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R Buettner

Publications and source records attributed to R Buettner.

At least 73 records · Page 4Linked to original sources
PubMed1997

Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta.

Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. We show here that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl-X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism we show that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6p12-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene.

Animals↗
PubMed1997

Melanoma-inhibiting activity, a novel serum marker for progression of malignant melanoma.

Melanoma-inhibiting activity (MIA) was isolated previously as a small soluble protein secreted from malignant melanoma cell lines in vitro. In vivo, highly restricted expression patterns in melanocytic tumors were identified. We therefore quantitated serum levels of MIA protein by means of a nonradioactive ELISA and investigated whether MIA provides a clinically useful parameter 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. Compared with S-100 and soluble intercellular adhesion molecule 1 serum levels in these patients, MIA was the most sensitive marker. Response to therapy in stage IV disease correlated with changes in MIA serum levels. Measuring repeatedly sera of 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 contrast, none of the patients with normal MIA serum levels developed metastases during the follow-up period of 6-12 months. In conclusion, MIA represents a novel serum marker for systemic malignant melanoma revealing the highest sensitivity and specificity among currently available markers. Useful clinical applications include staging of primary melanomas, detection of progression from localized to metastatic disease during follow-up, and monitoring therapy of advanced melanomas.

Adult↗
PubMed1997

Comparative analysis of AP-2 alpha and AP-2 beta gene expression during murine embryogenesis.

Transcription factor AP-2 has been identified as playing important roles during embryonic development of the neural tube, neural crest derivatives, skin, and urogenital tissues. Recently, we isolated a second AP-2 transcription factor, AP-2 beta, which is 76% homologous to the previously known AP-2 alpha gene, and showed that both genes are coexpressed in murine embryos at day 13.5 and 15.5 post coitum (pc). In the current study, we used specific cRNA probes to study comparatively AP-2 alpha and AP-2 beta expression by in situ hybridization of murine embryonic tissue sections. Our results reveal that expression of both genes starts at day 8 pc in the lateral head mesenchyme and extraembryonic trophoblast. The expression pattern was identical until day 10 pc but diverged significantly during later stages of development. From day 11 forward, specific expression patterns of AP-2 alpha and AP-2 beta mRNA were observed. Specific AP-2 beta signals were detected in the midbrain, sympathetic ganglia, adrenal medulla, and cornea. Specific AP-2 alpha signals were present in the limb buds, dorsal root ganglia, tooth germs, and Moll's and Meibom's glands. In contrast, expression of both genes occurred in skin, facial mesenchyme, spinal cord, cerebellum, and renal tubular epithelia. Our results indicate that both genes are expressed with different temporal and spatial patterns during embryonic development.

Animals↗
PubMed1997

Mouse CD-RAP/MIA gene: structure, chromosomal localization, and expression in cartilage and chondrosarcoma.

A cDNA encoding a novel protein has been previously isolated from two independent sources: melanoma cell cultures and chondrocytes. The protein from human melanoma cell lines and tumors is called melanoma inhibitory activity (MIA) (Blesch et al. [1994] Cancer Res. 54:5695-5701) and the protein from primary bovine chondrocytes and cartilaginous tissues is called cartilage-derived retinoic acid-sensitive protein (CD-RAP) (Dietz and Sandell [1996] J. Biol. Chem. 271:3311-3316). In order to investigate the gene regulation and function of CD-RAP/MIA, the mouse gene locus was isolated and analyzed. Developmental expression was determined by in situ hybridization to mouse embryos. Expression was limited to cartilaginous tissues and was initiated with the advent of chondrogenesis, remaining abundant throughout development. The mouse gene was isolated and sequenced from a 129Sv library and sequenced directly from an additional strain, B6C3Fe. The mouse CD-RAP/MIA gene is 1.5 kbp and consists of four exons. The promoter sequence of the gene contains many potential regulatory domains including 8 basic helix-loop-helix protein-binding domains and an AT-rich domain, both motifs shown to be present in the cartilage-specific enhancer of the type II procollagen gene. Other potential cis-acting motifs include binding sites for GATA-1, NF-IL6, PEA3, w-elements, NF kappa B, Zeste and Sp1. The gene, called cdrap, was localized to the end of an arm of chromosome 7 at the same site as the transforming growth factor beta 1 (Tgf-beta 1) and the glucose phosphate isomerase 1 (Gpi 1) genes. Potential mouse mutants that mapped to the same region of chromosome 7 were identified. Two of the potential mutants with skeletal phenotypes were sequenced, pudgy (pu) and extra toes with spotting (XsJ); however, no mutations were found in the coding sequence. To determine whether CD-RAP/MIA is associated with tumors of cartilage, mRNAs from a variety of rodent tissues and cell lines were screened. Expression was detected in a rodent tumor, the Swarm rat chondrosarcoma and a chondrosarcoma cell line derived from it, but not in other tissues or tumors of non-cartilage origin. Immunolocalization revealed CD-RAP/MIA protein localized in cartilage only. These results show that the normal expression of CD-RAP/MIA is limited to cartilage; however, pathologically, it is expressed both in melanoma and chondrosarcoma. The restricted expression of CD-RAP/MIA may provide an opportunity to monitor cartilage metabolic activity as well as the tumor activity of melanoma and chondrosarcoma.

Amino Acid Sequence↗
PubMed1997

Pigmented peripheral nerve sheath tumor of the oral cavity with expression of AP-2 beta and c-RET: a case report.

Pigmented peripheral nerve sheath tumor of the oral cavity is a rare lesion and may be difficult to distinguish clinically from true melanocytic tumors. We report a case of pigmented peripheral nerve sheath tumor with a high degree of melanocytic differentiation and describe its histologic and ultrastructural features. Immunohistochemistry and polymerase chain reaction findings support the neural crest origin of the tumor. The clinical course of our case indicates that local recurrence may be a significant problem in pigmented peripheral nerve sheath tumor.

Adult↗
PubMed1997

Ultraviolet A radiation-induced expression of human genes: molecular and photobiological mechanisms.

Increased gene expression as a consequence of environmental stress is typically observed in mammalian cells upon exposure to ultraviolet (UV) radiation. In previous years the cis- and trans-acting genetic elements responsible for gene induction by short wavelength UVC (< 280nm) and intermediate wavelength UVB (280 - 320 nm) radiation have been well characterized. More recently, progress has also been made in understanding the mechanisms by which long wavelength UVA (320 - 400 nm) radiation induces transcriptional activation of human genes. From these studies it is now known that the photobiological as well as the molecular mechanisms involved in UVA radiation-induced gene expression differ from those previously identified for UVB- or UVC-induced gene expression. In particular, the reactive oxygen species singlet oxygen was found to serve as the primary effector in UVA radiation-induced gene expression by inducing a signal transduction cascade that depends on activation of transcription factor AP-2. These studies indicate a previously unrecognized role of AP-2 in the mammalian stress response.

Animals↗
PubMed1996

The helix-loop-helix transcription factor SEF-2 regulates the activity of a novel initiator element in the promoter of the human somatostatin receptor II gene.

The effects of somatostatin hormones are mediated by a family of five different seven-helix transmembrane spanning receptors (SSTR1-5). The expression of the five different SSTR subtypes displays a complex temporal- and tissue-specific pattern. To investigate the molecular mechanisms controlling the different expression patterns of the SSTRs, we cloned the 5'-flanking region of the human SSTR2 gene. Characterization of the SSTR2 promoter resulted in the identification of a novel initiator element (SSTR2inr). Transcriptional activity of the SSTR2inr is dependent on the presence of a binding site (E-box) for basic helix-loop-helix (bHLH) transcription factors. By screening a mouse brain cDNA expression library we isolated a cDNA coding for the bHLH transcription factor SEF-2. SEF-2 binds to the E-box present in the SSTR2inr, both in vitro and in vivo and activates transcription from the SSTR2inr. A single point mutation within the E-box eliminates binding of SEF-2 and results in a complete loss of transcriptional activity of the SSTR2inr. Furthermore, DNA binding studies demonstrate that the basal transcription factor TFIIB can be tethered to the SSTR2inr through physical interaction with SEF-2. In summary, the SSTR2inr represents a novel type of initiator element that confers gene expression in the absence of a TATA-box or binding sites for other known initiator factors, like YY-1 or USF.

Amino Acid Sequence↗
PubMed1996

Structure and promoter analysis of the gene encoding the human melanoma-inhibiting protein MIA.

We have recently described the isolation of a novel protein, MIA, which is secreted from malignant melanoma cells and elicits growth inhibition on melanoma cells in vitro (Blesch, A., Bosserhoff, A. K., Apfel, R., Behl, C., Hessdörfer, B., Schmitt, A., Jachimczak, P., Lottspeich, F., Schlingensiepen, H., Buettner, R., and Bogdahn, U. (1994) Cancer Res. 54, 5695-5701). Here, we report the structure of the human MIA gene locus, describe its expression pattern in melanocytic tumors in vivo, and provide an initial characterization of the MIA promoter. The MIA gene is encoded by four exons, and the mRNA initiation site was identified 70 base pairs upstream from the translation start codon. MIA mRNA expression in vivo correlated with progressive malignancy of melanocytic lesions and was inducible in other cells by phorbol esters. To investigate mechanisms mediating this melanoma-associated expression pattern, we analyzed the promoter activity of the 1.3-kilobase genomic sequences located 5'-upstream of the MIA gene. The MIA promoter conferred high levels of gene activation specifically in human and murine melanoma cells, and its activity was further enhanced by treatment with phorbol esters. Site-directed mutation of an NF-kB site within the MIA promoter did reduce the basal promoter activity in melanoma cells but did not change significantly enhancement by phorbol esters.

Animals↗
PubMed1996

[Benign placental trophoblast nodule. Case report with overview of proliferative diseases of the intermediate stage trophoblast].

This report reviews proliferative diseases of intermediate trophoblast cells and summarises clinical findings and pathohistological and immunohistochemical features of a placental site nodule. This recently described entity is an endometrial or endocervical benign proliferation of intermediate trophoblast cells occurring after intrauterine gestation. Patients usually present with a history of abnormal vaginal bleeding and frequently cells that are present in their cervical smears are misinterpreted as squamous dysplasia. In such cases it is important to distinguish the placental site nodule from squamous lesions or decidual polyps; this is possible by means of immunohistochemistry.

Adult↗
PubMed1996

5 alpha-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro.

5 alpha-reductase (5 alpha R) activity in two human prostate cancer cell lines was compared to that in benign prostatic hyperplasia (BPH) tissue and COS cells transfected with and expressing the human genes for 5 alpha-reductase type 1 (5 alpha R1) and type 2 (5 alpha R2). Comparisons were based on pH profiles and sensitivities to selective inhibitors of 5 alpha-reductase In the cancer lines, activity was greatest over the pH range 7-8, compared to a sharp peak of activity between pH 5-5.5 in BPH tissue and COS cells expressing 5 alpha R2. Finasteride and SKF105,657 were potent inhibitors of 5 alpha-reductase activity in BPH tissue and COS cells expressing 5 alpha R2, but weak inhibitors in the cancer lines and in COS cells expressing 5 alpha R1. In contrast, UK117,026 was a more potent inhibitor of 5 alpha-reductase activity in the prostate cancer cell lines and in COS cells expressing 5 alpha R1. These data indicate that human prostate cancer cell lines express 5 alpha-reductase activity similar to that in COS cells transfected with 5 alpha R1, but different from that in BPH tissue. This may be a consequence of in vitro culture. Alternatively, it may reflect a change occurring as a result of neoplastic transformation in which case it will be important to select appropriate inhibitors in the clinic.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1995

Transcriptional activation by Myc is under negative control by the transcription factor AP-2.

The Myc protein binds to and transactivates the expression of genes via E-box elements containing a central CAC(G/A)TG sequence. The transcriptional activation function of Myc is required for its ability to induce cell cycle progression, cellular transformation and apoptosis. Here we show that transactivation by Myc is under negative control by the transcription factor AP-2. AP-2 inhibits transactivation by Myc via two distinct mechanisms. First, high affinity binding sites for AP-2 overlap Myc-response elements in two bona fide target genes of Myc, prothymosin-alpha and ornithine decarboxylase. On these sites, AP-2 competes for binding of either Myc/Max heterodimers or Max/Max homodimers. The second mechanism involves a specific interaction between C-terminal domains of AP-2 and the BR/HLH/LZ domain of Myc, but not Max or Mad. Binding of AP-2 to Myc does not preclude association of Myc with Max, but impairs DNA binding of the Myc/Max complex and inhibits transactivation by Myc even in the absence of an overlapping AP-2 binding site. Taken together, our data suggest that AP-2 acts as a negative regulator of transactivation by Myc.

Animals↗
PubMed1995

Characterization of selected strongly and weakly invasive sublines of a primary human melanoma cell line and isolation of subtractive cDNA clones.

Invasion of basement membranes is a key step in systemic spread of tumour cells. To analyze genetic mechanisms involved in this process, we have selected strongly and weakly invasive sublines with stable phenotypes from a primary human melanoma cell line by repeated passage through a reconstituted basement membrane in vitro. The sublines differed approximately 5-fold in their invasive potential. Invasiveness correlated with better attachment and overexpression of the integrin alpha v/beta 3 (vitronectin/laminin-receptor). Treatment with retinoic acid inhibited proliferation in both sublines and invasion in the weakly invasive cells but stimulated invasion in the strongly invasive subline. Northern-blot analyses revealed equal levels of mRNA expression regarding collagenase type-IV and retinoic-acid receptors but enhanced expression of TIMP-2 mRNA in weakly invasive cells. The 2 sublines differed significantly in their respective DNA ploidy when compared to the wild-type Mel Im cell line, suggesting that they represent heterogeneous clones present in the primary tumour. We have started to exploit this in vitro system for tumour heterogeneity to clone genes involved in invasion. By a subtractive cDNA cloning strategy, 12 partial cDNA clones were obtained that are specifically overexpressed in the strongly or weakly invasive subline. These results illustrate that stable genetic alterations lead to heterogeneous subpopulations within primary melanomas which differ in their ability to invade basement membranes and interact with components of the extracellular matrix.

Actins↗
PubMed1995

Cloning and characterization of a second AP-2 transcription factor: AP-2 beta.

AP-2 has been characterized previously as a unique 52 x 10(3) M(r) transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2 alpha expression was found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues.

Amino Acid Sequence↗
PubMed1995

In vitro and ex vivo expression of nucleolar proteins B23 and p120 in benign and malignant epithelial lesions of the prostate.

The expression of two specific nucleolar antigens, p120 and B23, has been investigated in the prostatic carcinoma cell line LNCaP as well as in 40 frozen and 40 formalin-fixed tissue samples of benign and malignant prostatic lesions (15 benign hyperplasias, 5 grade 1, 15 grade 2, and 5 grade 3 carcinomas). In vitro, immunoreactivity of p120 was confined to nucleoli of proliferating cells, with virtually no negative staining during S and G2/M phases. Unlike p120, B23 was expressed in the nucleoli of all LNCaP cells independently of growth and cell cycle phases. Hence, B23 was detectable in all stromal as well as in normal and malignant epithelial prostatic cells, both in fresh and in formalin-fixed tissue sections after microwave treatment. In contrast, the immunoreactivity of p120 was almost completely restricted to the nucleoli of prostate carcinoma cells: frozen sections of benign prostatic hyperplasia (n = 15) were either totally negative for p120 (n = 13) or had a low percentage of positively stained cells (labeling index = 3.3% in 3 cases). In the carcinoma group 76% (19/25) of the specimens were p120 positive, and there was a significant rise of labeling index from 18.1% in grade 1 to 82.2% in grade 3 carcinomas (P < 0.001). In contrast to B23, p120 could not be reliably demonstrated in formalin-fixed and paraffin-embedded tissue. We therefore conclude that anti-B23 is a general marker of nucleoli, whereas expression of p120 appears to correlate with "hyperactivity" of the nucleolus and provides a new tool for flow cytometrical and immunohistochemical assessment of nucleolar activity in tumor pathology.

Adenocarcinoma↗
PubMed1994

Cloning of a novel malignant melanoma-derived growth-regulatory protein, MIA.

Growth and progression of malignant melanoma cells is influenced by a complex network of growth-stimulating and -inhibiting factors produced by both the tumor cells and the local environment. Here we report the purification and molecular cloning of a novel growth regulating protein, designated melanoma inhibitory activity (MIA) and provide a preliminary functional characterization. MIA is translated as a 131-amino acid precursor and processed into a mature 107-amino acid protein after cleavage of a putative secretion signal. A murine complementary DNA was isolated that encoded a MIA-protein with 88% amino acid identity. MIA is secreted into the culture supernatant by several malignant melanoma cell lines as an M(r) 11,000 autocrine growth factor and acts as a potent tumor cell growth inhibitor for malignant melanoma cells and some other neuroectodermal tumors, including gliomas. MIA has no homology to any other known protein and, therefore, represents a novel type of growth-regulatory factor. Furthermore, we describe a molecular approach to express functionally active MIA in Escherichia coli, which might be attractive as a future antitumor therapeutical substance.

Amino Acid Sequence↗
PubMed1994

N-ras oncogene causes AP-2 transcriptional self-interference, which leads to transformation.

Genetic alterations in elements of normal signal transduction mechanisms are known to be oncogenic events often resulting in aberrant activation of programs of gene transcription. We have investigated the effect of N-ras oncogene-induced tumorigenic transformation on the transcription factor AP-2. N-ras oncogene-induced transformation of human teratocarcinoma cells PA-1 results in sixfold elevated AP-2 mRNA levels. However, the level of AP-2-mediated trans-activation is dramatically inhibited in these cells. We show here that the high-level expression of AP-2 ultimately results in transcriptional "self-interference". The activation domain of AP-2, when fused to the DNA-binding domain of GAL4, is sufficient for self-interference. Non-N-ras PA-1 cells constitutively expressing AP-2 or GAL4-AP-2 fusion protein from an SV40 promoter exhibit reduced AP-2-mediated transcriptional activation, inhibition of differentiation, and promotion of anchorage-independent growth, properties that are similar to N-ras-transformed PA-1 cells. Thus, AP-2 is placed in the N-ras signal transduction pathway, and many of the biological effects of N-ras can be accomplished by overexpression of AP-2. This is the first evidence that inhibition of the activity of a transcription factor by self-interference contributes to a physiological process.

Base Sequence↗
PubMed1994

The genomic structure of the human AP-2 transcription factor.

The transcription factor AP-2 is encoded by a gene located on chromosome 6 near the HLA locus. Here we describe the genomic organization of the AP-2 gene including an initial characterization of the promoter. We have mapped two mRNA initiation sites, the entire exon-intron structure and located two polyadenylation sites. The mature AP-2 mRNA is spliced from 7 exons distributed over a region of 18 kb genomic DNA. A recently cloned inhibitory AP-2 protein is generated by alternative usage of a C-terminal exon. The proline-rich transactivation motif is encoded by a single exon within the N-terminal region in contrast to the complex DNA binding and dimerization motif which involves amino acid residues located on four different exons. The sites of mRNA initiation are located 220 and 271 bases upstream from the ATG translation start site. Although the promoter contains no canonical sequence motifs for basal transcription factors, such as TATA-, CCAAT- or SP-1 boxes, it mediates cell-type-specific expression of a CAT reporter gene in PA-1 human teratocarcinoma cells and is inactive in murine F9 teratocarcinoma cells. We demonstrate that the promoter of the AP-2 gene is subject to positive autoregulation by its own gene product. A consensus AP-2 binding site is located at position -622 with respect to the ATG. This site binds specifically to bacterially expressed AP-2 as well as to multiple proteins, including AP-2, present in PA-1 and HeLa cell nuclear extracts. A partial AP-2 promoter fragment including the AP-2 consensus binding site is approximately 5-fold transactivated by cotransfection of an AP-2 expression plasmid.

Animals↗