Robert H. Bassin

Suppression of tropomyosin synthesis, a common biochemical feature of oncogenesis by structurally diverse retroviral oncogenes.

To identify proteins whose production may be altered as a common event in the expression of structurally diverse oncogenes, we compared two-dimensional electropherograms of newly synthesized proteins from NIH/3T3 cell lines transformed by a variety of retroviral oncogenes, from cellular revertant lines, and from a line (433.3) which expresses the v-ras oncogene in response to corticosteroids. Most alterations in the synthesis of specific proteins detected by this approach appeared to be the result of selection during prolonged cultivation and were probably unrelated to the transformation process. However, we detected seven proteins whose synthesis was strongly suppressed in cell lines transformed by each of the six retroviral oncogenes we studied and whose production was fully or partially restored in two cellular revertant lines. Suppression of two of these proteins was also correlated with the initial appearance of morphological alteration during corticosteroid-induced oncogene expression in 433.3 cells. These proteins (p37/4.78 and p41/4.75) were identified as tropomyosins, a group of at least five cytoskeletal proteins. Transformation by the papovaviruses simian virus 40 and polyomavirus caused no suppression of synthesis of these tropomyosins. This indicates that suppression of tropomyosin synthesis is not a nonspecific response by cells to being forced to grow with the transformed phenotype but is specifically associated with oncogenesis by diverse retroviral oncogenes. The results are consistent with the hypothesis that the different biochemical processes initiated by expression of structurally diverse retroviral oncogenes may converge on a limited number of common targets, one of which is the mechanism which regulates the synthesis of tropomyosins.

Normal dba/2 mouse cells synthesize a glycoprotein which interferes with mcf virus infection.

The mechanism of resistance to Friend leukemia virus-induced [mink cell focus-inducing, (MCF)-mediated] leukemogenesis in DBA/2 mice was investigated in cell culture systems. DBA/2 fibroblasts were found to be resistant to infection with MCF viruses but not to ecotropic or amphotropic murine leukemia viruses (MuLVs). Since this resistance has been correlated with the presence of an MCF virus-related gp70 constitutively present on the surface of DBA/2 cells, it seemed possible that the mechanism of resistance in this system involved the saturation of MCF-specific cell surface receptors with the gp70 in analogy to viral interference. Two inhibitors of glycoprotein synthesis, 2-deoxy-d-glucose and tunicamycin, which have been shown to reduce retrovirus-induced interference in productively infected cells, significantly decreased the resistance of DBA/2 cells to productive infection with MCF viruses. This decrease in resistance to MCF virus infection could be correlated with a decrease in the expression of MCF-related gp70 at the cell surface. Cells from several other mouse strains showed neither resistance to MCF virus infection nor enhancement of MCF infectivity following drug treatment. These data indicate that DBA/2 cells are resistant to MCF infection in vitro and, by implication, to MCF-mediated leukemogenesis in vivo by a process analogous to viral interference. Ecotropic pseudotypes of MCF virus were able to productively infect untreated DBA/2 cells, indicating that the cell surface interference-like process described here is the only restriction mechanism responsible for resistance to MCF infection in DBA/2 cells. The results are consistent with the idea that Friend leukemia virus actually causes disease via MCF intermediates.

Phenotypic mixing between N- and B-tropic murine leukemia viruses: Infectious particles with dual sensitivity to Fv-1 restriction

In effort to understand how N or B tropism is determined in murine leukemia virus (MuLV) particles, we analyzed the MuLV produced after dual infection of mouse cells by N- and B-tropic MuLV. The progeny MuLV from such a mixed infection are sensitive to Fv-1 restriction in both N- and B-type cells, but are still highly infectious for mouse cells which do not exhibit Fv-1 restriction. This dual sensitivity to Fv-1 restriction is a phenotypic property of MuLV produced by mixedly infected cells, since individual virus clones derived from this MuLV are either N- or B-tropic. In further experiments, we superinfected murine sarcoma virus (MSV)-transformed cells with mixtures of N- and B-tropic MuLVs. The rescued MSV is restricted in its ability to transforms both N- and B-type cells. The results suggest that N- and B-tropic MuLVs specify different determinants, which are incorporated into virions along with the viral genome and which are the recognition sites for Fv-1 restriction. The presence of a given determinant in a virion renders the virus sensitive to restriction in cells of the opposite Fv-1 type.

Inhibitors of glycosylation reverse retroviral interference

Abstract Cells which are productively infected with retroviruses are extremely resistant to superinfection with the homologous virus. We found that in certain cases, this resistance is largely or entirely eliminated by overnight treatment of the cells with either of two inhibitors of glycosylation (2-deoxyglucose or tunicamycin). This loss of viral interference was accompanied by a loss of gp70 from the cell surface, as demonstrated by surface labeling with 125 I and radioimmunoprecipitation. In other cell-virus combinations, these inhibitors gave much smaller reductions in viral interference and in the level of gp70 at the cell surface. The most likely explanation for these phenomena is that inhibition of glycosylation of the env precursor polyprotein prevents its subsequent processing into gp70 and transport to the cell surface; to the extent that the cellular pool of gp70 is depleted during an overnight treatment, cellular receptors become available for interaction with superinfecting virus particles.

Revertants of mouse cells transformed by murine sarcoma virus: I. Characterization of flat and transformed sublines without a rescuable murine sarcoma virus

Abstract Murine sarcoma virus (MSV)-transformed mouse clonal cell lines produced variants with some properties of nontransformed cells. Such variant cells were epithelioid, contact inhibited, and grew to low density, and their low cloning efficiency in soft agar was similar to that of normal parental 3T3 cells. However, they contained murine leukemia (MuLV) group-specific antigen(s) without demonstrable virus production and reverse transcriptase activity. MSV could no longer be rescued from these flat variant cells by superinfection with MuLV, by cocultivation with normal 3T3 cells or by transspecies rescue into cat cells. An enhancement of sensitivity to MSV and MuLV infection was observed in all flat variant cultures. Flat variant clones spontaneously gave rise to retransformed cells during extended cultivation. Morphology, saturation density, and cloning efficiency in soft agar of cloned spontaneous retransformed cell lines were similar to the original MSV-transformed cells. However, they failed to demonstrate MuLV gs antigen(s), virus production, reverse transcriptase activity and a rescuable MSV genome. The spontaneously retransformed cells were susceptible to MSV and MuLV infection. After treatment with 5-iododeoxyuridine (IUrd), reverse transcriptase activity and virus particles were only rarely induced in flat variant or spontaneously retransformed clones. These particles were not infectious for the original host cells and were not induced in normal 3T3 cells or a majority of the variant clones. Chromosome studies of these variants suggested that the partial or complete loss of expression of transformation in variants might have been associated with an imbalance in the number of chromosomes mediating expression or suppression in these cells.

Isolation of a continuous epithelioid cell line, HBT-3, from a human breast carcinoma.

Summary A continuous line of human breast carcinoma cells, HBT-3, was established in culture following collagenase treatment of a mucus producing adenocarcinoma. The cells are epithelioid in appearance, multiply rapidly, have a cloning efficiency of approximately 70%, and exhibit an abnormal karyotype with a mode of 66-69 chromosomes/cell and 3 markers. Tumor material and pathological diagnosis were provided through the courtesy of Dr. J. D. Mashburn, Director of Laboratories, Washington Sanitarium and Hospital, Takoma Park, MD. Dr. C. S. Stulberg, Senior Research Associate, The Child Research Center of Michigan, Detroit, kindly performed the immunofluorescence cell typing tests. A portion of these studies was carried out at Bionetics Research Laboratories, Inc., Bethesda, MD, under contract 69-2160 from the Special Virus Cancer Program of the National Cancer Institute. Technical assistance of Cathy K. Smith, Nancy Tuttle Fuller, and Diane Robertson is gratefully acknowledged. The authors thank Drs. Peter J. Fischinger and Tadao Aoki of the National Cancer Institute for their advice and many helpful discussions.

Ouabain sensitivity is linked to ras-transformation in human HOS cells

Mouse cells transformed by the retroviral oncogene v-Ki- ras are significantly more sensitive to the toxic effects of 1mM ouabain than are their nontransformed counterparts. We have extended these findings to a human cell line (HOS). HOS cells (ATCC CRL 1543) are relatively resistant to treatment with 1 microM ouabain while KHOS cells (transformed by Kirsten murine sarcoma virus) are extremely sensitive. Two flat revertant cell lines isolated from the KHOS line and lacking the v- ras gene sequences are resistant to ouabain. This effect may be observed morphologically and can also be demonstrated by dye exclusion and plating efficiency tests. In addition, the toxic effects of ouabain may be rapidly and efficiently quantitated in a 51Cr-release assay. This differential lethality may be used to enrich the proportion of non-transformed revertants in populations of mutagen-treated transformed cells.

Balb/3T3 cells chronically infected with N-tropic murine leukemia virus continue to express Fv-1b restriction

Abstract The ability of Fv-1 b mouse cells chronically infected with MuLV of the opposite tropism to exhibit Fv-1 restriction was examined. Balb/3T3 cells were infected at high m.o.i. with restricted (N-tropic) amphotropic MuLV, and a clone of virus-producing cells was established. Dose-response curves using N-ecotropic MuLV indicated that the Balb/3T3 clone chronically infected with amphotropic MuLV exhibited the same degree of restriction as did the uninfected Balb/3T3 line. Thus, Fv-1 restriction is expressed in cells which are at the same time producing restricted virus particles, indicating that Fv-1 restriction does not act on integrated viral DNA nor on any step in the virus replicative cycle after integration. Further implications of these findings are discussed.

Isolation and characterization of a unique C57BL B-tropic virus

Abstract A unique B-tropic virus (CS43) has been isolated from a C57BL mouse. This virus differs from those we have previously characterized and from several isolates we have obtained from normal and leukemic C57BL mice. Unlike other C57BL B-tropic viruses, CS43 does not have a p12 polypeptide homologous to that of the class II xenotropic virus, but does have a p12 homologous to that of AKR MuLV (class I). In common with the other C57BL B-tropic isolates, CS43 has an altered major core protein (p30), which competes only weakly in a class-specific assay that distinguishes ecotropic MuLV p30s from those of xenotropic origin. The data are compatible with a unique recombinational event in the generation of this isolate, which should prove useful in studying the origin of B-tropic viruses and the properties that determine N/B tropism.

STUDIES ON THE MECHANISM OF FV-1 RESTRICTION: PROPERTIES OF DETERMINANTS SPECIFYING VIRAL TROPISM

ABSTRACT. (1) Cells which are simultaneously infected with both N- and B-tropic murine leukemia virus (MuLV) produce infectious MuLV particles which are sensitive to both N- and B-type Fv-1 restriction. When these dually restricted particles are passaged at low moi, some give rise to N-tropic, and others to B-tropic progeny MuLV. These results indicate that N- and B-tropic MuLVs direct the synthesis of different determinants, and that several of these determinants are incorporated into each particle along with the viral genome. These determinants act as targets for Fv-1 restriction, since the presence of a given determinant in a virion renders the virus sensitive to restriction in cells of the opposite Fv-1 type.