Biomedical subjects

K Porvari

Publications and source records attributed to K Porvari.

9 recordsLinked to original sources

PubMed2002

17 beta-hydroxysteroid dehydrogenases and cancers.

17 beta-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between active 17 beta-hydroxysteroids and less-active 17-ketosteroids thereby affecting the availability of biologically active estrogens and androgens in a variety of tissues. The enzymes have different enzymatic properties and characteristic cell-specific expression patterns, suggesting differential physiological functions for the enzymes. Epidemiological and endocrine evidence indicate that estrogens play a key role in the etiology of breast cancer while androgens are involved in mechanisms controlling the growth of prostatic cells, both normal and malignant. Recently, we have developed, using LNCaP prostate cancer cell lines, a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition to more aggressive cells, able to grow in suspension cultures. Our results suggest that substantial changes in androgen and estrogen metabolism occur in the cells during the process. These changes lead to increased production of active estrogens during transformation of the cells. Data from studies of breast cell lines and tissues suggest that the oxidative 17HSD type 2 may predominate in human non-malignant breast epithelial cells, while the reductive 17HSD type 1 activity prevails in malignant cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered as estrogen target tissues such as colon. Our data show that the abundant expression of 17HSD type 2 present in normal colonic mucosa is significantly decreased during colon cancer development.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

The TMPRSS2 gene encoding transmembrane serine protease is overexpressed in a majority of prostate cancer patients: detection of mutated TMPRSS2 form in a case of aggressive disease.

The serine protease TMPRSS2 gene expression was studied by in situ hybridization using benign prostatic hyperplasia and prostate cancer tissue samples from 32 patients. Expression of TMPRSS2 gene was higher in cancer cells than that in benign cells in 84% of the specimens containing both benign and malignant tissues. The TMPRSS2 mRNA level was significantly increased in poorly differentiated (p = 0.014, n = 7) and untreated (p = 0.022, n = 13) primary prostate adenocarcinomas compared to benign tissues. In addition, androgen-deprivation therapy significantly decreased the expression of TMPRSS2 in benign prostate tissue (p = 0.07), which is in accordance with the androgen-inducible expression of the gene. The gene copy number of TMPRSS2, analyzed by competitively differential PCR, was duplicated in the malignant cells of about 38% of the prostate cancer patients analyzed. Thus, the increase in the gene copy number is probably not the primary reason for the detected overexpression of the TMPRSS2 gene. Mutations in the TMPRSS2 gene were screened using DNA isolated from paraffin-embedded prostate cancer tissues from 9 patients with aggressive prostate cancer and from 9 patients with nonaggressive disease. Thirteen exons covering the coding region were checked using enzymatic mutation detection and direct sequencing. One patient with aggressive prostate cancer carried a deletion and a stop codon in exon 11, leading to inactivation of the serine protease domain in TMPRSS2.

Humans↗

PubMed2000

Differentially expressed genes in two LNCaP prostate cancer cell lines reflecting changes during prostate cancer progression.

Prostate cancer tends to become transformed to androgen-independent disease over time when treated by androgen-deprivation therapy. We used two variants of the human prostate cancer cell line LNCaP to study gene expression differences during prostate cancer progression to androgen-independent disease. Production of prostate-specific antigen was regarded as a marker of androgen-dependence and loss of prostate-specific antigen was regarded as a marker of androgen-independence. mRNA from both cell lines was used for cDNA microarray screening. Differential expression of several genes was confirmed by Northern blotting. Monoamine oxidase A, an Expressed Sequence Tag (EST) similar to rat P044, and EST AA412049 were highly overexpressed in androgen-dependent LNCaP cells. Tissue-type plasminogen activator, interferon-inducible protein p78 (MxB), an EST similar to galectin-1, follistatin, fatty acid-binding protein 5, EST AA609749, annexin I, the interferon-inducible gene 1-8U, and phospholipase D1 were highly overexpressed in androgen-independent LNCaP cells. All studied genes had low or no expression in PC-3 cells. The EST similar to rat P044, the EST similar to galectin-1, follistatin, annexin I, and the interferon-inducible gene 1-8U were also expressed in benign prostatic hyperplasia tissue. The Y-linked ribosomal protein S4, Mat-8, and EST AA307912 were highly expressed in benign prostatic hyperplasia tissue. Additionally, both confirmation of differential expression in Northern blots and in situ hybridization were carried out for monoamine oxidase A, the EST similar to rat P044, the EST similar to galectin-1, fatty acid-binding protein 5, and the interferon-inducible gene 1-8U. We identified several potential prostate cancer markers, indicating that the method used is a useful tool for the screening of cancer markers, but other methods, such as in situ hybridization, are needed to further investigate the observations.

Animals↗

PubMed1999

Identification of the deoxyribonucleic acid-binding site of a regulatory protein involved in prostate-specific and androgen receptor-dependent gene expression.

In this study, we used a 5'-flanking region (-426/+28) of the rat prostatic probasin (rPB) gene shown to be sufficient to direct prostate-specific expression in transgenic mice in identifying the exact DNA-binding site of a putative prostate-specific transcription factor. Chloramphenicol acetyl transferase (CAT)-reporter gene analyses revealed that the construct pCAT PB -244/+52 was equally well induced by androgens in both prostatic LNCaP and nonprostatic COS-1, MCF-7, HEC-1, and HEP-1 cell lines, indicating that although the probasin gene region -244/+52 was important for androgen regulation, it was not regulated in a prostate-specific manner. Further studies suggested that the region -278/-240 was most crucial for prostate-specific expression. The sequence -426/-279 could be considered a silencer area, especially in nonprostatic cells. In deoxyribonuclease I footprinting, a protected 12-bp region was found between the nucleotides -251 and -240 only with nuclear extracts of prostatic origin. Deletion of this area decreased androgen induction significantly (P < 0.05) in transient transfections of prostatic cells compared with the wild-type reporter construct. Glucocorticoids were incapable of increasing the induction of the pCAT PB -278/+52 reporter construct compared with that of pCAT PB -244/+52 in the prostatic cell line LNCaP, suggesting that the putative prostate-specific protein acts as an inducer only when androgen and androgen receptor are present.

Androgen-Binding Protein↗

PubMed1997

Steroid-involved transcriptional regulation of human genes encoding prostatic acid phosphatase, prostate-specific antigen, and prostate-specific glandular kallikrein.

We have compared the steroid regulation of human genes encoding prostatic acid phosphatase (hPAP), prostate-specific antigen (hPSA), and prostate-specific glandular kallikrein (hK2) at the level of transcription. Reporter constructs of hPAP promoter covering the region -734/+467 were functional in both prostatic (LNCaP and PC-3) and nonprostatic (CV-1) cell lines in transient transfections. hPAP -231/+50 with eight identified transcription factor-binding sites showed the highest, and hPAP -734/+467 showed the lowest transcriptional activity in CV-1 cells. The hPAP promoter could not be induced with androgen, glucocorticoid, or progesterone, contrary to the hPSA (-620/+40) and hK2 (-493/+27) promoters in PC-3 cells cotransfected with the respective steroid receptor expression vector. Therefore, steroids cannot directly regulate hPAP gene expression via receptor binding to steroid response elements at -178 and +336, which have been shown to have androgen receptor-binding ability in vitro. Glucocorticoid was the most powerful activator of the hPSA construct at 10-nM steroid concentrations. On the contrary, glucocorticoid stimulation of the transcriptional activity of the hK2 construct was the weakest among the tested steroids. The results indicate that the steroid response elements in the proximal promoters of hPSA and hK2 genes are not androgen specific, offering the molecular basis for the expression of these genes outside the prostate in tissues containing steroid receptors.

Acid Phosphatase↗

PubMed1997

Human prostate-specific glandular kallikrein is expressed as an active and an inactive protein.

A polymorphism in the human prostate-specific glandular kallikrein (hKLK2) gene was described by direct sequencing (by PCR) of genomic DNAs isolated from prostatic cancer tissue, benign prostatic hyperplasia tissue, and blood leukocyte specimens. Results showed two forms of human prostate-specific glandular kallikrein protein (hK2), a consequence of a change from C to T at base 792 in the hK2 coding region. Producing the two forms as recombinant proteins in insect cells demonstrated that Arg226-hK2 (CC genotype) is an active protein and Trp226-hK2 (TT genotype) is inactive. Polymorphism studies of 36 patients with prostatic diseases identified only 1 with the TT genotype. The same kind of polymorphism was not detected in the human prostate-specific antigen (hKLK3) gene. Arg226-hK2 possessed only trypsin-like enzyme activity, whereas recombinant human prostate-specific antigen (hPSA) had only chymotrypsin-like activity. Monoclonal and polyclonal antibodies raised against hPSA purified from seminal plasma detected both active and inactive hK2. Thus, because inactive as well as stable hK2 protein may be present, a lack of trypsin-like activity in hPSA standards is not enough to confirm that the materials are free of hK2 contamination.

DNA↗

PubMed1995

Differential androgen regulation of rat prostatic acid phosphatase transcripts.

Steady-state levels of the mRNAs encoding rat prostatic acid phosphatase (rPAP) were measured from the ventral prostate of rats after castration and testosterone replacement. The longest and the most abundant mRNA molecule (4.9 kb) of the rPAP gene was quite resistant to the hormonal status of the prostate, whereas its medium-sized transcript (2.3 kb) and the shortest one (1.5 kb) were up-regulated by androgens. For comparison, we also analyzed the prostatic mRNA levels of rat lysosomal acid phosphatase (rLAP), whose gene expression is not considered to be hormonally dependent. Only modest variation was observed in the rLAP transcript after androgen withdrawal and replacement. Corresponding protein level investigations are analogous with our regulation studies, particularly in the case of 1.5-kb rPAP mRNA.

Acid Phosphatase↗

PubMed1994

Structural comparison of human and rat prostate-specific acid phosphatase genes and their promoters: identification of putative androgen response elements.

Structural comparison of human and rat prostate-specific acid phosphatase (hPAP and rPAP) genes indicate that the exon number is different between these species. The hPAP gene contains 10 exons, whereas the rPAP gene was 11 exons. However, exons 2-9 of the genes are identical in size. The 5' regions of the two genes show 71% identity in the most homologous region +1 to +340. The 5' untranslated regions of the human and rat genes are 50 and 49 nucleotides long, respectively. An Alu sequence is present upstream from the proximal promoter of the hPAP gene. Five putative androgen response elements altogether were localized in both the human and rat gene, one of which is conserved in location and sequence between the two genes. Two of these elements in both genes, the conserved one in the proximal promoter region and another one in intron 1, were shown to bind androgen receptor efficiently in vitro.

Acid Phosphatase↗

PubMed1993

Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A.

Acid Phosphatase↗