BACKGROUND: The role of Smad4 in carcinogenesis is important, because of its function as a central mediator of TGF-beta signaling. In the present study we analyzed the expressions of Smad4 mRNA and protein in human gastric cancer cell lines and tissues and we also analyzed their clinicopathological significance. METHODS: We used semi-quantitative RT-PCR for Smad4 mRNA expression in 13 cases of fresh gastric cancer tissues and two gastric cancer cell lines (MKN-28, SNU-1). We also used immunohistochemistry for Smad4 protein expression in 88 cases of formalin fixed gastric cancers tissues. RESULTS: The mRNA level of Smad4 was higher in MKN-28 cell line (intestinal type) than in the SNU-1 cell line (diffuse type). Fresh frozen gastric cancer tissues showed that the intestinal type of gastric cancer had higher Smad4 mRNA expressions than the diffuse type of gastric cancer (p<0.05). Immunohistochemical staining for Smad4 revealed that cytoplasmic and nuclear expressions of Smad4 were significantly correlated with histologic types of gastric cancer (p<0.05). That is, the intestinal type of gastric cancer showed more cytoplasmic and nuclear smad4 expressions than did the diffuse type of gastric cancer. Reduced cytoplasmic expressions and positive nuclear expressions of Smad4 were more prominent in the advanced gastric cancer than in the early gastric cancer. CONCLUSION: Taken together, we suggest that loss of Smad4 expression might be associated with the intestinal type of gastric cancer. Also reduced cytoplasmic Smad4 expressions and increased nuclear Smad4 expressions may be associated with the advanced stage of gastric cancer.
Adenocarcinoma* ; Carcinogenesis ; Cell Line ; Cytoplasm ; Formaldehyde ; Humans ; Immunohistochemistry ; RNA, Messenger ; Smad4 Protein ; Stomach Neoplasms ; Transforming Growth Factor beta

INTRODUCTION: A cleft palate is a common birth defect in humans with an incidence of 1/500 to 1/1,000 births. It appears to be caused by multiple genetic and environmental factors during palatogenesis. Many molecules are involved in palate formation but the biological mechanisms underlying the normal palate formation and cleft palate are unclear. Accumulating evidence suggests that transforming growth factor beta/bone morphogenetic proteins (TGF-beta/BMP) family members mediate the epithelial-mesenchymal interactions during palate formation. However, their roles in palatal morphogenesis are not completely understood. MATERIALS AND METHODS: To understand the roles of TGF-beta/BMP signaling in vivo during palatogenesis, mice with a palatal mesenchyme-specific deletion of Smad4, a key intracellular mediator of TGF-beta/BMP signaling, were generated and analyzed using the Osr2Ires-Cre mice. RESULTS: The mutant mice were alive at the time of birth with open eyelids and complete cleft palate but died within 24 hours after birth. In skeletal preparation, the horizontal processes of the palatine bones in mutants were not formed and resulted in a complete cleft palate. At E13.5, the palatal shelves of the mutants were growing as normally as those of theirwild type littermates. However, the palatal shelves of the mutants were not elevated at E14.5 in contrast to the elevated palatal shelves of the wild type mice. At E15.5, the palatal shelves of the mutants were elevated over the tongue but did not come in contact with each other, resulting in a cleft palate. CONCLUSION: These results suggest that mesenchymal Smad4 mediated signaling is essential for the growth of palatal processes and suggests that TGF-beta/BMP family members are essential regulators during palate development.
Animals ; Cleft Palate ; Congenital Abnormalities ; Eyelids ; Humans ; Incidence ; Mice ; Morphogenesis ; Palate ; Parturition ; Proteins ; Smad4 Protein ; Tongue ; Transforming Growth Factors

OBJECTIVETo clarify the relationship between the loss of expression of the deleted in pancreatic carcinoma locus 4 (DPC4) proteins and the pathogenesis of biliary tract carcinoma.

METHODS71 primary biliary tract carcinoma (BTCa), including 38 common bile duct (CBD) carcinomas, 18 gallbladder carcinomas, 15 hilar bile ducts (HBD) carcinomas were examined by immunohistochemical staining. In addition, the CBD carcinomas were divided into two groups: tumors with metastasis (M(+) group, 27 cases) and tumors without metastasis (M(-) group, 11 cases).

RESULTSThe frequency of loss of the expression of DPC4 protein was 32.8% in BTCa, 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, 13% in HBD carcinoma. Comparison of the frequency of loss expression of DPC4 was significant statistical difference in CBD carcinoma versus gallbladder carcinoma and HBD carcinoma (P < 0.01). The frequency of loss expression of DPC4 was 48.1% in the M(+) group and 45.4% in the M(-) group.

CONCLUSIONThere are a close relationship between pathogenesis of BTCa and inactivation of DPC4 and different frequencies of DPC4 gene alternation in various locations of the biliary tract, which are not significantly increased with tumor metastasis in BTCa.

Bile Duct Neoplasms ; Biliary Tract ; Carcinoma ; DNA-Binding Proteins ; metabolism ; Humans ; Pancreatic Neoplasms ; Smad4 Protein ; Trans-Activators

It was recently discovered that a subset of osteoblasts functions as a key component of the hematopoietic stem cells (HSC) niche in vivo, controlling HSC self-renewal and multi-lineage differentiation. Disruption of Smad4 gene specifically in osteoblasts leads to a remarkable decrease of osteoblast number and endosteal surface area. In order to elucidate if the osteoblast loss has any effect on hematopoietic activity, the bone marrow (BM) and extramedullary hematopoiesis in the osteoblast-specific Smad4 knockout mice were systematically analyzed, the proportions of mature hematocytes in BM, liver and spleen were detected by flow cytometry, the hematopoietic progenitor number in different stages was measured by colong-forming assay, CFU-S and analysis of LSK cells. The results indicated that the conditional mutant mice demonstrated normal BM hematopoiesis without sign of extramedullary hematopoiesis. Furthermore, the proportion of hematopoietic progenitor cells was normal, while cell number/body weight of the conditional knockout mice increased. It is concluded that hematopoiesis is normally maintained in osteoblast-specific Smad4 knockout mice, and osteoblast loss does not of necessity result in the decrease in BM hematopoiesis.
Animals ; Bone Marrow Cells ; cytology ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; physiology ; Mice ; Mice, Knockout ; Osteoblasts ; cytology ; physiology ; Smad4 Protein ; genetics

The effect of melatonin on juveniles with cardio fibrosis is poorly understood. We investigated whether HDACs participate in the anti-fibrotic processes regulated by melatonin during hypertrophic remodeling. Abdominal aortic constriction (AAC) was employed in juvenile rats resulting in pressure overload-induced ventricular hypertrophy and melatonin was subsequently decreased via continuous light exposure for 5 weeks after surgery. AAC rats displayed an increased cross-sectional area of myocardial fibers and significantly elevated collagen deposition compared to sham-operated rats, as measured by HE and Masson Trichrome staining. Continuous light exposure following surgery exacerbated the increase in the cross-sectional area of myocardial fibers. The expression of HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 genes were all significantly enhanced in AAC rats with light exposure relative to the other rats. Moreover, the protein level of TNF-α was also upregulated in the AAC light exposure groups when compared with the sham. However, Smad4 protein expression was unchanged in the juveniles' hearts. In contrast, beginning 5 weeks after the operation, the AAC rats were treated with melatonin (10 mg/kg, intraperitoneal injection every evening) or vehicle 4 weeks, and sham rats were given vehicle. The changes in the histological measures of cardio fibrosis and the gene expressions of HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 were attenuated by melatonin administration. The results reveal that melatonin plays a role in the development of cardio fibrosis and the expression of HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 in cardiomyocytes.
Animals ; Collagen ; Constriction ; Fibrosis* ; Gene Expression ; Heart ; Histone Deacetylases ; Hypertrophy ; Injections, Intraperitoneal ; Melatonin* ; Myocytes, Cardiac ; Rats* ; Smad4 Protein

BACKGROUNDTransforming growth factor beta (TGF-beta) plays an essential role in the regulation of normal physiologic processes of cells. TGF-beta has been shown to regulate several mitogen-activated protein kinases (MAPK) pathways in several epithelial cells. However, the effects of TGF-beta on soft tissue sarcoma are seldom reported. Our previous studies suggested that there should be some other signal transduction pathways besides Smads, which are important to regulate the growth of human embryonal rhabdomyosarcoma (RMS) cells. In the present study, we examined the expression and functional relations of extracellular signal-regulated kinase 2 (ERK2) and Smad4 in human RMS tissue and a RMS cell line, RD.

METHODSRD cells and normal human primary skeletal myoblasts (Mb) were treated with TGF-beta1 to establish the expression profile of ERK2 at the mRNA and protein levels detected by RT-PCR and immunofluorescence. Immunohistochemistry was used to detect the expression of ERK2 and Smad4 in 50 tissue specimens of human RMS and 23 specimens of normal skeletal muscles. Follow-up of specimens was performed 6 months to 70 months later.

RESULTSRD cells and human RMS tissues showed the higher expression of ERK2 and Smad4 than the normal control, either the protein level or the mRNA level. And, exogenous TGF-beta1 stimulation can lead to higher expression of ERK2 and its nuclear translocation, so TGF-beta1 can also activated MAPK (ERK2) pathway, resulting in a sustained activation of ERK2 for at least 2 hours. Immunohistochemistry analysis, however, showed that there was no correlation between ERK2 and Smad4 protein. The overexpression of ERK2 and Smad4 had no indicative effects on histological subtypes, histological grading, gender, age, and prognosis.

CONCLUSIONSIn RMS, signaling of TGF-beta1 from cell surface to nucleus can also be directed through the MAPK (ERK2) pathway besides the TGF-beta1/Smads pathway. The activation of ERK2 by TGF-beta1 may be Smad4 independent. Moreover, there may be some other tanglesome relationships between the TGF-beta1/Smads pathway and the MAPK pathway which takes part in the development, invasion and metastasis of tumor cells.

Cells, Cultured ; Humans ; Mitogen-Activated Protein Kinase 1 ; physiology ; Muscle, Skeletal ; metabolism ; RNA, Messenger ; analysis ; Rhabdomyosarcoma ; metabolism ; Signal Transduction ; Smad4 Protein ; physiology ; Transforming Growth Factor beta1 ; pharmacology

PURPOSE: Chromosomal instability of chromosome 18 and inhibition of the transforming growth factor beta (TGF-beta) signaling pathway, which is mediated through Smad4, play important roles in the tumorigenesis of colon cancer. This study evaluated the value of the expression of chromosome 18 monosomy in colon cancer as a prognostic factor and its correlations with the expressions of Smad4 and TGF-beta receptor II proteins. METHODS: We analyzed the rate of the expression of chromosome 18 monosomy in 66 colon cancers with using chromogenic in situ hybridization (CISH) and we evaluated its value as a prognostic factor by determining its correlation with the pathologic factors and immunohistochemical expressions of Smad4 and TGF-beta receptor II proteins. RESULTS: Of the 66 colon cancers, monosomy of chromosome 18, as determined by CISH, was observed in 18 cases (27.3%), and the decreased expression of Smad4 and TGF-beta receptor II proteins was observed in 30 cases (45.5%) and 25 cases (37.9%), respectively. The monosomy of chromosome 18 and the decreased expression of Smad4 proteins showed statistically significant correlations with the histologic differentiation, the presence of tumor emboli, the nodal status and the stage. The decreased expression of TGF-beta receptor II proteins had statistically significant correlations with the histologic differentiation, the T-stage, the nodal status and the stage. The monosomy of chromosome 18 showed a statistically significant correlation with the decreased expression of Smad4 and TGF-beta receptor II proteins. CONCLUSION: These results suggested that chromosome 18 monosomy may have prognostic value for colon cancer.
Carcinogenesis ; Chromosomal Instability ; Chromosomes, Human, Pair 18* ; Colon* ; Colonic Neoplasms* ; In Situ Hybridization ; Monosomy* ; Receptors, Transforming Growth Factor beta* ; Smad4 Protein ; Transforming Growth Factor beta*

OBJECTIVEto explore whether 6 tagging single nucleotide polymorphisms (SNPs) within SMAD4 gene were involved in the genetic susceptibility of coal worker's pneumoconiosis (CWP) by case-control study.

METHODSthis study consisted of 438 CWP patients and 448 controls. All study subjects were Han Chinese, underground coal miners and recruited from coal mines of Xuzhou Mining Business Group Co Ltd. The 5 ml venous blood sample was obtained from all studied subjects and extracted genome DNA from the isolated leucocytes. Six SNPs were selected from the HapMap and detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

RESULTSthe single SNP analyses showed that the genotype frequencies of SMAD4 (rs10502913) was significantly different from those in controls (P < 0.05). Multivariate logistic regression analyses revealed that SMAD4 (rs10502913) AA genotype was associated with increased risk of CWP (adjusted OR = 1.63, 95%CI = 1.00 - 2.69, P = 0.05) and this was evident among subgroups of those smoker (adjusted OR = 2.28, 95%CI = 1.09 ∼ 4.80, P < 0.05) and cases with stage I (adjusted OR = 2.42, 95%CI = 1.41 ∼ 4.14, P < 0.01). The SMAD4 (rs9304407) GG genotype was associated with an decreased risk of CWP (adjusted OR = 0.65, 95%CI = 0.43 ∼ 0.98, P < 0.05) and the further stratification analysis showed that the risk of CWP was decreased in nonsmoking groups.

CONCLUSIONSour results suggest that individuals with the SMAD4 (rs10502913) AA genotype was associated with an increased risk of CWP. However, carriers of SMAD4 (rs9304407) GG genotype have a protective effect on the developing CWP.

Aged ; Anthracosis ; genetics ; Case-Control Studies ; Coal Mining ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Smad4 Protein ; genetics

OBJECTIVETo investigate the effects of the plasma containing Qianlongtong Capsule (QLT)-containing plasma on the expression of the Smad4 gene in prostate stromal cells in vitro and provide some experimental evidence for the treatment of benign prostatic hyperplasia (BPH) with Chinese medicinal compound.

METHODSFifteen cases of BPH were equally randomized to three groups to be treated with QLT at a high dose (6 capsules once), a medium dose (3 capsules once), and a low dose (1.5 capsules once), tid, for 7 days consecutively. QLT-containing plasma was collected from the patients. Prostate stromal cells were identified by immunofluorescence when they became monolayered and cultured in the QLT-containing plasma for 24 hours, followed by detection of the expression of the Smad4 gene by real-time quantitative PCR and that of the Smad4 protein by Western blot.

RESULTSAfter treatment with the QLT-containing plasma, the expression of the Smad4 gene in the stromal cells was significantly increased in a dose-dependent manner as compared with the blank control and no-QLT groups (P < 0.01). The expression of the Smad4 protein was also markedly elevated after treatment. The differences were statistically significant between the blank control and medium-dose groups (P < 0.01), low-dose and medium-dose groups (P < 0.05), and high-dose and the other groups (P < 0.01), but not between the blank control and low-dose groups (P > 0.05).

CONCLUSIONQLT-containing plasma could inhibit the proliferation and improve the apoptotic index of prostate stromal cells in vitro, which was related to the elevation of the mRNA and protein expressions of Smad4.

Apoptosis ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Male ; Prostate ; drug effects ; metabolism ; RNA, Messenger ; genetics ; Smad4 Protein ; genetics ; metabolism ; Stromal Cells ; drug effects ; metabolism

OBJECTIVETo observe the expression of Smad4, the core of TGF-beta/Smads signal transduction pathway in different prostate cancer cell lines, and explore their molecular mechanism of bone metastatic potential.

METHODSThe Millicell polycarbonate filter coated with matrigel was used to confirm the invasive potency of LNCaP and ARCaP cell lines (IF11 and IA8). The expressions of the Smad4 protein and mRNA in these prostate cancer cells with different metastatic potentials were detected by Western blotting and RT-PCR, respectively.

RESULTSARCaP cell lines (IF11 and IA8) exhibited a stronger potency of invasion than LNCaP (P < 0.01). The Smad4 protein and mRNA highly expressed in the LNCaP cell line that was well-known with a low metastatic potential, but not in the ARCaP (IF11 or IA8) cells with high metastatic potentials (P < 0.01).

CONCLUSIONSmad4 expresses differently in LNCaP and ARCaP cell lines with different metastatic potentials and, as a tumor suppressive gene, its deficient expression may play an important role in the invasion and metastasis of advanced prostate cancer.

Cell Line, Tumor ; Humans ; Male ; Neoplasm Metastasis ; Prostatic Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics ; Smad4 Protein ; biosynthesis ; Transforming Growth Factor beta ; metabolism