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Molecular Cloning and Characterization of the Complementary DNA for the M_r 85,000 Protein Overexpressed in Adriamycin-resistant Human Tumor Cells¹

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo 170[Y. S., H. H., S. T., T. T.]; Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113 [K. N., T. T.]; and Tokyo Research Institute, Kyowa Hakko Kogyo Co., Ltd., Machida-shi, Tokyo 194 [K. Y., M. S.], Japan

An M_r 85,000 membrane protein was identified by a monoclonal antibody MRK20 raised against an Adriamycin-resistant subline of human myelogenous leukemia K562 (K562/ADM) cells. The M_r 85,000 protein was found to be overexpressed in both innate and acquired Adriamycin-resistant tumor lines. A complementary DNA (cDNA) clone coding for the M_r 85,000 protein was isolated by mixed oligonucleotide-primed polymerase chain reaction and further screening of a cDNA library from K562/ADM. Amino acid and nucleotide sequence analysis of the M_r 85,000 protein revealed that this protein is identical with CD36, a cell surface adhesion molecule of endothelium, platelets, and monocytes. We constructed an expression vector utilizing two different promoters, SV40 and MMTV, and two cDNAs for the M_r 85,000 protein that have different 3'-ends. DNA transfection experiments were carried out by the calcium phosphate method with a selectable marker using drug-sensitive human tumor lines KB3-1 and A2780 as recipient cells. We obtained transfectant clones expressing the M_r 85,000 protein stably or inducibly but found no resistance against Adriamycin or vincristine. Direct selection with Adriamycin or vincristine of tumor cells transfected with the SV40 promoter-regulated expression constructs also failed to yield drug-resistant clones. These results indicate that the M_r 85,000 protein/CD36 cannot confer drug resistance by itself, even though the protein can be an effective marker for Adriamycin resistance.

¹ This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan, and from the Ministry of Health and Welfare, Japan.

² To whom requests for reprints should be addressed.

Received 11/12/92. Accepted 3/26/93.