Detection of cytochrome P450 mRNA transcripts in prostate samples by RT-PCR.

BACKGROUND: The expression of cytochrome P450 (CYP)-dependent mono-oxygenases in the prostate is important, as it will determine the rate of activation of potential carcinogens as well as the metabolism of hormones with implications in diseases of the prostate. In addition, the levels of cytochromes P450 in prostatic tumours may well be determinants of the outcome of therapy involving P450 substrates such as anti-androgens. METHODS: The gene expression of 12 different CYP genes was measured by reverse transcription-polymerase chain reaction (RT-PCR) in a total of 28 human prostatic tumour and nontumour samples. RESULTS: Intriguingly, a large number of CYP mRNAs were detected in the prostate samples, including CYP1A2, -1B1, -2C19, -2D6, -3A4, -3A5, -3A7 and -4B1. CYP1B1 was consistently expressed and CYP3A5 and CYP4B1 were expressed in a majority of the samples tested. CONCLUSIONS: These data demonstrate a wide range of CYP genes being expressed in the prostate. The relative importance of these enzymes in the pathogenesis and treatment of prostatic disease remains an important theme for further study.

Messenger ribonucleic acid levels of steroid 5 alpha-reductase 2 in human prostate predict the enzyme activity.

Testosterone is converted to dihydrotestosterone by 5 alpha-reductase2 in the prostate. Dihydrotestosterone controls cell division, and interindividual differences in prostatic 5 alpha-reductase 2 expression and activity may be a determinant of the risk of developing prostate cancer. However, little is known about interindividual differences in intraprostatic hormonal activity in vivo. To determine whether 5 alpha-reductase-specific messenger RNA (mRNA) is predictive of 5 alpha-reductase activity in prostatic tissue, we analyzed 30 prostatic tissue specimens from 15 Caucasian patients, 47--82 yr old. The mRNA was measured by RT-PCR. Five specimens consisted of cancer, whereas the remaining 25 were derived from benign prostate hyperplasia (BPH). We found a strong association between enzyme activity at pH 5.5 and the 5 alpha-reductase 2-specific mRNA expression when expressed on the basis of beta-actin [Spearman's rank-correlation coefficient (r(s)) = 0.81; 95% confidence interval, 0.64-0.91; P < 0.0001]. The expression of 5 alpha-reductase 2-specific mRNA in the cancer specimens was significantly lower than in the BPH tissue (P = 0.03). There was no difference in the expression of 5 alpha-reductase 1-specific mRNA in the cancer specimens, compared with BPH (P = 0.56). The strong association between 5 alpha-reductase activity at pH 5.5 and the 5 alpha-reductase 2-specific mRNA expression makes it possible to predict prostatic 5 alpha-reductase activity using core needle biopsies.

Cytochrome P450 2D6 and glutathione S-transferase M1 genotypes and migraine.

BACKGROUND: Migraine is thought to be a disease of the brain and trigeminovascular system. Migraine patients often claim that stress, food, and beverages trigger their attacks. Chemical substances in these foodstuffs with the property of triggering migraine attacks have not yet been characterised. Cytochrome P450 2D6 (CYP2D6) and glutathione S-transferase M1 (GSTM1) are thought to be present in the brain. They metabolise numerous environmental compounds. The genes exhibit genetic polymorphism that is associated with altered enzyme activity. The aim of this study was to determine if the genotypes of these two enzymes are associated with migraine. MATERIALS AND METHODS: The study included 100 female patients and 245 female controls from the general population. Genomic DNA was isolated from whole blood. Allele specific PCR methods were used to identify the normal CYP2D6*1 allele and the mutated CYP2D6*3 and CYP2D6*4 alleles. Initially all samples were genotyped only for GSTM1 plus (+) and GSTM1 null (-) variants. All samples positive for GSTM1 were further analysed for the presence of allelic variants GSTM1*A and GSTM1*B. RESULTS: None of the CYP2D6 and GSTM1 genotypes was associated with migraine. We observed an odds ratio (OR) for the poor metaboliser genotype of CYP2D6 of 1.4 (95% CI = 0.5-3.6) and for the GSTM1 null genotype of 1.0 (95% CI = 0.6-1.5). CONCLUSION: The results of this study indicate that deficient metabolism because of mutated CYP2D6 alleles or GSTM1 allele variants is not important in the aetiology of migraine.

Quantitation of cytochrome P450 mRNAs in patients with suspected liver diseases as assessed by reverse transcriptase-polymerase chain reaction.

The cytochrome P450 system (CYP) is vital for the oxidation and detoxification of numerous drugs and other xenobiotics in the liver. Many of the CYP enzymes are polymorphically expressed and may be induced or inhibited by xenobiotics including drugs and alcohol. The measurement of gene expression is thus important in studies of the mechanisms of interaction with and function of the CYP system. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for the study of the mRNA expression of three CYP enzymes--2E1, 1A2, and 3A4--in snap-frozen percutaneous liver biopsy samples. The method was made quantitative by the introduction of a recombinant RNA internal standard that contains a transcript of the beta-globin gene and sequences specific for the studied CYP enzymes. The method allows the analysis of mRNA expression of several enzymes in as little as 5 mg of liver tissue. Liver tissue specimens from 19 patients with suspected liver disease were analyzed for CYP-specific mRNA expression. The mean mRNA concentrations for CYP1A2, 2E1, and 3A4 were 0.16, 0.74, and 0.32 amol of specific mRNA per nanogram of poly (A+) mRNA, respectively, but a large interindividual variation was observed. CYP3A7 primers were included in the internal standard. However, because of low expression it was not possible to quantitate the enzyme. This quantitative RT-PCR method is of value for studies of the mechanisms of variation and interactions with the members of the CYP enzyme family in healthy and diseased liver and other organs.

Differential gene expression of steroid 5 alpha-reductase 2 in core needle biopsies from malignant and benign prostatic tissue.

Androgens are implicated in the development of prostate cancer (CAP) and benign prostate hyperplasia. The conversion of testosterone to the more potent metabolite dihydrotestosterone by prostatespecific steroid 5 alpha-reductase type 2 (5 alpha-red2) is a key mechanism in the action of androgens in the prostate and is important in the promotion and progression of prostate diseases. Manipulation of the turnover of androgens is thus fundamental in the pharmacological treatment strategy. We have developed a sensitive solution hybridization method for quantification of the gene expression of 5 alpha-red2 in core needle biopsies of the prostate. The 5 alpha-red2-specific messenger RNA (mRNA) levels were measured in 50 human prostate transrectal ultrasound-guided core biopsies obtained from 31 outpatients (median age 72, range 67-88 yr) undergoing biopsy for diagnostic purposes. Significant differences were observed in the gene expression of 5 alpha-red2 between cancerous and noncancerous tissue. In the 14 biopsies judged cancerous, the median 5 alpha-red mRNA levels were 3.5 amol/ng total RNA compared with 12.0 amol/ng total RNA in the biopsies showing no cancer (P = 0.0018). The median 5 alpha-red2 mRNA level in noncancerous tissue was thus 3.4 times higher than in the cancerous specimens.

Differential effects of neuroleptic agents on hepatic cytochrome P-450 isozymes in the male rat.

We report the effects of various dopamine receptor-blocking drugs on gene and protein expression, as well as the activity of several hepatic cytochrome P-450 (CYP) enzymes in the male Sprague-Dawley rat. At equipotent doses (with respect to receptor blockade and behavioural tests), the dopamine D2-receptor selective sulpiride and remoxipride gave a conspicuous down-regulation of CYP2C11 and its associated androstenedione 16 alpha-hydroxylation activity as well as of the CYP2C11-specific mRNA. The average immunoidentified CYP2C11 levels correlated with the CYP2C11-specific mRNA levels in all treatment groups (r = 0.994), indicating a transcriptional mechanism. The CYP3A protein was also selectively down-regulated. In contrast, androstenedione 5 alpha-reduction was significantly increased. Clozapine, a non-selective neuroleptic, gave the same effects on the steroid metabolism as sulpiride and remoxipride. In contrast, diverging effects were observed for clozapine, compared to sulpiride and remoxipride, on the immunoidentified CYP1A2, CYP2B1, and CYP3A. These proteins were elevated by clozapine, and down-regulated by sulpiride and remoxipride. Our results are of interest for the interpretation of preclinical dose ranging toxicity tests of neuroleptic agents in rats. They may also be relevant in relation to certain interactions and adverse reactions observed in the clinical use of these drugs. The down-regulation of certain CYP enzymes is most likely mediated by an interaction with the growth hormone secretion.

Biochemical evidence for a mature phenotype in morphologically poorly differentiated neuroblastomas with a favourable outcome.

Neuroblastoma is an embryonal tumour of the sympathetic nervous system with marked heterogeneity in terms of histological maturity and clinical course. A previous study revealed that high tumour levels of the csrc protein, particularly its neuronal isoform (pp60csrcN), correlated with favourable outcome. To test whether this feature reflects a higher degree of neuronal maturation in these tumours, an extended series (47 consecutive neuroblastomas and 10 ganglioneuromas) were analysed for levels of csrc protein isoforms, neuron-specific enolase, and synaptophysin. Immunoblotting and radioimmunoassay techniques were employed. The results were compared with conventional histological signs of neuronal maturation. High pp60csrcN levels were specific for prognostically favourable neuroblastomas and correlated with high neuronal marker levels. However, signs of histological maturation correlated poorly with these parameters. It is therefore concluded that low stage tumours are highly differentiated in biochemical terms despite their frequently immature histology. Furthermore, the clinical usefulness of these biochemical parameters as prognostic markers was compared with established parameters in a multivariate analysis. Stage 4 disease, MYCN amplification, and age above 18 months at diagnosis was the most powerful combination of variables found for predicting a poor outcome. As expected, none of the neuronal differentiation markers investigated could add to the prediction of aggressive disease when compared with this model. However, high expression of pp60csrcN appeared to be useful in predicting long-term survival in high stage infant neuroblastoma.

The expression profile of alternatively spliced neuronal c-src RNA distinguishes between human tumours of the sympatho-adrenal lineage.

Human neuronal and neuroendocrine tumour specimens and cell lines were analysed regarding proteins and transcripts coded by the proto-oncogene c-src. At the protein level, most of the neuroblastomas and phaeochromocytomas expressed the neuronal c-src form, pp60c-srcN. None of the other neuroendocrine tumours, i.e. paragangliomas, neuroendocrine pancreatic tumours, or carcinoid tumours and small-cell lung carcinomas of different types, appeared to express the neuronal form. In the brain, c-src is transcribed into 3 differently spliced mRNA variants, c-src, c-srcNI, and c-srcNI+NII. The expression of these transcripts was analysed by PCR amplification of fragments covering the mini-exons I and NII of the corresponding cDNAs. The PCR products were analysed by Southern hybridization and characterized by determination of their sequences. Neuroblastomas, paragangliomas, retinoblastomas and the phaeochromocytomas expressed neuronal c-src splice variants. However, whereas neuroblastomas and retinoblastomas contained all 3 transcripts, the phaeochromocytomas and paragangliomas expressed, with 2 exceptions, only the c-src and the c-srcNI+NII mRNA species. To assess whether neuroblastomas display adrenal chromaffin characteristics, they were analysed regarding expression of the chromaffin marker enzyme, phenylethanolamine-N-methyl transferase. Whereas phaeochromocytomas were positive, all neuroblastomas were immuno-chemically negative for this enzyme. These results and the c-src expression profile suggest that neuroblastomas, including those with an adrenal location, do not originate from the adrenal chromaffin differentiation lineage. The data further suggest neuronal c-srcNI mRNA as a marker for sympathetic neuronal cells of the sympatho-adrenal lineage.

Expression of the neuronal form of pp60c-src in neuroblastoma in relation to clinical stage and prognosis.

The expression of the protooncogene c-src has been studied in specimens of childhood tumors with special reference to neuroblastoma and other tumors of neuronal origin. For comparison c-src gene expression was studied in seven neuroblastoma and neuroepithelioma cell lines. The structurally distinct neuronal product of the gene, pp60c-srN, expressed during normal development in neuroblasts and neurons, was identified by immunoblotting technique together with the fibroblast form, pp60c-src. While pp60c-src was found in most tumors studied, the neuronal form was restricted to neuroblastomas (23 of 27) and retinoblastomas (3 of 3) and could not be detected in the other childhood tumors. A dominance of the neuronal form, pp60c-srcN, was exclusively found in the infant cases of neuroblastoma (9 of 12), estimated to have good prognosis. These results indicate that pp60c-srcN might be a diagnostic marker in primitive childhood tumors. When expressed in higher amounts than pp60c-src, pp60c-srcN may be a positive prognostic marker in neuroblastoma, especially useful in the evaluation of infants. In addition, lack of pp60c-srcN seems to be incompatible with low stage neuroblastoma.

Human neuroblastoma cells in culture: a model for neuronal cell differentiation and function.

The human neuroblastoma cell line, SH-SY5Y, differentiates into neuron-like cells according to morphological, biochemical and functional criteria when treated with biologically active phorbol-esters. The differentiated phenotype is described and alterations in proto-oncogene expression in connection with growth control and differentiation are presented and discussed.

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.

Expression of c-src in cultured human neuroblastoma and small-cell lung carcinoma cell lines correlates with neurocrine differentiation.

Human cell lines with neuronal and neuroendocrine features were examined for their expression of pp60c-src, the cellular homolog of the transforming gene product pp60v-src of Rous sarcoma virus. Four neuroblastoma (LA-N-5, SH-SY5Y, Paju, and SK-N-MC) and three small-cell lung carcinoma (U-2020, U-1690, and U-1285) cell lines were selected on the basis of their stage of neurocrine differentiation, as determined by the expression of neuron-specific enolase. In an immune complex protein kinase assay, all seven cell lines displayed c-src kinase activity which was considerably higher than that found in nonneurocrine cells (human diploid fibroblasts, glioma, and non-small cell lung carcinoma cell lines). Furthermore, the c-src kinase activity, as determined by autophosphorylation or phosphorylation of an exogenous substrate, enolase, correlated with the stage of neurocrine differentiation. There was an approximately 30-fold difference in c-src kinase autophosphorylation activity between the cell lines representing the highest and lowest stages of neurocrine differentiation. A similar variation was found in the steady-state levels of the c-src protein of these cell lines. Highly differentiated neuroblastoma cells expressed two forms of the src protein. Digestion by Staphylococcus aureus V8 protease did reveal structural diversity in the amino-terminal ends of these c-src molecules. In summary, we found a clear correlation between c-src kinase activity and the stage of neuronal and neuroendocrine differentiation. Thus, the phenotypic similarity between neurons and neuroendocrine cells includes high c-src expression.

Different regulation of N- and c-myc expression during phorbol ester-induced maturation of human SH-SY5Y neuroblastoma cells.

The mRNA expression of c-myc and N-myc in the human neuroblastoma cell line SH-SY5Y was found not to change appreciably during the cell cycle and was also unaffected by proliferative inhibition induced by serum starvation or polyamine depletion. However, an early (0.5-8.0 h post-induction) transient reduction of c- and N-myc transcripts were observed in these cells upon induction to differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of these neuroblastoma cells with TPA for longer periods (1-8 days), which induces morphological and functional differentiation and growth arrest, was followed by decreased expression of both myc genes. However, the rate of disappearance differed considerably. The N-myc mRNA level was slightly decreased after 4 days and was still detectable 8 days after induction, whereas the c-myc transcript was down-regulated much faster. In contrast, when the cells were exposed to retinoic acid, which results in a maturation along an alternative pathway, the inhibition of N-myc and c-myc expression was similar. The c-fos mRNA expression increased in TPA-treated SH-SY5Y cells and remained high during extended exposure to the drug. The highest c-fos transcript level in induced cells coincided in time with the transient reduction of N-myc and c-myc. Thus, the TPA-induced neuronal differentiation of SH-SY5Y cells was compatible with high c-fos and a substantial N-myc mRNA expression.