Dlk is a transmembrane protein that possesses six epidermal growth factor-like sequences at the extracellular domain, a single transmembrane domain and an intracellular tail. The extracellular EFG-like region of Dlk can be released by action of an unknown protease that cuts the extracellular region near the cell membrane. Dlk belongs to the EGF-like homeotic protein family and has received many names: pG2, FA-1, Pref-1, SCP-1, ZOG and Dlk. All the proteins are identical or polymorphic products of a single gene. Dlk has been involved in several differentiation processes, such as adipogenesis, hematopoiesis and neuroendocrine differentiation. Dlk is also known as the preadipocyte factor-1 (Pref-1), is highly expressed in preadipocytes but is completely abolished in adipocytes. Pref-1 may function in the maintenance of the preadipocyte state and is a negative regulator of adipocyte differentiation. Dlk is expressed in tumors with neuroendocrine features, such as human neuroblastoma, rat pheochromocytoma, and a subset of Small Cell Lung Cancer (SCLC) cell lines. The Dlk expression is probably associated with some differentiation stages because the most undifferentiated cells were lacking expression of Dlk. The finding suggests that Dlk plays an important role in differentiation and tumorigenesis of several cell types. The study was designed to examine the influence of dlk overexpression on tumorigenicity of hepatoma cells. We constructed the mammalian expression vectors for full-length dlk, dlk extracellular domain, which were transfected into SK-Hep-1 cells for generation of stable clones. The transgene expressions in selected stable clones were verified by QRT-PCR and western blot analysis. Our results indicated that overexpression of extracellular domain significantly promoted the viability of SK-Hep1 cells during serum deprivation. In SCID mice, injection of full-length dlk clones led to increased tumor growth compared with the control groups. However, the migration ability was reduced in Dlk stable clones. In summary, these results suggested full-length Dlk promoted the tumor growth but reduced the migration ability of SK-Hep1 cells.

Hepatocellular carcinoma (HCC) is one of the most common cancerous diseases worldwide. The annual occurrences exceed one million peoples affected. Currently, the treatment modalities for HCC include surgical resection, trans-arterial embolization (TAE) and chemotherapy. However, these modalities are not completely effective, underscoring the need for development of novel therapeutic approaches. PTEN, a tumor suppressor that antagonizes the PI3K pathway, is frequently mutated or deleted in various human cancers. Loss of PTEN occurs in 40-50% of surgical resected HCC samples and predicts poor prognosis for HCC patients, suggesting PTEN restoration may constitute a treatment alternative for HCC. Since PTEN increased ethanol-induced cytotoxicity in hepatoma cells, PTEN gene delivery may serve as an adjuvant therapy in conjunction with ethanol TAE for HCC. In the present study, we evaluated the efficacy of PTEN gene therapy and its combination with ethanol in a syngenic Novikoff hepatoma model by implantation of N1-S1 cells into livers of Sprague Dawley rats. Adenovirus encoding PTEN (Ad-PTEN) or green fluorescent protein (Ad-GFP) was generated for gene delivery studies. The optimal condition for adenovirus vectors to infect N1-S1 cells was determined at multiplicity of infection (MOI) of 100-200. Infection of N1-S1 cells with Ad-PTEN, but not Ad-GFP, increased PTEN levels and led to 40-50% inhibition of cell proliferation via cell cycle arrest. Besides, the half maximal -inhibitory concentration (IC50) for ethanol in N1-S1 cells was determined at 6%. Combination with PTEN gene delivery further augmented the cytotoxicity of ethanol in N1-S1 cells from 40% to 70% inhibition. To evaluate the prevention efficacy of PTEN gene delivery, N1-S1 cells were infected with adenovirus vectors then implanted into livers of Sprague-Dawley rats to induce Novikoff hepatoma. Injection of PBS- or Ad-GFP-treated N1-S1 cells led to large hepatoma (with an average size of 3-4 cm) with tumor incidence of 80-90%. In contrast, injection of Ad-PTEN-infected N1-S1 cells only induced one hepatoma (with size of 0.1 cm) in six rats, suggesting that pretreatment with PTEN gene delivery effectively abolished the tumorigenic potential of N1-S1 hepatoma cells in vivo. In summary, these results validate the feasibility of PTEN gene delivery as a new promising therapeutic strategy for the treatment of orthotopic hepatoma in immune-competent rats.

Hepatocellular carcinoma (HCC) is the most common and devastating malignant tumor in Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC have been explored in recent years. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. HDGF (hepatoma-derived growth factor) is a novel growth factor, identified from conditioned medium of hepatoma cell line. HDGF has growth stimulating activity for fibroblast and some hepatoma cells. The high homology of protein sequence to HMG (high mobility group) protein but with distinct structure indicate it is a novel growth factor with mitogenic effect. Recently, elevated HDGF expression was found in developing kidneys but little in adult kidney. Besides, HDGF expression was found to be correlated with angiogenic status of tissues. Thus, it is speculated that HDGF plays a role during embryonic development and angiogenesis. Besides, HDGF also plays a role in cell-to-cell interaction and cell movement. HDGF is a growth factor that is involved in stimulating vascular smooth muscle cells (SMCs) proliferation during development and in disease. HDGF contains a true bipartite nuclear localization sequence necessary for nuclear targeting. It is required for HDGF stimulation of DNA replication and cell proliferation in vascular smooth muscle cell. In present studies, have transfected HDGF cDNA to 3T3 cell and the HDGF expression was verified by Western blot analysis. HDGF expression altered the morphologies and growth rate of 3T3 cells by 2-fold. Besides, HDGF-expressing cells were more resistant to serum-starvation. Injection of 3T3-HDGF cells, but not 3T3 cells, resulted in tumor formation in nude mice, suggesting that the angiogenic and mitogenic functions of HDGF might contribute to carcinogenesis. By using various reagents including H2O2, dexamethasome, taxol, cisplatin, CoCl2 and KCN, the cellular stress studies revealed differential responses between in 3T3 and 3T3-HDGFH. analyzed dose and time-dependent effects of UV irradiation and found that 3T3-HDGF cells are more sensitive to UV irradiation than 3T3 cells and susceptible to apoptosis. hope these experiments will bring further insights into the cellular function of HDGF, particularly during angiogenic process, thereby enable to evaluate its role during HCC progression and its potential as clinical marker and therapeutic target for HCC.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2004. / Vita. Includes bibliographical references (leaves 181-227).

Thesis (M. Phil.)--University of Hong Kong, 1993.

Thesis (M.S.)--Eastern Illinois University, 2009. / Includes bibliographical references (leaves 66-72).

Apie vėžį yra žinoma jau daug ir įvairios informacijos, taip pat netrūksta žinių ir kokiais būdais bei vaistais galima kovoti su šia liga. Farmacijoje yra atrasta ir sukurta nemažai aktyvių antivėžinių chemoterapinių preparatų vėžiui gydyti ar skirtų bent šios ligos slopinimui ir gyvenimo trukmės prailginimui. Todėl kita svarbi ir aktuali problema yra vaistų patekimas į reikiamą vietą – taikinį, kaip padėti vaistui patektį būtent į vėžines ląsteles ar audinį ir sumažinti riziką, kad vaistas pažeis normalius, sveikus audinius ir organus ir nepakenks jų funkcionavimui. Šiame tiriamajame darbe ir stengiamasi atsakyti į šią problemą, pasitelkus du metodus, kurie palengvina medžiagų patekimą į ląsteles – elektroporaciją ir sonoporaciją. Šių metodų funkcija yra panaši, tiek elektroporacijos, tiek sonoporacijos metu ląstelių plazminėse membranose susiformuoja poros, pro kurias gali patekti įvairios medžiagos (vaistai, dažai, baltymai, genai), kurios natūraliu būdu į ląstelę nepatenka arba patenka labai maži jų kiekiai, jei pernašoje dalyvauja ląstelės membranoje esantys baltymai nešikliai. Šiame darbe naudojami šie du, jau minėti metodai siekiant palengvinti chemoterapijoje naudojamo vaisto bleomicino patekimą į vėžines ląsteles in vivo. Tyrimams in vivo objektu buvo pasirinktos CBA – klono linijos pelės, kurioms į viršutinę nugaros dalį buvo įskiepyta MH22A hepatoma. Eksperimento duomenys rodo, kad tiek elektroporacija, tiek sonoporacija ir suminis abiejų metodų poveikis yra... [toliau žr. visą tekstą] / There is a lot of various information about cancer as well as plenty of knowledge and types of drugs to treat this disease. Pharmacy has discovered and developed a number of active anti-cancer chemotherapeutic agents for cancer treatment, or at least useful for reducing the spread of cancer. Therefore, another important and urgent problem is development of new method that would help to deliver directly into cancer cells or tissue. This in turn would allow to reduce side effects of the drug and preserve healthy tissues and organs. In this experimental work we attempted to answer this problem, using two methods that facilitate the transfer of materials into the cells - electroporation and sonoporation. These methods are based on similar principle: both electroporation and sonoporation affects the cell membranes to form pores that allow for various products (medicines, dyes, proteins, genes) to get access into the cell. The cell naturally is not accessible for those materials or the permeability is very small, in case special or nonspecific transport systems exist for this specific molecule. In this study we used the following two methods in order to facilitate the anticancer drug bleomycin delivery into cancer cells in vivo. The subject of these experiments in vivo is CBA – line clone mice bearing MH22A hepatoma tumors, transplanted on upper part of the flank. Experimental data showed that both electroporation and sonoporation and combination of both methods are effective in... [to full text]

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: This study investigated the effect of several hormones on the rat corticosteroid binding globulin proximal promotor and for the first time showed that modulation occurs at the promotor level and can be correlated with changes in corticosteroid binding globulin mRNA and protein levels. The effect of various physical and psychological stressors on rat liver corticosteroid binding globulin mRNA levels was also tested and it was shown that voluntary running had no effect on rat corticosteroid binding globulin levels but that involuntary swimming and immobilization decreased rat corticosteroid binding globulin mRNA levels. Glucocorticoid responsiveness of the corticosteroid binding globulin promoter was investigated further by using truncated contructs of the corticosteroid binding globulin proximal promoter. Glucocorticoid responsiveness was delineated to between -296 and -145bp from the transcription start site an area that contains putative binding sites for D-site binding protein, hepatic nuclear factor-3 and CAAT/enhancer binding protein suggesting that these transcription factors may be involved in glucocorticoid responsiveness of the corticosteroid binding globulin proximal promoter. The dissociative glucocorticoid activity of medroxyprogesterone acetate and Compound A, both putative dissociated glucocorticoids, was compared to standard glucocorticoids by examining transactivation of glucocorticoid response element-containing reporter constructs and transrepression of corticosteroid binding globulin gene expression in hepatic cell lines. Results showed that medroxyprogesterone acetate, but not Compound A, trans activates only in the presence, but not in the absence, of co-transfected glucocorticoid receptor. Medroxyprogesterone acetate down modulated dexamethasone transactivation while the modulatory effect of Compound A depends on the order of addition of Compound A. If added together Compound A has no effect on dexamethasone transactivation, however, if Compound A was added before dexamethasone, Compound A significantly decreased dexamethasone transactivation. Both medroxyprogesterone acetate and Compound A, like glucocorticoids, transrepressed the rat corticosteroid binding globulin proximal promoter. The potency of repression was similar but Compound A repressed with a higher efficacy than medroxyprogesterone acetate. We conclude that Compound A is a completely dissociated glucocorticoid in contrast to medroxyprogesterone acetate that displays only partial dissociation, which is dependent on glucocorticoid receptor levels. / AFRIKAANSE OPSOMMING: Tydens hierdie ondersoek is die effek van verskeie hormone op die rot kortikosteroied bindings globulien proksimale promoter ondersoek en vir die eerste keer is getoon dat modulering plaasvind op promoter-vlak en dat repressie korrileer met die verandering in kortikosteroied bindings globulien mRNA-en proteinvlakke. Die effek van verskeie fisiese en fisiologiese stressors op rotlewer kortikosteroied bindings globulien-mRNAvlakke is ook getoets en daar is getoon dat willekeurige hardloop geen effek op rot kortikosteroied bindings globulien-mRNA-vlakke het nie maar dat gedwonge swem en immobilisering rot kortikosteroied bindings globulien-mRNA-vlakke verlaag. Glukokortikoied responsiewiteit van die kortikosteroied bindings globulien proksimale promoter is verder ondersoek deur verkorte konstrukte van die kortikosteroied bindings globulien te toets. Glukokotikoied responsiewiteit is afgebaken tot tussen -296 en - 145bp vanaf die transkripsie beginplek 'n area wat beweerde bindings setels vir D-setel bindings protein, hepatosiet faktoor-3 en CCAAT-bindings protein-2 bevat en dus suggereer dat hierdie transkripsie faktore betrokke mag wees met glukokortikoied effekte op die kortikosteroied bindings globulien-proksimale promoter. Die dissosiatiewe glukokortikoied aktiwiteit van medroksiprogesteroon asetaat en Verbinding A, beide beweerde dissosiatiewe glukokortikoiede, relatief tot standaard glukokortikoiede is vergelyk deur transaktivering van glukokortikoied reseptor e1elment-bevattende konstrukte en onderdrukking van kortikosteroied bindings globulien geen ekspressie in lewersellyne te bestudeer. Medroksiprogesteroon asetaat, maar nie Verbinding A nie, transaktiveer slegs in die teenwoordigheid, maar nie in die afwesigheid, van ko-getransfekteerde glukokortikoied reseptore. Medroksiprogesteroon asetaat moduleer deksametasoon transaktivering afwaarts terwyl die modulerende effek van Verbinding A afhanklik van die orde van Verbinding A byvoeging is. Indien saam bygevoeg het Verbinding A geen effek op deksametasoon transaktivering nie, maar indien Verbinding A voor deksametasoon bygevoeg word verlaag Verbinding A deksametasoon transaktivering. Beide medroksiprogesteroon asetaat and Verbinding A, soos glukokortikoiede, onderdruk die rot kortikosteroied bindings globulien-proksimale promoter. Die sterkte van onderdrukking is dieselfde maar Verbinding A onderdruk met 'n hoër effektiwiteit as medroksiprogesteroon asetaat. Ons toon dat Verbinding A 'n totale dissosiatiewe glukokortikoied is in teenstelling met medroksiprogesteroon asetaat, wat slegs gedeeltelik dissosiatief is afhangende van glukokortikoied reseptor-vlakke.

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression during melanoma carcinogenesis remains unclear. In this study, adding exogenous HDGF stimulated the invasion and colonies formation of B16-F10 melanoma cells. Adenovirus vectors encoding HDGF and HDGF-RNAi were generated and characterized to up- and down-regulated HDGF expression in B16-F10 melanoma cells. It was found that HDGF overexpression stimulated the proliferation, invasiveness, anchorage-independent growth of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects. In lung-metastasis model, intravenous injection of HDGF-overexpressing melanoma cells resulted in increased metastasis while HDGF-downregulated melanoma cells caused decreased metastasis. Similarly, in primary melanoma model, subcutaneous injection of HDGF-overexpressing melanoma cells enhanced while HDGF-downregulated melanoma cells reduced the tumor burden in mice. Histological analysis revealed increased tumor proliferation and neovascularization with concomitant reduction of apoptosis in HDGF-overexpressing melanoma. Moreover, HDGF-overexpressing melanoma also exhibited enhanced propensity to metastasize from the primary tumors to lymph node and lung. Finally, it was found that HDGF overexpression increased nuclear factor kappa B (NF£eB) activities and Akt phosphorylation up and down stream alternation like PI3K, PTEN, I£eB and it¡¦s subunit IKK£\, IKK£], IKK£^ in melanoma cells. It also found that HDGF overexpression influenced MITF and HIF1£\ in melanoma after gene delivery. HDGF also altered EMT changes like E,N-cadherin, vimentin, and £],£^-catenin. The present study provides conclusive evidence that HDGF upregulation promotes the growth and metastasis of melanoma by promoting the survival and vascularization. Besides, HDGF knockdown may constitute a novel strategy for melanoma control.

published_or_final_version / Biochemistry / Master / Master of Philosophy