Immortalization and neoplastic transformation of normal diploid cells by defined cloned DNA fragments of herpes simplex virus type 2.

Abstract

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...

Increased Mutation Frequency after Herpes Simplex Virus Type 2 Infection in Non-permissive XC cells

“…The putative relationship between HSV-2 and this type of cancer was initially suggested by the results of seroepidemiological studies, showing that contact with the virus increased the risk of cancer (Rawls et al, 1968;Naib et al, 1966). It was later found that HSV particles (Duff & Rapp, 1973;Kessous et al, 1979) and some HSV DNA fragments could transform rodent cells in vitro (Jariwalla et al, 1983;Galloway et al, 1980;Reyes et al, 1979). In some instances, viral nucleic acids were detected (Bibor-Hardy et al, 1979;Frenkel et al, 1976;Galloway et al, 1980), and various HSV antigens could be found in transformed cells (Flannery et al, 1977;Gupta et al, 1980).…”

Characterization of primary human fibroblasts transformed by human papilloma virus type 16 and herpes simplex virus type 2 DNA sequences

“…Since the c-myc protooncogene is present on chromosome 8 (Neel et al, 1982) and alterations of this gene have been associated with HPV-16 and transformation (Riou et al, 1987;Durst et al, 1987 a;Crook et al, 1990) (Jariwalla et al, 1983), it could not immortalize R-30 cells by itself. This suggests that requirements for immortalizing human cells are unique with respect to primary rodent cells.…”

“…Inactivated HSV-2 can also immortalize human cells (epithelial as well as primary fibroblasts) but virus is not recovered (Takahashi & Yamanishi, 1974;Kucera & Gusdon, 1976). Within the HSV-2 genome there are two unique morphological transforming regions (mtr) designated as mtr II (map unit 0.585 to 0-63) and mtr III (map unit 0.42 to 0.58) (Galloway & McDougall, 1981 ;Jariwalla et al, 1983). The HSV-2 mtr III contains a minimal transforming fragment (TF) of 486 bp (486 TF) which apparently does not encode an oncogenic protein (Jones et al, 1986).…”

Expression of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for virus growth and neoplastic transformation

“…Cultures surviving exposure to inactivated virus developed into established lines (Manak et al,198 l). Transformation by ICP 10 expression vectors was studied in AE cells because ICP10-encoding DNA sequences transform immortalized but not diploid cells (Jariwalla et al, 1983;Hayashi et al, 1985). Cells were serially passaged (1:10 to 1:20 split ratios) beginning at 40 to 44 h post-transfection and assayed for anchorageindependent growth at PTPD 13 to 26.…”