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

Jacob J. Steinberg, Gary W. Oliver, Nazih Farah, Payman Simoni, Raz Winiarsky, Antonio Cajigas

Research output: Contribution to journalArticle

2 Citations (Scopus)

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-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).

Original languageEnglish (US)
Pages (from-to)333-341
Number of pages9
JournalJournal of Chromatography B: Biomedical Applications
Volume694
Issue number2
DOIs
StatePublished - Jul 4 1997
Externally publishedYes

Fingerprint

Bromodeoxyuridine
Tumors
Tissue
DNA
Labeling
Polyetherimides
Phosphorylation
Cellulose
Acetic Acid
Nucleotides
Chemical analysis

Keywords

  • 5-Bromo-2'-deoxyuridine
  • DNA
  • HeLa cells

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

In vivo determination of 5-bromo-2'-deoxyuridine incorporation into DNA tumor tissue by a new 32P-postlabelling thin-layer chromatographic method. / Steinberg, Jacob J.; Oliver, Gary W.; Farah, Nazih; Simoni, Payman; Winiarsky, Raz; Cajigas, Antonio.

In: Journal of Chromatography B: Biomedical Applications, Vol. 694, No. 2, 04.07.1997, p. 333-341.

Research output: Contribution to journalArticle

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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-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).",
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