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Biomedical subjects

U R Rapp

Publications and source records attributed to U R Rapp.

At least 163 records · Page 9Linked to original sources
PubMed1990

Complete coding sequence of a human B-raf cDNA and detection of B-raf protein kinase with isozyme specific antibodies.

A 2.2 kb cDNA clone which presumably includes the complete human B-raf coding sequence was isolated from a human testes cDNA library. Sequence analysis showed the presence of all three conserved regions CR1, CR2 and CR3 previously identified in raf protein kinases. The cDNA was expressed in E. coli yielding a B-raf fusion protein which reacts with antibodies specific for B-raf raised against the C-terminal peptide as well as with monoclonal antibodies which map into the kinase domain.

Amino Acid Sequence↗
PubMed1989

Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor.

We have examined the interaction between the serine/threonine kinase proto-oncogene product Raf-1 and the tyrosine kinase PDGF beta-receptor. Raf-1 tyrosine phosphorylation and kinase activity were increased by PDGF treatment of 3T3 cells or CHO cells expressing wild-type PDGF receptors but not mutant receptors defective in transmitting mitogenic signals, suggesting that the increase in Raf-1 kinase activity is a significant event in PDGF-induced mitogenesis. Concurrent with these increases, Raf-1 associated with the ligand-activated PDGF receptor. Furthermore, both mammalian Raf-1 and Raf-1 expressed using a recombinant baculoviral vector, associated in vitro with baculoviral-expressed PDGF receptor. This association was markedly decreased by prior phosphatase treatment of the receptor. Following incubation of partially purified baculoviral-expressed PDGF receptor with partially purified Raf-1, Raf-1 became phosphorylated on tyrosine and its serine/threonine kinase activity increased 4- to 6-fold. This is the first demonstration of the direct modulation of a protein activity by a growth factor receptor tyrosine kinase.

Animals↗
PubMed1989

An altered v-raf is required in addition to v-myc in J3V1 virus for acceleration of murine plasmacytomagenesis.

We have isolated and molecularly cloned a highly pathogenic virus variant, J3V1, from murine plasmacytomas induced by a combination of pristane and the weakly transforming recombinant retrovirus J3. J3 virus is a derivative of the v-raf/v-myc-carrying J2 virus that was generated by a frameshifting deletion inactivating v-raf in J2. J3V1 contains an additional deletion of 334 base paris in gag, which restores the correct reading frame for v-raf and results in the expression of a p57 gag-raf fusion protein. Reactivation of v-raf in J3 is required for efficient plasmacytoma acceleration in pristane-conditioned BALB/cAn mice.

Amino Acid Sequence↗
PubMed1989

raf/myc-infected erythroid cells are restricted in their ability to terminally differentiate.

A comparison was made of the in vitro erythroid colony-forming abilities of v-raf-, v-myc-, and v-raf/v-myc-containing retroviruses. In methylcellulose, v-raf efficiently produced colonies of well-differentiated hemoglobin-synthesizing erythroid cells, whereas v-raf/v-myc-infected erythroid cells were inhibited from terminally differentiating but retained the ability to replicate extensively. In contrast, v-myc was unable to stimulate the formation of erythroid colonies.

Animals↗
PubMed1989

Susceptibility to raf and raf/myc retroviruses is governed by different genetic loci.

The susceptibility to tumors induced by raf and raf/myc retroviruses was investigated in BALB/c, C57BL/6, (BALB/c x C57BL/6)F1 and (BALB/c x C57BL/6) backcross mice. Newborn mice were susceptible to neoplasms generated by both viruses, but resistance to raf-induced leukemia developed rapidly in all mice as they matured. Older C57BL/6 mice were also resistant to raf/myc lymphomas, whereas BALB/c mice remained susceptible to the virus at all ages, indicating that different genes control susceptibility to raf and raf/myc tumors. From these data and the susceptibility of C x B recombinant inbred strains, it appears that very few genes (perhaps even a single gene) may govern susceptibility to raf/myc lymphomas and that resistance is the dominant trait.

Age Factors↗
PubMed1989

Tyrosine kinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells through signaling pathways involving c-myc: conditional regulation of c-myc transcription by temperature-sensitive v-abl.

Retroviral expression vectors carrying the tyrosine kinase oncogenes abl, fms, src, and trk abrogate the requirements of murine myeloid FDC-P1 cells for interleukin-3 (IL-3). Factor-independent clones constitutively express c-myc in the absence of IL-3, whereas in parental cultures c-myc transcription requires the presence of the ligand. To directly test the effect of a tyrosine kinase oncogene on c-myc expression, retroviral constructs containing three different temperature-sensitive mutants of v-abl were introduced into myeloid IL-3-dependent FDC-P1 and 32D cells. At the permissive temperature, clones expressing temperature-sensitive abl behaved like wild-type abl-containing cells in their growth properties and expressed c-myc constitutively. Temperature shift experiments demonstrated that both IL-3 abrogation and the regulation of c-myc expression correlated with the presence of functional v-abl. Induction of c-myc expression by reactivation of temperature-sensitive v-abl mimicked c-myc induction by IL-3 in that it did not require protein synthesis and occurred at the level of transcription, with effects on both initiation and a transcription elongation block. However, v-abl-regulated FDC-P1 cell growth differed from IL-3-regulated growth in that c-fos and junB, which are normally induced by IL-3, were not induced by activation of v-abl.

Animals↗
PubMed1989

Expression of raf oncogenes activates the PEA1 transcription factor motif.

PEA1 (AP1) motif transcription enhancer activity was stimulated by v-raf and more efficiently by activated c-raf-1 or A-raf than by their normal counterparts, in agreement with a role for PEA1 in transformation by raf. Mutations in the ATP-binding site of v-raf prevented activation, suggesting that phosphorylation is somehow required.

Adenosine Triphosphate↗
PubMed1989

Loss of heterozygosity at the c-raf locus in small cell lung carcinoma.

The c-raf-1 oncogene is located at chromosome 3p25, near a region known to be specifically deleted in patients with renal cell carcinoma and small cell lung carcinoma (SCLC). From cytogenetic analyses of SCLC cell lines, we have estimated that one c-raf-1 allele was deleted in approximately 80% of the cases. However, c-raf-1 was generally thought to be distal to the most common deletion in SCLC, 3p14-23. Using restriction site polymorphisms (RFLPs) located within the c-raf-1 locus, we have examined DNA from 84 human lung carcinomas. In an analysis of 11 paired (normal versus tumor) SCLC DNA samples, all five informative cases showed loss of heterozygosity at this locus in the corresponding tumor sample. Analysis of 73 unpaired lung carcinoma DNAs showed that out of 31 non-SCLC samples, 19% were heterozygous for the BglI polymorphism and 25% showed heterozygosity with TaqI. However, all of the 42 SCLC samples were homozygous for both of these RFLPs. This striking loss of heterozygosity at the c-raf-1 locus in SCLC indicates that one allele of c-raf-1 is deleted in SCLC. The kinase activity of the c-raf protein appears to be constitutively activated in these cells. Whether this apparent activation results from genetic or epigenetic events is under investigation.

Carcinoma, Small Cell↗
PubMed1988

In vitro proliferation of human large granular lymphocytes with v-raf/v-myc recombinant retrovirus.

The effect of infection with a retrovirus carrying v-raf/v-myc oncogenes (J2 virus) on the in vitro proliferation of human large granular lymphocytes (LGL) was investigated. LGL infected with J2 virus (J2LGL), unlike uninfected cells, grew with a proliferation peak eight days after infection. Such cells retained the morphology and functional properties typical of LGL. Furthermore, 5% of J2LGL produced virus the day after infection, whereas non-virus production was detectable five days later. These data indicate that J2 virus provides a transient mitogenic signal for LGL.

Cell Division↗
PubMed1988

Phorbol ester stimulates the synthesis of sphingomyelin in NIH 3T3 cells. A diminished response in cells transformed with human A-raf carrying retrovirus.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [14C]choline-labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerPCho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of TPA (2-100 nM) had progressively less effect on CerPCho synthesis which correlated with increased hydrolysis of precursor PtdCho. In cells transformed with human or mouse A-raf carrying retroviruses TPA-stimulated PtdCho hydrolysis, but not CerPCho synthesis, suggesting independent regulation of these processes by the TPA-stimulated signal transduction system.

Animals↗
PubMed1988

Expression of human c-raf-1 oncogene proteins in E. coli.

Full length and truncated versions of the human c-raf-1 cDNA were cloned into the inducible E. coli expression vector pJL6. C-raf proteins of 73 kD, 57 kD and 39 kD were produced upon induction. p73 differs from normal p73 c-raf by deletion of the two first N-terminal amino acids and their replacement by 16 amino acids encoded by the vector. The p57 and p39 represent N-terminal deletions which leave the transforming protein kinase domain intact. These proteins could be readily purified from E. coli lysates by immunoprecipitation with raf-specific antisera.

Amino Acids↗
PubMed1988

Expression of the c-raf protooncogene, gamma-glutamyltranspeptidase, and gap junction protein in rat liver neoplasms.

Female Harlan Sprague-Dawley and F-344 rats were subjected to a 70% hepatectomy and 18 h later given one dose of 30 mg diethylnitrosamine/kg body weight. Beginning 1 week later, the animals were fed a diet containing 0.05% phenobarbital. Groups of rats were sacrificed 6 and 15 months later, and the livers were either frozen for cryostat sectioning or used to isolate RNA. Primary liver tumors present in these animals were used for RNA isolation, and a portion was taken for histopathological analysis. Eleven of 13 primary lesions, consisting of either neoplastic nodules or hepatocellular carcinomas, showed elevated levels of mRNA for the c-raf protooncogene. Increased c-raf mRNA in these tumors appeared to be unrelated to their cellular proliferative status inasmuch as the levels of c-raf mRNA did not correlate with levels of H4 histone mRNA. Decreased expression of the major rat liver gap junction protein mRNA was observed in all of the primary tumors. Immunocytochemical analysis using an anti-gap junction antibody revealed a decrease in gap junction immunoreactivity in some but not all preneoplastic focal lesions. All preneoplastic foci having positive gamma-glutamyltranspeptidase enzyme staining also exhibited a marked increase in gamma-glutamyltranspeptidase mRNA as determined by in situ hybridization. The possible relation of alterations of the mRNA levels of c-raf and the gap junction protein to the further development of preneoplastic foci is discussed.

Animals↗
PubMed1988

Preparation of raf-oncogene-specific antiserum with raf protein produced in E. coli.

In this report we describe the expression of v-raf protein in E. coli using a tryptophan-promoter-driven expression vector and its immunological characterization by anti-peptide sera. The purified recombinant protein was used to produce raf-specific antibodies which are suitable for studying v-raf and c-raf proteins in vitro and in vivo in a variety of species ranging from mouse to man.

Amino Acid Sequence↗
PubMed1988

A single point mutation in the envelope gene is responsible for replication and XC fusion deficiency of the endogenous ecotropic C3H/He murine leukemia virus and for its repair in culture.

The molecular basis has been determined for differences in infectivity and XC phenotype of endogenous ecotropic murine leukemia virus of the low-leukemia mouse strain C3H/He, its relative in the high-leukemia mouse strain AKR, and highly infectious, XC-positive C3H virus variants selected in vitro. Endogenous ecotropic type C virus induced by iododeoxyuridine from the nontransformed C3H/10T1/2 cell line is XC negative and replication deficient. In contrast, viruses produced late after iododeoxyuridine induction in chemically transformed C3H/10T1/2 cells (MCA5) are XC positive and infectious. XC-negative viruses can be converted to XC-positive viruses by being grown in certain transformed cell lines. We have cloned the endogenous ecotropic provirus of C3H/He from MCA5 cells, which is XC negative and replication deficient, as well as two XC-positive C3H proviruses derived by in vitro conversion. Fragment exchange between the XC-negative molecular clone p110 and the XC-positive AKR virus clone p623 revealed that the defect in p110 lies 3' of the SalI site located in the pol region. Nucleotide sequencing established that the C3H p110 provirus was integrated within the R region of an endogenous VL30 long terminal repeat (LTR) in reverse orientation and that the virus differed from the infectious AKR p623 provirus by a point mutation, substituting Lys for Arg at the potential precursor cleavage site for gp70 and p15E. In vitro-converted XC-positive C3H proviral clones 3211 and 4211 are identical to XC-negative C3H p110, except that they have Arg at this site and the normal cleavage site is thus regenerated in these clones. The XC-negative C3H p110 was blocked in processing of Pr85env, whereas clones 3211 and 4211 had normal cleavage of the env precursor into gp70. Both the XC-negative C3H provirus and the in vitro-converted XC-positive C3H proviruses had a single copy of a 99-base-pair enhancer element in the LTR, whereas two copies of this sequence are present in the AKR proviral LTR. Substitution of Arg for Lys at the envelope precursor processing site of C3H p110 by site-directed mutagenesis is sufficient by itself to convert the virus to the XC-positive replication-competent phenotype. Thus, we have established that a single point mutation at the processing site of the envelope precursor protein Pr85 is responsible for the difference in the infectivity and XC phenotype of endogenous ecotropic murine leukemia virus from C3H/He and AKR mice and that the basis for in vitro conversion is a mutation at this site.

AKR murine leukemia virus↗