Biomedical subjects

J Trapman

Publications and source records attributed to J Trapman.

At least 109 records · Page 6Linked to original sources

PubMed1989

The human prostatic carcinoma cell line LNCaP and its derivatives. An overview.

The FGC (fast growing colony) line, a derivative of the LNCaP cell line shares all the main characteristics, including its androgen dependence, described for the original LNCaP cultures. A number of sublines originated from the FGC line which were characterized with respect to their response to steroid-depleted serum and to the synthetic androgen, R1881. After subcloning the FGC line a series of clones was isolated with distinct patterns of androgen-responsiveness. Among the sublines and clones studied, the FGC, FGC-JB and FGC clone-9 were androgen-dependent, whereas subline LNO, R and presumably also FGC clone-22 were androgen-independent. Distinct morphological differences were observed between the cells of the various sublines and between clone-9 and 22. The LNCaP cell line, its descending sublines and clonal derivatives provide a suitable in vitro model for studying different aspects of androgen-responsiveness of human prostate cancer.

Androgens↗

PubMed1989

Follicle-stimulating hormone regulates androgen receptor mRNA in Sertoli cells.

Follicle-stimulating hormone (FSH) and testosterone stimulate the production of a variety of proteins by immature Sertoli cells. A highly purified Sertoli cell preparation was incubated for 3 days with FSH and testosterone. Both androgen receptor protein and mRNA concentrations were markedly increased by FSH. Testosterone also increased the androgen receptor protein concentration, but did not increase the expression of the androgen receptor mRNA. It is concluded that FSH plays a role in the responsiveness of Sertoli cells to testosterone.

Animals↗

PubMed1989

The N-terminal domain of the human androgen receptor is encoded by one, large exon.

Using specific cDNA hybridization probes, the first coding exon of the human androgen receptor gene was isolated from a genomic library. The exon contained an open reading frame of 1586 bp, encoding an androgen receptor amino-terminal region of 529 amino acids. The deduced amino acid sequence was characterized by the presence of several poly-amino acid stretches of which the long poly-glycine stretch (16 residues) and the poly-glutamine stretch (20 residues) were most prominent. Androgen receptor cDNAs from different sources contained information for poly-glycine stretches of variable size (23 and 27 residues, respectively). The androgen receptor amino-terminal domain was found to be hydrophilic and have a net negative charge. Combined with the previously described, partially overlapping cDNA clone 7A2M27 (Trapman et al. (1988) Biochem. Biophys. Res. Commun. 153, 241-248), the complete human androgen receptor was deduced to have a size of 910 amino acids.

Amino Acid Sequence↗

PubMed1989

Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor.

Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.

Animals↗

PubMed1989

Structural organization of the human androgen receptor gene.

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.

Amino Acid Sequence↗

PubMed1988

Molecular cloning and characterization of novel prostate antigen cDNA's.

Three different prostate antigen cDNA's were isolated from a PC 82 prostate tumor cDNA library. PA 75 has a size of 1.4 kb and contains the almost complete information for the 35 kD prostate antigen preproprotein. The 1.6 kb PA 525 cDNA lacks about 0.2 kb of the 3'-non coding region and contains an additional internal 0.4 kb fragment as a result of alternative splicing. PA 424 represents a 0.6 kb variant of PA 75. It contains a 0.15 kb internal fragment and a poly(A) tail preceded by an AAGAAA motive at the 3'-end. The predicted protein products of PA 525 and PA 424 will be different from PA 75 at the C-terminal end. In RNA preparations of two human prostate tumors (PC 82 and PC EW) seven different prostate antigen transcripts can be detected ranging in size from 0.5 kb to 5.6 kb. PA 75 cDNA represents the major 1.5 kb mRNA. PA 424 correlates with a 0.9 kb transcript and PA 525 with a 1.9 kb mRNA species.

Amino Acid Sequence↗

PubMed1988

Interferon expression in the testes of transgenic mice leads to sterility.

A plasmid containing the mouse interferon-alpha 1 gene under control of the mouse metallothionein-I promoter was used for the construction of transgenic mice. Four transgenic mice (two males and two females) were obtained containing 1 to over 10 copies of the introduced DNA. Both males appeared to be sterile. One of the female mice founded a transgenic strain in which the foreign DNA was transmitted to her offspring in a Mendelian fashion. In this strain most male animals are sterile or turn sterile with time. Northern blot analysis of several tissues of these animals shows that expression of the introduced interferon gene occurs only in the testis. In some of the animals biologically active interferon could also be detected in testes homogenates. Histological examination of testis tissue shows an ongoing degeneration of spermatogenic cells leading to calcium deposits and complete atrophy of the seminiferous tubules.

Animals↗

PubMed1988

Cloning, structure and expression of a cDNA encoding the human androgen receptor.

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.

Amino Acid Sequence↗

PubMed1988

Interferon-alpha-(IFN) producing CHO cell lines are resistant to the antiproliferative activity of IFN: a correlation with gene expression.

CHO cell lines that constitutively produce the murine interferon-alpha (IFN-alpha) subspecies alpha 4 and alpha 6 were constructed. The producer cell lines were protected against viral (vesicular stomatitis virus) infection by the IFN species secreted, but were resistant to the growth inhibitory activity of the IFN species. As compared with alpha 4, the alpha 6 protein displayed a high antiproliferative activity when added to normal CHO cells, which correlates completely with the high antiviral activity of alpha 6 on these cells. Three messenger ribonucleic acid (mRNA) species, which are normally induced in CHO cells by IFN treatment (1-8, 2-5A synthetase, and ISG 15) were constitutively present in CHO producer cell lines. The level of another mRNA (ISG 54), however, was very low in the producer cells as compared with its expression in short-term IFN-treated cells. These data indicate that 1-8, 2-5A synthetase and ISG 15 are not involved in the antigrowth activity of IFN in this system, but rather suggest a function of ISG 54 in this respect.

Animals↗

PubMed1988

Effects of FSH and IGF-I on immature rat Sertoli cells: inhibin alpha- and beta-subunit mRNA levels and inhibin secretion.

Effects of follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) on inhibin production by cultured Sertoli cells from 21- to 23-day-old rats were studied. The expression of inhibin alpha- and beta-subunit mRNAs, and inhibin immunoreactivity and in vitro bioactivity were estimated. Using a cDNA probe corresponding to the alpha-subunit of bovine inhibin, specific hybridization with a 1.5-1.7 kilobase (kb)mRNA species was observed. Addition of FSH to the cultured Sertoli cells for 24 h markedly increased the level of this mRNA in a dose-dependent way. IGF-I had no effect on the intensity of the hybridization. Using a cDNA probe corresponding to the beta B-subunit of human inhibin, 3.5 and 4.2 kb mRNA species were detected. FSH and IGF-I had no effect on the hybridization signal. No hybridization was observed with a cDNA probe corresponding to the beta A bovine inhibin subunit. Inhibin activity was detected in cells and medium by immunoassay, and in the medium by in vitro bioassay. FSH stimulated both immunoreactivity and in vitro bioactivity, whereas IGF-I had no effect at all. The present effect of FSH on inhibin alpha-subunit mRNA expression in cultured Sertoli cells indicates that regulation of inhibin production by FSH includes an effect at the transcriptional level. However, this does not exclude additional translational and posttranslational effects.

Animals↗

PubMed1988

Structure-function analysis of murine interferon-alpha: antiviral properties of novel hybrid interferons.

As described earlier the protein products of the murine interferon (IFN) genes MuIFN-alpha 1, -alpha 2, and -alpha 4 differ in their antiviral activity on hamster (CHO) and mouse (L929) cells. For structure-function analysis, hybrids were prepared between the three genes using common restriction enzyme sites. Natural and hybrid genes were transiently expressed in monkey COS cells. Under the conditions used IFN constituted 20-30% of the total amount of secreted proteins. Using a panel of hybrids either between alpha 1 and alpha 2 or between alpha 1, alpha 2, and alpha 4, the amino-terminal region of the protein, from amino acids 10 to 58, was found to determine its antiviral activity on hamster cells. On mouse cells, the antiviral activities of hybrids between alpha 4 and either alpha 1 or alpha 2 were compared. The high activity of alpha 4 (five to ten times that of alpha 1 or alpha 2) was not transmitted to hybrids having the amino-terminal part of alpha 4, but coincided with the presence of the alpha 4 carboxy-terminal region in all but one hybrid construct. The deletion of five amino acids (positions 103-107) located in this region of alpha 4 did not affect antiviral activity when introduced into MuIFN-alpha 2 and a MuIFN-alpha 42 hybrid by site-directed mutagenesis.

Animals↗

PubMed1988

Structure-function analysis of mouse interferon alpha species: MuIFN-alpha 10, a subspecies with low antiviral activity.

A mouse interferon alpha gene (MuIFN-alpha 10) was isolated from a BALB/c cosmid genomic library. The gene was located on a 1.8 kb HindIII fragment and a 5.1 kb EcoRI fragment. The coding region and parts of the 5' and 3' non-coding regions were sequenced. The results showed that the MuIFN-alpha 10 gene encoded a protein of 167 amino acids. Like most other MuIFN-alpha species it contained a putative N-glycosylation site at amino acid positions 78 to 80. It also possessed cysteine residues at positions 1, 29, 86, 99 and 129. In the signal peptide, in addition to cysteine 21, which is present in all MuIFN-alpha species sequenced so far, a cysteine was found at position 22. At the amino acid level MuIFN-alpha 10 showed strong homology to MuIFN-alpha 1 (only 15 out of 167 amino acids were different). The MuIFN-alpha 10 gene was transiently expressed in monkey COS cells under the direction of the simian virus 40 early promoter. The protein product secreted by COS cells was equally active on mouse (L929) and hamster (CHO) cells. However, as compared to MuIFN-alpha 1 and MuIFN-alpha 4 the specific activity on mouse cells of the protein was 10- to 100-fold lower. To find out which region of its structure was responsible for this low activity, hybrids of the genes encoding MuIFN-alpha 10 and MuIFN-alpha 1 were constructed using the two common XmmI sites which correspond to positions between amino acids 67 and 68 and 123 and 124, respectively. The data showed that hybrid constructs which were MuIFN-alpha 1-like from amino acid 68 or MuIFN-alpha 10-like from position 124 to the C terminus possessed high antiviral activity. Other hybrid constructs were hardly active at all. This implied that the amino acid 68 to 123 region was mainly responsible for the low antiviral activity of MuIFN-alpha 10. In this part of the molecule MuIFN-alpha 1 and MuIFN-alpha 10 differed in only five amino acids. A serine at position 110 and a valine at 85 were unique to MuIFN-alpha 10 as compared to all known MuIFN-alpha and human IFN-alpha subspecies.

Amino Acid Sequence↗

PubMed1987

Two domains in alpha interferons influence the efficacy of the antiviral response.

Murine interferon-alpha 1 and murine interferon-alpha 4 share 80% of their amino acids, yet the proteins differ considerably in their ability to protect mouse or hamster cells against viral infection. With the aim of localizing areas within these proteins which influence the biological response we have constructed hybrid alpha 1 alpha 4 genes by means of homologous recombination of the parent genes. When the antiviral activities of these proteins were compared, it appeared that there are at least two domains that affect the biological response to these proteins: area A (amino acids 10-20) and area B (amino acids 55-67). These areas are presumably involved in the interaction between ligand and receptor. Most interestingly, hybrids in which area A from IFN-alpha 1 is combined with area B from alpha 4, have antiviral activities on homologous cells that are one to two orders of magnitude higher than those of the parent proteins.

Amino Acid Sequence↗

PubMed1986

Characterization of monoclonal antibodies raised against the prostatic cancer cell line PC-82.

For production of monoclonal antibodies (McAbs), hybrid cells were prepared by fusion of spleen cells of BALB/c mice immunized with the human prostatic cancer cell line PC-82 and the P3-X6(3)Ag8.653 murine myeloma cell line. Supernatants of approximately 500 hybrid clones were screened for prostate specific antibodies using an ELISA on PC-82 cells. A selection of antibodies was further tested for their specificity on a large series of different tissues. A broad cross reactivity pattern was obtained. Most cross reactivity was with pancreatic tissue, kidney, and bowel. One antibody turned out to react with prostate stromal cells. Two McAbs (ER-Pr 1 and ER-Pr 2) reacted solely with prostatic epithelium. Monoclonal antibody affinity chromatography combined with SDS-PAGE showed that both antibodies were directed against a 35-kD protein. Immunoblotting revealed that this protein is identical to prostatic antigen (PA). The epitope detected by ER-Pr 1 and ER-Pr 2 was largely preserved after formalin-fixation of prostatic tissues which renders these antibodies very suitable for routine examination of tissue sections.

Animals↗

PubMed1986

Transient expression of murine interferon-alpha genes in mouse and monkey cells.

The coding regions of murine interferon-alpha (IFN-alpha) genes were combined with promoter and 3'-noncoding sequences from other eukaryotic genes. Transient expression of these fusion genes was achieved in monkey COS cells and in a mouse cell line (TOP cells) expressing polyoma virus (Py) large T antigen constitutively. The efficiency of the different expression plasmids was determined by measuring the amount of IFN secreted into the medium. Replacement of the 3'-noncoding region of an IFN-alpha gene by that of the rabbit beta-globin gene resulted in a fourfold higher IFN-alpha production. The SV40 early promoter and the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) produced similar amounts of IFN-alpha in COS cells. However, a tandem combination of the SV40 enhancer/early promoter and the mouse metallothionein-I promoter appeared fivefold more active than the SV40 early promoter. In TOP cells the MoMLV LTR was found to be threefold more active than the Py early promoter.

Animals↗

PubMed1986

Properties of natural and hybrid murine alpha interferons.

Four natural murine interferon-alpha genes (MuIFN-alpha 1, -alpha 2, -alpha 4 and -alpha 6) and four hybrid genes (alpha 1 alpha 4, alpha 2 alpha 4, alpha 4 alpha 1 and alpha 4 alpha 2) were transiently expressed in monkey COS cells under the transcriptional control of the simian virus 40 early promoter. The proteins were labelled with [35S]methionine during a 16 h incubation and proteins secreted by the cells during this period were separated by polyacrylamide gel electrophoresis and subsequently visualized by fluorography. Under the conditions used, the IFNs represented 5 to 10% of the total amount of secreted proteins. All genes were found to encode biologically active IFN subspecies, including alpha 4 which has a deletion of five amino acids. When the specific activities of the proteins were compared, it appeared that the specific antiviral activity of alpha 4 on mouse cells was three- to sixfold higher than the activities of the other natural IFN subspecies. The specific activities of the hybrid proteins were similar to those of the natural proteins, except for the alpha 2 alpha 4 hybrid which had a higher specific activity than the original proteins. The ability of the natural and hybrid subspecies to protect hamster cells against viral infection was determined using MuIFN-alpha 1 as a standard. Large differences in activity were found, with alpha 6 as the most and alpha 4 as the least active subspecies.

Animals↗

PubMed1985

Expression of cellular oncogenes in human prostatic carcinoma cell lines.

Prostatic cancer is one of the most frequent forms of malignancy in Western countries. Initially, growth of the majority of prostate tumors can be manipulated by endocrine therapy. However, ultimately androgen independent tumors continue to grow. We studied the expression of oncogenes in four different human prostatic carcinoma cell lines: PC 3, PC 133, PC 135, which are androgen independent, and the hormone dependent PC 82 cell line. Large amounts of Ha-ras and myc mRNA were present in all cell lines. Transcripts of fes, int-1 and abl were never detected. In some of the cell lines the presence of N-ras, Ki-ras, myb, fos, fms and sis mRNA was observed. The PC 82 cell line showed, in addition to myc and Ha-ras high levels of fos expression. Inhibition of tumor cell proliferation by withdrawal of androgen was accompanied by a tenfold reduction of the fos mRNA level and a twofold reduction of Ha-ras transcripts. In contrast, the expression of myc was not changed.

Animals↗

PubMed1985

The effect of murine interferon-alpha/beta on an established Rauscher murine leukemia virus-induced erythroleukemia in BALB/c mice.

Rauscher murine leukemia virus (R-MuLV) induces a rapidly developing erythroleukemia in BALB/c mice. Previously, we have shown that mouse interferon-alpha/beta (Mu IFN-alpha/beta) applied shortly after virus inoculation efficiently inhibits the leukemic process (Hekman et al., 1981). Here we describe the effect of Mu IFN-alpha/beta on an established leukemia. Varying doses of Mu IFN-alpha/beta were injected over 3 days, starting 8 to 12 days after virus inoculation. The effect of Mu IFN-alpha/beta on the leukemic process was monitored by measuring the spleen weight, reverse transcriptase activity in the serum and, in selected experiments, by microscopic examination of sections of the spleen using standard histological and immunological staining techniques. Depending on the spleen weight at the start of its application (maximal about 450 mg), Mu IFN-alpha/beta caused a dramatic reduction in the number of virus-infected erythroleukemic cells in the spleen. Also, R-MuLV disappeared from the serum within 3 days. If Mu IFN-alpha/beta was injected into R-MuLV-infected mice with an already 10-fold enlarged spleen, it could only stop further development of leukemia. Results obtained with crude Mu IFN-alpha/beta preparations were confirmed with absolutely pure Mu IFN-beta.

Animals↗