Probes for PTEN

Abstract

BACKGROUND:

Loss or mutation of PTEN alleles at 10q23 in combination with 8q24 amplification (encompassing MYC) are common findings in aggressive, human prostate cancer. Our group recently developed a transgenic murine model of prostate cancer involving prostate-specific Pten deletion and forced expression of MYC under the control of the Hoxb13 promoter. MYC overexpression cooperated with Pten loss to recapitulate lethal, human prostate cancer.

METHOD:

We now report on the generation of two mouse prostate cancer cell lines, BMPC1 and BMPC2, derived from a lymph node, and liver metastasis, respectively.

RESULTS:

Both cell lines demonstrate a phenotype consistent with adenocarcinoma and grew under standard tissue culture conditions. Androgen receptor (AR) protein expression is minimal (BMPC1) or absent (BMPC2) consistent with AR loss observed in the BMPC mouse model of invasive adenocarcinoma. Growth in media containing charcoal-stripped serum resulted in an increase in AR mRNA in BMPC1 cells with no effect on protein expression, unless androgens were added, in which case AR protein was stabilized, and showed nuclear localization. AR expression in BMPC2 cells was not effected by growth media or treatment with androgens. Treatment with an anti-androgen/castration or androgen supplemented media did not affect in vitro or in vivo growth of either cell line, irrespective of nuclear AR detection.

DISCUSSION:

These cell lines are a novel model of androgen-insensitive prostatic adenocarcinoma driven by MYC over-expression and Pten loss.

Abstract

Objectives

Recent commercialization of methods for in situ hybridization using Z-pair probe/branched DNA amplification has led to increasing adoption of this technology for interrogating RNA expression in formalin-fixed, paraffin-embedded (FFPE) tissues. Current practice for FFPE block storage is to maintain them at room temperature, often for many years.

Methods

To examine the effects of block storage time on FFPE tissues using a number of RNA in situ probes with the Advanced Cellular Diagnostic’s RNAscope assay.

Results

We report marked reductions in signals after 5 years and significant reductions often after 1 year. Furthermore, storing unstained slides cut from recent cases (<1 year old) at –20°C can preserve hybridization signals significantly better than storing the blocks at room temperature and cutting the slides fresh when needed.

Conclusions

We submit that the standard practice of storing FFPE tissue blocks at room temperature should be reevaluated to better preserve RNA for in situ hybridization.