William A. Strohl

The response of BHK21 cells to infection with type 12 adenovirus

Transformation of a clonal line of BHK21 cells by type 12 adenovirus (Ad 12) is described. A transformation rate of approximately 2 × 10−5 per initially infected cell was observed after high multiplicity infection. A study of the fate of the abortively infected cells revealed, however, that only a small fraction survived the infection and initiated growth of a colony. Of these rare surviving colonies, nearly half contained transformed cells. Of three BHK21 sublines tested, one yielded transformed cells only from colonies growing in soft agar suspension, a second yielded transformants only as foci in monolayers, while the third did not yield detectable transformants by either method. The adenovirus-transformed cells were distinguished from the parental BHK21 cells by the following characteristics: 1) a marked morphological alteration; 2) the synthesis of adenovirus tumor antigen; 3) growth to high cell density in the presence of a low concentration of serum; 4) induction, in hamsters, of tumors identical to those induced by inoculation of type 12 virus; 5) restriction in the ability to support the multiplication of type 2 adenovirus (Ad2). a marked morphological alteration; the synthesis of adenovirus tumor antigen; growth to high cell density in the presence of a low concentration of serum; induction, in hamsters, of tumors identical to those induced by inoculation of type 12 virus; restriction in the ability to support the multiplication of type 2 adenovirus (Ad2). The latter property is most likely due to blockage of a step late in the adenovirus replicative cycle, since 88% of the cells, which synthesized Ad2-specific structural antigens did not yield infectious virus. A similar shift from a productive to a largely abortive response to Ad 2 was seen in BHK21 cells simultaneously infected with Ad 12 and Ad2. This finding supports the view that the restriction of Ad2 replication in Ad 12-transformed cells, like the other characteristics mentioned, is associated with continued activity of at least part of the Ad12 genome.

The response of BHK21 cells to infection with type 12 adenovirus: II. Relationship of virus-stimulated DNA synthesis to other viral functions☆

Abstract BHK21 cells were shown by autoradiography to be arrested in G 1 after growth in 0.2% fetal calf serum. Infection with Ad12 at high multiplicity resulted in the initiation of DNA synthesis in over 90% of the cells, beginning at 10–12 hours after infection. Quantitative correlations revealed that one infecting particle was sufficient to stimulate DNA synthesis, and did so with an efficiency equal to that of the initiation of T antigen (T Ag) synthesis. Simultaneous study of the same cells by autoradiography and immunofluorescence after low multiplicity infection demonstrated that DNA synthesis was initiated only in cells in which T Ag had also been synthesized. This fact implicated the transcription of a viral gene or genes in the initiation of DNA synthesis. Chromosome preparations showed that the infection was followed, beginning at 22 hours, by a sharp increase in mitotic activity. Initially, all resulting metaphase figures were intact except for a small number of chromatid breaks. Starting at 30 hours after infection such relatively “normal” metaphase figures disappeared, to be replaced by a continuing accumulation of cells which were in what appeared to be a highly disorganized mitosis with extensively fragmented or overcondensed chromosomes. By 48 hours after infection, 25% of the cells in the culture were in this condition. These results are taken to indicate that the virus initiates chromosomal DNA synthesis but that infection eventually results in extensive destruction of the cell genome, and thus the death of the cell.

Properties of cells derived from adenovirus-induced hamster tumors by long-term in vitro cultivation: I. Clonal stability of three biological characteristics

Abstract After several hundred generations in vitro, HT cells originally derived from type 12 adenovirus (Ad.12)-induced hamster tumors have retained their malignant character, the capacity to synthesize Ad.12-specific antigen(s), and a significantly reduced capacity, compared with that of normal hamster cells, to produce infectious type 2 adenovirus (Ad.2) in response to superinfection. Of four independently derived cell lines studied, one (HT2) consistently produces less Ad.2 than the other three lines. No evidence has been obtained for “rescue” of Ad.12 among the yields from Ad.2-superinfected HT cells. The low yield from such cells is not due to poor adsorption of inoculum virus. Thirteen clones of two cell lines (HT2 and HT8) have been established after microdrop isolation of single cells. The fact that all clones tested have retained the three characteristics suggests genetic homogeneity of the parental populations. In particular, the differing extent to which synthesis of Ad.2 is depressed in the two parental lines is reflected in the response of their derived clones. The evidence argues against the idea that HT cell populations consist of a majority of genetically stable “resistant” and a minority of stably “susceptible” cells.

The response of BHK21 cells to infection with type 12 adenovirus: I. Cell killing and T antigen synthesis as correlated viral genome functions☆

Abstract BHK21 cell killing by type 12 adenovirus (Ad12) has been shown (1) to be a viral genome function, (2) to require only one successfully infecting virus particle, and (3) to be induced with an efficiency somewhat greater than the initiation of viral early protein synthesis, measured as Ad12 tumor-specific antigen (T Ag). When the multiplicity of infection is increased beyond that required to initiate T Ag synthesis in 98% of the cells, a persistent surviving fraction of cells is found, the magnitude of which is dependent on the extent and rate of cell multiplication during the first 24 hours after infection. Analysis of T Ag synthesis in clones produced by infected cells indicates that while infected cells can divide initially, the capacity to synthesize T Ag is not replicated, since the multiplying cells are all T Ag (−) even though they may be the offspring or sisters of T Ag (+) cells. While single T Ag (+) cells may persist for several days after infection, most of these detach from the coverslip within 24–48 hours after infection and can no longer give rise to clones. The increased survival of cells infected at high multiplicity while in logarithmic growth cannot be accounted for only by dilution of the infecting genomes and is not due to a genetically resistant cell type in the population.

Adenovirus Tumorigenesis: Role of the Viral Genome in Determining Tumor Morphology

Adenovirus type 12 transforms the fibroblastic BHK21 (baby hamster kidney) cell line into rounded or cuboidal cells that give rise in hamsters to undifferentiated small cell sarcomas indistinguishable from those induced in newborn hamsters by inoculation of the virus itself. In contrast. cells from this line transformed by polyoma virus retain their fibroblastic morphology and induce fibrosarcomas in hamsters. This suggests that the morphology of tumors induced by the adenovirus-transformed cells from this line may be determined by the viral genome and that such mechanism may also explain the remarkably uniform microscopic appearance which seems to characterize tumors induced in hamsters by direct inoculation of adenovirus type 12.

Encapsulation of high molecular weight DNA in large unilamellar phospholipid vesicles: Dependence on the size of the DNA

Liposomes, phospholipid vesicles, have been used for the past several years as a gentle and simple means of introducing into mammalian cells biologically active agents for which the cell surface membrane is not permeable. Multilamellar vesicles and small unilamellar vesicles have been used as carriers for small molecules, drugs and enzymes (reviewed in [ 11). A preparation similar to multilamellar vesicles has also been used to entrap DNA [2] and as a carrier for chromosomes [3]. Recently two procedures have been described for the preparation of large unilamellar vesicles [4,5]. Using these techniques, globin mRNA was introduced into differentiated mammalian cells resulting in the production of a globin-like prdtein [6,7]. In addition, after encapsulation intact poliovirus and purified poliovirus RNA were shown to be infectious in cells normally resistant to infection with poliovirus [8,9]. We report here that large unilamellar phosphatidylserine vesicles (LUV) are capable of encapsulating fragments of phage T7 DNA, prepared by digestion with restriction endonucleases with mol. wt 0.27-14.19 X 106. The encapsulated DNA is resistant to nuclease digestion. The efficiency of encapsulation of DNA is shown to be dependent upon the size of the DNA molecules.

Properties of cells derived from adenovirus-induced hamster tumors by long-term in vitro cultivation: II. Nature of the restricted response to type 2 adenovirus

Abstract The response of cultured cells derived from type 12 adenovirus-induced hamster tumors (HT cells) to superinfection with type 2 adenovirus (Ad.2) has been studied in detail. The low yield of infectious Ad.2 from such cultures has been shown, by infectious center and mass yield analyses, to be due to the presence of a small number of virus-producing cells. However, all superinfected HT cells are capable of producing viral structural antigen(s), provided the input multiplicity of Ad.2 is at least of the order of 10–100 PFU per cell. The restricted production of infectious progeny is not mediated by a transmissible interfering factor such as interferon. HT cells are as competent to produce vesicular stomatitis virus as are normal hamster cells. SV40-transformed hamster cells and normal hamster cells produce comparable (large) amounts of type 2 adenovirus. The evidence is consistent with the hypothesis that the response of HT cells to superinfection with Ad.2 is controlled by persistent genetic material of type 12 adenovirus responsible for the initiation of the malignant change. Several considerations are discussed which suggest that the response to superinfection is a more specific indicator of genome persistence than other identified properties of HT cells and that, operationally, the relation of the Ad.12 genome to HT cells may be analogous to that of a defective prophage to its bacterial host.

Quantitative studies of natural and experimental adenovirus infections of human cells: II. Primary cultures and the possible role of asynchronous viral multiplication in the maintenance of infection

Abstract Various quantitative characteristics of tonsil and adenoid specimens naturally and experimentally infected with adenovirus have been studied, particularly with the use of cultures prepared from trypsinized surgical specimens. Thirteen of twenty specimens studied yielded adenoviruses. In positive specimens, less than 1 per 107 cells plated proved to be infected although the incidence of cells susceptible to infection with type 2 adenovirus was at least 4000 times greater (1 per 250 cells). Virus multiplication followed the same time course in cultures initiated with cell suspensions or tissue fragments, despite the absence of virus neutralizing activity (presumably antibody) after trypsinization. A considerable asynchrony in the time of appearance of progeny virus was observed in naturally infected specimens, to the extent that 50% of the cells which ultimately yielded virus did not contain detectable PFU before 5 days. The possible relevance of the observed asynchrony to persistent adenovirus infections in vivo is discussed. The maximum frequency of susceptible cells was found by infectious center measurements on exogenously infected primary cell suspensions to be 0.4 to 2 × 10−2 per cell. This proportion was correlated with the frequency of cells giving rise to fibroblastic cells in cultures made from the primary suspension. The cells of the lymphocytic series appeared to be refractory to virus. That cells susceptible to adenovirus were present before initiation of cultures was suggested both by the infectious center results and by the characteristics of viral growth curves on cells in the primary suspension. The similarity of viral multiplication in specimens with and without a simultaneous naturally occurring infection indicated that natural, persistent adenovirus infections of human tonsils and adenoids were not accompanied by alterations in susceptibility of cells to virus.

Quantitative Studies of Natural and Experimental Adenovirus Infections of Human Cells. I. Characteristics of Viral Multiplication in Fibroblasts derived by Long-Term Culture from Tonsils.

Abstract Fibroblastic cells derived from suspensions of trypsinized human tonsil S have been subcultured for several months, and their properties with respect to infection with type 2 adenovirus have been studied. Plaques were obtained with an efficiency of 10–20% of that on KB cells. Correlated with this was the fact that adsorption on tonsil-derived fibroblasts was not measurable under conditions permitting greater than 95% uptake of virus by KB cells. Following infection at high input multiplicity, the yield of virus per cell was found to be 10 4 PFU, and the latent period about 18 hours. On the other hand, study of the virus yields from single cells infected at a low input multiplicity (10 −2 PFU KB per cell) revealed the existence of marked asynchrony in the appearance of infectious virus, such that 40% of the infected cells had not yielded virus after 5 days.

The response of BHK21 cells to infection with type 12 adenovirus: VI. Synthesis of virus-specific RNA

Abstract When G1-arrested BHK21 cells are infected with type 12 adenovirus (Ad12), some of the newly synthesized RNA is virus specific. RNA-DNA hybridization-competition experiments have demonstrated that this RNA corresponds to up to 60% of those “early” mRNA sequences which are transcribed in productively infected human embryonic kidney (HEK) cells. No “late” Ad12 mRNA was detected in BHK21 cells. Comparison of Ad12-specific RNA sequences transcribed in Ad12-infected BHK21 cells and in Ad12-induced hamster tumor cells (line HT 2 ) by hybridization-competition reaction indicated that no virus-specific sequences are transcribed in HT 2 cells which are not also present in BHK21 cells abortively infected with Ad12. Attempts to detect any Ad12 DNA synthesis in BHK21 cells by DNA-DNA hybridization, under conditions of minimal background cellular DNA synthesis, were unsuccessful. Correspondingly, immunofluorescent staining experiments failed to detect Ad12 capsid protein synthesis in over 10 4 infected cells.