Introduction

Repeated chemotherapeutic treatment frequently induces, or selects for, chemoresistance of remaining cancer cells by altering gene expression and inducing genomic instability because of mutation, recombination, and gene amplification events. Deregulation of DNA-repair enzymes is partly involved in this phenomenon (e.g. p53 gene, BRCA1/2, UBE2N, APEX, and Rad51). Furthermore, enzymes that metabolize and bioactivate drugs [e.g. dihydrofolate reductase (DHFR) (1) and NQO1 (2)] or proteins that transport cytotoxic agents (e.g. multidrug resitance protein (MDR1, Ref. 3] often contribute to chemoresistance.

During the implementation of a long-term screening program for inhibitors of chemoresistance, BVDU (3) was the only substance of clinical relevance we identified. It inhibited 2-amino-6-mercaptopurine-induced SV40 amplification (4) in Chinese hamster cells, and abrogated triethylene-melamine-induced recombination in yeast (5) . In Friend mouse erythroleukemia cells, treatment with DOX induced Mdr1 gene amplification and drug resistance, which was prevented by simultaneous treatment with BVDU (6) . Because the mode of action of BVDU in respect to these effects is unknown, this study aimed to elucidate underlying mechanisms. The preclinical data we obtained were a prerequisite for the start of clinical trials.

We performed in vitro experiments with AH13r rat hepatosarcoma and mouse 3T6 cells, and in vivo experiments with DMBA-induced SD-rat fibrosarcomas and adenocarcinomas. To test BVDU in a second in vivo cancer model, we injected AH13r cells into SD-rats for tumor induction.

As most antineoplastic drugs eliminate tumor cells by apoptosis, cancer cells can evade cell death by virtue of overactivated survival mechanisms. Thus, chemoresistance mechanisms can involve antiapoptotic traits. Therefore, we investigated several survival mechanisms, as well as the activated STAT3, and JUN-D. Moreover, we tested the activity of NQO1, an activating enzyme for anticancer drugs like MMC, MXA, or DOX, which is down-regulated in multidrug-resistant AH130 tumor cells. To identify a more comprehensive spectrum of BVDU-influenced proteins, we performed a two-dimensional gel electrophoresis and identified proteins that are differentially expressed in response to BVDU using MALDI-MS.