Heteroploid mouse NIH 3T3 fibroblasts and several rat fibroblast strains (Rat-1, Rat-2 and REF-52) are cell lines of special interest in the field of carcinogenesis because of their extensive use as normal cells in transformation assays for putative cancer-causing genes. Exposure of these cells to carcinogenic chemicals or oncogenic DNA produces anchorage-independent cells with retracted cytoplasms that lack actin cables. All human fibroblast strains, normal and transformed, synthesize two electrophoretic forms of actin (beta- and gamma-actin). In contrast, we discovered that early-passage mouse and rat strains synthesize abundant amounts of each of the three electrophoretic forms of actin (alpha-, beta- and gamma-actin) but mouse and rat cancer cells express only beta- and gamma-actins. We now show that in NIH 3T3 and Rat-2 fibroblasts a third actin, the smooth muscle alpha isoform, is abundantly co-expressed with beta- and gamma-actin. In every instance tested following transformation to tumorigenicity, the accumulation of alpha-actin messenger RNA and alpha-actin synthesis was greatly inhibited. Shutdown of alpha-actin expression thus appears to be a reproducible transformation-sensitive marker in rodent fibroblasts.

Inactivated herpes simplex virus type 2 (HSV-2) (1, 2) and low concentrations of native, noninactivated viral DNA (3, 4) induce the neoplastic transformation of mammalian cells in culture. Transformation appears to be mediated by fragment(s) of the HSV-2 genome: (i) mechanically sheared HSV-2 DNA (Mr 9 X 106) induces neoplastic transformation at a frequency identical to that observed with native DNA (4), (ii) hamster cells transformed by UV-irradiated HSV-2 retain a fraction (8-32%) of the viral DNA sequences (5), and (iii) virus transforming activity is more resistant than its infectivity to radiation damage (1, 2). However, the oncogenic fragment in the HSV-2 genome has not yet been identified.Provided that oncogenic activity does not depend on distant noncontiguous genes and that suitable restriction endonucleases can yield appropriate fragments of HSV-2 DNA that do not express lytic functions, it seems reasonable to propose that neoplastic transformation should be demonstrable with a restriction endonuclease fragment. The data described in this report support the validity of this proposal.MATERIALS AND METHODS Cells and Virus. Syrian hamster embryo (SHE) cells were grown in ERM medium with 10% fetal bovine serum (3). Vero and HEp-2 cells were grown in medium 199 with 10% fetal bovine serum. HSV-2 strain S-1, a human cervical tumor isolate (6), was plaque-purified and grown in HEp-2 cells at a low (0.1 plaque-forming unit/cell) multiplicity of infection. HSV-2 strain 333 was obtained from R. Duff.DNA Purification. Viral DNA was purified from Vero cells infected with HSV-2 strains S-1 or 333 by buoyant density centrifugation in NaI gradients containing ethidium bromide as described (3, 7). To isolate fragments, we crushed gel slices in buffer (10 mM Tris-HCl/1 mM EDTA/0.1% NaDodSO4, pH 8.0) and incubated them overnight at 45°C. Agarose was removed by centrifugation at 10,000 rpm for 20 min, and the supernatant was extracted with phenol/chloroform/isoamyl alcohol and dialyzed against Hepes-buffered saline, pH 7.05 (9). DNA concentration was determined by UV absorbance at 260 nm. Recovery was approximately 75%.

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Diploid Syrian hamster embryo (SHE) cells were passaged after transfection with recombinant plasmids containing herpes simplex virus type 2 (HSV-2) DNA inserts Bgl II focusforming fragment N, Bgl II transforming fragment C, and EcoRI/ HindIll fragment AE. Cultures transfected with salmon DNA or with 0.1-5.0 ,ug of Bgl HI fragment N reached crisis and senesced. Those transfected with 0.1-0.5 ,ug of Bgl H fragment C or its lefthand 64% subclone EcoRI/HindIll fragment AE escaped senescence and formed continuous lines. At early passages, these lines as well as isolated clones grew in 2% serum but formed small (0.1 mm) colonies in 0.3% agarose and were nontumorigenic. Serial passaging of Bgl II fragment C-induced cultures and isolated clones resulted in the appearance of large (>0.25 mm) colonies in agarose followed by tumorigenicity. This behavior was not exhibited by the EcoRI/HindIH fragment AE-induced cultures that remained nontumorigenic after 53 passages. DNA from normal SHE cells exhibited homology to Bgl HI fragment C but, under relatively stringent conditions, DNAs from transformed and tumor-derived lines exhibited discrete hybridizing -bands comigrating with authentic viral fragments. These results indicate that neoplastic transformation of normal diploid SHE cells by HSV-2 DNA fragments involves at least two distinct steps-i.e., immortalization and conversion to tumorigenicity. EcoRI/Hindlll fragment AE representing the left 64% of Bgl II fragment C is sufficient to induce immortalization. However, DNA sequences from both lefthand 64% and right-hand 36% subfragments of Bgl HI fragment C are required for tumorigenic transformation.Inactivated herpes simplex virus type 2 (HSV-2) (1-4), herpes simplex virus type 1 (HSV-1) (5), and native, uncleaved HSV-2 DNA (6) induce the neoplastic transformation of normal diploid mammalian cells in culture. Transfection with isolated restriction-enzyme fragments of viral DNA has identified one set of transforming sequences in HSV-1 DNA (7). On the other hand, two distinct transforming regions were identified in HSV-2 DNA (8-10). One of these, the Bgl II fragment C, was found to transform normal diploid Syrian hamster embryo (SHE) cells in the continuous passage assay (8). The other (Bgl II fragment N) was identified on established, heteroploid murine cell lines and Wistar rat cells by a direct one-step assay [focus-formation in liquid medium (9, 10) or colony-formation in methocel (10)].Our studies were designed to determine whether HSV-2 DNA sequences that affect initial events in the transformation process-namely, escape from senescence (immortalization)-are identical to those that induce tumorigenic potential. Accordingly, we selected to study SHE cells that, unlike the established cell lines, exhibit a normal diploid karyotype and have a defined life-span in vitro. They were transfected with defined cloned viral DNA fragments and continuously passaged as described (4,6,8). The findings described in this report indicate that distinct fragments of HSV-2 DNA affe...