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A Gazit

Publications and source records attributed to A Gazit.

At least 109 records · Page 6Linked to original sources
PubMed1991

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

Animals↗
PubMed1991

Tyrphostins inhibit PDGF-induced DNA synthesis and associated early events in smooth muscle cells.

Tyrphostins are low-molecular-weight synthetic inhibitors of protein tyrosine kinase, which block cell proliferation. Since platelet-derived growth factor (PDGF) is thought to figure prominently in disorders of vascular smooth muscle cells (VSMC), such as atherosclerosis, hypertension, and restenosis, we examined whether tyrphostins would inhibit PDGF-induced mitogenesis in VSMC. In this communication, we demonstrate that tyrphostins with the benzenemalononitrile nucleus inhibited PDGF-dependent growth of VSMC as well as PDGF-dependent DNA synthesis in these cells, with the concentrations for 50% inhibition ranging from 0.04 to 9 microM. Up to 30-fold higher tyrphostin concentrations were required to inhibit serum-stimulated DNA synthesis of VSMC. The effect of the tyrphostins is reversible, since on their removal a normal proliferative response to PDGF was resumed. Tyrphostins also inhibited PDGF-receptor autophosphorylation and PDGF-induced phosphorylation of intracellular substrates, including the phosphorylation of phospholipase C-gamma, with a potency ratio similar to their antimitogenic activity. The expression of c-fos mRNA, a mitogenic nuclear signal, was also reduced in PDGF-stimulated VSMC treated with tyrphostins at concentrations which inhibit PDGF-induced mitogenesis. It is concluded that tyrphostins are potent reversible inhibitors of PDGF-induced mitogenesis which act by inhibiting the tyrosine kinase activity of the PDGF receptor and the subsequent signaling cascade. Tyrphostins may be useful in the study and treatment of VSMC proliferation disorders.

Animals↗
PubMed1991

Activation of phospholipase C in human B cells is dependent on tyrosine phosphorylation.

Cross-linking of the surface antigen receptor on B lymphocytes has been demonstrated to lead to activation of phospholipase C (PLC) with subsequent increases in production of inositol phosphates and diacylglycerol. In turn, these second messengers increase cytosolic free calcium [( Ca2+]i) and activate the serine threonine phosphotransferase protein kinase C (PKC). These processes are thought to play a major role in B cell activation and proliferation. However, the mechanism linking the B lymphocyte antigen receptor to phospholipase C remains to be identified. We demonstrate herein that activation of the antigen receptor on human lymphocytes, in addition to activation of PLC, increases tyrosine phosphorylation of specific substrates. Tyrphostins, a new class of tyrosine kinase inhibitors which compete for substrate binding site of specific tyrosine kinases have recently been synthesized. Preincubation of B lymphocytes with two different tyrphostins blocked anti-IgM-induced proliferation, oncogene expression, tyrosine phosphorylation, increases in [Ca2+]i, and production of inositol phosphates. The same inhibitors were without effect on B cell proliferation induced by phorbol esters and cation ionophores which directly activate PKC and increase [Ca2+]i thus bypassing PLC. These findings strongly indicate that tyrphostins do not exhibit significant nonspecific toxicity and suggest that they act proximal to PLC. The ability of the tyrphostins to block increases in [Ca2+]i and inositol phosphate production, after activation of the B cell antigen receptor, indicates that a tyrosine kinase acts as an essential link between the B cell antigen receptor and PLC.

B-Lymphocytes↗
PubMed1990

Identification of sequences encoding the equine infectious anemia virus tat gene.

Equine infectious anemia virus (EIAV), a lentivirus, encodes a trans-activator (tat) which stimulates gene expression directed by the viral long terminal repeat (LTR). This function has been previously shown by us and others to be encoded by sequences within the middle region of the EIAV genome in which two short open reading frames, S1 and S2, reside. In the present study, by using in vitro mutagenesis, we show that disruption of S1, but not S2, completely abolished trans-activation. Addition of oligonucleotides complementary to S1 to cells transfected with a tat expression vector resulted in inhibition of trans-activation. EIAV cDNAs were isolated from a library of EIAV-infected cells constructed by using a eukaryotic expression vector. One cDNA clone which contained S1 sequences was able to trans-activate the EIAV LTR. Sequence analysis of this cDNA clone revealed that, in addition to S1, two other open reading frames were present. The cDNA still retained its activity when the latter two sequences were deleted.

Amino Acid Sequence↗
PubMed1990

An in-vivo infectivity assay for cloned retroviruses lacking a susceptible cell culture.

The lymphoproliferative disease virus (LPDV) of turkeys is the retroviral agent of etiology of a rapidly developing, naturally occurring, lymphoproliferative process. Recently we have molecularly cloned the viral genome. The lack of a susceptible cell culture which can sustain LPDV replication hampered the analysis of the infectious capability of the cloned genome. Based on the efficient in-vivo replication of LPDV we have developed a sensitive in-vivo approach aimed at establishing the infectious capability of the cloned provirus. According to this approach, peripheral leukocytes withdrawn from 3-week-old turkeys were transfected with the cloned DNA and the transfected leukocytes were re-injected into the turkey from which they had been obtained. The injected leukocytes enabled the efficient expression of the viral genome and the release into the blood stream of LPDV virions, which thereafter could travel to their appropriate in-vivo target lymphoid cells and start multiple replication cycles, resulting in the development of a detectable viremia. The applicability of this in-vivo assay for other cloned viral genomes is discussed.

Animals↗
PubMed1990

Equine infectious anemia virus derived from a molecular clone persistently infects horses.

A full-length molecular clone of equine infectious anemia virus (EIAV) was isolated from a persistently infected canine fetal thymus cell line (Cf2Th). Upon transfection of equine dermis cells, the clone, designated CL22, yielded infectious EIAV particles (CL22-V) that replicated in vitro in both Cf2Th cells and an equine dermis cell strain. Horses infected with CL22-V developed an antibody response to viral proteins and possessed viral DNA in peripheral blood mononuclear cells, as determined by polymerase chain reaction assays. In addition, horses infected with CL22-V became persistently infected and were capable of transmitting the infection by transfer of whole blood to uninfected horses. However, CL22-V, like the parental canine cell-adapted virus, did not cause clinical signs in infected horses. Reverse transcriptase assays of CL22-V- and virulent EIAV-infected equine mononuclear cell cultures indicated that the lack of virulence of CL22-V was not due to an inability to infect and replicate in equine mononuclear cells in vitro.

Animals↗
PubMed1990

Pattern of transcription of the genome of equine infectious anemia virus.

The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be expressed at relatively low levels, was difficult to detect consistently, and appeared to be generated by a single splicing event which linked the 5' exon to the 3' region of pol. The 4.0-kb transcript was concluded to be the env mRNA on the basis of its hybridization pattern with the various probes and its abundance. The 2-kb species was found to be multiply spliced and was encoded by sequences derived from orf2 but was not detected by probes representing 3'-env/3'-orf sequences. The 1.8-kb species was shown to consist of sequences representing orf1, part of orf2, and the 3'-orf/env and may represent the message for the EIAV trans-activator gene.

Amino Acid Sequence↗
PubMed1989

Tyrphostins inhibit the epidermal growth factor receptor-mediated breakdown of phosphoinositides.

In response to epidermal growth factor (EGF) and the Ca2+ ionophore A23187, the total phosphatidylinositides (IPT) increased in A431 human epidermoid carcinoma cells 1.8- and 2.0-fold and in the EGF-dependent A431/Clone 15-2 cells 3.0- and 8.0-fold, respectively, over basal levels. Both responses were inhibited by the antiproliferative agents tyrphostins, but the EGF-induced increase in IPT was inhibited to a much greater extent than that induced by the ionophore. Tyrphostins which are potent EGF-receptor kinase inhibitors were also potent in blocking the EGF-induced production of phosphoinositides. The less potent tyrphostins were found to inhibit the EGF-dependent IPT formation more weakly. These results support the notion that phospholipase C is activated through its phosphorylation by the EGF receptor.

Calcimycin↗
PubMed1989

Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyrosine kinase activity in living cells and EGF-stimulated cell proliferation.

Synthetic compounds called tyrphostins were examined for their effects on cells which are mitogenically responsive to epidermal growth factor (EGF). We studied in detail the effects of two tyrphostins on EGF binding, tyrosine phosphorylation in intact cells, EGF-receptor internalization, and mitogenesis. These compounds inhibited EGF-stimulated [3H]thymidine incorporation in a specific manner and the degree of selectivity varied. Both compounds inhibited EGF-stimulated receptor autophosphorylation and tyrosine phosphorylation of endogenous substrates in intact cells at doses that correlated with the IC50 for [3H] thymidine incorporation. These results are consistent with the notion that tyrosine phosphorylation is a crucial signal in transduction of the mitogenic message delivered by EGF. The compound RG50864 demonstrated specificity at inhibiting EGF-stimulated cell growth compared with stimulation with either platelet-derived growth factor or serum. For both compounds RG50864 and RG50810, long term exposure (16 h) of cells to tyrphostins was required for optimal inhibition because of the instability and slow action of these compounds. Tyrphostins did not alter cell surface display of EGF-receptor, EGF binding or EGF-induced internalization, degradation, and down-regulation of EGF receptors. These novel synthetic inhibitors, specific for EGF-receptor kinase, offer a new method to inhibit EGF-stimulated cell proliferation which may be useful in treating specific pathological conditions involving cellular proliferation, including different types of cancers.

Animals↗
PubMed1989

Tyrphostins I: synthesis and biological activity of protein tyrosine kinase inhibitors.

A novel class of low molecular weight protein tyrosine kinase inhibitors is described. These compounds constitute a systematic series of molecules with a progressive increase in affinity toward the substrate site of the EGF receptor kinase domain. These competitive inhibitors also effectively block the EGF-dependent autophosphorylation of the receptor. The potent EGF receptor kinase blockers examined were found to competitively inhibit the homologous insulin receptor kinase at 10(2)-10(3) higher inhibitor concentrations in spite of the significant homology between these protein tyrosine kinases. These results demonstrate the ability to synthesize selective tyrosine kinase inhibitors. The most potent EGF receptor kinase inhibitors also inhibit the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on EGF independent cell growth. These results demonstrate the potential use of protein tyrosine kinase inhibitors as selective antiproliferative agents for proliferative diseases caused by the hyperactivity of protein tyrosine kinases. We have suggested the name "tyrphostins" for this class of antiproliferative compounds which act as protein tyrosine kinase blockers.

Benzylidene Compounds↗
PubMed1989

Generation of fibrosarcomas in vivo by a retrovirus that expresses the normal B chain of platelet-derived growth factor and mimics the alternative splice pattern of the v-sis oncogene.

A retrovirus containing the entire human platelet-derived growth factor B-chain (PDGF-B) gene was constructed in order to investigate the in vivo biological activity of its encoded growth factor. When this virus was introduced into newborn mice, it reproducibly generated fibrosarcomas at the site of inoculation. Proviruses in each fibrosarcoma analyzed had lost 149 nucleotides downstream of the PDGF-B coding region. This deletion originated from an alternative or aberrant splice event that occurred within exon 7 of the PDGF-B gene and mimicked the v-sis oncogene. Thus, deletion of this region may be necessary for efficient retrovirus replication or for more potent transforming function. Evidence that the normal growth factor coding sequence was unaltered derived from RNase protection studies and immunoprecipitation analysis. Tumors were generally polyclonal but demonstrated clonal subpopulations. Moreover, tumor-derived cell lines became monoclonal within a few tissue culture passages and rapidly formed tumors in vivo. These findings argue that overexpression of the normal human PDGF-B gene product under retrovirus control can induce the fully malignant phenotype.

Abelson murine leukemia virus↗
PubMed1989

Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag.

To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques. The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits. When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test, there was 98.7% concordance among the assays. By using the ELISA it was possible to specifically detect antibodies earlier after experimental infection of horses with EIAV than with the other two tests. A competition ELISA developed in order to detect EIAV gag antigens was found to be approximately 15 times more sensitive than the radioimmunoassay for EIAV p15gag. Antigens of other animal lentiviruses as well as those of the prototype oncovirus failed to compete in this assay.

Animals↗
PubMed1989

Analysis of regulatory elements of the equine infectious anemia virus and caprine arthritis-encephalitis virus long terminal repeats.

We analyzed the equine infectious anemia virus (EIAV) long terminal repeat (LTR) for sequences that influence its promoter activity and ability to be trans-activated by the EIAV tat gene product. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 (SV40) regulatory sequences were used for these studies. We were able to identify the EIAV promoter region and showed that sequences within the U3 region significantly inhibited LTR-directed transcription. However, when placed in a heterologous context (SV40 promoter) these U3 sequences functioned as an enhancer. trans-activation of the EIAV LTR was found to depend upon sequences downstream of the transcription initiation site and also within U3. Deletion mutagenesis experiments showed that the major downstream element was present in a 46-nucleotide stretch (+4 to +50). An SV40 promoter construct containing these sequences could be trans-activated in cells expressing the EIAV tat gene product. For comparative purposes we also examined the LTR of another animal lentivirus, caprine arthritis-encephalitis virus (CAEV), for positive and negative transcriptional regulatory elements and demonstrated the presence of an enhancer within its U3 sequence. There is evidence that trans-activation of the CAEV LTR requires U3 sequences. When the EIAV U3 region was replaced by the CAEV U3 sequence, the promoter activity of the EIAV LTR was markedly elevated, but the responsiveness to the EIAV trans-activator could not be restored.

Animals↗
PubMed1989

Molecular cloning of an oncogenic replication-competent virus that causes lymphoproliferative disease in turkeys.

The lymphoproliferative disease virus of turkeys was molecularly cloned, structurally mapped, and shown to represent a distinct class of retroviruses evolutionarily related to the avian leukemia-sarcoma virus group. The cloned provirus did not contain any known oncogene or other cellularly derived sequences and was established as a replication-competent oncogenic entity capable of inducing the disease in the absence of any associated transforming counterpart.

Animals↗
PubMed1988

Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors.

A systematic series of low molecular weight protein tyrosine kinase inhibitors were synthesized; they had progressively increasing affinity over a 2500-fold range toward the substrate site of epidermal growth factor (EGF) receptor kinase domain. These compounds inhibited EGF receptor kinase activity up to three orders of magnitude more than they inhibited insulin receptor kinase, and they also effectively inhibited the EGF-dependent autophosphorylation of the receptor. The most potent compounds effectively inhibited the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on the EGF-independent proliferation of these cells. The potential use of tyrosine protein kinase inhibitors as antiproliferative agents is demonstrated.

Binding, Competitive↗
PubMed1988

Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat.

We used the Escherichia coli chloramphenicol acetyltransferase gene (cat) to study sequences that influence expression of the equine infectious anemia virus (EIAV) genome. The EIAV long terminal repeat (LTR) directed CAT activity in a canine cell line, but at levels much lower than those achieved with other eucaryotic viral promoters. In the same cells infected with EIAV or cotransfected with molecularly cloned EIAV genomic DNA, LTR-directed activity was markedly enhanced. Comparison of cat mRNA and protein levels in these cells indicated that this trans-activating effect could be accounted for by a bimodal mechanism in which both transcriptional and posttranscriptional events are enhanced. trans-Activation but not promoter activity was abolished by deletion of the R-U5 region of the EIAV LTR. EIAV sequences responsible for the trans-activating function could be localized to a region encompassing the 3' and 5' termini of the pol and env genes, respectively (nucleotides 4474 to 5775). Interestingly, this stretch harbors a short open reading frame with some amino acid sequence similarity to the human immunodeficiency virus type I tat gene product.

DNA Mutational Analysis↗