Biomedical subjects

H Diggelmann

Publications and source records attributed to H Diggelmann.

At least 37 records · Page 2Linked to original sources

PubMed1993

The mouse mammary tumor virus long terminal repeat encodes a 47 kDa glycoprotein with a short half-life in mammalian cells.

The mouse mammary tumor virus proviral DNA contains an open reading frame in the 3' long terminal repeat which can code for a 36 kDa polypeptide with a putative transmembrane sequence and five N-linked glycosylation sites. This gene is known to code for a superantigen which deletes a specific subset of CD4+ T lymphocytes in vivo. The superantigen encoded by the exogenous mouse mammary tumor virus of the GR strain acts specifically on V beta 14 bearing T cells. We produced recombinant vaccinia viruses to express either the complete or a truncated ORF protein after infection of primate cells in culture. The complete ORF gene in mammalian cells leads to the production of a 47 kDa protein which is specifically detected with an anti-ORF-peptide antiserum. The 47 kDa protein can be labeled with D-[2-3H]mannose and its synthesis is inhibited by tunicamycin, an N-linked glycosylation inhibitor, indicating that it is a glycoprotein. The truncated ORF protein beginning at the second ATG of the open reading frame is also modified, but the C-terminal half of ORF, starting at the fifth ATG, has the expected size of the non modified polypeptide. Pulse-chase experiments indicate that the ORF protein has a short half-life of about 1.5-2 hr.

Animals↗

PubMed1993

Detection of bcl-2/JH rearrangement in follicular and diffuse lymphoma: concordant results of peripheral blood and bone marrow analysis at diagnosis.

The capacity to detect t(14;18) breakpoints in non-Hodgkin's lymphoma (NHL) peripheral blood and bone marrow was studied by DNA PCR. We studied 33 patients with follicular lymphoma (FL) (Working Formulation subtypes B, C, D) and 38 patients with intermediate-grade NHL (subtypes F, G). In the FL subgroup, 86% of the morphologically-positive bone marrow patients had amplifiable t(14;18) breakpoints by PCR. Remarkably, of 19 FL patients with 'negative' bone marrows, 11 (58%) were PCR-positive. In addition, half of the early clinical stage patients (I and II) had detectable breakpoints in their bone marrow DNA. Samples from NHL patients with intermediate-grade disease exhibit the same phenomena but at a considerably lower frequency. Paired peripheral blood and bone marrow samples were available at diagnosis in a subset of 56 patients. The concordance between bone marrow and peripheral blood PCR findings was high, with peripheral blood of 55/56 showing the same PCR results as the corresponding bone marrow.

Base Sequence↗

PubMed1992

Expression pattern of mouse mammary tumor virus in transgenic mice carrying exogenous proviruses of different origins.

To study the tissue specificity of mouse mammary tumor virus (MMTV) gene expression, we developed two series of transgenic mice, containing the MMTV proviral DNA of mammary (GR) and kidney (C3H-K) origin. The expression pattern in the MMTV(GR) transgenic mice is very similar to that observed in infected animals, e.g., a strong preference for viral expression in the lactating mammary glands and lower levels of expression in salivary glands, lymphoid tissues, and male reproductive organs. One line of transgenic mice carrying the C3H-K provirus has a similar expression pattern, indicating that MMTV(C3H-K), despite a striking alteration in the U3 region of its long terminal repeat, can be expressed in the same tissues as the wild-type MMTV.

Animals↗

PubMed1992

Expression of the mouse mammary tumor virus ORF gene in cultured cells.

We have recently shown that expression vectors harboring the open reading frame of the long terminal repeat region of mouse mammary tumor virus direct the synthesis of a product which acts as a superantigen in transgenic mice. The detection of the ORF protein has been hampered by the extremely low levels of expression observed in these mice, as estimated from the low levels of specific mRNA. To study the properties of the ORF protein, we attempted its expression in different cell types in culture. The experiments performed in yeast show that the ORF gene product is a glycoprotein of approximately 45 kDA. As expected from the derived primary sequence, the unglycosylated product made in the presence of tunicamycin has a molecular weight of 36 kDA. No secretion of the glycosylated protein was observed. Curiously, the full-length molecule was made in lower amounts than a truncated version which contains only the C-terminal half of the protein. Transfection experiments in different mammalian cells suggest that high expression of the ORF protein might have an adverse effect on survival of cells in culture.

Amino Acid Sequence↗

PubMed1990

Tissue-specific factors and glucocorticoid receptors present in nuclear extracts bind next to each other in the promoter region of mouse mammary tumor virus.

Nuclear extracts from different mouse tissues have been used to study the interaction of factors with the steroid-inducible promoter of mouse mammary tumor virus. In addition to the glucocorticoid receptor that interacts with the distal region of the promoter, several tissue-specific proteins were found to bind to the 5' flanking region of the receptor-binding site and to the basal promoter region. The differences in the pattern of protection observed suggest that tissue-specific factors might co-operate with steroid receptors and result in a cell-type-dependent modulation of hormone action.

Animals↗

PubMed1990

Cellular and tumoural heterogeneity of EGFR gene amplification in human malignant gliomas.

It has been demonstrated that the epidermal growth factor receptor (EGFR) gene, the normal human counterpart of the viral erb-B oncogene is amplified and overexpressed in over 50% of human malignant gliomas (HMGs). In the present study, analysis of the immunohistological staining characteristics of 57 HMGs using an anti-EGFR monoclonal antibody (mab) showed positive staining in 65% of the tumours with large cellular and regional differences in staining pattern and intensity. Screening a smaller number of HMGs with molecular hybridization techniques revealed 10/21 glioblastomas (48%) amplified for the gene; of 11 glioblastomas studied by Northern blot hybridization, 7 tumours with gene amplification showed RNA overexpression, the remaining 4 without amplification did not. Regional differences in DNA levels were observed by Southern blot in 2 tumours: in one particular case, amplification and overexpression were found to be localized to one half of a single HMG, the other half showing neither EFGR gene amplification nor overexpression.

Blotting, Northern↗

PubMed1989

Dual function of a nuclear factor I binding site in MMTV transcription regulation.

Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk- fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element.

Animals↗

PubMed1989

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.

Animals↗

PubMed1988

Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus.

We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells.

Base Sequence↗

PubMed1987

Prognostic implications of expression of the cellular genes myc, fos, Ha-ras and Ki-ras in colon carcinoma.

Messenger RNA levels of the c-fos, c-myc, c-Ha-ras and c-Ki-ras genes were studied in 39 tissue samples obtained from 17 patients undergoing surgery for colon carcinoma and other colon diseases. DNA extracted from the same samples was studied by Southern analysis. The tissues were tumors and grossly normal mucosa from each case and in some instances benign polyps and metastases. Our results indicate: (1) that 50% of cases studied show an increase in expression of at least one of the oncogenes studied; (2) that over-expression is not random, some cases over-expressing several of the genes studied; (3) that the expression pattern of the oncogenes studied varies between primary tumor and metastases; (4) that amplification is a rare event, being limited to one instance in which c-myc was amplified in a metastasis; (5) that cases which exhibit high levels of mRNA in one or more genes studied correlate with biologically aggressive tumors; and (6) that "non-expressors" are at higher risk for local recurrence based on correlations with mucin histochemistry.

Adenocarcinoma↗

PubMed1986

Functional analysis of the glucocorticoid regulatory elements present in the mouse mammary tumor virus long terminal repeat. A synthetic distal binding site can replace the proximal binding domain.

Transcription of mouse mammary tumor virus DNA is stimulated by steroid hormones. The DNA sequences involved in this regulation are located in the viral long terminal repeat between positions -200 and -50 with respect to the transcription initiation site. In this region four, one distal and three proximal, in vitro binding sites for the glucocorticoid hormone-receptor complexes have been identified. We have prepared a series of 5' and 3' deletions of this region, using the exonuclease ExoIII. Combination of suitable 5' and 3' fragments enabled us to reconstitute the entire long terminal repeat with small internal deletions. The mutated long terminal repeats linked to the coding region of the Herpes simplex virus thymidine kinase gene were introduced into LTK- aprt- cells by transfection. Transcription from the mouse mammary tumor virus promoter in the presence or absence of hormone was assayed by nuclease S1 mapping. Deletion of the proximal in vitro binding sites resulted in a decrease in hormonal inducibility. When a synthetic oligonucleotide harboring the sequence of the distal in vitro binding site was inserted at the site of the proximal ones, hormone response was restored. This indicated that the distal binding site can replace the proximal ones in their hormone-regulatory function. However, insertion at the same site of an oligonucleotide containing the sequence 5' TGTTCT 3' found in all four binding sites, did not restore the hormone response, indicating that sequences flanking the TGTTCT motif are required for hormone response. Insertion of an unrelated DNA fragment at the site of the proximal binding element deletion completely abolished the hormone response. Analyses of different proximal binding-site deletion and insertion mutants suggested the presence of a transcriptional element located downstream from the most proximal hormone-receptor binding site.

Animals↗

PubMed1986

A new site of integration for mouse mammary tumor virus proviral DNA common to BALB/cf(C3H) mammary and kidney adenocarcinomas.

The BALB/cf/Cd substrain of mice, developed by inbreeding and selection, shows a 70% incidence of spontaneous kidney adenocarcinomas. Initially foster-nursed on C3H mothers, these mice no longer produce mammary tumors, but there is evidence that mouse mammary tumor virus (MMTV) is involved in the formation of these renal carcinomas. We identified a chromosomal region called int-41, representing a locus interrupted by the integration of an exogenous MMTV provirus in a BALB/c mammary tumor. We found a DNA rearrangement and transcriptional activation in the domain specified by the int-41 probe in one primary kidney adenocarcinoma. A 5.2-kb int-41 specific mRNA was detected in the kidney tumor cell line established from a transplantable renal adenocarcinoma. This mRNA hybridized with MMTV and int-41 specific probes suggesting that it is a hybrid molecule composed of MMTV-LTR sequences covalently linked to host cell RNA. This mRNA was strongly stimulated by the presence of glucocorticoid hormone in the culture medium. Our data are compatible with the hypothesis that the int-41 chromosomal domain is involved in the neoplastic transformation of epithelial cells from different organs.

Adenocarcinoma↗

PubMed1986

Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat.

Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses.

Adenocarcinoma↗

PubMed1986

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

Animals↗

PubMed1984

[Mechanisms of activation and function of oncogenes].

Transforming genes (oncogenes) were first identified and characterized as genetic elements of tumorviruses. It has been demonstrated that these genes are derived from cellular genes by transduction. The corresponding cellular genes are ubiquitous in nature and have been well conserved during evolution. It is therefore very likely that they are important elements in normal regulation of cell growth, e.g. during development. Several mechanisms of oncogene activation have been observed. Qualitative as well as quantitative changes in oncogene expression lead to cell transformation. According to their mechanism of action, at least 4 functional groups of oncogenes can be distinguished. It has recently been shown that the transformation of a normal cell into a tumor cell proceeds in several steps involving different oncogenes.

Animals↗

PubMed1984

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

Cell Line↗

PubMed1984

Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.

Adenine Phosphoribosyltransferase↗

PubMed1983

Nucleotide sequence of the 5' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5' splicing site for subgenomic mRNAs.

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).

Amino Acid Sequence↗