Immortalization of normal human oral keratinocytes with type 16 human papillomavirus

Abstract

Primary human oral keratinocytes were transformed by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA, and two transformed cell lines named human oral keratinocytes-16A and -16B (HOK-16A and HOK-16B) were established. While normal cells and cells transfected with vector only exhibited a limited lifespan, the HOK-16A and HOK-16B lines demonstrated immortality and altered morphology from their normal counterpart. The HOK-16A and HOK-16B lines contained approximately 40 and approximately 25 copies of intact HPV-16 DNA as integrated form per cell respectively, and both cell lines expressed several viral specific poly(A+) RNAs. Notably these cell lines also overexpressed cellular myc proto-oncogene in comparison with the normal counterpart. However, the immortalized cell lines were not able to produce tumors in nude mice, indicating that the cells are partially transformed. The HOK-16A and HOK-16B lines are, therefore, useful for investigating the multistep molecular events of oral carcinogenesis.

ΔNp63α Protein Triggers Epithelial-Mesenchymal Transition and Confers Stem Cell Properties in Normal Human Keratinocytes

“…In the current study, we compared the relative telomere lengths in NHEK before and after EMT by the qPCR method. HOK-16B, an immortalized counterpart of NHOK (39), demonstrated lower telomere length compared with NHEK, consistent with our prior report (40) (Fig. 7D).…”

Gene amplification and overexpression of protein phosphatase 1α in oral squamous cell carcinoma cell lines

“…Normal human oral keratinocytes were cultured from tissues obtained from patients undergoing uvulopalatopharyngoplasty at the University of Pittsburgh Medical Center (consent obtained through the Head and Neck SPORE at the University of Pittsburgh under their Institutional Review Board Guidelines) as described (Park et al, 1991).…”

Increased ICAM-1 expression in transformed human oral epithelial cells: molecular mechanism and functional role in peripheral blood mononuclear cell adhesion and lymphokine-activated-killer cell cytotoxicity.

“…The tissues were washed in calcium-and magnesium-free Hank's Balanced Saline Solution (HBSS, Life Technologies, Gaithersburg, MD), digested with dispase and collagenase to separate the epithelial layer, then digested with trypsin to separate individual cells. The dissociated epithelial cells were grown to confluency in keratinocyte growth medium (KGM) supplemented with 30 μg/ml bovine pituitary extract, 0.1 ng/ml human epidermal growth factor, 5 μg/ml bovine insulin, 0.5 μg/ml hydrocortisone, 50 μg/ml gentamicin and 50 ng/ml amphotericin-B (Clonetics Corp., San Diego, CA), at a density of 5 × 10 4 cells per 28 cm 2 (26). …”