doc-1-mediated apoptosis in malignant hamster oral keratinocytes

Purpose: Cell cycle mediators involved in inducing apoptosis are frequently deregulated during carcinogenesis. Deleted in oral cancer-1 (doc-1) is an S-phase regulator that is inactivated during oral carcinogenesis. Transfection of doc-1 into malignant oral keratinocytes lends to increased cell loss. It is hypothesized that ectopic expression of doc-1 in hamster oral cancer cells induces apoptosis. Materials and Methods: Malignant hamster oral keratinocytes (wt-HCPC-1), which lack measurable doc-1 mRNA and protein, were previously transfected with either a CMV-doc-1 expression vector construct (doc-HCPC-1) or the parental control vector pcDNA3 (cv-HCPC-1). A trypan blue exclusion assay was performed to examine cell death in the parental or wild-type HCPC-1 keratinocytes, HCPC-1 transfected with the parental pcDNA3 vector, and the doc-1 transfected HCPC-1 cells. To examine whether ectopic expression of doc-l mediates gross cellular changes consistent with apoptosis, toluidine blue-safranin differential staining and the quantitative fluorescent microscopy assays were performed. To identify early apoptotic cytochemical changes observed in the cell membrane and nucleus, annexin V/propidium iodide (PI) fluorescence-activated cell sorter (FACS) analysis and the terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL) assay were performed. Results: Doc-HCPC-1 showed elevated numbers of dead cells over wt-HCPC-1 and cv-HCPC-1 in the trypan blue exclusion assay. Toluidine blue-safranin staining and quantitative fluorescent microscopy showed significant morphologic changes in the doc-1 transfectants consistent with apoptosis (P < .05). TUNEL assays (P < .05) and annexin V/PI FAGS analysis (P < .05) also showed early cytochemical changes in the doc-HCPC-1 transfectants, confirming that ectopic expression of doc-1 induces apoptosis. Conclusions: These data suggest that doc-1 induces apoptosis in malignant hamster oral keratinocytes. It is hypothesized that doc-1 is a mediator of apoptosis that is inactivated during hamster oral carcinogenesis.