[The STAT5 signaling in the expression of alpha-subunit of interleukin-2 receptor in human blood lymphocytes].

The comparative study of the STAT3 and STAT5 activity (as assessed by tyrosine phosphorylation level) and the expression of a α-subunit of interleukin-2 receptor (as examined by cytophotometric evaluation of the number of CD25+ cells) during the phytohemagglutinin (PHA)-induced proliferation of human blood lymphocytes (HBL) have been made. It has been revealed that the level of STAT3 phosphorylation is high in both res ting and competent HBL and remains unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen in both resting and competent HBL. We observed phosphorylation of STAT5 no earlier than 5 h after PHA stimulation and the maximum phosphorylation was detected following 24 h. Exogenous IL-2 induced high level of STAT5 phosphorylation in the competent HBL as early as at 30 min and this level of STAT5 phosphorylation kept in the next 24-48 h. The correlation between alterations in tyrosine phosphorylation level of STAT5 and the expression of CD25 has been established. WHI-P131, an inhibitor of JAK3 kinase, prevents STAT5 activation, cell surface expression of CD25 and lymphocyte proliferation. It has been concluded that JAK3/STAT5 signaling via IL-2 receptor is necessary to maintain the long-term expression of the high-affinity αßγ(c)-receptor of IL-2 and optimal proliferation of HBL.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. I. The role of JAK and Src tyrosine kinases as revealed by inhibitory analysis].

The long-lasting expression of an alpha-subunit of interleukin-3 receptor (IL-2Ralpha) was found to accompany the PHA-induced proliferation of human blood lymphocytes (HBL), so that to the end of the second day of mitogenic stimulation only, the large blasts may express the high affinity alphabetagamma(c) receptor for IL-2. With the selective pharmacological drugs to JAK (WHI-P131) and Src (PP2) it is shown that the non-receptor tyrosine kinases are involved in the surface CD25 expression. It is revealed that the PP-2-inhibitable expression of CD25 is timely associated with the initial stage of T cell activation, whereas WHI-P131-inhibitable expression was present during the whole G0/G1/S transition. These data indicate that at the early, antigen-dependent stage the expression of IL-2Ralpha is induced via Src-dependent signaling pathway, and prolonged increase in IL-2Ralpha expression is regulated by IL-2/IL-2 receptor interaction via JAK-dependent signaling pathway.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. II. The role of interleukin-2].

The expression of alpha-subunit of interleukin-2 receptor (IL-2Ralpha) was assessed by quantifying activation-induced upregulation of CD25 in human blood lymphocytes (HBL) stimulated by interleukin-2 (IL-2). It was established that exogenous IL-2 induced no surface expression of CD25 neither proliferation at 48 h of IL-2 action. In component HBL, pretreated by sub-mitogenic doses of phytohemagglutinin (PHA), 5-15 % of cell population was revealed to represent the CD2t+ cells, and in the competent cells only, exogenous IL-2 induced the surface expression of CD25 as well as the growth and the proliferative response, which was comparable with those to mitogenic doses of PHA. The JAK3 inhibitor WHI-P131 eliminated IL-2-dependent CD25 expression without influencing the CD25 expression in competent cells. Unlike, PP2 was found to inhibit the IL-2-dependent CD25 expression in a lesser extent than WHI-P131, however this drug was effectively inhibited CD25 expression in PHA-pretreated, competent HBL. These data suggest that Src-dependent signaling participate in the early IL-2Ralpha expression that precedes the IL-2-dependent cell cycle progression of activated HBL. It is concluded that in normal T cells, the IL-2Ralpha expression in firstly induced by antigen (mitogen) and thereafter it is held IL-2 through JAK-dependent signaling pathway.

[Specific function of STAT5 in regulation of proliferation of chronic leukemia K562 cells: inhibitory effect of WHI-P131].

In this study, we examined the possible role of JAK/STAT signaling pathway in regulation of proliferation of chronic leukemia cells K562. The thyrosine phosphorylation of STAT3 and STAT5 was used as a marker of an activation status of STAT proteins. We demonstrate that in growing culture of K562 both STAT3 and STAT5 are constitutively activated. To determine the significance of STATs activity in maintaining the high level of K562 proliferation we tested two JAK inhibitors: AG-490 (JAK2 and JAK3 inhibitor) and WHI-P131 (a new specific JAK3 inhibitor). We showed that in long-tern cultures (48 h) of K562 cells with AG-490 or WHI-P132 the cells remain viable. It was found that treatment with WHI-P131 (30-100 microM) decreased the thyrosine phosphorylation of STAT5 being without effect on the high level of STAT3 phosphorylation. In proliferating K562 cells, AG-490 (25-50 microM) did not influence STAT3 and STAT5 phosphorylation. The flow cytometry analysis revealed a dose-dependent decrease in G1 and S phases and an increase in G2/M phases in WHI-P131-treated K562 cell cultures and no changes in cell cycle structure in AG-490-treated cells. Thus, our findings indicate a preferential role for STAT5 (not constitutively active STAT3) in proliferation of leukemia to other JAK drugs which stimulate apoptosis and decrease proliferation, WHI-P131 prevents K562 cells growth by arresting in G2/M phases of cell cycle.