Ad/CMV-hTGF-β1 treats rabbit intervertebral discs degeneration<Emphasis Type="Italic">in vivo</Emphasis>

Summary  To investigate therapeutic efficiency of Ad/CMV-hTGF-β1 gene for rabbit intervertebral dise degeneration model. 60 Japanese white rabbits were selected to form the L5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral dise degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 μl (10×10⁶ pfu) of Ad CMV-hTGF-β1 gene, constituted the therapy group, 12 were injected with 20 μl (10×10⁶ pfu) of Ad CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group, 3 weeks after injection, examples were taken for investigation of HE staining, MRI. Western Blotting and immunohistochemical research TGF-β1. Wide distribution of TGF-β1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-β1 content in intervertebral dises treated with TGF-β1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral dise pathological degree improved. Ad CMV-hTGF-β1 gene transfection is a potential method to increase TGF-β1 content and reverse intervertebral dise degeneration. ZHAN Zirui, male, born in 1970 Associate Professor The Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education P.R.C. (No. [2001]345).     

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure-activity relationships of the 4-aryl group

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC(50) of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI(50) values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI(50) value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.     

Biomedical informatics: development of a comprehensive data warehouse for clinical and genomic breast cancer research

The Windber Research Institute is an integrated high-throughput research center employing clinical, genomic and proteomic platforms to produce terabyte levels of data. We use biomedical informatics technologies to integrate all of these operations. This report includes information on a multi-year, multi-phase hybrid data warehouse project currently under development in the Institute. The purpose of the warehouse is to host the terabyte-level of internal experimentally generated data as well as data from public sources. We have previously reported on the phase I development, which integrated limited internal data sources and selected public databases. Currently, we are completing phase II development, which integrates our internal automated data sources and develops visualization tools to query across these data types. This paper summarizes our clinical and experimental operations, the data warehouse development, and the challenges we have faced. In phase III we plan to federate additional manual internal and public data sources and then to develop and adapt more data analysis and mining tools. We expect that the final implementation of the data warehouse will greatly facilitate biomedical informatics research.     

活血补气方对胎鼠宫内发育迟缓的防治效果及其机制

目的:探讨活血补气方(丹参、川芎、当归、黄芩和黄芪)对胎鼠宫内发育迟缓(IUGR)的防治效果及其机制,为该方剂的临床应用提供实验依据.方法:被动吸烟法建立孕鼠IUGR妊娠模型.IUGR妊娠鼠被随机分为4组(每组10例),即中药高、中、低剂量干预组和未干预组,每天分别给予活血补气方汤药3、1、0.3 ml/100g和生理盐水1 ml/100 g.另选10只正常妊娠鼠作为正常对照组,每天给予生理盐水1 ml/100g.于妊娠第21天处死孕鼠并剖腹取胎,测定胎鼠体重、鼻-臀长,并计算胎仔重量系数.分别用硝酸还原酶法、放射免疫分析法(RIA)和酶免疫法(EIA)测定妊娠晚期孕鼠血NO、ET-1含量和尿8-epi-PGF2α含量.结果:与未干预组比较,各干预组的胎鼠体重、鼻-臀长及胎仔重量系数均明显增加,差异具有显著性意义(均为P＜0.01);干预组的孕鼠血NO水平升高,血ET-1水平降低,NO/ET-1比值升高,尿8-epi-PGF2α水平降低,差异具有显著性意义(均为P＜0.01).结论:IUGR妊娠时,孕鼠体内脂质过氧化作用增强,并存在着NO/ET-1失衡,可导致胎鼠IUGR的发生;活血补气方具有抗氧化的功能,能抑制IUGR妊娠的脂质过氧化作用和调节NO/ET-1平衡,对胎鼠IUGR具有较好的防治效果.     

CC chemokine ligand 25 enhances resistance to apoptosis in CD4+ T cells from patients with T-cell lineage acute and chronic lymphocytic leukemia by means of livin activation

We investigated CD4 and CD8 double-positive thymocytes, CD4(+) T cells from typical patients with T-cell lineage acute lymphocytic leukemia (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of induction of resistance to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. We found that CCL25 selectively enhanced resistance to TNF-alpha-mediated apoptosis in T-ALL and T-CLL CD4(+) T cells as well as in MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. One member protein of the inhibitor of apoptosis protein (IAP) family, Livin, was selectively expressed in the malignant cells at higher levels, particularly in T-ALL CD4(+) T cells, in comparison with the expression in CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 and apoptotic induction with TNF-alpha, the expression levels of Livin in these malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) ligation, selectively activated Livin to enhance resistance to TNF-alpha-mediated apoptosis in c-jun-NH(2)-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in distinct types of cells. CD4 and CD8 double-positive thymocytes used CCR9/CCL25 for migration, homing, development, maturation, selection, cell homeostasis, whereas malignant cells, particularly T-ALL CD4(+) T cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation.     

NF-kappaB activity blockade impairs the angiogenic potential of human pancreatic cancer cells

The effect of blockade of NF-kappaB activity on human pancreatic cancer angiogenesis was determined in an orthotopic xenograft model. Highly metastatic L3.3 human pancreatic cancer cells, which expressed an elevated level of constitutive NF-kappaB activity, were transfected with a mutated IkappaBalpha (IkappaBalphaM). After implantation in the pancreas of nude mice, parental (L3.3) and control vector-transfected (L3.3-Neo) cells produced rapidly growing tumors and liver metastases, whereas IkappaBalphaM-transfected (L3.3-IkappaBalphaM) cells had decreased tumorigenicity and metastatic potential. NF-kappaB signaling blockade significantly inhibited the in vitro and in vivo expression of the major proangiogenic molecules vascular endothelial growth factor and interleukin-8 and decreased tumor vascular formation. These events were correlated with retarded tumor growth and suppression of metastasis. Collectively, these data suggest that suppression of tumorigenicity and metastasis by NF-kappaB blockade is due to impaired angiogenic potential of tumor cells.     

Treatment of colorectal cancer metastasis: The role of chemotherapy

5-Fluorouracil (5-FU) has been the main chemotherapeutic agent for the treatment of colorectal cancer for four decades with modest efficacy. Modulation of 5-FU by leucovorin or continuous infusion improves the response rate, but overall survival duration remains approximately 12 months. Many oral fluoropyrimidines have been studied, including capecitabine, UFT, S-1, and Eniluracil. Capecitabine has demonstrated equivalent efficacy with 5-FU and has been approved as first line treatment. The combinations of capecitabine with CPT-11 or oxaliplatin are being developed. CPT-11 demonstrated non-crossover resistance with 5-FU and was proven to be effective treatment for patients who received prior 5-FU. CPT-11 in combination with 5-FU has demonstrated improved response rate and overall survival duration over 5-FU or CPT-11. Oxaliplatin plus 5-FU has offered another effective treatment option for colorectal cancer. Both 5-FU plus leucovorin in combination with CPT-11 or oxaliplatin are widely used first-line chemotherapies for advanced colorectal cancer. Optimal combinations and sequences of treatment are being studied, since several effective regimens have become available.     

Discovery of Ca2+-relevant and differentiation-associated genes downregulated in esophageal squamous cell carcinoma using cDNA microarray

To identify genes that are differentially expressed in human esophageal squamous cell carcinoma (ESCC), we have developed a cDNA microarray representing 34 176 clones to analyse gene expression profiles in ESCC. A total of 77 genes (including 31 novel genes) were downregulated, and 15 genes (including one novel gene) were upregulated in cancer tissues compared with their normal counterparts. Immunohistochemistry and Northern blot analysis were carried out to verify the cDNA microarray results. It was revealed that genes involved in squamous cell differentiation were coordinately downregulated, including annexin I, small proline-rich proteins (SPRRs), calcium-binding S100 proteins (S100A8, S100A9), transglutaminase (TGM3), cytokeratins (KRT4, KRT13), gut-enriched Krupple-like factor (GKLF) and cystatin A. Interestingly, most of the downregulated genes encoded Ca(2+)-binding or -modulating proteins that constitute the cell envelope (CE). Moreover, genes associated with invasion or proliferation were upregulated, including genes such as fibronectin, secreted protein acidic and rich in cystein (SPARC), cathepsin B and KRT17. Functional analysis of the alteration in the expression of GKLF suggested that GKLF might be able to regulate the expression of SPRR1A, SPRR2A and KRT4 in ESCC. This study provides new insights into the role of squamous cell differentiation-associated genes in ESCC initiation and progression.