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R Buettner

Publications and source records attributed to R Buettner.

89 records · Page 5Linked to original sources
PubMed1994

Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes.

We have cloned a protein from bovine brain, brain-derived voltage-dependent anion channel 1 (BR1-VDAC), that is identical to a recently sequenced plasmalemmal-bound porin from human lymphocytes. mRNA hybridization indicates that BR1-VDAC is widely distributed throughout nervous and nonnervous tissues. In situ localization substantiated that the BR1-VDAC is associated with the plasmalemma of astrocytes. A monoclonal antibody that recognizes the N terminus of the BR1-VDAC protein completely blocks an astrocytic high-conductance anion channel that has electrophysiological similarities with the mitochondrial VDAC. Since the high-conductance anion channel in astrocytes has been shown to respond to hypoosmotic solutions, its molecular identification provides the basis for a better understanding of volume regulation in brain tissue.

Animals↗
PubMed1994

[Molecular cloning of a new AP-2 transcription factor, AP-2beta, and its function in cell differentiation].

Transcription factor AP-2 has been previously shown to play an important function in embryonal development and cell differentiation. We have investigated the possibility that AP-2 function in embryonic development is exerted by a multigene family of AP-2 related transcription factors. Here we describe the molecular cloning of such an AP-2 related gene, AP-2 beta, and prove that it encodes for a functional transcription factor. In situ hybridizations of murine embryo sections revealed a temporally restricted and tissue-specific expression pattern that indicates a function of AP-2 beta in the development of the midbrain in the differentiation of sensory neurons for taste, olfaction and palpation.

Animals↗
PubMed1993

Cloning and characterization of a fourth human somatostatin receptor.

We have isolated a gene coding for a fourth human somatostatin (somatotropin release-inhibiting factor) receptor. This additional somatostatin receptor (hSSTR4) is specifically expressed in human fetal and adult brain and lung tissue. The deduced amino acid sequence of the receptor displays both sequence and structural homology to three cloned somatostatin receptors as well as to other members of the family of GTP-binding-protein-coupled seven-helix transmembrane-spanning receptors. Pharmacological characterization of the expressed receptor reveals specific, high-affinity binding of somatostatin 14 and somatostatin 28. Surprisingly, several well-characterized synthetic somatostatin analogs fail to exhibit high-affinity binding to hSSTR4, indicating the existence of pharmacologically different receptor subtypes. Our data suggest that the diverse biological effects exerted by somatostatin are mediated by a family of receptors with discrete patterns of expression and different pharmacological properties.

Amino Acid Sequence↗
PubMed1993

An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.

AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spanning the entire AP-2 gene proves that AP-2A and AP-2B transcripts are alternatively spliced from the same gene. Both transient and stable transfection experiments show that AP-2B inhibits AP-2 transactivator function, as measured by an AP-2-responsive chloramphenicol acetyltransferase reporter plasmid. Furthermore, constitutive AP-2B expression in PA-1 cells causes a retinoic acid-resistant phenotype, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, in a fashion similar to transformation of these cells by oncogenes. To determine the mechanism by which AP-2B exerts its inhibitory function, we purified bacterially expressed AP-2A and AP-2B proteins. While bacterial AP-2B does not bind an AP-2 consensus site, it strongly inhibits binding of the endogenous AP-2 present in PA-1 cell nuclear extracts. However, DNA sequence-specific binding of bacterially expressed AP-2A cannot be inhibited by bacterially expressed AP-2B. Therefore, inhibition of AP-2 activity by the protein AP-2B may require an additional factor or modification supplied by nuclear extracts.

Alternative Splicing↗
PubMed1993

[In vivo and ex vivo expression of nucleolar proliferation-associated antigens (p120, B23) in the prostate].

Expression of two nucleolar antigens, p120 and B23, was studied in a prostatic carcinoma cell line (LNCaP) and in frozen and paraffin embedded tissue sections of 40 benign and malignant prostatic lesions. The percentage of p120 negative G0/G1 phase cells rose significantly during transition from exponential to plateau growth phase in vitro (from 9% to 32%). In contrast, B23 was equally expressed throughout different cell cycle and growth phases. Thus, nucleoli of almost all stromal and epithelial cells were stained by B23 in tissue sections. P120, however, selectively stained nucleoli of proliferating prostatic epithelium. Whereas 88% (13/15) of benign hyperplasia were p120 negative this was the case in only 24% (6/25) of carcinomas. Using microwave procedure both MoAbs reacted in paraffin sections, but the percentage of p120 negative cases doubled. A routine application to formalin fixed and paraffin embedded tissue cannot be recommended thus far.

Cell Cycle↗
PubMed1993

[Differential regulation of androgen receptor promoter in hormone-sensitive and -insensitive prostate carcinoma cells].

The sensitivity of prostate epithelial cells to androgens is mediated by the androgen receptor (AR). Analyses of cell lines in vitro and prostate carcinomas in vivo provide evidence that loss of AR expression parallels the loss of sensitivity to androgens. The aim of our work is to analyze the genetic control elements of the AR gene and answer the question of why these elements fail to function in hormone-insensitive prostate cancer cells. Therefore, we have cloned and fully sequenced a 5700 base pair sequence 5'-upstream of the AR gene. This DNA fragment contains promoter and enhancer elements that are active in androgen-sensitive but inactive in androgen-insensitive prostate cancer cell lines.

Androgens↗
PubMed1993

[AP-2: a nuclear effector of malignant transformation by ras oncogene].

We have applied a series of cell clones established from the human teratocarcinoma cell line PA-1 to study the effect of malignant transformation by ras-oncogenes on the regulation of cell growth and differentiation. A particular aim of this study was to identify nuclear gene-regulatory factors that are affected by ras-transformation. We show that a key nuclear target of ras is the transcription factor AP-2. AP-2 function is inhibited through the ras-controlled signal transduction cascade by at least two different mechanisms, i.e. inhibition of an AP-2 coregulatory factor and by expression of an alternatively spliced inhibitory AP-2 protein.

Alternative Splicing↗
PubMed1993

[Acute arterial occlusion of the upper extremity by tumor embolism].

We present a case of acute occlusion of the right branchial artery in a 56-year-old male caused by bronchogenic tumor embolus as the first manifestation of malignancy. The primary tumor was shown to be inoperable and rapidly formed metastases in skin and liver. The case presented demonstrates, that embolic events originating from malignant tumor cells are characteristic for organ-exceeding tumor growth. Thus the prognosis is generally poor.

Arm↗
PubMed1992

The effect of retinoic acid on chemosensitivity of PA-1 human teratocarcinoma cells and its modulation by an activated N-ras oncogene.

Combination of chemotherapeutic drugs with agents that induce cell differentiation is a possible means of improving cancer chemotherapy. To explore this approach we used 4 cell lines established from the human teratocarcinoma-derived cell line PA-1; 2 retinoic acid (RA)-sensitive lines compared to 2 RA-resistant lines transformed by an activated N-ras oncogene. Equal numbers of colony-forming cells were exposed for 72 hr to 10(-6)M RA and subsequently to a range of concentrations of cisplatinum, etoposide or bleomycin. Enhanced cytotoxicity of cisplatin and etoposide (3- to 5-fold) was observed in the N-ras-transformed cell lines compared to the non-transformed lines. Treatment with RA caused an increase in the cytotoxicity of all 3 drugs to the 2 RA-sensitive cell lines. In contrast, a reduction of cytotoxicity was observed in the 2 N-ras-transformed lines. Our results indicate that sensitivity to cytotoxic agents can be increased by RA in RA-sensitive cells, but the opposite effect is seen in N-ras transformed, RA-resistant cells. Therefore, a general rationale for combination therapy with RA and cytotoxic drugs cannot be inferred.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

Identification of a retinoic acid-inducible endogenous retroviral transcript in the human teratocarcinoma-derived cell line PA-1.

Retinoic acid (RA), a developmental morphogen, causes activation of a transcript of an endogenous retrovirus-related element in the human teratocarcinoma-derived cell line PA-1. This provirus is defective, and the provirus-related sequences exist as multicopy elements (more than 20 copies) in human DNA. This is the first human endogenous retroviral mRNA that is known to be transcriptionally activated by RA. The nucleotide sequence of the 3,357 bp of this viral cDNA was determined and shows a strong homology to the type C-related human endogenous retroviral proviruses ERV3 and 4-1. This cDNA contains 'R-U5-delta pol-env-U3-R sequences of the provirus. Adjacent to the putative 5' long terminal repeat of this provirus there is an 18-bp sequence complementary to the 3' end of isoleucine tRNA. We named this RA-responsive virus RRHERV-I.

Base Sequence↗
PubMed1991

Alteration of homeobox gene expression by N-ras transformation of PA-1 human teratocarcinoma cells.

We used a series of cell clones from a human teratocarcinoma cell line, PA-1, to study the effect of transformation by an activated N-ras oncogene on the expression of genes involved in retinoic acid (RA)-induced differentiation and growth regulation. Recently, it has been shown that expression of human HOX 2 genes is sequentially activated by RA beginning from Hox 2.9 at the 3' end of the HOX 2 cluster (A. Simeone, D. Acampora, L. Arcioni, P. W. Andrews, E. Boncinelli, and F. Mavilio, Nature [London] 346:763-766, 1990). We now report that six different genes of the cluster HOX 1 are sequentially induced by RA in a similar temporal pattern, beginning with genes at the 3' end of the cluster. However, in N-ras-transformed cell clones, RA-induced expression of these homeobox genes is delayed. Hox 1.4 and Hox 1.3, genes abundantly induced in nontransformed clones after 3 days of RA treatment, are expressed in N-ras-transformed cells only after 10 days of RA treatment. At this time, the cells' growth is arrested at very high density, and no differentiated morphologic characteristics are observed. Constitutive expression of a transfected Hox 1.4 gene under the control of a simian virus 40 promotor leads to differentiated cell morphology similar to that of the RA-induced phenotype and restores the growth-inhibitory effects of RA in N-ras-transformed cells. These observations provide evidence that enhanced proliferation in N-ras-transformed cells compromises teratocarcinoma cell differentiation by a mechanism that transiently suppresses homeobox gene induction and implies a central role for homeobox genes in RA-induced cell differentiation. We conclude that stimulation of a putative growth factor signal pathway, associated with ras-induced proliferation, transiently suppresses the induction of transcription factors functionally involved in cell growth and differentiation.

Amino Acid Sequence↗
PubMed1991

Modulation of differentiation in PA-1 human teratocarcinoma cells after N-ras oncogene-induced tumorigenicity.

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.

Blotting, Northern↗
PubMedDate not supplied

Increase in telomerase activity during progression of melanocytic cells from melanocytic naevi to malignant melanomas.

During successive cell divisions of mortal cells the length of the telomeres (TTAGGG repeats in vertebrates) at the end of chromosomes decreases. It has been suggested that this process is responsible for cellular senescence. Expression of the ribonucleoprotein telomerase appears to prevent shortening of telomeres in germ-line cells and cancer cells. The purpose of this study was to investigate telomerase activity in melanocytic lesions and its possible role in the multi-step tumor progression model of malignant melanoma. To quantify the level of telomerase activity both in cultured cells and in fresh tissue samples the TRAP (telomeric repeat amplification protocol) ELISA was used. Eight cell lines of malignant melanoma, 3 primary cultures of fibroblasts, 36 melanocytic naevi, 5 atypical melanocytic naevi, 3 Spitz's naevi, 31 primary malignant melanomas and 13 metastases of malignant melanomas were investigated. Also 34 samples of skin (22 samples of perilesional skin and 12 samples of normal skin) were analysed. In our experiments all melanoma cell lines were strongly positive, whereas in fibroblasts telomerase activity could not be detected. Of the primary melanomas and metastatic melanomas, 90.3% and 92.3%, respectively, were strongly positive, and of the atypical melanocytic naevi, 80% were positive. Of the 36 common melanocytic naevi only 10 (27.7%) expressed weak telomerase activity and of the 34 samples of human skin, 4 (11.7%) expressed very weak telomerase activity. Our results indicate that telomerase activity increases from benign melanocytic naevi to atypical naevi and further to malignant melanoma and metastatic melanoma cells, and therefore may play a role in tumour initiation and progression.

Adolescent↗
PubMedDate not supplied

Comparison of two prognostic markers for malignant melanoma: MIA and S100 beta.

It has recently been shown that the serum level of melanoma-inhibitory protein (MIA) provides useful information for the therapy and follow-up of patients with malignant melanoma. Previously, S100 beta has been described as a useful tumor marker for malignant melanoma. In this study, we compare the significance of the two markers in follow-up, therapy outcome and prognosis by measuring MIA and S100 beta serum levels in 50 melanoma patients. Serum levels were measured in patients with malignant melanomas of stages I-IV with at least 3 time points of measurement. Serial MIA and S100 beta measurements were obtained from 32 patients with stage IV disease in parallel to chemotherapy and from 18 patients with a history of stage I and stage II disease during follow-up. The response to chemotherapy in stage IV disease and relapse of melanoma during follow-up correlated with changes in MIA and S100 beta serum levels. In comparison, MIA revealed slightly higher specificity and sensitivity. In conclusion, both markers are useful for detection of progression from localized to metastatic disease during follow-up and for monitoring therapy of advanced melanomas.

Adult↗
PubMedDate not supplied

MIA, a novel serum marker for progression of malignant melanoma.

MIA was isolated previously as a small soluble protein secreted from malignant melanoma cell lines in vitro. Highly restricted expression patterns in melanocytic tumors were identified in vivo. We therefore quantitated serum levels of MIA protein by means of a non-radioactive ELISA and investigated whether MIA provides clinically relevant parameters in patients with malignant melanomas. Here we report enhanced MIA serum levels in 13% and 23% of patients with stage I and II disease, respectively, and in 100% with stage III or IV disease. Response to therapy in stage IV disease correlated with changes in MIA serum levels and surgical removal of metastases led to normalization of serum values. Repeated measuring of sera from 350 patients with a history of stage I or II melanoma during follow-up, we detected 32 patients developing positive MIA values. At the time of serum analysis 15 of them had developed metastases and one presented with metastatic disease 6 months later. In conclusion, MIA represents a novel serum marker for systemic malignant melanoma revealing the highest sensitivity and specificity among currently available markers.

Adult↗