cancerres.aacrjournals.org/conte … /7544.full
some highlights supporting methylation theory:
We tested whether PSA, a known AR target gene, can be induced by R1881 treatment of AI cells, which we showed to have a functional AR. In AD cells used as a positive control, treatment with R1881, which led to an increase in AR level (Fig. 2, A ⇓ , top panel), produced a concomitant 12-fold up-regulation of the PSA protein (Fig. 2, A ⇓ , middle panel). However, in the AI cells the level of PSA remained undetectable despite R1881 treatment and a constitutively active, androgen-responsive AR (Figs. 1A ⇓ and 2A ⇓ , top panel). Several mechanisms may be responsible for the lack of PSA expression, among them are a mutation or loss of the gene, transcriptional silencing of the gene, or lack of necessary transcription factor(s) or cofactors. To distinguish between these possibilities, we first tested whether R1881 will activate a PSA-promoter in AI cells transfected with a PSA-promoter-luciferase construct; transfected AD cells served as a positive control. The basal expression of the PSA reporter gene in AI cells was 2-fold greater than in AD cells (Fig. 2B) ⇓ , possibly because of the higher levels of AR, and this activity was additionally stimulated by exposure of both cell types to 10 nm of R1881. The AD cells required cotransfection with an AR expression plasmid for full stimulation, possibly because the PSA promoter reporter was competing for transcription factors with the endogenous PSA promoter. In AD cells cotransfected with a plasmid expressing the mutant AR found in LNCaP cells (25 , 26) , the PSA-reporter gene was similarly induced by R1881 treatment (results not shown). These results showed that R1881 can activate the transcription of a transfected PSA promoter in AI cells, suggesting that it is the endogenous gene that is either silenced or mutated. A common mechanism of gene silencing is hypermethylation of the promoter (34) . To test whether this was the case, we incubated AI cells with 5-aza for 3, 5, or 7 days. This treatment is known to inhibit DNA-methylation and cause re-expression of genes silenced by hypermethylation (35) . Using RT-PCR detection we showed that 3-day treatment with 5-aza caused a dose-dependent re-expression of the endogenous PSA gene (Fig. 2C) ⇓ , suggesting that the gene is silenced by hypermethylation (5- and 7-day treatment with 5-aza produced a similar induction; results not shown). We do not know whether PSA gene silencing is the result of chronic androgen deprivation or just an epi-phenomenon of this treatment, but in several additional cell lines derived in a similar way, PSA was also undetected (results not shown). At present, there is no published evidence that the PSA gene is hypermethylated in hormone refractory prostate cancer, but investigation of this possibility in human samples might explain the discrepancy between PSA levels and tumor burden often observed in patients with advanced disease.
And something else…
We began to explore the link between the AR and p21WAF1 in AD cells. We reasoned that if p21WAF1 functions as an inhibitor of cell cycle progression, R1881 treatment, which stimulates AD cell growth and increases the level and the activation of AR (Fig. 1, A and B) ⇓ , should reduce the p21WAF1 level. Indeed, we found that in R1881-treated AD cells, the p21WAF1 protein level was reduced 8-fold compared with an untreated control …These results indicate that down-regulation of p21WAF1 is linked to activation of AR and that it is not cell-type specific .
Perhaps we can test for high levels of p21waf1? No idea really, over my head a bit… hopefully someone else can take a look at the whole article and decipher it
