Francia was funded by the Sunnybrook and Women's College Health Sciences Centre Trust Fellowship. This work was supported by a grant to R. S. Kerbel from the National Cancer Institute of Canada.