Abraham Karpas

Identification and Characterization of a Novel Gene, C13orf25, as a Target for 13q31-q32 Amplification in Malignant Lymphoma

The amplification at 13q31-q32 has been reported in not only hematopoietic malignancies but also in other solid tumors. We identified previously frequent amplification of chromosomal band 13q31-q32 in 70 cases of diffuse large B-cell lymphoma patients by conventional comparative genomic hybridization analysis. In an attempt to identify a candidate gene within this region, we used array comparative genomic hybridization and fluorescent in situ hybridization to map the 13q31-q32 amplicon. We then screened the 65 expressed sequence tags and Glypican 5 (GPC5) by reverse transcription-PCR and Northern blotting. As a result, we identified a novel gene, designated Chromosome 13 open reading frame 25 (C13orf25), which was overexpressed in B-cell lymphoma cell lines and diffuse large B-cell lymphoma patients with 13q31-q32 amplifications. However, GPC5, which has been reported to be a target gene for 13q31-q32 amplification, was truncated in one cell line, Rec1, possessing the amplification, and its expression in various cell lines with amplification at 13q31-q32 was not significantly different from that in other cell lines without amplification, suggesting that GPC5 is not likely to be the candidate gene. Additional analysis identified two major transcripts in the C13orf25 gene. The two transcripts A and B predicted open reading frames of 32 and 70-amino acid polypeptides, respectively. The former has been reported as bA121J7.2, which is conserved among species. Transcript-B also contained seven mature microRNAs in its untranslated region. These results suggest that the C13orf25 gene is the most likely candidate gene for the 13q31-q32 amplicon found in hematopoietic malignancies.

Inhibition of HIV replication by amino-sugar derivatives

The plant alkaloids castanospermine, dihydroxymethyldihydroxypyrrolidine and deoxynojirimycin have recently been shown to have potential anti‐HIV activity [(1987) Proc. Natl. Acad. Sci. USA 84, 8120–8124; (1987) Nature 330, 74–77; (1987) Lancet i, 1025–1026]. They are thought to act by inhibiting α‐glucosidase I, an enzyme involved in the processing of N‐linked oligosaccharides on glycoproteins. We report here the relative efficacy of a spectrum of amino‐sugar derivatives as inhibition of HIV cytopathicity. Several α‐glucosidase inhibitors and α‐fucosidase inhibitors were found to be active at concentrations which were non‐cytotoxic.

The establishment and cytological, cytochemical and immunological characterisation of human haemic cell lines: Evidence for heterogeneity☆

Abstract Continuous tissue culture lines were established from the bone marrow or peripheral blood of 28 patients with haematological malignancies. The morphological and immunological properties of these newly established cell lines were studied after periods of 6 months to 2 years growth in liquid culture. Apart from a single T-cell line, all lines tested proved to be EBNA positive, and all but the T-cell lines and two others showed cell surface characteristics similar to those of blastoid lines of lymphoid origin. Nevertheless, many of the cell lines showed cytochemical positivity for alkaline phosphatase, not normally found in lymphoid precursors, most showed at least some positivity to both α-naphthol AS-D acetate and chloro-acetate esterases at near neutral pH, although not to α-naphthyl butyrate or chloro-acetate at pH 8. Three cell lines produced lysozymes and 10 of 13 lines tested showed phagocytic activity towards bacteria. Lysozyme production and phagocytic activity were found only in cell lines derived from patients with acute myeloid leukaemias, and alkaline phosphatase activity was more common in, though not confined to, such lines. The evidence indicates a wider enzymatic and functional diversity among established human haemopoietic cell lines than has hitherto been suspected.

HUMAN PLASMACYTOMA WITH AN UNUSUAL KARYOTYPE GROWING IN VITRO AND PRODUCING LIGHT-CHAIN IMMUNOGLOBULIN

A new human plasmacytoma line (Karpas 707) was established from the cells of the bone-marrow and peripheral blood of a patient with Bence-Jones-positive multiple myeloma. The cells of this line resembled the patients malignant cells and fresh myeloma cells from nine other patients in being Epstein-Barr viral nuclear antigen negative. They looked like plasma cells and they secreted only lambda light chains. Both these cells and the patients fresh cells were hypodiploid and contained the Philadelphia chromosome and several other markers. This line seems to be the first human plasmacytoma line since the IgE lambda-producing line (U266) established in 1968, and it may be suitable for the production of human monoclonal antibodies.

GPC5 is a possible target for the 13q31-q32 amplification detected in lymphoma cell lines

AbstractComparative genomic hybridization (CGH) analyses have detected gains of copy number on 13q, especially at 13q31-q32, in cell lines and primary cases of various types of lymphoma. Since amplification of chromosomal DNA is one of the mechanisms that can activate tumor-associated genes, and because 13q amplification had been reported in various other types of tumors as well, we attempted to define by fluorescence in situ hybridization (FISH) a common region at 13q31-q32 in which to explore genes that might be targets for the amplification events. Although the commonly amplified region we defined was relatively large (approximately 4 Mb), only one true gene, GPC5, was found there. GPC5 was over-expressed in lymphoma cell lines that had shown amplification, in comparison with those that had not. Our findings suggest that GPC5 is a likely target for amplification, and that over-expression of this gene may contribute to development and/or progression of lymphomas and other tumors.

Recovery of the genome of murine sarcoma virus (msv) after infection of cells with nuclear dna from msv transformed non-virus producing cells.

Abstract The hamster (HT- 1 line) and the mouse (D- 243 line) cultures are murine sarcoma virus (MSV) transformed cells which do not produce infectious virus. Nuclear DNA was isolated from these cells. This DNA was used to treat normal mouse embryo cells ( 3 T 3 line) and Rauscher leukaemia virus (RLV) infected 3 T 3 cells. Both cell cultures became transformed, but only the RLV infected 3 T 3 cells released infectious MSV. The nuclear DNA lost its biological activity following treatment with D Nase. These experiments indicate that vertical transmission of mammalian C-type RNA virus is carried by the nuclear DNA of the viral transformed cells.

Release of spleen focus-forming virus (SFFV) from differentiation inducible promyelocytic leukemia cell lines transformed in vitro by Friend leukemia virus.

Abstract Three clonally derived sublines of acute promyelocytic leukemia (APML) induced in mouse long-term marrow cultures by Friend leukemia virus (FLV) were examined for release of spleen focus-forming virus (SFFV) and differentiation to mature granulocytes following in vitro exposure to chemical-inducing agents or transfer in vivo to diffusion chambers in irradiated rats or mice. Two APML lines derived from NIH Swiss mouse marrow cultures infected with FLV-anemia-inducing strains (FLV-A) and one line from C3H/HeJ mouse marrow cultures infected with a clonal Rauscher helper virus pseudotype of a cloned SFFV derived from FLV-polycythemia inducing strain (FLV-P) released biologically active SFFV after 18 months growth in suspension culture. In contrast, none of 75 cultures of NIH Swiss or C3H/HeJ bone marrow infected with Rauscher helper virus alone generated permanent APML cell lines or released detectable SFFV from short-term suspension cultures of granulocytic cells. Each line was comprised of 95–99% promyelocytes which required WEHI-3 dialyzed conditioned medium (DCM) for replication in suspension or colony formation in semisolid medium. NIH Swiss APML lines were normal diploid 38XX while the C3H/HeJ line was hypodiploid with 39 chromosomes. Differentiation to mature polymorphonuclear leukocytes was not detected with chemical inducers in vitro but was detected after exposure to irradiated rat plasma or growth in diffusion chambers in vivo. The release of SFFV from FLV-transformed clonal APML cells provides evidence for involvement of this virus in malignant transformation of the granulocyte pathway.

Human retroviruses in leukaemia and AIDS: reflections on their discovery, biology and epidemiology.

The study of retroviruses has had a profound impact by unveiling an unusual form of viral replication: the multiplication of RNA viruses via a proviral DNA, for which Jan Svoboda provided the experimental model over forty years ago. In 1970 Temin, Mizutani and Baltimore discovered that this group of viruses contains a unique enzyme catalysing the synthesis of a DNA copy of the viral RNA: reverse transcriptase (RT). The discovery of RT has itself had an enormous impact on molecular biology in general, but also stimulated many premature claims of its detection in human disease. Claims by Gallos laboratory that the cytoplasm of human leukaemia cells contained RT proved to be unfounded, as did his report in collaboration with Weiss that myeloid leukaemia contained HL23 virus, this organism proving not to be human but a laboratory contaminant of three monkey viruses. Conclusive demonstration of a retroviral involvement in human leukaemia was first provided in 1981 by Hinuma and his associates, showing that adult T‐cell leukaemia (ATL), a rare form of leukaemia endemic to south‐west Japan, is caused by a new retrovirus (ATLV). Other publications in December 1980 and through 1981 claimed the discovery of a new human T‐cell leukaemia virus involved in mycosis fungoides (MF) and Sézarys syndrome (SS). This virus was termed HTLV by Gallo. The nucleotide sequence of ATLV is strongly conserved, that of my 1983 isolate from a black British ATL patient being practically identical with the Japanese virus isolates.

Canine Tracheobronchitis Isolation and Characterization of the Agent with Experimental Reproduction of the Disease

Summary The isolation of herpescanis virus from the respiratory tract of naturally infected dogs is reported. Several animals from which the virus was isolated exhibited the characteristic symptoms of canine tracheobronchitis while others were not clinically ill. Except for differences in in vivo and in vitro cytopathogenicity, the virus possesses similar physical, chemical, and morphological characteristics of the herpes group of viruses. It was positively identified by serologic methods as herpescanis virus. Experimental reproduction of the respiratory disease was observed following intranasal inoculation of susceptible dogs, while inoculated pups died of acute hemorrhagic disease following intraperitoneal inoculation. The pathogenicity of herpescanis virus is evaluated on the basis of our studies and those of other workers.

A new human plasma cell line, Karpas 620, with translocations involving chromosomes 1, 11 and 14

We report here the establishment of a new cell line, Karpas 620 (K620), from the peripheral blood of an elderly woman with an IgG‐kappa plasma cell leukaemia (PCL). The line has the same hypotetraploid karyotype as the fresh cells from the patient. The cultured cells have the ultrastructural appearance of plasma cells with abundant rough endoplasmic reticulum (RER) and secrete kappa light chain. They are positive for surface antigens HLA DR, and WR17 (CD 37) and negative for CD1, CD3, CD4 and CD8. Using high resolution (HR) cytogenetic analysis it has been possible to identify all the marker chromosomes including several rearrangements commonly seen in malignancies of B cell lineage. These are a 14q + marker with a typical ‘Burkitt’morphology der(14)(pter→q32.3::8q24.1→qter) but with no reciprocal 8q–, and three translocations involving chromosome 11 at q 13 with partners other than chromosome 14, namely 1q32.1, 8q24.22 and 13q14.3. An earlier report of molecular studies on the DNA of K620 has shown a rearrangement near the region on 11q13 designated BCL‐1 (Rabbitts et al, 1988). This is the first report of a rearrangement in the region of 11q 13 in a cell line originating from a case of plasma cell leukaemia.