Restoration of Transforming Growth Factor-β Signaling Enhances Radiosensitivity by Altering the Bcl-2/Bax Ratio in the p53 Mutant Pancreatic Cancer Cell Line MIA PaCa-2

Mansoor M Ahmed,Rachael A Alcock,Damodaran Chendil,Swatee Dey,Anindita Das,Kolaparthi Venkatasubbarao,Mohammed Mohiuddin,Luzhe Sun,William E Strodel,James W Freeman

doi:10.1074/jbc.m110168200

Abstract

In this study, we investigated whether lack of transforming growth factor beta (TGF-beta) type II receptor (RII) expression and loss of TGF-beta signaling played a role in radiation resistance of pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene. Transfection of this cell line with a RII cDNA led to a stimulation of the transcriptional activity of p3TP-Lux, a TGF-beta-responsive reporter construct. The RII transfectants (MIA PaCa-2/RII) showed a significant increase in sensitivity to radiation when compared with MIA PaCa-2/vector cells. The increase in sensitivity to radiation was reversed by neutralizing antibodies to TGF-beta, indicating that these changes were dependent on TGF-beta signaling. Compared with MIA PaCa-2/vector cells, MIA PaCa-2/RII cells showed a greater than 3-fold increase in apoptosis after radiation. Enhanced radiation sensitivity of MIA PaCa-2/RII cells was associated with an induction of Bax mRNA and protein that was followed by a release of cytochrome c and activation of caspase-3 and poly(ADP-ribose) polymerase cleavage after radiation exposure. Overexpression of Bcl-x(L) or treatment with antisense oligodeoxynucleotides targeted against Bax significantly inhibited radiation-induced apoptosis in MIA PaCa-2/RII but not in MIA PaCa-2/Vector cells, suggesting that Bax induction is necessary for radiation-induced TGF-beta signaling-mediated apoptosis. Thus, restoration of TGF-beta signaling sensitized these cells to ionizing radiation, although these cells possess a mutated p53 gene. In addition, disruption of RII function by dominant negative mutant of RII inhibited the radiation-induced TGF-beta signaling and apoptosis in primary cultures of mouse embryonic fibroblasts. Together, these observations imply that RII is an important component of radiation-induced TGF-beta signaling, and loss of function of RII may enhance resistance to radiation-induced apoptosis.

Highlights

We investigated whether lack of transforming growth factor ␤ (TGF-␤) type II receptor (RII) expression and loss of TGF-␤ signaling played a role in radiation resistance of pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene
We determined whether transfection of MIA PaCa-2 cells with RII cDNA might restore TGF-␤ signaling in these cells
Three of the transfected clones tested showed appreciable levels of RII mRNA as analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) (Fig. 1, lanes 3, 4, and 6), and all three clones showed restoration of TGF-␤ signaling as determined by p3TP-Lux assay

Summary

Introduction

We investigated whether lack of transforming growth factor ␤ (TGF-␤) type II receptor (RII) expression and loss of TGF-␤ signaling played a role in radiation resistance of pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene. Transfection of this cell line with a RII cDNA led to a stimulation of the transcriptional activity of p3TP-Lux, a TGF-␤؊responsive reporter construct. Role of TGF-␤ Type II Receptor in Radiation Sensitivity complex associates with other co-factors including Fast-1, p300/CBP, and AP-1, forming a transcriptional active complex (14 –17)

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