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 eoplasia, cancer therapeutics, cell differentiation and HIV-1 entry. We dentified 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. Although the minus cells grew slower than the plus cells (about 2-fold at 48 hours) the percentage of BrdU pulse labeled cells was slightly higher (73 vs 61%) suggesting significant loss of minus cells by apoptosis. These cells have undetectable levels of hTERT and hTERT mRNA; SP1 and c-Myc were downregulated (about 2-fold) while Mad1 was upregulated more than 3-fold compared to the plus clones. Treatment with differentiation agents as sodium butyrate, retinoic acid or TPA decreased significantly telomerase activity in all clones and affected protein expression: c-Myc was generally decreased although in some cases was increased; SP1 was slightly upregulated and Mad1 remained unchanged in most cases; induction of apoptosis was faster and more extensive in the minus clones. In the minus clones 27 genes were statistically significantly upregulated and 14 - downregulated more than 5-fold as measured by the Affymetrix human genome array containing 12,000 sequences of full-length genes. Very high differential upregulation (50-fold) was observed for Id2. This raises the interesting possibility for a dual function of Id2 in the minus cells as an enhancer of both differentiation and proliferation - it has been previously shown that Id2 is expressed in fresh acute myeloid leukemic samples having more differentiated blasts and is dominant-negative antagonist of proteins of the retinoblastoma family that suppress the G1 to S phase transition. The downregulation of the myc oncoproteins (which are known to upregulate Id2) in the minus cells suggests the existence of another yet to be identified pathway(s) of Id2 and telomerase regulation that is not correlated with proliferation rates. These findings and the wealth of information provided by the gene expression profiles for this model system may not only help in the elucidation of the mechanisms that regulate telomerase activity but also in the understanding of the complex networks controlling hematopoietic cell differentiation, apoptosis and neoplasia. We developed a new method for analysis of oligonucleotide microarray data specifically for the signal distribution and normalization. With the new method we analyzed three different sets of experiments, including our own data on U937 clones and HIV-1 dynamics in patients under potent antiretroviral therapy. During the last year we continued to further develop a model of cell dynamics in presence of telomerase inhibitors under well defined conditions. We previously found that with a relatively simple set of differential equations we can quantitatively fit simultaneously several independent sets of experimental data; the data were best fitted if we assume the existence of two subpopulations of cells - rapidly dividing and very slowly dividing cells. Currently, we are trying to further characterize these disctinct cell populations and develop more complex and realistic models of their dynamics. We also continued to analyze cell dynamics in patients with HIV infections. A major previous result was that T lymphocytes contain two subpopulations of cells - rapidly and slowly dividing. The number of rapidly dividing cells, but not that of slowly dividing cells, correlated with the amount of HIV in the blood plasma suggesting that the proliferation of the short lived cells is caused by immune activation due to viral antigens. This finding has potential implications for understanding HIV pathogenesis and specifically the mechanisms of CD4 cell number decline with the progression of the disease. Currently, we are continuing this analysis in an attempt to characterize the disctinct cell populations. We have been also analyzing several cohorts of patients treated with potent combination of antiretroviral drugs and confirmed our previous finding for a single cohort of children that by measuring HIV dynamics during the first week of treatment one can predict with 80-90% certainty the treatment efficacy. This result is very important because it allows early change in therapy if inefficient thus avoiding potential development of drug resistant mutants and drug toxicity. Currently we are continuing to improve the accuracy of the prediction. Z01 BC 10042-04
