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P Vihko

Publications and source records attributed to P Vihko.

At least 37 records · Page 2Linked to original sources
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↗
PubMed2000

Expression of mouse 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7 in the ovary, uterus, and placenta: localization from implantation to late pregnancy.

Rodent 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7 (17HSD/KSR7) catalyzes the conversion of estrone (E1) to estradiol (E2) and is abundantly expressed in the ovaries of pregnant animals in particular. In the present work we demonstrate cell-specific expression of 17HSD/KSR7 in the ovaries, uteri, and placentas of pregnant and nonpregnant mice using in situ hybridization. The results show that mouse 17HSD/KSR7 (m17HSD/KSR7) messenger RNA is distinctly and exclusively expressed in a proportion of corpora lutea (CLs). During pregnancy, expression of m17HSD/KSR7 is most abundant around embryonic day 14.5 (E14.5), when the ovaries are filled with CLs expressing 17HSD/KSR7. In the uterus, m17HSD/KSR7 is first detected on E5.5, when expression surrounds the implantation site on the antimesometrial side. As gestation progresses, m17HSD/KSR7 is expressed in the decidua capsularis on E8 and E9.5, disappearing thereafter from the antimesometrial decidua. On E9 onward, m17HSD/KSR7 messenger RNA expression takes place at the junctional zone of the developing placenta. On E12.5 and E14.5, m17HSD/KSR7 is abundantly expressed in the spongiotrophoblasts, where expression gradually declines toward parturition. In conclusion, m17HSD/KSR7 expression in the CL is related to the life span of the CL. Moreover, spatial and temporal expression of m17HSD/KSR7 in the uterus suggests that locally produced E2 plays a role in implantation and/or decidualization. Finally, the results indicate that mouse placenta is capable of converting E1 to E2 in situ, and that the synthesized E2 may be effective in a paracrine, autocrine, and/or intracrine manner and be involved in placentation.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies.

We have expressed recombinant human vitamin D receptor and its ligand-binding domain in Spodoptera frugiperda (Sf9) insect cells with a 30-litre bioreactor. Both proteins were purified to apparent homogeneity with yields of 0.5-3.5 mg/l. Gel-filtration analyses indicated that the purified human vitamin D receptor and its ligand-binding domain were present as monomers in solution. The purified vitamin D receptor and its ligand-binding domain were demonstrated to bind 1alpha,25-dihydroxyvitamin D(3) with high affinity, the K(d) values ranging from 0.9 to 1.2 nM. Neutron scattering studies of the ligand-binding domain demonstrated that the samples are homogeneous and contain monomeric species of polypeptides. The purified vitamin D receptor binds to the vitamin D response elements of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor-alphaDeltaAB and we were able to purify these complexes in quantities sufficient for crystallization studies. The results indicate that we can produce biologically active human vitamin D receptor and its ligand-binding domain in insect cells and purify them for functional and structural studies.

Animals↗
PubMed1999

Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.

Acid Phosphatase↗
PubMed1999

Three-dimensional structure of a mammalian purple acid phosphatase at 2.2 A resolution with a mu-(hydr)oxo bridged di-iron center.

The crystal structure of purple acid phosphatase from rat bone has been determined by molecular replacement and the structure has been refined to 2.2 A resolution to an R -factor of 21.3 % (R -free 26.5 %). The core of the enzyme consists of two seven-stranded mixed beta-sheets, with each sheet flanked by solvent-exposed alpha-helices on one side. The two sheets pack towards each other forming a beta-sandwich. The di-iron center, located at the bottom of the active-site pocket at one edge of the beta-sandwich, contains a mu-hydroxo or mu-oxo bridge and both metal ions are observed in an almost perfect octahedral coordination geometry. The electron density map indicates that a mu-(hydr)oxo bridge is found in the metal center and that at least one solvent molecule is located in the first coordination sphere of one of the metal ions. The crystallographic study of rat purple acid phosphatase reveals that the mammalian enzymes are very similar in overall structure to the plant enzymes in spite of only 18 % overall sequence identity. In particular, coordination and geometry of the iron cluster is preserved in both enzymes and comparison of the active-sites suggests a common mechanism for the mammalian and plant enzymes. However, significant differences are found in the architecture of the substrate binding pocket.

Acid Phosphatase↗
PubMed1999

Structure of the ternary complex of human 17beta-hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin) and NADP+.

Excess 17beta-estradiol (E2), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which catalyzes the reduction of inactive estrone (E1) to the active 17beta-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E2 in breast tumor tissues. We present here the structure of the ternary complex of 17beta-HSD1 with the cofactor NADP+ and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an equine estrogen used in estrogen replacement therapy. The ternary complex has been crystallized with a homodimer, the active form of the enzyme, in the asymmetric unit. Structural and kinetic data presented here show that the 17beta-HSD1-catalyzed reduction of E1 to E2 in vitro is specifically inhibited by equilin. The crystal structure determined at 3.0-A resolution reveals that the equilin molecule is bound at the active site in a mode similar to the binding of substrate. The orientation of the 17-keto group with respect to the nicotinamide ring of NADP+ and catalytic residues Tyr-155 and Ser-142 is different from that of E2 in the 17beta-HSD1-E2 complex. The ligand and substrate-entry loop densities are well defined in one subunit. The substrate-entry loop adopts a closed conformation in this subunit. The result demonstrates that binding of equilin at the active site of 17beta-HSD1 is the basis for inhibition of E1-to-E2 reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E2 levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.

Amino Acid Sequence↗
PubMed1999

Microsatellite instability: impact on cancer progression in proximal and distal colorectal cancers.

Whilst individual planning of treatment and follow-up in every colorectal cancer case is an increasing demand, prognostic markers are needed for predicting cancer progression in the primary phase. We studied the effect of replication error (RER)-positivity on colorectal cancer progression by analysing 255 colorectal cancer specimens by polymerase chain reaction (PCR) and fragment analysis and correlating the results with the clinical and histological features of the tumour and with patient outcome. RER-positivity was detected in 12% (28/235) of cases. It was associated with proximal location of the tumour (P < 0.001), poor differentiation (P = 0.001) and large tumour size (P = 0.009). The 5-year cumulative survival rate of the patients with RER-positive cancer of the proximal colon was markedly better (100%) than that of those with RER-negative proximal cancer (74%), whilst in cases of cancer of the distal colon or rectum, RER-positivity (21%) indicated poorer survival than RER-negativity (57%). Thus, it is suggested that RER-positivity has an opposite impact on cancer progression in cases of proximal and distal cancers. RER-positivity appears to indicate improved prognosis only in cases of proximally located cancer, in which it could accordingly be useful as a prognostic marker.

Adolescent↗
PubMed1999

17Beta-hydroxysteroid dehydrogenases in normal human mammary epithelial cells and breast tissue.

17Beta-hydroxysteroid dehydrogenase activity represents a group of several isoenzymes (17HSDs) that catalyze the interconversion between highly active 17beta-hydroxy- and low activity 17-ketosteroids and thereby regulate the biological activity of sex steroids. The present study was carried out to characterize the expression of 17HSD isoenzymes in human mammary epithelial cells and breast tissue. In normal breast tissues 17HSD types 1 and 2 mRNAs were both evenly expressed in glandular epithelium. In two human mammary epithelial cell lines, mRNAs for 17HSD types 1, 2 and 4 were detected. In enzyme activity measurements only oxidative 17HSD activity, corresponding to either type 2 or type 4 enzyme, was present. The role of 17HSD type 4 in estrogen metabolism was further investigated, using several cell lines originating from various tissues. No correlation between the presence of 17HSD type 4 mRNA and 17HSD activity in different cultured cell lines was detected. Instead, oxidative 17HSD activity appeared in cell lines where 17HSD type 2 was expressed and reductive 17HSD activity was present in cells expressing 17HSD type 1. These data strongly suggest that in mammary epithelial cell lines the oxidative activity is due to type 2 17HSD and that oxidation of 17beta-hydroxysteroids is not the primary activity of the 17HSD type 4 enzyme.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Three independently deleted regions at chromosome arm 16q in human prostate cancer: allelic loss at 16q24.1-q24.2 is associated with aggressive behaviour of the disease, recurrent growth, poor differentiation of the tumour and poor prognosis for the patient.

Loss of heterozygosity at chromosome arm 16q is a frequent event in human prostate cancer. In this study, loss of heterozygosity at 16q was studied in 44 prostate cancer patients exhibiting various clinical features. Fifteen polymorphic polymerase chain reaction (PCR) markers were used to identify the separately deleted areas and the findings were compared with clinicopathological variables and 5-year survival of the patients. The results indicated that there are at least three independently deleted regions at 16q. Allelic losses at the central and distal areas were associated significantly with aggressive behaviour of the disease (16q24.1-q24.2, P < 0.01, and 16q24.3-qter, P < 0.05), and the central area of deletion was further significantly associated with poorly differentiated tumour cells (P < 0.05) and with recurrent (P < 0.01) growth of the tumour. During the follow-up period, 28% of the patients initially with M0 disease developed distant metastases. Of the patients showing allelic loss at 16q24.1-q24.2, distant metastasis were found in 45% during the 5-year follow-up period, and 31% of the patients showing loss at 16q21.1 also developed distant metastases. After the 5-year follow-up period, 14 (32%) of the patients remained alive, whereas 19 (43%) had died because of their prostate cancer. The overall survival rate of the patients showing allelic loss at 16q21.1 or 16q24.1-q24.2 was significantly lower than that of the patients with retained heterozygosity.

Alleles↗
PubMed1999

Loss of heterozygosity at 18q21 is indicative of recurrence and therefore poor prognosis in a subset of colorectal cancers.

Adjuvant therapies are increasingly used in colorectal cancers for the prevention of recurrence. These therapies have side-effects and should, thus, be used only if really beneficial. However, the development of recurrence cannot be predicted reliably at the moment of diagnosis, and targeting of adjuvant therapies is thus based only on the primary stage of the cancer. Loss of heterozygosity (LOH) in the long arm of chromosome 18 is suggested to be related to poor survival and possibly to the development of metastases. We studied the value of LOH at 18q21 as a marker of colorectal cancer prognosis, association with clinicopathological variables, tumour recurrence and survival of the patients. Of the 255 patients studied, 195 were informative as regards LOH status when analysed in primary colorectal cancer specimens using the polymerase chain reaction (PCR) and fragment analysis. LOH at 18q21 was significantly associated with the development of recurrence (P = 0.01) and indicated poor survival in patients of Dukes' classes B and C, in which most recurrences (82%) occurred. An increased rate of tumour recurrence is the reason for poor survival among patients with LOH at 18q21 in primary cancer. These patients are a possible target group for recurrence-preventing adjuvant therapies.

Adult↗
PubMed1999

Primary structure of a sperm cell anion exchanger and its messenger ribonucleic acid expression during spermatogenesis.

Chloride/bicarbonate (Cl-/HCO(3)-) exchangers are a family of proteins (anion exchanger [AE] gene family) that regulate many vital cellular processes such as intracellular pH, cell volume, and Cl- concentration. They may also be involved in the regulation of sperm cell motility and acrosome reaction during fertilization, as these two phenomena are bicarbonate dependent, and we have previously shown that a polypeptide immunologically related to erythrocyte band 3 is expressed in mammalian sperm cells. We have now identified this putative sperm cell anion exchanger as the AE2 isoform of this gene family. First, we determined its complete primary structure from the human testis lambda gt 11 cDNA library. The cloned sequence was found to consist of 3896 base pairs (bp) with an open reading frame of 3726 bp, and to be almost identical to the previously published human genomic AE2 sequence. Only four amino acid disparities were found between these two sequences. Second, our in situ hybridization analyses showed that AE2 mRNA is expressed in developing sperm cells, indicating that the cloned sequence corresponds to the sperm cell AE. Our reverse transcription-polymerase chain reaction analyses suggested further that the expression of AE2 mRNA was variable to some extent during the epithelial cell cycle. Strongest expression was observed at stages VII-XIV except for stage X, i.e., when major structural and morphological changes take place. These results suggest that the full-length AE2 isoform regulates HCO(3)- transport in mature sperm cells and thus their motility in vivo.

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↗
PubMed1999

Identification of essential subelements in the hHSD17B1 enhancer: difference in function of the enhancer and that of the hHSD17BP1 analog is due to -480C and -486G.

The function of the gene encoding human 17beta-hydroxysteroid dehydrogenase (17HSD) type 1, the hHSD17B1 gene, is regulated by a cell-specific enhancer at position -662 to -392. The adjacent hHSD17BP1 gene, whose function is not known, contains an analogous region in its 5'-flanking region. The identity between the hHSD17B1 enhancer and the hHSD17BP1 equivalent is as high as 98%, i.e. they differ by only five nucleotides. Results from reporter gene analyses showed that the hHSD17BP1 analog, a pseudoenhancer, has only 10% the activity of the hHSD17B1 enhancer. Furthermore, the results indicate that the reduced function of the pseudoenhancer is a consequence of the presence of G and A at positions -480 and -486, whereas the hHSD17B1 enhancer contains -480C and -486G. In addition, three protected areas were localized to regions -495/-485 (FP1), -544/-528 (FP2), and -589/-571 (FP3) in deoxyribonuclease I footprinting analysis of the hHSD17B1 enhancer. Replacement of the footprinted regions with a nonsense sequence demonstrated that the FP2 region is the most critical for enhancer activity. Mutations of FP2 or a short palindromic region within it led to almost complete abolishment of enhancer activity. We have identified several subelements that are essential for appropriate function of the hHSD17B1 enhancer. The results also show that the hHSD17B1 and hHSD17BP1 genes operate differently despite the high homology between them.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Tartrate-resistant bone acid phosphatase: large-scale production and purification of the recombinant enzyme, characterization, and crystallization.

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed in bone-resorbing osteoclasts and certain tissue macrophages in human tissues. The functions of TRAP in biological systems are not known. Elucidation of the three-dimensional structure of the active site could yield important information about the physiological substrate(s) of the enzyme. We have produced recombinant rat bone TRAP using a baculovirus expression vector system. The production was scaled up to a 30-l bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypeptide chain. Trypsin digestion resulted in a preparation where two subunits of approximately 23 kDa and approximately 16 kDa appeared after disulfide reduction. Trypsin digestion activated the enzyme. We generated monoclonal antibodies against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzyme could be separated by Mono-S cation-exchange chromatography. Crystals of TRAP have been obtained with ammonium sulfate/polyethylene glycol as precipitant. They belong to space group P212121 or P21212 with unit cell dimensions a = 57.2 A, b = 69.5 A, and c = 87.2 A and diffract to at least 2.2 A resolution. A packing density value of 2.55 A3/Da is consistent with one subunit in the asymmetric unit.

Acid Phosphatase↗
PubMed1998

Androgen-sensitive human prostate cancer cells, LNCaP, produce both N-terminally mature and truncated prostate-specific antigen isoforms.

To characterize prostate-specific antigen (PSA) produced by cancer cells, different isoforms of PSA secreted by the human prostate cancer cells, LNCaP, were purified. LNCaP-PSA production was induced by synthetic androgen, R1881. LNCaP-PSA was separated into four pools. The molecular mass of LNCaP-PSA isoforms in these pools was 34 kDa under reducing conditions and 29 kDa under nonreducing conditions on SDS/PAGE. pI of LNCaP-PSA isoforms varied from 6.8 to 8.2. Pool A had the highest specific activity, 37 nmol/(min x mg). All the pools formed stable complexes with alpha1-antichymotrypsin and alpha2-macroglobulin. The pools contained 10-60% of N-terminally correctly processed LNCaP-PSA isoforms. According to the molecular modelling, the addition or deletion of two or four N-terminal amino acids could affect the three-dimensional structure and thereby remarkably reduce the enzyme activity of LNCaP-PSA.

Amino Acid Sequence↗
PubMed1998

Studies on the protein tyrosine phosphatase activity of tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is an enzyme with unknown biological function. In human tissues, its expression is restricted to bone-resorbing osteoclasts and activated macrophages. Osteoclasts secrete TRAP to the circulation during bone resorption. Reduction of the enzyme's binuclear iron center is important in regulating its activity. The purple form of the enzyme is inactive and contains two ferric ions. Mild reduction activates it to a pink form containing one ferric and one ferrous ion. Instead, strong reduction removes the iron content, resulting in a colorless, inactive enzyme. We describe spontaneous activation of the purple form to the pink form upon incubation at +37 degrees C. Further incubation results in slow inactivation of the enzyme and color change to yellowish. The enzyme purified from osteoclasts is a mixture of the purple and pink forms, but the enzyme purified from serum represents the yellowish form. We suggest that the newly synthesized enzyme is purple and reduced in the cell to the functionally active pink form. After fulfilling its biological function in the cell, the enzyme is further reduced to the yellowish form and secreted into the circulation. In the serum, further reduction would dissociate the iron content. The enzymes from osteoclasts and macrophages had similar catalytic properties, both being active as a protein tyrosine phosphatase (PTPase). The acid phosphatase (AcP) and PTPase activities were similar, and the preferred AcP substrate, pNPP, was processed in the same active site as phosphotyrosine. Our results suggest that redox-regulated PTPase activity may be a major function of TRAP in vivo.

Acid Phosphatase↗
PubMed1998

The protein kinase C activator, phorbol ester, elicits disparate functional responses in androgen-sensitive and androgen-independent human prostatic cancer cells.

The protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) activated cell death in androgen-sensitive LNCaP cells but not in androgen-independent DU-145 or PC-3 cells, whose growth was significantly decreased by PKC inhibitors staurosporine and H7. All cell lines had similar levels of total PKC activities which, however, differed on their dependency on Ca2+ ions and lipid and were regulated differently by TPA. Furthermore, expression of the immediate early genes c-fos and c-jun was up-regulated by TPA only in LNCaP and DU-145 cells, whereas PC-3 cells failed to express c-fos mRNA. The regulation of the c-myc mRNA by TPA correlated inversely with activation of cell death being down-regulated in LNCaP cells, and slightly increased in the androgen-independent cell lines. These results suggest that the PKC signal transduction pathway functions differently in androgen-sensitive and insensitive prostatic cells.

Androgens↗