Radioimmunoassay of mammalian type C viral proteins. 3. Detection of viral antigen in normal murine cells and tissues.

A radioimmunoassay specific for a murine leukemia virus structural protein, the gs antigen, detects an antigenic reactivity in normal murine cells in culture and natural tissues. The assay was shown to measure an antigen that is highly related to the virion protein as shown by absorption tests, immunoadsorbent chromatography, and by analysis of linearized dose-response curves. These findings combined with the finding of viral-specific RNA indicate that portions of the viral genome are being expressed with a much greater frequency than previously appreciated.

Direct association of fibronectin and actin molecules in vitro.

Affinity chromatography with actin-Sepharose conjugates of purified human fibronectin, normal human plasma, or serum-free culture fluid from human fibroblasts showed that fibronectin molecules can directly bind to actin. A quantitative recovery of soluble human fibronectin was accomplished by chromatography on actin immobilized on Sepharose beads. Human fibronectin molecules bound to actin-Sepharose were eluted with 0.25--0.35 M potassium bromide, and these molecules competed in a species-specific radioimmunoassay for human fibronectin. The subunits of fibronectin isolated by actin-Sepharose chromatography comigrated in SDS polyacrylamide gel electrophoresis with those of electrophoretically homogeneous fibronectin purified by conventional procedures. The efficient direct binding of fibronectin to actin suggests that interactions between these proteins might also take place in vivo but further studies are needed to elucidate the biological significance of this affinity.

A scanning electron microscope study of surface features of viral and spontaneous transformants of mouse Balb-3T3 cells.

Cells of the mouse line Balb/3T3 as well as three virus-induced transformants and two spontaneous transformants grown in vitro have been studied for their topography by scanning electron microscopy. The parent cell in confluent culture closely resembles an endothelial cell in its form and in the structure of its association with adjacent cells. The tumorigenic transformants produced by SV40, murine sarcoma virus, or polyoma viruses are fusiform to pleomorphic and distinctly different from the cell of origin. They show relatively smooth surfaces except for blebs and marginal microvilli. Perhaps most surprising is the similarity they bear to one another. This is made the more singular by the very different form shown by the tumorigenic transformants of spontaneous origin. One of these, S2-4, possesses a thickened rather than the lamellar form of the parent A31 cell and is covered by long microvilli and many spherical blebs. The other, TuT(3), more closely resembles the cell of origin but shows extensive ruffling at its margins. All transformants grow without evidence of contact inhibition. The significance of the surface morphologies and the factors influencing cell form are discussed.

Specificity of in vitro binding of primate Type C viral RNA and the homologous viral p12 core protein.

The binding of type C viral p12 proteins to purified viral RNA has been examined in vitro with the use of a family of closely related infectious primate type C viruses--the woolly monkey (SSAV) and gibbon (GALV) group. This in vitro protein-RNA binding is type specific. The system should serve as a model for studies of the evolution of nucleic acid binding proteins.

Primate type C virus p30 antigen in cells from humans with acute leukemia.

Antigens related to the major structural protein (p30) of type C viruses isolated from a woolly monkey and a gibbon ape were found in peripheral white blood cells from five patients with acute leukemia.

Basis for the acquisition of malignant potential by mouse cells cultivated in vitro.

Balb/c mouse embryo lines maintained in culture for over 200 generations under conditions that minimize cell-cell contact do not become tumorigenic. Lines cultivated under conditions where there is extensive cell contact become tumor-producing within 30 generations. The tissue-culture property that correlates best with tumorigenicity is the loss of contact inhibition of cell division.

Susceptibility of human diploid fibroblast strains to transformation by SV40 virus.

A quantitative system has been developed for the study of transformation of human diploid fibroblasts in culture by two oncogenic viruses, SV40 and the E46 strain of adeno 7-SV40 "hybrid" virus. Seven of the eleven cell strains derived from human skin biopsies when infected with SV40 (10(9) tissue culture infective doses per milliliter) gave rise to transformed colonies with approximately the same frequency (0.03 percent). Two strains derived from patients with Fanconi's anemia, an autosomal recessive disease associated with a high incidence of chromosome abnormalities and spontaneous neoplasms, gave values more than ten times higher. Two strains from persons heterozygous for this gene were also considerably more susceptible to viral transformation.

Induction of murine C-type viruses from clonal lines of virus-free BALB-3T3 cells.

Each clone of BALB/c mouse embryo cells that has been tested can be induced to form C-type virus. The individual cells therefore contain a complete copy of the genetic information for making the murine RNA tumor viruses.

Evolution of type C viral genes: origin of feline leukemia virus.

Reiterated gene sequences related to the RNA of feline leukemia virus (FeLV) are detected in all tissues of domestic cats and their close Felis relatives but not in more distantly related Felis species. Partially homologous viral gene sequences are found in rodent, and particularly rat, DNA. Together with the immunologic relationships observed between FeLV and endogenous rodent type C viruses, the results lead to the conclusion that FeLV-related genes were transmitted from a rodent to cat ancestor and have been perpetuated in the germ line of cats.

Reverse transcriptases of primate viruses as immunological markers.

Antibodies were prepared against the DNA polymerases (reverse transcriptases) of three potentially oncogenic RNA viruses of primates. Two type C viruses, isolated from a woolly monkey fibrosarcoma and from a gibbon ape lymphosarcoma, have polymerases that are immunologically related to each other and are distinct from the type C viruses isolated from other mammals.

Superinduction of endogenous type C virus by 5-bromodeoxyuridine from transformed mouse clones.

Certain transformed subclones of the mouse cell line BALB/3T3 release 5 to 15 times more type C virus per cell than the parent cell line after treatment with 5-bromodeoxyuridine. Virus release begins within 8 hours and exponentially increases for the first 24 to 48 hours. Superinducibility is associated with the transformed phenotype.

Mammalian cells in culture frequently release type C viruses.

Cell cultures commonly used in animal cell research, both cell strains and continuous cell lines from various mammalian species, spontaneously produce type C RNA viruses.

Transformation induced by Abelson murine leukemia virus involves production of a polypeptide growth factor.

Rat embryo fibroblasts transformed by Abelson murine leukemia virus (MuLV) produce and release a transforming growth factor (TGF). Production of this factor is correlated with a tyrosine-specific protein kinase that is functionally active and is associated with the major Abelson MuLV gene product, P120. Transformation-defective mutants of Abelson MuLV do not transform cells, do not have their virus coded transforming gene product phosphorylated in tyrosine, and do not induce TGF production. Abelson MuLV-induced TGF morphologically transforms cells in culture, competes with 125I-labeled epidermal growth factor (EGF) for binding to cell receptors, and induces phosphorylation of tyrosine acceptor sites in the 160,000-dalton EGF membrane receptor. After purification to homogeneity, Abelson virus-induced TGF migrates as a single polypeptide with an apparent size of 7400 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Structure and function of human amphiregulin: a member of the epidermal growth factor family.

The complete amino acid sequence of amphiregulin, a bifunctional cell growth modulator, was determined. The truncated form contains 78 amino acids, whereas a larger form of amphiregulin contains six additional amino acids at the amino-terminal end. The amino-terminal half of amphiregulin is extremely hydrophilic and contains unusually high numbers of lysine, arginine, and asparagine residues. The carboxyl-terminal half of amphiregulin (residues 46 to 84) exhibits striking homology to the epidermal growth factor (EGF) family of proteins. Amphiregulin binds to the EGF receptor but not as well as EGF does. Amphiregulin fully supplants the requirement for EGF or transforming growth factor-alpha in murine keratinocyte growth, but it is a much weaker growth stimulator in other cell systems.

Bovine leukemia virus genes in the DNA of leukemic cattle.

Reverse transcripts of the rna genome of the bovine leukemia virus (BLV) as well as 125I-labeled BLV RNA hybridize to the DNA of tissues from leukemic cattle with the adult form of the disease but not to bovine thymic lymphoma or normal bovine tissues.

Rat transforming growth factor type 1: structure and relation to epidermal growth factor.

The complete amino acid sequence of rat transforming growth factor type 1 has been determined. This growth factor, obtained from retrovirus-transformed fibroblasts, is structurally and functionally related to mouse epidermal growth factor and human urogastrone. Production of this polypeptide by various neoplastic cells might contribute to the continued expression of the transformed phenotype.

Stimulation of bone resorption in vitro by synthetic transforming growth factor-alpha.

Experiments were conducted to test the hypothesis that tumor-derived transforming growth factor-alpha (TGF-alpha) is responsible for the increased bone resorption and hypercalcemia seen in some malignant diseases. Homogeneous synthetic TGF-alpha prepared by the solid-phase synthesis method stimulated bone resorption directly in vitro in a concentration-dependent manner. Incubation times of 72 hours or more were required to stimulate resorption, which is similar to the time course of bone resorption by epidermal growth factor.

Epithelial wound healing enhanced by transforming growth factor-alpha and vaccinia growth factor.

Epidermal regeneration following middermal injuries to skin requires both proliferation and migration of keratinocytes. Epidermal growth factor (EGF) stimulates the proliferation of keratinocytes in culture, and topical administration of EGF accelerates epidermal regeneration of partial thickness burns or split-thickness incisions in vivo. Transforming growth factor-alpha (TGF-alpha) and vaccinia growth factor (VGF) have substantial sequence homology with EGF, and all appear to bind to the same receptor protein. Whether TGF-alpha or VGF can affect epidermal wound healing in vivo is not known. The present studies show that topical administration of TGF-alpha or VGF in antibiotic cream to partial thickness burns (second degree) accelerated epidermal regeneration in comparison with untreated or vehicle-treated burns. Low levels of both TGF-alpha and VGF (0.1 microgram per milliliter) appeared to be more effective than EGF in stimulating epidermal regeneration. Regenerated epithelium from burns treated with TGF-alpha or VGF appeared normal histologically. This finding suggests that topical application of selected growth factors may be useful in accelerating healing of partial thickness injuries.

Developmental expression of rat transforming growth factor-alpha mRNA.

Expression of the gene encoding transforming growth factor-alpha (TGF alpha) was examined in developing rat embryos by using a cloned TGF alpha cDNA as a hybridization probe. Northern blot analysis of RNA isolated from whole fetuses revealed that TGF alpha mRNA was present at relatively high levels in 8- through 10-day-old embryos and then declined to the low or undetectable level, which is characteristic of adult tissues before birth. The level of TGF alpha mRNA present during early gestation was similar to that present in retrovirus-transformed cells in culture, suggesting that TGF alpha expression is not highly localized in the embryo. These observations are consistent with the hypothesis that TGF alpha plays a role in development, possibly as a fetal growth factor.

The amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity.

We have isolated the gene for a novel growth regulator, amphiregulin (AR), that is evolutionarily related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is a bifunctional growth modulator: it interacts with the EGF/TGF-alpha receptor to promote the growth of normal epithelial cells and inhibits the growth of certain aggressive carcinoma cell lines. The 84-amino-acid mature protein is embedded within a 252-amino-acid transmembrane precursor, an organization similar to that of the TGF-alpha precursor. Human placenta and ovaries were found to express significant amounts of the 1.4-kilobase AR transcript, implicating AR in the regulation of normal cell growth. In addition, the AR gene was localized to chromosomal region 4q13-4q21, a common breakpoint for acute lymphoblastic leukemia.