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B Theisinger

Publications and source records attributed to B Theisinger.

At least 19 recordsLinked to original sources
PubMed1999

CAPN 8: isolation of a new mouse calpain-isoenzyme.

Recent molecular biological approaches indicate that calpain, also named CANP for calcium-activated neutral protease and originally characterized as an intracellular cytoplasmatic nonlysosomal cysteine protease that requires calcium ions for activity, constitutes a large superfamily consisting of ubiquitous and tissue specific homologues, which are widely distributed in cells of various organisms from human to fungus. Due to the increasing number of substrates along with the involvement of calpain isoenzymes in mammalian diseases, especially in malignancies, members of the calpain superfamily seem to be important biomodulators in physiological as well as pathological cell function. Here we report the characterisation of a new calpain, named CAPN 8 with a different C-terminal domain, implicating a putative new regulatory mechanism. Northern blot analysis revealed an ubiquitous expression with different RNA levels in all tissues examined. Highest levels were found in brain, kidney, and digestive tract, suggesting a specific regulatory function of CAPN 8 in these tissues.

Amino Acid Sequence↗
PubMed1999

Expression of calpain I messenger RNA in human renal cell carcinoma: correlation with lymph node metastasis and histological type.

Calpain, also named CANP (for calcium-activated neutral protease), is an intracellular cytoplasmatic non-lysosomal cysteine endopeptidase that requires calcium ions for activity. Many substrates of the calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumor supressor protein p53, protein kinase C, pp60c-src and the adhesion molecule integrin, have been implicated in the pathogenesis of different human tumors, suggesting an important role of the calpains in malignant diseases. We now report differential expression of the calpain I gene (CL I) in a variety of tumors, extending our study to a larger series of renal cell carcinomas. Using Northern-blot analysis, we studied calpain I expression in 30 renal cell carcinomas as compared with matched healthy tissues. Tumor samples were classified according to their histological type: 21 clear cell carcinomas, 4 chromophobe carcinomas, 3 papillary carcinomas and 2 oncocytomas. In renal tumor samples, calpain I gene mRNA was expressed at highly variable levels, significantly depending on the different histological types. Moreover, there was a correlation of higher calpain I expression with increased malignancy: within the clear cell carcinoma subset, tumor samples with advanced nodal status (N1 and N2) showed a significantly higher calpain I expression than tumors without metastasis to regional lymph nodes. Our data suggest an important role of calpain isoenzymes in carcinogenesis and tumor progression.

Biomarkers, Tumor↗
PubMed1998

Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A. Two old enzymes combine for the novel Nm23 protein phosphotransferase function.

We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH.Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteins in vitro. The molecular mass of the highly purified recombinant GAPDH.Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.

Creatine Kinase↗
PubMed1997

Isolation and characterization of new microsatellites at the nm23-H1 and nm23-H2 gene loci and application for loss of heterozygosity (LOH) analysis.

The nm23-H1 gene has been suggested to be a metastasis suppressor gene. Studies about the events of loss of heterozygosity (LOH) at the nm23 locus and its correlation to metastasis are controversially discussed. To optimize detection of LOH at the nm23 locus, we screened two P1 clones for additional microsatellites. Tumor and normal DNA from 37 colorectal, 16 gastric, and 8 germ cancer patients were examined for LOH. We found two new CA repeats, one 5' to nm23-H1 and another 3' to nm23-H2. Using these nm23 locus-specific CA repeats and five other chromosome 17 loci (D17S1522, D17S1566, D17S855, D17S515, and TP53), allele loss was observed in 4/32 (12.5%) patients with colon cancer, 2/14 (14.3%) with gastric cancer, and 1/7 (14%) with germ cancer. No isolated LOH of the nm23 region was observed.

Aged↗
PubMed1996

A second trefoil protein, ITF/hP1.B, is transcribed in human breast cancer.

Trefoil proteins form a specific group of stable secreted polypeptides. They are expressed in a lot of human cancers and during inflammatory processes of the gastrointestinal tract. Recently a new human trefoil protein, ITF/hP1.B, was isolated. Until now no studies of the activity of this gene in human solid tumors exist. In our examination we show for the first time that this gene is transcribed in human breast cancer. In contrast to another trefoil protein, pS2, the expression of ITF/hP1.B is not under control of estrogen in the human breast cancer cell line MCF-7. We suggest that the gene activity of ITF/hP1.B in addition to pS2 expression may be an improved prognostic marker in human breast cancer.

Biomarkers, Tumor↗
PubMed1995

A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleoside-diphosphate kinase.

Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.

Amino Acids↗
PubMed1994

Presence of regulatory sequences within intron 4 of human and murine c-myb genes.

The molecular mechanisms that modulate c-myb mRNA transcription in hematopoietic cells appear to involve intron regulatory sequences. We have characterized the fourth of ten introns from both human and murine c-myb genes in regard to nucleotide sequence and specific protein binding. For this approach complete genomic c-myb intron 4 fragments were isolated from mouse and human DNA using PCR amplification with flanking exon-primers derived from the mouse gene. Comparison of the obtained sequences revealed strong homology between the two species. Using crude nuclear protein extracts from mouse and human myb expressing cells (70Z/3B; Molt4) and gel shift experiments we found specific protein interaction for both introns and to determine the protein binding site in detail, we performed DNase I footprinting. Our results indicate that the binding factor is absent in control cell lines without c-myb transcriptional activity, suggesting a possible positive regulatory function of the DNA-protein complex. To confirm these findings we introduced the human c-myb intron 4 DNA sequence into the EcoRI site of the pCAT-Promoter plasmid and transfected Molt4 cells with this chimeric construct. The transient expression studies revealed that intron 4 sequences possess enhancer activity. Thus, we have demonstrated that intron 4 sequences can be important for the regulation of c-myb proto-oncogene expression.

Animals↗
PubMed1994

Expression pattern of breast-cancer-associated protein pS2/BCEI in colorectal tumors.

Recently, several carcinomas of the gastrointestinal tract were tested for pS2/BCEI activity, a gene isolated from breast-cancer cells and coding for a small secreted peptide. In the latter tumors, its activity is under estrogen control; surprisingly, it was also found expressed in carcinomas of the stomach, biliary tract and pancreas. We have now investigated the expression of this gene in 64 colorectal carcinomas, 31 adenomas and 13 polyps in comparison with their matrix tissues by applying molecular (RNA analysis) and immunohistochemical (pS2 antibody) techniques. Positive pS2 immunostaining (ranging from focal to strong immunoreaction) was noted in 89% of human colon cancers, while 11% remained negative. Furthermore, all 40 transitional mucosae were strongly positive, whereas normal mucosa was negative. Of hyperplastic polyps, 68.2% displayed a significant immunoreaction, and 80.6% of adenomas were focally positive. Finally, 6 out of 16 cases showed significant pS2 transcription in Northern blot analysis. These data clearly indicate that the breast-cancer-associated pS2 protein also plays an as yet undetermined role in the tumorigenesis of human colorectal carcinomas.

Breast Neoplasms↗
PubMed1994

Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1.

Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-K beta) shows that exon-intron boundaries are well conserved between these two species.

Amino Acid Sequence↗
PubMed1993

High levels of nm23-H1 and nm23-H2 messenger RNA in human squamous-cell lung carcinoma are associated with poor differentiation and advanced tumor stages.

Expression of the candidate metastasis-suppressor gene nm23-H1 has been shown to correlate inversely with metastatic potential in some human tumors, but not in all. Until now, few studies have been carried out on the activity of the homologous nm23-H2 gene in human cancer. No nm23 transcription studies exist for human lung cancer so far. To determine whether the nm23 genes could have a metastasis-suppressor function in non-small-cell lung carcinoma (NSCLC), pulmonary sarcoma and carcinoids, we analysed both nm23-HI and nm23-H2 mRNA levels in 37 tumor samples obtained from patients who underwent potentially curative resection between 1986 and 1990, and in 4 metastatic tumors obtained from autopsy. As compared to corresponding healthy lung parenchyma, both nm23-HI and nm23-H2 transcript levels were elevated in 37 of 41 tumors. The increases in nm23 mRNA expression were stronger in advanced stages of squamous-cell carcinoma, large-cell carcinoma, sarcoma and carcinoids than in early stages of the respective tumor types. Within stages I and II of squamous-cell carcinoma, significantly higher nm23 mRNA levels were found in poorly differentiated tumors than in moderately differentiated ones. Moreover, an inverse correlation between nm23 expression and disease-free survival of the patients was observed. In conclusion, our results indicate that the increased nm23 expression in the analysed tumors is not consistent with the proposed metastasis-suppressor function, but the 2 nm23 genes nevertheless may be implicated in the mechanism of tumor progression.

Adenocarcinoma↗
PubMed1993

Retrospective analysis of prognostic significance of the estrogen-inducible pS2 gene in male breast carcinoma.

BACKGROUND: The estrogen-inducible pS2 gene, originally isolated from a breast cancer cell line, is correlated with hormone-dependent female breast tumors and its expression is associated with longer overall and disease-free survival. METHODS: The authors have investigated 38 samples of carcinomas of the male breast for pS2 expression by using a monoclonal antibody. The immunostaining was compared with clinical data, in particular, to the progesterone receptor status, to assess a possible prognostic value of this parameter. RESULTS: Although most cases (27 of 38) were immunopositive (i.e., above the 5th percentile of immunoreactive cells), no correlation with tumor grade and survival was notable. CONCLUSIONS: Therefore, on the contrary to the situation in female breast cancer, pS2 activity failed to constitute a new prognostic parameter in male breast carcinomas.

Adult↗
PubMed1992

Assignment of the gene for human spasmolytic protein (hSP/SML1) to chromosome 21.

A human cDNA corresponding to the porcine pancreatic spasmolytic protein (PSP) was isolated, and the recombinant clone was originally termed hSP for human spasmolytic protein. Later, the term SML1 for spasmolysin was suggested for the human gene. This protein shows a remarkable sequence homology to pS2, a protein coded by an estrogen-induced gene isolated from the breast carcinoma cell line MCF-7. Although, at the DNA level, the gene sequences pS2 and hSP/SML1 display insufficient homology for cross-hybridization, their expression in tumor cells occurs with remarkable coordination. The human pS2 gene sequence has been assigned to chromosome 21, and we have therefore attempted to map the hSP/SML1 gene by using cDNA and Southern blotting of genomic DNAs from a panel of human-rodent somatic cell hybrids carrying different complements of human chromosomes. Interestingly, the hSP/SML1 gene is also localized on chromosome 21.

Blotting, Southern↗
PubMed1992

Association of the human spasmolytic polypeptide and an estrogen-induced breast cancer protein (pS2) with human pancreatic carcinoma.

The human pS2 gene, isolated from the breast carcinoma cell line MCF-7 and shown to be under estrogen transcriptional control in a subclass of breast cancer cells was reported to be secreted in normal stomach surface epithelial cells, whereas additional gastrointestinal tissues like pancreas and colon do not secrete pS2 at all. In porcine pancreas, a spasmolytic polypeptide (sharing domains of homology with pS2) was observed; a corresponding human gene (hSP) was shown to be active in normal stomach mucosa. hSP and pS2 gene activity in normal and neoplastic pancreas tissues was then compared. Whereas both genes are inactive in normal pancreatic cells, activation of the pS2 sequence in a primary pancreatic carcinoma cell culture and in 23 tumor tissues was noted when investigated by immunostaining. In all cases when pS2 showed a regular 0.6 kb transcript, hSP displayed a transcript of 0.7 kb. Six of these tumors showed a reduced pS2 immunoreactivity and, at the same time, aberrant pS2 mRNA bands and a complete shut-down of the hSP gene were noted. In one case, whereas normal pancreas remained negative, the corresponding tumor and its metastasis displayed regular transcripts of pS2 and hSP. This remarkably high correlation suggests that pS2 and hSP expression in the pancreatic tumors, but not in their corresponding healthy tissue is significantly linked to molecular steps leading to tumorigenesis.

Adenocarcinoma↗
PubMed1991

Expression of the breast cancer associated gene pS2 and the pancreatic spasmolytic polypeptide gene (hSP) in diffuse type of stomach carcinoma.

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.

Blotting, Northern↗
PubMed1990

The cellular myb oncogene is amplified, rearranged and activated in human glioblastoma cell lines.

The human c-myb gene which encodes a DNA binding protein and which is rarely amplified in neoplastic cells was found to be altered in four human glioblastoma cell lines. It exists in multiple copies in 2 out of 4 cases studied. The degree of amplification as determined by densitometry was about 10-fold, a rearrangement within the coding region and an enhanced gene activity of c-myb were noted. The observation of c-myb oncogene amplification and activity in glioblastoma cell lines presents the first report of this effect in human brain tumor cells.

Brain Neoplasms↗