Multistep transformation by defined fragments of herpes simplex virus type 2 DNA: oncogenic region and its gene product.

Abstract

("immortalization"). However, the cells remained nontumorigenic, thereby raising obvious questions pertaining to the biological effect of the right-hand side ofBgl II C, which is now represented by the cloned BamHI E fragment (pGH17a, 0.533-0.584 m.u., Fig. 1).The present study was designed to (i) determine whether Bgl II C-induced neoplastic transformation of normal diploid SHE cells is a multistep process and (it) identify the sequences that impart tumorigenic potential. The data indicate that a 4.4-kilobase (kb) subfragment located within the BamHI E fragment of HSV-2 DNA (0.554-0.584 m.u.) and overlapping 16% of the right hand of the Bgi II C fragment converts cells immortalized by fragment EcoRI/HindIII AE to a fully tumorigenic phenotype. The 4.4-kb subfragment encodes a 144-kDa protein that is antigenically and structurally similar to a HSV-2-infected cell protein (ICP 10) that appears to be at least one of the components of the viral ribonucleotide reductase (5). A tumor derivative established with these transformed cells expresses the c-myc oncogene. MATERIALS AND METHODSCells. SHE cells, prepared from 13-day embryo, were obtained from T. Okeda and grown in IBR modified Dulbecco's Eagle reinforced medium with 10% fetal calf serum. HEp-2 cells were grown in medium 199 with 10% calf serum. K562 cells were grown in suspension as described (6).Recombinant Plasmid DNA. Plasmids derived from pBR322 that contain fragments of HSV-2 (333) DNA in the 0.416-0.584 m.u. region (Fig. 1) were obtained from G. S. Hayward (The Johns Hopkins University, Baltimore, MD). The plasmids were propagated in Escherichia coli HB 101 and the DNA was purified from cleared lysates by CsCl/ethidium bromide density-gradient centrifugation.Restriction Enzymes and Gel Electrophoresis. Enzymes were purchased from New England Biolabs and used as recommended by the manufacturer. Cleaved DNAs were electrophoresed on horizontal slab gels of 0.7% agarose in Tris borate buffer (89 mM Tris borate/89 mM boric acid/2 mM EDTA) containing 0.5 Ag of ethidium bromide per ml at 25 V for 15 hr. The DNA was visualized with long-wave UV light. DNA-containing agarose bands were electroeluted and extracted with phenol/chloroform/isoamyl alcohol.Establishment of Immortal/Tumorigenic Lines. HindIfllinearized plasmid DNAs (HindIII cuts once within pBR322) were mixed with 5 ,ug of salmon sperm DNA and precipitated with calcium phosphate, and aliquots (0.5 ml) were applied to subconfluent monolayers of normal SHE cells (passage 4; 2 x 105 cells per 60-mm dish) (3). Cultures were refed fresh medium at 4 hr after transfection and were serially passaged (1:10 or 1:20 split ratio) at confluency. Phenotypic alterations associated with transformation were determined as described (3, 7). Colonies in 0.3% agarose were counted at 3 wk and scored as medium (0.1-0.2 mm) or large (> 0.2 mm) by using a calibrated microscope eyepiece. Cloning efficiency (CE) was expressed as the (number of colonies x 100)/number of seeded cells. Tumorigenic potential was determin...

Herpes Simplex Virus and Human Cytomegalovirus: Their Role in Morphological Transformation and Genital Cancers

“…Clear evidence that transformation by HSV leads to oncogenically transformed cells is not in dispute (Galloway & McDougall, 1983;Cameron et al, 1985;Hayashi et al, 1985;. Two facts arising from morphological transformation studies with HSV-2 are also generally accepted.…”

“…As a result tsD can produce some early polypeptides at non-permissive temperature in addition to the IE ones. Both tsK and tsD induce the large subunit of RR but its transforming function has been associated only with established cells Hayashi et al, 1985). Macnab (1974Macnab ( , 1975Macnab ( , 1979 and Cameron et al (1985) used primary cells, and therefore the large subunit of RR is not likely to be the transforming function.…”

“…Further analysis of this system will be awaited with great interest as the cell sequence may encode a cellular oncogene. (Hayashi et al, 1985;Iwasaka et al, 1985), or prostaglandin E2 and protein kinase C activity (Krebs et al, 1987;L. S. Kucera, personal communication, l lth International Herpesvirus Workshop, 1986).…”

“…The PstI C Sail B fragment is the smallest (480 bp) Jariwalla et al, 1986) found to transform Rat-2 cells in the focus assay system of JariwaUa et al (1986). The subfragment of BamHI e labelled H oncogenically transforms hamster cells (Hayashi et al, 1985).…”

Enhanced malignant transformation induced by expression of a distinct protein domain of ribonucleotide reductase large subunit from herpes simplex virus type 2.

“…1). DNA sequences from BamHI-E and its transforming subclones overlap with the coding region of the ribonucleotide reductase (RR) large subunit (RR1) and induce the tumorigenic conversion of immortalized Rat2 and Syrian hamster cell lines (1)(2)(3). Molecular analysis of the BamHI-E region showed that small DNA subfragments, mapping within the N-terminal region of RR1 ( Fig.…”