Molecular cloning and characterization of the complementary DNA for the M(r) 85,000 protein overexpressed in adriamycin-resistant human tumor cells.
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 or 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.
Sugimoto
Y
Y
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.
Hamada
H
H
Tsukahara
S
S
Noguchi
K
K
Yamaguchi
K
K
Sato
M
M
Tsuruo
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002277:Carcinoma / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004317:Doxorubicin; D004351:Drug Resistance / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry*; D010051:Ovarian Neoplasms / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry; D012334:RNA, Neoplasm / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D004317:Doxorubicin