With AZD1480 1 mM for 72 h, cells were harvested, washed twice in cold phosphate buffered saline, 1% bovine serum albumin, and stained with anti B7 H1, B7 DC or mouse IgG1 isotype control antibodies PE conjugated for 20 min. The stained cells were washed Calcium Channel review twice in cold phosphate buffered saline, fixed in 1% paraformaldehyde phosphate buffered saline and then applied to the flow cytometer. A total of 20 000 events were analyzed. Data were collected on a FACSCalibur flow cytometer, using FlowJo software as described previously. 8 Each result is the mean of three independent experiments. Bar graphs show the mean of three independent experimentss. e. m. Measurement of cytokines and chemokines HL cell lines were incubated with AZD1480 or dimethyl sulfoxide for 72 h.
Supernatants Avasimibe were then collected and examined for production of IL 13, IL 6, IP 10, RANTES and IL 8 by enzyme linked immunosorbent assay, by using the human cytokine 30 plex panel and for production of TARC by using the Human TARC ELISA kit, according to the manufacturers, instructions. Each experiment was performed in triplicate, and each result is the mean of three independent experimentss. e. m. Statistical analyses The effectiveness of the drugs used in this study, alone and in combination, was analyzed by using CalcuSyn software. The combination index was calculated according to the Chou Talalay method. 17,18 CI?1 indicates an additive effect of two drugs, CI o1 indicates a synergistic effect, and CI 41 indicates antagonism between the two drugs.
Procedures to determine the effects of certain conditions on cell proliferation, cell cycle, apoptosis and cytokine production were performed in three independent experiments. The two tailed Student,s t test was used to estimate the statistical significance of the differences between results from the three experiments. Significance was set at Po0. 05. Results Expression of activated JAK2 in cultured HL cells To explore the potential therapeutic value of the JAK2 inhibitor AZD1480 in HL, we initially examined the expression pattern of its protein target, the active, phosphorylated form of JAK2 Y1007/1008, in cultured HL cells. We hypothesized that cells with high levels of p JAK2 would be more sensitive to the antiproliferative effect of AZD1480 than cells with lower levels of p JAK2. We found p JAK2 to be expressed in two of the four HL cell lines.
None of the HL cell lines expressed p JAK1 Y1022/1023, two cell lines expressed p TYK2 Y1054/1055, and only one cell line expressed p JAK3 Y980. Thus, the activation pattern of the JAK family members is rather heterogeneous in the HL cell lines. Next, we investigated the expression pattern of the active phosphorylated form of downstream. We found p STAT3, p STAT5 and p STAT6 to be expressed in the three Figure 1 Baseline JAK/STAT pathway activation status and effects of AZD1480 in HL cell lines. Baseline activation of the JAK/STAT pathway in the HL cell lines HD LM2, L 428, KM H2 and L 540. PBMCs from three healthy donors were used for comparison. Western blot assay of the four HL cell lines treated with increasing doses of AZD1480 for 72 h. MTS assay of the four HL cell lines treated with AZD1480. Cells were incubated with increasing concentrations of AZD1480 for 24, 48 and 72 h. The value f
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