Biomedical subjects

Tong-Chuan He

Publications and source records attributed to Tong-Chuan He.

29 records · Page 2Linked to original sources

PubMed2003

Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling.

Bone formation is presumably a complex and well-orchestrated process of osteoblast lineage-specific differentiation. As members of the TGFbeta superfamily, bone morphogenetic proteins (BMPs) play an important role in regulating osteoblast differentiation and subsequent bone formation. Several BMPs are able to induce de novo bone formation. Although significant progress has recently been made about the transcriptional control of osteoblast differentiation, detailed molecular events underlying the osteogenic process remain to be elucidated. In order to identify potentially important signaling mediators activated by osteogenic BMPs but not by non-osteogenic BMPs, we sought to determine the transcriptional differences between three osteogenic BMPs (i.e., BMP-2, BMP-6, and BMP-9) and two inhibitory/non-osteogenic BMPs (i.e., BMP-3 and BMP-12). Through the microarray analysis of approximately 12,000 genes in pre-osteoblast progenitor cells, we found that expression level of 203 genes (105 up-regulated and 98 down-regulated) was altered >2-fold upon osteogenic BMP stimulation. Gene ontology analysis revealed that osteogenic BMPs, but not inhibitory/non-osteogenic BMPs, activate genes involved in the proliferation of pre-osteoblast progenitor cells towards osteoblastic differentiation, and simultaneously inhibit myoblast-specific gene expression. BMP-regulated expression of the selected target genes was confirmed by RT-PCR, as well as by the CodeLink Bioarray analysis. Our findings are consistent with the notion that osteogenesis and myogenesis are two divergent processes. Further functional characterization of these downstream target genes should provide important insights into the molecular mechanisms behind BMP-mediated bone formation.

Adenoviridae↗

PubMed2003

Myoid differentiation and prognosis in adult pleomorphic sarcomas of the extremity: an analysis of 92 cases.

BACKGROUND: The results of a recent study demonstrated an association between myoid differentiation and an adverse prognosis in adult patients with pleomorphic sarcoma, as determined by 5-year metastasis-free survival rates. METHODS: To confirm the importance of muscle differentiation on prognosis in a well controlled clinical context, 92 samples from patients with pleomorphic sarcoma of the extremity from a single institution were immunostained with 4 monoclonal antibodies believed to be correlated with myoid differentiation: alpha-smooth muscle actin, muscle-specific actin, desmin, and myoglobin. RESULTS: Forty-two cases were positive for at least 1 muscle marker and 50 cases were uniformly negative. Between the two groups, there was no significant difference in tumor size, tumor extent, or patient age found; however, histologic grade was significantly higher (P = 0.038) in the myoid tumors. The 5-year survival differed significantly between patients with myoid tumors (35%) and those without myoid tumors (65%) (P = 0.0054). Myoid differentiation remained an adverse prognostic indicator after adjusting for clinically significant factors (i.e., histologic grade, tumor size, tumor extent, and patient age) (P = 0.01) (hazard ratio, 2.39; 95% confidence interval, 1.24-4.63). Furthermore, there was an inverse relation found between the number of myoid markers present and survival (P = 0.004). CONCLUSIONS: Myoid differentiation was found to be an independent indicator of adverse prognosis in adult patients with pleomorphic spindle cell sarcoma of the extremity.

Actins↗

PubMed2003

[Potent and specific inhibition of hepatitis B virus antigen expression by RNA interference].

OBJECTIVE: To develop an RNAi approach that specifically targets the core gene sequence of hepatitis B virus by synthesizing short interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on HBV replication. METHODS: The eukaryotic expression plasmid pHBV1.3, which contains 1.3-fold-overlength genome of HBV, were cotransfected into HepG2 cells with either the RNAi plasmid pSIHBV/C or unrelated control plasmid pSI. At 24, 48, 72 hours post transfection (p.t.), the levels of HBsAg and HBeAg in the cell culture medium were determined with Abbott MEIA Kits. The expression of intracellular viral proteins was also determined by immunofluorescence staining. RESULTS: Successfully constructed expressing siRNA vector pSIHBV which targeted the core gene of Hepatitis B virus.The introduction of RNAi plasmid was shown to efficiently and specifically inhibit the synthesis of surface and e antigen of HBV by Abbot MEIA kits, with inhibitory rates at 92%, 85% peaking at 24 hours p.t. Immunofluorescence staining showed that intracellular synthesis of viral antigen was sharply reduced to nearly background levels when the ratio of pSIHBV/C and pHBV1.3 was at 1:20, whereas the control vector did not exhibit any inhibitory effect on the replication and expression of HBV. CONCLUSION: Our results demonstrate that the short interfering RNA targeting HBV core gene exerts robust inhibition on HBV viral replication, suggesting that RNAi-based anti-HBV replication strategy may represent a potentially efficacious approach to the clinical management of HBV infection.

Fluorescent Antibody Technique↗

PubMed2003

[The expression of Recombinant adenovirus IkappaBalphaM in human hepatocarcinoma HepG2 and it's inhibitive effect to the activity of NF-kappaB].

OBJECTIVE: To investigate the expression of recombinant IkappaBalphaM (AdIkappaBalphaM) in human hepatocarcinoma cell line HepG(2) and the inhibiting effect to NF-kappaB. METHODS: To test the virus titer in 293 cells and the infective efficiency virus titer in HepG(2) cells with GFP and limited dilution method, then to assay the IkappaBalphaM expression of recombinant adenovirus and the alteration after induction of TNF-alpha in 293 cells and HepG(2) by Western blotting, furthermore, to observe NF-kappaB activity in HepG(2) before and after treatment of TNF-alpha by EMSA. RESULTS: The titer of AdIkappaBalphaM is 2 x 10(8) pfu/L, MOI equals to 20. AdIkappaBalphaM could be expressed stably and efficiently in HepG(2) and will not degrade by induction of TNF-alpha; but IkappaBalpha in the uninfection cell as well as AdIkappaBalpha control was increased at first and then decreased. EMSA demonstrated that the infected cells showed no activation of NF-kappaB before and after the treatment of TNF-alpha, but the cells of uninfected and infected with AdIkappaBalpha appeared excessive activation of NF-kappaB. CONCLUSION: AdIkappaBalphaM could be amplified in 293 cell and effectively infect to target cells HepG(2), and could be expressed stably in cells, and wouldn't be degrade with treatment of TNF-alpha, also it can effectually inhibit the excessive activation of NF-kappaB in HepG(2). The result of our research indicates the theoretical value of IkappaBalphaM as a super inhibitor, inhibition activity of NF-kappaB with IkappaBalphaM super-suppressor aided with routine anti-tumor therapy would become an effective method.

Adenoviridae↗

PubMed2003

Tyrosine kinase inhibitor STI-571/Gleevec down-regulates the beta-catenin signaling activity.

Beta-Catenin is a critical transducer of the Wnt signal pathway and plays an important role in many developmental and cellular processes. Deregulation of beta-catenin signaling has been observed in a broad range of human tumors. In this report, we investigated whether tyrosine kinase inhibitor STI-571 could inhibit the beta-catenin signaling activity and hence suppress cell proliferation. Our results demonstrated that STI-571 effectively inhibited the constitutive activity of beta-catenin signaling in human colon cancer cells as well as the Wnt1-induced activation of beta-catenin signaling in HOS, HTB-94, and HEK 293 cells. Furthermore, STI-571 was shown to effectively suppress the proliferation of human colon cancer cells. Finally, we demonstrated that the Wnt1-mediated activation of a GAL4-beta-catenin heterologous transcription system was effectively inhibited by STI-571. Thus, our findings suggest that tyrosine phosphorylation may play an important role in regulating beta-catenin signaling activity, and inhibition of this signaling pathway by STI-571 may be further explored as an important target for alternative/adjuvant treatments for a broader range of human cancer.

Adenoviridae↗

PubMed2003

Potential use of Sox9 gene therapy for intervertebral degenerative disc disease.

STUDY DESIGN: A new recombinant adenoviral vector expressing Sox9, a chondrocyte-specific transcription factor, was tested in a chondroblastic cell line and primary human intervertebral disc cells in vitro. Direct infection of intervertebral disc cells then was assessed in a rabbit model. OBJECTIVES: To deliver a potentially therapeutic viral vector expressing Sox9 to degenerative human and rabbit intervertebral discs cells, and to assess the effect of Sox9 expression on Type 2 collagen production. SUMMARY OF THE BACKGROUND DATA: The concentration of competent Type 2 collagen, an essential constituent of the healthy nucleus pulposus, declines with intervertebral disc degeneration. Recent studies suggest that Sox9 upregulates Type 2 collagen production. Interventions that augment Type 2 collagen production by intervertebral disc cells may represent a novel therapeutic method for patients with degenerative disc disease. METHODS: Adenoviral delivery vectors expressing Sox9 and green fluorescent protein were constructed using the AdEasy system. The chondroblastic cell line, HTB-94, and cultured human degenerated intervertebral disc cells were infected with the vectors. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses were performed to document increased Type 2 collagen expression. The AdSox9 virus then was injected directly into the intervertebral discs of three rabbits. After 5 weeks, the injected discs were evaluated histologically. RESULTS: The AdSox9 virus efficiently transduced HTB-94 cells and degenerated human disc cells. Western blot analysis confirmed increased Sox9 production. Increased Type 2 collagen production was demonstrated in infected HTB-94 and human disc cells using both reverse transcriptase-polymerase chain reaction and immunohistochemical staining. In the rabbit model, cells infected with AdSox9 maintained a chondrocytic phenotype, and the architecture of the nucleus pulposus was preserved over a 5-week study period compared to control discs. CONCLUSIONS: A novel adenoviral vector efficiently increased Sox9 and Type 2 collagen synthesis in cultured chondroblastic cells and human degenerated disc cells. In a rabbit model, sustained Sox9 production preserved the histologic appearance of the nucleus pulposus cells in vivo. These findings suggest a potential role for Sox9 gene therapy in the treatment of human degenerative disc disease.

Adenoviridae↗

PubMed2003

Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs).

BACKGROUND: Bone morphogenic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway to evaluate the ability of certain BMPs to promote fracture-healing and spinal fusion. The optimal BMPs to be used in different clinical applications have not been elucidated, and a comprehensive evaluation of the relative osteogenic activity of different BMPs is lacking. METHODS: To identify the BMPs that may possess the most osteoinductive activity, we analyzed the osteogenic activity of BMPs in mesenchymal progenitor and osteoblastic cells. Recombinant adenoviruses expressing fourteen human BMPs (BMP-2 to BMP-15) were constructed to infect pluripotent mesenchymal progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, and osteoblastic TE-85 cells. Osteogenic activity was determined by measuring the induction of alkaline phosphatase, osteocalcin, and matrix mineralization upon BMP stimulation. RESULTS: BMP-2, 6, and 9 significantly induced alkaline phosphatase activity in pluripotential C3H10T1/2 cells, while BMP-2, 4, 6, 7, and 9 significantly induced alkaline phosphatase activity in preosteoblastic C2C12 cells. In TE-85 osteoblastic cells, most BMPs (except BMP-3 and 12) were able to induce alkaline phosphatase activity. The results of alkaline phosphatase histochemical staining assays were consistent with those of alkaline phosphatase colorimetric assays. Furthermore, BMP-2, 6, and 9 (as well as BMP-4 and, to a lesser extent, BMP-7) significantly induced osteocalcin expression in C3H10T1/2 cells. In C2C12 cells, osteocalcin expression was strongly induced by BMP-2, 4, 6, 7, and 9. Mineralized nodules were readily detected in C3H10T1/2 cells infected with BMP-2, 6, and 9 (and, to a lesser extent, those infected with BMP-4 and 7). CONCLUSIONS: A comprehensive analysis of the osteogenic activity of fourteen types of BMPs in osteoblastic progenitor cells was conducted. Our results suggest an osteogenic hierarchical model in which BMP-2, 6, and 9 may play an important role in inducing osteoblast differentiation of mesenchymal stem cells. In contrast, most BMPs are able to stimulate osteogenesis in mature osteoblasts.

Adenoviridae↗

PubMed2002

Cytoplasmic and/or nuclear accumulation of the beta-catenin protein is a frequent event in human osteosarcoma.

The molecular events that precede the development of osteosarcoma, the most common primary malignancy of bone, are unclear, and concurrent molecular and genetic alterations associated with its pathogenesis have yet to be identified. Recent studies suggest that activation of beta-catenin signaling may play an important role in human tumorigenesis. To investigate the potential role of beta-catenin deregulation in human osteosarcoma, we analyzed a panel of 47 osteosarcoma samples for beta-catenin accumulation using immunohistochemistry. Potential activating mutations were investigated by sequencing exon 3 of the beta-catenin gene in genomic DNA isolated from tumor samples. Our findings revealed cytoplasmic and/or nuclear accumulation of beta-catenin in 33 of 47 samples (70.2%); however, mutation analysis failed to detect any genetic alterations within exon 3, suggesting that other regulatory mechanisms may play an important role in activating beta-catenin signaling in osteosarcoma. In our survival analysis, beta-catenin deregulation conferred a hazard ratio of 1.05, indicating that beta-catenin accumulation does not appear to be of prognostic value for osteosarcoma patients. When analyzed against other clinicopathologic parameters, beta-catenin accumulation correlated only with younger age at presentation (26.4 vs. 39.8 years). Nevertheless, our results demonstrate that the deregulation of beta-catenin signaling is a common occurrence in osteosarcoma that is implicated in the pathogenesis of osteosarcoma.

Adult↗

PubMed2002

Elevated protein expression of cyclin D1 and Fra-1 but decreased expression of c-Myc in human colorectal adenocarcinomas overexpressing beta-catenin.

Mutations of the adenomatous polyposis coli tumor suppressor gene, or its downstream target beta-catenin, have been implicated in the initiation of most sporadic human colorectal epithelial neoplasms. These mutations, in turn, lead to aberrant nuclear accumulation of beta-catenin and subsequent activation of the beta-catenin/Tcf transcription factor complex. In vitro studies utilizing cultured human colon cancer cell lines have identified c-myc, cyclin D1 and fra-1 as target genes of beta-catenin/Tcf signaling. In our study, 12 cases of human colorectal adenocarcinomas were examined by Western immunoblotting analysis and immunohistochemical staining to specifically investigate whether the protein expression of these target genes was indeed altered in vivo by beta-catenin dysregulation. The results show that the protein level of beta-catenin was significantly increased in all 12 tumors (3.4 +/- 1.0-fold increase compared to the control normal mucosa by Western immunoblotting, p < 0.05), and this increase was associated with positive nuclear staining by immunohistochemistry in 10 cases. Increased levels of expression of cyclin D1 and Fra-1 proteins were also demonstrated in every tumor (9.0 +/- 2.7 and 3.3 +/- 0.9-fold increases compared to normal mucosa, respectively). Surprisingly, the protein level of c-Myc was significantly decreased in all tumors examined by 49 +/- 19% (p < 0.05), but the c-myc mRNA level was increased in 8 of 12 tumors when compared to that in normal mucosa by RT-PCR. Immunohistochemical staining performed on these carcinomas and additional 27 colorectal carcinomas further demonstrated that the protein expression level of c-Myc and beta-catenin nuclear localization were not correlated. Moreover, 15 of 20 colorectal adenomas exhibited positive nuclear beta-catenin immunostaining, among which 11 also exhibited increased c-Myc protein expression. These data thus support the notion that upregulation of cyclin D1 and Fra-1 in human colorectal adenocarcinomas is driven by abnormally expressed beta-catenin. However, the regulation of c-myc expression in colorectal tumors appears to be more complex. While dysregulated beta-catenin may cause a transcriptional upregulation of the c-myc gene, the c-Myc protein expression appears to be further regulated by a posttranscriptional mechanism(s) during the process of neoplastic progression.

Adenocarcinoma↗

PubMed2002

Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma.

PURPOSE: This study was designed to investigate whether nuclear receptor agonists can be used as potential differentiation therapy agents for human osteosarcoma. EXPERIMENTAL DESIGN: Four osteosarcoma cell lines (143B, MNNG/HOS, MG-63, and TE-85) were treated with proliferator-activated receptor (PPAR)gamma agonists, troglitazone and ciglitazone, and a retinoid X receptor (RXR) ligand, 9-cis retinoic acid. The proliferation and induction of apoptosis in the treated cells were assessed, as was the induction of alkaline phosphatase, a differentiation marker of osteoblasts. RESULTS: The expression of PPARgamma was readily detected in all tested osteosarcoma lines. On treatment with the PPARgamma and RXR ligands, all four osteosarcoma lines exhibited a significantly reduced proliferation rate and cell viability. Among the four lines, 143B and MNNG/HOS were shown to be more sensitive to ligand-induced apoptosis, as demonstrated by the Crystal Violet and Hoechst staining assays. Of the three tested ligands, troglitazone was shown to be the most effective in inducing cell death, followed by 9-cis retinoic acid. Moreover, a strong synergistic effect on the induction of cell death was observed when both troglitazone and 9-cis retinoic acid or ciglitazone and 9-cis retinoic acid were administered to osteosarcoma cells. Troglitazone was shown to effectively induce alkaline phosphatase activity, a well-characterized hallmark for osteoblastic differentiation. CONCLUSIONS: Our findings suggest that PPARgamma and/or RXR ligands may be used as efficacious adjuvant therapeutic agents for primary osteosarcoma, as well as potential chemopreventive agents for preventing the recurrence and metastasis of osteosarcoma after the surgical removal of the primary tumors.

Alitretinoin↗

PubMedDate not supplied

Gene therapy for spinal fusion.

Spinal fusion will continue to be an important part of the surgical treatment of spinal pathology for the foreseeable future. Traditional challenges to successful spinal fusion surgery include autograft donor site morbidity and pseudoarthrosis. Recent advances in the understanding of the biology of bone formation have allowed the development of therapeutic biologics. Although recombinant bone morphogenetic proteins delivered to the arthrodesis site will stimulate fusion, these proteins have been less successful in more challenging fusion situations (posterolateral), require supraphysiologic doses to promote fusion in humans, and are quite expensive. Gene therapy may represent the easiest method for the application of bone-forming biologic agents to promote spinal fusion. Both in vivo and ex vivo techniques of delivery of therapeutic genes have been used effectively to promote fusion in lower animals. Considerable research is required to identify gene therapy techniques and vectors with acceptable safety profiles and high fusion rates.

Animals↗