Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA

Abstract

Immortalization of human keratinocytes (HKc) by human papillomavirus type 16 (HPV16) is reproducible at a high frequency, is due directly to the presence of the viral sequences in the cells, and occurs independently from the genetic characteristics of the host cells. Ten human keratinocyte strains, each derived from a different individual, were transfected with pMHPV16d and selected with G418. Eight became established lines. Two strains, which failed to grow shortly after successful G418 selection, were negative for HPV16 DNA. No lines were established following transfection of the same HKc strains with vector sequences only. The immortalized lines maintained a constant number of copies of the viral genome integrated into the cellular DNA. Each line showed a unique integration pattern of HPV16 sequences into the cellular genome, but expressed similar patterns of viral messages. Sublines able to grow in the absence of growth factors (epidermal growth factor and bovine pituitary extract), and others which became resistant to differentiation stimuli (serum and calcium) were obtained by selection in growth factor-free medium and serum-supplemented medium, respectively. The establishment of continuous cell lines is a direct consequence of the presence of viral sequences; however, because none of these lines formed tumors in nude mice, additional events must be necessary for progression of malignancy. HPV16-immortalized human keratinocyte lines can be used to investigate and identify the viral factors involved with the modification of growth and differentiation control by HPV16.

Human keratinocytes and tumor-derived cell lines express alternatively spliced forms of transforming growth factor-α mRNA, encoding precursors lacking carboxyl-terminal valine residues

“…However, results obtained in the CHO cells may not necessarily re¯ect the properties of these variants in other cell types. Therefore, we are currently exploring the proteolytic processing of wt and variant TGF-a in normal HKc and HKc/HPV16 in our in vitro model of multi-step carcinogenesis (Pirisi et al, 1987(Pirisi et al, , 1988 as well as in human epithelial cells derived from a variety of human tumors. The only signi®cant di erence in the primary structure among the three forms of TGF-a precursors is that the two carboxyl-terminal valine residues in wt TGF-a are replaced by ®ve (GCRLY) or four (ATLG) amino acids in TGF-a VaI and variant II, respectively (Figure 1c).…”

“…We have previously observed, in our in vitro model of HPV16-mediated human cell carcinogenesis (Pirisi et al, 1987(Pirisi et al, , 1988Zyzak, et al, 1994;DiPaolo et al, 1989DiPaolo et al, , 1990) that human keratinocytes immortalized with human papillomavirus type 16 DNA (HKc/ HPV16) secrete 50 ± 100-fold less mature TGF-a than normal human keratinocytes (HKc), despite the fact that mRNA levels for TGF-a are either unchanged or slightly increased (Zyzak et al, 1994). These results may indicate that HPV16-transformed cells have lost the enzyme activity required for TGF-a processing, or that the cells express isoforms of proTGF-a that are incompletely processed.…”

“…CHO K1 cells stably transfected with the expression vector pcDNA3.1 (Invitrogen) only, or with pcDNA3.1 containing TGF-a cDNA sequences were maintained in the same medium in the presence of 300 mg/ml G418 (Calbiochem or Gibco). HKc, HKc/HPV16 (Pirisi et al, 1988), W12 clone 20861, and clone 20863 (Jeon et al, Figure 6 Clonal growth assays of CHO cells expressing wt and variant TGF-a precursors. Assays were performed as described in the text, and the total colony area in each dish was measured by a computerized image analysis system.…”

“…Alternatively spliced forms of TGF-a X Xu et al 1995) were cultured in complete serum-free MCDB153-LB medium (CM) (Gibco) (Pirisi et al, 1988). HKc/GFI were maintained in CM lacking EGF and BPE (Pirisi et al, 1988).…”

“…HKc/GFI were maintained in CM lacking EGF and BPE (Pirisi et al, 1988). HKc/DR were maintained in CM supplemented with 5% FBS and 1 mM calcium chloride.…”