Characterisation of high-level cisplatin-resistant cell lines established from a human hepatoma cell line and human KB adenocarcinoma cells: cross-resistance and protein changes.
Human liver carcinoma cells (BEL-7404) and human KB adenocarcinoma cells were selected by stepwise increases in cisplatin. Drug sensitivity assays indicated that the IC50 value for 7404-CP7.5 cells was 49 micrograms ml-1 cisplatin, 111-fold higher than for the parental hepatoma cells. The IC50 value for KB-CP10 cells was 38 micrograms ml-1 cisplatin, which is 1152-fold higher than for the parental KB cells. The 7404-CP7.5 cells were cross-resistant to methotrexate (39 x), 5-fluorouracil (23 x) and 6-mercaptopurine (13 x), but were sensitive to drugs which are known substrates for the multidrug transporter (P-glycoprotein), including colchicine, vinblastine and actinomycin D. Similar cross-resistance patterns were observed for KB-CP10 cells. No evidence of DNA amplification or expression of the MDR1 gene was found. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed increases in 52 kDa protein(s) in both the soluble cytosolic and crude membrane fractions in 7404-CP(r) cells and in KB-CP(r) cells. The amount of 52 kDa protein was proportional to the degree of resistance of the 7404-CP(r) cells to cisplatin. Two-dimensional gel analysis demonstrated that two polypeptides of molecular mass 52 and 50 kDa were overexpressed in the membrane fractions in both 7404-CP20 and KB-CP20 cells. Using amino acid microsequencing and Western blotting, major 52 kDa protein was identified as the mitochondrial heat shock protein hsp60. Two-dimensional gels of [35S]methionine-labelled polypeptides showed many other changes, including reduction in soluble proteins of approximately 57 kDa molecular weight in KB-CP20 cells, and of 35 kDa in both 7404-CP20 and KB-CP20 cells. These results suggest that alterations of certain proteins occur commonly in cisplatin-resistant cells, particularly proteins of molecular weight 52 and 50 kDa.
Shen
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DW
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Akiyama
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Schoenlein
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Pastan
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I
Gottesman
M M
MM
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000633:toxicity*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000633:toxicity*; D002999:Clone Cells; D003600:Cytosol / Q000378:metabolism; D004351:Drug Resistance; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007624:KB Cells; D008113:Liver Neoplasms; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D008715:Methionine; D002945:Cisplatin