Androgen receptor interacts with telomeric proteins in prostate cancer cells

Abstract

The telomeric complex, shelterin, plays a critical role in protecting chromosome ends from erosion, and disruption of these complexes can lead to chromosomal instability culminating in cell death or malignant transformation. We reported previously that dominant-negative mutants of one of the telomeric proteins called TIN2 cause death of androgen receptor (AR)-negative but not AR-positive prostate cancer cells, raising the question of a possible role of AR in the structural stability of telomeric complexes. Consistent with this possibility, in the present study, we observed that the AR antagonist Casodex (bicalutamide) disrupted telomeric complexes in AR-positive LNCaP cells but not in AR-negative PC-3 cells. Immunofluorescent studies revealed colocalization of TIN2 and AR. Reciprocal immunoprecipitation studies showed association of AR with telomeric proteins. Furthermore, telomeric proteins were overexpressed in prostate cancer cells compared with normal prostate epithelial cells, and sucrose density gradient analysis showed co-sedimentation of AR with telomeric proteins in a shelterin-like mega complex. Together, these observations suggest an allosteric role of AR in telomere complex stability in prostate cancer cells and suggest that AR-antagonist Casodex-mediated cell death may be due to telomere complex disruption.

FIGURE 1.

Casodex disrupts telomeric complexes in AR-positive prostate cancer cells. A and B, LNCaP cells treated with 100 μm Casodex for 48 h (A and B) or with 20 μg/ml etoposide for 1 h (B) were co-immunostained with antibodies against 53BP1 and TRF2. LNCaP (C) or PC-3 (D) cells were treated with or without 100 μm Casodex for 48 h and then immunostained with 53BP1 antibody. 53BP1 foci were counted, and data are presented as the percentage of cells with 0–5, 6–10, 11–20, or >20 foci/cell. Immunostaining and confocal microscopy were performed as described under “Experimental Procedures.” 80 cells in each treatment group were scored in three separate experiments.

FIGURE 2.

Casodex disrupts telomeric complexes without affecting the expression of telomeric proteins. A, LNCaP cells treated with 100 μm Casodex for 0, 3, 10, 24, or 48 h were immunostained for 53BP1. 53BP1 foci were counted, and cells were categorized as having ≤5 or >5 foci/cell. ∼200 cells in each treatment group were scored in three independent experiments. B, total RNA from LNCaP cells treated with Casodex was extracted using TRIzol (Invitrogen), and RT-PCR was performed as described previously (32) to measure mRNA levels. Sequence-specific primers for TIN2, TRF1, TRF2, TPP1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (11), and for AR, 5-tcagttcacttttgacctgctaa-3′ (forward) and 5′-gtggaaatagatgggcttga-3′ (reverse) primers were used. C, whole cell lysates of LNCaP cells treated with Casodex were subjected to Western blot analysis to determine TIN2, TRF1, TRF2, AR, and glyceraldehyde-3-phosphate dehydrogenase (loading control) protein levels.

FIGURE 3.

AR is colocalized with TIN2 in telomeres. LNCaP cells were co-immunostained with antibodies against AR and TIN2. The yellow spots identified by white arrows in the “Merge” panel represent AR and TIN2 co-localization.

FIGURE 4.

AR is associated with telomeric proteins in LNCaP cells. A, telomeric proteins are overexpressed in prostate cancer cells. Whole cell lysates prepared from prostate cancer cells (LNCaP, PPC-1, and PC-3) and normal prostate epithelial cells (PrEC) were subjected to Western blot analysis to determine TRF1, TRF2, TIN2, AR, and β-actin (loading control) protein levels. B, AR and TRF2 in LNCaP cell lysates were individually immunoprecipitated (IP) using monoclonal antibodies, and immunoprecipitates and starting lysate (2% of input) were subjected to Western blot (WB) analysis of AR, TRF1, and TRF2. C, cell lysates were prepared from LNCaP cells transfected with HA-tagged TRF1 as described previously (13), and HA-TRF1 in cell lysates was immunoprecipitated using antibodies against the HA epitope. Immunoprecipitates and unprecipitated lysate (10% of input) were subjected to Western blot analysis of AR and TIN2. D, sucrose density gradient analysis of telomeric proteins and AR in LNCaP cells. Nuclear extract (NE) prepared from exponentially growing LNCaP cells was subjected to sucrose density gradient centrifugation, and the gradient was resolved into 12 fractions, which were subjected to Western blot analysis to identify the distribution of TRF1, TRF2, TIN2, TPP1, and AR in the gradient. Lamin B was used as a control. Top, top of the gradient; Bottom, bottom of the gradient; NE, Nuclear extract loaded onto the gradient.