MDM2 Is Required for Suppression of Apoptosis by Activated Akt1 in Salivary Acinar Cells

Abstract

Chronic damage to the salivary glands is a common side effect following head and neck irradiation. It is hypothesized that irreversible damage to the salivary glands occurs immediately after radiation; however, previous studies with rat models have not shown a causal role for apoptosis in radiation-induced injury. We report that etoposide and gamma irradiation induce apoptosis of salivary acinar cells from FVB control mice in vitro and in vivo; however, apoptosis is reduced in transgenic mice expressing a constitutively activated mutant of Akt1 (myr-Akt1). Expression of myr-Akt1 in the salivary glands results in a significant reduction in phosphorylation of p53 at serine¹⁸, total p53 protein accumulation, and p21^WAF1 or Bax mRNA following etoposide or gamma irradiation of primary salivary acinar cells. The reduced level of p53 protein in myr-Akt1 salivary glands corresponds with an increase in MDM2 phosphorylation in vivo, suggesting that the Akt/MDM2/p53 pathway is responsible for suppression of apoptosis. Dominant-negative Akt blocked phosphorylation of MDM2 in salivary acinar cells from myr-Akt1 transgenic mice. Reduction of MDM2 levels in myr-Akt1 primary salivary acinar cells with small interfering RNA increases the levels of p53 protein and renders these cells susceptible to etoposide-induced apoptosis in spite of the presence of activated Akt1. These results indicate that MDM2 is a critical substrate of activated Akt1 in the suppression of p53-dependent apoptosis in vivo.

We thank Rachel Henderson of the Transgenic Mouse Facility for assistance in generating the founder mice and Mary E. Reyland and David O. Quissell for discussions and comments on the manuscript. Scott A. Weed provided assistance with microscopy and imaging, Sean W. Limesand provided assistance with quantitative RT-PCR protocols, and Carol Wehling assisted with immunohistochemistry. We also thank Kathy Barzen, Linda Sanders, and Yoon Joo Shin for technical support during these studies and James DeGregori and Bob Sclafani for discussions on the project.

This work was supported in part by the NIH (PO DE 12798 and R01-DE1354) to S.M.A. and by a fellowship grant from the Sjögren's Syndrome Foundation (PN0101-097) to K.H.L. K.H.L. was also the recipient of NRSA Fellowship DE 14315 and is the current recipient of K22 Award DE 16096. The Transgenic Mouse Facility at the University of Colorado Cancer Center is supported by a Cancer Center grant from the National Cancer Institute (CA 46934).