Marian Fogel

The activation of virus synthesis in polyoma-transformed cells

Abstract The activation of virus synthesis has been studied in two polyoma virus (PV) transformed cell lines obtained after infection of cell cultures from rat embryo muscles with a small plaque (SP) and a large plaque (LP) strain of PV. The isolation of clones from these two cell lines in the presence of anti-PV antiserum has shown, that although there was no detectable virus synthesis in 30 clones derived from the SP transformed line, 92 out of 100 clones derived from the LP transformed line continued to produce virus synthesizing cells. The virus produced by the virus synthesizing cells was of the LP type. The results indicate, that there can be a “spontaneous” activation of virus synthesis in clones from the LP transformed line, that this ability for “spontaneous” activation can be transmitted to progeny clones, and that clones with “spontaneous” activation can segregate some clones that appear to be no longer activatible under the same conditions. The “spontaneous” activation of virus synthesis generally occurred in about 1 in 10 4 cells. It was shown that the frequency of virus synthesizing cells in clones with “spontaneous” activation can be increased about 100 fold by X-irradiation, and in multinucleate cells formed after fusion of LP transformed and normal mouse cells with Sendai virus.

THE INDUCTION OF FORSSMAN-ANTIGEN SYNTHESIS IN HAMSTER AND MOUSE CELLS IN TISSUE CULTURE, AS DETECTED BY THE FLUORESCENT-ANTIBODY TECHNIQUE.

Abstract The fluorescent-antibody technique has been used to determine whether hamster and mouse cells that are not synthesizing Forssman (F) antigen in vivo can be induced to synthesize this antigen by plating in vitro , and whether different normal and neoplastic cell types can be induced under these conditions. In primary cultures of normal hamster embryos, there was no detectable F antigen in cells spread on the coverslips at one day after cell plating in vitro . After two days the antigen appeared as scattered points in the cytoplasm of some cells, and after three days it was found in nearly all cells. In secondary hamster-embryo cultures, and in cultures made from F-positive tumors, the antigen was already detectable when the cells were examined at one day after plating. The results indicate that F-antigen synthesis after plating primary cultures in vitro was due to antigen induction in cells with originally no detectable antigen, and not due to the selective growth of F-positive cells. Induction occurred even when cell division was inhibited by X-irradiation. Induction of F antigen was also observed both in the epithelial and fibroblast-like cells of hamster kidneys, and in mouse-embryo cells. In contrast to the results with hamsters, a proportion of F-negative cells were found in the mouse cultures even after twelve days in vitro .

Induction of virus synthesis in polyoma-transformed cells by DNA antimetabolites and by irradiation after pretreatment with 5-bromodeoxyuridine

Polyoma-virus (PV)-transformed cells which exhibited a spontaneous induction rate of about 1 × 10−4 cells for polyoma viral capsid (V) antigen synthesis, producing 100 plaque-forming units (PFU) per induced cell, were shown to be inducible for PV synthesis by various DNA antimetabolites. Mitomycin C (MC), nitrogen mustard (NH2), and N-nitrosomethylurea (NMU) induced 43, 24, and 9% of cells, respectively, for V antigen synthesis. The yields of infectious virus in the treated cultures were increased by a factor of up to 5 × 102, as compared to untreated cultures. The inducibility of the cells by MC, NH2, and NMU was shown to be dose-dependent increasing with the concentration of the inducing compounds to a maximum which varied for each inducer. Among the induced cells, some did not produce infectious virus, but produced V antigen; in the cultures which exhibited maximum cell inducibility for V antigen synthesis, only about 38–47% of the V antigen-containing cells produced virus. The synthesis of V antigen and of infectious virus was shown to occur in cells irreversibly blocked for cell division by the inducing agents. Bromodeoxyuridine, iododeoxyuridine, and fluorodeoxyuridine induced V antigen production in 0.8–1.4% cells, the induced cells yielding an average of 12–50 PFU per cell. Pretreatment of the cells with bromodeoxyuridine increased the inducibility of the cells for V antigen production by UV and X-irradiation, by factors of up to 10 and 15, respectively, and for infectious virus synthesis by factors of up to 5 and 6, respectively. The results suggest that the cellular DNA is the target for induction of virus synthesis in these cells and that damage of the cellular DNA results in activation of the virus genome.

Polyoma virus synthesis in tumor cells as measured by the fluorescent antibody technique.

Abstract A study has been made, by use of the fluorescent antibody technique, of virus synthesis in tumors induced by polyoma in different species. It was found that the proportion of fluorescent cells increased after plating in vitro . There was a high percentage (up to 89 %) of fluorescent cells in primary mouse tumors, a lower percentage in primary rat tumors, and a very small amount of fluorescence in primary hamster tumors. The percentage of fluorescent cells in the hamsters increased considerably (up to 42%) in tumors that had been grown in vivo as cell transplants. Evidence was obtained in all three species of an abortive multiplication cycle in tumor cells, resulting in the production of fluorescent viral antigen without complete infective virus. The use of fluorescent antibodies can thus be of value in demonstrating the existence of viral genetic material in tumor cells that do not produce infective virus.

Integration of viral into chromosomal deoxyribonucleic acid in an inducible line of polyoma-transformed cells

Abstract RNA-DNA hybridization has shown that in poiyoma-transformed rat cells which can be induced to synthesize infectious virus (LPT cells), polyoma DNA sequences are associated with chromosomal DNA RNA complementary to the viral DNA (cRNA) was synthesized in vitro, using purified viral DNA as a template and Escherichia coli RNA polymerase. High-molecular-weight chromosomal DNA was fractionated from linear and supercoiled viral DNA molecules by centrifugation of whole cells through alkaline glycerol gradients. Hybridization carried out between the cRNA and fractionated chromosomal DNA showed that the amount of RNA hybridized to the LPT DNA was two to three times larger than the amount hybridized to DNA from normal rat cells. cRNA was also hybridized, under the same conditions, with mixtures containing a constant amount of normal cell DNA and varying quantities of purified viral DNA. These assays have established that a linear relationship exists between the amount of cRNA specifically hybridized with a given sample of DNA and the quantity of viral DNA in the sample. Using this relationship, it is estimated that LPT chromosomal DNA contains 6–9 genome-equivalents of polyoma DNA per cell. This quantity represents 18–29% of the amount of polyoma DNA found in the cells, as determined by hybridization of cRNA with unfractionated LPT DNA. To exclude the possibility that the chromosomally associated viral DNA is an artifact due to incomplete removal of the extrachromosomal viral DNA, control experiments were performed in which the cells were superinfected with polyoma virus (m.o.i.-500; 3 hr infection). In these experiments, less than 1% of the viral DNA introduced into the cells by the superinfecting virus were found by the same techniques to be associated with chromosomal DNA. Other experiments show that LPT cells do not contain significant amounts of complex viral DNA molecules which sediment in the vicinity of chromosomal DNA. It is therefore suggested that viral and chromosomal DNA are bound to each other by bonds which cannot be disrupted by alkali treatment.

Phenotypic changes in cultured hamster embryonic liver cells as studied by immunofluorescence

Abstract Dedifferentiation of cultured hamster embryo liver cells with respect to the liver-specific antigen (LSA) was studied by means of the immunofluorescent technique. Depending on the intercellular relations, conditioned by the method used for culture, liver cells may either preserve or lose LSA. LSA disappeared from the cells after they were grown in monolayer cultures for a few days. Conversion of the cells from LSA plus to minus was due to phenotypic changes and not to selection. Various factors restricting division of the cells in monolayer cultures affected unequally their capacity to synthesize LSA. LSA was preserved in cells maintained at 24°C, but disappeared from those in which division was stopped by X-irradiation. Contact among like cells prevents the disappearance of LSA, and its synthesis may be restored in organ-cultured pellets of cells which have been precultured in monolayers for no longer than 5–6 days.

Infection of human cells with polyoma virus

Abstract Human cells which are resistant to infection with polyoma virus (PV) and PV-DNA by the conventional virus-absorption method were infected by microinjection of the virus or the viral-DNA into the cell cytoplasm. Induction of PV-tumor (T) antigen production and synthesis of cellular DNA were found in a large number of the infected cells, but no viral capsid (V) antigen was detected. Among 3 × 103 virus-injected cells, none developed into a transformed cell colony. Multinucleated human muscle cells (myotubes), in which DNA synthesis and mitosis are irreversibly repressed after microinjection of PV, do not incorporate [3H]thymidine but produce T-antigen.

Synthesis of an antigenic material (RAM) in rat cells cultured in vitro and in rat organs in vivo

The induction of the synthesis of an antigenic material (RAM) in in vitro cultivated rat cells and the appearance of such material in intact organs in vivo has been studied by means of the fluorescent antibody technique. Cells from organized structures that did not contain detectable RAM in vivo could be induced to synthesize this antigen when cultured in vitro. In primary cultures from embryonic livers and hearts of new-born rats no antigen was present, except in occasional cells, after 1 day of growth in vitro. In cultures from whole embryos about one-third of the cells gave a faint fluorescent after 1 day. The conversion of most of the cells in which the RAM was undetectable during the first 24 hr of cultivation in vitro to those that contained the antigen occurred after 1 day in cultures from whole embryos, after 2 days in the cardiac cells and approximately after 3 days in liver cultures. The results indicate that the appearance of the antigenic material was due to the synthesis of this substance in cells in which the antigen was formerly not detectable and not due to a selective growth of RAM containing cells. In embryonic tissue fragments explanted in vitro the antigen appeared mainly on the periphery of the pieces and in the flat-elongated cells that wandered out from the tissue fragments. The central part of such tissue fragments remained predominantly negative after several days of culture in vitro. In a proportion of pieces, fluorescence appeared in the intercellular spaces either on one part or throughout the upper surface of the whole tissue fragment. In embryo monolayer cultures 5–6 days after explantation colonies composed of spheroid cells with increased cellular contact appeared. In such colonies the antigen was either localized in the intercellular spaces or was not present at all. The distribution of the antigenic material in different rat organs in vivo was not uniform. It was undetectable in the liver, and in other organs it was confined to certain regions mainly revealing an interstitial localization. RAM continued to be present for about two years in eight in vitro propagated cell clones obtained from a polyoma-induced rat kidney tumor, and in one cell line derived from rat embryo mass culture. The distribution of RAM in rats in vivo, the conditions favoring its appearance in vitro, and its localization in monolayer cultures and in cultivated tissue fragments suggests that this antigenic substance might correspond to the Forssman type antigen in Forssman positive species. The possible role of this antigenic material is discussed.

Polyoma virus-transformed rat cell lines inducible for viral capsid antigen synthesis

Abstract A number of polyoma virus (PV)-transformed lines of rat, mouse and hamster origin established in the course of this study were investigated for virus induction by mitomycin C. Neither infectious virus nor V-antigen was detected in six mouse and four hamster lines. Out of 14 rat lines, three were found inducible for infectious virus, and one could be induced only for V-antigen synthesis. Two lines, designated as RPA and RPB differ from the others with respect to their response to mitomycin C and elevated temperature (40°). The proportion of V-antigen-containing cells increased 50- and 60-fold in the RPA and RPB lines, respectively, when the mitomycin C-treated cultures were subsequently incubated at 40° for 24 hr, as compared to the cultures kept continuously at 37°. No infectious virus was detected in the mitomycin C-treated RPA cultures incubated at either temperature. The RPB line can be induced for infectious virus production at a cell frequency of 1 100 V-antigen-containing cells when incubated at 37°. Though the proportion of V-antigen-containing cells increased in the heated cultures of this line 60-fold, the number of plaques produced by the cell extracts and the proportion of virus-producing cells were similar both in the heated and unheated cultures. Fifty percent of clones isolated from the RPA line and about 80% of RPB clones are inducible, exhibiting, except for one RPB clone, a similar pattern of response to the inducing agents as the parental lines. In heterokaryon cultures of untreated mouse embryo cells and RPA or RPB cells pretreated with mitomycin C and heat, a proportion of multinucleated cells was shown to contain V-antigen. However, virus maturation was not detected in the heterokaryon cultures with RPA cells and was not significantly enhanced in the cultures with RPB cells. We also studied the effect of elevated temperature (40°) on PV induction by mitomycin C in highly inducible PV-transformed lines. In contrast to the RPA and RPB lines, in the highly inducible lines, the elevated temperature, though exerting a relatively weak enhancing effect on induction by mitomycin C, increased the proportion of V-antigen-containing cells and the virus yields to an almost equal extent. These results would indicate that the lack of virus production in the V-antigen-containing RPA and in most RPB cells is due to a deficiency of the viral genome persisting in these cells and that a temperature-sensitive factor is involved in the process of V-antigen induction in these lines.