CYTOFLUOROMETRIC DETERMINATION OF EXCISION REPAIR OF UV-INDUCED PYRIMIDINE DIMERS IN EHRLICH ASCITIC CANCER CELLS

Abstract

The ability of excision repair of a single [mouse] Ehrlich ascitic cancer cell was quantitated by cytofluorometry of unrepaired pyrimidine dimer-FITC [1st important soluble component] complex/single nuclear Feulgen DNA. UV-irradiated Ehrlich ascitic cancer cells reacted with antibodies to denatured UV-DNA using the antisera which were absorbed with methylated bovine serum albumen and heat denatured DNA. Then the immunofluorescent staining specific for pyrimidine dimers was combined with subsequent Feulgen nuclear reaction. Simultaneous cytofluorometric measurement of the amounts of pyrimidine dimer-FITC complex and nuclear Feulgen DNA was performed on a single tumor cell at various times of incubation after UV-irradiation. Before the occurrence of excision repair of UV-DNA lesions, the amount of pyrimidine dimers/unit amount of nuclear DNA was equal in all tumor cells at various stages of cell cycle. After incubation for 3 h, unrepaired pyrimidine dimers were largest in amount in polyploid cells and least in diploid cells; some diploid cells had no nuclear fluorescence of FITC with a faint fluorescence in the cytoplasm. It might indicate their completion of excision repair of UV-induced pyrimidine dimers followed by transport of them to the cytoplasm within the period. Thus the potency of excision repair of a cell in S phase which has not been demonstrated by the detection of unscheduled DNA synthesis could be quantitated using the immunofluorescent-Feulgen double staining. The order of the increase in the magnitude of excision repair in Ehrlich ascitic cancer cells at different stages of cell cycle was polyploid cells < G2 and S phase cells < diploid G1 cells. When non-absorbed antisera were used, a significant fluorescence was found in the nuclei of some unirradiated large Ehrlich ascitic cancer cells. The specific fluorescence was suspected to have some special relation to naturally occuring single-stranded DNA in the tumor cells.