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

Abstract

Human papilloma virus type 16 (HPV-16) and herpes simplex virus type 2 (HSV-2) are human viruses implicated in the development of cancer, in particular cervical cancer. The ability of HSV-2 and HPV-16 to transform early passage human cells was examined in this report. For these studies, gingival fibroblasts were utilized. One gingival cell strain was derived from a normal individual (N-16). The second cell strain was derived from hyperplastic gingival tissue of an epileptic individual (R-30) treated with phenytoin, an antiseizure drug. A common side effect of phenytoin is the induction of gingival overgrowth. R-30 cells contained a stable chromosomal translocation between chromo-

Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.

“…Expression of two viral proteins, E6 and E7, is consis tently found in the majority of cervical carcinomas and derived cell lines (Schwarz et al 1985;Smotkin and Wettstein 1986;Baker et al 1987;Wilczynski et al 1988). Expression of these two viral proteins is sufficient to efficiently inmiortalize human keratinocytes and ex tend the normal life span of human fibroblasts (HawleyNelson et al 1989;Mimger et al 1989a;Watanabe et al 1989;Dhanwada et al 1992). The HPV-16 E6 gene prod uct binds and aids in the degradation of p53 (Scheffner et al 1990;Wemess et al 1990) while the E7 gene product binds a set of proteins that includes hypophosphorylated pRb, pl07, pl30, and cyclin A (Dyson et al 1989(Dyson et al , 1992Davies et al 1993;Tommasino et al 1993).…”

Characterization of human keratinocytes transformed by high risk human papillomavirus types 16 or 18 and herpes simplex virus type 2

“…DNA preparation and Southern blot hybridization. Protocols for the preparation of high M r DNA and Southern blot hybridization were described previously (Dhanwada et al, 1992).…”

“…High M r DNA was prepared from various cell lines as previously described (Dhanwada et al, 1992) and digested with EcoRI overnight at 37 °C. Oligonucleotide primers, 21 bases in length, were chosen to amplify a target sequence of 366 bp located within the HSV-2 mtr III 486 TF sequence.…”

“…The probe used to detect the amplified sequence was the 486 TF sequence (486 bp). The protocol for PCR analysis was previously described in detail (Dhanwada et al, 1992).…”

“…Six of eight tumours (Di Luca et al, 1987) and all of eight tumours (Di Luca et al, 1989) contained both HSV-2 and HPV-16 or -18 DNA sequences. Previous studies demonstrated that primary human fibroblasts transfected with HPV-16 and HSV-2 morphological transforming region III (mtr III) are more aneuploid than fibroblasts immortalized with HPV-16 alone and that HSV-2 DNA sequences are retained in these transformed cells (Dhanwada et al, 1992). Furthermore, mtrII of HSV-2 can convert cervical keratinocytes immortalized with HPV-16 to a tumorigenic cell line .…”