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Characterization of primary human keratinocytes transformed by human papillomavirus type 18
1Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Abstract:
Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo, and cell strains were generated by selecting in G418. One cell strain (FE-A), which exhibits an extended life span, is currently in its 30th passage. In comparison, control cultures can only be maintained up to the seventh passage. Southern blot analysis revealed the presence of at least one intact, integrated viral genome in these cells. FE-A cells showed altered growth properties, characterized by a change in morphology, and clonal density. Differentiation markers analyzed by Western blotting (immunoblotting), such as cytokeratins and involucrin, indicated that the cells resembled a partially differentiated epithelial population. Increased expression of the 40-kilodalton cytokeratin was observed in FE-A cells, similar to that observed in simian virus 40-immortalized human keratinocytes (M. Steinberg and V. Defendi, J. Cell Physiol. 123:117-125, 1985). FE-A cells were also found to be defective in their response to terminal differentiation stimuli. Calcium and 12-O-tetradecanoyl-phorbol-13-acetate treatment induced normal epithelial cells to differentiate, whereas the human papillomavirus 18 (HPV-18)-containing keratinocytes were resistant to these signals, indicating their partially transformed nature. These cells were not able to induce tumors in nude mice over a period of up to 8 months. A second cell strain, FE-H18L, also generated by transfecting HPV-18, also exhibited an extended life span and similar alterations in morphology. Viral RNA transcribed from the early region of HPV-18 was detected in both cell strains by Northern (RNA) blot analysis. These cell strains should provide a useful model for determining the role of HPV in carcinogenesis.
Insights
Human epithelial cells infected with human papillomavirus 18 (HPV-18) showed extended lifespan and altered growth properties. These HPV-18 cells resist differentiation, offering a model for studying HPV
Area of Science:
- Oncology
- Virology
- Cell Biology
Background:
- Human papillomavirus (HPV) is implicated in various cancers.
- Understanding the early events of HPV infection is crucial for cancer research.
- Investigating cellular changes induced by HPV can reveal mechanisms of carcinogenesis.
Purpose of the Study:
- To generate and characterize human epithelial cell lines immortalized by human papillomavirus 18 (HPV-18).
- To investigate the in vitro biological properties of HPV-18-immortalized cells.
- To establish a model system for studying the role of HPV in cancer development.
Main Methods:
- Primary human epithelial cells were cotransfected with pHPV-18 and pSV2neo.
- Cell strains were selected using G418 and cultured for extended periods.
- Southern blot, Western blotting (immunoblotting), and Northern (RNA) blot analyses were performed.
- Cells were treated with differentiation stimuli (calcium, 12-O-tetradecanoyl-phorbol-13-acetate) and assessed for tumor formation in nude mice.
Main Results:
- Two cell strains, FE-A and FE-H18L, exhibited extended lifespans (up to 30 passages) compared to controls (up to 7 passages).
- FE-A cells displayed altered morphology, clonal density, and increased expression of 40-kilodalton cytokeratin.
- HPV-18-containing keratinocytes were resistant to terminal differentiation stimuli and showed defective responses.
- Integrated HPV-18 genomes and transcribed viral RNA were detected; cells did not form tumors in nude mice.
Conclusions:
- HPV-18 immortalizes human epithelial cells, conferring extended lifespan and altered growth characteristics.
- These HPV-18-immortalized cells exhibit partial transformation, indicated by resistance to differentiation.
- The generated cell strains serve as a valuable in vitro model for investigating HPV's role in carcinogenesis.