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Biomedical subjects

Lin Hou

Publications and source records attributed to Lin Hou.

At least 19 recordsLinked to original sources
PubMed2007

[Effect of siRNA transfection targeting VEGF gene on proliferation and apoptosis of human breast cancer cells].

AIM: To explore the effect of RNA interference-mediated vascular endothelial growth factor (VEGF) gene silencing on proliferation and apoptosis of human breast cancer MCF-7 cells. METHODS: Small interfering RNA (siRNA) targeting VEGF gene was designed and DNA template was synthesized, then siRNA was obtained by in vitro transcription. After ds-siRNA was transfected into MCF-7 cells with Lipofectamine, the proliferation of MCF-7 cells was detected by MTT colorimetry, and the apoptosis was measured by Hoechst33258 staining. The change of cell cycle was analyzed by flow cytometry (FCM). The expression of VEGF on mRNA and protein level was analyzed by RT-PCR and immunohistochemical technology respectively. RESULTS: The siRNA targeting human VEGF gene effectively inhibited the proliferation of MCF-7 cells, induced cell apoptosis, arrested the cell cycle in G(0)/G(1) phase, and decreased the expression of VEGF on both mRNA and protein level. But these effects did not appear in scrambled siRNA (siRNA(SCR)) control group. CONCLUSION: The siRNA targeting human VEGF gene could reduce VEGF expression, inhibit cellular proliferation and induce apoptosis in MCF-7 cells in vitro.

Apoptosis↗
PubMed2006

DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation.

NAT10 (N-acetyltransferase 10) is a protein with histone acetylation activity and primarily identified to be involved in regulation of telomerase activity. The presented research shows its transcriptional activation by genotoxic agents and possible role in DNA damage. NAT10 mRNA could be markedly increased by using hydrogen peroxide (H2O2) or cisplatin in a dose- and time-dependent way, and the immunofluorescent staining revealed that the treatment of H2O2 or cisplatin induced focal accumulation of NAT10 protein in cellular nuclei. Both H2O2 and cisplatin could stimulate the transcriptional activity of the NAT10 promoter through the upstream sequences from -615 bp to +110 bp, with which some nuclear proteins interacted. Ectopic expression of NAT10 could enhance the number of survival cells in the presence of H2O2 or cisplatin. The above results suggested that NAT10 could be involved in DNA damage response and increased cellular resistance to genotoxicity.

Acetyltransferases↗
PubMed2006

Enhanced expression of halp gene confers cellular resistance to H2O2 induced senescence.

OBJECTIVE: To investigate the H2O2-induced expression of human histone acetyltransferase-like protein (hALP), a telomerase regulation-associated gene, and its effects on the stress-triggered cellular senescence. METHODS: The induced expression of hALP was measured by semi-quantitative RT-PCR and immunofluorescent histochemistry after treatment of HeLa cells by H2O2. The effects of hALP expression on cellular responses to H2O2 were analyzed by MTT, flowcytometry, and SA-beta-gal staining, respectively. RESULTS: hALP mRNA could be dose-dependently induced by treatments of 0.2-1.6 mmol/L H2O2, and the induction could be observed after 6 hours and kept for 36 hours in the presence of 0.4 mmol/L H2O2. Meanwhile, the immunofluorescent staining showed marked stronger nuclear intensity of hALP protein in H2O2-treated HeLa cells. In the treatment of H2O2, the ectopic expression of hALP enhanced continuous growth and overcame G2/M arrest as well as decreased senescence-associated beta-gal staining. On the contrary, the transfected clones with antisense or blank vector and original He-La cells presented growth suppression, G2/M delay and higher percentage of SA-beta-gal activities in the presence of H2O2. CONCLUSIONS: The expression of hALP could be up-regulated by treatment of H2O2, and elevated expression could enhance cellular resistance to H2O2-induced cellular senescence. The data might be of references to elucidation of basic biological function of hALP gene and its associated telomerase activity.

Asparaginase↗
PubMed2006

[Inhibitory effect of p53 gene on telomerase activity and proliferative activity in salivary adenoid cystic carcinoma cells].

OBJECTIVE: To evaluate the inhibitory effect of p53 gene on salivary adenoid cystic carcinoma (SACC) cells. METHODS: Adenoviral vector pDeltaE1-p53 was constructed and transfected into SACC-83 cells. The enhanced p53 expression was measured by reverse transcription polymerase chain reaction (RT-PCR), and the effects of transfected p53 on SACC-83 cells were analyzed by TRAP-PCR-ELISA, luciferase reporter, flow cytometry (FCM), soft agar assay and tumorigenicity test. RESULTS: The expression of p53 gene in SACC-83 cells was increased after introduction of pDeltaE1-p53. The telomerase activity and the transcriptional activity of hTERT promoter were inhibited. The cells cycles of transfected SACC-83 were arrested in G(1) phase and the rate of colony-formation was decreased, and similarly the tumorigenicity in nude mice was also decreased. CONCLUSIONS: The introduction of wild-type p53 by adenoviral vector could suppress the telomerase activity and malignant phenotypes of salivary adenoid cystic carcinoma cells.

Animals↗
PubMed2005

[Expression of SARS-CoV in various types of cells in lung tissues].

OBJECTIVE: To investigate the cell types infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in lung tissues and explore the mechanism of lung injury in SARS. METHODS: In-situ hybridization(ISH) and immunohistochemistry(IHC) double staining was applied to study the lung tissues from 7 SARS cases of Beijing and one of Anhui province. According to SARS-CoV genome sequence, the cDNA probe was synthesized and labelled by digoxin. Immunohistochemically, antibodies of cytokeratin(CK), CD34, CD68, Vimentin and CD3 were applied to demonstrate bronchial epithelial cells, type II pneumocytes, endothelial cells, macrophages, fibroblasts and T cells respectively. RESULTS: The positive results of in-situ hybridization showed that the lung tissues of all cases expressed SARS-CoV RNA, and positive signals displayed in cytoplasms (purple-blue, NBP-BCIP. ISH-IHC double staining showed that positive signals of both ISH (purple-blue NBT-BCIP and IHC (red-brown, AEC expressed in the cytoplasms (purple and red). The positive results of double staining indicated that bronchial epithelial cells, type II pneumocytes, endothelial cells, macrophages, fibroblasts and T lymphocytes were diffusely infected by SARS-CoV. CONCLUSION: This study of ISH-IHC double staining in lung tissues of SARS patients showed that bronchial epithelial cells, type II pneumocytes, endothelial cells, macrophages, T lymphocytes and fibroblasts were attacked diffusely in SARS lungs. Various types of cells damaged by SARS-CoV and inflammatory mediators released by those cells play an important role in the pathogenesis of lung injury in SARS.

Adult↗
PubMed2005

Transcriptional upregulation of DNA polymerase beta by TEIF.

The overexpression of DNA polymerase beta (beta-pol) has been identified in lots of human cancers, but the mechanism has seldom been investigated. Telomerase transcriptional element-interacting factor (TEIF) can bind to hTERT promoter, stimulating its transcription and telomerase activities. Here, we report that TEIF could also enhance the expression of beta-pol at transcription level. TEIF could specifically activate transcription of beta-pol promoter, but not that of DNA polymerase alpha or delta promoter. The responsible sequences for binding of TEIF were revealed as GC-rich elements dispersing from +19 to -29 nt of beta-pol promoter, which due to mutations caused decreasing in binding of TEIF and apparent losing of transactivation activity. The in vivo interaction between TEIF and beta-pol promoter was identified by chromatin immunoprecipitation assay. Besides, ectopic expression of TEIF in HeLa cells could upregulate both levels of endogenous beta-pol mRNA and protein, and consequently increases resistance to the oxidative stress of H2O2. The data may provide new clue to the elucidation of beta-pol overexpression in cancers and also a functional link between beta-pol and telomerase.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Severe acute respiratory syndrome associated coronavirus is detected in intestinal tissues of fatal cases.

OBJECTIVES: A significant percentage of confirmed severe acute respiratory syndrome (SARS) patients experienced gastrointestinal symptoms, and the viral sequence was detectable in the stool of most patients. At present, the knowledge of the pathology of the digestive system in SARS patients is limited. Because a resurgence of the SARS epidemic is constantly possible, there is an urgent need to understand the involvement of the digestive system in this new disease. METHODS: We performed seven SARS autopsies in which samples of alimentary tract and digestive glands were examined with routine pathology, electron microscopy (EM), in situ hybridization (ISH), immunohistochemistry, and real-time polymerase chain reaction (PCR). RESULTS: The main histopathological finding was atrophy of the mucosal lymphoid tissue. A few mucosal epithelial cells and lymphocytes in the intestine were positively stained for coronavirus with ISH. SARS-coronavirus (CoV)-like particles were found in the mucosal epithelial cells under EM and mild focal inflammation was detected in the alimentary tract. One patient who experienced severe diarrhea had pseudomembranous enteritis of the ileum. Fatty degeneration and central lobular necrosis were observed in the liver. No evidence of direct viral infection was found in the esophagus, the stomach, the salivary gland, the liver, or the pancreas. CONCLUSIONS: In addition to the lungs, the gastrointestinal tract is another target of SARS-CoV infection, as the intestinal epithelial cells and mucosal lymphoid tissue are infected. The findings provide possible explanations for the gastrointestinal symptoms and the presence of virus in the stool of SARS patients.

Adult↗
PubMed2005

Expression of a telomerase-associated gene in normal, atrophic, and tumorous testes.

OBJECTIVE: To evaluate the expression of telomerase transcriptional elements-interacting factor (TEIF) in human testis under different status and its relation with human telomerase reverse transcriptase (hTERT) expression. METHODS: Specific antisera against TEIF were generated by immunization of rabbits with purified recombinated partial TEIF. Samples were assigned to three groups according to their pathological types, including 16 normal testes, 8 atrophic testes, and 6 testicular seminomas. They were subjected to immunohistochemical staining of TEIF and hTERT. Results from both TEIF and hTERT were analyzed semi-quantitatively and compared. RESULTS: The expressions of TEIF and hTERT were detected in all samples of normal, atrophic testes, and seminomas. No differences of TEIF expressions among these three groups were observed (P > 0.05). On the contrary, the expressions of hTERT were significantly lower in atrophic testes compared with those of normal testes and seminomas (both P < 0.05). Nevertheless, co-expressions of TEIF with hTERT were revealed to be in normal and malignant cases (P < 0.05) but not in atrophic testes, which generally presented TEIF expression. The cellular distributions of both proteins were similar and mainly in spermatocytes and some Sertoli cells, while were all negative in the interstitial cells and other stromal cells. Conclusions The uniform expressions of TEIF in all these specimens suggest that it may be a marker of testis and its related diseases. The strong expression of hTERT in normal testes and testicular seminomas comparing with the low expression in atrophic testes may suggest a role for telomerase in maintaining proliferation of germ cells.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

[An analysis of induced expression and function of telomerase-regulation associated hALP gene on genotoxic agents].

OBJECTIVE: To confirm the responses and function of hALP, a telomerase-regulation associated gene, in DNA damage. METHODS: HeLa and Hep2 cells were treated by genotoxic agents H2O2 and cisplatin, and the induced expression of hALP was measured by quantitative RT-PCR and immunofluorescent histochemistry. The alterations in transcriptional activity of hALP promoter were estimated by luciferase reporter assays. The effects of genotoxic agents on cells in different status of hALP expression were analyzed by MTT method. RESULTS: The level of hALP mRNA could be increased when treated by 0.2 - 1.6 mmol/L H2O2 and reach a peak in concentration of 0.4 mmol/L. The induction could be observed after 6 h in the treatment of 0.4 mmol/L H2O2 and the higher level can be retained for 36 h. Similarly, cisplatin induced hALP mRNA expression is also dose and time dependent. The immunofluorescent staining showed that the treatment of 0.2 or 0.4 mmol/L H2O2, 0.2 or 0.5 micromol/L cisplatin increased the intensity of hALP protein in cellular nuclei. The luciferase assays demonstrated that both H2O2 and cisplatin could up-regulate hALP promoter activity through its upstream - 705 - +20 nt region. In cell survivor assay, the HeLa cells expressing sense hALP gene could grow continuously in the presence of 0.4 mmol/L H2O2 or 0. 5 micromol/L cisplatin while cells with antisense hALP or control cells were slower in growth. CONCLUSIONS: The expression of hALP gene could be up-regulated by DNA damage through activating transcription of its promoter, and increase cellular resistance to genotoxic agents.

Antineoplastic Agents↗
PubMed2004

Molecular cloning and characterization of a human gene involved in transcriptional regulation of hTERT.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase in human, has been identified as the rate-limiting factor in telomerase activity, but its detailed mechanism of transcription regulation remains unclear. In this investigation, a novel human gene telomerase transcriptional elements-interacting factor (TEIF) was isolated from HeLa cell based on hTERT promoter yeast one-hybrid assay. TEIF has a 2358-bp open reading frame encoding a predicted protein of 786 amino acids, which is functionally conserved in general eukaryotic species. The transcription of TEIF was detected in cells and expressed an 86-kDa native protein, distributing mainly in cellular nuclei. Its interaction with hTERT promoter was identified and the DNA binding potential was credited to C-terminus of TEIF. Transfection of TEIF into HeLa cells presented marked transactivation of hTERT promoter and stimulated both endogenous hTERT transcription and telomerase activities. These results suggest that TEIF protein might be a transcription regulator and take part in the activation of hTERT.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

The evidences of human orphan receptor COUP-TFII inhibiting telomerase activity through decreasing hTERT transcription.

Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells, but the details of regulation of hTERT need to be elucidated. By screening HeLa cDNA library with hTERT promoter-based yeast one-hybrid assay, one of positive clones, which potentially interacted with hTERT promoter, contained the full sequences of COUP-TFII cDNA. EMSA showed that the prepared His-tagged COUP-TFII could firmly bind to -201 to +35 fragment of hTERT promoter. The precise binding sites were confirmed by DNase I footprint analysis and proved to involve the E-box motif of hTERT promoter. Luciferase reporter assays indicated that COUP-TFII could suppress the transcription of hTERT promoter, and the inhibition to some extent could be reversed by co-transfection of c-myc. Stable introduction of COUP-TFII into HeLa cells decreased both endogenous hTERT expression and telomerase activity. The results suggested that the human COUP-TFII could specifically interact with hTERT promoter, in part via the E-box, and suppress the expression of hTERT.

Biological Assay↗
PubMed2004

Development of single-domain recombinant antibodies to reverse transcriptase domain of human hTERT.

This paper describes the development of single-domain recombinant antibodies against human telomerase core protein. A His-tagged hTERT spanning main reverse-transcriptase domain of hTERT was purified from host E. coli and used to immunize BALB/c mice. The VHs (heavy chain variable region genes) were amplified by PCR from total RNA of splenocytes and further induced random mutagenesis by DNA shuffling to enrich the repertoire of VH library. All VHs were cloned into phagemid vectors and displayed to generate 4 x 10(10) phage libraries. The candidates carrying VH domains against hTERT were primarily screened through three times of panning procedure on His-tagged hTERT coated microplates, and specific antibodies were further selected by West-Western blot. Two clones, designated as a3 and b8, were confirmed to interact with the target in the solid-phase assay. DNA sequencing proved their mouse VH origin. The purified single-domain antibody of b8 could not only recognize native hTERT, but also neutralize human telomerase activity on inhibitory assay and b8 showed the stronger suppressive efficacy compared with a3. The data demonstrated that the developed single-domain recombinant antibodies were hTERT-specific with high potential of binding and activity inhibition.

Amino Acid Sequence↗
PubMed2004

[Expression of pRb and E2F-1 and telomerase activity in ameloblastoma].

OBJECTIVE: To investigate the expression of pRb and E2F-1, and the association between their expression and the activity of telomerase (hTERT) or cyclin E in human ameloblastoma (AB), and to explore the clinical biological characteristics of AB. METHODS: The expressions of pRb, E2F-1, cyclin E and hTERT mRNA in human AB were detected by in situ hybridization or immunohistochemistry (SP method). RESULTS: The positive expression ratio of pRb in the cell nucleus of AB was 20.4% (11/54). The positive ratio of E2F-1, cyclin E and hTERT mRNA was 92.6% (50/54), 66.7% (36/54) and 94.4% (51/54), respectively. With AB recurrence and malignant transformation, the expression of hTERT, E2F-1, cyclin E was up-regulated. hTERT and cyclin E or E2F-1 mRNA had high positive relation (Spearsman'r(s) = 1.000, P = 0.0001). CONCLUSIONS: The regulatory pathway of Rb/E2F-1 is associated with the cell proliferation and in differentiation of AB. The activity or release of telomerase may be related to the lower expression of Rb and higher expression of E2F-1, and is up-regulated in G(1) late phase by cyclin E.

Adolescent↗
PubMed2004

Inhibitory role of transcription factor COUP-TFII in expression of hTERT in HeLa cells.

OBJECTIVE: To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity. METHODS: The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA. RESULTS: COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity. CONCLUSION: The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.

COUP Transcription Factor II↗
PubMed2004

[Research on the expression of c-myc mRNA in ameloblastoma].

PURPOSE: To study the expression of c-myc mRNA in ameloblastoma (AB) and odontogenic keratocyst (OKC) and to investigate the genesis, development and biological characteristics of AB and OKC. METHODS: We detected c-myc mRNA in 54 cases of AB (31 primary ABs, 19 recurrent Abs and 4 malignant ABs), 16 cases of OKC and 7 cases of normal oral mucosa (NOM) by in situ hybridization. Chi2 test was used to analyze the results. RESULTS: The positive rate of AB, OKC and NOM was 81.5% (44/54), 75.0% (12/16) and 14.3% (1/7), respectively. There was statistical difference among three groups (chi2=15.488, P<0.05). Comparing the expression of primary AB (71.0%) with recurrent (94.7%), malignant AB (100.0%), there was a significant statistical difference (chi2=16.912, P<0.05). CONCLUSIONS: The expression of c-myc is important to the genesis and development of AB; following the change of primary, recurrent and malignant AB, the expression of c-myc mRNA increased significantly.It is suggested that c-myc mRNA may be an effective marker to evaluate the prognosis of AB.

Ameloblastoma↗
PubMed2004

[The gene flow of population genetic structure].

The difference of population genetic structure is one of the important embodiments of genetic diversity. There is a long history of the study of population genetic structure, and the study of gene flow of population genetic structure is aroused more and more importance. It has an important effect on population genetics, evolution biology, conservation biology and ecology. Although the level of gene flow is estimated by traditional population genetics, there is a large restriction in its precision. With the development of biological technology, the methods of the research on gene flow reach the molecular level. Methods of protein electrophoresis and molecular markers (RAPD, RFLP, VNTR, ISSR and mitochondrial DNA) are used to research gene flow among populations. This paper introduces not only some models of population genetic structure: Continent-Island Model, Island Model, Stepping-Stone Model, Isolation-By-Distance Model and Hierarchical Model; but also the study methods, function and role of gene flow is based on models of population genetic structure, research achievements in recent years and the restriction of the methods.

Alleles↗
PubMed2004

[Expression of telomerase activity and c-myc and stimulatory protein 1 in human ameloblastoma].

OBJECTIVE: To study the oncogene transcriptor c-myc, stimulatory protein 1 (SP1) expression in ameloblastoma (AB) and their relation with telomerase reverse transcripase (hTERT), and to investigate the clinical biological characteristics of AB. METHODS: The expression was observed in AB by in situ hybridization and SP method. RESULTS: The positive rates of c-myc mRNA, hTERT mRNA and SP1 protein were 81.5% (44/54), 94.4% (51/54) and 83.3% (45/54), respectively. Their positive rates increased as AB recurred and transformed malignantly. A strong correlation was found between hTERT and c-myc, hTERT and SP1 (rs = 0.853, P < 0.001; rs = 0.900, P < 0.001). CONCLUSION: Activity of telomerase plays an important role in the tumorigenesis development of AB. Increasing of hTERT expression may be related to c-myc and SP1. The expression of these three parameters has a significant correlation with the clinical biological characteristics of AB.

Ameloblastoma↗