Volume 48 (2002) No. 1
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Review
Microdissection Techniques for Cancer Analysis
A. CÖR1,2, N. VOGT2, B. MALFOY2...............................................3
1Institute for Histology and Embryology, Medical Faculty, Ljubljana, Slovenia
2Institut Curie, UMR 147, Cytogenetique Moleculaire et Oncologie, Paris, France
Corresponding Author: Andrej Cör, Institute for Histology and Embryology, Medical Faculty, Korytkova 2,
1000 Ljubljana, Slovenia. Tel.: +386 (1) 543-7381; Fax.: +386 1) 543-7361; e-mail: andrej.coer@mf.uni-lj.si.
Abstract.
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Articles
Chromosomal Mapping of HCaRG, a Novel Hypertension-Related, Calcium-Regulated Gene
N. SOLBAN1, P. DUMAS1, F. GOSSARD1, Y. SUN1, M. PRAVENEC2,3, V. KREN2,3,
R. LEWANCZUK4, P. HAMET1, J. TREMBLAY1..........................9
1Centre Hospitalier de l’Université de Montréal, Montréal, Canada
2Institute of Physiology, Academy of Sciences, Prague, Czech Republic
3Institute of Biology and Medical Genetics, 1st Medical Faculty, Charles University, Prague, Czech Republic
4Department of Endocrinology, University of Alberta, Edmonton, Canada
Corresponding author: Johanne Tremblay, Laboratory of Cellular Biology of Hypertension, Centre Hospitalier de l’Université de Montréal (CHUM), Campus Hôtel-Dieu 3840 St. Urbain St., Montréal, Québec H2W 1T8, Canada. Tel.: (514) 890-8000 (ext. 12721); fax: (514) 412-7152; e-mail: johanne.tremblay@umontreal.ca.
Abstract.
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Protooncogene Bcl-2 Induces Apoptosis in Several Cell Lines
M. KALBAČOVÁ, M. VRBACKÝ, Z. HUMLOVÁ, Z. MĚLKOVÁ......................15
Department of Pathological Physiology, 1st Medical Faculty, Charles University, Prague, Czech Republic
Corresponding author: Zora Mělková, Department of Immunology and Microbiology, Charles University, 1st Medical Faculty, Studničkova 7, 128 00, Prague 2, Czech Republic. Fax : +420 (2) 24913110; e-mail: zmelk(zavináč)lf1.cuni.cz.
Abstract.
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The Regulatory Region of Prague C v-Src Inhibits the Activity of the Schmidt-Ruppin A v-Src
Kinase Domain
J. BRÁBEK, D. MOJŽITA, L. HAMPLOVÁ, P. FOLK.....................................28
Department of Physiology, Charles University, Prague, Czech Republic
Corresponding author: Petr Folk, Department of Physiology, Charles University, Viničná 7, 128 00 Praha 2, Czech Republic. Tel.: +420 (2) 2195 3147; Fax: +420 (2) 2195 3242; e-mail: dicty@mbox.cesnet.cz.
Abstract.
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Short Communication
Genomic Organization of the Mouse src Gene. Sequencing of src Introns Revealed a New Chromosome 2 Microsatellite Marker
V. FUČÍK1, J. BERAN1, Z. ČERNÝ1, J. MÁCHA2, J. JONÁK1.......................34
1Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
2Faculty of Science, Charles University, Prague, Czech Republic
Corresponding author: Vladimír Fučík, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, Prague 6, Czech Republic. Fax: +420 (2) 33331274; e-mail: fucik@img.cas.cz.
Abstract.
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Monoclonal Antibody Register
New Monoclonal Antibody to Human Apolipoprotein J
J. ČAPKOVÁ, G. GEUSSOVÁ, J. PĚKNICOVÁ
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author: Jana Čapková, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic. Fax: 420 (2) 444 717 07; e-mail: capkova@biomed.cas.cz.
Abstract.
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Microdissection Techniques for Cancer Analysis
A. CÖR, N. VOGT, B. MALFOY
One difficulty in studying molecular changes of tumours has been the inability to isolate DNA and RNA from a homogeneous cell population. The combination of several new technologies should help overcome these hurdles. Microdissection is a technique for rapid and easy procurement of a pure cellular subpopulation away from its complex tissue milieu. Laser-assisted microdissection has recently been identified as a quick, simple and effective method by which microdissection of complex tissue specimens can be routinely performed for molecular analysis. With the advent of laser microdissection, cDNA libraries can be developed from pure cells obtained directly from stained neoplastic tissue, and microarrays of thousands of genes can now be used to examine gene expression in microdissected tumour tissue samples. This review will concentrate on the application of different microdissection techniques in the area of cancer research.
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Chromosomal Mapping of HCaRG, a Novel Hypertension-Related, Calcium-Regulated Gene
N. SOLBAN, P. DUMAS, F. GOSSARD, Y. SUN, M. PRAVENEC, V. KREN,
R. LEWANCZUK, P. HAMET, J. TREMBLAY
We recently identified a novel gene that is negatively regulated by extracellular calcium concentration with higher
levels of transcripts in hypertensive animals (SHR). We named this gene HCaRG (Hypertension-related, Calcium-Regulated Gene). In this work we report the chromosomal localization of the HCaRG gene among
different species. We identified a BglII RFLP between BN.lx and SHR rats. We then analysed the strain distribution pattern of this RFLP in 31 RIS, originating from BN.lx and SHR rats, and compared it to the segregation of 475 markers localized in the rat genetic map. Hcarg localizes to the rat chromosome 7 between the markers Mit3 and Mit4. This region is homologous to human chromosome 8q21-24. We identified three clones in Genbank that contain the sequence of HCaRG. It was therefore possible to narrow down the localization of human HCaRG to chromosome 8q24.3. Furthermore, a suggestive localization of mouse Hcarg based on conservation of linkage between human and mouse is on chromosome 15. We previously identified a putative calcium-binding motif (EF-Hand) and a nuclear receptor-binding domain (LxxLL) in the rat sequence of the HCaRG protein. Sequence comparison between five different species showed that these domains are highly conserved. Furthermore, a search of ESTs in Genbank for homologous sequences showed that HCaRG is expressed only in eukaryotes, particularly in mammals.
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M. KALBAČOVÁ, M. VRBACKÝ, Z. HUMLOVÁ, Z. MĚLKOVÁ
Using a recombinant vaccinia virus expressing protooncogene Bcl-2, we demonstrate opposite effects of the expressed Bcl-2 in two cell lines: apoptosis induction in BSC-40 cells and apoptosis prevention in HeLa G cells. The apparent molecular weight of the expressed Bcl-2, its amounts and its effects on the mitochondrial membrane potential are comparable in both cell lines, suggesting that the consequences of Bcl-2 expression depend on cellular environment. To further support these findings we demonstrate the pro-apoptotic effect of the expressed Bcl-2 in several other cell lines.
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Kinase Domain
J. BRÁBEK, D. MOJŽITA, L. HAMPLOVÁ, P. FOLK
Existing variants of the oncogene v-src differ in their transforming potential as well as in the range of their hosts. We compared the protein kinase activities of two Prague C v-Src variants (PRC and H19), reported to be of low oncogenic potential (Plachý et al., 1995), with the highly oncogenic Schmidt-Ruppin A v-Src (SRA). We employed in vitro kinase assays of affinity-purified proteins expressed in rabbit reticulocyte lysate and in S. cerevisiae. In both systems used, the specific kinase activity of the Prague C v-Src kinases amounted to only ca 20% of the activity of SRA. This positions the PRC Src close to activated c-Src, despite the lack of the regulatory C-terminal tail in PRC. We constructed chimeras between PRC and SRA v-Src and tested them for specific kinase activity in S. cerevisiae. Remarkably, the regulatory N-terminal part of PRC, when fused to the SRA-derived kinase domain, lowered the chimeras’ PK activity to ca 20%, suggesting that it is the regulatory part of PRC that is responsible for its low phosphotransferase activity.
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Genomic Organization of the Mouse src Gene. Sequencing of src Introns Revealed a New Chromosome 2 Microsatellite Marker
V. FUČÍK, J. BERAN, Z. ČERNÝ, J. MÁCHA, J. JONÁK
Ten introns interrupting the coding sequence of the mouse src protooncogene were sequenced (in total 11260 bp) and their general characteristics compared with the homologous genes in human and chicken. While the study of genome organization of the src gene was performed only in the inbred mouse strain BALB/cHeA (Mus musculus domesticus), one special region in the intron 5 was also sequenced in additional mouse strains (M. musculus musculus and M. spretus), because the discovered CA and GT repeat array differences could serve as a new polymorphic marker in the chromosome No. 2 and help elucidate some evolutionary relations between mouse strains.
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New Monoclonal Antibody to Human Apolipoprotein J
J. ČAPKOVÁ, G. GEUSSOVÁ, J. PĚKNICOVÁ
Apolipoprotein J (apoJ) is a 70 kDa glycoprotein associated with high-density lipoproteins (HDL) in human plasma (de Silva et al., 1990a). HDL transport more than 80% of the plasma cholesterol from extrahepatic tissues to the liver for excretion (Glomset, 1968).
Wide tissue distribution of apoJ was determined by isolation of apoJ mRNA from a variety of human tissues. The presence of apoJ mRNA was mainly detected in the steroidogenic tissues, testis and ovary, and also in the brain (de Silva et al.,1990b).
The apoJ molecule is a dimer which consists of two disulfide-linked subunits designated apoJalfa (34–36 kDa) and apoJbeta (36–39 kDa) (de Silva et al., 1990c). For purification and characterization of the apoJ molecule, monoclonal antibodies were utilized. Six monoclonal antibodies specific for apoJ were generated and described (de Silva et al., 1990a, 1990c).
ApoJ has extensive similarities to the human protein designated as SP-40,40 (or complement lysis inhibitor (CLI)) (Choi et al., 1989) and human TRPM-2/clusterin (Wong et al., 1994). The presence of SP-40,40 was also demonstrated within human seminal plasma at levels comparable to those in serum (Kirszbaum et al., 1989).
Both these proteins, SP-40,40 and TRFM-2/clusterin, have been implicated in a variety of physiological processeses, including sperm maturation, lipid transport, membrane remodelling and inhibition of the complement cascade (Wong et al., 1994).
ApoJ is also secondarily incorporated into the sperm membrane, as sperm travel through the male reproductive tract, and belongs to the sperm-coating proteins. Among monoclonal antibodies against human sperm proteins that we generated, a monoclonal antibody that specifically recognized apoJ was found. The monoclonal antibodies was designated Hs-3.
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