Androgenrefractory Outgrowth of LNCaP Cells under Androgendeprived Conditions

In order to understand the molecular mechanism for the survival ofPCA cells under androgen ablation conditions, LNCaP cells were cultured in charcoal-stripped serum (CSS)-medium. Very limited death of LNCaP cells was detected under these culture conditions. Indeed, cells began to undergo neuroendocrine cell differentiation (NED) three days after androgen withdrawal. Cells at this stage are called LNCaP-tNED since NED is a transient process. After being cultured in CSSmedium for 10 weeks, LNCaP cells started to re-grow, and an androgen-refractory subline LNCaP-Rf was established eventually from parental LNCaP cells (Figure 1).

Figure 1. Morphological and biochemical analyses ofLNCaP cells grown under acute and chronic androgen-deprived culture conditions.

A. Morphology of LNCaP, LNCaP-tNED, and LNCaP-Rf cells.

B. Northern analysis of NSE and PSA in LNCaP, LNCaP-tNED, and LNCaP-Rf cells.

C. Western analysis of AR and Bcl-2 in LNCaP, LNCaP-tNED, and LNCaP-Rf cells.

Figure 1. Morphological and biochemical analyses ofLNCaP cells grown under acute and chronic androgen-deprived culture conditions.

A. Morphology of LNCaP, LNCaP-tNED, and LNCaP-Rf cells.

B. Northern analysis of NSE and PSA in LNCaP, LNCaP-tNED, and LNCaP-Rf cells.

C. Western analysis of AR and Bcl-2 in LNCaP, LNCaP-tNED, and LNCaP-Rf cells.

The parental LNCaP, LNCaP-tNED and LNCaP-Rf cells showed distinct morphological features (Fig. 1A). LNCaP-tNED cells exhibited neuroendocrine-like features, including long slender processes and compact cell body, while the untreated LNCaP cells show their typical fusiform morphology. ,LNCaP-Rf cells maintained an intermediate morphological appearance between parental LNCaP and LNCaP-tNED cells. Consistent with morphological observations, LNCaP-Rf cells expressed more mRNA ofneuron-specific enolase (NSE), a specific neuroendocrine marker, than parental LNCaP cells, but less than LNCaP-tNED cells (Figure 1B), In Figure 1C, levels ofthe androgen receptor (AR) protein were transiently decreased in LNCaP-tNED cells. However, LNCaP-Rf showed comparable AR protein levels to untreated LNCaP cells. It is worth noting that more AR protein became hypophosphorylated following androgen ablation (lane 2 and 3, Figure 1C). In contrast, the AR protein was present mainly in a phosphorylated state under normal culture conditions (lane 1, Fig. 1C). The AR protein became hyperphosphorylated after androgen treatment (lane 4, Fig. 1C). The expression levels of prostate-specific antigen (PSA) were decreased largely in LNCaP-Rf cells in comparison to those in LNCaP cells (Fig. 1B). These experiments indicate that following androgen ablation, LNCaP cells transiently differentiate into neuroendocrine-like cells, and eventually evolve into androgen-refractory cells. Establishment of LNCaP-tNED and LNCaP-Rf from LNCaP cells provides excellent cellular models to explore the mechanism for survival and re-growth ofLNCaP cells under androgen deprivation conditions.

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