5 and 2. 2 fold increases in OVCAR 3 and SKOV 3 cell BrdU incorporation respectively, in compar ison to the untreated cells or CCL25 plus anti CCR9 anti body treated cultures. In general, CCL25 treatment abrogated the growth inhibition of OVCAR 3 and SKOV 3 cell lines caused by cisplatin in a CCR9 dependent fash ion. CCL25 induced cisplatin resistance of OvCa cell lines Treatment of OVCAR 3 and SKOV 3 cell lines with cispl atin alone resulted in 96% and 95% respective increases in apoptosis relative to the untreated cells. CCL25 treatment significantly lowered the percentage of apop totic OVCAR 3 and SKOV 3 cells. However, when the OvCa cell lines were treated with anti CCR9 antibody or CCL25 anti CCR9 antibody, the percentage of apop totic cells was restored to levels of observed with cisplatin treatment alone.

Apoptosis was also assessed under the same conditions by TUNEL assay. OvCa cell lines treated with cisplatin alone resulted in 130% increase in apop tosis relative to the untreated cells. The per centage of apoptotic cells was significantly lower than controls when cells were treated with cisplatin and CCL25. However, this CCL25 mediated survival was sig nificantly reduced by CCR9 blockade. CCL25 CCR9 interactions impact on PI3Kp85 phospho Tyr and Akt Ser473 activation To determine the CCR9 mediated signals involved in OvCa cell survival, we performed FACE assays for PI3Kp85 phosphorylation. CCL25 induced a significant increase in PI3Kp85 activation within 5 min utes. This increase was lower after 10 minutes, but was still significantly higher than levels displayed by untreated cells.

As expected, the PI3K inhibitor, wortmannin, reduced this increase. However, CCL25 treated OvCa cells co incubated with the FAK inhibitor, PF 573, 228, continued to activate PI3Kp85. When treated with cisplatin alone, there was no differ ence in PI3Kp85 activity in comparison to Brefeldin_A the untreated cells. However, PI3Kp85 phosphorylation during cisplatin treatment significantly increased 10 minutes post CCL25 co incubation. Similarly, cisplatin treatment had no effect on PI3Kp85 phosphorylation in the FAK inhibited cells, while the cisplatin and CCL25 combination induced an immediate rise in PI3K activation, followed by a slight decrease. CCL25 also induced a gradual increase in Akt phosphorylation 5 and 10 minutes after treatment.

Wort mannin treatment abrogated this increase, but CCL25 treated OvCa cells co incubated with the FAK inhibitor continued to activate Akt at significantly high levels. Cis platin treatment did not affect Akt phosphorylation, but CCL25 plus cisplatin treatment caused significant increases in Akt phosphorylation. While wortmannin pretreatment inhibited this CCL25 mediated Akt activity, cisplatin plus FAK inhibitor treated and CCL25 co incu bated cells had the same level of enhance Akt phosphory lation total protein levels as OvCa cells treated with CCL25 alone.