Human Prostate Tumor Cell Lines Research Articles

Presence of 11 β‐hydroxysteroid dehydrogenase enzyme in the human prostate tumor cell line LNCaP

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is the enzyme that catalyzes the reversible oxidation of the biologically active steroid cortisol and corticosterone to their inactive metabolites cortisone and dehydrocorticosterone. We report its presence in significant levels in the human prostate carcinoma cell line LNCaP cultured in medium RPMI-1640 with 10% fetal calf serum (FCS). The 11-dehydrogenase activity of 11 beta-HSD is present, while the 11-reductase activity is undetectable in these tumor cells under the present culture conditions. The enzyme activity is found to be linear with time of incubation, and is proportional to plated cell density. The enzymatic activity can be determined in cultures maintained for longer times. Carbenoxolone, the potential inhibitor of the 11 beta-HSD, inhibits 95% of the dehydrogenase activity of the tumor cells when used in nM concentration. The presence of this enzyme in tumor cell line indicates that 11 beta-HSD plays an important role in maintaining the active glucocorticoid levels in the prostate.

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens

The human prostate tumor cell line LNCaP containd an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor proteon and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induced reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.

The Promoter of the Prostate-Specific Antigen Gene Contains a Functional Androgen Responsive Element

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.

Effect of etidronate disodium on the development of bone lesions in an animal model of bone metastasis using the human prostate cancer cell line PC‐3

We have established an animal model of bone metastasis using the PC-3 human prostate tumor cell line. In order to assess whether inhibition of bone resorption would prevent the development of bone metastasis, the diphosphonate etidronate (EHDP) was administered to 20 mice at a dose of 30 mg/kg subcutaneously daily starting 2 days prior to injection of tumor cells. Control mice received daily injections of the saline vehicle. In the EHDP-treated mice, there was no significant reduction in the incidence of bone metastasis, the size of the lesions, or the number of bone lesions per mouse. Approximately 50% of the mice developed bone metastasis, which was similar to the control group and similar to what was observed in earlier studies with this animal model. Histomorphometric analysis of bones showed marked inhibition of mineralization in EHDP-treated mice, thus indicating biological effect on the bone. Therefore, the use of EHDP in biologically effective doses failed to reduce the incidence, size, or number of bone metastases in this animal model.

The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines

The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT coVon encoding a threonine residue to GCT, encoding alanine.

Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: High affinity for progestagenic and estrogenic steroids

LNCaP tumor cells, derived from a metastatic lesion of a human prostatic carcinoma, are androgen-sensitive in cell culture. Although increase in growth rate is observed with low doses of progestagens or estradiol, these cells contain exclusively androgen receptors. In the present study the binding affinity of different ligands for both non-DNA- and DNA-binding (transformed) forms of the androgen receptor were analyzed. The cytosolic (non-transformed) form of the receptor displayed an abnormal high affinity for progestagens and estradiol when compared with the cytosolic androgen receptor from other sources. Subsequently the non-transformed forms of the androgen receptor obtained from LNCaP cell nuclei was studied. A high binding affinity was found not only for dihydrotestosterone, but also for progesterone and the synthetic progestagen R5020 (relative binding affinity 42% and 10% of dihydrotestosterone). The binding characteristics of the transformed androgen receptor were examined in intact cells at 37°C. LNCaP cells were compared in this respect with COS cells containing the cloned human androgen receptor, normal human skin fibroblasts and PC3 (prostate) and NHIK (cervix) human tumor cell lines. The affinity of the transformed androgen receptors for the progestagen R5020 in LNCaP cells was significantly higher than in the other cell systems, although the differences were less pronounced than for the non-transformed receptor form. In conclusion: the LNCaP tumor cells contain an androgen receptor with an abnormal binding site. This might be due to a mutation and/or a post-transcriptional effect.

The Proliferative Effect of “Anti-Androgens” on the Androgen-Sensitive Human Prostate Tumor Cell Line LNCaP

The effect of steroidal and nonsteroidal "anti-androgens" on the proliferative capacity of androgen-sensitive LNCaP-FGC human prostate tumor cells in culture was studied using charcoal-dextran stripped human serum-supplemented media. Cyproterone and medroxyprogesterone acetates, flutamide, hydroxyflutamide, and anandron (R23908) were administered alone at concentrations between 3 X 10(-12) and 3 X 10(-6) M. Results indicated that although medroxyprogesterone induced maximal proliferation at 3 X 10(-9) M, the other "anti-androgens" (with the exception of flutamide that was ineffective) were effective at 3 X 10(-8) M and higher concentrations; the amplitude of the proliferative response by these compounds was comparable to that elicited by estradiol-17 beta (3 to 5-fold over control). None of the anti-androgens tested triggered the shutoff effect characteristic of androgen action. When 3 X 10(-10) M DHT and the above mentioned anti-androgens were administered simultaneously, a synergistic pattern was seen; on the contrary, 3 X 10(-8) M DHT cancelled the proliferative effect of each of the anti-androgens when administered simultaneously. The relative binding affinity of these anti-androgens to androgen receptors present in LNCaP-FGC cells did not correlate well with their proliferative efficiency. The data collected were interpreted within the premises of the negative control hypotheses for the regulation of cell proliferation in metazoans. Within those premises, results became compatible with the notion that first, "anti-androgens" elicited the proliferation of androgen-sensitive cells by neutralizing the effect of a serum-borne inhibitor (androcolyone-I); this event seems not to be mediated by androgens receptors. Second, anti-androgens did not trigger a proliferative shutoff response like androgens do, i.e. the proliferative pattern induced by anti-androgens was comparable to that elicited by estrogens and progestins. Third, when administered simultaneously with 3 X 10(-10) M DHT, anti-androgens behaved synergistically. Fourth, the DHT-induced shutoff effect consistently overrode the proliferative effect generated by anti-androgens and estrogens when added alone. Finally, taken together these results raise important questions regarding the therapeutic role of anti-androgens in prostate cancer.

Identification by C-banding of two human prostate tumour cell lines, 1013L and DU 145.

Two human prostate carcinoma cell lines are easily distinguished by C-banding. DU 145 has one metacentric chromosome with a large centromeric band, 1 metacentric and 2 acrocentric chromosomes with interstitial C-bands. The Y chromosome was present in 48% and 42% of the cells at passages 83 and 106, respectively, whereas in 1013L only one metacentric chromosome contained distinct extra C bands--one at the centromere and one interstitially. The Y chromosome was present in 72% of the cells at passage 15 and in 20% at passage 22, but by passage 44 it had disappeared. Karyotype analysis using G-banding revealed that DU 145 had retained several of its original markers, and demonstrated multiple marker chromosomes in 1013L.

Mechanism of androgen action: Recent observations on the domain structure of androgen receptors and the induction of EGF-receptors by androgens in prostate tumor cells

In this paper two different aspects of androgen action are reviewed. Polyacrylamide gel electrophoresis of androgen receptors, photoaffinity labeled with R 1881 showed that receptors isolated from both human prostate cells and calf uterine cytosol cells are proteins with a molecular mass of approx 110 kD. Purification to homogeneity of this form of the receptor from calf uterus also yielded a 110 kD protein. A molecular model for the DNA-binding form of the receptor is presented in which one polypeptide comprises three active domains: one for ligand binding, one for interaction with nuclear acceptor sites, and a third domain which modulates nuclear interaction. Mild digestion with chymotrypsin or a protease from rat prostates removes the modulating domain and leaves the ligand binding and nuclear interaction domain intact. Trypsin treatment yields a fragment of lower molecular mass containing the ligand binding domain with some affinity for RNA, but not DNA. In vitro studies with a human prostate tumor cell line (LNCaP), suggest that androgens not only directly effect cell growth, but also act indirectly. Both epidermal growth factor (EGF) and androgens stimulate cell growth. In addition androgens stimulate synthesis of receptors for EGF. Thus androgens effect tumor cell growth by autocrine or paracrine mechanisms by making the cells more sensitive for growth factor mediated stimuli.

Androgen-Dependent Growth Regulation of and Release of Specific Protein(s) by the Androgen Receptor Containing Human Prostate Tumor Cell Line LNCaP

Androgen-Dependent Growth Regulation of and Release of Specific Protein(s) by the Androgen Receptor Containing Human Prostate Tumor Cell Line LNCaP

Possible mechanism for differences in sensitivity to cis-platinum in human prostate tumor cell lines

Two human prostate tumor cell lines which exhibit a 2.4-fold difference in sensitivity to cis-platinum (cis-Pt) were found to possess slightly different sulphydryl contents, and the presence of a metallothionein-like zinc-binding protein was demonstrated in the line exhibiting relative resistance to cis-Pt. Although these factors have been postulated to play a role in the mechanism(s) of resistance to cis-Pt in other cell types, preliminary data in this report suggest that differences found in drug uptake and subsequent binding to DNA are most likely responsible for variations in cis-Pt sensitivity displayed by these prostate tumor cell lines.

Androgen‐dependent growth regulation of and release of specific protein(s) by the androgen receptor containing human prostate tumor cell line LNCaP

Hormone sensitivity, as indicated by the presence of steroid hormone receptors and by the effect of androgens and anti-androgens on the release of proteins by cultured cells of the human prostate tumor cell line lymph node carcinoma of the prostate-fast growing colony (LNCaP-FGC), has been studied. The growth of the LNCaP-FGC cells were stimulated by androgens in a dose-dependent way. Under optimal conditions the synthetic nonmetabolizable androgen 17 beta-hydroxy-17 alpha-methyl-(3H)-estra-4,9,11-trien-3-one (R1881) (0.1 nM) stimulated cell growth by approximately 2.3 times. Increasing doses of R1881 (1-100 nM) partly decreased the stimulation of the cell growth. The anti-androgen cyproterone acetate exerted inhibitory effects on cell growth. The nuclear extract of the LNCaP-FGC cells contained 17,000 +/- 2,500 (mean +/- SD) KCl-extractable, nuclear androgen receptor sites/cell. Estrogen and progesterone receptors were not detectable in the nuclear extracts nor in cytosol, indicating that these receptors were absent. The release of proteins in the culture medium was studied using incorporation of the 35S-methionine, sodium dodecyl sulfate (SDS)-gel electrophoresis, and fluorography. Cells grown in media containing charcoal-stripped fetal calf serum released significantly lower amounts of a protein with an apparent molecular mass of 42,000 daltons. The release of this 42-kD protein could be restored in cells cultured in the presence of 5 alpha-dihydrotestosterone (0.1-1 microM) or R1881 (0.1-100 nM), whereas the addition of estrogens or corticosteroids had no effect. In the presence of anti-androgens, such as cyproterone acetate and 5,5-dimethyl-3-(4-nitro-3-(trifluoro-methyl)-phenyl)-2,4-imidasolidin edione, inhibitory effects on the release of the 42-kD protein were observed. The observed parallel between the effects of (anti)-androgens on the growth of the LNCaP prostate cells and the release of the 42-kD protein suggests that this protein is involved in the regulation of malignant prostate cell growth.

The androgen responsive human prostate tumor cell line LNCaP: Androgen receptors and the effect of androgens on the release of proteins

The androgen responsive human prostate tumor cell line LNCaP: Androgen receptors and the effect of androgens on the release of proteins

In vitro responses of human prostate tumour cell lines to a range of antitumour agents.

Four human prostate tumour continuous cell lines were employed for in vitro drug sensitivity testing of nine cytotoxic drugs, X-irradiation and two hormonal preparations. Responses were assessed by measuring cell survival as judged by colony formation in agarose. A marked heterogeneity of responses was noted to many of the agents studied, including X-irradiation. However, in terms of ID70 values for 24-h drug exposures, comparable responses in all the cell lines tested, with values falling within a 1.4- to 2.9-fold range, were noted with actinomycin D, adriamycin, m-AMSA, dibromodulcitol, 5-fluorouracil, hydroxyurea and ICRF-159. In contrast, mixed responses to cis-platinum and methotrexate were particularly marked, with ID70 values differing by factors of 3.8 and 7.5 respectively. In addition, when higher drug concentrations of m-AMSA, cis-platinum, dibromodulcitol and ICRF-159 were employed, significant deviations in the four survival curves became apparent. Continuous exposure to diethylstilboestrol and medroxyprogesterone acetate caused negligible cell kill. Due to these variable responses it was not possible to predict that any agent tested might have specific value in the treatment of prostate cancer. However, preliminary in vitro screening with such a panel of cell lines may complement in vivo studies with models of prostatic cancer and prove useful in investigations of mechanisms of drug resistance.