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Biomedical subjects

G Winberg

Publications and source records attributed to G Winberg.

At least 37 records · Page 2Linked to original sources
PubMed1995

Parallel existence of Epstein-Barr virus (EBV) positive and negative cells in a sporadic case of Burkitt lymphoma.

In the Burkitt lymphoma line Oma-BL1, EBV positive and negative cells coexist. We demonstrate that EBV positive and negative subclones are identical with respect to chromosome markers and HLA type and that the same c-myc rearrangement occurs in all the subclones. This shows that the tumor cells are derived from the same patient and are of monoclonal origin. In the positive subclones, the EBV genome was stably maintained in the episomal form. The EBV negative subclones could be derived from previously uncloned tumor cells in early passage, but not from the EBV positive subclones.

Adolescent↗
PubMed1995

A group of NotI jumping and linking clones cover 2.5 Mb in the 3p21-p22 region suspected to contain a tumor suppressor gene.

The chromosomal region 3p21.2-p22 has been shown to be involved in the development of several forms of solid tumors. Such deletions, translocations, and rearrangements presumably result in the disturbance or loss of a critical gene function. Pulsed-field gel electrophoresis (PFGE), using NotI linking clones as a probe represent a powerful tool for analyzing such rearrangements. A NotI linking clone, AP20 (D3S1641), was localized by in situ hybridization to 3p21.3-p22. Two NotI jumping clones adjacent to this clone were isolated, clone J32-612 covering 0.5 Mb and clone J31-611 covering approximately 1 Mb. Clone J31-611 crosses the border of the deletion present in hybrid cell line MCH939.2, which contains a deleted 3p21 region. For these jumping clones, corresponding NotI linking clones, NLJ3 (D3S1642) and NL3-003, were isolated. Altogether, linking and jumping clones from the AP20 locus hybridize to NotI fragments totaling 2.5 Mb in length. These NotI-containing clones detect expressed sequences in several human tissues. Clone NLJ3 possesses homology to the human platelet-derived endothelial cell growth factor gene and may represent a new member of this gene family. Another clone (AP20) revealed 66% sequence similarity to rat skeletal muscle voltage-sensitive sodium channel subtype 2. Therefore, this group of clones will be useful not only for analyzing rearrangements in tumors, but also for the isolation of new genes from the 3p21.3-p22 region.

Base Sequence↗
PubMed1995

Differences in the growth pattern and clinical course of EBV-LMP1 expressing and non-expressing nasopharyngeal carcinomas.

All low differentiated or anaplastic forms of nasopharyngeal carcinoma (NPC) carry multiple copies of EBV-DNA and express EBNA1. The major membrane protein, LMP1, is only expressed in 65% of the tumours. The physiological function of LMP1 in the viral life cycle is unknown, but it has been shown to transform established rodent fibroblasts and immortalised human keratinocytes in vitro, and to increase the likelihood of a malignant transformation. We studied 74 cases collected from the Shanghai and Guanzhou areas in China. LMP1 expression was assessed in tumour biopsies by immunoblotting. Clinical and follow-up data were evaluated according to the classification of WHO. The laboratory and the clinical data were assembled in a mutually independent double blind fashion. Our findings indicate that the LMP1-positive tumours grew faster and more expansively than LMP1-negative tumours, but nevertheless had a better prognosis. LMP1-negative tumours recurred at a higher frequency, and showed an increased tendency to metastasise.

Carcinoma↗
PubMed1995

Antibodies to LMP2A/2B in EBV-carrying malignancies.

Antibodies to the Epstein-Barr virus (EBV)-encoded membrane proteins, LMP2A and LMP2B, were assayed in 540 individuals, including 154 patients with nasopharyngeal carcinoma, 16 with African Burkitt's lymphoma, 113 with Hodgkin's disease, 14 with EBV-carrying gastric carcinoma, 14 with oral hairy leucoplakia (HIV+ patients), 37 with non-Hodgkin's lymphoma, 49 with tumours of the head/neck, 19 with infectious mononucleosis, 62 with chronic illnesses with EBV titres consistent with re-activations, and 62 healthy controls. A novel assay, mouse monoclonal enhanced indirect immunofluorescence assay (MIFA) was designed and used to test the sera for antibodies to the LMP2A and 2B proteins, expressed in human keratinocytes. Antibody to both LMP2A and LMP2B was strikingly specific to NPC. Virtually all (99 of 101) of the LMP2 antibody positive individuals were NPC patients, 95% of whom had antibodies that reacted both with the LMP2A- and LMP2B-transfected indicator cells, while the remaining 5% reacted only with the LMP2B expressing cells.

Antibodies, Viral↗
PubMed1995

Coupled transcription of Epstein-Barr virus latent membrane protein (LMP)-1 and LMP-2B genes in nasopharyngeal carcinomas.

The presence of transcripts of the Epstein-Barr virus genes for Epstein-Barr nuclear antigen (EBNA)-1 and EBNA-2 and for latent membrane protein (LMP)-1, LMP-2A and LMP-2B was investigated in 24 nasopharyngeal carcinoma (NPC) biopsies of Chinese origin and two NPC-derived solid tumour lines, CAO and C15, of Chinese and north African origin respectively, propagated by serial transplantation in nude mice. Transcripts were detected by PCR amplification of cDNA. EBNA-1 transcripts were present in all biopsies tested and originated exclusively from the FQ promoter while the C and W promoters were inactive. Using nested primers, LMP-1 and LMP-2B RNAs were found to be co-ordinately expressed in 22 of the 24 biopsies, while the two remaining tumours were negative for both. LMP-2A transcription was detected in 17 of the 22 LMP-1-positive biopsies. In summary, the following patterns of viral gene expression were observed in the tumour biopsies: (i) LMP-1-LMP-2B and LMP-2A positive (17 biopsies); (ii) LMP-1-LMP-2B positive but LMP-2A negative (five biopsies); (iii) no viral gene other than EBNA-1 expressed (two biopsies).

Animals↗
PubMed1995

A subpopulation of normal B cells latently infected with Epstein-Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1.

Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.

Animals↗
PubMed1994

NotI linking clones as a tool for joining physical and genetic maps of the human genome.

To study the connection among NotI linking clones, CpG islands, and genes, the sequence surrounding 143 NotI sites was determined. These NotI linking clones were isolated from human chromosome 3-specific libraries and contain an average C + G content of 65%. These clones represent sequence-tagged sites that can be positioned onto chromosome maps and used for generating a long-range NotI map of the human genome. A majority (about 90%) of these clones contain transcribed sequences, as detected by Northern blot hybridization, providing an efficient link between physical and functional (genetic) maps. The GenBank nucleotide database was searched with sequences from these NotI linking clones. For many clones, homology was found to human and other vertebrate genes. About 20 clones contained various repeats in their sequences and may represent microsatellite loci. Most of these NotI linking clones therefore represent evolutionarily conserved DNA fragments and also can be used for comparative genome mapping of other mammalian species. In addition, approximately 20% of all sequenced human CpG island-containing genes and more than 12% of all well-characterized human genes were found to possess NotI restriction sites. This is at least 2-5 times more than has been previously estimated and suggests that NotI sites have a much stronger association with genes.

Animals↗
PubMed1994

Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue.

Epstein-Barr virus (EBV)-encoded LMP1 gene derived from a nude mouse passaged nasopharyngeal carcinoma (NPC) of Chinese origin (C-LMP1) and its B cell (B95-8 prototype)-derived counterpart (B-LMP1) were compared for their ability to induce tumour rejection in a mouse mammary adenocarcinoma system. Each of the two LMP1 genes was introduced individually by retroviral vectors into a non-immunogenic mammary carcinoma line, S6C, that originated in an ACA (H-2f) mouse. Syngeneic ACA mice were immunised for 3 consecutive weeks with irradiated B- or C-LMP1 expressors or control cells. The immunised and control mice were then challenged with graded numbers of viable cells from the corresponding cell line. Only the B-LMP1 expressing cells were highly immunogenic. Up to 10(5) cells were rejected in pre-immunised mice, whereas at least 10(2) cells grew in non-immunised controls. No rejection response was detected against the C-LMP1 expressing cells which grew equally well in control and immunised mice, with a minimum inoculum of 10(2) cells in the majority of the clones. In a previous study, we found numerous sequence differences between B- and C-LMP1. The question of whether any of these differences is related to the non-immunogenicity of C-LMP1 needs further investigation. Meanwhile, our findings raise the possibility that the NPC cells may escape host rejection by the development of a non-immunogenic LMP1 variant under the impact of immunoselection.

Animals↗
PubMed1993

Lambda SK diphasmids: phage lambda vectors for genomic, jumping, linking and cDNA libraries.

Fifteen new phage lambda vectors are discussed: lambda SK4, lambda SK6, lambda SK10, lambda SK16, lambda SK17, lambda SK20, lambda SK21, lambda SK22, lambda SK23, lambda SK24, lambda SK25, lambda SK27, lambda SK28, lambda SK40 and lambda SK41. Their structural and functional features facilitate the implementation of a number of new strategies and cloning procedures which simplify, economize and vastly improve the utility and efficiency of library construction in lambda vectors. Such improved strategies and examples of their application, with particular reference to jumping and linking libraries, are presented.

Bacteriophage lambda↗
PubMed1993

Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1.

Two isolates of the EBV-LMP1 gene were compared for their ability to induce phenotypic changes in a non-tumorigenic human keratinocyte line, Rhek-1, immortalized with an adenovirus 12-SV40 hybrid virus. One isolate, designated B-LMP1, was derived from B95-8, a B-cell line of marmoset origin, that carries a viral strain from a mononucleosis patient. The other, designated C-LMP1, originated from a nude mouse passaged Chinese NPC tumor, CAO. Both types of transfectants were less serum dependent than the non-transfected and the vector-transfected controls. The ability to grow on low serum increased with increasing LMP1 expression. All transfectants were more highly clonable than the non-transfected or vector-transfected controls. Clonability in soft agarose increased with increasing LMP1 expression. Nine of 24 C-LMP1 transfectants produced tumors in SCID mice. Seven of them grew invasively into the surrounding tissue. Only one of 12 B-LMP1 transfected Rhek-1 clones was tumorigenic. It did not grow invasively. All tumorigenic transfectants expressed LMP1 at high or moderate levels. All tumors were found to express LMP1. Transfectants with low LMP1 expression did not produce tumors. The untransfected Rhek-1 cells and six vector control clones failed to produce tumors.

Adenoviridae↗
PubMed1991

The epstein-Barr-virus-encoded membrane protein LMP but not the nuclear antigen EBNA-1 induces rejection of transfected murine mammary carcinoma cells.

The EBV-encoded membrane protein (LMP) and the nuclear antigen EBNA-1 were compared for their capacity to induce rejection of transfected murine mammary carcinoma cells in syngeneic hosts. The tumorigenic potential of stable LMP and EBNA-1 expressing sublines of the ACA (H-2f)-derived mammary carcinoma line S6C was tested in pre-immunized syngeneic and semi-syngeneic animals. LMP expressing S6C cells elicited a strong rejection response as demonstrated by the lower tumor take and slower growth in immunized vs. control mice. In contrast, EBNA-1-expressing cells were non-immunogenic in syngeneic hosts and in one semi-syngeneic F1-hybrid. Rejection in 2 additional F1-hybrids did not appear to be due to EBNA-1-specific immune responses. Our findings support the hypothesis that the escape of EBV carrying tumor cells from EBV-specific immune surveillance may be facilitated by the fact that viral gene expression is limited to EBNA-1.

Animals↗
PubMed1991

Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma.

The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.

Amino Acid Sequence↗
PubMed1991

The Epstein-Barr virus BNLF-1 membrane protein LMP1 induces homotypic adhesion mediated by CD11a/CD18 in a murine B-cell line, mimicking the action of phorbol ester.

Expression of the Epstein-Barr virus BNLF1 gene (LMP1) or treatment with 4B-phorbol-12,13-dibutyrate in the murine pro-B-cell line A/J-95 leads to a five- to sevenfold enhancement of homotypic adhesion without significant changes in adhesion molecule expression. Antibody to CD11a inhibits aggregation, indicating that CD11a/CD18-mediated adhesion is a common target for both agents.

Animals↗
PubMed1990

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.

Animals↗
PubMed1990

Expression of the Epstein-Barr virus (EBV)-encoded membrane antigen (LMP) increases the stimulatory capacity of EBV-negative B lymphoma lines in allogeneic mixed lymphocyte cultures.

Epstein-Barr virus (EBV)-negative Burkitt lymphoma (BL) lines are poor stimulators in allogeneic mixed lymphocyte cultures compared to EBV-transformed lymphoblastoid cell lines derived from the same individuals. We have previously shown that the stimulatory capacity of the tumor cells is increased after EBV conversion (Avila-Carino et al., Int. J. Cancer 1987. 40: 691). As a first step towards the identification of the viral gene product responsible for this change we have studied the influence of the EBV latent membrane protein (LMP) on the stimulatory capacity of the EBV-negative BL lines BL41 and DG75 and the B lymphoma line BJAB. Four LMP-transfected sublines of BL41, four DG75 LMP transfectants and one LMP-transfected subline of BJAB showed a significantly stronger stimulatory capacity than the original line. The effect was directly proportional to the amount of LMP detected in each transfectant but was not due to reactivation of LMP-specific memory cells since lymphocytes from EBV-seropositive and -seronegative individuals responded equally. In order to define the relation between LMP expression and induction of stimulatory capacity, DG75 was transfected with constructs containing the LMP gene under the control of an heat-shock promoter. The peak of LMP expression in heat shock-treated cells preceded the appearance of stimulatory capacity by 6-12 h suggesting that critical amounts of the protein may be required to induce the phenotypic change recognized by the T cells. LMP influenced in a dose-dependent manner the expression of the adhesion molecules LFA-1, LFA-3 and ICAM-1 and B cell activation markers CD23 and CD39 in transfected sublines of BL41, but did not affect the expression of these markers in the DG75 and BJAB cell line. All LMP-expressing transfectants showed an increased capacity to form conjugates with unprimed allogeneic lymphocytes.

Antibodies, Monoclonal↗
PubMed1989

Efficient retroviral transfer of a mouse c-myc construct into HL60.

We introduced an LTR-driven mouse c-myc second and third exon, Tn5Neo gene construct into the inducible human leukemia line HL60 using an amphotropic retroviral vector system. Over 90% of the cells became neo-resistant and the transfected myc gene was transcribed in several neomycin resistant clones. Making use of the simultaneous presence of the different myc genes in the same cell, we compared expression of the corresponding mRNAs after differentiation and their decay mechanisms.

Animals↗