Involvement of Compensatory CD44+ Stem Cells Following BCL-2 Suppression by Antisense Oligonucleotides.

2590 Background: Gene therapy is in theory specific but encounters difficulties in practice. Suitable targets are found in many pathways and tumors do express altered patterns of expression. However the actual activity of most regulatory genes are similar to normal. Resistance develops because biochemical pathways are complex, regulated by stimulatory and inhibitory factors, each possibly affected by therapy. It’s suggested that tumors alter dependence on targeted gene products for growth by relying upon others, through compensation. Antisense oligos have targeted regulatory proteins in both in vivo and in vitro prostate cancer models. Cells treated with antisense directed against bcl-2 compensated by suppressing caspase-3 (an apoptosis promoter) and enhancing androgen receptor (AR), (co-activating) p300 and IL-6 expression. This suggests that in LNCaP a progression to increased androgen sensitivity accompanies bcl-2 suppression with a pattern of co-activation associated with more advanced prostate tumors. Methods: We evaluated mono- and bispecific oligos which targeted and equally suppressed bcl-2 expression in LNCaP cells. To further evaluate compensatory mechanisms related to tumor resistance we evaluated the level of CD44 expression employing RT-PCR and agarose gel quantification. Bands representing pcr product were photographed, converted to black and white and assessed by MIPAV software. Results: Comparable amounts of RNA from LNCaP cells treated with either mono- or bispecific oligos directed against bcl-2 (and EGFR in the bispecifics) were evaluated by RT-PCR using primers directed against CD44. When background intensity was subtracted, the relative intensity of the bands corresponding to CD44, and representing cells treated with MR4, MR24 and MR42 (compared to controls) were respectively increased 3.0% ± 33.6 (NS), suppressed 16.4% ± 49.1 (NS) and suppressed 9.2% ± 26.5 (NS). These results were pooled from both duplicate PCR runs and gels, and indicate no significant changes in CD44 expression was produced by any oligo type. Conclusions: Stem cells expressing this marker were unaffected by treatment, suggesting this population is not altered by suppressive bcl-2 therapy.