Association between endogenous gene expression and growth regulation induced by TGF-beta1 in human gastric cancer cells.
OBJECTIVE
To investigate the association between endogenous gene expression and growth regulation including proliferation and apoptosis induced by transforming growth factor-beta1 (TGF-beta1) in human gastric cancer (GC) cells.


METHODS
Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the main components of the TGF-beta1/Smads signal pathway in human poorly differentiated GC cell line BGC-823. Localization of Smad proteins was also determined using immunofluorescence. Then, the BGC-823 cells were cultured in the presence or absence of TGF-beta1 (10 ng/mL) for 24 and 48 h, and the effects of TGF-beta1 on proliferation and apoptosis were measured by cell growth curve and flow cytometry (FCM) analysis. The ultrastructural features of BGC-823 cells with or without TGF-beta1 treatment were observed under transmission electron microscope. The apoptotic cells were visualized by means of the terminal deoxynucleotidyl transferase (TdT)-mediated dTUP in situ nick end-labeling (TUNEL) method. Meanwhile, the expression levels of endogenous p15, p21 and Smad7 mRNA and the corresponding proteins in the cells were detected at 1, 2 and 3 h after culture in the presence or absence of TGF-beta1 (10 ng/mL) by semi-quantitative RT-PCR and Western blot, respectively.


RESULTS
The TGF-beta1/Smad signaling was found to be intact and functional in BGC-823 cells. The growth curve revealed the most evident inhibition of cell proliferation by TGF-beta1 at 48 h, and FCM assay showed G1 arrest accompanied with apoptosis induced by TGF-beta1. The typical morphological changes of apoptosis were observed in cells exposed to TGF-beta1. The apoptosis index (AI) in TGF-beta1-treated cells was significantly higher than that in the untreated controls (10.7+/-1.3% vs 0.32+/-0.06%, P<0.01). The levels of p15, p21 and Smad7 mRNA and corresponding proteins in cells were significantly up-regulated at 1 h, but gradually returned to basal levels at 3 h following TGF-beta1 (10 ng/mL) treatment.


CONCLUSIONS
TGF-beta1 affects both proliferation and apoptosis of GC cells through the regulation of p15 and p21, and induces transient expression of Smad 7 as a negative feedback modulation of TGF-beta1 signal. Our results suggest a novel functional role of p21 as an accelerant of TGF-beta1-mediated apoptosis in GC cells.
Li
Xue
X
Laboratory of Medical Genetics, Harbin Medical University, Harbin 150086, Heilongjiang Province, China.
Zhang
Yun-Yan
YY
Wang
Qi
Q
Fu
Song-Bin
SB
D000230:Adenocarcinoma / Q000235:genetics*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D050762:Cyclin-Dependent Kinase Inhibitor p15; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051906:Smad7 Protein; D013274:Stomach Neoplasms / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
C493956:CDKN1A protein, human; C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; C497806:SMAD7 protein, human; D051906:Smad7 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins