6 research outputs found
The human prostatic cancer cell line LNCaP and its derived sublines: An in vitro model for the study of androgen sensitivity
__Abstract_
The LNCaP-FGC (fast growing colony) cell line, a subline derived from the LNCaP cell line, shares all the main characteristics, including its androgen sensitivity, described for the parental line. A number of sublines originating from the FGC line were characterized with respect to their response to steroid-depleted medium and to the synthetic androgen R1881. The growth of FGC cells in DCC medium with 0.1 nM R1881 was stimulated 2–3-fold compared to growth in DCC medium only. FGC cells that were continuously grown in DCC medium did not die, but their growth rate was clearly slowed down, and the cells remained responsive to androgen. These cells, therefore, have the androgen-sensitive, rather than the androgen-dependent phenotype. As cells of the subline FGC-JB could not be maintained in DCC medium, these cells better represent the androgen-dependent cell type. In contrast to the FGC line, cells of the R line, grew equally well in medium with complete or DCC serum. Under none of these culture conditions, R cells could significantly be stimulated further with R1881. Further analysis of the LNCaP-FGC sublines should provide valuable information concerning the development of androgen resistance in human prostate cancer
Kinetics of neuroendocrine differentiation in an androgen-dependent human prostate xenograft model
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
G0-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 CgA-positive 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 G0-phase-arrested
cells
Androgen-independent expression of adrenomedullin and peptidylglycine alpha-amidating monooxygenase in human prostatic carcinoma
Most of the locally advanced and metastatic prostate carcinomas (PCs) treated with antiandrogenic therapy eventually become refractory to this treatment. Locally produced factors may control prostate tumor biology after androgen withdrawal. Adrenomedullin (AM) is expressed in the prostate and could control cell growth in androgen-independent conditions. AM needs to be amidated by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM) to become fully active. The objective of the present study was to analyze whether the expression of preproadrenomedullin (preproAM) and PAM in PC is regulated by androgens. For this purpose, human in vitro and in vivo PC models were grown in the presence or absence of androgens, and the expression of AM and PAM was examined by immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. Furthermore, immunohistochemical analysis of AM in clinical specimens was performed to test if its expression is related to Gleason score and antiandrogenic therapy. In PC cell lines and xenografts, mRNA and protein AM levels were similar in the presence or absence of androgens. PAM expression seemed to be induced by androgen-withdrawal. Our results in clinical samples showed no relationship between AM expression and Gleason score or antiandrogenic treatment. In conclusion, our results demonstrate that preproAM and PAM expression in the human prostate is androgen-independent. In addition, we also report for the first time the expression of a novel PAM transcript in PC, which has not been previously described in other tissues
Peptidylglycine alpha-amidating monooxygenase- and proadrenomedullin-derived peptide-associated neuroendocrine differentiation are induced by androgen deprivation in the neoplastic prostate
Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide-amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP- or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy
Differentially expressed genes in androgen-dependent and -independent prostate carcinomas
Differential gene expression between androgen-dependent (LNCaP-FGC) and
androgen-independent (LNCaP-LNO) prostate cancer cells has been
investigated using RNA arbitrarily primed and differential display PCR of
mRNA. Four differentially expressed cDNA transcripts were identified, of
which differential expression was confirmed by Northern blot analysis.
Sequence analysis revealed two unknown (JC19 and GC79) and two known genes
[B-cell translocation gene 1 and UDP-glucuronosyltransferase 2B15
(UGT2B15)]. JC19, GC79, and B-cell translocation gene 1 were more highly
expressed in LNCaP-FGC cells compared with LNCaP-LNO cells, whereas
UGT2B15 was only expressed in LNCaP-LNO cells. Androgens and
1,25-dihydroxyvitamin D3 were able to down-regulate UGT2B15 mRNA in
LNCaP-LNO cells. For GC79 mRNA, down-regulation was only observed with
androgens in LNCaP-FGC cells. Expression of JC19 mRNA was studied using a
panel of human prostate cancer xenografts. In androgen-dependent
xenografts, expression of JC19 mRNA was much higher compared with
androgen-independent xenografts, in which significant expression was
hardly detectable. The mRNA expression pattern in the xenografts is in
good agreement with that observed in the cell culture system. In
conclusion, the differential display technique used in the present study
allows analysis of gene expression in vitro and in vivo and can be used
for the identification of important genes involved in androgen-independent
prostate cancer development
Androgen deprivation of the PC-310 [correction of prohormone convertase-310] human prostate cancer model system induces neuroendocrine differentiation
Neuroendocrine (NE) cells are androgen-independent cells and secrete
growth-modulating neuropeptides via a regulated secretory pathway (RSP).
We studied NE differentiation after androgen withdrawal in the
androgen-dependent prostate cancer xenograft PC-310. Expression patterns
of chromogranin A, secretogranin III, and prohormone convertase-1 were
analyzed at both protein and mRNA level to mark the kinetics of NE
differentiation both in vivo and in vitro. PC-310 tumor-bearing nude mice
were killed at 0, 2, 5, 7, 14, and 21 days postcastration. PC-310C
cultures initiated from collagenase-treated tumor tissue could be
maintained up to four passages, and androgen-deprivation experiments were
performed similarly. PC-310 tumor volumes decreased by 50% in 10 days
postcastration. Proliferative activity and prostate-specific antigen (PSA)
serum levels decreased to zero postcastration, whereas PSA levels in
PC-310C culture media first decreased and subsequently increased after 5
days. In vivo, androgen receptor (AR) expression decreased initially but
returned to control level from 5 days postcastration on. CgA,
secretogranin III, and secretogranin V expression increased in vivo from 5
days postcastration on. Subsequently, prohormone convertase-1 and peptidyl
alpha-amidating monooxygenase as well as the vascular endothelial growth
factor were expressed from 7 days postcastration on, and, finally, growth
factors such as gastrin-releasing peptide and serotonin were expressed in
a small part of the NE cells 21 days postcastration. The PC-310 tumors did
not show colocalization of the AR on the NE cells in the tumor residues
after 21 days. As in the PC-310 xenograft, NE differentiation was induced
and AR expression relapsed after prolonged androgen suppression in
PC-310C. For PC-310C cells, this relapse was associated with the secretion
of PSA. PC-310C is the first culture of human prostatic cancer cells
having the NE phenotype. The PC-310 model system is a potential
androgen-dependent model for studying the role of NE cells in the
progression of clinical prostate cancer. Androgen deprivation of
NE-differentiated prostate cancer may induce the formation of both NE- and
AR-positive dormant tumor residues, capable of actively producing NE
growth factors via a RSP, possibly leading to hormone refractory disease