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H Klocker

Publications and source records attributed to H Klocker.

At least 109 records · Page 6Linked to original sources
PubMed1995

Tumor-infiltrating T lymphocytes from renal-cell carcinoma express B7-1 (CD80): T-cell expansion by T-T cell co-stimulation.

B7-1 (CD80) provides co-stimulation for T-cell activation by interacting with CD28 or CTLA4. Here we demonstrate the expression of B7-1 in freshly isolated and cultured lymphocytes from renal-cell carcinoma. In fresh preparations of lymphocytes infiltrating renal-cell-carcinoma tissue, B7-1 mRNA could readily be detected by reverse transcription PCR, and 2-color flow-cytometry analysis revealed substantial B7-1 expression on T cells from these isolates. As expected, tumor-derived T cells also expressed CD28, the B7 receptor. While B7-1 expression of tumor-derived T cells was maintained during culture in interleukin-2-supplemented medium, CD28 expression was further enhanced. We also show that B7-1 is functionally involved in T-cell expansion: anti-B7-1 MAb inhibited the PHA-induced proliferation of tumor-derived B7-1+ T cells (35%) in the absence of exogenous antigen-presenting cells, indicating that B7-1 mediates T-T cell co-stimulation (self-co-stimulation). Our data demonstrate that T cells infiltrating renal-cell carcinoma express B7-1, and that mutual co-stimulation via the B7-1/CD28 pathway contributes to the interleukin-2-driven expansion of tumor-derived T cells in vitro. The frequency of B7-1+ T cells in tumor lesions and the level of B7-1 on these cells may determine the time course of T-cell expansion in vivo. Self-co-stimulation, however, might also represent one mechanism leading to the state of suppression or anergy characteristic of tumor-infiltrating lymphocytes.

B7-1 Antigen↗
PubMed1995

Distant metastases from prostatic carcinoma express androgen receptor protein.

Nearly all primary prostatic carcinomas have been found to express the androgen receptor (AR) protein, which is the intracellular mediator of androgen action. To gain a better insight into the mechanisms of androgen independence of advanced prostatic carcinoma, it is important to know whether the AR is also present in metastases of androgen-independent tumors. We have assessed the status of the AR and the prostate-specific antigen in 22 metastases of 18 patients with progressive prostate cancer. In 18 cases, the metastases were localized in bone, in 3 cases in the epidural space, and in 1 case in the periosteum. All but one patient had received some kind of endocrine treatment for prostatic carcinoma. Paraffin-embedded tissue sections were stained for the AR following a streptavidinbiotin-peroxidase protocol with the polyclonal antibody PG-21, which is directed against amino acids 1 through 21 of the rat and the human AR. The percentage of AR-positive cells was evaluated on the basis of an arbitrary 4-point scale. All 22 tumor metastases displayed AR positivity. One AR-positive metastatic lesion did not stain for prostate-specific antigen, but in all other metastases, this protein was detected by means of immunohistochemistry. The present study provides evidence that, unlike androgen-independent prostatic carcinoma cell lines, distant prostatic carcinoma metastases do express the AR. These findings indicate that the AR may be involved in the progression of prostate cancer.

Bone Neoplasms↗
PubMed1995

A single amino acid exchange abolishes dimerization of the androgen receptor and causes Reifenstein syndrome.

A single exchange of an alanine to a threonine at amino acid position 596 in the androgen receptor has been identified as an inheritable trait in patients with Reifenstein syndrome. This exchange is a result of a germ line mutation in the genomic DNA sequences that make up the D-loop of the receptor. The D-loop and sequences in the hormone binding domain together provide the interacting surfaces for receptor dimer formation and subsequent binding to DNA. Here we show that the single amino acid exchange abolishes dimerization of the receptor. With this finding we demonstrate that the destruction of dimerization of the androgen receptor is one of the causes of Reifenstein syndrome.

Alanine↗
PubMed1995

Activation of the androgen receptor by polypeptide growth factors and cellular regulators.

The polypeptide growth factors insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha); second-messenger cyclic adenosine monophosphate (cAMP): protein kinase activators; and neurotransmitters were found to activate the estrogen (ER), progesterone (PR), and glucocorticoid receptor (GR) either in the absence of their natural ligands or synergistically with the respective hormone. There is now evidence of coupling of signaling pathways involving the androgen receptor (AR). Three polypeptide growth factor, IGF-I, keratinocyte growth factor (KGF), and EGF, stimulated AR-mediated reporter-gene transcription in the absence of androgen in DU-145 cells, which were cotransfected with the reporter gene and an AR expression vector. IGF-I effects were observed irrespective of the promoter driving the reporter gene. This growth factor increased the prostate-specific antigen (PSA) level in LNCaP cells, which contain endogenous AR. In CV-1 cells, which transiently express the AR, second-messenger cAMP potentiated effects of testosterone in stimulation of AR-mediated reporter-gene activity. Inhibition of androgen-stimulated chloramphenicol acetyltransferase (CAT) activity in the LNCaP cell line was achieved with retinoic acid. Stimulation and inhibition of prostatic carcinoma cell growth by polypeptide growth factors and cellular regulators may depend on the presence of the AR in an androgen-depleted environment.

Animals↗
PubMed1995

Comparison of different prostate-specific antigen cutpoints for early detection of prostate cancer: results of a large screening study.

OBJECTIVES: This study was designed to compare the usefulness of the normal prostate-specific antigen (PSA) level and the age-referenced PSA level in a large screening study for early detection of prostate cancer. METHODS: A total of 21,078 subjects (aged 45 to 75 years) were participants in a 1-year prostate cancer screening project with PSA as the initial test. Of the volunteers, 1618 (8%) showed an elevated PSA level according to age-specific reference ranges, and using the normal PSA cutoff point (4.0 ng/mL), 1872 (9%) had elevated PSA levels between 4.0 and 6.5 ng/mL. RESULTS: Biopsies in both groups were performed if the PSA level was elevated. We evaluated the effect on biopsy rate and cancer detection. A PSA cutoff point of 2.5 ng/mL in men 45 to 49 years old and a PSA cutoff point of 3.5 ng/mL in men 50 to 59 years old with normal digital rectal examination findings resulted in an 8% increase in the number of biopsies (66 of 778) and an 8% increase in organ-confined cancer detection. An increasing cutoff of 4.5 ng/mL in men 60 to 69 years old and 6.5 ng/mL in men 70 to 75 years old resulted in 21% fewer biopsies (205 of 983) and would have missed 4% of organ-confined tumors (8 of 220). CONCLUSIONS: We conclude that the use of PSA age-specific reference ranges increases the detection of clinically important and organ-confined cancers in young men and decreases the number of biopsies in older men.

Aged↗
PubMed1995

Androgen receptor alterations in patients with disturbances in male sexual development and in prostatic carcinoma.

The androgen receptor, a ligand-activated nuclear transcription factor belonging to the large superfamily of nuclear receptors, mediates the intracellular action of androgens. It plays a central role in male sexual development and in prostatic carcinoma as a target of endocrine therapy. We have looked for androgen receptor mutations as a cause of male sexual ambiguity and as a possible reason for failure of androgen ablation therapy on prostatic carcinoma. In 5 patients of 2 families with perineoscrotal hypospadia and undescended testes, we have identified a mutation ala596-->thr in the DNA-binding domain of the androgen receptor. This mutation interferes with DNA binding of the receptor. Reactivation of this mutant receptor by binding of an antibody or by interaction with other proteins and by exchange of the amino acid thr602-->ala indicates that the dimerization step is affected. A point mutation ser703-->gly was detected in a newborn male child with perineoscrotal hypospadias. This mutation decreased receptor-hormone affinity. As a consequence its transactivation activity was dependent on the androgen concentration. Although the molecular mechanisms of these two mutations are completely different, both resulted in partial androgen insensitivity and interfered with virilization in the affected patients. A different kind of mutation was present in a tumor specimen derived from an advanced therapy-resistant prostatic carcinoma. This point mutation resulted in exchange of valine-->methionine at amino acid position 715 in the receptor protein. In contrast to the former two mutations this receptor showed a gain in function.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

In vivo LDL receptor and HMG-CoA reductase regulation in human lymphocytes and its alterations during aging.

The LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play primary roles in the regulation of cellular cholesterol metabolism. To investigate the transcriptional regulation of lipid metabolism under physiological conditions ex vivo and its alterations during aging, we analyzed both the activity and mRNA concentration of the LDL receptor and HMG-CoA reductase in freshly isolated lymphocytes from healthy young and elderly donors. Data from fluorescent reverse transcriptase-polymerase chain reaction indicated that not only plasma LDL but also plasma HDL downregulates lymphocyte LDL receptor mRNA. Downregulation by HDL was three times more effective than that by LDL and presumably involved specific HDL binding sites. There was coordinate regulation of HMG-CoA reductase mRNA with LDL receptor mRNA that was independent of plasma lipoprotein concentrations. Despite elevated plasma concentrations of LDL, lymphocytes from elderly donors paradoxically expressed increased levels of the LDL receptor (P = .030) and HMG-CoA reductase mRNA (P = .062). The age-related dysregulation of the LDL receptor was predominantly due to impaired downregulation by plasma LDL rather than by HDL. Thus, not only LDL but also HDL and age significantly influences the transcriptional regulation of the LDL receptor in extrahepatic cells in vivo.

Adult↗
PubMed1994

Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor.

Aberrant activation of the androgen receptor (AR) may be one of the mechanisms which contribute to progression of prostatic carcinoma to an androgen-independent stage. We investigated effects of growth factors on stimulation of the AR-mediated gene transcription in human prostatic tumor cell lines. DU-145 cells, which do not contain endogenous AR, were cotransfected with an androgen-inducible chloramphenicol acetyltransferase (CAT) reporter gene and an AR expression vector. The reporter gene (CAT) was driven either by artificial promoters consisting of one or two androgen-responsive elements in front of a TATA box or by the promoter of the prostate-specific antigen (PSA) gene, a naturally occurring androgen-inducible promoter. Insulin-like growth factor-I (IGF-I), at a concentration of 50 ng/ml, stimulated AR-mediated reporter gene transcription to the same extent as the synthetic androgen methyltrienolone. This growth factor was effective irrespective of the nature of the androgen-inducible promoter. Keratinocyte growth factor (KGF) and epidermal growth factor (EGF), at concentrations of 50 ng/ml, activated CAT reporter gene transcription only in experiments in which the artificial promoter with two androgen-responsive elements was used. Insulin-like growth factor-II and basic fibroblast growth factor displayed no effect on AR-mediated gene transcription. None of the growth factors stimulated reporter gene activity in control experiments when added to cells cotransfected with the CAT gene and an empty expression vector. AR activation by IGF-I, KGF, and EGF was completely inhibited by the pure AR antagonist casodex, showing that these effects are AR mediated. Activation of endogenous AR by growth factors was studied in the LNCaP cell line by determination of PSA secretion. IGF-I, at a concentration of 50 ng/ml, increased the PSA level in the supernatant of this cell line 5-fold. Again, the IGF-I effect on PSA secretion was blocked by casodex. Our results provide evidence that IGF-I, KGF, and EGF directly activate the AR in the absence of androgens, which means that the androgen-signaling chain may be activated by growth factors in an androgen-depleted environment. These findings may have implications for endocrine therapy for metastatic prostatic carcinoma.

Androgen Antagonists↗
PubMed1994

Androgen receptor alterations in prostatic carcinoma.

Intracellular action of androgens is mediated by the androgen receptor (AR), which is a key element of the androgen signal transduction cascade and a target of endocrine therapy for prostatic carcinoma. Therefore, the qualitative and quantitative alterations of AR expression in prostatic carcinomas and their possible implications for tumor progression and treatment are of great interest. Findings in prostatic tumor cell lines of rat and human origin suggest a reduction of AR protein expression accompanied by an increase in tumor malignancy. However, immunohistochemical studies and binding assays demonstrated presence of ARs in all histological types of prostatic tumors, in therapy-responsive as well as in therapy-unresponsive ones. AR content of prostatic tumor specimens did not correlate with outcome of endocrine therapy of advanced prostatic carcinoma in these studies. Solely the degree of heterogeneity of AR expression may be useful as an indicator of responsiveness to therapy. AR mutations have been detected in the LNCaP cell line and in three primary prostatic tumor specimens. Three of them are point mutations in the hormone-binding domain of the AR, the fourth mutation is a CAG-microsatellite depression in the N-terminus. Evidence coming from studies on AR in prostatic cancer highlights the possibility that AR structural alterations may have significance in tumor progression.

Animals↗
PubMed1994

Androgen signal transduction and prostatic carcinoma.

In the prostate, androgen action affects the growth of the gland, its morphology, and regulation of protein expression. Endocrine therapy of non-organ-confined tumors is based on the androgen dependence of the vast majority of prostatic carcinomas. Although initial response rates are high, this therapy is only temporarily effective. Critical molecular changes ultimately resulting in androgen independence of tumor cells are unknown at this time. The androgen signal-transduction cascade and its central element, the androgen receptor (AR), are possible targets for such changes. Immunohistological analysis using anti-AR antibodies has revealed the presence of AR in a vast majority of therapy-responsive as well as therapy-unresponsive prostatic carcinomas, indicating that loss of AR expression is not the reason for androgen independence. On the other hand, molecular-biology studies have revealed qualitative and quantitative impairment of AR expression in prostatic tumor cell lines that represent very late stages of prostatic carcinoma development. Mutant ARs were detected in the prostatic tumor cell line LNCaP and in two specimens from primary prostatic tumors. The LNCaP mutant AR as well as mutant AR715met, one of the mutant receptors detected in tumor tissue, show a gain of function as compared with the wild-type receptor. In addition to androgens, the natural activators of the AR, the LNCaP receptor is activated also by progestagenic and estrogenic steroids and by the nonsteroidal antiandrogen flutamide. AR715met is activated by adrenal androgens and progesterone in addition to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1994

Cytogenetics of prostate cancer. Consensus Conference on Diagnosis and Prognostic Parameters in Localized Prostate Cancer. Stockholm, Sweden, May 12-13, 1993.

This report summarizes the current knowledge with respect to genetic changes associated with the development of prostate cancer. The relation between the occurrence of these changes and the stage of the disease is by far not clear yet, albeit that some tendencies become more or less evident. In Fig. 2 these are summarized. Whereas changes on 7q and loss or gain of the X chromosome are not consistently found by either RFLP or ISH analysis, other changes are (8p-, 10p-, 10q, 16q-, and 18q-). Clearly the picture is far from complete and even more the relevant genes on these chromosomes are not defined. E-cadherin was considered a good candidate and indeed the value as progression marker is great. However, the studies so far indicate that E-cadherin does not behave as a classical type I suppressor gene and the relation with 16q loss remains to be established. The data available to date regarding chromosomal changes in prostate cancer are limited. Many studies have to be pursued to identify the consistent changes, and moreover, to map the relevant loci harbouring the genes that are implicated in the development of this disease. This knowledge will be critical in the design of appropriate diagnostic methods and possibly clues towards therapy.

Cadherins↗
PubMed1993

DNA sequence of the androgen receptor in prostatic tumor cell lines and tissue specimens assessed by means of the polymerase chain reaction.

Essentially all prostatic carcinomas relapse to an androgen-independent stage during androgen ablation therapy. The underlying genetic changes are still unclear. Such changes are suspected to affect the androgen-signalling pathway as well as growth promoting and inhibiting factors. This study was undertaken to test for structural changes of the androgen receptor in prostatic tumor cell lines and primary tumors. Complementary DNA (cDNA) fragments of the androgen receptor (AR) were isolated from the cell lines LNCaP, PC-3, and DU 145, ten tissue specimens obtained by radical prostatectomy, and five fine-needle biopsies by means of the polymerase chain reaction (PCR) technique. Fragments encoding the hormone- and DNA-binding domains were analyzed by DNA sequencing. The PCR technique is highly sensitive and especially recommended for the analysis of small tissue samples, such as those obtained by fine-needle aspiration. No alterations were detected in the tissue specimens and the five fine-needle aspirates. In the three tumor cell lines that represent late stages of prostatic tumor, different findings were obtained. The androgen-independent DU 145 cells did not express androgen receptors, whereas the PC-3 cells, which are also androgen-independent, expressed very low levels of normal AR. In contrast to this, the androgen-dependent LNCaP cells expressed high levels of structurally abnormal androgen receptors. These results suggest that androgen receptor mutations are probably uncommon molecular events in the early stages of prostatic cancer. Qualitative and quantitative changes, however, seem to occur in advanced prostatic cancer.

Base Sequence↗
PubMed1993

Characterization of two point mutations in the androgen receptor gene of patients with perineoscrotal hypospadia.

Perineoscrotal hypospadia is a major sign of sexual ambiguity due to inadequate androgen action in genetic and gonadal males. In patients showing these symptoms we have detected two androgen receptor gene mutations. In consequence we characterized the properties of the mutant receptors with respect to hormone-binding, transactivation and DNA-binding. An amino acid substitution alanine-596-->threonine in the D-box of the androgen receptor was detected in 3 and 2 brothers, respectively. This mutant receptor, AR-thr596, bound ligand in a normal fashion. It showed a promoter-dependent defect of transactivation and was unable to induce transcription of a promoter containing one androgen responsive element but showed almost wild-type transactivation of a promoter containing two closely spaced androgen-responsive elements. The complex promoter of the human prostate-specific antigen gene was induced with intermediate efficiency. In electrophoretic mobility shift assays AR-thr596 was unable to form a complex with oligonucleotides containing 1 or 2 androgen responsive elements, however its DNA-binding activity was restored by an anti-androgen receptor antibody in the presence of ligand. A point mutation which caused substitution of serine-703 in the hormone-binding domain with glycine was detected in a new-born male with ambiguous genitalia. This mutant receptor, AR-gly703, showed a reduced ligand affinity. The total amount of specific androgen binding sites in genital fibroblasts of the patient was reduced. Transactivation activity of AR-gly703 was dependent on hormone concentration. It was inactive at low levels of androgens but was fully activated in the presence of high androgen concentrations. The nature of the promoter had no effect on transactivation properties of this mutant androgen receptor. Its DNA-binding activity in gel shift experiments was normal.

Base Sequence↗
PubMed1993

A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors.

Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors. Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.

Alanine↗
PubMed1993

Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone.

Structural changes of the androgen receptor (AR) may contribute to the development of resistance to endocrine therapy in prostatic carcinoma. We have isolated AR cDNA fragments from seven tumor specimens derived from patients with advanced metastatic prostatic tumors. In one specimen obtained from a patient who failed to respond to endocrine and cytotoxic therapy we have detected a point mutation in the hormone-binding domain of the receptor. This AR mutation is a guanine-to-adenine transition at nucleotide 2671 that leads to substitution of methionine for the wild type valine at position 715. It is a somatic mutation because it was not present in the AR genomic DNA fragments isolated from prostatic and testicular tissues of the same patient. The mutant AR was recreated in an expression vector and transiently expressed in COS-7 and CV-1 cells. Hormone-binding assays revealed that the mutant receptor does not differ from the wild type receptor in its ability to bind androgen. The dissociation constant for the synthetic androgen mibolerone was 3 nM for both receptors. There was also no significant difference in binding of other steroids and nonsteroidal antiandrogens as revealed by competition binding assays. However, transfection experiments to determine the trans-activation potential of the mutant receptor produced differences in the action of this receptor compared to the wild type receptor. Dihydrotestosterone and the synthetic androgens methyltrienolone (R1881) and mibolerone were equally proficient in conferring trans-activation activity to both the mutant and wild type receptors. Adrenal androgens such as dehydroepiandrosterone and androstenedione, as well as progesterone mediated a higher trans-activation through the mutant than through the wild type receptor. These data demonstrate that the exchange of a single valine into methionine at position 715 in the AR promoters trans-activation not only by testicular but also by adrenal androgens and progesterone. This pattern of ligand-dependent trans-activation may have significance in the process controlling the progression of prostatic carcinoma.

Adrenal Glands↗
PubMed1993

[The androgen receptor. New aspects in intersexuality patients].

Androgen resistance is thought to vary phenotypically from a normal female to an infertile male. At the functional level a variety of defects have been detected in cultured skin fibroblasts in these patients. Since the isolation and cloning of the 5 alpha-reductase and the androgen receptor gene, several new disorders have been characterized at the molecular level, including major gene rearrangements, deletions and defects in RNA splicing, and single amino acid substitutions. These studies have provided insight into the spectrum of mechanisms that can alter receptor function and serve to increase our understanding of the etiology in patients with androgen resistance.

Androgens↗
PubMed1992

Point mutation in the DNA binding domain of the androgen receptor in two families with Reifenstein syndrome.

Inadequate androgen action in genetic and gonadal males causes an intersex phenotype. We have analyzed the androgen receptor (AR) gene in male pseudohermaphrodites with normal specific binding of dihydrotestosterone in their genital skin fibroblasts. In five patients with Reifenstein syndrome we have detected a point mutation in the DNA binding domain. They are from two unrelated families and presented with perineoscrotal hypospadias and undescended testes. After puberty they showed small testes, no palpable prostate, micropenis, azoospermia, and gynecomastia. The mutation was discovered when cDNA fragments from three brothers were sequenced. For rapid detection of the mutation in heterozygous and hemizygous carriers, allele-specific PCRs and restriction-analysis techniques have been developed. Relatives of the patients, a group of normal blood donors, and other patients were screened with these methods. Among 41 intersex patients with incomplete virilization, another two brothers presenting with this mutation were identified. The mutation is a guanine-to-adenine transition at nucleotide 2314, which changes the alanine codon (GCC) immediately after the first cysteine of the second zinc finger motif of the AR into a threonine codon (ACC). The mutation was recreated in an AR expression vector, and wild-type as well as mutant ARs were expressed in COS-7 cells. Cotransfection experiments were made using a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene. The ability of the mutant receptor to stimulate transcription of the reporter gene was reduced by about two-thirds, as compared with the wild-type receptor.

Alleles↗