Malignant Transformation of Human Cells by Constitutive Expression of Platelet-derived Growth Factor-BB

Marta Martinez,Jeffrey Schechner,Cynthia Cohen,Asha Shah,Jose M. Cuezva,Chun-Yang Fan,Jun Chung,Arthur M. Mercurio,Rhoda Alani,Jack L. Arbiser,Byungwoo Ryu,Baskaran Govindarajan,Rebecca S. Arnold

doi:10.1074/jbc.m500411200

Abstract

Platelet-derived growth factors (PDGFs) comprise a family of growth factors strongly implicated in human oncogenesis. A number of human tumors overexpress PDGF family members or have translocations activating PDGF receptors. Whereas the epidemiologic evidence implicating PDGF in human tumors is strong, malignant transformation of human cells by overexpression of PDGF has not been demonstrated. We have previously developed a human cell line by the sequential introduction of large T cells and telomerase, and we have demonstrated that these cells express functionally active PDGF receptor (PDGFR) beta. In order to determine whether growth factor-mediated transformation of human cells could occur, these cells were transduced with a retrovirus encoding PDGF-BB. Constitutive expression of PDGF-BB led to malignant transformation in nude mice. This is the first demonstration of constitutive signaling causing malignant transformation of human cells. Some of the changes that occur because of constitutive growth factor expression can be reversed by the clinically approved tyrosine kinase inhibitor Glivec, whereas other changes are not reversible by tyrosine kinase inhibitors. Our model allows the assessment of epigenetic changes that occur during human carcinogenesis. In addition, these studies provide insight into the clinical failure of tyrosine kinase inhibitors as monotherapy for advanced malignancy.

Highlights

Introduction of Id1 Accelerates Tumorigenesis in Vivo SV7tert Platelet-derived growth factors (PDGFs) cells were infected with retroviruses encoding pLXSN or a constitutively active Id-1 [15]
Constitutive expression of PDGF-BB led to a significant induction of luciferase activity, indicating that a major mode of VEGF regulation by PDGF-BB in this system is at the level of transcription
These studies elegantly demonstrate the steps required for full transformation of human cells and allow the assessment of epigenetic events that occur over long term human carcinogenesis

Summary

Introduction

1 Accelerates Tumorigenesis in Vivo SV7tert PDGF cells were infected with retroviruses encoding pLXSN or a constitutively active Id-1 [15]. The unselected cells were pooled and were used for further in vivo and in vitro experiments. Overexpression of Id-1 was confirmed by Western analysis (Fig. 9). For Id-1 immunoblot experiments, 30 – 40-␮g aliquots of cell and tissue lysates prepared as above were separated by 15% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Immobilon P, Millipore). The membrane was blocked and incubated with primary antibody (Id-1 polyclonal antibody from Santa Cruz Biotechnology, SC-488) in TNET buffer (10 mM Tris-HCl, pH 7.5, 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20) overnight at 4 °C. Antigen-antibody complexes were detected and visualized after incubation for 1 h with appropriate horseradish peroxidase-labeled secondary antibodies

Methods

Results

Conclusion