Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: Parental MDA-PCa-2b (MDA2b) and chronic IL-1 subline cells (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2) were treated acutely for 3 days (A, C) or 6 days (B) with vehicle control (V), 25 ng/ml IL-1α (a), or 25 ng/ml IL-1β (b) and analyzed for cell viability using MTT (A, B) or protein accumulation by western blot (C). Acute IL-1 exposure reduces cell viability and proliferation, reduces full-length PARP (indicative of cell death activation), induces SOD2 and LCN2 protein accumulation (canonical IL-1-induced genes), and reduces AR and NKX3.1 (canonical AR target gene) protein accumulation in MDA-PCa-2b parental cells, but has little to no effect on the chronic IL-1 sublines. Thus, the IL-1 sublines evolved insensitivity to IL-1. Error bars, ± STDEV of 4 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. Fold MTT optical density (OD) is normalized to treatment control. β-actin is the western blot loading control.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Western Blot, Activation Assay

Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: (A) MDA-PCA-2b parental (MDA2b) and chronic IL-1 subline (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2) cells were treated acutely for 3 days with vehicle control, 25 ng/ml IL-1α, or 25 ng/ml IL-1β and analyzed by RT-qPCR for mRNA levels of the IL-1 receptor, IL-1R1 . Acute IL-1 exposure does not increase IL-1 receptor ( IL-1R1 ) mRNA levels in parental cells, suggesting basal IL-1R1 levels are sufficient to mediate IL-1 signaling. Furthermore, IL-1 does not show a differential effect on IL-1R1 mRNA levels in parental versus subline cells, suggesting subline insensitivity is independent of IL-1R1 levels. (B) Vehicle control treated cells were compared for basal mRNA levels of IL-1R1 and of canonical IL-1-induced genes, LCN2 , NOX1 , and SOD2 . IL-1R1, LCN2 , NOX1 , and SOD2 basal mRNA levels are comparable across the parental and subline cells, suggesting chronic IL-1 exposure does not induce constitutive activation of canonical IL-1 intracellular signaling. These data suggest that MDA-PCa-2b cell lines evolve insensitivity to exogenous chronic IL-1 exposure independent of IL-1R1 levels or constitutive activation of intracellular IL-1 signaling. Error bars, ± STDEV of 3 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. For IL-1-treated cells, mRNA levels are normalized to vehicle control for each cell line. For basal expression, mRNA levels are normalized to the parental cell line.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Quantitative RT-PCR, Activation Assay, Expressing

Journal: Journal of cellular signaling

Article Title: Chronic IL-1 Exposed AR + PCa Cell Lines Show Conserved Loss of IL-1 Sensitivity and Evolve Both Conserved and Unique Differential Gene Expression Profiles

doi:

Figure Lengend Snippet: Parental (MDA-PCA-2b (MDA2b), LNCaP) and subline (MDA-αs1, MDA-αs2, MDA-βs1, MDA-βs2, LNas1, LNbs1) cells were treated acutely for 3 days with vehicle control, 25 ng/ml IL-1α, or 25 ng/ml IL-1β and analyzed by RT-qPCR for mRNA levels (A, B, C). Acute IL-1 exposure increases LCN2 , NOX1 , and SOD2 mRNA levels in parental MDA-PCa-2b and LNCaP cells, but acute IL-1 exposure has attenuated or no effect on mRNA levels in the subline cells. Thus, both LNCaP and MDA-PCa-2b cell lines show conserved intracellular response to acute IL-1-induced changes mRNA levels and evolve chronic IL-1 insensitivity independent of constitutive canonical IL-1 intracellular signaling. Error bars, ± STDEV of 3 biological replicates; p-value, *≤ 0.05, **≤ 0.005, ***≤ 0.005, NS = not significant. For IL-1-treated cells, mRNA levels are normalized to vehicle control for each cell line.

Article Snippet: MDA-PCa-2b cells were maintained in HPC1/20% FB Essence (FBE) containing 0.5 ng/ml IL-1α (Gold Bio, St. Louis, MO; 1110–01A-10) or IL-1β (Gold Bio, St. Louis, MO; 1110–01B-10) for approximately 4 months; and the proliferative colonies that emerged were expanded and termed MDA-PCa-2b IL-1α subline (MDA-αs) and MDA-PCa-2b IL-1β subline (MDA-βs).

Techniques: Quantitative RT-PCR