Genetic changes in the genesis of gastrointestinal carcinoma.
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Breast Neoplasms↗
Biomedical subjects
C Welter
Publications and source records attributed to C Welter.
Nonradioactive southwestern analysis using chemiluminescent detection.
Replacing radioactively labeled probes by nonradioactive ones and detection by chemiluminescence instead of colorimetry allows a nonhazardous handling and offers the possibility of easily reprobing filters in Southwestern analysis. Using the described procedure, we were able to determine the molecular weight of DNA-binding proteins and achieve a high signal-to-noise ratio.
Animals↗
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↗
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↗
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↗
Generating highly labeled oligonucleotides for DNA-protein interaction.
We developed a new strategy to prepare double-stranded oligonucleotides containing recognition sites for specific binding proteins to examine DNA-protein interactions in various assays (gel mobility shift, UV-crosslinking, and affinity chromatography). The advantages of our procedures are as follows. Only one strand needs to be synthesized using a commercial oligonucleotide synthesizer. The probes can be labeled to a high specific activity and the exact position of labeling can be chosen, which is necessary for UV-crosslinking studies. Furthermore, multimeric binding sites for efficient DNA affinity chromatography can easily be generated. It is also possible to precisely place modified bases without the need for chemical precursors. Using this protocol, more detailed information about the binding protein factors and their behavior in interaction with recognition sites can be obtained.
Base Sequence↗
A rapid protocol for the purification of mitochondrial DNA suitable for studying restriction fragment length polymorphisms.
When analyzing mitochondrial DNA (mtDNA) from various normal and malignant human tissues, it became necessary to enhance mtDNA isolation for improved yields and quality. The method described here consists of rapid and simple-to-perform steps, avoiding complicated instrumentation. It was designed for preparation of undegraded mtDNA and is highly useful when limited amounts of tissues, cells and unique biopsies of tumors (fresh or frozen) are available. The resulting mtDNA is sufficiently pure for restriction analysis, subcloning, labeling and various types of hybridization. Using Sau3A and MspI, restriction analysis revealed new restriction-fragment length polymorphisms for Caucasians, independent of the DNA source, and hence excluding tissue-specific DNA modifications.
Centrifugation↗
DNA curvature in front of the human mitochondrial L-strand replication origin with specific protein binding.
DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA-replication, site specific recombination, and DNA packaging. In the human mitochondrial DNA we have found a DNA curvature structure within the 3'-region of ther URF2 sequence in front of the L-strand origin of replication. This structure interacts specifically with a protein factor isolated from mitochondria. Based on the localization of this DNA curvature structure and the known function of such structures the data suggest a model in which this DNA signal sequence and its specific protein binding is involved in the regulatory initiation event of L-strand replication.
Base Sequence↗
Alteration of mitochondrial DNA in human oncocytomas.
Oncocytoma, a benign solid tumor, occurs in a number of organs but most frequently in the kidneys. Its oncocytes display an intensely eosinophilic cytoplasm due to presence of numerous mitochondria. Mitochondrial DNA analysis of six renal cell oncocytomas and adjacent renal tissue was performed using five restriction endonucleases. In the Hinfl restriction pattern, an additional 40 bp band was noted. This new band was demonstrated in all oncocytomas but in none of the corresponding renal tissues or additionally tested renal carcinomas. By reisolating and hybridizing this band to the mitochondrial genome, its sequence was localized within the cytochrome c oxidase subunit I gene. The data suggest that a molecular alteration of mitochondrial DNA is specific for oncocytomas, and this constitutes the first observed mitochondrial DNA change in a human solid tumor.
Adenoma↗
[Tumor-specific changes in the mitochondrial genome of oncocytomas].
Two astrocytomas, 5 colon carcinomas, 3 clear cell carcinomas of kidney, 5 renal oncocytomas and 1 oxyphile adenoma of thyroid were examined for mitochondrial DNA-alterations applying 5 different restriction enzymes (Alu I, Hae III, Hpa II, Rsa I, Hinf I). Only by applying Hinf I restriction enzyme digestion we were able to prove alterations of the mitochondrial DNA in oncocytomas (2 additional bands of about 55 or 35 base pairs). This was the first proof of mitochondrial DNA-alteration in a human tumor. The present sequence analyses of DNA-alteration will help to explain which importance is attached to the alterations regarding genesis of oncocytic tumors.
Adenoma↗
An efficient salt-chloroform extraction of DNA from blood and tissues.
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Cells, Cultured↗
Renal oncocytoma. A phenotypic and genotypic entity of renal parenchymal tumors.
The light and electron microscopic morphology of two renal parenchymal tumors was consistent with the diagnosis of renal oncocytoma. Both tumors had a mosaic chromosome pattern of cells with normal and abnormal karyotypes. No recurrent chromosome aberration and also no rearrangement of chromosome 3p was found. Restriction analysis of the mitochondrial DNA revealed a new autoradiographic band at about 50 basepairs in size occurring exclusively in oncocytomas. The possible use of these findings in the diagnosis of renal oncocytomas is discussed.
Adenoma↗
Rapid step-gradient purification of mitochondrial DNA.
A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5'-end labeling, gel retention assays, and various types of hybridization.
Animals↗
Increased amount of a mitochondria-associated 21 x 10(3) dalton protein in human gastrointestinal tumors.
Differences in the structure and number of mitochondria in tumor cells were found. Using isoelectrofocusing two-dimensional polyacrylamide gel electrophoresis which allows detection of alterations in the protein pattern of tumor mitochondria, we studied both quantitative and qualitative changes in the mitochondrial protein pattern of human gastrointestinal tumors and corresponding normal matrix tissues. One low molecular protein spot was found to be quantitatively changed in the tumors. The approximate molecular weight was 21 x 10(3) daltons and the pI value 5.7.
Adult↗
[Congenital posterior dislocation of the radius head among members of one family].
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Adult↗
Expression of the c-erbB2 proto-oncogene in male breast carcinoma: lack of prognostic significance.
Increase of gene activity of the proto-oncogene erbB2 which codes for the transmembrane kinase receptor p185erbB2 has been observed in > 30% of female breast and gynecological carcinomas. This overexpression was shown to be correlated with poor prognosis. We have investigated 38 samples of carcinomas of the male breast for p185erbB2 expression by using tumor thin sections and a monoclonal antibody. The immunostaining was compared to clinical data to assess a possible prognostic value of this parameter. Although most cases were immunopositive (36/38), no correlation to tumor grading and survival spans was notable. Therefore, erbB2 activity fails to add a new prognostic parameter in male breast carcinomas.
Biomarkers, Tumor↗
Overexpression of nm23-H4 RNA in colorectal and renal tumours.
A new member of the nm23 gene family, designated nm23-H4, was cloned recently. Nm23-H4 is a mitochondrial protein. To determine whether nm23-H4 could have a function in tumour progression like nm23-H1 or nm23-H2, we analysed nm23-H4 expression in 18 renal tumours and 42 colorectal carcinomas obtained from patients who underwent curative resection. As compared to the corresponding healthy tissue, 14 renal tumours and 41 colorectal carcinomas showed increased nm23-H4 mRNA expression. While the increase was only moderate in renal cell carcinoma, a strong overexpression of nm23-H4 was noted in most colorectal carcinomas, possibly indicating a role of this gene in tumour genesis. The nm23-H4 transcript levels did not correlate with tumour stage, grade of differentiation or lymph node involvement.
Adenocarcinoma, Clear Cell↗