Immunological quantitation of thymidylate synthase using the monoclonal antibody TS 106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines.
Thymidylate synthase (TS) (EC 2.1.1.45) is an important cellular target for the fluoropyrimidine cytotoxic drugs that are widely used in the treatment of solid tumors. Using the TS 106 monoclonal antibody to human TS, we have compared the immunological quantitation of TS by Western immunoblot and immunofluorescent techniques to the conventional biochemical 5-fluorodeoxyuridine monophosphate binding assay in a panel of 5-fluorouracil (5-FU)-sensitive and -resistant human cancer cell lines. Densitometric quantitation of TS 106-labeled Western immunoblot analysis of cell lysates from two 5-FU-resistant colon carcinoma cell lines, NCI H630R1 and NCI H630R10, revealed 12.8- and 16-fold increases in TS, respectively, compared to the parent 5-FU-sensitive NCI H630 colon cell line. By biochemical analysis, the TS level was 15- and 23-fold higher, respectively, in these resistant cell lines. Similarly, immunoblot analysis of cell lysates from two 5-FU-resistant breast cancer cell lines, MCF-Ad5 and MCF-Ad10, detected a 2.3- and 6.3-fold increase in TS, respectively, compared to the parent MCF-7 cell line. By biochemical assay the TS activity was 1.8- and 7.0-fold higher in these resistant breast cell lines. Western immunoblotting analysis revealed a 35-fold range of TS protein concentrations among the 10 cell lines examined, compared to a 38-fold range demonstrated by the biochemical assay. Direct comparison of Western blotting and the biochemical assay revealed a highly significant correlation (r2 = 0.93) between the two assays. Moreover, using the monoclonal antibody TS 106, the Western blotting technique was capable of detecting TS protein levels as low as 0.3 fmol in cellular lysates. Quantitation of TS in intact cells by immunofluorescent TS labeling and flow cytometric analysis was performed using three of the cell lines, NCI H630, NCI H630R10, and MCF-Ad10. This revealed a 26-fold increase in TS in the 5-FU-resistant NCI H630R10 line compared to the parent NCI H630 line and a 3.5-fold increase in TS compared to the 5-FU-resistant MCF-Ad10 breast cell line. The 5-FU-resistant MCF-Ad10 breast cell line, in turn, displayed a 7.7-fold increase in TS, compared to the 5-FU-sensitive NCI H630 cell lines. TS immunofluorescent analysis was capable of measuring TS within individual cells. The development of these immunological assays using an anti-TS monoclonal antibody will facilitate the quantitation of TS in cell lines and tissue samples.
Johnston
P G
PG
NCI-Navy Medical Oncology Branch, Bethesda, Maryland 20892.
Drake
J C
JC
Trepel
J
J
Allegra
C J
CJ
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology; D003110:Colonic Neoplasms / Q000201:enzymology; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D005455:Fluorescent Antibody Technique; D005468:Fluorodeoxyuridylate; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D008970:Molecular Weight; D013274:Stomach Neoplasms / Q000201:enzymology; D013940:Thymidylate Synthase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000911:Antibodies, Monoclonal; D005468:Fluorodeoxyuridylate; D004317:Doxorubicin; D013940:Thymidylate Synthase; D005472:Fluorouracil