Increased levels of cytokines in the salivary glands have been associated with the loss of secretory cells and reduced salivary function. It has been demonstrated that interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) treatment of a human submandibular gland (HSG) cell line causes growth arrest in the G0/G1 phase of the cell cycle, followed by apoptosis. To stimulate DNA synthesis and reverse this growth arrest, we used an adenovirus vector to overexpress the transcription factor E2F1 in HSG cells. Initially, cells were synchronized by a double thymidine block and then infected with recombinant adenovirus (AdE2F1) expressing E2F1. Cells were harvested at intervals and analyzed by flow cytometry. Greater than 50% of synchronized cells infected with AdE2F1 were in S phase by 18 hours postinfection (hpi) compared to 12% of uninfected cells. Similarly, AdE2F1 infection of HSG cells arrested by IFN-gamma and TNF-alpha treatment caused a fivefold increase in S-phase cells by 48 hpi. However, by 72 hpi, AdE2F1-infected cells showed increases in the subdiploid cell population. Forty-one percent of AdE2F1-infected cells labeled positive by TUNEL, compared to fewer than 6% for controls. Additionally, AdE2F1-infected cells (84 hpi) had low forward-angle and high side scatter light characteristics, similar to apoptotic lymphocytes. These results suggest that E2F1 accumulation in growth-arrested salivary gland cells can stimulate DNA synthesis and overcome a G0/G1 block in the cell cycle. However, E2F1 overexpression did not lead to complete mitosis in HSG cells but, rather, diverted cells into an apoptotic pathway.