Due to the complexities of the HPV viral life cycle and its tight link with epithelial differentiation the virus is difficult to study in the laboratory
environment. Until fairly recently the only methods of studying HPV gene action were by either transiently or stably expressing virus proteins in transformed cells. While this is good enough for binding studies, protein/protein interactions and for functional studies on the individually expressed proteins it does not allow any functional information from the natural system where multiple viral proteins are expressed at any one time in a background of changing cellular protein expression. In order to get an idea of the functions of the viral proteins at various stages in the virus life cycle, a system that allows for differentiation of infected epithelial cells is required.
There are three cell culture systems that are used when studying the
papillomavirus life cycle; namely organotypic raft culture systems; HPV stably transformed NIKS cells and W12 cells. Raft cultures are systems that provide cells with a 3D support system allowing the cells to fully differentiate in culture.
The epidermal cells are grown on a matrix of collagen on top of a wire grid at the liquid air interface, where any nutrients must diffuse to the upper layers via a concentration gradient similar to the in vivo system. The rafts are grown for around two weeks to allow the cells to differentiate. If the epithelial cells contain HPV genomes they can produce infectious virus. Raft cultures are
excellent systems for studying the complete virus life cycle or late viral proteins that require differentiation for production. However, they are relatively time consuming and cumbersome to handle in the laboratory. While these systems are the best for inducing complete differentiation and efficient virus production, other less cumbersome systems are often routinely used where almost complete differentiation of the cells and less efficient virus production is acceptable.
One such system for studying the virus life cycle is the NIKS system. NIKS stands for Normal Immortal KeratinocyteS and were originally isolated by Professor Allen-Hoffmann in 1999 from the BC-1-Ep strain of human neonatal foreskin keratinocytes (Allen-Hoffmann et al., 2000). The NIKS cells spontaneously arose after passaging normal foreskin keratinocytes until senescence (Allen-Hoffmann et al., 2000). The cells that survived this crisis were termed NIKS. NIKS cells retain the ability to differentiate in culture similar to the parental cells (Allen- Hoffmann et al., 2000). They also have wild type p53 levels and do not undergo anchorage independent growth (Allen-Hoffmann et al., 2000). The major
identifiable difference between the NIKS and the parental cells is the
appearance of an extra copy of the long arm of chromosome 8 in the NIKS cells (Allen-Hoffmann et al., 2000). Significantly the c-Myc gene is located on chromosome 8, however steady state levels of c-Myc RNA in NIKS are not increased compared to the parental cells (Allen-Hoffmann et al., 2000).
growth in human cells as an extra chromosome 8 is often found in cancers
including breast and prostate cancers (Allen-Hoffmann et al., 2000). These NIKS cells were then stably transfected with HPV16 genomes taken from the W12 cell line (see below) and a GFP expression plasmid conferring G418 resistance (Flores et al., 1999). These cells were shown to fully differentiate in raft culture and therefore could complete a productive viral infection (Flores et al., 1999). The NIKS cells could not only support the HPV16 viral life cycle but also other HPV types such as HPV31b (Flores et al., 1999). The ability of these cells to support a productive virus infection has made them ideal for studying papillomavirus life cycles. Importantly Flores et al were able to detect virus like particles in the nucleus of the transfected NIKS in raft culture suggesting the virus can indeed undergo a productive virus life cycle (Flores et al., 1999). The only caveat for using these cells in my view is that the genomes have been transfected and NIKS have not been infected “naturally”, however these cells can be passaged so will be continuously available whereas stocks of other naturally occurring cell lines, such as the W12 line, will eventually run out.
In our laboratory we use the W12 model system. W12 cells are cervical
epithelial cells that were originally isolated from a low grade cervical lesion by Professor Margaret Stanley in 1989. W12E (clone 20863) cells contain around 100 episomal copies of the HPV16 genome and can differentiate in monolayer and raft culture allowing completion of a productive viral infection (Stanley et al., 1989, Jeon et al., 1995). As these cells were isolated from a low grade lesion, the HPV16 genomes are naturally occurring virus genomes and the cells have been immortalised by the virus itself making the system arguably much more like the natural infectious situation. The W12 system was evolved to produce a second cell line which contains integrated HPV16 genomes, called W12G cells
(clone 20861) (Jeon et al., 1995). W12E and W12G cells can both differentiate in culture and are not transformed. They require murine J2 3T3 feeder layer cells and mitogens including EGF, Cholera toxin and hydrocortisone for growth. In our laboratory two more cells lines were sequentially derived from W12G cells. W12GPX were derived by growing W12G cells without feeder layer cells. Following crisis and cell death, a new cell line, W12GPX was established that was able to grow in the absence of feeder cells. W12GPXY cells were then derived from W12GPX cells by growing them in the absence of mitogens in the medium (Aasen et al., 2003). W12GPX and W12GPXY cells display a transformed phenotype and are invasive in raft cultures. Moreover, when injected into nude mice, W12GPXY cells formed large squamous cell carcinomas (Aasen et al., 2003). W12GPX and W12GPXY cells have lost the ability to differentiate as when grown in organotypic raft culture there was no differentiation (Aasen et al., 2003). Finally, while W12GPX cells grow in monolayer culture in large colonies similar to untransformed epithelial cells, W12GPXY cells grow in small colonies. The W12 model system comprising these four cell lines allows the study of both the productive viral life cycle and the HPV16-induced transformation of the cervical epithelial cells. Like other well known cervical cancer cell lines
W12GPX and W12GPXY cells only express the HPV16 E6 and E7 proteins, however the benefit of using the W12 cell lines is that we have a series of cell lines
derived from a common ancestor cell with varying phenotypes relating to increased transformation with W12G cells being non-transformed and the W12GPXY cells are fully transformed with the W12GPX cells in an intermediate state of transformation. A flow chart showing the derivation of the W12 cell lines is shown in Figure 1.7.
Figure 1.7 Flow chart of W12 cell line. W12E cells containing episomal copies
of the HPV16 genome and W12G cells containing integrated copies of the HPV16 genome were isolated from a low grade CIN 1 lesion: both are immortalised but not transformed. W12E cells allow the study of the virus life cycle. W12GPX and W12GPXY cells were further derived from W12G cells and are transformed cervical epithelial cells. W12G, W12GPX and W12GPXY cells allow the study of HPV16 induced cellular transformation.