Tissue homeostasis results from a balance in the amounts of cellular proliferation and programmed cell death, or apoptosis. Irradiation of the head and neck region of rodents has been shown to result in salivary gland hypofunction similar to that observed in humans receiving ionizing irradiation (49
). In the rat model, salivary gland function is diminished 50 to 70% at 6 to 9 months following exposure to gamma irradiation; however, previous studies reported that only 2% of cells were apoptotic as evidenced by the presence of condensed nuclei on hematoxylin- and eosin-stained sections (61
). Because of the disparity between the extent of apoptosis and the extent of salivary gland hypofunction, some authors have concluded that apoptosis is not causally related to radiation-induced salivary gland hypofunction (68
). Although the apoptotic index observed in a tissue reflects the balance between the induction of apoptosis and the clearance of apoptotic cells, our data indicate that there is far more apoptosis at 8 and 24 h following irradiation with a single dose of 5 Gy than previously suggested by quantifying the number of condensed nuclei. Better understanding of gamma-irradiation-induced damage to salivary glands is clinically relevant to improving the care of patients undergoing treatment for head and neck cancer.
The ability of Akt to suppress apoptosis induced by various stimuli is well established (28
). Primary salivary acinar cells isolated from myr-Akt1 transgenic mice consistently had lower levels of DNA damage-induced apoptosis than did control cells isolated from age-matched FVB mice. Importantly, this suppression of apoptosis by myr-Akt1 was also observed in vivo following exposure of the head and neck region to gamma irradiation treatment (Fig. ). Following DNA damage, p53 undergoes several posttranslational modifications, and the stability of the p53 protein increases dramatically (55
). We determined the levels of total p53 and serine18
phosphorylated p53 by immunoblot analysis and quantitated the levels of p53-responsive genes (p21WAF1
and Bax) by RT-PCR. Levels of total p53 and phosphorylated p53 (serine18
) were increased in primary cells from FVB mice following exposure to either etoposide or gamma irradiation (Fig. ), resulting in the induction of p21WAF1
and Bax expression in these cells (Fig. ). The induction of p21WAF1
was observed following low doses of etoposide or gamma irradiation, and this reaction could contribute to the sensitivity of the salivary glands to ionizing radiation in vivo. In contrast, primary salivary acinar cells from myr-Akt1 transgenic mice were less sensitive to etoposide and gamma irradiation-induced apoptosis and had reduced levels of total p53, phosphorylated p53, p21WAF1
RNA, and Bax RNA following exposure to DNA-damaging agents.
It has recently been reported that Akt negatively regulates the levels of p53 protein via activation of MDM2 (54
). Normally, p53 is maintained at low levels in the cell due to its short half-life, which is in part regulated by MDM2 (66
). MDM2 is an E3 ubiquitin ligase that targets p53 for ubiquitination and subsequent degradation by the proteosome (56
). Analysis of the Akt/MDM2/p53 pathway has largely been accomplished by transfection of established cell lines with activated mutants of Akt. In transgenic mice expressing constitutively activated Akt1, there is an increase in total MDM2 protein and MDM2 phosphorylation at serine163
which correlates with a dramatic reduction in total p53 protein, suggesting that Akt regulates p53 protein expression in vivo. We extended this observation to show that phosphorylation of MDM2 is Akt dependent and that MDM2 is required for myr-Akt1-induced suppression of apoptosis induced by DNA damage in primary salivary acinar cells from myr-Akt1 transgenic mice. It is interesting to note that phosphorylation of Chk1 at serine345
, the putative phosphorylation site for ATM/ATR, occurs under conditions of reduced MDM2 expression in myr-Akt1 cells. This may indicate a feedback mechanism by which phosphorylation of Chk1 is dependent on the level of total p53 protein.
It has been suggested that the p53 homologs p63 and p73 are required for p53-dependent apoptosis induced following DNA damage; however, p63 and p73 are not required for p53-dependent apoptosis in T cells (32
). Full-length versions of p73 and p63 (termed TA to indicate presence of a transactivation domain) have been shown to induce apoptosis. Deletion of the N-terminal transactivation domain of p73 or p63, through utilization of the second transcriptional start site (termed ΔN), produces a protein that serves as a dominant-negative molecule that suppresses action of the respective full-length isoforms, as well as of p53 (94
). Loss of p63 and p73 cooperates with the loss of p53 to accelerate tumor formation, suggesting that p63 and p73 may function as tumor suppressors in some tissues, including salivary glands (31
). For these reasons it was important to examine the levels of all p53 family members in the salivary glands of myr-Akt1 transgenic mice. The basal levels of p53, p63, and p73 were all reduced in myr-Akt1 mice (Fig. ). Regulation of p73 activity may occur by MDM2-dependent translocation without degradation or may involve another putative Akt substrate, YAP (9
); however, neither of these molecules can explain the reduced levels of p73 RNA we have observed. Modulation of p63 protein levels by activated Akt has not been previously observed and cannot be explained by the phosphorylation of MDM2, as p63 and MDM2 do not interact (91
). We are currently conducting additional studies to understand the regulation of p63 by Akt and to investigate whether YAP is phosphorylated in the salivary glands of myr-Akt1 transgenic mice.
Significant efforts have focused on the identification of Akt substrates that suppress apoptosis in tissue culture cells (13
). Analysis of transgenic mice that express activated Akt1 has confirmed the importance of some of these substrates. For example, Wendel et al. demonstrated the chemoresistance of a B-cell lymphoma that overexpresses Eμ-Myc
; activated Akt1 was dependent on mTOR and eIF4E, and an mTOR inhibitor, rapamycin, reversed the Akt-induced chemoresistance in these cells (92
). This result was somewhat surprising given the number of Akt substrates regulating apoptosis and emphasizes that the critical substrates for Akt may differ with the stimuli and cell type examined. Our analysis of activated Akt1 overexpression in vivo has revealed a universal reduction in the expression of the p53 family of proteins in salivary acinar cells, although the mechanism underlying this observation may differ for each p53 family member. It is well known that mutations in p53 and/or overexpression of MDM2 occurs in many cancers, and numerous studies have focused on the dysregulation of p53 in cancer (47
). Disruption of the MDM2/p53 regulation pathway shows great promise in reactivating wild-type p53 in cancer cells (85
), as well as sensitizing tumors to radiation therapy (69
). Our studies have demonstrated that MDM2 is a critical substrate of Akt in suppression of apoptosis caused by DNA damage. These studies also suggest that the resistance of tumors expressing activated Akt to chemotherapy and radiation therapies could be reversed by targeting MDM2's function or expression. Future studies will determine whether targeted disruption of MDM2 in the salivary glands sensitizes myr-Akt1 transgenic mice to gamma irradiation in vivo.