Biomedical subjects

M Chedid

Publications and source records attributed to M Chedid.

At least 37 records · Page 2Linked to original sources

PubMed1994

Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin.

In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P-labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P-induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P-dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.

Animals↗

PubMed1994

Prokaryotic expression cloning of a novel human tyrosine kinase.

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, sequencing, chromosomal localization and expression of mouse p21 (Waf1).

The recent discovery that expression of Waf1 (p21), an inhibitor of cyclin-dependent kinases, is induced by the tumor suppressor p53 provides an important linkage between growth suppression and the cell cycle. We report here the cloning and sequencing of a mouse p21 cDNA that contains the entire coding region. Hybridization of the mouse p21 probe in Southern blot analyses confirms that p21 is a single-copy gene and that the corresponding locus, Waf1, lies proximal to H-2 on mouse chromosome 17. In northern analyses, the expression of p21 is found in most normal mouse tissues, but a surprising lack of correlation is found between mRNA levels of p21 and p53. In order to determine which regions of p21 are most evolutionarily conserved, we have compared the cDNA sequences for the entire p21 coding region in 13 different mouse strains or species and the human p21 sequence. We conclude that two regions (corresponding to human codons 21-60 and 130-164) are strongly conserved in p21 and that these regions may represent domains that are especially critical to a functional p21 protein.

Amino Acid Sequence↗

PubMed1994

A single nucleotide substitution at codon 31 (Ser/Arg) defines a polymorphism in a highly conserved region of the p53-inducible gene WAF1/CIP1.

The recently discovered WAF1/CIP1 gene is a mediator of p53 tumor suppressor activity. To analyse WAF1/CIP1 for possible mutations, polymerase chain reaction (PCR) amplified cDNAs from several tumor cell lines were cloned and sequenced. A single point mutation which changes codon 31 from AGC to AGA (Ser to Arg) was found. This change resulted in the loss of a Bpu1102I and gain of an Esp3I restriction site, allowing for rapid screening of this mutation in human DNAs. Analysis of genomic DNAs from 50 randomly selected individuals revealed that this base pair substitution represents a polymorphism with an allelic frequency of 0.14. Transfection studies demonstrated that the expression of the Arg allele of WAF1/CIP1 was not associated with loss of tumor suppressor activity. Moreover, screening of 22 tumor DNA samples revealed no association between the tumor phenotype and the Arg allele of WAF1/CIP1 (two out of 22 tumor DNAs contained the Arg31 allele). This polymorphism will be a useful molecular marker in the analysis of loss of heterozygosity in human cancers, and further studies using a larger panel of tumors may reveal an association between this polymorphism and specific types of cancer.

Amino Acid Sequence↗

PubMed1993

Fibroblast growth factor receptor 4 is a high affinity receptor for both acidic and basic fibroblast growth factor but not for keratinocyte growth factor.

A cDNA predicted to encode a transmembrane tyrosine kinase receptor with sequence features characteristic of known fibroblast growth factor (FGF) receptors was isolated from an expression library constructed from the human mammary epithelial cell line B5/589. This cDNA, designated cl44, encodes a product of 803 amino acid residues and was readily distinguishable from known FGF receptors. During the course of our studies, Partanen et al. (Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson, W. L., and Alitalo, K. (1991) EMBO J. 10, 1347-1354) isolated a new FGF receptor, designated FGFR4, from the human leukemia cell line, K562. Its amino acid sequence is identical to that of cl44 with the exception of 1 residue. The 5'-untranslated sequences of the two cDNAs diverged far upstream of the initiation codon. A myoblast line, L6E9, which lacks FGF receptors, was utilized to express high levels of FGFR4. We found, in contrast to Partenen et al., who reported only binding of acidic FGF, that FGFR4 bound both acidic and basic FGF with dissociation constants of 10-15 and 120 pM, respectively. No detectable binding of keratinocyte growth factor was observed. In studies aimed to determine whether FGF receptors contribute to the development of human tumors, we screened RNAs prepared from cell lines derived from a variety of solid tumors. High levels of the cl44 transcript were detected in 8 of 14 and 6 of 9 human mammary and kidney carcinomas, respectively, but only infrequently in other types of tumors. In contrast, FGFR1 was found to be frequently expressed in kidney, but not in breast tumor cells, suggesting a possible role for FGFR4 in human mammary cancer.

Amino Acid Sequence↗

PubMed1993

Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product.

Using an expression cDNA cloning approach, we examined human tumor cell lines for novel oncogenes that might evade detection by conventional techniques. We isolated a transforming sequence that was highly efficient in transforming NIH 3T3 mouse fibroblasts. DNA sequence analysis identified the gene as the human homolog of a recently cloned alpha subunit of mouse GTP-binding protein G alpha 12. NIH 3T3 cells transfected with G alpha 12 cDNA grew in soft agar and were tumorigenic in nude mice. There were no apparent mutations in the cloned cDNA in comparison with a G alpha 12 cDNA clone isolated from a normal human epithelial cell library, implying that overexpression alone was sufficient to cause NIH 3T3 cell transformation. The observed altered growth properties mediated by G alpha 12 showed a certain degree of dependency on serum factors, and its mitogenic potential was also potently inhibited by suramin treatment.

3T3 Cells↗

PubMed1993

Isoforms of human HGF and their biological activities.

Hepatocyte growth factor (HGF) is a plasminogen-like protein thought to be a humoral mediator of liver regeneration. It is produced by stromal fibroblasts and acts as a mitogen for a variety of target cells. Scatter factor, a stimulant of epithelial and vascular endothelial cell motility, is the same as HGF. HGF mediates its effects by activation of a membrane-spanning tyrosine kinase, recently identified as the met proto-oncogene product. We purified a naturally occurring hepatocyte growth factor variant, whose predicted sequence extends only through the second kringle domain of this plasminogen-related molecule. This smaller molecule, derived from an alternative HGF transcript, lacked mitogenic activity but specifically inhibited HGF-induced mitogenesis. Cross-linking studies demonstrated that the truncated molecule competes with HGF for binding to the c-met proto-oncogene product. Thus, the same gene encodes both a growth factor and its direct antagonist.

Amino Acid Sequence↗

PubMed1993

Expression cDNA cloning of a serine kinase transforming gene.

By ectopic expression of cDNAs derived from a Ewing sarcoma cell line in NIH3T3 cells, we isolated a transforming gene (est). Sequence analysis revealed homology to the cot oncogene, which encodes a novel serine kinase. Whereas the cot product was truncated at its carboxy-terminal end as a result of gene rearrangement during transfection, est encodes the normal cot product. Thus, this gene can be activated as an oncogene by overexpression as well as by gene rearrangement. NIH3T3 cells transfected with est formed progressively growing colonies in soft agar and were tumorigenic in nude mice. The 3.2-kb est transcript was expressed at low level in both human fibroblasts and epithelial cells. Addition of the tumor promoter, okadaic acid (OA), or cytokine, interleukin 1 (IL-1), but not serum or platelet-derived growth factor (PDGF), induced increased expression of the est transcript. Using fluorescence in situ hybridization, we localized the est gene to the short arm of human chromosome 10 at band p11.2.

3T3 Cells↗

PubMed1991

Identification of a competitive HGF antagonist encoded by an alternative transcript.

We identified a naturally occurring hepatocyte growth factor (HGF) variant, whose predicted sequence extends only through the second kringle domain of this plasminogen-related molecule. This smaller molecule, derived from an alternative HGF transcript, lacked mitogenic activity but specifically inhibited HGF-induced mitogenesis. Cross-linking studies demonstrated that the truncated molecule competes with HGF for binding to the HGF receptor, which has been identified as the c-met protooncogene product. Thus, the same gene encodes both a growth factor and its direct antagonist.

Amino Acid Sequence↗

PubMed1991

Activation of AP-1 by IL-1 and phorbol esters in T cells. Role of protein kinase A and protein phosphatases.

We have examined the regulation of the AP-1 transcription complex in the IL-1-responsive murine T cell thymoma cell line EL-4 6.1 C10. Our results demonstrate that AP-1-mediated gene expression in T cells may be regulated by several signaling pathways and factors, including IL-1, protein kinase C, protein kinase A (PKA), and one or more serine/threonine-specific protein phosphatases. The activation of protein kinase C results in an increase in nuclear AP-1 DNA binding activity, as well as enhanced gene expression. IL-1 and agents that elevate intracellular cAMP levels do not, by themselves, induce AP-1 activation, but they synergize with phorbol esters. IL-1 and forskolin may enhance AP-1 function by different mechanisms, because forskolin enhanced gene expression without producing an increase in nuclear AP-1 DNA binding, whereas IL-1 increased AP-1-binding activity and gene expression. These observations, in conjunction with the lack of a demonstrable effect of IL-1 on cAMP production in EL-4 cells, are consistent with the view that IL-1 enhances AP-1 activation by a pathway that does not directly involve cAMP and PKA. However, the induction of AP-1 activity by IL-1 and phorbol esters is dependent upon the presence of PKA, as evidenced by the loss of AP-1 inducibility in cells transfected with a cDNA encoding protein kinase inhibitor, a specific inhibitor of PKA. The effect of protein kinase inhibitor on AP-1 activation in response to IL-1 and tetradecanoyl-phorbol-13-acetate was reversed in the presence of the serine/threonine protein phosphatase inhibitor okadaic acid. Thus, the level of AP-1 activity in T cells may be determined by the balance between the activities of several serine/threonine protein kinases and phosphatases.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Involvement of cyclic AMP-dependent protein kinases in the signal transduction pathway for interleukin-1.

Expression of a highly specific protein inhibitor for cyclic AMP-dependent protein kinases in interleukin-1 (IL-1)-responsive cells blocked IL-1-induced gene transcription that was driven by the kappa immunoglobulin enhancer or the human immunodeficiency virus long terminal repeat. This inhibitor did not affect protein kinase C-mediated gene transcription, suggesting that cyclic AMP-dependent protein kinases are involved in the signal transduction pathway for IL-1 in a number of responsive cell types.

Carrier Proteins↗

PubMed1989

Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase.

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.

Adenosine Diphosphate Ribose↗

PubMed1989

Interleukin 1 and cyclic AMP induce kappa immunoglobulin light-chain expression via activation of an NF-kappa B-like DNA-binding protein.

Interleukin 1 (IL-1) induces the synthesis of kappa immunoglobulin light chains and the expression of surface immunoglobulin in the murine pre-B-cell line 70Z/3 (J. G. Giri, P. W. Kincade, and S. B. Mizel, J. Immunol. 132:223-228, 1984). In the present study, we found that these effects of IL-1 are mimicked by cyclic AMP (cAMP) analogs and cAMP-elevating drugs. The induction of kappa immunoglobulin light-chain gene expression by IL-1 was associated with an increase in intracellular cAMP levels. Incubation of 70Z/3 cells with IL-1 or cAMP resulted in the activation of the kappa immunoglobulin enhancer, as detected by the induction of chloramphenicol acetyltransferase (CAT) in cells transfected with a kappa enhancer-CAT expression plasmid. In contrast, CAT plasmids lacking a kappa immunoglobulin enhancer were inactive in the presence of IL-1 or cAMP. Furthermore, IL-1 and cAMP analogs and inducers were found to induce the activation of a NF-kappa B-like DNA-binding protein that exhibited specificity for the kappa immunoglobulin enhancer. These results suggest that cAMP may play an important role as a second messenger for IL-1 in the induction of kappa immunoglobulin light-chain synthesis in pre-B cells via the activation of a DNA-binding protein that is similar or identical to NF-kappa B.

Animals↗

PubMed1988

Characterization of the human interleukin 1 receptor on human polymorphonuclear leukocytes.

Interleukin 1 (IL-1) is a mediator of inflammation with multiple proinflammatory and immunologic enhancing activities. Human polymorphonuclear leukocytes (PMN) also play a major role in the inflammatory response. We have found that PMN possess a single type of high affinity receptor for human recombinant (r) IL-1 alpha with an apparent dissociation constant of 0.28 nM. Approximately 700 receptors are present per cell. Binding is rapid with 50% of maximal binding occurring within 20 min at 4 degrees C. Internalization of the receptor occurs within 25 min after shifting the cells to 37 degrees C. The receptor exhibits an apparent molecular weight of approximately 60-70 kDa. Electron microscopic autoradiography studies reveal that the 125I-rIL-1 alpha localized in the nucleus within 180 min after shifting cells to 37 degrees C. The accumulation of relatively high levels of 125I-rIL-1 alpha in the nucleus is consistent with earlier observations on the nuclear localization of IL-1 in T lymphocytes. The possibility that IL-1 may exert a direct action in the nucleus remains to be determined.

Cross-Linking Reagents↗

PubMed1988

Cyclic AMP--an intracellular second messenger for interleukin 1.

We demonstrated that interleukin 1 (IL-1), a potent peptide mediator in immune and inflammatory responses, stimulates the synthesis of cAMP in a variety of IL-1-responsive cell targets. We also showed that cAMP analogs and cAMP-inducing agents can replace IL-1 in the induction of interleukin 2 receptors on lymphocytes as well as in phytohemagglutinin-induced murine thymocyte proliferation. By use of IL-1 and the cAMP-inducer, forskolin, a direct correlation between the induced level of cAMP and the degree of lymphocyte interleukin 2 receptor expression or thymocyte proliferation was established. Our results indicate that cAMP may be an important intracellular second messenger for IL-1.

Animals↗

PubMed1987

The major histocompatibility complex of primates.

The major histocompatibility complex (MHC) encodes cell surface glycoproteins that function in self-nonself recognition and in allograft rejection. Among primates, the MHC has been well defined only in the human; in the chimpanzee and in two species of macaque monkeys the MHC is less well characterized. Serologic, biochemical and genetic evidence indicates that the basic organization of the MHC linkage group has been phylogenetically conserved. However, the number of genes and their linear relationship on the chromosomes differ between species. Class I MHC loci encode molecules that are the most polymorphic genes known. These molecules are ubiquitous in their tissue distribution and typically are recognized together with nominal antigens by cytotoxic lymphocytes. Class II MHC loci constitute a smaller family of serotypes serving as restricting elements for regulatory T lymphocytes. The distribution of class II antigens is limited mainly to cell types serving immune functions, and their expression is subject to up and down modulation. Class III loci code for components C2, C4 and Factor B (Bf) of the complement system. Interspecies differences in the extent of polymorphism occur, but the significance of this finding in relation to fitness and natural selection is unclear. Detailed information on the structure and regulation of MHC gene expression will be required to understand fully the biologic role of the MHC and the evolutionary relationships between species. Meanwhile, MHC testing has numerous applications to biomedical research, especially in preclinical tissue and organ transplantation studies, the study of disease mechanisms, parentage determination and breeding colony management. In this review, the current status of MHC definition in nonhuman primates will be summarized. Special emphasis is placed on the CyLA system of M. fascicularis which is a major focus in our laboratory. A highly polymorphic cynomolgus MHC has been partially characterized and consists of at least 14 A locus, 11 B locus, 7 C locus class I allelic specificities, 9 Ia-like class II antigens and 6 Bf (class III) variants.

Animals↗

PubMed1987

Differential expression of the HLA-DR genes in various melanoma cell lines treated with interferon-gamma: methylation of the HLA-DR alpha gene in these lines is not correlated with its expression.

Melanoma cell lines treated with or without interferon-gamma were tested for the presence of DR alpha mRNA and protein. The six lines examined fell into three general categories: two that expressed high levels of DR alpha mRNA and protein before and after interferon-gamma treatment, one that expressed very low levels before treatment with interferon-gamma, but was induced to express high levels after interferon-gamma treatment, and three that expressed very low levels before treatment, and were only slightly inducible after treatment with interferon-gamma. The presence of DR-alpha protein on the melanoma cell surface was always positively correlated with the presence of DR alpha mRNA in the cells. Furthermore, in the cell line that was interferon-gamma-inducible, the time at which DR alpha mRNA and protein appeared and the doses of interferon-gamma needed to induce this appearance were directly correlated. Methylation patterns of the DR alpha gene in these cell lines were also studied in order to determine whether the degree of DR alpha gene methylation among the lines correlated with expression of the gene. Digestion of DNA with the restriction enzyme MspI, which recognizes the sequence 5'CCGG3' and 5'CmCGG3', led to the appearance of a 3.1 kb band from all lines tested. Hpa II digestion, which recognizes 5'CCGG3', but not 5'CmCGG3', led to the appearance of 3.1, 4.4, and 6.7 kb bands in all lines tested except for DUMEL 8, which showed only the 3.1 kb band. Interestingly, DUMEL 8 expressed very low levels of DR alpha mRNA and protein before and after interferon-gamma treatment. We conclude that interferon-gamma has a regulatory effect on DR alpha genes of various melanoma cell lines to varying degrees. This may reflect an effect of interferon-gamma on certain subpopulations of melanocytes in vivo. Our data also indicate that partial methylation of the DR alpha gene does not inhibit its expression. Furthermore, interferon-gamma does not appear to induce expression of the DR gene by altering methylation patterns within the region recognized by our probe.

Cell Line↗

PubMed1985

Long-term culture and characterization of alloreactive T-cell infiltrates from renal needle biopsies.

Long-term T-cell lines have been established by culturing renal needle biopsies from kidney recipients undergoing graft rejection. These cultures are maintained in the presence of Interleukin 2, with weekly addition of irradiated donor lymphocytes as a source of antigen. The cells express T-cell markers, T3, T4, and T8, T-cell growth factor (IL2) receptor, and HLA-DR antigens. In one cell line, J2, the T4 and T8 antigens are found on two separate cell populations. This line is also shown to specifically kill Epstein Barr Virus transformed donor cells. This cytotoxicity is directed against HLA-B35 determinant and has been maintained in vitro for over 6 months.

Antibodies, Monoclonal↗