Synchronized prostate cancer cells for studying androgen regulated events in cell cycle progression from G₁ into S phase

Abstract

Androgen-ablation is a most commonly prescribed treatment for metastatic prostate cancer but it is not curative. Development of new strategies for treatment of prostate cancer is limited partly by a lack of full understanding of the mechanism by which androgen regulates prostate cancer cell proliferation. This is due, mainly, to the limitations in currently available experimental models to distinguish androgen/androgen receptor (AR)-induced events specific to proliferation from those that are required for cell viability. We have, therefore, developed an experimental model system in which both androgen-sensitive (LNCaP) and androgen-independent (DU145) prostate cancer cells can be reversibly blocked in G(0)/G(1) phase of cell cycle by isoleucine deprivation without affecting their viability. Pulse-labeling studies with (3)H-thymidine indicated that isoleucine-deprivation caused LNCaP and DU145 cells to arrest at a point in G(1) phase which is 12-15 and 6-8 h, respectively, before the start of S phase and that their progression into S phase was dependent on serum factors. Furthermore, LNCaP, but not DU145, cells required AR activity for progression from G(1) into S phase. Western blot analysis of the cell extracts prepared at regular intervals following release from isoleucine-block revealed remarkable differences in the expression of cyclin E, p21(Cip1), p27(Kip1), and Rb at the protein level between LNCaP and DU145 cells during progression from G(1) into S phase. However, in both cell types Cdk-2 activity associated with cyclin E and cyclin A showed an increase only when the cells transited from G(1) into S phase. These observations were further corroborated by studies using exponentially growing cells that were enriched in specific phases of the cell cycle by centrifugal elutriation. These studies demonstrate usefulness of the isoleucine-deprivation method for synchronization of androgen-sensitive and androgen-independent prostate cancer cells, and for examining the role of androgen and AR in progression of androgen-sensitive prostate cancer cells from G(1) into S phase.

Androgen regulated TRPM8 expression: A potential mRNA marker for metastatic prostate cancer detection in body fluids

“…5). We reported previously that inhibition of AR activity with Casodex prevents LNCaP cells from entering S phase (22,23). In the present study we observed that Casodex also suppressed TRPM8 expression in LNCaP cells (Fig.…”

“…Synchronization of LNCaP cells using isoleucine-free medium was essentially as described by Cifuentes et al (23). Cells arrested in G 0 /G 1 phase were released into complete medium containing 10% FCS and 10 nM testosterone.…”

“…Since AR plays an important role in LNCaP cell proliferation (22,23), we tested whether androgen/AR regulated TRPM8 expression is associated with the progression of prostate cancer cells from G 1 to S phase. We employed isoleucine-deprivation to reversibly block LNCaP cells in G 0 /G 1 phase (23). Cells released from isoleucine-block progressed synchronously from G 1 to S phase (Fig.…”

Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells

“…Isoleucine deprivation was done by our previously published method (29). The cells were grown to 60% to 70% confluence, and then the medium was replaced with isoleucine-free RPMI 1640 supplemented with 6% dialyzed FCS (Life Technologies), 2.5 mmol/L glutamine, 100 Ag/mL streptomycin, 100 units/mL penicillin, and 10 nmol/L testosterone.…”

“…Whole-cell extracts (Cell Lysate ) were incubated with either mAb (mAb 441 ) to a synthetic peptide comprising residues 301 to 317 in the middle of the ATD of AR, pAb (pAb N20 ) to a synthetic peptide comprising residues 1 to 20 of the ATD of AR, or immunoprecipitation buffer lacking antibody (No Ab). The mixtures were then treated with an immunoglobulin adsorbent (proteinblock (29). In these cells, AR levels fluctuated during the cell cycle from a low at G 0 -G 1 (0-4 hours after release from isoleucine block) to a maximum just as S phase begins (12-16 hours after release from isoleucine block; Fig.…”