Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The genomic and the cDNA clones of human glandular kallikrein (hK2), a member of the kallikrein family, have been isolated; however, the hK2 protein has not yet been identified and characterized. The deduced sequence of hK2 is highly homologous to prostate specific antigen (PSA), a widely accepted prognostic indicator of prostate carcinoma. Also, hK2 mRNA, like PSA mRNA, is exclusively expressed in prostatic epithelia. These two properties make hK2 a potentially useful marker for studying prostate cancer. In this paper, we describe for the first time the overexpression of the entire hK2 protein (pre-pro hK2:pphK2) in the E. coli system. Our system yields high levels of authentic pphK2 (as determined by partial amino acid sequence analysis) comprising about 40% of total cellular protein. pphK2 was purified to near homogeneity by preparative SDS/PAGE and used to generate anti-pphK2 antibodies in rabbits. The antibodies recognize the recombinant hK2 protein and a major band of approximately 34 kDa in seminal fluid.
Mol Cell Endocrinol 1995 Apr 01
PMID:Overexpression of a human prostate-specific glandular kallikrein, hK2, in E. coli and generation of antibodies. 766 87

LNCaP, a human prostate cancer cell line, metabolizes testosterone into a variety of 5 alpha-reduced C19 steroids, such as dihydrotestosterone (DHT), androstane-3 alpha, 17 beta-diol (3 beta, 17 beta-DIOL), and androsterone (ADT). Recent reports also suggest that 5 alpha-reduced C19 steroid glucuronides can be detected in the medium. The purpose of this work was to characterize by liquid chromatograph ion spray mass spectroscopy (LCMS) the metabolites formed by LNCaP during incubation with testosterone and its 5 alpha-reduced C19 steroids. Time course studies using 10 nM labeled testosterone, 3 alpha-DIOL, 3 beta-DIOL, or ADT showed that a large proportion of polar steroids were produced by LNCaP. Identification of metabolites produced by LNCaP was carried out by LCMS using 1 microM substrates. Analysis of testosterone metabolism indicated that testosterone glucuronide was formed at 77 +/- 2% after 96 h of incubation. Using DHT as substrate, 3 alpha-DIOL-G and DHT-G were the major metabolites, accounting for 46 +/- 4% and 38 +/- 3%, respectively, of the total radioactivity in the medium; ADT-G accounted for 8 +/- 1%. Further analysis by LCMS also indicated that the glucuronide group in 3 alpha-DIOL-G was at position 17-carbon, 3 alpha-DIOL-G (86 +/- 3%) was the prominent metabolite formed from 3 alpha-DIOL, a minor product was detected at 7 +/- 1% and identified by mass spectrometry to correspond to a trihydroxylated C19 steroid.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1995 Feb
PMID:Glucuronosyltransferase activity in human cancer cell line LNCaP. 776 24

The present study was designed to investigate the effects of 13-cis-retinoic acid (13-cis-RA) (100 micrograms/mouse/day) and androgen ablation (castration) alone and in combination on growth of a human prostatic carcinoma line (LNCaP) transplanted to athymic nude mice as an experimental model. The results of these studies suggest that; (1) androgen ablation (castration) significantly decreased the size of LNCaP xenograft as compared to untreated animals; (2) when 13-cis-RA was administered to nude mice carrying established tumors (0.51 +/- 0.04 cm3), the tumor size was significantly reduced as compared to untreated controls (0.65 +/- 0.06 cm3 versus 1.63 +/- 0.12 cm3). About 50% of the animals in this group showed xenografts necrosis followed by complete regression of tumors by five months; (3) the combination of androgen ablation and 13-cis-RA treatment to nude mice carrying tumors showed synergistic effect in decreasing the tumor size. These results indicate that combination therapies based on androgen ablation and retinoid administration may be a useful approach for the treatment of prostate cancer.
Biochem Mol Biol Int 1995 Mar
PMID:Regression of LNCaP human prostate tumor xenografts in athymic nude mice by 13-cis-retinoic acid and androgen ablation. 777 85

Hereditary peculiarities in individual responses to environmental chemicals are a common occurrence in human populations. Genetic variation in glutathione S-transferase, CYP1A2, N-acetyltransferase, and paraoxonase exemplify the relationship of metabolic variation to individual susceptibility to cancer and other toxicants of environmental origin. Heritable receptor protein variants, a subset of proteins of enormous pharmacogenetic potential that have not thus far been extensively explored from the pharmacogenetic standpoint, are also considered. Examples of interest that are considered include receptor variants associated with retinoic acid resistance in acute promyelocytic leukemia, with paradoxical responses to antiandrogens in prostate cancer, and with retinitis pigmentosa. Additional heritable protein variants of pharmacogenetic interest that result in antibiotic-induced deafness, glucocorticoid-remediable aldosteronism and hypertension, the long-QT syndrome, and beryllium-induced lung disease are also discussed. These traits demonstrate how knowledge of the molecular basis and mechanism of the variant response may contribute to its prevention in sensitive persons as well as to improved therapy for genetically conditioned disorders that arise from environmental chemicals.
Environ Mol Mutagen 1995
PMID:Influence of heredity on human sensitivity to environmental chemicals. 778 56

The proliferation of the human prostatic cancer JCA-1 cells showed a complex response to different concentrations of TPA. Whereas cells exposed for 24 h had growth reduction which was proportional to the concentration of TPA added to the culture media, they showed resistance to low (0.016 and 0.16 nM) but not high (> or = 1.6 nM) doses of TPA after 72 h. Growth-inhibited, treated cells also displayed a distinct cell morphology compared with the controls. Since c-myc expression has previously been shown to be correlated with cellular proliferation, we determined changes in its steady-state level in control and treated cells by Northern analysis. Following a 24h, 48h, and 72h treatment, with 16 nM TPA, c-myc mRNA was suppressed by 91%, 83%, and 78%, respectively, in good agreement with the extent of growth reduction observed. At the low dose of TPA (0.16 nM), however, the c-myc mRNA expression remained inhibited by 85% even though cell growth was only reduced by 10-14%. No difference in the total amount of c-myc protein could be detected between control and treated cells by Western analysis.
Biochem Mol Biol Int 1994 Aug
PMID:Regulation of cell growth and the c-myc proto-oncogene expression by phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) in the androgen-independent human prostatic JCA-1 cells. 784 24

The metabolism of androgens in prostatic carcinoma has not been sufficiently studied so far, mainly because of the difficulty in obtaining human tissue specimens. The availability of LNCaP (lymph node carcinoma of the prostate) cells which retain most of the characteristics of the original carcinoma (dependence on androgens, presence of androgen receptors, production of acid phosphatase, etc.) has allowed the present in vitro study designed to characterize the metabolic pathways through which testosterone (T) is metabolized in malignant cells. LNCaP cells have been incubated in the presence of different labelled androgenic precursors to quantitate the formation of the respective metabolites, as indicators of the specific activities of the enzymes involved in such conversions; whenever possible, the kinetic constants (Km and Vmax) of the enzymes have also been calculated. It has been observed that, when [14C] T is used as substrate, the cells form both dihydrotestosterone (DHT) and androst-4-en-3,17 dione (delta 4) with the prevalence of the latter. When [14C] delta 4 is the substrate, T and 5 alpha-androstan-3,17-dione (5 alpha-A) are found with 5 alpha-A representing the major product. In addition, the cells form diols and 5 alpha-A from [14C]DHT as well as androsterone (A) and DHT from [14C]5 alpha-A; there is a prevalence of diols in the former case, and of A in the latter one. The yields of the different metabolites recovered after 2 h of incubation of the cells with the appropriate labelled substrates are therefore in descending order of magnitude: 5 alpha-A > A > diols > delta 4 > DHT > T. These results are also confirmed by the analysis of the rate of production of the different steroids. Taken together the present results suggest that: (a) qualitatively LNCaP cells possess all the major key enzymes involved in androgen processing; (b) the metabolism of androgens in this cell line resembles quantitatively that found in prostatic cancer tissue; all the metabolic steps which contribute to DHT degradation exceed the ones leading to its accumulation; (c) 5 alpha-reductase shows a significantly higher affinity for delta 4 than for T; (d) LNCaP cells may represent a suitable in vitro model for the study of factors controlling the formation and the degradation of androgens in prostatic carcinoma, thus permitting a better understanding of the metabolic processes involved in prostatic benign or malignant (carcinoma) transformation.
J Steroid Biochem Mol Biol 1994 Oct
PMID:Androgen metabolism in the human prostatic cancer cell line LNCaP. 794 55

The purpose of this study was to examine the effects of 13-cis-retinoic acid (13-cis-RA) on the growth regulation of DU-145 human prostatic cancer cells. The results of these experiments demonstrate that cell growth and metastatic potential of DU-145 cells were significantly inhibited by 13-cis-RA (10 microM). In order to elucidate the possible molecular mechanisms of 13-cis-RA action on prostate cancer cells, we examined the expression of nuclear receptor genes (hRXR alpha) and found that 13-cis-RA treated cells showed higher mRNA expression for hRXR alpha nuclear receptors compared to untreated cells. To elucidate further the possible biochemical mechanisms associated with these alterations, we analyzed the phosphorous metabolites by MR spectroscopy and found that the major metabolites were PME, (PC, PE) and DPDE (UDP-GalNAc, UDP-GLcNAc). The DU-145 cells and xenografts, which were both treated with 13-cis-RA, showed a two-fold decrease in DPDE's, compared to their controls. The higher resolution spectra of perfused cells revealed that phosphocholine levels were twice as high in 13-cis-RA-treated DU-145 cells as compared to untreated cells. These investigations demonstrate for the first time that 13-cis-RA inhibits the growth of human prostatic cancer cells, and this inhibition is associated with an increase in hRXR alpha nuclear receptor gene expression and alterations in phosphorous metabolites detected by 31P MR spectroscopy.
Biochem Mol Biol Int 1994 Jan
PMID:13-cis-retinoic acid-mediated growth inhibition of DU-145 human prostate cancer cells. 801 74

Incubation of whole LNCaP cells in suspension with tritium labeled cortisol revealed two major and one minor radioactive product. Of the major products, one migrated with an Rf value identical to cortisol (Kendall's compound "F"), and the second migrated with an Rf value similar to nonradioactive cortisone (Kendall's compound "E"); the third minor product comigrated with 21-acetylated cortisol. The conversion of cortisol to cortisone was linear with respect to cell number, and conversion reached a plateau after 120 min of incubation at 37 degrees C. One half of the cortisol was converted to cortisone within 2 h of incubation at 37 degrees C. This conversion was nicotine amide dinucleotide (NAD) dependent. Low levels of transcription activation by cortisol were documented in LNCaP cells transfected with glucocorticoid and androgen responsive mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene (MMTV-CAT). Hormone binding assay and transactivation analysis revealed the presence of a functional mineralocorticoid receptor in LNCaP cells. Treatment of transfectants with F in the presence of carbenoxolone, a potent inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), resulted in a two orders of magnitude increase in measurable CAT activity. The addition of the reduced form of nicotine amide dinucleotide (NADH) in the presence of 10(-7) M E stimulated measurable CAT activity in LNCaP cells. In conferring aldosterone specificity in mineralocorticoid target tissues, 11 beta-HSD may have an important role as "gate keeper" in allowing a specific androgen response in hormone responsive LNCaP prostate cancer cells.
J Steroid Biochem Mol Biol 1994 Jun
PMID:11 beta-Hydroxysteroid dehydrogenase and tissue specificity of androgen action in human prostate cancer cell LNCaP. 803 14

The effects of androgens, antiandrogens, and other steroid hormones on growth of the human prostate cancer cell line LNCaP were studied. Despite the absence of receptors for progesterone and estradiol, the growth rate of the androgen responsive LNCaP-FGC cells increased when cultured in the presence of either estrogens or progestagens. In addition, most antiandrogens were also growth stimulators. This aberrant response was due to a threonine to alanine substitution at amino acid position 868 in the steroid binding domain of the androgen receptor (AR). Only the antiandrogen ICI 176,334 could block transcription and cell growth by the mutant receptor. By immunoprecipitation of the AR from LNCaP cells with the specific antibody F39.4.1 and Western blotting, three types of heat-shock proteins co-precipitated: hsp90, hsp70 and hsp56. This co-isolation could be prevented by pre-incubating the cells with androgens or with the antiandrogen hydroxyflutamide. Only the antiandrogen ICI 176,334 could block the effect of androgens on complex dissociation and prevent tight nuclear binding of the AR. Hydroxyflutamide could only inhibit tight nuclear binding of the wild-type AR. Therefore, in LNCaP cells the mutation in the steroid binding domain of the AR prevents a blockade of receptor function by most antiandrogens, but not by ICI 176,334, probably because of a different mechanism by which this compound blocks receptor function.
J Steroid Biochem Mol Biol 1994 Jun
PMID:Studies on the human prostatic cancer cell line LNCaP. 804 98

Testosterone (T) is the major exogenous stimulus for the growth of prostatic carcinoma. It is believed that the proliferative action of T may be mediated by locally expressed growth modulatory factors. Recent evidence from our laboratory suggests that a LHRH (or a LHRH-like) loop might be expressed in human prostatic tumor cells. To verify this hypothesis, we have studied whether a mRNA for LHRH is expressed in the human androgen-responsive prostatic cancer cell line LNCaP, using the reverse transcription-polymerase chain reaction technique in the presence of a pair of specific oligonucleotide primers. A cDNA band of the expected size was obtained from LNCaP cells; this band hybridized with a 32P-labeled LHRH oligonucleotide probe and its sequence showed a complete match with the reported sequence of the human placental LHRH cDNA. These observations indicate that the mRNA coding for LHRH is expressed in LNCaP cells and suggest that a LHRH (or a LHRH-like) peptide might be produced by these cells. To clarify the possible action of this peptide, LNCaP cells were grown in a steroid-free medium and treated with a LHRH antagonist. The treatment resulted in a significant increase of tumor cell growth. These data clearly indicate that the LHRH system expressed in LNCaP cells plays an inhibitory role on cell proliferation, and that this system seems to be regulated in a negative way by steroids. An EGF/TGF alpha autocrine stimulatory loop (peptides, receptors, intracellular signals) is also functional in these cells. Treatment of LNCaP cells grown in serum-free conditions (i.e. in the absence of exogenous growth factors) with a monoclonal antibody against the EGF receptor, or with immunoneutralizing antibodies against EGF or TGF alpha, resulted in a significant decrease of cell proliferation. T positively regulates this EGF/TGF alpha system by increasing the concentration of EGF binding sites. The present data indicate that an inhibitory LHRH (or LHRH-like) system is expressed in LNCaP cells and participates in the local mechanisms regulating tumor cell proliferation together with an EGF/TGF alpha stimulatory loop. Both systems appear to be modulated by T.
J Steroid Biochem Mol Biol 1994 Jun
PMID:Androgen-dependent prostatic tumors: biosynthesis and possible actions of LHRH. 804 99


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