Alternative splicing of the neurotrophin-3 gene gives rise to different transcripts in a number of human and rat tissues.

The mouse neurotrophin-3 (NT-3) gene has been shown to contain two exons (exon 1A and exon 1B) upstream of the single coding exon (exon 2). These upstream exons are alternatively spliced to the coding exon, generating two different NT-3 transcripts. We investigated whether alternative splicing of two upstream exons also occurs in the human and rat NT-3 gene. It was found that the human and rat NT-3 gene also contains two exons upstream of the main coding exon and that alternative splicing of these upstream exons generates two different NT-3 transcripts : transcript 1A and transcript 1B (TR1B). These two transcripts were widely expressed in several human and rat tissues. Also, a third transcript, transcript 1A1C, derived from splicing of exon 1A to exon 1B before splicing to the coding exon was seen in a small number of rat tissues. Previous quantification of neurotrophin-3 mRNA has not been transcript-specific. Here we describe a transcript-specific semiquantitative RT-PCR method allowing the quantification of TR1B in human tissues. We show that this is the major NT-3 transcript and that expression of this transcript was much higher in the adult when compared with the corresponding fetal tissues.

Molecular determinants of the estrogen receptor-coactivator interface.

Transcriptional activation by the estrogen receptor is mediated through its interaction with coactivator proteins upon ligand binding. By systematic mutagenesis, we have identified a group of conserved hydrophobic residues in the ligand binding domain that are required for binding the p160 family of coactivators. Together with helix 12 and lysine 366 at the C-terminal end of helix 3, they form a hydrophobic groove that accommodates an LXXLL motif, which is essential for mediating coactivator binding to the receptor. Furthermore, we demonstrated that the high-affinity binding of motif 2, conserved in the p160 family, is due to the presence of three basic residues N terminal to the core LXXLL motif. The recruitment of p160 coactivators to the estrogen receptor is therefore likely to depend not only on the LXXLL motif making hydrophobic interactions with the docking surface on the receptor, but also on adjacent basic residues, which may be involved in the recognition of charged residues on the receptor to allow the initial docking of the motif.

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.

The estrogenic activity of phthalate esters in vitro.

A large number of phthalate esters were screened for estrogenic activity using a recombinant yeast screen. a selection of these was also tested for mitogenic effect on estrogen-responsive human breast cancer cells. A small number of the commercially available phthalates tested showed extremely weak estrogenic activity. The relative potencies of these descended in the order butyl benzyl phthalate (BBP) > dibutyl phthalate (DBP) > diisobutyl phthalate (DIBP) > diethyl phthalate (DEP) > diisiononyl phthalate (DINP). Potencies ranged from approximately 1 x 10(6) to 5 x 10(7) times less than 17beta-estradiol. The phthalates that were estrogenic in the yeast screen were also mitogenic on the human breast cancer cells. Di(2-ethylhexyl) phthalate (DEHP) showed no estrogenic activity in these in vitro assays. A number of metabolites were tested, including mono-butyl phthalate, mono-benzyl phthalate, mono-ethylhexyl phthalate, mon-n-octyl phthalate; all were wound to be inactive. One of the phthalates, ditridecyl phthalate (DTDP), produced inconsistent results; one sample was weakly estrogenic, whereas another, obtained from a different source, was inactive. analysis by gel chromatography-mass spectometry showed that the preparation exhibiting estrogenic activity contained 0.5% of the ortho-isomer of bisphenol A. It is likely that the presence of this antioxidant in the phthalate standard was responsible for the generation of a dose-response curve--which was not observed with an alternative sample that had not been supplemented with o,p'-bisphenol A--in the yeast screen; hence, DTDP is probably not weakly estrogenic. The activities of simple mixtures of BBP, DBP, and 17beta-estradiol were assessed in the yeast screen. No synergism was observed, although the activities of the mixtures were approximately additive. In summary, a small number of phthalates are weakly estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vivo; this will require tests using different classes of vertebrates and different routes of exposure.

AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors.

Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.

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.

Mutated human androgen receptor gene detected in a prostatic cancer patient is also activated by estradiol.

Androgens are necessary for the development of prostatic cancer. The mechanisms by which the originally androgen-dependent prostatic cancer cells are relieved of the requirement to use androgen for their growth are largely unknown. The human prostatic cancer cell line LNCaP has been shown to contain a point mutation in the human androgen receptor gene (hAR), suggesting that changes in the hAR may contribute to the abnormal hormone response of prostatic cells. To search for point mutations in the hAR, we used single strand conformation polymorphism analysis and a polymerase chain reaction direct sequencing method to screen 23 prostatic cancer specimens from untreated patients, 6 prostatic cancer specimens from treated patients, and 11 benign prostatic hyperplasia specimens. One mutation was identified in DNA isolated from prostatic cancer tissue, and the mutation was also detected in the leukocyte DNA of the patient and his offspring. The mutation changed codon 726 in exon E from arginine to leucine and was a germ line mutation. The mutation we found in exon E of the hAR gene does not alter the ligand binding specificity of the AR, but the mutated receptor was activated by estradiol to a significantly greater extent than the wild-type receptor. The AR gene mutation described in this study might be one explanation for the altered biological activity of prostatic cancer.

Expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale.

We have expressed human prostate-specific antigen (PSA) on a pilot-scale in Spodoptera frugiperda Sf9 insect cells using recombinant baculovirus system. Infected cells secreted PSA into culture medium at a concentration of 2-4 mg per liter. PSA was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. The N-terminus of active PSA was correctly cleaved; two amino acids of the propeptide remained, however, at the N-terminus of the inactive PSA. Purified recombinant PSA showed a chymotrypsin-like activity with the synthetic substrate MeO-Suc-Arg-Pro-Tyr-pNA, but did not have a trypsin-like activity when Pro-Phe-Arg-pNA was used. The molecular mass of active PSA was 31.0 kDa in reduced SDS-PAGE, 26.0 kDa in nonreduced SDS-PAGE and 26.5 kDa in ion spray mass spectrometry. The active protein formed complexes with alpha 1-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M) in vitro similar to the commercial PSA purified from human seminal fluid.

Prostate-specific antigen and human glandular kallikrein: two kallikreins of the human prostate.

Prostate-specific antigen (PSA) is a 33 kD protein synthesized in the epithelial cells of the prostate gland. It is a serine protease that belongs to the subgroup of kallikreins, among which it is very similar to a putative enzyme called human glandular kallikrein (hGK-1). Although the hGK-1 enzyme remains to be characterized in vivo, the hGK-1 gene is expressed in the same prostatic epithelial cells as the PSA gene. Expression of the PSA gene is under complex control and the steady-state level of PSA mRNA is increased by androgens, and decreased by epidermal growth factor and activation of protein kinase C. This suggests the existence of several regulatory elements within the cis-acting control elements of the PSA gene. As a seminal serine protease, PSA has been shown to digest the high molecular weight seminal vesicle protein, seminogelin. However, it is likely that this does not constitute the only natural substrate of PSA, as PSA has been shown to degrade insulin-like growth factor-binding protein-3. Serum PSA concentrations are frequently increased in patients with prostatic cancer, but this is also the case in patients with benign prostatic hyperplasia. Thus, PSA measurements alone are not useful as a screening tool for undiagnosed prostatic cancer. However, serum PSA concentrations can be successfully used together with other methods in diagnosing prostatic diseases and in monitoring the successfulness of treatments for prostatic cancer.

Evaluation of PAP and PSA gene expression in prostatic hyperplasia and prostatic carcinoma using northern-blot analyses, in situ hybridization and immunohistochemical stainings with monoclonal and bispecific antibodies.

In this report we have investigated levels of prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) gene expression in prostatic carcinoma (Ca) and benign prostatic hyperplasia (BPH) specimens. Northern-blot analyses of total prostatic mRNA indicated that there was a tendency towards lower amounts of PAP mRNA and PSA mRNA in the Ca specimens than in the BPH specimens, although, because of the great variation in the expression levels of both mRNAs, these differences were not statistically significant. In situ hybridization analyses clearly showed that both PAP and PSA mRNAs were confined to the columnar epithelial cells and that stromal cells were devoid of these mRNAs. In addition, PAP and PSA mRNAs were more abundant in BPH tissue than in adjacent Ca tissue within the same specimen. The levels of PAP and PSA enzymes were analyzed immunohistochemically using a bispecific antibody having high affinity for both PAP and PSA, and the results were compared with those obtained using monoclonal anti-PAP and anti-PSA antibodies. All 3 antibodies stained only epithelial cells and BPH tissue consistently gave more intense staining than Ca tissue. Furthermore, the anti-PSA and the bispecific anti-PAP-PSA antibodies stained well or moderately differentiated Ca tissues more strongly than poorly differentiated Ca tissues. No PSA staining was detected in 3 and no PAP staining in 5 of the moderately or poorly differentiated carcinomas (grades II or III). Our results show that, in comparison with BPH tissue, prostatic Ca tissue is associated with significantly lower levels of mRNAs coding for the prostatic marker enzymes PAP and PSA, as well as with lower concentrations of these enzymes. Furthermore, dedifferentiation of prostate Ca is associated with a decrease in the level of intraprostatic PSA.

Growth factor regulation of gene expression in the human prostatic carcinoma cell line LNCaP.

In order to characterize the effects of growth factors on the regulation of expression of the genes coding for prostatic differentiation markers, prostatic acid phosphatase and prostate-specific antigen, we studied changes occurring in the biosynthesis of these enzymes in LNCaP prostatic cancer cells treated with growth factors. Epidermal growth factor was found to reduce the secretion of prostatic acid phosphatase and prostate-specific antigen by the cells, as the result of lowered steady-state levels of the corresponding messenger RNAs (mRNAs). In addition, epidermal growth factor (EGF) interfered with the androgen regulation of these genes. EGF evoked these changes in a concentration- and time-dependent fashion, in both the presence and absence of serum and most likely through interactions with the epidermal growth factor receptor, inasmuch as similar effects were achieved by treating the cells with transforming growth factor alpha. The regulation of the human glandular kallikrein 1 gene was quite similar to the regulation of the prostate-specific antigen gene. In addition to the expression of the genes coding for prostatic secretory proteins, the amount of the human androgen receptor mRNA was down-regulated by EGF. This reduction was more pronounced than the autologous down-regulation of human androgen receptor (hAR) mRNA by androgen and could be maintained for at least 5 days. In the presence of androgen, some of the effects of EGF and transforming growth factor alpha on the levels of androgen-regulated mRNAs may be due to down-regulation of the expression of the hAR gene. Transforming growth factor beta 1, which blocked the growth induction of LNCaP cells by EGF, increased the level of prostatic acid phosphatase and hAR mRNAs, but when given to the cells together with EGF its up-regulatory effect could not be discerned. In summary, regulation of the prostatic acid phosphatase and prostate-specific antigen genes is a complex matter, inasmuch as androgens and growth factors regulate the levels of the mRNAs originating from them. Furthermore, the interactions between the androgen-regulatory system and the growth factor-regulatory systems are likely to be at multiple levels in prostatic cells, as suggested by the modulation of the hAR gene expression by these growth factors.

Steroids inversely affect the biosynthesis and secretion of human prostatic acid phosphatase and prostate-specific antigen in the LNCaP cell line.

In order to elucidate the mechanism of androgen-regulation of genes expressed only in the prostate gland, the effects of steroid hormones on the biosynthesis and secretion of human prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were studied in the human prostatic carcinoma cell line, LNCaP. This cell line produces PAP and PSA, both of which were found to be similar to the proteins purified from and located in human prostatic tissue, as shown by Western blot analysis. The synthetic androgen, R1881, regulated the biosynthesis of these two important tumour marker proteins inversely: the amount of PSA released into the medium was increased to 506% +/- 100 of the control levels, while that of PAP was decreased to 26% +/- 3, in 7 days. These effects were dependent on the concentration of the steroid in the growth medium. The androgen-dependent changes observed in the amounts of the secreted proteins were correlated with alterations in their intra-cellular levels. LNCaP cells were found to have very different capacities for secreting PAP and PSA. Whereas the measurable, cellular amounts of PSA and PAP were of similar magnitudes, much larger amounts of PSA than PAP were secreted into the medium. PSA was also found to be more stable than PAP in the culture medium of the LNCaP cells. Other steroids could elicit effects on PAP and PSA biosynthesis similar to those induced by R1881, and the combined effects of effective concentrations of these steroids were undistinguishable from those caused by each one of them separately, suggesting that all these compounds compete for binding to the same modified androgen receptors of the LNCaP cells. Thus, our results confirm the observations of the altered nature of the LNCaP androgen receptors, and demonstrate the ability of these ligands to produce changes in the expression of androgen-dependent prostatic genes. The fact that the changes observed at the protein level were accompanied by increased levels of PSA mRNAs and by decreased levels of PAP mRNA in steroid-treated cells, suggests that one of the targets of androgen and steroid action in the regulation of these genes is at the mRNA level.

Androgens up-regulate the human prostate-specific antigen messenger ribonucleic acid (mRNA), but down-regulate the prostatic acid phosphatase mRNA in the LNCaP cell line.

To better understand androgen-regulated gene expression in the prostate, we have used Northern blot analysis to study the effects of androgens and other steroid hormones on the steady state levels of several human prostatic mRNAs in the LNCaP cell line. Dihydrotestosterone (40 nM) as well as a synthetic androgen, R1881 (0.1 nM), increased the amounts of prostate-specific antigen (PSA) and human glandular kallikrein mRNAs; at the same time, the level of prostatic acid phosphatase (PAP) mRNA was down-regulated. Incubation of LNCaP cells with medium containing 0.1 or 1 microM R1881 for 3, 7, or 13 days resulted in up-regulation of PSA and human glandular kallikrein mRNAs and down-regulation of PAP mRNA. Thus, the two clinically important prostate-specific marker proteins are inversely regulated in this cell line. The level of human androgen receptor mRNA was also repressed by the androgen treatments. 17 beta-Estradiol and progesterone had effects similar to those of R1881 on gene expression in LNCaP cells. Our results show that the decrease in the amount of secreted PAP and the increase in the amount of secreted PSA caused by androgens and other steroid hormones in the LNCaP cells of epithelial origin are mediated by changes in the levels of the corresponding mRNAs.

Expression of the gene coding for human prostate-specific antigen and related hGK-1 in benign and malignant tumors of the human prostate.

Human glandular kallikrein-1 gene (hGK-1) is closely related to the gene of human prostate-specific antigen (PSA) and both genes are expressed in the human prostate. We have studied PSA and hGK-1 mRNAs in human prostatic tissue samples from patients with benign prostatic hyperplasia (BPH) or adenocarcinoma (CA), using Northern and slot-blot analysis in order to gain insight into the expression of these highly similar genes. Multiple mRNAs were found to originate from both genes. The major mRNA species of 1.6 kb accounted for 57% to 76% of the total coding capacity for PSA in different tissue specimens, but a variant mRNA species of 1.9 kb was also abundant. Most of the BPH samples contained marked amounts of an aberrant 0.9 kb mRNA, and long PSA mRNAs of 6.1 kb, 4.5 kb and 3.1 kb were found in elevated amounts in some of the CA samples. The amount of PSA mRNAs that would produce aberrant PSA proteins if translated into protein varied from 18% to 38% in these tissue samples. The major mRNA species originating from hGK-1 was of 1.6 kb, but other less abundant mRNA species could also be observed. The amount of PSA and hGK-1 mRNAs was determined from slot blots hybridized with specific oligonucleotide probes. No significant differences could be found in the PSA gene expression between BPH and CA samples. The total amount of the PSA mRNAs in all the different BPH specimens was fairly similar, but there was a 3-fold difference between the highest and lowest PSA mRNA levels in the CA samples. The hGK-1 mRNA levels in the BPH specimens studied demonstrated greater variance than the PSA mRNA levels in the same samples. The correlation between PSA gene and hGK-1 expression in the BPH samples was good, suggesting that there are similarities in the regulation of these genes. However, the lack of correlation between the amounts of PSA and hGK-1 mRNAs in the CA samples except in sample C1 indicates that there are also differences in the gene regulation. The observed 3.7- to 6.5-fold excess of PSA mRNAs as compared with the amount of hGK-1 mRNAs present in the same tissue specimen also indicated differences in the cis- or trans-acting regulatory elements of these genes.

cDNA coding for the entire human prostate specific antigen shows high homologies to the human tissue kallikrein genes.

We have isolated four cDNAs encoding the entire preproprotein of prostate specific antigen from human prostatic cDNA libraries. Comparison of the coding regions of prostate specific antigen with human pancreatic kallikrein (1-3) and human glandular kallikrein (4) showed 73%-84% nucleotide and 61-77% amino acid homologies, respectively, between these enzymes. Also the 3' noncoding regions of these genes were conserved. The close resemblance of prostate specific antigen, a marker for prostatic cancer, to glandular kallikrein suggests related immunogenic properties for them.

High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase.

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.

Gene structure for the alpha 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon.

Two overlapping human genomic clones that encode a short-chain collagen, designated alpha 1(XIII), were isolated by using recently described cDNA clones. Characterization of the cosmid clones that span approximately equal to 65,000 base pairs (bp) of the 3' end of the gene established several unusual features of this collagen gene. The last exon encodes solely the 3' untranslated region and it begins with a complete stop codon. The 10 adjacent exons vary in size from 27 to 87 bp and two of them are 54 bp. Therefore, the alpha 1-chain gene of type XIII collagen has some features found in genes for fibrillar collagens but other features that are distinctly different. Previous analysis of overlapping cDNA clones and nuclease S1 mapping of mRNAs indicated one alternative splicing site causing a deletion of 36 bp from the mature mRNA. The present study showed that the 36 bp is contained within the gene as a single exon and also that the gene has a 45-bp -Gly-Xaa-Xaa- repeat coding exon not found in the cDNA clones previously characterized. Nuclease S1 mapping experiments indicated that this 45-bp exon is found in normal human skin fibroblast mRNAs. Accordingly, the data demonstrate that there is alternative splicing of at least two exons of the type alpha 1(XIII)-chain gene.

Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase.

lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases.