aphidicolin and Lymphoma--Large-B-Cell--Diffuse

HOB1/VCR, a multidrug-resistant subline of the immunoblastic B lymphoma cell line, was established by sequential selection in increasing concentrations of vincristine. The expression of the human mdr l gene, as analyzed by reverse transcription and polymerase-chain reaction (RT-PCR), revealed a 10-15-fold overexpression in this resistant cell line. A complete inhibition of vincristine resistance by verapamil was observed in the vincristine-resistant HOB1/VCR cells, which suggests that acquired resistance may be mainly due to P-glycoprotein. HOB1/VCR cells also developed a 67-fold cross-resistance to the anticancer drug cis-diamminedichloroplatinum (cisplatin). DNA repair of the resistant and the parental cell lines was investigated by in situ detection with a cisplatin-DNA adduct-specific antibody and by measurement of repair-associated host cell reactivation of damaged plasmid DNA. HOB1/VCR cells exhibited a 2-fold decrease in the level of cisplatin-DNA adducts, compared to the parental cells. The DNA repair rate following peak accumulation of cisplatin-DNA adducts (which took approximately 4 h) was also enhanced in the resistant cells. This was supported by the measurement of the cisplatin level remaining in cells by atomic absorption spectrophotometry, which showed a 2.7-fold reduction in the resistant cells. In addition, the acquired resistance and enhanced DNA repair in HOB1/VCR cells were partially reversed by nontoxic aphidicolin, a DNA polymerase-alpha and DNA repair inhibitor. Inhibition of the intracellular level of glutathione by DL-buthionine-[S,R]-sulfoximine demonstrated that cell viability was inhibited 4-fold more in the resistant cells than in the parental cells. The results suggest that the reduced formation of cisplatin-DNA adducts and the increased glutathione content of the multidrug-resistant cells play a major role in phenotypic cross-resistance to cisplatin.

Topics: Antineoplastic Agents; Aphidicolin; Cell Survival; Cisplatin; Colchicine; DNA Adducts; DNA Repair; DNA, Neoplasm; Doxorubicin; Drug Resistance, Neoplasm; Enzyme-Linked Immunosorbent Assay; Gene Expression; Genes, MDR; Humans; Lymphoma, Large B-Cell, Diffuse; Male; Mitomycin; Polymerase Chain Reaction; Puromycin; Spectrophotometry, Atomic; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Verapamil; Vincristine

The effects of protein and DNA synthesis inhibitors on in vitro tumor cell invasion was studied in a quantitative invasion system using human amnion. Highly invasive M5076 tumor cells were incubated on the basement membrane (BM) of the amnion in the presence of cycloheximide, aphidicolin or sodium butyrate. Tumor cell penetration through the entire thickness of the amnion was measured after 24 h. Protein synthesis in the M5076 cells was inhibited 95% within 1 h by 10(-6)M cycloheximide while DNA synthesis was unaffected. Treatment with cycloheximide reduced the number of spontaneously invading cells by 82% and by 66% in the presence of the chemoattractant, formylmethyl leucine-phenylalanine (FMLP), known to stimulate invasiveness. DNA synthesis in the M5076 tumor cells was selectively inhibited with aphidicolin (10 micrograms/ml). Although the DNA synthesis was greatly reduced, no significant effect on invasiveness with or without FMLP was observed. A less specific inhibitor of DNA synthesis, sodium butyrate (1 mM), arrested cell proliferation in the G1 phase of the cell cycle. When the butyrate-treated M5076 cells were tested in the invasion assay, a 50% increase in the number of invasive cells was observed. All three inhibitors were used in concentrations that did not affect cell viability or cell attachment to the amnion. These data indicate that protein synthesis but not DNA synthesis and not cell proliferation are required for tumor cells to invade native connective tissue barriers in vitro.

Topics: Amnion; Animals; Aphidicolin; Butyrates; Butyric Acid; Cell Division; Cell Line; Collagen; Cycloheximide; Diterpenes; DNA, Neoplasm; Humans; Lymphoma, Large B-Cell, Diffuse; Mice; N-Formylmethionine Leucyl-Phenylalanine; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms, Experimental