Transformation of hamster embryo fibroblasts by a specific fragment of the herpes simplex virus genome (original) (raw)
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Journal of General Virology, 1985
We have determined the herpes simplex virus (HSV) type 2 DNA sequences responsible for the initiation of morphological transformation and have investigated the retention and expression of these sequences in morphologically transformed cells and in tumours derived from these cells. All the transformed cells analysed were selected by a focus formation assay and are oncogenic in the inbred host rat. Cloned HindIII and BglII fragments from the HSV-2 genome were assayed for the ability to initiate morphological transformation of rat embryo cells. Only the HindIII a (map units 0.52 to 0-72) and the BgllI n (0'582 to 0.612) clones gave transformed foci. This shows that the BglII n region is responsible for initiation of transformation. Southern blot analysis of DNA extracted from these transformed cells and from turnouts derived from these transformed cells revealed that neither the BgllI n fragment nor fragments of 500 bp mapping within it are detected at the level of one copy per cell and therefore need not be retained in the cell to maintain the oncogenic phenotype. In addition there was no evidence of expression of the HSV-specified ribonucleotide reductase activity which is partially encoded within the BglII n fragment of HSV-2. We also analysed DNA from rat embryo cells transformed by ts mutants of HSV-2 (HG52) or HSV-1 (HFEM or 17) at non-permissive temperature or by virus at supraoptimal temperature or by sheared virus DNA and DNA from tumours derived from lines of these transformed cells. In addition, we cloned both transformed and tumour cell lines and analysed these similarly. In no case could we detect HSV DNA sequences at the level of one copy per cell.
Alterations in the cellular phenotype induced by herpes simplex viruses
Journal of Medical Virology, 1990
Numerous studies have shown that herpes simplex virus types 1 and 2 (HSV-1, HSV-2) are able to transform the morphological phenotype of rodent cells. Unlike other DNA tumor viruses the transformed cells did not consistently retain or express a given set of viral genes. In fact, transformation could be obtained using fragments of viral DNA that did not wholly encode viral proteins. Of interest within the transforming fragments were sequences which could assume a secondary structure like that of insertion elements. The failure to detect viral DNA in transformed cells led to the hit-and-run hypothesis of HSV transformation. The mechanism by which HSV induces transformation is not understood. Various lines of investigation have shown that HSV is able to cause mutations-both point mutations and gene rearrangements. HSV is also able to induce gene amplification, particularly of sequences harboring an origin of replication such as SV40 or papillomaviruses. Other experiments have shown that HSV can activate the expression of endogenous type C retroviruses. More broadly, HSV has been shown to activate cellular transcription or to switch on the synthesis of host cell proteins not normally expressed in untransformed cells. The failure to detect viral DNA in a high proportion of human anogenital tumors made it difficult to implicate HSV in the etiology of those neoplasias, but it is consistent, however, with the observations on the mode of HSV transformation in vitro, and suggests that HSV could be involved in a multistage process of on cog en ic transform at i on.
Analysis of viral DNA sequences in hamster cells transformed by herpes simplex virus type 2
Proceedings of the National Academy of Sciences, 1980
Herpes simplex virus type 2 (HSV-2) DNA was treated with four restriction endonucleases (EcoRI, HincIll, BgJ II, and Xba I) and eight fragments were purified and labeled with 32P in vitro. The kinetics of renaturation of each of the fragments was measured in the presence of DNA extracted from 333-8-9, a hamster cell line transformed by UV light-inactivated HSV-2 strain 333, and from a series of cloned derivatives and their tumor lines. All of the lines examined contained a partial set of viral sequences present at only a few copies per cell. Passage of the cell lines in tissue culture or in animals re-suIted in partial loss of viral DNA. Two blocks of sequences were present in most of the lines examined; those mapping at positions 21-33 of the HSV-2 genome were detected in seven of seven cell lines tested and those at positions 60-65 were detected in six of eight. Other sequences from the L component can also be present in the DNA of HSV-2-transformed hamster cells.
Cancer Research, 1981
A newly developed experimental model system was used to determine in vitro transformation-specific parameters which correlate with tumorigenicity. The data suggested that clonal herpes simplex virus type 2-transformed syngeneic rat embryo cells with intermediate, transformed rat embryo fibroblasts (t-REF-G-1) or high, rat fibrosarcoma tumorigenic potential in syngeneic rats could be differentiated from clonal transformed nontumorigenic (t-REF-G-2) and nontransformed rat embryo fibroblast cells by their growth to increased saturation density and cloning efficiency in soft-agar medium. All clonal herpes simplex virus type 2-transformed cells, regardless of tumorigenie potential, possessed an increased rate of hexose trans port and plasminogen activator activity and were less fibroblastoid in morphology compared to nontransformed rat em bryo fibroblast cells. There was no significant difference in celldoubling time or total phospholipid composition between clonal transformed tumorigenic, nontumorigenic, and nontransformed cells. However, all clonal herpes simplex virus type 2-trans formed cell lines possessed a decreased percentage of arachidonic acid and an increased percentage of monounsaturates compared to percentages in nontransformed rat embryo fibro blast cells. Saturation density, cloning efficiency, and tumori genicity in newborn and adult rats of one nontumorigenic clonal cell line (t-REF-G-2) increased with cell passage in tissue culture. Other transformation-specific biochemical parameters (hexose transport, plasminogen activator activity, and fatty acid composition) remained stable with in vitro passage of the clonal cell lines. Five subclonal cell lines established from 12-Otetradecanoylphorbol-13-acetate-treated clonal t-REF-G-2 (low passage) cells showed increased cloning efficiencies com pared to 2 subclonal cell lines from mock-treated cells; also, all 5 subclonal cell lines from 12-O-tetradecanoylphorbol-13acetate-treated clonal cells were tumorigenic in newborn rats. Subclonal cell lines from mock-treated clonal cells were non tumorigenic. It was clear from the results that the absolute cloning efficiency did not correlate with tumorigenicity. This is an important conclusion, since it suggests that quantitation of transformation-specific characteristics at a given point in time will not necessarily be indicative of the tumorigenic potential of the cells.
Cancer research, 1981
A newly developed experimental model system was used to determine in vitro transformation-specific parameters which correlate with tumorigenicity. The data suggested that clonal herpes simplex virus type 2-transformed syngeneic rat embryo cells with intermediate, transformed rat embryo fibroblasts (t-REF-G-1) or high, rat fibrosarcoma tumorigenic potential in syngeneic rats could be differentiated from clonal transformed nontumorigenic (t-REF-G-2) and nontransformed rat embryo fibroblast cells by their growth to increased saturation density and cloning efficiency in soft-agar medium. All clonal herpes simplex virus type 2-transformed cells, regardless of tumorigenie potential, possessed an increased rate of hexose trans port and plasminogen activator activity and were less fibroblastoid in morphology compared to nontransformed rat em bryo fibroblast cells. There was no significant difference in celldoubling time or total phospholipid composition between clonal transformed tumorigenic, nontumorigenic, and nontransformed cells. However, all clonal herpes simplex virus type 2-trans formed cell lines possessed a decreased percentage of arachidonic acid and an increased percentage of monounsaturates compared to percentages in nontransformed rat embryo fibro blast cells. Saturation density, cloning efficiency, and tumori genicity in newborn and adult rats of one nontumorigenic clonal cell line (t-REF-G-2) increased with cell passage in tissue culture. Other transformation-specific biochemical parameters (hexose transport, plasminogen activator activity, and fatty acid composition) remained stable with in vitro passage of the clonal cell lines. Five subclonal cell lines established from 12-Otetradecanoylphorbol-13-acetate-treated clonal t-REF-G-2 (low passage) cells showed increased cloning efficiencies com pared to 2 subclonal cell lines from mock-treated cells; also, all 5 subclonal cell lines from 12-O-tetradecanoylphorbol-13acetate-treated clonal cells were tumorigenic in newborn rats. Subclonal cell lines from mock-treated clonal cells were non tumorigenic. It was clear from the results that the absolute cloning efficiency did not correlate with tumorigenicity. This is an important conclusion, since it suggests that quantitation of transformation-specific characteristics at a given point in time will not necessarily be indicative of the tumorigenic potential of the cells.
Journal of General Virology, 1977
Four cell lines biochemically transformed by u.v.-irradiated herpes simplex virus contain virus DNA fragments ranging from 3 to 22 ~o of the HSV genome. Of five revertant clones selected for aH-TdR or BrdUrd resistance, four had lost all detectable virus DNA while the fifth, selected for BrdUrd resistance, retained the entire virus fragment but there was a reduction of virus copies per cell from 5 to I. Three 'supertransformed' revertant cell lines contained virus DNA fragments ranging from I2 to 28 %. The number of virus DNA fragments per cell ranged from I to 5 and clearly indicated that a single copy of the virus thymidine kinase gene is adequate for biochemical transformation. The determination of the base composition of the transforming virus DNA fragment indicated that the transforming DNA has a base composition approximately the same as the HSV genome and does not constitute a low GC virus DNA region. Cross hybridization between HSV-I transformed cells and HSV-2 DNA is very slight, indicating that the DNA found in clone I39 is not entirely composed of the HSV-I and HSV-2 common sequences.
Restricted Replication of Herpes Simplex Virus Type 1 in Murine Embryonal Carcinoma Cells
Journal of General Virology, 1987
Herpes simplex virus type i (HSV-1) has a broad host range but the KOS strain of HSV-1 did not replicate efficiently in murine embryonal carcinoma (EC) cells. The yield of infectious HSV-1 from EC cells was 100-to 1000-fold lower than that from fibroblast cell lines of mouse, monkey or human origin. The thymidine kinase (TK) gene of HSV-1 is expressed early during the infectious cycle. The levels of TK mRNA and of TK activity in infected EC ceils were only two-to threefold lower than levels from infected fibroblast cells. Infected EC cells supported replication of about half as much HSV-1 DNA as did fibroblast cells. The reduced yield of infectious virus was consistent with a paucity of virions in infected EC cells examined by electron microscopy, suggesting a major block late during the HSV-I infectious cycle. We isolated a variant strain of HSV-1, called KOSEC, which replicated as efficiently in EC cells as in mouse fibroblasts. KOSEC infected EC and fibroblast cells, synthesized more TK mRNA, more TK enzyme, and more HSV-1 DNA than did the same cells infected with the KOS stain. Both HSV-1 strains induced similar levels of synthesis of gD, an early viral glycoprotein. By co-infection of EC cells with the KOS and KOSEC virus, both the elevated virus yield and the elevated TK synthesis seen in KOSECinfected cells appeared to be recessive. Apparently a viral mutation that affects expression of some early viral functions can also overcome the EC cell restriction to HSV-1 replication.
Proceedings of The National Academy of Sciences, 1986
The transforming potential of the herpes simplex virus type 2 (HSV-2) BamHI fragment E (map position 0.533-0.583) encoding the 140-kDa ribonucleotide reductase was assayed by transfection in established Rat-2 cells. Foci of refractile, morphologically distinguishable cells were induced at lower efficiency and after a longer incubation period as compared to the human tumor oncogene EJ-Ha-ras. Focus-derived BamHI fragment E-transformed cell lines formed medium-to-large (0.1-0.25 mm) colonies in soft agar and were tumorigenic in immunocompetent syngeneic rats. Southern blot analysis of normal rat DNA after EcoRI digestion revealed specific DNA segments homologous to HSV-2 BamHI fragment-E DNA. In BamHI fragment E-transformed and tumor-derived lines, about 8- to 30-fold amplification was detected in a subset of the specific HSV-related DNA segments. In addition, extrachromosomal DNA was isolated from transformed cells by plasmid rescue and contained the left-hand 70% of HSV-2 BamHI fragment E fused to rat DNA. These results indicate the presence in normal cells of nonrepetitive DNA segments, related to the transforming HSV-2 fragment, that can be targeted for genetic alterations associated with neoplastic transformation.