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Simultaneous Nuclear Antigen and DNA Content Quantitation Using Paraffinembedded Colonic Tissue and Multiparameter Flow Cytometry¹

Department of Pathology, Northwestern University, McGaw Medical Center, Chicago, Illinois 60611 [K. D. B., C. V. C., R. K. E., T. M., D. G. S.], and Department of Pathology, University of Southern California, Los Angeles, California 90033 [A. L. E.]

The simultaneous quantitation of nuclear antigens and DNA content is presented using monoclonal antibodies and flow cytometric analysis, with paraffin-embedded human colonic pathology specimens utilized as source material. The monoclonal antibodies evaluated were shown by immunogold electron microscopy to recognize nuclear proteins preferentially associated with interchromatin (p105) and heterochromatin (p34) regions. Indirect immunofluorescence analysis of p105 revealed two distinct G₁-G₀ cell subpopulations in cells from normal colonic epithelium and colonic adenocarcinomas. In addition, enhanced levels of both p105 and p34 were observed in aneuploid DNA content stemlines, relative to diploid cells. Cell-sorting experiments performed on cells sorted on the basis of p105 and DNA contents reveal the capability of this method for identifying morphologically heterogeneous cell subpopulations. Other data suggest that p105 is differentially expressed in well-differentiated versus poorly differentiated tumor regions. The potential utility of this approach for the retrospective study of proliferation-associated antigens and protooncogene protein products is discussed.

¹ This research was supported by USPHS Grants CA37413 and SO7RR0537022 awarded by the National Cancer Institute, Department of Health and Human Services. The Northwestern University Flow Cytometry Program is supported by a generous gift from the Coleman Foundation. Portions of this work were presented at the International Conference on Analytical Cytology XI, sponsored by the Society for Analytical Cytology, Hilton Head, SC, November 17–22, 1985.

² To whom requests for reprints should be addressed, at Northwestern University, McGaw Medical Center, Wesley Pavilion, Room 530, 250 East Superior Street, Chicago, IL 60611.

³ Graduate student in the Northwestern University Tumor Cell Biology Program and recipient of partial support from the Nellie Fox Scholarship Fund.

Received 4/ 2/85. Revised 1/29/86. Accepted 1/30/86.

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