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Biomedical subjects

F van Roy

Publications and source records attributed to F van Roy.

51 records · Page 3Linked to original sources
PubMed1996

Growth inhibition in clonal subpopulations of a human epithelioid sarcoma cell line by retinoic acid and tumour necrosis factor alpha.

Epithelioid sarcoma is a highly malignant soft tissue tumour that is refractory to conventional chemotherapy and irradiation. Since permanent cell lines of this tumour are extremely rare, in vitro data on compounds with significant antiproliferative effects are still lacking. Therefore, we investigated the effects of retinoic acid (RA) and tumour necrosis factor alpha (TNF-alpha) on tumour cell proliferation of three different clonal subpopulations (GRU-1A, GRU-1B, GRU-1C) derived from the same human epithelioid sarcoma cell line, GRU-1. In GRU-1A both RA (P=0.01) and TNF-alpha (P=0.002) exhibited highly significant and dose-dependent growth inhibitory effects, which could further be increased by a combined application of both compounds (P<0.006). GRU-1B proved to be sensitive to RA (P=0.006), whereas no response to TNF-alpha was observed. GRU-1C was resistant to both RA and TNF-alpha. The antiproliferative effect of TNF-alpha was mediated by TNF receptor 1(TNF-R1) and correlated positively with both the number of TNF-R1 per cell and receptor affinity. No correlation was detected between RA-induced growth inhibition and the expression pattern of the RA receptors (RARs) RAR-alpha, RAR-beta, and RAR-gamma. Plating efficiency, however, could exclusively be reduced by RA in GRU-1B, the only cell line expressing RAR-alpha. Taken together, these data are the first showing significant antiproliferative effects in human epithelioid sarcoma by RA and TNF-alpha. Whereas the TNF-alpha response seems to depend on the expression of TNF-R1, no simple correlation could be found between RA sensitivity and the expression pattern of RARs.

Antineoplastic Agents↗
PubMed1995

E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers.

Compelling experimental evidence exists for a potent invasion suppressor role of the cell-cell adhesion molecule E-cadherin. In addition, a tumour suppressor effect has been suggested for E-cadherin. In human cancers, partial or complete loss of E-cadherin expression correlates with malignancy. To investigate the molecular basis for this altered expression we developed a comprehensive PCR/SSCP mutation screen for the human E-cadherin gene. For 49 breast cancer patients the occurrence of tumour-specific mutations in the E-cadherin gene was examined. No relevant DNA changes were encountered in any of 42 infiltrative ductal or medullary breast carcinoma samples. In contrast, four out of seven infiltrative lobular breast carcinomas harboured protein truncation mutations (three nonsense and one frameshift) in the extracellular part of the E-cadherin protein. Each of the four lobular carcinomas with E-cadherin mutations showed tumour-specific loss of heterozygosity of chromosomal region 16q22.1 containing the E-cadherin locus. In compliance with this, no E-cadherin expression was detectable by immunohistochemistry in these four tumours. These findings offer a molecular explanation for the typical scattered tumour cell growth in infiltrative lobular breast cancer.

Base Sequence↗
PubMed1995

Cloning and characterization of the human invasion suppressor gene E-cadherin (CDH1).

E-cadherin is a Ca(2+)-dependent epithelial cell-cell adhesion molecule. Downregulation of E-cadherin expression often correlates with strong invasive potential and poor prognosis of human carcinomas. By using recombinant lambda phage, cosmid, and P1 phage clones, we isolated the full-length human E-cadherin gene (CDH1). The gene spans a region of approximately 100 kb, and its location on chromosome 16q22.1 was confirmed by FISH analysis. Detailed restriction mapping and partial sequence analysis of the gene allowed us to identify 16 exons and a 65-kb-long intron 2. The intron-exon boundaries are highly conserved in comparison with other "classical cadherins." In intron 1 we identified a 5' high-density CpG island that may be implicated in transcription regulation during embryogenesis and malignancy.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of a human pseudogene (CTNNAP1) for alpha E-catenin (CTNNA1): assignment of the pseudogene to 5q22 and the alpha E-catenin gene to 5q31.

A pseudogene (CTNNAP1) for the human alpha E-catenin gene was isolated from a human genomic phage library. The pseudogene sequence shows 90% similarity to the alpha E-catenin mRNA at the nucleotide level. Thirty-eight stop codons in all three reading frames and multiple other mutations were found, indicating that the pseudogene does not encode a functional protein. No introns were found in the region corresponding to the open reading frame of the alpha E-catenin cDNA, and two direct repeats flank this same region. Hence, the pseudogene can be classified as a processed pseudogene. Polymerase chain reaction with pseudogene-specific primers on genomic DNA and cDNA from human cell lines and healthy blood donors demonstrated the general occurrence of the pseudogene and the lack of its transcription. By fluorescence in situ hybridization the pseudogene was mapped to human chromosome 5q22 and the alpha E-catenin gene to the formerly disputed locus 5q31. This is the first report of a pseudogene for a member of the cadherin-catenin cell-cell adhesion complex.

Base Sequence↗
PubMed1994

Downregulation in vivo of the invasion-suppressor molecule E-cadherin in experimental and clinical cancer.

The invasion-suppressor molecule E-cadherin (E-CAD) can be regulated at multiple levels: synthesis, processing and stability of mRNA; synthesis, processing and stability of protein; localization and posttranslational modification of protein; binding to catenins (E-CAD-associated proteins); and size and charge of cell surface glycosaminoglycans. Loss of E-CAD antigen and of E-CAD function in vivo has been observed with cell lines that homogeneously expressed functional E-CAD in vitro. These observations led to the idea that factors in the host may downmodulate E-CAD on the cancer cells, thereby promoting cell invasion. Nude mouse cancers that were homogeneously E-CAD-positive and noninvasive in vitro, formed by epithelioid MDCK or NMuMG cells, stained heterogeneously for E-CAD; such cancers were invasive and metastatic. The in vivo downmodulation appeared to be transient. Ex vivo cultures from primary cancers, as well as from metastases, produced homogeneously E-CAD-positive and noninvasive cells. Downmodulation did not occur when cells were micro-encapsulated and then implanted in the mouse, suggesting a role for immediate cancer cell-host cell contact. Similar in vitro/in vivo/ex vivo experiments with mouse MO4 fibrosarcoma cells, transfected with E-CAD cDNA under the control of a b-actin promotor, showed downregulation at the transcriptional or mRNA stability level. This downregulation was rapidly reversible upon ex vivo culture of the tumor cells. TGF-bl and IGF-I were found, respectively, to downregulate and upregulate the expression or the function of E-CAD. We speculate that IGF-1 restores the function of E-CAD through interaction of the IGF-I tyrosine kinase receptor with the catenin-actin cytoskeletal complex. In human cancers, immunohistochemistry has revealed changes in E-cadherin that agree with the experimental data on transient downmodulation of the invasion-suppressor function of E-cadherin by host factors.

Animals↗
PubMed1993

Enhancement of tumor necrosis factor cytotoxicity by lithium chloride is associated with increased inositol phosphate accumulation.

We have previously reported that LiCl increases considerably the cytotoxic activity of TNF towards some transformed cell lines such as L929. Here we show that treatment of these cell lines with the combination of TNF and LiCl leads to the prolonged accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, whereas treatment with TNF or LiCl alone did not. In contrast, both a LiCl-unresponsive TNF-sensitive cell line and TNF-resistant cell lines did not respond with increased accumulation of inositol phosphates (IPn) upon treatment with the combination of TNF and LiCl. Furthermore, the combination of TNF and LiCl induced a transient increase in cytidine diphosphate-diacylglycerol in L929 cells. Increased IPn and cytidine diphosphate-diacylglycerol accumulation preceded the onset of cell killing by approximately 1 h. TNF-mediated cytotoxicity and TNF-induced IPn accumulation were equally sensitive to inhibition by the phospholipase inhibitor neomycin and to stimulation by the protein kinase inhibitor staurosporine. Characterization of the inositol bisphosphate isomers by HPLC analysis revealed that the TNF + LiCl-induced increase in IPn levels was due to activation of a phospholipase C and not of a phospholipase D. In contrast to TNF, several other cytotoxic agents did not increase IPn production upon application in the presence of LiCl. The TNF + LiCl-induced increase in inositol triphosphate suggests a role for intracellular Ca2+ mobilization in TNF action. Moreover, several agents that lower the intracellular Ca2+ concentration inhibited TNF cytotoxicity. In conclusion, our data provide evidence that TNF cytotoxicity and its enhancement by LiCl are mediated by increased IPn accumulation resulting in Ca2+ mobilization.

Animals↗
PubMed1991

Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role.

A cDNA encoding the cell-cell adhesion molecule E-cadherin was transfected into highly invasive epithelial tumor cell lines of dog kidney or mouse mammary gland origin. Transfectants with a homogeneously high expression of E-cadherin showed a reproducible loss of activity in two types of in vitro invasion assays. Invasiveness of these transfectants could be reinduced specifically by treatment with anti-E-cadherin antibodies. In vivo, they formed partly differentiated tumors, instead of fully undifferentiated tumors. Alternatively, a plasmid encoding E-cadherin-specific anti-sense RNA was introduced into noninvasive ras-transformed cells with high endogenous E-cadherin expression. The resulting down-regulation, albeit partial, rendered the cells invasive. These data provide direct evidence that E-cadherin acts as an invasion suppressor molecule.

Animals↗
PubMed1990

Modulation of cellular susceptibility to the cytotoxic/cytostatic action of tumor necrosis factor by adenovirus E1 gene expression is cell type-dependent.

Primary baby rat kidney cells, primary human embryonic retinoblast cells, established NIH3T3 and established normal rat kidney (NRK) cells, expressing E1A and/or E1B gene regions of adenovirus 5 (Ad5) or Ad12, were investigated for susceptibility to the cytotoxic/cytostatic action of Tumor Necrosis Factor (TNF). In the primary cells and in the NRK cells, there was no correlation between TNF sensitivity and E1 gene expression; neither did sensitivity to TNF correlate with the oncogenicity of the Ad serotype. In contrast, the expression of Ad E1 gene regions in NIH3T3 cells was found to enhance TNF sensitivity of this cell line. Differences in E1A expression levels between cell types cannot explain this discrepancy regarding modulation of TNF sensitivity by E1A.

Adenovirus Early Proteins↗
PubMed1986

Genome organisation of the FBR-osteosarcoma virus complex: identification of a subgenomic fos-specific message.

The FBR murine virus complex together with the FBJ murine virus complex are known to be bone tumor inducers in newborn mice. Both transforming viruses have transduced c-proto-fos-derived sequences in their genome. FBR-MuSV was molecularly cloned as a biologically active 10-kbp EcoRI fragment from non-productively transformed rat embryo fibroblasts into Charon phage 4A (lambda MOL503) and subsequently subcloned in plasmid pBR322 (pMOL503). Its natural associated helper FBR-MuLV, excized as an internal 8.2-kbp PstI proviral DNA fragment from chronically infected NIH/3T3 cells, was cloned into the unique PstI site of pBR322. Comparative analysis of the restriction maps of FBR-MuSV and FBR-MuLV together with the electron microscopic analysis of heteroduplex DNA molecules formed between both molecular clones suggested that FBR-MuLV is the parental virus of FBR-MuSV. fos- and fox-specific DNA hybridisation probes identified a genomic sized 3.3-kb mRNA and a subgenomic 2.2-kb messenger RNA. Using a 5'-gag hybridisation probe, only the genomic 3.3-kb RNA molecule was detected, demonstrating that a donor splice site is present upstream of the gag sequences and used to generate the fos-specific 2.2-kb subgenomic mRNA.

Animals↗
PubMed1983

Improved localization of phosphorylation sites in simian virus 40 large T antigen.

The location of phosphorylation sites in the large T antigen of simian virus 40 has been studied both by partial chemical cleavage and by partial proteolysis of various forms of large T. These included the full-size wild-type molecule with an apparent molecular weight of 88,000, deleted molecules coded for by the mutants dl1265 and dl1263, and several shortened derivatives generated by the action of a cellular protease. These molecules differed from each other by variations in the carboxy-terminal end. In contrast, a ubiquitous but minor large T form with a molecular weight of 91,000 was found to be modified in the amino-terminal half of the molecule. In addition to the phosphorylation of threonine at position 701 (K.-H. Scheidtmann et al., J. Virol. 38:59-69, 1981), two other discrete domains of phosphorylation were recognized, one at either side of the molecule. The amino-terminal region was located between positions 81 and 124 and contained both phosphothreonine and phosphoserine residues. The carboxy-terminal region was located between approximate positions 500 and 640 and contained at least one phosphoserine residue but no phosphothreonine. The presence in the phosphorylated domains of large T of known recognition sequences for different types of protein kinases is discussed, together with possible functions of large T associated with these domains.

Antigens, Viral↗
PubMed1978

Interference with simian virus 40 DNA replication by adenovirus type 2 during mixed infection of monkey cells.

Infection of monkey cells with human adenovirus (Ad) is abortive, but the infection can be enhanced by coinfecting with simian virus 40 (SV40). However, in the coinfected monkey cells, Ad interferes strongly with SV40 DNA biosynthesis. This interference was found to be a reproducible, delicately controlled phenomenon that was proportional to the multiplicity of infection of Ad and dependent on the active expression of the Ad genome. Newly synthesized SV40 DNA was not broken down in cells after delayed superinfection with Ad, and several early events of SV40 infection such as adsorption, penetration, uncoating, induction of cellular DNA synthesis, and enhancement of Ad infection were not markedly influenced by Ad-mediated interference. It is unlikely that interference is simply due to competition between SV40 and Ad for metabolites, enzymes, or replication sites. The interference effect could be partially neutralized by an increase in the multiplicity of coinfecting SV40 or by an increase in the time interval between SV40 infection and Ad coinfection. Interference was shown to be due to the activity of an Ad early gene product. However, the detailed mechanism of this Ad interference is still unclear.

Adenoviruses, Human↗