7TD1 cells were grown in complete culture medium as described previously [22,23]. The cells were cultured with 4 ng/ml recombinant human IL6 (Upstate Biotechnology, Lake Placid, NY). Chronically treated cells (7TD1-Dxm) were continuously cultured in the presence of dexamethasone (85 μM) and in the absence of IL6 over a period of three months to generate the resistant subline, 7TD1-Dxm. This subline was maintained in culture with dexamethasone (85 μM) and without IL6. Dexamethasone was dissolved in ethanol at 1 mg/ml stock solution. HA-14-1 (Sigma Aldrich, St.Louis, MO) was dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 50 mM. Appropriate vehicle controls were included in experiments. Cellular proliferation was monitored using the MTT assay [22,23] as described previously [24]. All experimental procedures were carried out with a prior wash-out period of 24 hrs during which both the cell lines were incubated without IL6 or Dxm. All experiments utilized parent 7TD1 cells between passages 30 and 60.

As previously described [22], cells were harvested by centrifugation at 200 × g for 6 minutes and whole cell lysates prepared using a lysis buffer containing 20 mM Tris-HCl (pH 7.6), 100 mM sodium chloride, 2% Triton X-100, 10 mM EDTA (Ethylene diamine tetraacetic acid), 100 mM sodium fluoride, 60 mM sodium pyrophosphate, 0.2% sodium azide, 0.2 mM sodium orthovanadate, 1 mM PMSF (Phenylmethanesulfonyl fluoride), 20 µg/ml leupeptin, and 2 µg/ml aprotonin. Lysates were subsequently sonicated for 5 seconds and centrifuged at 5200 × g for 10 minutes; the resulting supernatant was mixed with 3× SDS loading buffer and incubated at 90˚C for 3 minutes. The proteins were then separated on a 10% (w/v) polyacrylamide gel. Separated proteins were transferred to a PVDF membrane by electroblotting at 100 V for 1 hr. After transferring, the PVDF membrane was blocked in a solution containing 5% milk powder and 2.5% bovine serum albumin (BSA) in 1× Tris-buffered saline with 0.05% tween (TBST) at 4˚C for 6 hrs and then washed with 1× Tris-buffered saline (TBS) and 1× TBST for four and three times, respectively. After washing, the membrane was probed with primary antibody for total Bcl-2 protein and Bcl-X_L (1:1000 dilutions, Cell Signaling Technology, Danvers, MA) at 4˚C overnight, followed by washing with 1× TBS and 1× TBST for four and three times, respectively. The membrane was then probed with Horse-Radish Peroxidase (HRP) labeled goat anti-rabbit IgG secondary antibody (1:2500 dilutions, KPL, Gaithersburg, MD). Blots were then processed by Pierce chemiluminiscence kit (Thermo Scientific, Rockford, IL) as recommended by the commercial vendor and autoradiographed on an X-ray film (RPI corp, Mt. Prospect, IL). Western blot analysis was carried out in triplicates.

Apoptotic cells were quantified using the DNA fragmentation TUNEL assay (Molecular Probes, Eugene, OR). Briefly [22], 7TD1 and 7TD1-Dxm cells were subjected to various specified treatments, incubated for 48 hrs, washed with 1× PBS, fixed in a mixture of ice-cold 1× PBS: 70% ethanol, and kept at −20˚C for 12 - 18 hrs. The cells were washed with 1× PBS, stained using the APOBrdu TUNEL assay kit according to the manufacturer’s protocol, and analyzed using a flow cytometer (Becton Dickinson, San Jose, CA).

Cytochrome c release into cytoplasm was quantified using a cytochrome c release assay kit (Calbiochem, La Jolla, CA). Briefly, 7TD1 and 7TD1-Dxm cells were subjected to various specified treatments, incubated for 24 hrs, washed twice with 1× PBS, resuspended in 300 µl permeabilization buffer and incubated on ice for 10 minutes. After permeabilization, the cells were fixed in 8% paraformaldehyde in PBS for a period of 20 minutes at room temperature. After fixing, the cells were washed twice in 1× wash buffer and resuspended in 250 µl of blocking buffer and kept at room temperature for 1 hr. After incubation, 250 µl of anti-cytochrome c antibody working solution was added to each sample and incubated for 1 hr at room temperature. The cells were then washed twice with 1× wash buffer. They were then resuspended in 500 µl of anti-IgG FITC and incubated again for 1 hr at room temperature. They were then washed with 1× washed buffer and resuspended in 400 µl of wash buffer and analyzed using flow cytometer (Becton Dickinson, San Jose, CA).

MMP was analyzed using two Mitotracker dyes Mitotracker red and Mitotracker green (Molecular probes, Eugene, OR). Mitotracker red binds to non-depolarized mitochondria, and Mitotracker green binds to all mitochondria. Thus, a ratio of non-depolarized to total mitochondria was calculated to estimate the MMP. Briefly, 7TD1 and 7TD1-Dxm cells were subjected to various specified treatments, and incubated for 24 hrs. At the end of indicated time point, Mitotracker red and Mitotracker green dyes were added for a final concentration of 250 nM and 150 nM, respectively. They were then incubated at 37^ºC for 30 minutes, centrifuged at 200 × g for 6 minutes, washed and resuspended in 1× ice cold, PBS and analyzed by flow cytometer (Becton Dickinson, San Jose, CA). The ratio of mean red and green fluorescence intensities is expressed relative to the control group.

Statistical significance of experimental results was analyzed by univariate analysis of variance followed by Tukey’s post-hoc test with a minimum significance level set at P < 0.05. In figures, individual letters identify those treatment groups where the differences are statistically significant as indicated by ANOVA (e.g., all treatment groups identified by a letter “a” are statistically significantly different from those treatments identified by all other letters, “b”, “c”, etc.).

Consistent with our previous observations [22], 7TD1- Dxm cells were resistant to inhibition of proliferation by dexamethasone treatment as compared to parent 7TD1 cells. To examine the role of inhibition of Bcl-2, an antiapoptotic protein, on proliferation of 7TD1-Dxm cells, we initially treated 7TD1-Dxm cells with HA-14-1, a Bcl-2 specific inhibitor. Figure 1 shows that treatment with HA-14-1 inhibited proliferation of 7TD1-Dxm cells in concentration-dependent manner with maximum inhibition observed at 20 µM (a 4-fold decrease was observed).

We next examined the effect of HA-14-1 on induction of apoptosis. Figure 2 shows that treatment of both sensitive and resistant cells with 20 µM HA-14-1 alone had a significant impact on induction of apoptosis (40% - 60% apoptotic cells were seen as compared to control) in both the lines. Addition of dexamethasone with HA-14-1 did not have further effect on induction of apoptosis as compared to treatment with HA-14-1 alone.

This finding suggests that inhibition of Bcl-2 at the concentration required for maximum inhibition of prolif-

eration was sufficient to induce apoptosis but did not alter the resistance of 7TD1-Dxm cells to dexamethasone treatment.

Since inhibition of Bcl-2 by HA-14-1 blocked proliferation and induced apoptosis in both parent 7TD1 and 7TD1-Dxm cells, we next examined Bcl-X_L and Bcl-2 protein expression. Figure 3(a) shows that treatment with AG490, a JAK inhibitor, alone at 50 µM or dexamethasone alone at 85 µM had an effect of reducing protein levels of Bcl-2 in parent 7TD1 cells. 7TD1-Dxm

cells were completely resistant to reduction of Bcl-2 protein upon treatment with Dxm. This effect of reducing Bcl-2 appears to be specific as Bcl-X_L was unaffected by Dxm treatment (Figure 3(b)). This observation led us to propose a role for dexamethasone in differential regulation of the anti-apoptotic proteins downstream to JAK/ STAT3 signaling pathway in the process of acquisition of the Dxm-resistant phenotype and confirmed our assumption about the involvement of mitochondrial pathway of apoptosis induction in the development of resistance in these cells. Parent 7TD1 cells were treated with IL6 at 4 ng/ml which was used as a negative control. The densitometric analysis of the results obtained from Bcl-2 western blot analysis (Figure 3(c)) shows that while in the parent 7TD1 cells the protein levels of Bcl-2 significantly decreased upon treatment with dexamethasone alone, in 7TD1-Dxm cells Bcl-2 protein levels were resistant to reduction by dexamethasone.

We next examined the effect of dexamethasone on cytochrome c release from mitochondria into the cytoplasm which is a downstream event that occurs after reduction of Bcl-2 protein. Histogram analysis of the data obtained from flow cytometric analysis suggested while there was a shift in the peak observed in parent 7TD1 cells treated with dexamethasone alone at 85 µM when compared to control, suggesting a release in the cytochrome c into the cytoplasm, no shift was observed in 7TD1-Dxm cells treated with dexamethasone alone as compared to control (Figures 4(a) and (b)). Figures 4(c) shows that treatment with HA-14-1 at 20 µM alone and in combination with 85 µM dexamethasone induced significant release of cytochrome c into the cytoplasm (up to 70% labeled cells were observed) indicating the role of Bcl-2 in regulating the release of cytochrome c from the inter-membranal space of mitochondria. Upon treatment with dexamethasone alone, 7TD1-Dxm cells showed resistance to release of cytochrome c while the parent 7TD1 cells showed a significant labeling of the cells (40% labeled cells observed compared to control) suggesting release of cytochrome c into the cytoplasm. This result is consistent with the effect of Dxm treatment on Bcl-2 protein expression and suggests a role for mitochondria in the development of resistance by these cells.

Since cytochrome c release was not observed in 7TD1- Dxm cells upon dexamethasone treatment, we further determined the MMP as its disruption can be a prior step for release of cytochrome c. We treated both cell lines

with IL6 (4 ng/ml), AG490 at 50 µM, HA-14-1 at 20 µM, alone and in combination with dexamethasone (85 µM) or with dexamethasone alone at 85 µM for a period of 24 hrs. Figure 5 shows that both parent 7TD1 cells and 7TD1-Dxm cells were resistant to inhibition of MMP suggesting that MMP disruption was not responsible for releasing cytochrome c. Thus, other mechanism(s) are likely involved. Similar results were obtained even when the incubation period was extended to 48 or 72 hrs.