Sulindac sulfide induces several subpopulations of colon cancer cells, defined by PCNA/Ki-67 and DNA strand breaks

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We assessed the effect of sulindac sulfide (SS), a colon cancer chemopreventive agent, on the proliferation and apoptosis in the colon cancer cell lines HCT-15 and HT-29. We applied a triparameter flow cytometric analysis that simultaneously determined DNA content, expression of Ki-67 or proliferating cell nuclear antigen (PCNA), and extent of DNA strand breaks by TUNEL (TdT-mediated dUTP nick end labeling). HCT-15 and HT-29 cells were exposed to SS 200 μM and 175 μM, respectively, for up to 72 h. As expected, SS inhibited proliferation and induced apoptosis. SS also induced several subpopulations of cells defined by their expression of proliferation markers and DNA strand breaks. By 72 h the rapidly proliferating cells [PCNA/Ki-67(+)/TUNEL(−)] were reduced from >90% to about one third. Of the remaining cells, about one third were apoptotic [PCNA/Ki-67(−)/TUNEL(+)] and one third were quiescent [PCNA/Ki-67(−)/TUNEL(−)]. Another subpopulation was detected that was PCNA/Ki-67(+)/TUNEL(+), some had a dominant subdiploid peak and over half were in S or G 2/M phases by DNA content. Thus, a subpopulation of apoptotic cells strongly expressed PCNA and Ki-67, suggesting that their specificity as proliferation markers may need reassessment. Similar results were obtained with the HL-60 promyelocytic cell line.