Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines.
A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.
Chu
E
E
Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Drake
J C
JC
Koeller
D M
DM
Zinn
S
S
Jamis-Dow
C A
CA
Yeh
G C
GC
Allegra
C J
CJ
D015139:Blotting, Southern; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance / Q000502:physiology; D004790:Enzyme Induction; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D013940:Thymidylate Synthase / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012313:RNA; D004317:Doxorubicin; D013940:Thymidylate Synthase; D005472:Fluorouracil