Author: Dr. Dimiter S. Dimitrov
Laboratory of Experimental and Computational Biology (LECB)
National Cancer Institute at Frederick, MD, USA.

This talk is supported by the Connectionist-Based Information Systems
Emerging Research Theme (CBIS) - Professor N Kasabov, Department of
Information Science

Title: The analysis of microarray data : Telomere dynamics, cell
proliferation, apoptosis and differentiation.


Telomeres are DNA repeat sequences at the end of chromosomes and their
binding proteins including telomerase which ensure the complete replication
of the 3' end of chromosomal DNA and protect chromosome ends from
recombination, fusion and degradation. Telomerase is a large
ribonucleoprotein complex containing an RNA subunit and several proteins
including telomerase reverse transcriptase (TERT) with a major function to
elongate telomeres. It is activated in 80-90% of all human tumors and in
actively proliferating cells but not in most somatic tissues. We developed
improved methods for measuring telomere lengths and found that telomeres
shorten much more rapidly in infants than in adults, and also in HIV
infected humans and monkeys than in uninfected. The results suggest an
increased cell turnover in infants that may explain their rapid progression
to AIDS. They do not support the hypothesis for exhaustion of the
regenerative ability of the immune system as a cause of CD4 cell decline. We
also developed mathematical models of the telomere and cell dynamics in
tumors in presence of telomerase inhibitors and demonstrated that due to
telomere heterogeneity the inhibitory delay would be much shorter than
expected. Interestingly, we found that two subpopulations of cells, rapidly
and slowly dividing, need to exist to fit the model to the data.

To further understand cell dynamics in cancer we examined in detail the
human leukemic U937 cell line which 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 downregulated and upregulated,
respectively. In the minus cells 27 genes were statistically significantly
upregulated and 14 - downregulated more than 5-fold as measured by an
Affymetrix human genome array containing 12,000 sequences of full-length
genes. Very high differential upregulation (50-fold) was observed for Id2
which was confirmed by Western blotting. Id3 was also highly upregulated and
Ets1,2 downregulated as measured by Western blotting. One can speculate that
the interplay between c-Myc-Max/Mad1 and members of the Id and Ets families
of proteins ensures high rates of cell division and apoptosis combined with
low telomerase activity in the minus cells suggesting complex regulation of
telomerase activity in these U937 clones not correlated with division rates.
These findings and the wealth of 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.

I will also discuss the methodologies we are currently developing for
analysis of the Affymetrix gene chip data.