Cell cycle phase dependent emergence of thymidylate synthase studied by monoclonal antibody (M-TS-4).
A method of identifying thymidylate synthase (TS) at the cellular level was developed using anti-TS monoclonal antibody (M-TS-4), a monoclonal antibody created against purified TS from a HeLa cell line. In HeLa cells and four human glioma cell lines (U-251, U-87, 343-MGA, and SF-188), TS was identified primarily in the cytoplasm. Autoradiographic and flow cytometric studies showed that TS appeared mainly in the G1 phase and subsided early in the S phase; thus, the G1 phase can be divided into TS-positive and -negative fractions. Nuclear TS was not demonstrated unequivocally with M-TS-4, and the relationship between nuclear TS and DNA synthesis could not be determined. Although the percentage of TS-positive cells was larger than the S-phase fraction measured by autoradiography after a pulse of tritiated thymidine or by the immunoperoxidase method using BUdR, the ratios were within a similar range (1.2-1.4) in all cell lines studied. Therefore, the S-phase fraction can be estimated indirectly from the percentage of TS-positive cells measured by M-TS-4. Because the emergence of TS detected by our method is cell cycle dependent, M-TS-4 may be useful for biochemical studies of TS and for cytokinetic analysis.
Shibui
S
S
Department of Neurological Surgery, School of Medicine, University of California, San Francisco.
Hoshino
T
T
Iwasaki
K
K
Nomura
K
K
Jastreboff
M M
MM
D000911:Antibodies, Monoclonal; D001345:Autoradiography; D001973:Bromodeoxyuridine; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D005434:Flow Cytometry; D005910:Glioma; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D013194:Staining and Labeling; D013940:Thymidylate Synthase / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D004247:DNA; D013940:Thymidylate Synthase; D001973:Bromodeoxyuridine