Elsevier

Lung Cancer

Volume 31, Issues 2–3, March 2001, Pages 181-191
Lung Cancer

Alteration of cell growth and morphology by overexpression of transforming growth factor β type II receptor in human lung adenocarcinoma cells

https://doi.org/10.1016/S0169-5002(00)00169-0Get rights and content

Abstract

TGF-β is a potent inhibitory regulator of cell growth, which is transduced through interaction between type I (RI) and type II (RII) receptors that form heteromeric kinase complexes. Abnormal expression of these receptors has been identified in several human epithelial cancers and has been shown to be highly associated with resistance to TGF-β. In this study, we investigated the expression of RI and RII in 13 human non-small cell lung cancer cell lines (NSCLCs) and demonstrated decreased or loss of RII expression in five lung cancer cell lines, but not of RI. Of these cell lines, the role of RII in NCI-H358 adenocarcinoma, which lacks RII and is insensitive to TGF-β, was investigated by transducing this cell line with a recombinant retrovirus expressing full-length TGF-β RII. Stably transfected cells showed significant increase in RII mRNA and protein expression. These cells responded to exogenous TGF-β1 with suppressed proliferation in a dose-dependent manner and G1 arrest accompanied by morphological change distinct from control cells. We also investigated whether overexpression of dominant-negative RII (dnRII) in NCI-H441 adenocarcinoma, which is sensitive but expresses low levels of RII, could block signaling through the receptor complex. The overexpression of this kinase-domain-truncated RII by expressing the retroviral dnRII construct led to loss of the ability to respond to TGF-β1 and an exhibition of uncontrolled growth. These results suggest a close association between the loss of the expression of wild-type TGF-β RII and carcinogenesis in human lung cancer cells.

Introduction

Transforming growth factor-β (TGF-β) is a multifunctional peptide growth factor, which controls not only cell proliferation but other cellular processes such as differentiation, chemotaxis and the formation of extracellular matrix in various cell types and human tissues [1]. TGF-β, is secreted as a latent form (LTGF-β), and is activated when cleaved from LTGF-β by acidification and proteolysis. The active form of TGF-β exerts these biological effects by binding to sets of two specific membrane proteins called receptors type I (RI) and type II (RII). Each possesses an extracellular region, a single transmembrane segment and a cytoplasmic serine/threonine kinase domain [2], [3]. RI recognizes TGF-β-bound RII and acts as a heteromeric kinase complex in the cascade signal pathway [4], which suggests that the expression of both receptors is crucial for the growth-inhibitory effect of TGF-β on proliferating epithelial cells.

Mutation of RII or the absence of RII expression is known in a wide spectrum of cancers, such as colon and gastric cancer [5], [6], breast cancer [7], head and neck cancer [8], hereditary nonpolyposis colorectal cancer (HNPCC) [9] and thyroid cancer [10]. In addition, the abnormal expression of RII in the case of lung cancer which includes both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) has been reported [11], [12], [13]. More recently, It has been found that the overexpression of wtRII receptor in prostate cancer, which lacked RII expression, restored the inhibitory effect of TGF-β [14]. Therefore, we investigated if TGF-β RII could behave as an important tumor suppressor gene in human lung adenocarcinoma cells through retroviral transduction system.

In this study, we found an absence or reduction of RII mRNA in the NSCLC analyzed. RI mRNA expression was not reduced in the NSCLC. Furthermore, it was found that retroviral transduction of wild-type TGF-β RII in NCI-H358 adenocarcinoma, lacking in RII expression and resistant to TGF-β, restored sensitivity to TGF-β, significantly reduced the growth rate and changed the cell morphology. Conversely, it was also found that overexpression of dnRII in NCI-H441 adenocarcinoma, sensitive to TGF-β, blocked the inhibitory effect of TGF-β. These results indicate that tumor development in human NSCLC is associated with the abnormal expression of TGF-β RII.

Section snippets

Cell lines and cell culture

The thirteen NSCLC cell lines used in this study were obtained and cultured in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) and Penicillin/Streptomycin at 37°C in an atmosphere of 5% CO2.

Generation of RII expressing cell line

NCI-H358 and NCI-H441 cells (lung adenocarcinoma cell lines) were transduced with recombinant retrovirus produced from the PT67 packaging cell line, which was transfected with MFG-full length TGF-β RII or MFG-dominant-negative (devoid of cytoplasmic serine/threonine kinase domain) TGF-β RII vector

Reduced TGF-β RII expression in NSCLC

To investigate whether the expression of functional TGF-β RII and RI was associated with the growth inhibitory effect of TGF-β1, we examined the expression of each receptor in 13 NSCLC cell lines by Northern blot analysis. Fig. 1 shows the absence or reduction of RII mRNA in five NSCLC cell lines; NCI-H460, -H520, -H522, -H709 and -H358. The other five NSCLC cell lines expressed relatively high level of RII mRNA.

Growth suppression in NCI-H358 expressing wild-type TGF-β RII

NCI-H358, lacking in TGF-β RII and insensitive to TGF-β1, was transduced with

Discussion

Several studies have recently showed that tumor development, caused by a dysfunctional TGF-β signaling pathway, is attributed to either a loss or mutation of the TGF-β RII receptor [15], [16], [17], [18], [19], which implies that the loss of RII function is one of the mechanisms involved in regulating cell proliferation. We first examined 13 NSCLC for RI and RII mRNA expression. Five of 13 NSCLC cell lines showed loss or great reduction of RII expression but not of RI. These findings are

Acknowledgements

We thank Dr Seong-Jin Kim (National Cancer Institute, NIH, Bethesda, MD) for the gift of MFG-RII, MFG-dnRII vector, pGEX-4T3-RII and pGEX-4T3-RI.

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