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Histone H3 Messenger RNA in Situ Hybridization Correlates with in Vivo Bromodeoxyuridine Labeling of S-Phase Cells in Rat Colonic Epithelium¹

Departments of Gastrointestinal Medical Oncology and Digestive Diseases [H. K., G. S., G. A. G., K. F.], Experimental Radiotherapy [N. H. A. T.], Biomathematics [J. J. L., J. A. D.], and Clinical Investigation [W. N. H.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and DAKO Corporation, Carpinteria, California [D. S., L. C.]

Measurements of cell cycle phase fractions, particularly S-phase, are useful for studies of cell biology and carcinogenesis. Up-regulation of histone gene expression is tightly coupled to the G₁-S-phase transition of the cell cycle, and mRNA levels rise 30–100-fold during S-phase. Labeling of histone H3 mRNA using in situ hybridization (ISH) was assessed as a measure of S-phase cells and compared with that found using in vivo 5-bromodeoxyuridine (BrdUrd) labeling in formalin-fixed rat colonic crypts under baseline, modified 72-h starvation, and 24-h refeeding conditions. The labeling index scored in single-labeled sections by histone H3 ISH tightly correlated with that found by in vivo BrdUrd labeling (r = 0.99, P < 0.0001) and clearly discriminated between the control, starved, and refed states (P < 0.001). In 180 crypt sections double labeled using histone H3 ISH and BrdUrd, 92% of 1572 labeled cells exhibited both nuclear BrdUrd and cytoplasmic histone H3 label. It is concluded that histone H3 ISH is an accurate measure of the S-phase fraction and provides an alternative to in vivo BrdUrd labeling in rat colon. This finding warrants validation in human studies.

¹ This work was supported in part by NIH Grant CA06294.

² To whom requests for reprints should be addressed, at Department of Gastrointestinal Medical Oncology and Digestive Diseases, University of Texas M. D. Anderson Cancer Center, Box 78, 1515 Holcombe Boulevard, Houston, TX 77030.

Received 11/16/95. Accepted 12/14/95.

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