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In vivo determination of 5-bromo-2'-deoxyuridine incorporation into DNA tumor tissue by a new 32P-postlabelling
J J Steinberg1, G W Oliver, N Farah
1Department of Pathology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, 10467, USA.
Abstract:
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-alpha-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 10(6) 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).