Biomedical subjects

A Wennborg

Publications and source records attributed to A Wennborg.

14 recordsLinked to original sources

PubMed2001

Effects of HsRad51 overexpression on cell proliferation, cell cycle progression, and apoptosis.

Expression of the DNA repair and recombination protein human Rad51 (HsRad51) is increased in transformed cells and in cancer cell lines. In order to study the effects of acute HsRad51 ectopic overexpression on cell proliferation, cell cycle progression, and apoptosis, we generated clones of the human fibrosarcoma cell line HT1080 carrying a HsRad51 transgene under a repressible promoter. The HsRad51-overexpressing cells showed decreased plating efficiency and growth rate in a dose-dependent manner with regard to the degree of overexpression. An accumulation of HsRad51-overexpressing cells in G(2) was observed following release of cells after synchronization with double thymidine block. Moreover, the fraction of apoptotic cells measured by annexin V-FACS increased with the time of HsRad51 overexpression. In the light of these observations, sustained increased levels of HsRad51 may contribute to tumor progression by causing a selection for cells tolerant to the growth-suppressive and apoptosis-inducing effects of acute HsRad51 overexpression.

Annexin A5↗

PubMed2001

Novel candidate genes for atherosclerosis are identified by representational difference analysis-based transcript profiling of cholesterol-loaded macrophages.

OBJECTIVES: To analyze the early gene expression in macrophages accompanying the phenotypic changes into foam cells upon exposure to oxidized low-density lipoprotein. To identify candidate genes and markers for further studies into the pathogenesis of atherosclerosis. METHODS: Cells of the monocytic cell line THP-1 were activated by PMA and exposed to oxidized low-density lipoprotein. Gene expression profiles were investigated after 24 h, using a solid phase cDNA representational difference analysis (RDA) method and shotgun sequencing. Results were verified by microarray hybridization, and analyzed in the virtual chip display of a novel software tool for transcript profile exploration. RESULTS: By comparing transcript profiles of exposed/unexposed cells, 1,984 transcript sequences, representing a total of 921 genes with altered expression levels in response to oxidized low-density lipoprotein exposure, were identified. Genes that are central to cell cycle control and proliferation, inflammatory response, and of pathways not previously implicated in atherosclerosis were identified. The data obtained is also made available on-line at http:// biobase.biotech.kth.se/thp1a for further exploration. CONCLUSION: The identification of new candidate genes for atherosclerotic disease through RDA-based transcript profiling facilitates further functional genomic studies in coronary artery disease. Candidate genetic polymorphism markers of potential clinical relevance can be identified by filtering information in genome variation databases through the virtual chip analysis of the transcript profiles and subsequently tested in association studies.

Arteriosclerosis↗

PubMed2000

Identification of in vivo mutations in exon 5 of the human HPRT gene in a set of pooled T-cell mutants by constant denaturant capillary electrophoresis (CDCE).

Constant denaturant capillary electrophoresis (CDCE), based on co-operative DNA melting equilibria, has the resolving power to separate single nucleotide mutants from wild type sequences. We used this technique to study mutations in a 70-bp isomelting domain of the human HPRT gene, which included the entire exon 5 and its flanking splice donor and acceptor sites. Pooled samples of 6-thioguanine selected T-cell clones from 51 healthy donors representing a total of approximately 1000 individual HPRT mutants were analysed. Slow moving peaks from the heteroduplex part of the CDCE electropherograph were collected and subjected to a second round of PCR and CDCE analysis, followed by DNA sequencing. Five independent mutations were detected. Four were splicing errors; one insertion of CC and two G-->A transitions in the splice donor site of intron 5, and one G-->C transversion in the splice acceptor site of intron 4. The fifth mutation was a missense transversion, T389>G. A reconstruction experiment, in which DNA with known mutation was mixed with wild type DNA, showed the sensitivity of mutation detection to be better than 1:100 under the conditions used in this study. These results demonstrate the high sensitivity of the CDCE-method for mutation screening.

Base Sequence↗

PubMed2000

Expression profile viewer (ExProView): a software tool for transcriptome analysis.

A software tool, Expression Profile Viewer (ExProView), for analysis of gene expression profiles derived from expressed sequence tags (ESTs) and SAGE (serial analysis of gene expression) is presented. The software visualizes a complete set of classified transcript data in a two-dimensional array of dots, a "virtual chip," in which each dot represents a known gene as characterized in the transcript databases Expressed Gene Anatomy Database or UniGene. The virtual chip display can be changed between representations of different conceptual systems for gene/protein classification and grouping. Four alternative projections are currently available: (i) cellular role, (ii) subcellular compartment, (iii) chromosome localization, and (iv) total UniGene display. However, the chip can be adapted to any other desired layout. By selecting dots, further information about the represented genes is obtained from the local database and WWW links. The software thus provides a visualization of global mRNA expression at the descriptive level and guides in the exploration of patterns of functional expression, while maintaining direct access to detailed information on each individual gene. To evaluate the software, public EST and SAGE gene expression data obtained from the Cancer Genome Anatomy Project at the National Center for Biotechnology Information were analyzed and visualized. A demonstration of the software is available at http://www.biochem.kth. se/exproview/.

Data Display↗

PubMed2000

Caspase-3 mediated cleavage of HsRad51 at an unconventional site.

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.

Adenocarcinoma↗

PubMed1998

Radiation induced chromosomal instability in human T-lymphocytes.

Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G0-lymphocytes exposed to gamma-irradiation at both high dose rate (45 Gy h-1) and low dose rate (0.024 Gy h-1) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G0 after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.

Chromosome Aberrations↗

PubMed1998

Proteolytic cleavage of HsRad51 during apoptosis.

The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.

Antibodies, Monoclonal↗

PubMed1996

RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft Windows.

The use of algorithms for calculation and analysis of RNA secondary structures has largely been limited to mainframe computers, mainly due to the 16-bit memory restrictions imposed by MS-DOS. The program presented here, RNAdraw, moves calculations to the 32-bit Microsoft Windows environments with an intuitive user interface with extensive viewing, editing and printing possibilities. The algorithms for secondary structure/basepair probability matrix/heat curve calculation have been ported directly from a 32-bit Unix environment. RNAdraw also offers novel features such as the options to edit energy parameters, extract structures of different probability levels, create de novo secondary structures interactively, and combine viewing of structures and basepair probabilities.

Algorithms↗

PubMed1995

A human RNase E-like activity that cleaves RNA sequences involved in mRNA stability control.

We have detected an endoribonucleolytic activity in human cell extracts that processes the Escherichia coli 9S RNA and outer membrane protein A (ompA) mRNA with the same specificity as RNase E from E. coli. The human enzyme was partially purified by ion-exchange chromatography, and the active fractions contained a protein that was detected with antibodies shown to recognize E. coli RNase E. RNA containing four repeats of the destabilizing motif AUUUA and RNA from the 3' untranslated region of human c-myc mRNA were also found to be cleaved by E. coli RNase E and its human counterpart in a fashion that may suggest a role of this activity in mammalian mRNA decay. It was also found that RNA containing more than one AUUUA motif was cleaved more efficiently than RNA with only one or a mutated motif. This finding of a eukaryotic endoribonucleolytic activity corresponding to RNase E indicates an evolutionary conservation of the components of mRNA degradation systems.

Base Sequence↗

PubMed1995

Downregulation of c-myc expression after heat shock in human B-cell lines is independent of 5' mRNA sequences.

The effect of heat-shock on the expression of c-myc genes in different chromosomal contexts was investigated in a panel of human B-lymphoid cell lines. Burkitt's lymphoma cell lines with c-myc translocation breakpoints upstream of the first exon, within the exon itself, or in the first intron showed downregulation of c-myc levels as did a cell line without any translocation. The c-myc mRNA of cell lines with translocation breakpoints within the c-myc gene have previously been reported to have prolonged half-lives. After heat shock, the levels of these mRNA species were reduced with similar kinetics as the normal c-myc mRNA. An exception was an RNA species where the only c-myc sequences are derived from exon 1, showing that sequences from this part of the c-myc gene are not sufficient to mediate the rapid downregulation. Nuclear run-on analysis did not show reduced transcription of c-myc after heat shock and a comparison of cytoplasmic and total RNA did not indicate accumulation of longer, unspliced c-myc mRNA species. These observations suggest a posttranscriptional, cytoplasmic downregulation targeting exons 2 and/or 3. B-lymphoma lines transfected with a hsp70 promoter-linked c-myc gene were deficient in their ability to reinitiate proliferation after heat shock, providing a physiological rationale for the normal downregulation of c-myc after this type of physical stress.

B-Lymphocytes↗

PubMed1993

Analysis of c-Myc domains involved in stimulating SV40 replication.

We have demonstrated previously that overproduction of c-Myc, N-Myc and, to a lesser extent, L-Myc facilitates the replication of simian virus 40 (SV40)-based vectors in human lymphoid cells. Using a series of c-myc deletion mutants, we investigated which c-Myc regions are important in stimulating SV40 replication. The ability of c-Myc to promote SV40 replication was significantly reduced by deletions in the second exon domain, formerly shown to be crucial for c-Myc's transforming capacity. The c-myc mutants with a disrupted basic region (b) or leucine zipper (Zip) motif were also unable to stimulate SV40 replication. These regions are implicated in protein-DNA and protein-protein interactions, respectively, suggesting that the c-Myc protein might be associated with the DNA-protein replication complex. We present data obtained from gel mobility shift assays and from an immunocomplex-binding assay substantiating this hypothesis.

Base Sequence↗

PubMed1993

Mapping of the 19p13 breakpoint in an ovarian carcinoma between the INSR and TCF3 loci.

Chromosome rearrangements involving band 19p13 have been described in about half of all ovarian carcinomas. Four ovarian carcinomas with translocations of 19p13 were investigated with 11 DNA probes for markers localized to this band. All markers exhibited normal restriction patterns, suggesting that they were not rearranged. The 19p13 breakpoint of one tumor was mapped to a location between the INSR and the TCF3 loci.

Carcinoma↗

PubMed1988

Surface marker characterization of EBV-carrying cells in blood of healthy seropositive individuals.

We have determined the phenotype of EBV-carrying cells in human blood by establishing spontaneous LCLs from populations selected according to three B cell markers, B1, B2, and 35.1C5. LCLs grew from the B1-positive, B2-positive, B2-negative and 35.1C5-positive population. Thus, EBV-carrying cells in blood of seropositive individuals are restricted to the B cell lineage. These B cells may or may not express the B2 epitope, and they may be derived from the lymph node mantle zone or the splenic marginal zone. These in vivo EBV-carrying cells could enter the viral productive cycle and transform coresident B cells during the initial 3 days in vitro. The characteristic of the LCLs with regard to these 3 B cell markers did not correspond to the original B cell population from which they were derived.

Adult↗

PubMed1987

Conversion of the lymphoma line "BJAB" by Epstein-Barr virus into phenotypically altered sublines is accompanied by increased c-myc mRNA levels.

The steady-state level of c-myc proto-oncogene mRNA was investigated in the EBV-negative human B-lymphoma line BJAB and 2 sublines that have been converted by EBV into stable EBV-genome-carrying and EBNA-positive status. The EBV-converted sublines expressed c-myc at a 2- to 6-fold higher level than the original BJAB during exponential growth. The EBV-positive BJAB lines are known to differ from the parent line in several phenotypic characteristics, including increased agarose clonability, lower serum requirement and, in one case, increased tumorigenicity in nude mice. The pattern of increased c-myc expression accompanying EBV conversion was not observed in the myc/Ig translocation-carrying Burkitt lymphoma line RAMOS or in 2 of its EBV-converted sublines.

Actins↗