In vivo determination of 5-bromo-2'-deoxyuridine incorporation into DNA tumor tissue by a new ³²P-postlabelling thin-layer chromatographic method

The halopyrimidine 5-bromo-2'-deoxyuridine (BUDR) can serve as one of many indicators of tumor malignity, complementary to histologic grade. We have developed a thin-layer chromatographic (TLC) technique that can assess tumor DNA base composition and analogue (BUDR) incorporation which vies with immunochemistry for BUDR. This requires post-labeling DNA by nick-translation and radioactive 5'-phosphorylation of representative ³²P-α-dNMPs (deoxynucleotide monophosphates). Subsequent 3'-monophosphate digest exchanges a radioactive ³²PO₄ for the neighboring cold nucleotide. Separation in two dimensional PEI-cellulose TLC is carried out in acetic acid, (NH₄)₂SO₄, and (NH₄)HSO₄. TLC of dNMPs was applied to control HeLa DNA, and HeLa cells receiving BUDR. BUDR is detected in 10⁶ HeLa cells after 12-72 h incubations. Findings in HeLa DNA demonstrate normal TLC retention factors for all ³²P-dNMPs. Two dimensional R(F) (x,y axes in cm) demonstrate: dAMP = 1.4, 9.4; dCMP = 10.0, 13.5; dGMP = 4.6, 4.4; dTMP = 9.0, 7.4; and BUDRMP 6.4, 6.6. This technique quantifies BUDR-which parallels tumor S phase, and serves as an indicator of labelling index (LI).