Limb-Bud and Heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of functional TCF/LEF binding sites in the LBH locus and rapid beta-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/beta-catenin signaling is inhibited by Wnt7a, which in limb development signals through a non-canonical pathway involving Lmx1b. Furthermore, we show that Lbh is aberrantly overexpressed in mammary tumors of MMTV-Wnt1 transgenic mice and in aggressive basal-subtype human breast cancers that display Wnt/beta-catenin hyperactivation. Deregulation of LBH in human breast cancer appears to be Wnt/beta-catenin dependent as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. RNAi mediated knockdown of LBH in basal breast cancer cell lines resulted in loss of CD44high/CD24low tumor cells, luminal differentiation, reduced cell growth, reduced colony forming ability, and increased apoptosis, suggesting a novel pro-survival and stem cell maintenance function of LBH in breast cancer. Reciprocal overexpression studies in the basal breast carcinoma line BT549 resulted in increased tumorigenicity in vitro, suggesting that LBH overexpression is indeed oncogenic. Finally, we further characterized LBH protein expression patterns and post-transcriptional regulation. Collectively, this thesis demonstrates that LBH is a direct Wnt target gene in both development and basal breast cancer that promotes the undifferentiated phenotype and survival of basal breast tumor cells.

No description available.

No description available.

Gliomas are the most common human brain tumors and are divided into four stages by WHO classification scheme. Benign gliomas are defined as grades I (Pilocytic astrocytomas) and II (Astrocytomas), whereas grade III (Anaplastic Astrocytomas, AA) and grade IV (Glioblastoma Multiforme, GBM) are malignant. Although both grades III and IV are malignant, the prognoses for these tumors are quite different. The 2-year survival rate for grade III gliomas is 50%, and grade IV is < 20 %. Mechanisms of tumorigenesis are not exactly elucidated in brain tumor cells. The thesis is to study the role of p27 kip1 in human malignant brain tumors. The experimental methods include ribonuclease protection assay (RPA), western blotting, immunohistochemical staining and immunocytochemical staining. mRNAs of p130, p107, Rb, p53 and p27 kip1 in normal brain tissues and brain tumors were overexpressed in most case. The p27kip1 mRNA were expressed in all astrocytomas and GBM, and mRNA quantity of p27kip1 were more in brain tumors than in normal brain tissues. PI3K/Akt pathway regulates several cellular functions such as cell survival and cell proliferation. Active Akt can phosphorylate p27kip1 that may contribute cell cycle from G1 phase to S phase. Skp2 identifies phospho-p27kip1 and promotes p27kip1 degradation. We found p27kip1 overexpression and Akt activation in astrocytomas and GBM. The expression of p-Akt were found in 20 %, 87 % and 71 % in normal brain tissues, astrocytomas and GBM, respectively. Expression of p27kip1 and p-Akt has shown significant correlation in GBM (P = 0.0236). Overexpression of p27kip1 mRNA in brain tumors may be consequence of p-Akt and Skp2.

The growth and metastasis of solid tumors are dependent on angiogenesis. An endogenous angiogenesis inhibitor, vasostatin, is the proteolytic fragment derived from the N-terminal 180 residues of calreticulin. Previous studies indicated that vasostatin specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth. However, continuous administration of vasostatin is difficult and expensive to facilitate, thereby underscoring the need to develop gene delivery approach. Adenovirus vectors possess advantages for gene delivery including high titer, high infection efficiency and broad host range. The aim of the present study was to generate and characterize recombinant adenovirus vectors encoding vasostatin (Ad-VS) or Igk-fused vasostatin (Ad-Igk-VS), thereby to evaluate the efficacy of anti-angiogenesis gene therapy for tumor suppression. Recombinant Ad-VS and Ad-Igk-VS were generated and verified by PCR and western blot analysis. In addition, adenovirus encoding angiostatin was also produced as positive control for angiogenesis assays. Adenovirus-mediated vasostatin gene delivery specifically inhibited the proliferation of bovine aortic endothelial cells (BAEC), but not non-endothelial cells such as Hela or NIH3T3 cells. Moreover, vasostatin gene delivery potently inhibited the proliferation, migration and tube formation, but not secretion of matrix metalloproteinases (MMPs), in endothelial cells. Flow cytometry analysis indicated that vasostatin gene delivery induced apoptosis in BAEC. Using western blot analysis, it was revealed that gene delivery of vasostatin increased the levels of Fas and FADD in BAEC. In conclusion, adenovirus-mediated vasostatin gene delivery inhibited various angiogenesis processes at least via induction of Fas/FasL pathway and may hold potential for cancer therapy.

Cancer has become the first among the 10 major death causing factors in Taiwan. Glioblastoma multiforme (GBM) is the most common malignant tumor in adult human brain tumors. Previously, heat shock or all trans retinoic acid (ATRA) treatment has been shown to be effective in inducing cell apoptosis and cell cycle arrest in several cancer cell lines. In this study, human brain tumor cell line GBM8401 was exposed to 43¢J for 30 min followed by incubation with ATRA. The treatment resulted in up to 50% inhibition of cell growth rate and 50% reduction of cell survival rate . Analysis of cell apoptosis related gene expression and protein expression with RT-PCR and Western blot has showen that p21, p27, pro-caspase 3, phospho-JNK and phospho-p38 were overexpressed after treatment of tumor cells with 43¢J for 30 min followed by addition of ATRA for 15 min to 8 hr. The immunocytochemistry assay revealed that overexpression of phospho-p53 in the nuclei after tumor cells were treated with 43¢J for 30 min followed by addition of ATRA for 8 hr. Results from this study show that treating tumor cells with heat shock before incubation with ATRA will enhance cell apoptosis and inhibit cell growth.

No description available.

No description available.

Abstrate: Ras proto-oncogenes are members of the superfamily of GTP-binding proteins. Many tyrosine kinase receptors, including those for epidermal growth factor, insulin, and nerve growth factor, signal through RAS proteins. The product of members of this oncogene family (H-, K-, N-ras) is a 21 kD protein with nucleotide binding activity, involved in the transduction of information from the cell surface to the nucleus. The three RAS proteins exit in two states: a resting state in which they are bound to GDP and an active state in which they bind GTP. The most common form of mutational activation of Ras oncogenes in human tumors is through single base substations affecting either the GTP-binding of main (codons 12 and 13) or the GTPase domain (codon 61) of the protein. Thus, mutant RAS proteins result in constitutive activation of the downstream signaling cascade because their affinity for GTP is increased or their GTPase activity is decreased, so that the protein cannot return to the resting state. To investigate we have screened 89 thyroid tumor specimens, which include 8 follicular carcinomas (FC), 42 papillary carcinoma (PTC), 2 anaplastic carcinoma (AC),5 Hurthle cell adenoma (HA), 12 follicular adenoma (FA) and 20 nodular goiter (NG), for mutation in three Ras genes using PCR and automatic sequencing. Four tumors contained Ras gene mutation. Of these, three were identified among FC (37.5%), which mutation were in the codon 61 of each Ras genes. One mutation were at codon 61 of N-ras in FA specimens (8.3%). In addition, 33.7% (30/89) of specimens contain H-ras codon 27 polymorphism. In conclusion, our data indicated that the prevalence rates of Ras gene mutation were 5.8% and 2.7% in thyroid carcinoma and thyroid benign adenoma, respectively. Other environmental or genetic factors might also involved in the thyroid tumorigenesis and worth further investigation. The data were further confirmed using the combination of the PCR and denaturing gradient gel electrophoresis (DGGE). Four more cases of possible Ras mutation were detected which did not revealed by automatic sequencing , indicating that DGGE is a more sensitive method in detecting single nucleotide mutation. DGGE analysis should increase the detection rate of Ras gene mutation in our analysis.

bstract Cancer has been the first lethal factor in Taiwan, and hepatitis B is also a serious problem in our country. We have identified that the extracts of Taiwanese Schisandraceous plants, including Schizandra arisanensis, Kadsura matsudai and K. japonica, have inhibitory effect on type B hepatitis surface antigen (HBsAg) and e-antigen (HBeAg). Besides, we also find that the extracts of Echinops grijsii can inhibit the growth of human cancer cell line, KB and Hela. To purify active compounds by using column chromatography and high-performance liquid chromatography, we have furnished fifty-eight compounds from above Schisandraceous plants, including thirty-seven C18 lignans composed of [6.8.6]-dibenzocyclooctadiene skeleton, nine C19 homolignans, four triterpenes, three steroids and five cyclic-aromatic compounds. In the case of Echinops grijsii, we got thirteen major compounds containing eight thiophenes, four triterpenes, and a sterol. Structural elucidation of the novel homolignan and ten lignans are based on the spectral and chemical analyses, mainly by using two-dimensional nuclear magnetic resonance (NMR) of 1H and 13C nucleus. From the anti-HBsAg and anti-HBeAg assay, we found that four of new C18 lignans and one known (+)-gomisin K3 (33) lignan exhibited the significant inhibitory effects on surface antigen of hepatitis B virus. Moreover, to modify the known lignans, kadsurarin with different halogens and functional group having nitrogen atom, as well as (+)-gomisin K3 with several kinds of phenyl compounds and sulfur functional groups are processed. The preliminary structures and bioactivity relationships (SAR) studies demonstrated that (+)-gomisin K3 with sulfuric functional group could decrease cytotoxicity and increase inhibitory effect on surface antigen and e-antigen of hepatitis B virus. In the part of studying synthesis by using inactive 5¡¦-(but-3-en-1-ynyl)-2,2¡¦ -bithiophene (1) from Echinops grijsii, the yne-ene moiety of its structure was hydrolyzed into carbonyl group and then was attached a serious of various carbon number of hydroxy groups. Besides the above experiment, £\-trithienyl (1) was served as a substrate and made it into unsaturated carbonyl group, which then was linked with a series of hydroxy groups. Cytotoxicity datum showed that the derivatives of bi-thiophene have better anticancer activity than derivatives of tri-thiophene. And the assay results also exhibited that the bi-thiophene derivatives with hydroxy group with less than three carbon numbers have better inhibitory activity against cancer cells.