We have been interested in the fundamental mechanisms that control cell dynamics including proliferation, differentiation and death as related to cancer and AIDS, specifically in the biochemical networks regulating telomerase activity and other cancer-related genes. The human leukemic U937 cell line has been widely used to study neoplasia, cancer therapeutics, and cell differentiation. We identified U937 (minus) clones exhibiting more than 100-fold lower telomerase activity, shorter telomeres (4 vs 20 kbp), and more apoptotic cells (8.3 vs 3.3 %) than other (plus) clones, but similar percentages (about 70%) of BrdU labeled cells. In these cells c-Myc and Mad1 were 2-3-fold down-regulated and up-regulated, respectively. Treatment with differentiation agents (sodium butyrate, retinoic acid or TPA) decreased significantly telomerase activity in all clones and further decreased expression of c-Myc and increased Mad1; minus cells were more sensitive to differentiation agent-induced apoptosis. In the minus cells 27 genes were statistically significantly up-regulated and 14 genes were down-regulated more than 5-fold as measured by an Affymetrix human genome array containing 12k sequences of full-length genes. Very high differential up-regulation (50-fold) was observed for Id2, which was confirmed by Western blotting. Id3 was also highly up-regulated and Ets1/2 down-regulated as measured by Western blotting. Using a novel PCR-based DNA-protein interaction detection approach, we showed that c-Myc and Sp1 from plus cell lysate bound to hTERT promoter with much higher efficiency than from minus cell lysate. On the other hand, Mad1 from minus cell lysate showed much higher binding. Ets binding to hTERT promoter as revealed by this protocol suggested a new element in hTERT regulation. This data reinforced the hypothesis that interplay between c-Myc-Max/Mad1 and Sp1 are involved in the transcriptional regulation of hTERT. The lack of apparent difference in ETS binding to hTERT promoter, as well as the different expression in Id family proteins between plus and minus cells shed some light on the mechanism of complex regulation of telomerase activity in these U937 clones not correlated with division rates. These findings and the information provided by the gene expression profiles for this model system may also help in the understanding of the complex networks controlling hematopoietic cell differentiation, apoptosis, and proliferation. We have developed a new method for analysis of oligonucleotide microarray data specifically for the signal distribution and normalization. With the new method we analyzed several new sets of experiments, including data on retinoic acid induced differentiation of U937 clones. Our analysis of experimental data of HIV infected patients obtained by our collaborators J. Kovacs, R. Lempicki and C. Lane supported the hypothesis for existence of dynamically distinct subpopulations of cells and differential effects of HIV leading to increased turnover of short-lived cells and lack of effect on the turnover of long-lived cells.
