Expression and copy number analysis of TRPS1, EIF3S3 and MYC genes in breast and prostate cancer.

The long arm of chromosome 8 is one of the most common regions of amplification in cancers of several organs, especially carcinomas of the breast and prostate. TRPS1, MYC and EIF3S3 genes are located in one of the minimal regions of amplification, 8q23-q24, and have been suggested to be the target genes of the amplification. Here, our goal was to study copy number and expression of the three genes in order to investigate the significance of the genes in breast and prostate cancer. By using fluorescence in situ hybridisation (FISH), we first found that TRPS1 and EIF3S3 were amplified together in about one-third of hormone-refractory prostate carcinomas. Next, we analysed the mRNA expression of the three genes by real-time quantitative RT-PCR and the gene copy number by FISH in six breast and five prostate cancer cell lines. Breast cancer cell line, SK-Br-3, which contained the highest copy number of all three genes, showed overexpression of only EIF3S3. Finally, the expression levels of TRPS1, EIF3S3 and MYC were measured in freshly frozen clinical samples of benign prostate hyperplasia (BPH), as well as untreated and hormone-refractory prostate carcinoma. The TRPS1 and MYC expression levels were similar in all prostate tumour groups, whereas EIF3S3 expression was higher (P=0.029) in prostate carcinomas compared to BPH. The data suggest that the expression of EIF3S3 is increased in prostate cancer, and that one of the mechanisms underlying the overexpression is the amplification of the gene.

Vitamin D and vitamin D analogs in cancer treatment.

The secosteroid hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is a key player in the regulation of bone mineralization and calcium homeostasis. In addition, 1,25-(OH)2D3 has antiproliferative and prodifferentiation effects on various cells in vitro and in vivo. The growth-inhibitory properties of 1,25-(OH)2D3 could be harnessed in the treatment of cancer. However, its use as an anti-cancer drug is limited because of the calcemic effects of pharmacological doses. In an attempt to dissociate the antiproliferative and calcemic effects, numerous vitamin D3 analogs were developed. The mechanisms by which 1,25-(OH)2D3 and 1,25-(OH)2D3 analogs exert their growth-inhibitory effects are not clear but include effects on cell differentiation, apoptosis, cell cycle regulation, metastases, and angiogenesis. In the current review aspects involved in the tumor suppressive activity of 1,25-(OH)2D3 and 1,25-(OH)2D3 analogs will be addressed. The use of vitamin D3 compounds, alone or in combination with other drugs, in cancer treatment and the potential drawbacks will also be discussed.

Structure and function of GC79/TRPS1, a novel androgen-repressible apoptosis gene.

Expression of death-signaling genes induces many biochemical cascades resulting in elimination of cells via apoptosis or programmed cell death. GC79/TRPS1 is a novel apoptosis associated gene that encodes a multitype zinc finger GATA-type transcription factor. Expression of GC79/TRPS1 is repressed in the rat ventral prostate and significantly elevated after androgen withdrawal by castration. Castration leads to regression of the prostate caused by apoptosis of androgen-dependent prostate cells. Prostate cancer consists of androgen-dependent and androgen-independent cells. The androgen-independent cells, usually present in the prostate of advanced prostate cancer patients do not have the ability to undergo apoptosis after androgen withdrawal. GC79/TRPS1 expression in androgen-dependent prostate cancer cells is repressed by androgens, while GC79/TRPS1 expression is hardly detectable in androgen-independent prostate cancer cells under cell culture conditions. This suggests that lack of GC79/TRPS1 expression could be a mechanism for the inability to induce the apoptotic pathway in androgen-independent prostate cancer cells after androgen withdrawal. This review will focus on the current knowlegde of the structure and function of GC79/TRPS1, a novel androgen-repressible apoptosis gene.

A novel gene on human chromosome 2p24 is differentially expressed between androgen-dependent and androgen-independent prostate cancer cells.

Identification of genes involved in the transition from androgen-dependent to androgen-independent prostate cancer is important to extend our current knowledge of the disease. Using differential display RT-PCR analysis between androgen-dependent and androgen-independent prostate cancer cells, we have identified a novel gene, designated GC109. GC109 harbours a putative Cys-His cluster, a nuclear localisation signal, a leucine zipper and a ret finger protein (rfp)-like domain. GC109 mRNA expression in normal human tissues was found not to be restricted to the prostate. However, using a variety of 15 human cancer cell lines, GC109 mRNA was preferentially expressed in androgen-dependent LNCaP-FGC, compared with androgen-independent LNCaP-LNO, DU145 and PC3 human prostate cancer cells. Finally, the GC109 gene was mapped on human chromosome 2p24. Based on its protein domain structure and chromosomal localisation, we hypothesise that GC109 may be involved in chromosomal rearrangements in prostate cancer.

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells.

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.

Identification of a gene on human chromosome 8q11 that is differentially expressed during prostate-cancer progression.

Using differential-display RT-PCR analysis between androgen-dependent LNCaP-FGC and androgen-independent LNCaP-LNO human prostate-cancer cells, we have identified a gene not previously described as being expressed in prostate. The gene is more highly expressed in androgen-independent than in androgen-dependent LNCaP prostate-cancer cells. Sequence analysis showed that the gene has already been cloned as a transcript present in embryonic brain, with unknown functions. Expression of the gene was found not to be restricted to the prostate, and not regulated by androgens in androgen-independent prostate-cancer cells. In concert with the cell-culture system, Northern-blot analysis of gene expression in vivo, using a panel of human prostate-cancer xenografts, demonstrated that the gene is more highly expressed in androgen-independent than in androgen-dependent prostate-cancer xenografts. The gene could be mapped on human chromosome 8q11. The 8q arm is known to be frequently amplified during prostate-cancer progression and harbors several proto-oncogenes potentially involved in cancer development. Since expression of the gene is positively correlated with prostate-cancer progression and its 8q11 chromosomal localization, we hypothesize that the gene may be involved in the development and progression of prostate cancer.

Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer.

The beta 1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the beta 1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of beta 1-subunit protein, but not of the alpha 1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent.

Involvement of amino acid residues 423-429 of human protein S in binding to C4b-binding protein.

Human protein S binds to C4b-binding protein (C4BP) both in plasma and in a system using purified proteins. Amino acid residues 420-434 of the first disulfide loop of the sex hormone binding globulinlike domain of protein S are involved in the interaction of protein S with C4BP. To define the involvement of specific polar amino acids within residues 420-434, we studied in parallel synthetic protein S peptides and recombinant protein S variants containing the same amino acid replacements, K423E, E424K, Q427E and K429E. Synthetic peptide analogs of peptide PSP-420 (residues 420-434) were assayed for binding C4BP and as inhibitors of complex formation. The PSP-420 peptide and the analogous peptide with the substitution E424K, but not the peptides containing the substitutions K423E and K429E, were able to bind C4BP. Recombinant proteins with mutations of K423E, Q427E and K429E showed reduced affinity for C4BP compared to plasma protein S, recombinant wild type protein S, or E424K-protein S. These results suggest that Lys-423, Gln-427 and Lys-429 of protein S are important for normal binding to C4BP. The anti-protein S monoclonal antibody LJ-56, raised against peptide PSP-420, recognizes only free protein S and inhibits complex formation with C4BP. Antibody LJ-56 recognized the E424K and Q427E peptides but not the K423E or K429E peptides. Similarly, the E424K and Q427E protein S mutants were recognized by LJ-56, whereas the K423E and K429E protein S mutants were not recognized. This suggests that both in the peptide PSP-420 and in protein S, Lys-423 and Lys-429 significantly contribute to binding to antibody LJ-56. These results demonstrate that protein S residues 423, 427 and 429, but not residue 424, are involved in binding to both the antibody LJ-56 and to C4BP. When peptides PSP 420 and SL-6 (residues 447-460) with carboxyterminal amide or carboxylate moieties were compared to their ability to inhibit C4BP-protein S complexation, PSP-420-amide was the most potent. This finding together with the other results described here supports the hypothesis that the residues 420 and 434 in protein S provides a major binding site for C4BP.

Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain.

Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To define the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (mini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wild-type recombinant protein S and mini-protein S with similar affinities, whereas a monoclonal antibody that recognizes an epitope in the SHBG domain did not detect mini-protein S. Mini-protein S did not bind to C4BP in a solid-phase binding assay, and the cofactor activity of mini-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no difference in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S molecule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP binding and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system using purified components. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.

Differentially expressed genes in androgen-dependent and -independent prostate carcinomas.

Differential gene expression between androgen-dependent (LNCaP-FGC) and androgen-independent (LNCaP-LNO) prostate cancer cells has been investigated using RNA arbitrarily primed and differential display PCR of mRNA. Four differentially expressed cDNA transcripts were identified, of which differential expression was confirmed by Northern blot analysis. Sequence analysis revealed two unknown (JC19 and GC79) and two known genes [B-cell translocation gene 1 and UDP-glucuronosyltransferase 2B15 (UGT2B15)]. JC19, GC79, and B-cell translocation gene 1 were more highly expressed in LNCaP-FGC cells compared with LNCaP-LNO cells, whereas UGT2B15 was only expressed in LNCaP-LNO cells. Androgens and 1,25-dihydroxyvitamin D3 were able to down-regulate UGT2B15 mRNA in LNCaP-LNO cells. For GC79 mRNA, down-regulation was only observed with androgens in LNCaP-FGC cells. Expression of JC19 mRNA was studied using a panel of human prostate cancer xenografts. In androgen-dependent xenografts, expression of JC19 mRNA was much higher compared with androgen-independent xenografts, in which significant expression was hardly detectable. The mRNA expression pattern in the xenografts is in good agreement with that observed in the cell culture system. In conclusion, the differential display technique used in the present study allows analysis of gene expression in vitro and in vivo and can be used for the identification of important genes involved in androgen-independent prostate cancer development.

Internalization and degradation of recombinant human coagulation factor VIIa by the human hepatoma cell line HuH7.

Previous studies demonstrated that several normal and transformed cultured human cell lines specifically bind human coagulation factors VII and VIIa via tissue factor. In the present study, we show that 125I-radiolabeled recombinant human factor VIIa (125I-rFVIIa) binds to a human hepatoma cell line (HuH7). In the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG, binding of 125I-rFVIIa to the HuH7 cells was decreased approximately 60%, suggesting of tissue factor-independent binding sites for 125I-rFVIIa on these cells. The binding isotherm of 125I-rFVIIa for the HuH7 cells in the presence of anti-tissue factor IgG exhibited a hyperbolic profile and was time-, temperature- and calcium-dependent. Furthermore, binding at 4 degrees C was specific, dose-dependent and saturable. Scatchard analysis of the binding data demonstrated a single class of binding sites with a dissociation constant (Kd) of 3.2 nM and 27,000 binding sites per cell. At 4 degrees C, 125I-rFVIIa bound to, and eluted from, the cell was indistinguishable from offered 125I-rFVIIa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. The molecular properties of the tissue factor-independent binding protein were studied by using the cleavable cross-linking agent 3,3'-dithiobis(sulfosuccinimidylpropionate). A cross-linking product of 125I-rFVIIa and a cell surface protein with an apparent M(r) approximately 100,000 was observed. The cross-linking reaction was strongly inhibited by a 100-fold molar excess of unlabeled rFVIIa, but not by rabbit polyclonal anti-human tissue factor apoprotein IgG, indicating that cross-linking does not involve the extracellular domain of tissue factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and characterization of thrombin-resistant variants of recombinant human protein S.

Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa. Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment. All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the interaction between human protein S and human C4b-binding protein using deletion variants of recombinant human protein S.

Human protein S interacts noncovalently with human C4b-binding protein (C4BP). We have studied this interaction using deletion variants of recombinant human protein S. Two deletion variants were constructed by restriction enzyme digestion and in vitro site-specific mutagenesis of the human protein S cDNA. The variants were stably expressed in C127 cells. Recombinant proteins were purified using Fast Flow Q anion-exchange chromatography. The activated protein C (APC) cofactor activity, C4BP binding properties and reactivity to different monoclonal antibodies against human protein S were examined. The first variant (E variant), which has a deletion of the third epidermal growth factor (EGF)-like domain (deletion of exon VII, corresponding to amino acid residues ASP-160 to Asp-202) expresses normal APC cofactor activity in a plasma system. This activity was inhibited by the addition of purified C4BP. The second variant (L variant), which has a deletion of the C-terminal loop of the sex hormone binding globulin (SHBG)-like domain (deletion of exon XV, corresponding to amino acid residues Asp-583 to Ser-635) also expresses normal APC cofactor activity in plasma. This activity could only be partially inhibited by the addition of purified C4BP. Binding of the recombinant proteins to C4BP was studied in a system using purified proteins. The E variant binds to C4BP with the same affinity similar as recombinant wild type protein S (apparent Kd approximately 10(-10) M). The L variant, however, shows a markedly reduced affinity for binding to C4BP (apparent Kd approximately 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and characterization of recombinant human protein S in heterologous cells--studies of the interaction of amino acid residues leu-608 to glu-612 with human C4b-binding protein.

Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments.

Human tissue-type plasminogen activator (t-PA) consists of five domains designated (starting from the N-terminus) finger, growth factor, kringle 1, kringle 2, and protease. The binding of t-PA to lysine-Sepharose and aminohexyl-Sepharose was found to require kringle 2. The affinity for binding the lysine derivatives 6-aminohexanoic acid and N-acetyllysine methyl ester was about equal, suggesting that t-PA does not prefer C-terminal lysine residues for binding. Intact t-PA and a variant consisting only of kringle 2 and protease domains were found to bind to fibrin fragment FCB-2, the very fragment that also binds plasminogen and acts as a stimulator of t-PA-catalyzed plasminogen activation. In both cases, binding could completely be inhibited by 6-aminohexanoic acid, pointing to the involvement of a lysine binding site in this interaction. Furthermore, the second site in t-PA involved in interaction with fibrin, presumably the finger, appears to interact with a part of fibrin, different from FCB-2.

Enhanced plasminogen activator production of Syrian hamster embryo cells transformed by chemicals or the c-Ha-ras oncogene: type of plasminogen activators involved and their contribution to the transformed phenotype.

The plasminogen activator (PA) activity produced by Syrian hamster embryo (SHE) cells in different stages of neoplastic conversion was analysed. PA activity was characterized immunologically and by SDS-PAGE. Normal SHE cells had a very low PA activity. Although activity of either the tissue type of PA (t-PA) or the urokinase type (u-PA) or both were found to be increased in most immortal or transformed SHE cells, there was no correlation between enhanced production of a particular PA type and the development of the immortal or transformed phenotype. However, within a group of cell lines clonally derived from a culture of immortal cells, a positive correlation was found between extracellular t-PA, but not u-PA, activity and cellular growth rate. For the Syrian hamster PA species, crossreacting with anti-human u-PA, a mol. wt of 39 kd was observed. For the Syrian hamster PA species, crossreacting with anti-human t-PA, multiple species were found with mol. wts of 98, 72 and 59 kd respectively. Evidence was obtained that the 72-kd species represents the intact enzyme, the 59-kd species a partial digestion product thereof and the 98 kd species, which often appears as a doublet, a complex of either of these species with an inhibitor, likely to be secreted by the same cells. Finally, our data suggest a novel mechanism for the enhancement of t-PA activity of transformed cells, namely by a decrease in the effective extracellular amount of putative inhibitor.

Effect of extract of Hirudo Medicinalis L. against adherence of calcium oxalate crystals to acid-injured bladder mucosa.

The effect of extract of Hirudo Medicinalis L. on preventing the adhesion of calcium oxalate crystals to 0.1 M hydrochloric acid-injured bladder urothelium of the rat was studied. It was found that in this species the extract coated to the bladder mucosa after it was instilled into the chemically injured bladder; and the adhesion of calcium oxalate crystals was prevented. In regard to the anti-adhesion property the Hirudo extract appears more effective than heparin, a documented glycosaminoglycan.

Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin.

Human tissue-type plasminogen activator (t-PA) catalyses the conversion of inactive plasminogen into active plasmin, the main fibrinolytic enzyme. This process is confined to the fibrin surface by specific binding of t-PA to fibrin and stimulation of its activity by fibrin. Tissue-type plasminogen activator contains five domains designated finger, growth factor, kringle 1, kringle 2 and protease. The involvement of the domains in fibrin specificity was investigated with a set of variant proteins lacking one or more domains. Variant proteins were produced by expression in Chinese hamster ovary cells of plasmids containing part of the coding sequence for the activator. It was found that kringle 2 domain only is involved in stimulation of activity by fibrin. In the absence of plasminogen and at low concentration of fibrin, binding of t-PA is mainly due to the finger domain, while at high fibrin concentrations also kringle 2 is involved in fibrin binding. In the presence of plasminogen, fibrin binding of the kringle 2 region of t-PA also becomes important at low fibrin concentrations.

Quantification of tissue-type plasminogen activator (t-PA) mRNA in human endothelial-cell cultures by hybridization with a t-PA cDNA probe.

We describe the construction of a recombinant DNA plasmid, consisting of the vector pBR322 and full-length tissue-type plasminogen-activator (t-PA) cDNA, by using polyadenylated RNA from cultured Bowes melanoma cells as substrate. A 1280-base-pair PstI restriction fragment, covering the 3' untranslated region and part of the coding region for the t-PA L-chain, was used as a radiolabelled probe to determine the size and the number of t-PA mRNA molecules in cultured endothelial cells of different origin from the same individual. Northern blotting showed that in all these cells a t-PA mRNA is synthesized of about 2500 nucleotides, indicating that transcriptional initiation, splicing and polyadenylation is similar. The number of t-PA mRNA molecules per cell measured, by using a dot-blotting technique and t-PA mRNA made in vitro, with a plasmid DNA preparation harbouring a specific promotor of the Salmonella typhimurium bacteriophage SP6, t-PA cDNA and SP6 RNA polymerase as standard, is approx. 10,000 in all cultured endothelial cells from adult vessels. However, the amount of t-PA antigen synthesized and/or secreted differs by a factor of 6-20. Relatively large amounts of t-PA antigen secreted were detected in conditioned medium from vena-cava-derived cells, whereas low amounts were found in conditioned medium from arteria-iliaca-derived cells.

Assignment of the human tissue-type plasminogen activator gene (PLAT) to chromosome 8.

Using 1.2kb 3'-terminal Pst-I fragment of a full length tissue-type plasminogen activator (t-PA) cDNA clone (ptPA-8FL) and a set of rodent human somatic cell hybrids, the corresponding human gene PLAT was localized on chromosome 8.