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doc-1--mediated apoptosis in malignant hamster oral keratinocytes
S J Cwikla1, T Tsuji, J McBride
1Harvard School of Dental Medicine, Boston, MA 02115, USA.
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 leads 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-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.
Insights
Deleted in oral cancer-1 (doc-1) gene reintroduction into oral cancer cells induced apoptosis. This suggests doc-1 is a key apoptosis mediator inactivated during oral carcinogenesis.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Cell cycle regulators are often altered in cancer.
- Deleted in oral cancer-1 (doc-1) is an S-phase regulator that is inactivated during oral carcinogenesis.
- doc-1 reintroduction into oral cancer cells increases cell death.
Purpose of the Study:
- To investigate if ectopic doc-1 expression induces apoptosis in hamster oral cancer cells.
- To confirm doc-1's role as an apoptosis mediator in oral carcinogenesis.
Main Methods:
- Transfected malignant hamster oral keratinocytes with a doc-1 expression vector.
- Assessed cell death using trypan blue exclusion, toluidine blue-safranin staining, and quantitative fluorescent microscopy.
- Identified early apoptotic changes via annexin V/propidium iodide (PI) fluorescence-activated cell sorter (FACS) analysis and terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL) assay.
Main Results:
- Doc-1 transfected cells exhibited significantly higher cell death compared to controls.
- Morphological and cytochemical analyses confirmed apoptosis induction in doc-1 expressing cells.
- TUNEL and FACS assays demonstrated early apoptotic markers in doc-1 transfectants.
Conclusions:
- Ectopic doc-1 expression effectively induces apoptosis in malignant oral keratinocytes.
- doc-1 is a crucial mediator of apoptosis that becomes inactivated during oral cancer development.