Transformed Hamster Cells Research Articles

Immunological analysis of plasminogen activators from normal and transformed hamster cells. Evidence that the plasminogen activators produced by SV40 virus-transformed hamster embryo cells and normal hamster lung cells are antigenically identical.

Rabbits were immunized against the plasminogen activator released by SV4- virus-transformed hamster embryo cells. The resulting antiplasminogen activator immunoglobulin (APA-IgG) inhibited the enzymatic activity of the plasminogen activator produced by SV40-transformed hamster cells, and the plasmin-catalyzed release of these cells from the tissue culture dish. APA-IgG was not cytotoxic for these cells even in the presence of complement and did not inhibit their release of plasminogen activator. APA-IgG formed a single precipitin line in immunodiffusion plates using highly purified plasminogen activator as antigen. APA-IgG inhibited the plasminogen activator produced by newborn hamster lung cells and by an established diploid line (DON) of hamster lung cells, but did not inhibit plasminogen activators produced by normal or transformed hamster kidney cells or by cells of other species (mouse and human). We derive three major conclusions from these data: (a) There are several immunologically distinguishable forms (isozymes) of plasminogen activators in normal hamster tissues. (b) The plasminogen activators produced by normal hamster lung cells and by SV40 virus-transformed hamster embryo cells share antigenic determinants and are presumably the same isozyme. (c) The plasminogen activators produced by different hamster tumor cells do not share antigenic determinants and are presumably different isozymes.

Presence of Chicken Cell Surface Antigen on Rous Virus Activated in Heterokaryons of Transformed Non-permissive Hamster Cells and Chicken Cells

Incubation with antiserum to chick embryo (CE) cells, in the presence of complement, inactivates Rous sarcoma virus (RSV) produced by heterokaryons formed by non-permissive RSV-transformed hamster cells and CE cells as well as RSV produced by heterokaryons is observed following incubation with antiserum to the transformed hamster cells, plus complement. Hence, the envelope of RSV activated in heterokaryons, as that of RSV produced by CE cells, must contain a surface antigen of the CE cell, and the virus must mature only, or preferentially, at chicken-specific sites of the heterokaryon surface.

Lectin receptors on the cell surface membrane and the kinetics of lectin-induced cell agglutination

Agglutination of malignant transformed hamster cells by concanavalin A (ConA) and the lectins from wheat germ (WGA) and soybean (SBA) has been automatically quantitated, by measuring the amount of light transmitted through a cell suspension. The transformed hamster cells were agglutinated by SBA only after treatment with neuraminidase. The initial rate of agglutination and the concentration of lectin ( K c ) required for the half-maximum rate ( V m ) has been determined. The initial rate and V m were lower and more temperature-sensitive, and the K c was higher, for ConA than for WGA and SBA. There was no detectable temperature-dependent phase transition for the initial rate of agglutination. The total number of receptors was lower for ConA than for WGA and SBA and the apparent association constant between lectin molecules and cell surface receptors was higher for ConA (10 7M −1) than for WGA and SBA (1.6 × 10 6M −1). The half V m of agglutination required 75% saturation of the cell receptors for ConA, and only 13–17% saturation of the receptors for SBA and WGA. A 30% decrease in the number of SBA receptors present in agglutinable cells completely prevented their agglutination. The results indicate that there is heterogeneity of lectin receptors on the cell surface and that only a small proportion of the total number of WGA and SBA receptors have to be occupied for agglutination by these lectins.

Effect of Interferon on Induction of S Antigen by Polyoma Virus in BHK 21 Cells

A new surface antigen (S antigen) can be detected by immunofluorescence during the abortive transformation of hamster cells by polyoma virus. It was found that addition of interferon greatly reduces the percentage of cells positive for S antigen.

Transcription of the genome of adenovirus type 12. I. Viral mRNA in abortively infected and transformed cells.

In baby hamster kidney (BKH-21) cells abortively infected with adenovirus type 12, polysome-associated, virus-specific RNA could be detected starting 5 to 7 h after infection. The amount of this RNA reached a maximum between 10 to 12 h after infection and continued to be synthesized at a reduced level until late in infection (48 to 50 h.). In BHK-21 cells transformed by adenovirus type 12 (HB cells), 0.26% of the polysome-associated mRNA was virus specific. The size of the virus-specific mRNA isolated from polysomes of BHK-21 cells abortively infected with, or transformed by adenovirus type 12 was determined by electrophoresis in polyacrylamide gels in 98% formamide, i.e., under conditions which eliminated secondary structure or aggregation of RNA. In abortively infected hamster cells viral mRNA size classes of molecular weights 0.9 times 10-6 and 0.65 times 10-6 to 0.67 times 10-6 were predominant. A minor fraction of 1.5 times 10-6 daltons was consistently found and increased with time after infection. Late after infection (24 to 26 h), viral mRNA of 1.9 times 10-6 daltons was also observed. The size distribution of adenovirus type 12-specific mRNA from transformed hamster cells (HB line) was very similar to that in abortively infected cells, except that the relative amount of the viral mRNA fraction of 1.5 times 10-6 daltons was much higher. It is uncertain whether the viral mRNA of high-molecular-weight represents mixed transcripts derived from integrated viral genomes and adjacent host genes.

A histochemical study of acid phosphatase in normal and virus-transformed cultured fibroblasts

The distribution of acid phosphatase has been investigated in normal and virus-transformed cultured hamster and mouse fibroblasts. The enzyme was found to be present in lysosomes, autophagic vacuoles and elements of the Golgi apparatus. It was also found to be associated with a surface coat in some virus-transformed mouse cells and in the cytoplasm of both normal and transformed hamster cells.

Fucolipid Patterns of Cell Lines Transformed by Highly and Weakly Tumorigenic Simian Virus 40 and Herpes Simplex Virus

The fucolipids of simian virus 40 (SV40)-transformed Balb/c mouse and herpes simplex virus (HSV)-transformed hamster cells of varying degrees of tumorigenicity were examined. In the virus-transformed cell lines and in a spontaneously transformed cell line, as compared to non-transformed cell lines, there was a decrease in the incorporation of fucose radioactivity into fucolipid IV. Fucolipid IV is the least chromatographically mobile, presumably most complex fucolipid. Only in the highly transplantable SV40- and HSV-transformed cell lines was the decrease in incorporation of radioactivity in fucolipid IV as marked as that observed in oncornavirus-transformed cell lines. Similarly, only in the highly tumorigenic DNA-virus-transformed cell lines was there a significant increase in incorporation of radioactivity from radioisotopically-labeled fucose into fucolipid III as has been observed in oncornavirus-transformed cell lines.

Avian sarcoma virus transformed hamster cells made resistant to ethidium bromide.

ABSTRACT Hamster cells transformed with the Schmidt-Ruppin strain of avian sarcoma virus were selected for resistance to ethidium bromide (EB). The resistant cell lines proliferated in the presence of up to 30 μg/ml EB. From avian sarcoma virus-transformed hamster cells already resistant to bromodeoxyuridinu (BrdU), ethidium bromide-resistant cells which were able to grow in 10μg/ml EB were also prepared. These cells remain deficient in thymidine kinase activity and are suitable for selective preparation of hybrid cells. The EB resistance was genetically stable. The EB-resistant cell lines, and doubly resistant cells (BrdU, EB) showed no differences in mitochondrial ultrastructure compared with the original cell lines. Thymidine incorporation into mitochondrial DNA was not influenced by EB resistance. All resistant cell lines, including the doubly resistant cell line, contained the avian sarcoma virus genome. The number of cells needed for positive rescue experiments for avian sarcoma virus genome by cell fusion with permissive chicken embryo cells was the same as with the original cell lines. The single EB-resistant cell lines contained R-type virus-like particles, while in BrdU-rcsistant and doubly resistant cells the R-type particles were absent. The possible nature of EB resistance is discussed.

Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture

When Syrian hamster embryo cells were pretreated with a weak chemical carcinogen, methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS), or with a physical agent such as X-irradiation prior to being exposed to a potent cancer-producing chemical, transformation (crisscrossing of cells not seen in control) occurred up to nine times more often than when the cells were not pretreated. The degree of enhancement appears independent of carcinogen dose. The transformation frequency associated with the carcinogens benzo(a)pyrene (BP), dimethylbenz(a)anthracene (DMBA), 3-methylcholanthrene (MCA), N-acetoxy-2-acetylaminofluorene (AcAAF), and N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) was increased. There are similarities in the enhancement produced by pretreatment of hamster cells with X-irradiation and with alkylating agents: with both, maximum enhancement occurred approx. 48 h after treatment and lethality attributable to the pretreatment was 10–20% relative to control. However, enhancement produced by X-irradiation pretreatment was slightly greater than that obtained with MMS. The exact cause of the enhancement in transformation resulting from the interaction of these agents is not yet known, but the enhancement associated with MMS pretreatment cannot be related to partial cell synchronization or disruption in the cell cycle. Hamster cells pretreated with 250 μM of MMS demonstrated no alteration in normal cel DNA synthesis through 48-h post-treatment. Analysis of unscheduled DNA synthesis by autoradiography or by alkaline sucrose gradients indicated that the damaged DNA was rapidly repaired after treatment. Therefore, repair of DNA damage as it is now understood is probably not involved.

N-(Phosphonacetyl)-l-Aspartate, a Potent Transition State Analog Inhibitor of Aspartate Transcarbamylase, Blocks Proliferation of Mammalian Cells in Culture

Abstract N-(phosphonacetyl)-l-aspartate (PALA) is an analog of the activated complex for the reaction catalyzed by aspartate transcarbamylase and has previously been shown to be a potent and specific inhibitor of the enzymes from Escherichia coli and mouse spleen. PALA is now shown to be a potent inhibitor of de novo pyrimidine nucleotide biosynthesis in five lines of mammalian cells in culture. When the concentration of PALA in the medium is 10-4 m, simian virus 40-transformed hamster cells do not divide and, within about 2 days, have detached from the culture dishes and appear dead. The toxicity of 10-4 m PALA is prevented completely if 10-4 m uridine is present in the medium, showing that PALA inhibits uridylic acid biosynthesis specifically. The same effect can be observed with the other cell lines. When the external concentration of radioactive PALA is in the range 10-4 to 10-5 m, its concentration in the cytosol is approximately equal to the external concentration. Most of the PALA recovered from cells remains undegraded. In crude cell extracts, the Ki for PALA is about 1 x 10-9 m at 37° and pH 7.5. Addition of 1 x 10-7 m PALA in vitro to an extract of transformed hamster cells inhibits by 60% the incorporation of radioactivity from CO2 into UMP and CMP; approximately 10-6 m PALA is required to give comparable inhibition of incorporation from carbamyl-P. Higher concentrations give virtually complete inhibition in both cases. These results suggest that PALA may find clinical use in suppressing cellular proliferation. A small number of the virus-transformed hamster cells become resistant to 10-4 m PALA in culture and retain resistance after they are grown for many generations in the absence of the drug. Resistance is accompanied by a large increase in aspartate transcarbamylase activity.

Properties of SV40 rescued from actinomycin D-sensitive and actinomycin D-resistant transformed hamster cells

The SV40 plaque size variants SVLP and SVSP are characterized with respect toin vitro transformation, oncogenicity and TSTA induction. Compared to SVLP, SVSP virus transforms more efficiently hamster, mouse and monkey cellsin vitro, and is more oncogenic for hamsters. By the graft rejection test, the small plaque variant appears to be less immunogenic than the large plaque variant.

Incorporation of SV40-tumor specific transplantation antigen (SV40-TSTA) in vesicular stomatitis virus grown in SV40 transformed hamster cells (author's transl)

AbstractHighly purified and inactivated VSV (VSV/TSV‐5 C1.2) grown in SV40 transformed hamster cells, TSV‐5 C1.2, induces in hamster a transplantation immunity against host‐tumor cells. Immunization with VSV/TSV‐5 C1.2 gave more than a 10‐fold protection against TSV‐5 C1.2 tumor cells. The transplantation immunity confered by the immunization is specific for tumor cells which possess SV40‐TSTA. VSV/TSV‐5 C1.2 protects hamsters against another SV40‐transformed cell line, EHSVi‐C1.1. In contrast, immunization with VSV/EHB, a purified preparation of VSV grown in spontaneously transformed hamster cells, EHB, gave no protection to TSV‐5 C1.2 cell challenge. It was concluded that the presence of SV40‐TSTA activity in VSV/TSV‐5 C1.2 virions was due to incorporation of this tumor cell antigen in the viral envelope. The existence of cell antigens in VSV/TSV‐5 C1.2 and VSV/EHB preparations was also shown in vitro by Cr51 release assay. The anti‐VSV/TSV‐5 C1.2 rabbit serum reacted specifically with SV40‐transformed cells, TSV‐5 C1.2 and EHSVi‐C1.1.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. Transformation of hamster kidney cells by adenovirus 2 and the nondefective hybrid viruses.

Nonhybrid adenovirus 2 (Ad2) and five nondefective Ad2-simian virus 40 (SV40) hybrids (Ad2(+)ND(1)-Ad2(+)ND(5)) failed to produce tumors when injected into newborn hamsters. However, each of these agents transformed hamster kidney cells in vitro, and the transformed cell lines produced tumors when injected into suckling hamsters. Foci of cells transformed by the hybrids could not be distinguished from foci of cells transformed by nonhybrid Ad2. Histopathologically, tumors induced by the nondefective hybrid-transformed lines were of the adenovirus type with no areas of SV40-type fibrosarcoma. All of the cell lines studied contained Ad2 T antigen and Ad2 RNA. Only the B55HK(1) line contained the three early SV40 antigens, U, TSTA, and T. The ND(4)HK(1) line contained SV40 T and TSTA antigens (the latter antigen being detected only after 48 to 49 tissue culture passages) and SV40 RNA. The ND(2)HK(2) line contained SV40 RNA but no detectable SV40 antigens; the other hybrid-transformed lines contained no SV40 RNA. The transformation of hamster cells by the nondefective Ad2-SV40 hybrids seemed to be caused by the Ad2 genome and could not be attributed to the regions of the SV40 genome contained within the hybrids. Additionally, these results indicate that interactions between the genomes of the nondefective hybrid viruses and the hamster cells they transform are complex and could involve the site of integration, the size of the incorporated viral genome, the transcription or translation of the viral genome, or various combinations of these processes.

Detection of Specific Surface Antigens on Cells Transformed by Cytomegalovirus with the Techniques of Mixed Hemagglutination and 125I-Labeled Antiglobulin

Abstract Cytomegalovirus (CMV)-transformed hamster cells were examined for the presence of virus-specific membrane antigens by using rabbit antiserum prepared against CMV-infected human embryonic lung (HEL) cells. After absorption with sheep erythrocytes and heterologous hamster tumor cells the antiserum reacted specifically with the CMV-transformed cells in the mixed hemagglutination test. The absorbed antiserum did not react with hamster cells transformed by herpes simplex virus type 2 or PARA-adenovirus 7 or with untransformed hamster embryo fibroblasts. These results were confirmed with the 125I-labeled antiglobulin technique. Absorption with HEL cells rendered the antiserum specific for HEL cells infected with CMV. This reactivity was lost after absorption with 107 CMV-transformed cells but not with 107 hamster embryo fibroblasts. These results establish that CMV-transformed hamster cells express virus-related membrane antigen(s), and that the same antigen(s) is also found on CMV-infected human cells.

Surface membrane glycopeptides which coincide with virus transformation and tumorigenesis.

Glycopeptides from the surface of clones of hamster embryo cells were examined at various intervals after infection with polyoma virus. Two types of transformed cells were examined: (i) clones that showed delayed transformation or an initially low tumorigenicity, and (ii) clones that were rapidly transformed showing an initially high tumorigenicity. The glycopeptides were removed from the cell surface by trypsin and, after Pronase digestion, were examined by filtration through Sephadex G-50. With delayed transformation, a specific group of glycopeptides was increasingly evident over an 85-day period as the cells showed phenotypic properties of transformation and the ability to form tumors. In the other series, all but one clone of hamster embryo cells showed rapid transformation after infection with polyoma virus. This clone was less tumorigenic and showed little of the specific glycopeptides. In all cases of delayed or rapid transformation examined, the specific group of glycopeptides increased proportionately to the ability of the cells to form tumors. All of the cells derived from progressively growing tumors formed by injection of these transformed hamster cells into adult animals showed an abundance of this group of glycopeptides. These results suggest that specific surface membrane glycopeptides accompany viral transformation and tumorigenesis.

Studies of Hamster Cells Transformed by Adenovirus 2 and the Nondefective Ad2-SV40 Hybrids

Adenovirus 2 does not produce tumors when injected into newborn hamsters, and this failure is not corrected by the acquisition of the portion of the SV40 genome present in any of the nondefective Ad2-SV40 hybrid viruses. However, nonhybrid Ad2 will transform hamster cells in vitro, and these transformed cells will produce tumors when injected into newborn hamsters. This finding, taken in conjunction with other studies (Graham et al., this volume; McAllister et al., 1969; McDougall et al., this volume; Williams 1973), suggests that the categorization of Ad2 and Ad5 as "nononcogenic" viruses is a reflection of host response rather than an intrinsic property of the viruses. However, transformation of hamster cells by Ad2 is inefficient, requiring 10(7)-10(8) PFU to produce a focus of transformed cells. Transformation of hamster kidney cells by the nondefective hybrids cannot be associated with portions of the SV40 genome. During the transformation process, complex interactions must occur between the hamster cell genome and the viral genome of Ad2 or the nondefective hybrids. Thus while it appears that there is very little Ad2 DNA in one nonhybrid Ad2-transformed cell line, there is a larger amount of Ad2 DNA in all of the hybrid virus-transformed cell lines. Moreover, while one Ad2+ND2-transformed line has apparently lost the SV40 portion of the viral genome, it is present and transcribed in another line transformed by the same virus. Finally, in the lines of nondefective hybrid-transformed cells examined thus far, there is extensive transcription from the Ad2 H strand. This pattern of transcription differs from transcription of the viral genome in most hybrid Ad2-transformed hamster and rat cells in which L-strand transcripion predominates.

Blocks in Ganglioside Synthesis in Transformed Hamster Cells and Their Revertants

Abstract Hamster cells transformed by polyoma virus or by treatment with the chemical carcinogen dimethylnitrosamine and revertants from these transformed cells were examined for UDP-N-acetylgalactosamine:hematoside N-acetylgalactosaminyltransferase activity and for their types of gangliosides. Compared to normal cells, both the transformed and revertant cells showed 3 to 12% of the enzyme activity and a simplification of the ganglioside pattern. The presence of similar blocks in glycosyltransferase activity and ganglioside synthesis in these transformed cells and their revertants indicates that these blocks were not associated with the regulation of cell growth, but may be due to a common genetic change in transformed cells and their revertants that is not found in normal cells.

An antigen associated with messenger RNA in a transformed hamster cell line.

An antigen associated with messenger RNA in a transformed hamster cell line.

In vitro transformation of hamster embryo cells by a guinea pig herpes-like virus.

SummaryHamster embryo cells infected with GPHLV showed morphological cellular transformation. Virus strains isolated from leukemic guinea pigs showed somewhat higher transforming capacity than the isolates from “normal” Hartley guinea pigs. Virus specific antigen was detected in the cytoplasm of 7-20% of the transformed hamster cells. Infectious virus could be recovered from the transformed cells by co-cultivation with guinea pig kidney cells. Quantitative analysis showed that in these cell lines 12-47/100,000 transformed hamster cells yielded infectious virus.The authors are grateful to Dr. R. H. Green for his valuable suggestions during the preparation of this manuscript. The excellent technical assistance of JoEllyn Bradley and Judy Wnek is appreciated.

Thermosensitive events in adenovirus transformed cells. II. Helping of adenovirus-associated viruses.

Summary Using the indirect immunofluorescence assay, diffuse herpetic antigens were demonstrated in hamster cells infected with the virus at 35 ~ or at 37 ~ C. A similar picture was obtained with adenovirus transformed hamster cells after herpesvirus infection at 35 ~ C, but at 37~ fluorescence was restricted to the perinuelear region. Coinfection of the transformed cells with herpes simplex virus and adenovirusassociated virus type 3 (AAV3) produced the appearance of AAV3 antigens at 35 o C but not at 37 ~ C. The thermosensitive event which is necessary for helping AAV3 may be the same as that necessary for the appearance of herpes antigen(s) in the peripheral part of the cell. 1. Introduetion