Transformed Hamster Cells Research Articles

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.

Herpes simplex virus-directed overreplication of chromosomal DNA physically linked to the simian virus 40 integration site of a transformed hamster cell line

In the simian virus 40 (SV40)-transformed hamster cell line Elona herpes simplex virus (HSV) induces amplification of SV40 DNA sequences to high-molecular-weight head-to-tail concatemers indicating an extrachromosomal rolling circle replication. In order to enable investigations concerning intrachromosomal amplification of SV40 DNA sequences and flanking cellular sequences a genomic library of Elona DNA was constructed in phage λ. Clones harboring cellular DNA adjacent to the SV40 integration site were isolated. Plasmid subclones devoid of SV40 DNA sequences were used as hybridization probes against total DNA from HSV-infected cells. Thus the amplification of both flanking cellular sequences was demonstrated, indicating a bidirectional replication mode.

In vivo evolution of adenovirus 2-transformed cell virulence associated with altered E1A gene function.

Neoplastic cell populations may evolve to a state of higher virulence in immunocompetent hosts. Transforming gene involvement in this process of tumor progression was evaluated using adenovirus type 2 (Ad2)-transformed hamster cells that are highly susceptible to destruction by natural killer cells and activated macrophages, due to Ad E1A gene function, and are nontumorigenic in immunocompetent animals. Cells selected for increased tumorigenicity retained parental cell patterns of viral gene integration and methylation and expressed Ad2 E1A proteins but exhibited altered E1A function evidenced by decreased susceptibility to killer cell-mediated lysis and inability to support E1A(-) mutant virus replication. The data suggest that an interruption in cellular pathways of E1A expression may result in increased transformed cell virulence.

Inactivation of DNA-mediated transformation of hamster cells by γ-rays and deoxyribonuclease I

DNA damage has been induced in the mammalian expression vector pSV2- gpt by irradiation with X-rays or treatment with deoxyribonuclease I (DNAase I) under controlled conditions in vitro. The biological effect of such treatment was assessed by stable gene expression in Chinese hamster ovary (CHO) cells using DNA-mediated gene transfer. Induction of DNA double-strand breaks (dsbs), resulting from the interaction of independently-induced single-strand breaks (ssbs) under the present conditions, was measured by agarose gel electrophoresis of the treated vector. The correlation between radiation-induced gene inactivation and dsb induction mediated by OH radicals suggests that a dsb in the gene is a major inactivating lesion in this system. Individual radiation-induced ssbs and nucleotide damage are produced much more frequently than dsbs under these conditions, but the majority of these lesions do not appear to inactivate the gpt gene. DNAase I treatment, giving only simple 5′ P+_3′ OH breaks in the vector DNA, gave a correlation of approxiamately 1.5 dsb in the gpt gene per inactivating event, confirming little repair of dsbs in this system. Inactivation of the gpt gene without appreciable formation of dsbs was found however, when the vector was irradiated at high dose rate in the presence of the OH-radical scavenger Kbr. The nature of non-break damage causing inactivation requires further study.

Comparative analysis of DNA loop length in nontransformed and transformed hamster cells

The size of the looped, matrix-attached DNA domains was estimated in both nontransformed and transformed hamster cells. In BHK cells it was observed that in interphase as well as during mitosis, transformed cells, on the average, have shorter loops than nontransformed cells. In CHO cells, however, no alteration of the length of the looped DNA domains was found to accompany transformation. The implications of these observations are discussed in relation to the process of transformation.

Evidence for a shift in 5′-termini of early viral RNA during the lytic cycle of JC virus

We have used primer extension and S1 analysis to localize the 5'-termini of JC virus (JCV) early RNAs in infected primary human fetal filial cells at various times postinfection and in stable JCV-transformed hamster fetal filial cells. At early times postinfection (Days 1–5), two early transcripts are initiated at nucleotides 5122 and 5082. A major shift in 5′-ends at later times results in the synthesis of a new series of early mRNAs beginning upstream at nucleotide 35 and downstream at nucleotides 5047, 5037, and 5012. In the transformed hamster cells, however, only one RNA species was detected, starting at nucleotide 5122. The mechanism underlying the shift in the initiation site of JCV early RNAs during a lytic infection remains unclear but appears analogous to that which occurs in the SV40 lytic cycle. Since the shift occurs during DNA replication, when T-antigen is at maximal levels, it is possible that T-antigen binding to JCV DNA and/or alterations in chromatin structure contribute to this event.

Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line.

When the simian virus 40 (SV40)-transformed Syrian hamster cell line Elona is infected with herpes simplex virus type 1, an excessive amplification of SV40-specific DNA sequences occurs. Analysis of total DNA from herpes simplex virus-infected cells revealed that amplified DNA sequences were present predominantly in a high-molecular-weight form, consisting of a tandem array of many unit-length SV40 DNA molecules. Repeat units of amplified DNA were found to be very similar to standard SV40 DNA as was shown by restriction analyses, except for a small deletion close to the origin of replication, which could also be detected in the chromosomal DNA of uninfected cells. A procedure, devised for selective enrichment of amplified SV40 DNA molecules from the bulk of cellular and herpesviral DNA, allowed molecular cloning of single repeat units and nucleotide sequence analysis of the relative genomic region.

Distinctive transforming genes in x-ray-transformed mammalian cells.

DNAs from hamster embryo cells and mouse C3H/10T1/2 cells transformed in vitro by x-irradiation into malignant cells transmit the radiation transformation phenotype by producing transformed colonies (transfectants) in two mouse recipient lines, the NIH 3T3 and C3H/101/2 cells, and in a rat cell line, the Rat-2 cells. DNAs from unirradiated cells or irradiated and visibly untransformed cells do not produce transformed colonies. The transfectants grow in agar and form tumors in nude mice. Treatment of the DNAs with restriction endonucleases prior to transfection indicates that the same transforming gene (oncogene) is present in each of the transformed mouse cells and is the same in each of the transformed hamster cells. Southern blot analysis of 3T3 or Rat-2 transfectants carrying oncogenes from radiation-transformed C3H/10T1/2 or hamster cells indicates that the oncogenes responsible for the transformation of 3T3 cells are not the Ki-ras, Ha-ras, or N-ras genes, nor are they neu, trk, raf, abl, or fms, although quick blot analysis using 11 oncogene probes detected increased transcripts of c-abl and c-fms in the 3T3 transformants containing oncogenic sequences from the x-ray-transformed C3H/10T1/2 cells. The work demonstrates that DNAs from mammalian cells transformed into malignancy by direct exposure in vitro to radiation contain genetic sequences with detectable transforming activity in three recipient cell lines. The results provide evidence that DNA is the target of radiation carcinogenesis induced at a cellular level in vitro. The experiments indicate that malignant radiogenic transformation in vitro of hamster embryo and mouse C3H/10T1/2 cells involves the activation of unique non-ras transforming genes, which heretofore have not been described.

Loss of tumor-suppressive function during chemically induced neoplastic progression of Syrian hamster embryo cells.

Cell hybrids between normal, early-passage Syrian hamster embryo cells and a highly tumorigenic, chemically transformed hamster cell line, BP6T, were formed, selected, and analyzed. Tumorigenicity and anchorage-independent growth were suppressed in the hybrid cells compared to the tumorigenic BP6T cells. These two phenotypes segregated coordinately in these cells. To determine at what stage in the neoplastic process this tumor-suppressive function was lost, two chemically induced immortal cell lines were examined at different passages for the ability to suppress the tumorigenic phenotype of BP6T cells following hybridization. Hybrids of BP6T cells with the immortal, nontumorigenic cell lines at early passages were suppressed for tumorigenicity and anchorage-independent growth. This tumor-suppressive ability was reduced in the same cells at later passages and in some cases nearly completely lost, prior to the neoplastic transformation of the immortal cell lines. Subclones of the cell lines were heterogeneous in their ability to suppress tumorigenicity in cell hybrids; some clones retained the tumor-suppressive ability and others lost this function. The susceptibility to neoplastic transformation of these cells following DNA transfection with the viral ras oncogene or BP6T DNA inversely correlated with the tumor-suppressive ability of the cells. These results suggest that chemically induced neoplastic progression of Syrian hamster embryo cells involves at least three steps: induction of immortality, activation of a transforming oncogene, and loss of a tumor-suppressive function.

Molecular and biological properties of BK virus-IR, a BK virus variant isolated from a human tumor

We describe the molecular and biological properties of BK virus (BKV)-IR, a new BKV variant isolated from a human tumor of pancreatic islets. BKV-IR bears a 253-base-pair (bp) deletion and an 80-bp insertion in the early region of the genome. The deletion abolishes the expression of small-t antigen. The inserted sequences, grouped in four clusters, produce rearrangements in the first and second enhancer elements. They are bound by 12-bp direct repeats and could form a 217-base stem-loop structure suggestive of an insertion sequence. As compared with wild-type BKV, BKV-IR transformed hamster cells with a reduced efficiency and induced ependymomas in hamsters at a lower frequency and with a longer latency period. Tumors induced by BKV-IR, however, showed features of higher malignancy. The possible role of the insertion sequence-like element in transformation by BKV-IR is discussed.

Transfer of antibiotic resistance genes between yeast and mammalian cells under conditions favoring cell fusion.

Antibiotic resistance to G418 has been transferred into Chinese hamster cell lines via a plasmid vector. The same plasmid, which also contained the Leu2 gene, has been used to transform Leu2- yeast (strain MC16) to leucine prototrophy. Subsequent fusion between transformed yeast and untransformed hamster cells demonstrated that plasmid DNA could be transferred and its genes expressed within the mammalian cell genome. The fusion of transformed hamster cells with untransformed MC16 yeast cells demonstrated that DNA integrated within the mammalian cell genome could be transferred to correct the Leu2 deficiency and also confer G418 resistance on some yeast colonies.

Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1

We have examined the effect of DNA tumor virus transformation of primary hamster embryo cells on the tyrosyl kinase activity of pp60c-src. Our present study demonstrates that some clones of hamster embryo cells transformed by simian virus 40, adenovirus type 2, adenovirus type 12, or bovine papillomavirus 1 can possess elevated pp60c-src kinase activity when compared with normal hamster embryo cells. However, other clones of hamster embryo cells transformed by these same viruses were found to have normal levels of pp60c-src kinase activity. In those clones of transformed cells where pp60c-src kinase activity was elevated, the increased levels of kinase activity were the result of an apparent increase in the specific activity of the pp60c-src phosphotransferase rather than an increase in the amount of the src gene product. Additionally, pp60c-src was not found to be physically associated with tumor antigens known to be encoded by these viruses. These results indicate that elevated levels of pp60c-src kinase activity can be found in hamster embryo cells transformed by several different DNA tumor viruses and suggest that the molecular mechanism by which pp60c-src kinase activity is elevated may differ from that previously observed in polyomavirus-transformed cells. These results also imply that elevation of pp60c-src kinase activity is not required for the transformation of hamster cells by these viruses.

Enhanced induction of SV40 replication from transformed mammalian cells by fusion with UV-irradiated untransformed cells

DNA-damaging agents such as ultraviolet (UV) light are known to cause stimulation of virus replication in SV40-transformed hamster and human cells. The dose-response curves of UV-induced SV40 replication in transformed hamster cells resemble that obtained for UV-enhanced reactivation (ER) and UV-enhanced mutagenesis (EM) of SV40 or herpes viruses in mammalian cells. We have investigated whether UV-enhanced production of SV40 from transformed hamster (THK) and human (NB-E) cells belongs to the same category of conditional responses as ER and EM. To answer this question we have made use of the phenomenon that fusion of the SV40-transformed cells with monkey cells that are permissive to SV40 results in a considerable increase in the production of SV40 virus. When THK or NB-E cells were fused with UV-irradiated CV-1 cells at various times after irradiation, induction of SV40 was further increased with UV-irradiated CV-1 cells at various times after irradiation, induction of SV40 was further increased and reached a maximum value of 2–3 fold when fusion was delayed for 24–48 h after irradiation. The kinetics of enhanced SV40 induction resembled that of ER and EM, suggesting that teh UV-stimulated part of the induction represents one of the pleiotropic responses that are transiently induced in mammalian cells by DNA-damaging agents. Evidence is presented, showing that this transient effect can be induced only in cells that are permissive to SV40 replication. This suggests that the enhanced induction observed after fusion with irradiated monkey cells may be attributed to a transient increase in the activity of ‘permissiveness’ factors. No enhanced induction was found when the THK or NB-E cells were fused with irradiated rodent cells, that are not or only slightly permissive to SV40 replication.

Infectivity of Friend Murine Leukemia Virus for Hamster Cells

Hamster-adapted Friend murine leukemia virus (F-MuLV) infected hamster cells and produced an XC-positive progeny. However, the sensitivity of hamster-adapted F-MuLV's to hamster cells was about 100 times lower than that to mouse cells. Hamster cells chronically infected with F-MuLV in vitro produced progeny virus in titers comparable to those in similarly infected mouse cells. Xenotropic and amphotropic murine leukemia virus can infect hamster cells when phenotypically mixed with the F-MuLV. Murine sarcoma virus pseudotyped with the F-MuLV easily induced sarcomas in hamsters and transformed hamster cells in vitro.

Identification of a single chromosome in the normal human genome essential for suppression of hamster cell transformation.

Normal human fibroblasts were fused to carcinogen-transformed baby hamster kidney (BHK) cells and found to be able to suppress the anchorage-independent transformed phenotype of the hamster cells. This suppression was not due to interspecies incompatibility, for transformation could be effectively expressed in hybrids if either the human or the BHK parent had initially been transformed by a dominantly acting viral genome. Upon growth of suppressed hybrids, loss of human chromosomes was accompanied by the re-expression of transformation. Karyotype analysis indicated that only human chromosome 1 was retained in all hybrids that were suppressed and was lost in all hybrids in which transformation was re-expressed. Cytological evidence for the presence or absence of chromosome 1 was confirmed by electrophoretic identification of the human isozyme for phosphoglucomutase 1. Clones re-expressing transformation were isolated from two suppressed hybrids and in both cases loss of suppression was accompanied by the loss of human chromosome 1. Thus, the maintenance of suppression in these cross-species hybrids appears to require the continued presence of normal human chromosome 1. These findings raise the possibility that the frequent involvement of human chromosome 1 in potentially inactivating aberrations in human tumors may reflect a suppressor role for this chromosome in human malignancy.

Low molecular weight RNAs with homologies to cellular DNA at sites of adenovirus DNA insertion in hamster or mouse cells.

The adenovirus type 2 (Ad2)-transformed hamster cell line HE5 contains one or very few integrated copies of Ad2 DNA. At the site of insertion of Ad2 DNA, the cellular DNA sequence has been completely preserved and has homologies to small unpolyadenylated, cytoplasmic RNAs of 300 nucleotides in length and to minority populations of smaller RNAs present in HE5 cells and in normal hamster cells. The 300-nucleotide RNA is present on average in approximately 20 copies per cell. This RNA, and shorter RNAs, reveal homologies to the hamster DNA sequence of approximately 400 nucleotides to the right of the site of insertion of Ad2 DNA, which is present in one or very few copies per genome. The nucleotide sequence of the DNA segment homologous to this RNA does not contain open reading frames in excess of a sequence encoding 18 amino acids. Thus, it is unlikely that the small RNAs are actually translated and their function is unknown. The nucleotide sequence does not exhibit similarities to known low mol. wt. RNAs of eukaryotic origin. The low mol. wt. cellular RNA has been found in HE5 cells, in other hamster cell lines and organs, and also in mouse cells. There are differences with respect to size and abundance in the RNAs smaller than 300 nucleotides between HE5 cells and LSH hamster embryo cells. The adenovirus type 12 (Ad12)-induced mouse tumor CBA-12-1-T carries greater than 30 copies of integrated Ad12 DNA. The cellular DNA sequence at the site of Ad12 DNA insertion exhibits homologies to small RNAs (approximately 300 nucleotides long) from mouse cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli.

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.

Identification of a gene function of herpes simplex virus type 1 essential for amplification of simian virus 40 DNA sequences in transformed hamster cells

Infection with herpes simplex viruses (HSV) lead to a significant increase of the simian virus 40 (SV40) DNA content in the SV40-transformed hamster cell lines C0631 and Elona. Analysis of this gene-amplifying activity revealed (i) that it cosedimented with infectious herpesvirions in sucrose density gradients, (ii) that it was abolished by anti-HSV antibodies or (iii) by antiviral drugs acting on the HSV-induced DNA polymerase; and analysis of temperature-sensitive mutants showed that this DNA polymerase was an essential component of HSV-induced, gene-amplifying activity in SV40-transformed hamster cells.

Excision of amplified viral DNA at palindromic sequences from the adenovirus type 12-transformed hamster cell line T637.

In the DNA of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 approximately 20-22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease-sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold-back structures of DNA have been found. These snap-back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4-specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.

Adenovirus 2 early gene expression promotes susceptibility to effector cell lysis of hybrids formed between hamster cells transformed by adenovirus 2 and simian virus 40.

Weakly oncogenic adenovirus 2 (Ad2)-transformed LSH hamster cells are sensitive to lysis by spontaneously cytolytic lymphoid cells and activated macrophages, whereas highly oncogenic simian virus 40 (SV40)-transformed LSH cells are relatively resistant to these nonspecific effector cells. Somatic cell hybrids formed between Ad2- and SV40-transformed hamster cells, which expressed Ad2 tumor (T) antigens, exhibited an increased cytolytic susceptibility compared to Ad2 T antigen-negative cell hybrids or nonhybrid SV40-transformed cells. No correlation was found between the expression of SV40 T antigen in hybrid cells and cytolytic susceptibility. The results suggest the existence of a novel function for early Ad2 genome-encoded polypeptides (T antigens) expressed in transformed hamster cells--the induction of susceptibility to destruction mediated by immunologically nonspecific effector cells.