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