Abstract
2178
Objective: While the loss of CD3-ζ is a common observation in invasive ovarian cancer, the mechanisms responsible for this decreased expression remains unknown. We have identified a circulating 14kD ζ inhibitor that can mimic in vitro many of the cellular events associated with suppressed cellular immunity, corresponding to immunotrophism. Previous investigations have separately demonstrated links between CD3-ζ and JAK3 expressions, between induction of T-cell apoptosis and CD3-ζ expression, and between JAK3 expression and induction of T-cell apoptosis. Our hypothesis is that this ζ inhibitor represents a central regulatory site, modulating expression of CD3-ζ and JAK3. Methods of Study: Blood samples were obtained from normal female controls (n=15) and women with ovarian cancer (n=30). ζ inhibitory factor was partially purified using Sepharcyl S200, followed by RP-HPLC. Jurkat cells (106 cells/ml) were incubated in medium containing 10ng/ml of the serum-derived inhibitor. After 24, 48 and 96 hours, aliquots of cells were removed, the cells centrifuged, and the pellet washed and lyzed. The cellular expression of CD3-ζ, JAK3, bcl-2 and bax were evaluated by western immunoblotting. To examine activated JAK3 expression, Jurkat cells (2×106) were incubated with 10ng/ml ζ inhibitor alone or with 2μM phorbol 12,13 butyrate, 0.64μM ionomycin for 24 hours at 37°C. For apoptosis studies, Jurkat cells (5×106 cells) were incubated for 24 hours with media alone, media containing 10% control normal serum, media containing 10ng/ml ζ inhibitor (isolated from 3 ovarian cancer patients), anti-Fas antibody, or cisplatin (as positive controls) using a Cell Death ELISA. Results: The ζ inhibitor from ovarian cancer patients (at 10ng/ml) inhibited the expression of CD3-ζ (83.7% reduction, p <0.001), while sera from normal volunteers failed to significantly affect ζ expression. When the effect of the serum-derived inhibitor was assessed on cellular expression of JAK3, the presence of ζ inhibitor suppressed both the constitutive (decreased 34%, p<0.001) and induced (76%, p<0.001) expression of JAK3 in T-cells. The ζ inhibitor preparations appeared to induce a significant increase in the level of apoptosis observed within Jurkat cells (2.17 fold increase, p<0.0001). This finding paralleled our finding of increased DNA fragmentation in ζ inhibitor-treated Jurkat cells. Under the same treatment conditions, the pro-apoptotic protein (bax) was elevated 1.46 fold (p=0.001) by exposure to the ζ inhibitor, while the anti-apoptotic protein (bcl-2) was slightly decreased (1.24 fold, p=0.018). Conclusions: The presence of serum-derived ζ inhibitor correlates with the ability to inhibit both CD3-ζ and JAK3. The exposure of T cells to the ζ inihibitor induces apoptosis, apparently via the combination of enhanced expression of bax and supressed bcl-2, which are regulated by JAK3 levels.
- American Association for Cancer Research