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H E Varmus

Publications and source records attributed to H E Varmus.

At least 37 records · Page 2Linked to original sources
PubMed1998

Basic fibroblast growth factor induces cell migration and proliferation after glia-specific gene transfer in mice.

Basic fibroblast growth factor (bFGF) is overexpressed in most high-grade human gliomas, implying that it is involved in the pathogenesis of these tumors. To assess the biological effect of inappropriate production of bFGF in normal astrocytes, we developed a system for glia-specific gene transfer in transgenic mice. A transgene encoding the receptor for subgroup A avian leukosis virus and controlled by the astrocyte-specific glial fibrillary acidic protein promoter permits efficient glia-specific transfer of genes carried by subgroup A avian leukosis virus vectors. With this system, we have demonstrated that bFGF induces proliferation and migration of glial cells in vivo, without the induction of tumors.

Alkaline Phosphatase↗
PubMed1998

Genetic mapping of the cloned subgroup A avian sarcoma and leukosis virus receptor gene to the TVA locus.

A chicken gene conferring susceptibility to subgroup A avian sarcoma and leukosis viruses (ASLV-A) was recently identified by a gene transfer strategy. Classical genetic approaches had previously identified a locus, TVA, that controls susceptibility to ASLV-A. Using restriction fragment length polymorphism (RFLP) mapping in inbred susceptible (TVA*S) and resistant (TVA*R) chicken lines, we demonstrate that in 93 F2 progeny an RFLP for the cloned receptor gene segregates with TVA. From these analyses we calculate that the cloned receptor gene lies within 5 centimorgans of TVA, making it highly probable that the cloned gene is the previously identified locus TVA. The polymorphism that distinguishes the two alleles of TVA in these inbred lines affects the encoded amino acid sequence of the region of Tva that encompasses the viral binding domain. However, analysis of the genomic sequence encoding this region of Tva in randomly bred chickens suggests that the altered virus binding domain is not the basis for genetic resistance in the chicken lines analyzed.

Amino Acid Sequence↗
PubMed1997

Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a.

Members of the Wnt family of signaling molecules are expressed differentially along the dorsal-ventral axis of the developing neural tube. Thus we asked whether Wnt factors are involved in patterning of the nervous system along this axis. We show that Wnt-1 and Wnt-3a, both of which are expressed in the dorsal portion of the neural tube, could synergize with the neural inducers noggin and chordin in Xenopus animal explants to generate the most dorsal neural structure, the neural crest, as determined by the expression of Krox-20, AP-2, and slug. Overexpression of Wnt-1 or Wnt-3a in the neuroectoderm of whole embryos led to a dramatic increase of slug and Krox-20-expressing cells, but the hindbrain expression of Krox-20 remained unaffected. Enlargement in the neural crest population could occur even when cell proliferation was inhibited. Wnt-5A and Wnt-8, neither of which is expressed in the dorsal neuroectoderm, failed to induce neural crest markers. Overexpression of glycogen synthase kinase 3, known to antagonize Wnt signaling, blocked the neural-crest-inducing activity of Wnt-3a in animal explants and inhibited neural crest formation in whole embryos. We suggest that Wnt-1 and Wnt-3a have a role in patterning the neural tube along its dorsoventral axis and function in the differentiation of the neural crest.

Animals↗
PubMed1997

Rescue of osteoclast function by transgenic expression of kinase-deficient Src in src-/- mutant mice.

The Src tyrosine kinase has been implicated in a wide variety of signal transduction pathways, yet despite the nearly ubiquitous expression of c-src, src-/- mice show only one major phenotype-osteopetrosis caused by an intrinsic defect in osteoclasts, the cells responsible for resorbing bone. To explore further the role of Src both in osteoclasts and other cell types, we have generated transgenic mice that express the wild-type and mutated versions of the chicken c-src proto-oncogene from the promoter of tartrate resistant acid phosphatase (TRAP), a gene that is expressed highly in osteoclasts. We demonstrate here that expression of a wild-type transgene in only a limited number of tissues can fully rescue the src-/- phenotype. Surprisingly, expression of kinase-defective alleles of c-src also reduces osteopetrosis in src-/- animals and partially rescues a defect in cytoskeletal organization observed in src-/- osteoclasts. These results suggest that there are essential kinase-independent functions for Src in vivo. Biochemical examination of osteoclasts from these mice suggest that Src may function in part by recruiting or activating other tyrosine kinases.

Acid Phosphatase↗
PubMed1997

Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action.

Frizzled polypeptides are integral membrane proteins that recently were shown to function as receptors for Wnt signaling molecules. Here, we report the identification of a novel, secreted 36-kDa protein that contains a region homologous to a putative Wnt-binding domain of Frizzleds. This protein, called Frizzled-related protein (FRP), was first identified as a heparin-binding polypeptide that copurified with hepatocyte growth factor/scatter factor in conditioned medium from a human embryonic lung fibroblast line. Degenerate oligonucleotides, based on the NH2-terminal sequence of the purified protein, were used to isolate corresponding cDNA clones. These encoded a 313-amino acid polypeptide, containing a cysteine-rich domain of approximately 110 residues that was 30-40% identical to the putative ligand-binding domain of Frizzled proteins. A 4.4-kb transcript of the FRP gene is present in many organs, both in the adult and during embryogenesis, and homologs of the gene are detectable in DNA from several vertebrate species. In biosynthetic studies, FRP was secreted but, like Wnts, tended to remain associated with cells. When coexpressed with several Wnt family members in early Xenopus embryos, FRP antagonized Wnt-dependent duplication of the embryonic dorsal axis. These results indicate that FRP may function as an inhibitor of Wnt action during development and in the adult.

Amino Acid Sequence↗
PubMed1997

Absence of p53 in a mouse mammary tumor model promotes tumor cell proliferation without affecting apoptosis.

Loss or mutation of p53 may have multiple biological and genetic effects that result in accelerated tumor progression. Loss of p53 in some tumors has been correlated with a marked decrease in tumor cell apoptosis. p53 loss may also accelerate tumor growth through an increase in cell proliferation rates. To examine the effects of p53 loss on tumor progression in a controlled experimental context, we previously crossed p53-deficient mice to mammary tumor-susceptible Wnt-1 transgenic (TG) mice. The resulting female Wnt-1 TG offspring of this cross all developed mammary tumors, regardless of p53 status (p53+/+, p53+/-, or p53-/-). However, female p53-/- Wnt-1 TG mice developed tumors much sooner than their p53+/+ counterparts. In this report, we demonstrate that the average growth rates of tumors missing (p53-/-) or losing p53 (p53+/- with loss of heterozygosity) are accelerated compared to tumors with both wild-type p53 alleles (p53+/+). This accelerated growth rate appears to be due primarily to increases in rates of tumor cell proliferation. Tumor cell apoptotic levels were modest and were not measurably different in the presence or absence of wild-type p53. These results differ substantially from other mouse tumor models in which p53 loss was closely correlated with accelerated growth rates through attenuated apoptosis. Thus, the mechanisms by which p53 loss influences tumor progression may differ, depending on the tissue type and/or the oncogenic pathways involved.

Animals↗
PubMed1996

A characteristic bent conformation of RNA pseudoknots promotes -1 frameshifting during translation of retroviral RNA.

The structures of four different RNA pseudoknots that provide one of the signals required for ribosomal frameshifting in mouse mammary tumor virus have been determined by NMR. The RNA pseudoknots have similar sequences and assume similar secondary structures, but show significantly different frameshifting efficiencies. The three-dimensional structures of one frameshifting and one non-frameshifting RNA pseudoknot had been determined previously by our group. Here we determine the structures of two new RNA pseudoknots, and relate the structures of all four pseudoknots to their frameshifting abilities. The two efficient frameshifting pseudoknots adopt characteristic bent conformations with stem 1 bending towards the major groove of stem 2. In contrast, the two poor frameshifting pseudoknots have structures very different from each other and from the efficient frameshifters. One has linear, coaxially stacked stems, the other has stems twisted and bent, but in the opposite direction to the efficient frameshifters. Changes in loop size that favor bending (shorter loops) increase frameshifting efficiency; longer loops that allow linear arrangement of the stems decrease frameshifting. Frameshifting pseudoknots in feline immunodeficiency virus and simian retrovirus have different loop sequences, but the sequences at their stem junctions imply the same bent conformation as in the mouse mammary tumor viral RNA. The requirement for a precise pseudoknot conformation for efficient frameshifting strongly implies that a specific interaction occurs between the viral RNA pseudoknot and the host protein-synthesizing machinery.

Base Sequence↗
PubMed1996

Wild-type p53 transgenic mice exhibit altered differentiation of the ureteric bud and possess small kidneys.

Transgenic mice expressing wild-type murine p53 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) undergo progressive renal failure due to abnormal kidney development. Similar phenotypes are observed in two transgenic lines that express wild-type p53 within the ureteric bud but not in transgenic animals expressing a dominant-negative p53 mutant allele. Defective differentiation of the ureteric bud, as evidenced by altered marker expression during development, accompanies expression of the p53 transgene. At E17.5-18.5, metanephric mesenchymal cells undergo high rates of apoptosis, and fewer cells than normal are converted to tubular epithelium. As a result, p53 transgenic kidneys grow to only half of their expected size and contain about half of the normal number of nephrons, with compensatory hypertrophy of the glomeruli. In this setting, rather than arrest the cell cycle or induce apoptosis directly, abnormally high levels of wild-type p53 appear to alter cellular differentiation in embryonic ureteric buds and cause secondary effects (apoptosis and inefficient conversion to epithelium) in the adjacent undifferentiated mesenchyme.

Animals↗
PubMed1996

Deficiency of the Hck and Src tyrosine kinases results in extreme levels of extramedullary hematopoiesis.

Expression of the Src-family kinases--Src, Hck, and Fgr--increases dramatically during myeloid cell development. Src-deficient mice exhibit functional abnormalities in only one myeloid cell type, the osteoclast, resulting in impaired bone remodeling and osteopetrosis, while hck-/- or fgr-/- mice have few and subtle myeloid cell deficiencies. To determine whether these limited phenotypes are due to the coexpression of multiple Src-family kinases with overlapping functions, we have intercrossed src-/- mice to hck-/- and fgr-/- mutants to produce double mutants. Two thirds of hck-/- src-/- double mutants die at birth; surviving animals develop a severe form of osteopetrosis, resulting in extreme levels of splenic extramedullary hematopoiesis, anemia, and leukopenia. These hematopoietic defects are caused by abnormalities in the bone marrow environment because hck-/- src-/- mutant stem cells reconstitute a normal hematopoietic system in irradiated wild-type mice. In contrast, fgr-/- src-/- double mutants have no defects beyond those observed in src-/- animals. Cultured normal murine osteoclasts express abundant amounts of Src, Hck, and Fgr and Hck levels are increased in src-/- osteoclasts. These observations suggest that Hck and Src serve partially overlapping functions in osteoclasts and that the expression of Hck in src-/- osteoclasts ameliorates their functional defects.

Animals↗
PubMed1996

Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection.

The viral integrase (IN) protein is the only viral protein known to be required for integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host cell DNA, a step in the viral life cycle that is essential for viral replication. To better understand the relationship between in vitro IN activity and IN-mediated integration of viral DNA in an infected cell, we characterized the effects of 13 IN mutations on viral replication in cultured cells. Using HIV-1 genomes that express the hygromycin resistance gene and do not express the HIV-1 env gene, we generated stocks of pseudotype virus coated with the murine leukemia virus amphotropic envelope glycoprotein, containing either wild-type or mutant HIV-1 IN. All mutants produced normal amounts of physical particles, as measured by reverse transcriptase activity and capsid protein (p24) concentration, but they formed three groups based on infectious titer and synthesis of viral DNA. Changes at the three highly conserved acidic residues in the IN core domain (D-64, D-116, and E-152) impair provirus formation without affecting viral DNA synthesis or the accumulation of viral DNA in the nucleus of the infected cell, a phenotype predicted by each mutant's lack of in vitro integrase activity. Mutations at positions N-120, R-199, and W-235 minimally affect in vitro integrase activity, but infectious titers are severely reduced, despite normal synthesis of viral DNA, implying a defect during integration in vivo. Mutations in the zinc binding region (H12C, H16V, and H16C), S81R, and a deletion of residues 32 through 275 yield noninfectious particles that synthesize little or no viral DNA following infection, despite wild-type levels of reverse transcriptase activity and viral RNA in the particles. The two latter classes of mutants suggest that IN can affect DNA synthesis or integration during infection in ways that are not appreciated from currently used assays in vitro.

Animals↗
PubMed1996

Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation.

Activation of telomerase in human cancers is thought to be necessary to overcome the progressive loss of telomeric DNA that accompanies proliferation of normal somatic cells. According to this model, telomerase provides a growth advantage to cells in which extensive terminal sequence loss threatens viability. To test these ideas, we have examined telomere dynamics and telomerase activation during mammary tumorigenesis in mice carrying a mouse mammary tumor virus long terminal repeat-driven Wnt-1 transgene. We also analyzed Wnt-1-induced mammary tumors in mice lacking p53 function. Normal mammary glands, hyperplastic mammary glands, and mammary carcinomas all had the long telomeres (20 to 50 kb) typical of Mus musculus and did not show telomere shortening during tumor development. Nevertheless, telomerase activity and the RNA component of the enzyme were consistently upregulated in Wnt-1-induced mammary tumors compared with normal and hyperplastic tissues. The upregulation of telomerase activity and RNA also occurred during tumorigenesis in p53-deficient mice. The expression of telomerase RNA correlated strongly with histone H4 mRNA in all normal tissues and tumors, indicating that the RNA component of telomerase is regulated with cell proliferation. Telomerase activity in the tumors was elevated to a greater extent than telomerase RNA, implying that the enzymatic activity of telomerase is regulated at additional levels. Our data suggest that the mechanism of telomerase activation in mouse mammary tumors is not linked to global loss of telomere function but involves multiple regulatory events including upregulation of telomerase RNA in proliferating cells.

Animals↗
PubMed1995

Overexpression of the Sky receptor tyrosine kinase at the cell surface or in the cytoplasm results in ligand-independent activation.

Most receptor tyrosine kinases are activated by dimerization induced by their cognate ligands. Protein S, an abundant serum protein previously shown to be a potent anticoagulation factor, has been proposed to be a ligand for the Sky tyrosine kinase (Stitt et al., 1995). Here we show that Sky, when expressed to high levels, is tyrosine phosphorylated even in the absence of a ligand. Furthermore, a version of Sky (termed Sky delta SS) engineered to be overexpressed in the cytoplasm and thus in a ligand-free environement, can function as a dimeric tyrosine kinase. Sky delta SS can transform RatB1a fibroblasts and thus retains all the properties of the full-length Sky kinase. These data suggest that Sky, when overexpressed either at the cell surface or in the cytoplasm, is competent to form dimers even in the absence of its ligand. We also demonstrate that an isoform of Sky, originally reported as Brt and here termed Sky Isoform I, resides in the cytoplasm. Therefore, the activities of Sky delta SS we describe may reflect those of the naturally occurring Isoform I.

Animals↗
PubMed1995

A genetic screen identifies cellular factors involved in retroviral -1 frameshifting.

To identify cellular factors that function in -1 ribosomal frameshifting, we have developed assays in the yeast Saccharomyces cerevisiae to screen for host mutants in which frameshifting is specifically affected. Expression vectors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of the lacZ gene or the CUP1 gene so that the reporters are in the -1 frame relative to the initiation codon. These vectors have been used to demonstrate that -1 frameshifting is recapitulated in yeast in response to retroviral mRNA signals. Using these reporters, we have isolated spontaneous host mutants in two complementation groups, ifs1 and ifs2, in which frameshifting is increased 2-fold. These mutants are also hypersensitive to antibiotics that target the 40S ribosomal subunit. We have cloned the IFS1 gene and shown that it encodes a previously undescribed protein of 1091 aa with clusters of acidic residues in the carboxyl-terminal region. Haploid cells lacking 82% of the IFS1 open reading frame are viable and phenotypically identical to ifs1-1 mutants. This approach could help identify potential targets for antiretroviral agents.

Adaptor Proteins, Signal Transducing↗
PubMed1995

c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism.

We have explored the role of the tyrosine kinase c-Src in cellular adhesion. Fibroblasts derived from src-/- mice (src-/- fibroblasts) exhibit a reduced rate of spreading on fibronectin. These defect is rescued by expression of wild-type chicken c-Src. Analyses of mutants suggest that c-Src increases the rate of cell spreading in src-/- fibroblasts through a kinase-independent mechanism requiring both the SH3 and SH2 domains. To further address the role of c-Src in adhesion, we examined the activity and subcellular distribution of c-Src during the adhesion of fibroblasts on fibronectin. We observed a transient increase in the specific kinase activity of c-Src accompanied by the partial dephosphorylation of the negative regulatory site Y527. Activation of c-Src is followed by its redistribution to newly formed focal adhesions. These results suggest that the enzymatic activity and subcellular distribution of c-Src are coordinately regulated during cellular adhesion and that c-Src can affect adhesion by a kinase-independent mechanism.

Animals↗
PubMed1995

Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos.

Glycogen synthase kinase 3 (GSK-3) is homologous to the product of the Drosophila gene shaggy (zeste-white 3), which is required for signalling by wingless during Drosophila development. To test whether GSK-3 is also involved in vertebrate pattern formation, its role was investigated during early Xenopus development. It was found that dominant-negative GSK-3 mutants induced dorsal differentiation, whereas wild-type GSK-3 induced ventralization. These results indicate that GSK-3 is required for ventral differentiation, and suggest that dorsal differentiation may involve the suppression of GSK-3 activity by a wingless/wnt-related signal.

Animals↗
PubMed1995

Deficiency of p53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability.

By crossing mice that carry a null allele of p53 with transgenic mice that develop mammary adenocarcinomas under the influence of a Wnt-1 transgene, we have studied the consequences of p53 deficiency in mammary gland neoplasia. In Wnt-1 transgenic mice homozygous for the p53 null allele, tumors appear at an earlier age than in animals heterozygous or wild-type at the p53 locus. About half of the tumors arising in p53 heterozygotes exhibit loss of the normal p53 allele, implying selection for p53-deficient cells. Mammary tumors lacking p53 display less fibrotic histopathology and increased genomic instability with aneuploidy, amplifications, and deletions, as detected by karyotype analysis and comparative genomic hybridization. In one tumor, the amplified region of chromosome 7 had an ectopically expressed int-2/FGF3 proto-oncogene, a gene known to cooperate with Wnt-1 in the production of mammary tumors. These findings favor a model in which p53 deficiency relaxes normal restraints on chromosomal number and organization during tumorigenesis.

Adenocarcinoma↗