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BACKGROUND Androgen‐independent growth leads to progressive prostate cancer after androgen‐ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand‐independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells. METHODS We established and characterized the androgen‐independent FGC‐DCC from the androgen‐dependent LNCaP fast growing colony (FGC) cell line. The androgen‐independent DU‐145, FGC‐DCC, and PC‐3, and the androgen‐dependent LNCaP and PC‐346C cell lines were used to study growth modulation of gastrin‐releasing peptide (GRP), calcitonin (CT), serotonin (5‐HT), and vasoactive intestinal peptide (VIP) by 3H‐thymidine incorporation. Specificity of the growth‐modulating effects was tested with the anti‐GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides. RESULTS Androgen‐independent growth stimulation by neuropeptides was shown in DU‐145 and PC‐346C. 2A11 inhibited GRP‐induced 3H‐thymidine incorporation in DU‐145 and PC‐346C and inhibited proliferation of the FGC‐DCC and PC‐3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP‐induced growth effect in DU‐145 and PC‐346C, whereas oxadiazoloquinoxaline‐1‐one (ODQ) had no effect on 3H‐thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC‐DCC, or PC‐3. CONCLUSIONS GRP‐induced growth of DU‐145 and PC‐346C was specific and cAMP‐mediated. Androgen‐independent growth of FGC‐DCC cells was mainly due to an induction of Bcl‐2 expression and possibly through the activation of an autocrine and NE‐like pathway, as has been shown also for the PC‐3 cell line. Growth induction of non‐NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer. Prostate 42:34–44, 2000. © 2000 Wiley‐Liss, Inc.

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About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80°C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN ≥6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN <5: not reliable for demanding downstream analysis; 5 ≤RIN <6: suitable for RT-qPCR; 6 ≤RIN <8: suitable for gene array analysis; and RIN ≥8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN ≥6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made.

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It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G 0 -phase-arrested cells.Here we further characterized NE differentiation , androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0% , and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgApositive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days , respectively. However , no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G 0 -phase-arrested cells. 2-8 These cells produce various growth-modulating neuropeptides in a paracrine or autocrine way. -15Possible roles for NE cells in the prostate may be regulation of homeostasis and secretion of prostatic fluid, either actively or passively. NE cells can be identified by routine electron microscopy (dense core granules) or by immunohistochemistry with specific antibodies against secreted products, for example, serotonin, 16 or secretion-associated proteins, such as chromogranin A (CgA), [17][18][19] which is a marker for neuroendocrine differentiation. NE cells are considered to be nonproliferating cells and do not express the androgen receptor 20 and are therefore probably unaffected by androgen deprivation. Consequently, they will not undergo apoptosis under such circumstances. Therefore, it is relevant to assess whether or not CgA-positive cells co-express the antiapoptotic oncogene Bcl-2. 21 Moreover, NE cells show a heterogeneous cytokeratin expression pattern as there are basal, luminal, and intermediate NE cell types 16,22,23 and are often found near Bcl-2-positive prostate cancer cells. 24,25The single expression of CgA is not the only requisite for the determination of NE differentiated cells as there exists a regulated secretory pathway (RSP) in NE cells 26 next to the lysosomal and an exocrine constitutive pathway. Along the RSP pathway, secretion and processing of bioactive neuropeptides and growth hormones, such as insulin and glucagon in the pancreas, 27,28 are regulated. The RSP consists of a sequence of processes linked from transcription/translation of various factors ...

Androgen deprivation of the NE-differentiated PC-310 model induced the formation of NE-differentiated AR(minus sign) and non-NE AR(+) tumor residues. The NE-differentiated cells actively produced growth factors via an RSP that may lead to hormone-refractory disease. The dormant non-NE AR(+) tumor cells were shown to remain androgen sensitive even after long-term androgen deprivation. In the PC-310 xenograft, time-dependent NE differentiation and subsequent maturation were induced after androgen depletion. The androgen-dependent PC-310 xenograft model constitutes an excellent model for studying the role of NE cells in the progression of clinical prostate cancer.

The advent of molecular characterization of tissues has brought an increasing emphasis on the quality of biospecimens, starting with the tissue procurement process. RNA levels are particularly affected by factors in the collection process, but the influence of different pre-analytical factors is not well understood. Here we present the influence of tissue specimen size, as well as the transport and freezing protocols, on RNA quality. Large, medium, and smaller porcine liver samples were stored either dry, on moist gauze, or in salt solution for various times, and then frozen in either liquid nitrogen or in pre-cooled isopentane. Large and small human liver samples were frozen in pre-cooled isopentane either immediately or after one hour at room temperature. The small samples were stored dry, on moist gauze, or in salt solution. RNA was isolated and RIN values were measured. The RNA for six standard reference genes from human liver was analyzed by RT-qPCR, and tissue morphology was assessed for artifacts of freezing. Experiments using porcine liver samples showed that RNA derived from smaller samples was more degraded after one hour of cold ischemia, and that cooled transport is preferable. Human liver samples showed significant RNA degradation after 1 h of cold ischemia, which was more pronounced in smaller samples. RNA integrity was not significantly influenced by the transport or freezing method, but changes in gene expression were observed in samples either transported on gauze or in salt solution. Based on observations in liver samples, smaller samples are more subject to gene expression variability introduced by post-excision sample handling than are larger samples. Small biopsies should be transported on ice and snap frozen as soon as possible after acquisition from the patient.