doc-1--mediated apoptosis in malignant hamster oral keratinocytes.
OBJECTIVE
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 leads to increased cell loss. It is hypothesized that ectopic expression of doc-1 in hamster oral cancer cells induces apoptosis.


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-1 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 FACS 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.
Cwikla
S J
SJ
Harvard School of Dental Medicine, Boston, MA 02115, USA.
Tsuji
T
T
McBride
J
J
Wong
D T
DT
Todd
R
R
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002462:Cell Membrane / Q000737:chemistry; D002469:Cell Separation; D002610:Cheek; D006224:Cricetinae; D053938:DNA Fragmentation; D015972:Gene Expression Regulation, Neoplastic; D005809:Genes, Regulator; D016147:Genes, Tumor Suppressor / Q000235:genetics*; D020287:In Situ Nick-End Labeling; D015603:Keratinocytes / Q000166:cytology*; D008856:Microscopy, Fluorescence / Q000379:methods; D009062:Mouth Neoplasms / Q000235:genetics*; D011506:Proteins / Q000235:genetics; D016196:S Phase / Q000502:physiology; D013194:Staining and Labeling; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C096151:CDK2AP1 protein, human; D011506:Proteins; D025521:Tumor Suppressor Proteins