PMC

Replicative life span of parental and derived oral keratinocytes. Growth characteristics of OKF6, OKF6-D1, OKF6-dnp53, OKF6-D1/dnp53, OKF6-D1/dnp53/EGFR, and OKF6-D1/dnp53/EGFR plus EGF. The replicative life span was assessed by calculating the PDs of each cell line. Immortalization was assessed if cells grew at least three times beyond the life span of the parental cells.

Replicative life span of oral keratinocytes additionally overexpressing EGFR and c-myc. Growth characteristics of OKF6-D1/dnp53, OKF6-D1/dnp53/EGFR, OKF6-D1/dnp53/EGFR plus EGF, and OKF6-D1/dnp53/EGFR/c-myc cells (a), as well as OKM1, OKM1-D1/dnp53, OKM1-D1/dnp53/EGFR, OKM1-D1/dnp53/EGFR plus EGF, and OKM1-D1/dnp53/EGFR/c-myc cells (b) are displayed. The replicative life span was assessed by calculating the PD values.

Effects of EGFR overexpression in the generated oral keratinocytes. Equal amounts of protein for OKF6, OKF6-D1, OKF6-dnp53, OKF6-D1/dnp53, OKF6-D1/dnp53/empty, and OKF6-D1/dnp53/EGFR were separated on a 6% SDS/PAGE. The level of EGFR overexpression was compared by subsequent immunoblot analysis using a polyclonal EGFR antibody (1005). EGFR overexpression is indicated as fold increase compared to parental OKF6 cells.

Nude mouse tumors induced by generated oral cancer cells. Tumors induced by the generated human oral cancer cells contain histologic features of poorly differentiated squamous cell cancer mixed with spindle cells. Depicted are representative photomicrographs of tumors from OKF6-D1/dnp53/EGFR/c-myc (two individual clones) shown with hematoxylin/eosin staining. (Magnification, ×400.)

Western Blot analysis of AKT/phospho-AKT, ERK/phospho-ERK as well as c-myc in the generated oral keratinocytes. (a) Equal amounts of protein of OKF6-D1/dnp53, OKF6-D1/dnp53/empty, and OKF6-D1/dnp53/EGFR cells were separated with 10% SDS/PAGE, and a Western Blot was performed. The primary antibodies used were polyclonal AKT1/PKBa, monoclonal phospho-AKT (Ser-473), polyclonal ERK1/2 (C-14), and monoclonal phospho-ERK1/2 (E-4). (b) Equal amounts of protein for OKF6, OKF6-D1, OKF6-D1/dnp53, OKF6-D1/dnp53/EGFR, OKF6-D1/dnp53/EGFR/c-myc (two individual clones), and TE-12 were separated with 10% SDS/PAGE. The level of c-myc overexpression was compared by immunoblot analysis using a polyclonal c-myc antibody (N-262).

Telomerase activity and telomere length in parental and derived oral keratinocytes. (a) Cellular extracts (100 ng) of OKF6-hTERT, OKF6-D1, OKF6-dnp53, OKF6-D1/dnp53, OKF6-D1/dnp53/EGFR, as well as OKM1-D1/dnp53, OKM1-D1/dnp53/EGFR, and OKM1-D1/dnp53/EGFR/c-myc cells were assayed for telomerase activity using the PCR-based TRAP assay. Heat-treated (heat) samples served as negative control, and OKF6-hTERT served as a positive control. IC is an internal PCR control. (b) Telomere length for preimmortal OKF6-D1/dnp53 (100 PD), immortal OKF6-D1/dnp53 (200 PD), OKF6-D1/dnp53/EGFR, OKF6-D1/dnp53/EGFR/empty, and OKF6-D1/dnp53/EGFR/c-myc cells was analyzed by hybridization of genomic DNA with a specific oligonucleotide probe. Two different clones of OKF6-D1/dnp53/hTERT cells served as positive control.