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Biomedical subjects

J G Collard

Publications and source records attributed to J G Collard.

At least 19 recordsLinked to original sources
PubMed1992

Sublocalization of an invasion-inducing locus and other genes on human chromosome 7.

By somatic cell fusion studies between noninvasive mouse T-lymphoma cells and invasive human activated normal T-cells we have previously shown that the genetic information responsible for the induction of invasive and metastatic potential in interspecies T-cell hybrids is located on human chromosome 7. Apparently, genes derived from normal activated T-cells are dominantly expressed in the hybrids and control the invasive and, as a consequence, metastatic potential of these T-lymphoma cells. To sublocalize the invasion-inducing locus on chromosome 7 we have generated hybrids that harbor only specific regions of human chromosome 7 with or without a small fragment of human chromosome 21. Analysis of these hybrids revealed that the invasion-inducing locus maps to 7p12----cen. The human DNA complement of the hybrids was confirmed by Southern blot analysis using a large panel of chromosome 7-specific DNA probes. Several of these genes could be further sublocalized. These included: ARAF2 to 7p12----cen, D7S21 to 7pter----p12, ACTB to 7p15----p12, EGFR to 7p12, MDH2 to 7cen----q22, and PDGFA to 7pter----p15.

Blotting, Southern↗
PubMed1989

Genetic basis of T-lymphoma invasion.

We have discussed the use of BW5147 T-lymphoma cells as a model system to explore the genetic basis of invasion and metastasis. The degree of invasiveness of these cells in vitro is highly correlated with experimental metastasis formation in vivo upon tail vein injection in syngeneic AKR mice. A powerful in vitro selection system has been developed which allows to select rare invasive cell variants obtained by various experimental manipulations. We found that introduction of the human c-Ha-ras oncogene, the presence of human chromosome 7 from normal activated T-cells, DNA hypomethylation induced by 5-azacytidine treatment, and possibly also retrovirus insertional mutagenesis can convert noninvasive BW5147 T-lymphoma cells into invasive and metastatic cells. Several experimental approaches are discussed to identify the gene(s) involved.

Animals↗
PubMed1988

Invasiveness in hepatocyte and fibroblast monolayers and metastatic potential of T-cell hybridomas in mice.

Fusion of noninvasive, nonmetastatic BW5147 T-lymphoma cells with normal T-lymphocytes usually resulted in highly invasive and metastatic T-cell hybridomas, apparently due to properties derived from the normal T-cell. Occasionally hybrids arose that were non- or low invasive, probably by loss of relevant genes upon chromosome segregation, since these cells contained much less DNA than highly invasive hybrids. The metastatic potential of 20 representative T-cell hybridomas was tested by tail vein injection in syngeneic mice and cells were found to be either nonmetastatic (NM), low metastatic (LM), or high metastatic (HM). NM hybrids were tumorigenic but did not form metastases and HM hybridomas caused wide-spread metastasis. LM cells formed metastases in a limited number of mice and predominantly in lymphoid tissues. In hepatocyte cultures, NM cell lines were found to be the least invasive, HM cells the most, whereas LM hybrids exhibited intermediate levels. Invasiveness was not only measured in rat hepatocyte cultures but also in rat embryo fibroblast monolayers, and the relative invasive capacity in both model systems correlated well. Pertussis toxin inhibited invasion in both systems to 20-30% of control values. This suggests that the mechanisms of invasion into hepatocyte and fibroblast cultures are at least partially similar and that the fibroblast invasion assay is a relevant model to study aspects of lymphoma metastasis. We conclude that invasive potential is a prerequisite for T-cell hybridomas to colonize tissues from the bloodstream and that a minimum level of invasiveness is necessary for extensive and wide-spread metastasis formation.

Animals↗
PubMed1988

Genetic analysis of invasion and metastasis.

Metastasis formation is a multistep process that probably requires a complex interplay of a large and heterogeneous group of genes, including genes involved in cellular resistance to immunorejection and genes controlling the invasive potential of cells. Transfection experiments have shown that oncogenes of the ras gene family as well as oncogenes of the kinase group are able to induce invasive and metastatic properties in non-transformed cells as well as in tumorigenic, but non-metastatic, cells. However, these findings are not in agreement with observations on spontaneous human tumours in which no correlation was found between activation or increased expression of ras genes and the invasive and metastatic properties of these cells. Further studies have indicated that in particular cell types nuclear oncogenes like N-myc and adenovirus derived E1A may influence metastasis formation by trans-regulation of other genes, including class I genes of the major histocompatibility complex and genes coding for proteolytic enzymes. The many efforts to identify additional invasion and metastasis associated genes by transfection of high molecular weight metastatic tumour DNA met with little success. Somatic cell fusion studies, however, indicate that such genes exist and that one or more of them are located on human chromosome 7.

Genetic Techniques↗
PubMed1987

Location of genes involved in invasion and metastasis on human chromosome 7.

We have obtained evidence for the existence of genes controlling invasion and metastasis by somatic cell fusion studies. Noninvasive, nonmetastatic mouse BW5147 T-lymphoma cells were fused with invasive human T-cells. The human cells were either activated normal peripheral blood lymphocytes or leukemic T-lymphoblasts. Both fusions resulted in highly invasive human-mouse T-cell hybrids which metastasized in nude mice. Thus the genes derived from either malignant or nonmalignant but inherently invasive cells enable the T-cell hybridomas to metastasize. By continued in vitro selection for invasive cells employing monolayers of rat embryo fibroblasts followed by subcloning, we were able to isolate invasive hybrids that had lost all human chromosomes except chromosome 7. We present evidence that one or more genes residing on human chromosome 7 are necessary and sufficient for both the establishment and maintenance of invasiveness and metastatic potential of the interspecies T-cell hybrids.

Animals↗
PubMed1987

Invasive and metastatic potential induced by ras-transfection into mouse BW5147 T-lymphoma cells.

Noninvasive, nonmetastatic BW5147 T-lymphoma cells were transfected with the activated human c-Ha-ras oncogene and were examined subsequently for the acquisition of invasive properties in vitro and of metastatic potential in vivo. It was found that several transfectants harboring the ras gene had become invasive in vitro, as assessed in hepatocyte cultures, and metastatic after tail vein injection into syngeneic AKR mice. The induced level of both invasive and metastatic potential appeared to depend on the level of expression of the transfected ras gene. Those transfectants exhibiting an elevated level of ras expression, mostly cells containing a high copy number of the ras gene, showed the highest invasiveness (up to 30-fold increase) and produced widespread metastasis in all mice tested. Transfectants with a low level of ras expression were less invasive and formed metastases in a few mice only, limited to a few organs or even to a single deposit in one organ. Untransfected BW cells, control transfected cells without the ras gene, and ras transfectants that did not express the gene were noninvasive and nonmetastatic. No changes in number of ras gene copies were found between isolated metastases and the transfectants from which they were derived. However, RNA analysis of the cells from the isolated metastases revealed similar, as well as elevated or diminished levels of ras transcription when compared to the corresponding cell lines prior to injection, suggesting that a persistent high expression of the ras gene is not necessarily needed for the independent growth at the secondary site. Our results indicate that the activated human ras oncogene may confer metastatic potential onto lymphoid tumor cells, probably due to the induction of invasiveness.

Animals↗
PubMed1987

Efforts to produce human cytotoxic T-cell hybridomas by electrofusion and PEG fusion.

Cytotoxic human T cells from different sources were fused with different types of human T-lymphoma cells and mouse B-myeloma cells using variations of the polyethylene glycol (PEG) method and electrofusion. Both techniques yielded proliferating hybridomas. The frequency of wells with proliferating hybridomas depended on the tumor fusion partner used; the best results were obtained with HSB-1, whereas fusions with JURKAT-1 and HPB-1 did not yield any hybridomas. For one tumor cell line (HSB-1), considerably more hybridomas were obtained with electrofusion than with the PEG fusion (with or without heat shock). There was no consistent relationship between the presence or absence of cytotoxic activity of the T lymphocytes against the tumor fusion partner and the yield of hybridomas. In human-human as well as in human-mouse hybridomas most of the lymphocyte derived chromosomes were lost. Four of the more than 600 hybridomas tested showed transient cytotoxic activity, but in none of them this function could be immortalized. Two of the hybridomas obtained with CEM-1 as tumor fusion partner expressed low levels of lymphocyte-derived CD3 antigens. Two hybridomas obtained with HSB-1 were highly invasive in vitro in rat hepatocyte cultures, whereas HSB-1 tumor cells were not.

Animals↗
PubMed1986

Expression of Ly-6.2 and Thy-1 antigens on NIH 3T3 cells suppressed after transformation with activated human ras-oncogenes.

We have studied the expression of several cell surface antigens in NIH 3T3 cells after neoplastic transformation with activated human ras and myc oncogenes. The binding of monoclonal antibodies (MAbs), specific for mouse differentiation antigens Ly-6.2, Thy-1 and 9F3, to normal and transformed cells was assessed using a fluorescence-activated cell sorter (FACS IV). Significant reduction of Ly-6.2 and Thy-1 antigen expression was detected in cells transformed with either the N-ras, Ki-ras or H-ras oncogenes but not in c-myc transfected cells. 9F3 antigen expression remained at the original high level in all transfectants studied. Normal levels of Ly-6.2 and Thy-1 expression reappeared in revertants derived from unstable ras-transfectants. These data indicate that ras sequences did not preferentially transform cells that were deficient in Ly-6.2 and Thy-1 antigens. It was also shown that the reduced binding of anti-Ly-6.2 antibodies to ras-transfectants was not due to a masking effect of increased cell-surface sialylation occurring in ras-transfected cell lines. Other possible explanations of the detected phenotypic changes are discussed. The results extend the range of tumor-associated membrane alterations in NIH 3T3 cells following transfection with human tumor DNA containing activated ras oncogenes by a hitherto unreported alteration in the expression of Ly-6 and Thy-1 antigens.

Animals↗
PubMed1986

Construction and analysis of an EMBL-3 phage library containing partially digested human chromosome 21-specific DNA inserts (15-20 kb).

In the mouse-human hybrid cell line SCC 16-5, chromosome 21 is the only human chromosome present. Fractions highly enriched for this chromosome were obtained by applying the chromosome velocity sedimentation technique to this cell line. DNA prepared from these chromosomal fractions was partially digested with Mbo I, size fractionated on an NaCl gradient, and cloned in the EMBL-3 phage vector. The phage library thus prepared was highly enriched for human chromosome 21-specific recombinant DNA sequences 15-20 kb long. Of the approximately 21,000 phage clones obtained, at least 99% were recombinant. Following phage plaque filter hybridization and Southern blotting, it was found that half of the recombinants were positive for human repetitive DNA. Almost all phages harbored highly or middle repetitive human or mouse DNA sequences owing to the large size of the recombinant inserts. In this library, the human chromosome 21 is represented approximately four times. All human recombinants studied thus far contained DNA inserts originating from chromosome 21 only. The employed cloning strategy is discussed with regard to utility, purity, quality, and completeness of chromosome-specific recombinant DNA libraries.

Animals↗
PubMed1986

Sublocalization of c-myb to 6q21----q23 by in situ hybridization and c-myb expression in a human teratocarcinoma with 6q rearrangements.

We have sublocalized the human proto-oncogene c-myb by applying two different techniques: in situ hybridization of metaphase spreads and chromosome spot hybridization of flow-sorted chromosomes. For this we used a teratocarcinoma cell line carrying specific chromosome translocations involving the two chromosomes 6 and one chromosome 11. The distribution of the c-myb gene copies on the different translocation chromosomes revealed that c-myb is located in the region 6q21----q23. Because of the close proximity of the c-myb locus to the chromosomal breakpoints in the teratocarcinoma, we investigated whether c-myb was implicated in the development of this tumor. No rearrangement, deletion, or amplification of the gene was detected in the teratocarcinoma cells. Furthermore, the level of c-myb expression was comparable to that of other cell lines of nonhematopoietic origin. These results suggest that c-myb was not affected by the translocation and played no significant role in the development of this teratocarcinoma.

Cell Line↗
PubMed1986

Cell surface sialic acid and the invasive and metastatic potential of T-cell hybridomas.

T-cell hybridomas prepared by fusion of non-invasive non-metastatic BW5147 T-lymphoma cells and activated normal T-cells were found to be highly invasive in vitro and highly metastatic in vivo upon tail vein injection. By prolonged culturing and subcloning, non-invasive, non-metastatic hybrids were selected with modal DNA/cell contents close to the diploid value of both fusion partners. Since normal activated T-cells were invasive in vitro in hepatocyte cultures, these data suggest that invasiveness of the hybrids is derived from the parental normal T-cells and is one of the properties responsible for the metastatic potential of these cells. Analysis of a large panel of T-cell hybrids with fluorescein isothiocyanate conjugated lectins, specific for terminal galactose and/or N-acetylgalactosamine sugar residues, showed an inverse correlation between expression of lectin receptor sites and invasive and metastatic potential of the hybrids. Soybean agglutinin, as well as peanut agglutinin and Ricinus communis agglutinin, reacted strongly with non- or low-invasive hybrids but only weakly with invasive hybrids. The difference in lectin binding between both types of hybrids appeared to be due to masking of receptor sites by sialic acid. Removal of cell surface sialic acid by neuraminidase treatment unmasked the lectin receptor sites of invasive hybrids to the level of the corresponding sites of non- or low-invasive cells. This increase in active lectin binding sites was simultaneously accompanied by a striking decrease of invasiveness to the level of the low-invasive hybrids. Conversely, the blocking of R. communis agglutinin receptors by sialic acid allowed selection of invasive hybrids from segregating cell populations with the toxic lectin R. communis agglutinin. The results taken together indicate that sialylation of particular cell surface carbohydrate residues on the T-cell hybridomas is associated with the invasive and metastatic potential of these hybrids. The reduction of invasive potential after removal of cell surface sialic acid provides further evidence for a functional role of this sugar residue in invasiveness of the T-cell hybrids.

Animals↗
PubMed1986

Effect of cancer-related and drug-induced alterations in surface carbohydrates on the invasive capacity of mouse and rat cells.

The effect of alterations in cell surface carbohydrates on invasion of mouse and rat cells into embryonic chick heart fragments in organ culture was studied. Matching pairs of malignant and nonmalignant cells, including all categories of carcinogenic induction (i.e., viral, chemical, or oncogenic), were compared for their alterations in cell surface carbohydrates and invasive behavior. Glycopeptides derived from the surface of malignant cells expressed cancer-related changes in carbohydrate composition, demonstrated by gel filtration chromatography as a shift in size distribution in comparison with those from nonmalignant counterparts. This phenotypic property strictly correlated with the acquisition of the invasive capacity. Morphological transformation of cells without simultaneous alteration in surface carbohydrates was, however, insufficient for invasion. To test the possible mechanistic role of altered surface carbohydrates in the invasive capacity of cells, the surface molecules of noninvasive cells were modified by incubation with an alkyl-lysophospholipid (racemic 1-O-octadecyl-2-O-methyl glycero-3-phosphocholine). Alkyl-lysophospholipid induced an increase in surface sialylation resembling the changes found in malignant and invasive cells. After pretreatment with alkyl-lysophospholipid, morphologically transformed but nonmalignant and noninvasive cells became able to invade chick heart tissue. These findings indicate that alterations in cell surface carbohydrates, induced by entirely different mechanisms, endowed cells with invasive capacity.

Animals↗
PubMed1985

Transfection by human oncogenes: concomitant induction of tumorigenicity and tumor-associated membrane alterations.

NIH 3T3 cells were transfected with DNA derived from human bladder carcinoma, colon carcinoma and HL60 promyelocytic leukemia cells. The transfectants were examined for the presence of human oncogenes in relation to tumorigenic potential and composition of surface-located fucosyl glycopeptides by gel filtration, Concanavalin-A-binding and high-performance liquid chromatography. All transfectants, harboring 3 different human cellular ras genes, appeared to be tumorigenic in nude mice and displayed characteristically altered glycopeptides. The surface glycopeptides were consistently changed to higher apparent molecular weight due to enrichment in higher-branched sialic-acid-containing glycopeptides. Similar alterations have been found previously in virally- and chemically-transformed cells in vitro and tumors raised in vivo, and were designated as cancer-related or tumor-associated glycopeptides. Revertants derived from HL60-DNA-induced transfectants, which had lost the transfected human N-ras oncogene, simultaneously lost their tumorigenic potential and expression of cancer-related membrane glycopeptides. In addition, spontaneous transformants, exhibiting morphology and growth patterns indistinguishable from those of tumor-DNA-induced transfectants, neither contained transferred human DNA sequences nor expressed cancer-related glycopeptides. Nevertheless these cells were capable, after prolonged latency periods, of inducing tumors in nude mice. Cells derived from such tumors constantly displayed cancer-related glycopeptides on their surface, suggesting selection of tumorigenic cells from spontaneous transformants during passage in nude mice. In one of these tumors at least, an endogenous mouse ras-gene appeared to be activated. The results indicate a close correlation between the presence of activated ras-oncogenes in the genome of the transfected cells, the tumorigenic potential of these cells and the expression of surface-located cancer-related glycopeptides. The data suggest that functions provided by human ras-oncogenes contribute to the alteration of membrane glycopeptides on tumor cells.

Animals↗
PubMed1985

Identical chromosome translocations involving the region of the c-myb oncogene in four metastases of a mediastinal teratocarcinoma.

A 39-year-old white male presented with a disseminated mediastinal teratocarcinoma. Karyotyping was performed on two mature residual metastatic lesions in the lungs immediately following chemotherapy, on a recurring lung lesion after 5 months, and on a metastasis in the right thigh 5 months after salvage chemotherapy. All four lesions were pseudoeuploid and showed identical chromosomal abnormalities: a translocation with the two chromosomes #6 and one chromosome #11 involved, resulting in 46, XY, t (6;6;11) (q21;q23;q13). The breakpoint in chromosome #6 is in the region to which the oncogene c-myb has been localized, and the breakpoint in chromosome #11 is at a known fragile and possibly oncogenic site, suggesting that the translocations in this case may have played a crucial role in the development of the malignancy.

Adult↗
PubMed1985

Gene mapping by chromosome spot hybridization.

A method is described for the localization of cloned single-copy genes to flow-sorted chromosomes. Chromosomes were sorted directly onto nitrocellulose filters and the chromosomal DNA was subsequently hybridized with gene-specific radioactively labeled DNA probes. Mild aspiration of the filters during sorting was applied to collect the deflected chromosomes in a small spot. Sorting of 10,000-30,000 chromosomes was sufficient to detect gene-specific hybridization with single-copy DNA probes. Using this technique, we have sublocalized the human c-myb oncogene to 6q21-q23 by sorting translocated chromosomes with breakpoints in the q21 and q23 region of chromosome 6. Chromosome spot hybridization appears to be a rapid and simple method to assign cloned genes to chromosomes. Hybridization of an unlocalized gene probe to spots of chromosomes pre-enriched by velocity sedimentation can quickly narrow the choice of chromosomes which need to be sorted. Conversely, individual chromosomes in a flow karyotype can be identified by hybridizing sorted chromosomal DNA with chromosome-specific DNA probes.

Cell Fractionation↗
PubMed1985

Oncogene activation in human myeloid leukemia.

We have studied by means of DNA-mediated gene transfer the activation of protooncogenes in human myeloid leukemias that represent various stages of myeloid differentiation. DNA from three cell lines, HL-60 (promyelocytic leukemia), Rc2a (myelomonocytic leukemia), and KG-1 (acute myeloblastic leukemia), was capable of transforming NIH/3T3 cells. Hybridization analysis indicated that, in all three tumor cell lines, the N-ras oncogene was activated. The cell lines U-937 ("histiocytic lymphoma") and K-562 (erythroblastic leukemia) yielded no transforming DNA. Fresh leukemia cells derived from an acute myelomonocytic leukemia patient and from a juvenile chronic myelogenous leukemia patient contained an activated N-ras and c-Ki-ras oncogene, respectively. DNA from some other myelogenous leukemia patients was not able to transform NIH/3T3 cells. Our results indicate that hematopoietic tumors of the myeloid lineage may contain oncogenes active in NIH/3T3 cell transformation and that, in particular, the N-ras oncogene may be activated in tumors representing various stages of maturation.

Cell Line↗
PubMed1985

Invasiveness of T-cell hybridomas in vitro and their metastatic potential in vivo.

BW5147 lymphoma cells, which are noninvasive and nonmetastatic, were fused with normal T-lymphocytes. The invasiveness of the generated T-cell hybridomas was tested in hepatocyte cultures, and their metastatic potential was tested by tail vein injection. A total of 29 hybridomas generated from alloantigen-activated T-cells were all found to be invasive. One of these cell lines rapidly lost invasiveness in culture. Most hybridomas generated from nonstimulated spleen T-cells were also invasive, but 5 of 27 were not. Six invasive and four noninvasive hybridomas were injected into the tail vein of syngeneic mice. All invasive cell lines caused extensive and widespread tumor growth, particularly in the liver, which was usually severalfold enlarged; the spleen; kidneys; and ovaries. In contrast the noninvasive hybrids, which were tumorigenic upon s.c. injection, did not form any metastases. We conclude that properties derived from normal T-cells, when introduced into noninvasive T-lymphoma cells, cause them to become invasive as well as metastatic. Furthermore for this tumor cell type invasiveness as measured in hepatocyte cultures appears to be closely associated with the ability to colonize organs from the bloodstream.

Animals↗