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G Jenster

Publications and source records attributed to G Jenster.

30 records · Page 2Linked to original sources
PubMed1993

Nuclear import of the human androgen receptor.

Nuclear import of the human androgen receptor was investigated by immunocytochemical analysis of androgen receptor deletion and substitution mutants, which were transiently expressed in COS-1 cells. The signal responsible for nuclear import is encoded by amino-acid residues 608-625 and is functionally similar to the bipartite nucleoplasmin nuclear-localization signal. Although the subcellular distribution of androgen receptors mutated in the DNA-binding domain was unchanged compared with the wild-type androgen receptor, in the presence of ligand these mutations resulted in part of the receptor population forming clusters. Depending on the presence or absence of the bipartite nuclear localization signal, clusters were formed in the nucleus or in the cytoplasm, respectively. Expression of the wild-type androgen receptor in different cell lines revealed a cell-line-specific subcellular distribution of the unliganded receptor. The androgen receptor was predominantly nuclear when expressed in HeLa cells, whereas mainly cytoplasmic staining was observed when it was expressed in COS-1 cells. In the presence of hormone, the androgen receptor was located in the nucleus, independent of the cell line that was expressing the receptor. Anti-androgens and various steroid hormones induced the nuclear localization of the wild-type androgen receptor in a dose-dependent way, without activating transcription of an androgen-regulated reporter gene. This indicates that the inability of the tested compounds to activate transcription is not due to inhibited nuclear import.

Amino Acid Sequence↗
PubMed1992

The human androgen receptor: structure/function relationship in normal and pathological situations.

Discrete functions have been attributed to precise regions of the human androgen receptor (hAR) by expression of deletion mutants in COS and HeLa cells. A large C-terminal domain constitutes the hormone-binding region and a central basis, cysteine-rich domain is responsible for DNA binding. In addition, separate domains responsible for transactivation and nuclear translocation have been identified. In LNCaP cells (a prostate tumor cell line) the hAR is a heterogeneous protein which is synthesized as a single 110 kDa protein, but becomes rapidly phosphorylated to a 112 kDa protein. Metabolic labeling experiments using radioactive orthophosphate also indicated that the hAR is a phosphoprotein. Structural analysis of the AR gene in LNCaP cells and in 46, XY-individuals displaying androgen insensitivity (AIS) has revealed several different point mutations. In LNCaP cells the mutation affects both binding specificity and transactivation by different steroids. In a person with complete AIS a point mutation was identified in the splice donor site of intron 4, which prevents normal splicing and activates a cryptic splice donor site in exon 4. The consequence is a functionally inactive AR protein due to an in-frame deletion in the steroid-binding domain. In two unrelated individuals with complete AIS, two different single nucleotide alterations in codon 686 (Asp) were found. Both mutations resulted in functionally inactive ARs due to rapidly dissociating hormone-AR complexes. It is concluded that the hAR is a heterogeneous phosphoprotein in which functional errors have a dramatic impact on phenotype and fertility of 46, XY-individuals.

Amino Acid Sequence↗
PubMed1992

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens.

The human prostate tumor cell line LNCaP contains an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induce reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.

Binding Sites↗
PubMed1992

Functional domains of the human androgen receptor.

A series of human androgen receptor (AR) deletion mutants was constructed to study the relationship between various structural domains and their different functions in the AR protein. Immunoblots of wild type AR and AR mutants expressed in COS-1 cells, revealed a doublet appearance of all AR proteins. One exception was an AR mutant lacking amino acid residues 51-211 that migrated as a single protein band, possibly due to altered post-translational modification. The steroid binding domain was found to be encoded by approx. 250 amino acid residues in the C-terminal end. Deletions and truncations in this part of the receptor abolished hormone binding. The N-terminal domain was observed to be essential for transcriptional activation. AR mutants lacking large parts of this domain were transcriptionally inactive. Deletion of the hormone binding domain yielded a constitutively active AR protein, indicating that in the absence of hormone this domain displays an inhibitory function. In the absence of ligand the wild type AR expressed in COS-1 cells was distributed over nucleus and cytoplasm. The addition of hormone directed all androgen receptors to the nucleus. In contrast, an AR mutant lacking part of the DNA binding domain and part of the hinge region, was almost exclusively cytoplasmic in the absence of hormone. This mutant lacks a conserved region, homologous to the SV40 large T- and nucleoplasmin nuclear localization signal. Hormone induced transfer of this AR mutant to the nucleus, indicating the presence of a second, hormone dependent nuclear targeting mechanism.

Amino Acid Sequence↗
PubMed1991

Human bcr-abl gene has a lethal effect on embryogenesis.

The chimaeric bcr-abl oncogene is thought to have a crucial role in the development or maintenance of chronic myelogenous leukaemia. To study this oncogene in a more direct way, the bcr-abl gene encoding the P210 protein under control of the bcr gene promoter was introduced into fertilized one-cell embryos, which were then re-implanted into foster mothers. Our data, obtained after several experiments, demonstrate that no live transgenic progeny could be obtained using this bcr-abl construct. The bcr gene is expressed in the course of embryogenesis and the bcr-abl gene product appears to have a pleiotropic lethal effect during this period of development. In concordance, several gross abnormalities were observed while no evidence of neoplastic formation was found. These results suggest that the bcr-abl encoded protein severely affects the process of normal embryogenesis.

Animals↗
PubMed1991

Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization.

A series of human androgen receptor (AR) deletion mutants was constructed to study the relationship between the structural domains and their different functions in the AR protein. Human AR mutants were expressed in COS-1 and HeLa cells to investigate hormone binding, transcriptional activation, and subcellular localization. The wild-type human AR (AR 1-910) was expressed as a 110- to 112-kDa doublet, as revealed on immunoblots. All mutant AR proteins also migrated as doublets, except for one. This AR has a deletion from amino acid residues 51-211 and migrated as a single protein band, possibly due to altered posttranslational modification. The AR steroid-binding domain is encoded by approximately 250 amino acid residues in the C-terminal end. Deletions in this domain as well as truncation of the last 12 C-terminal amino acid residues abolished hormone binding. Cotransfection studies in HeLa cells showed that transcriptional activation of an androgen-regulated reporter gene construct was induced by the wild-type human AR. Mutational analysis revealed two regions in the N-terminal part, encoded by amino acid residues 51-211 and 244-360, to be essential for this transcriptional activation. Deletion of the hormone-binding domain yielded a constitutively active AR protein, indicating that in the absence of hormone this domain displays an inhibitory function. In the presence of its ligand, the wild-type AR was located in the cell nucleus. In the absence of androgens the receptor was mainly nuclear, but cytoplasmic localization was observed as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Substitution of aspartic acid-686 by histidine or asparagine in the human androgen receptor leads to a functionally inactive protein with altered hormone-binding characteristics.

We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution (with 15-20% of wild-type androgen-binding capacity), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (with normal androgen-binding capacity, but a rapidly dissociating ligand-receptor complex). The mutations eliminate a Hinfl restriction site. Screening for the loss of the Hinfl site in both families with the Asp----Asn mutation resulted in the recognition of heterozygous carriers in successive generations of each. Both mutant androgen receptors were generated in vitro and transiently expressed in COS and HeLa cells. The receptor proteins produced had the same altered binding characteristics as those measured in fibroblasts from the affected subjects. R1881-activated transcription of a GRE-tk-CAT reporter gene construct was strongly diminished by both mutant receptors and was only partially restored using a 100-fold higher concentration of ligand compared with wild-type receptor. Thus, aspartic acid-686 appears essential for normal androgen receptor function. Substitution of this amino acid residue, by either histidine or asparagine, results in androgen insensitivity and lack of androgen-dependent male sexual differentiation.

Adult↗
PubMed1990

A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens.

LNCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We have discovered in the LNCaP androgen receptor a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or Hela cells. Androgens, progestagens, estrogens and anti-androgens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene construct (GRE-tk-CAT). The mutation therefore influences both binding and the induction of gene expression by different steroids and antisteroids.

Binding Sites↗
PubMed1990

Acute leukaemia in bcr/abl transgenic mice.

The Philadelphia chromosome, widely implicated in human leukaemia, is the result of a reciprocal translocation t(9;22) (q34;q11) in which the abl oncogene located at 9q34 is translocated to chromosome 22q11, where it is fused head-to-tail with 5' exons of the bcr gene. In acute lymphoblastic leukaemia, some patients have a breakpoint within the major breakpoint cluster region of the bcr gene, whereas others have the break within its first intron. This second type of translocation results in the transcription of a 7.0-kilobase chimaeric bcr/abl messenger RNA translated into a bcr/abl fusion protein, p190, which has an abnormal tyrosine kinase activity and is strongly autophosphorylated in vitro. We have generated mice transgenic for a bcr/abl p190 DNA construct and find that progeny are either moribund with, or die of acute leukaemia (myeloid or lymphoid) 10-58 days after birth. This finding is evidence for a causal relationship between the Philadelphia chromosome and human leukaemia.

Animals↗
PubMed1990

Mutation of the human neu protein facilitates down-modulation by monoclonal antibodies.

Amplification and overexpression of the neu gene have been found in several human adenocarcinomas. We have obtained monoclonal antibodies to the human neu protein by immunizing a Balb/c mouse with a Balb/c cell line expressing the human neu gene by transfection. The monoclonal antibodies reacted with neu protein on intact cells by immunofluorescence and immunoprecipitated neu in metabolically labeled cells, also in the presence of tunicamycin. We tested possible down-modulating effects of these monoclonal antibodies on SKBR-3 mammary tumor cells, which express high levels of wild-type human neu protein. We also used NIH3T3 cells transfected with either a normal or a mutated human neu gene, encoding a protein with a valine to glutamic acid substitution in the transmembrane domain. Down-modulation of the normal cell-surface neu protein was inefficient. In contrast, the antibodies induced 50-65% down-modulation in NIH3T3 cells expressing the mutated human neu protein and could inhibit these cells to form colonies in soft agar. We propose that these differences are due to changed aggregation properties of the point-mutated protein.

Animals↗
PubMedDate not supplied

[Gene expression profiles of protein kinases and phosphatases obtained by hybridization with cDNA arrays: molecular portrait of human prostate carcinoma].

Hybridization with cDNA arrays was used to obtain expression profiles of 263 protein-tyrosine kinase (PTK), protein-tyrosine phosphatase (PTP), dual-specific phosphatase (DuSP), and other genes for the normal prostate tissue, primary prostate carcinomas (PC) of 84 patients, 7 xenografts, and 5 carcinoma cell lines. Analysis of 96 profiles revealed eight clusters of genes coexpressed in PC (coefficient of correlation r > 0.7). According to the known functions of their genes, the clusters were designated as proliferating-cell (CDC42, TOP2A, FGFR3, MYC, etc.), neoangiogenesis and blood-cell (LCK, VAV1, KDR, VEGF, MMP9, SYK, PTPRS, and FLT4), invasion-1 and invasion-2 (ADAM17, TRPM2, DUSP6, VIM, CAV1, CAV2, JAK1, PTPNS1, FYN, and PDGFB), HER2, and PSA/PSM/HER3. Basing on expression profiles of 66 genes, a molecular classification of PC was constructed and allowed discrimination between PC and cell lines or xenografts at 98.9% probability. The results suggested that, along with PSA, PSM (FOLH1), kallikrein-2, and a-2-macroglobulin, cell signaling genes EGFR, HER2, HER3, TOP2, KRT8, KRT18, VEGF, CD44, VIM, CAV1, and CAV2 may serve as diagnostic and prognostic markers in PC. The HER2, VEGF, and CD44 genes and the MMP and ADAM families were assumed to be promising targets for inhibitors of PC cell proliferation and metastasis.

Gene Expression Regulation, Neoplastic↗