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Volume 11 - 2007


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1-14

Colchicine-mediated focal adhesion formation promotes transient, lipoplex-mediated transfection of A549 cells

Author(s): Dr. Lindsay A., Schwarz,

Abstract: Colchicine, a microtubule-disrupting drug, enhances lipoplex-DNA-mediated transfection. Colchicine-mediated increases in transgene expression are dependent on interference with tubulin polymerization, as pretreatment with paclitaxel, a microtubule-stabilizing agent, significantly inhibited the enhancing effects of colchicine. In addition to its interference with tubulin polymerization, colchicine-treatment activates Rho family GTPases, integrin clustering and the non-receptor tyrosine kinase, focal adhesion kinase (FAK), all known to be involved in formation of focal adhesions. We show that colchicine-mediated enhancement of transgene expression required activation of a Rho GTPase, as Clostridium difficle toxin B inhibited enhancement. Activation of a Rho GTPase lead to engagement of integrins, as the RGD-sequence peptide, an inhibitor of integrin clustering, abrogated colchicine-enhanced transgene expression. Genistein, a tyrosine kinase inhibitor, and cytochalasin D, both capable of inhibiting stress fiber formation, abolished colchicine-induced increases in transgene expression and suppressed focal adhesion formation, suggesting enhanced transgene expression involved stress fiber and focal adhesion formation. FRNK is an endogenous regulator of the tyrosine kinase, focal adhesion kinase (FAK). In A549 cells stably overexpressing the negative regulator, FRNK, colchicine pre-treatment did not enhance transgene expression, suggesting a critical role for FAK. Moreover, PP1, a selective, src-family kinase inhibitor also suppressed the ability of colchicine to enhance transgene expression. We propose that Rho-regulated, integrin clustering stimulates FAK and src kinase activation, formation of both focal adhesions and stress fibers, all of which appear critical to colchicine-mediated enhancement of transgene expression, as transfected by lipoplexes.

Keywords: lipoplex, cell signaling, focal adhesion kinase, gene transfection



15-20

To investigate the in vitro inhibitory effect of rAd-ODC/Ex3as on human esophageal carcinoma cells. The infection rate of rAd-ODC/Ex3as was measured with the aid of GFP expression. Western Blot technique was used to observe the inhibition of ODC expression in infected tumor cells. The malignant phenotype of Eca109 cell line was assessed by growth curve. TUNEL was used to analyze cell apoptosis. Approximate 65% of Eca109 cell line were infected with rAd-ODC/Ex3as when MOI reached 50. The expression of ODC was inhibited in the infected tumor cells. rAd- ODC/Ex3as could inhibit Eca109 cell line growth and invasive ability at 20 of MOI. TUNEL proved that rAd- ODC/Ex3as can lead to cell apoptosis. rAd-ODC/Ex3as could inhibit effectively the expression of ODC gene and the growth of of esophageal squamous carcinoma cell line Eca109 in vitro, and induce apoptosis. It may be one of the promising medicines for antisense gene therapy in esophageal cancer.

Author(s): Dr. Hui, Tian,

Abstract: To investigate the in vitro inhibitory effect of rAd-ODC/Ex3as on human esophageal carcinoma cells. The infection rate of rAd-ODC/Ex3as was measured with the aid of GFP expression. Western Blot technique was used to observe the inhibition of ODC expression in infected tumor cells. The malignant phenotype of Eca109 cell line was assessed by growth curve. TUNEL was used to analyze cell apoptosis. Approximate 65% of Eca109 cell line were infected with rAd-ODC/Ex3as when MOI reached 50. The expression of ODC was inhibited in the infected tumor cells. rAd- ODC/Ex3as could inhibit Eca109 cell line growth and invasive ability at 20 of MOI. TUNEL proved that rAd- ODC/Ex3as can lead to cell apoptosis. rAd-ODC/Ex3as could inhibit effectively the expression of ODC gene and the growth of of esophageal squamous carcinoma cell line Eca109 in vitro, and induce apoptosis. It may be one of the promising medicines for antisense gene therapy in esophageal cancer.

Keywords: Ornithine decarboxylase; Adenovirus vector; esophageal neoplasms; Eca109 cell line; Gene therapy



21-26

Autologous stem cell transplantation for primary refractory or relapsing Hodgkin’s disease: comparison between CD34+ immunoselected and unselected stem cells graft

Author(s): Dr. Federica, Sora,

Abstract: Autologous stem cells transplantation (ASCT) is widely accepted in the treatment of high risk Hodgkin’s disease (HD) either resistant or relapsing after first-line chemotherapy. Since the presence of Hodgkin/Reed- Sternberg cells in peripheral blood stem cells (PBSC) collection was demonstrated, and CD34+ antigen is not expressed on HD cells, positive selection of CD34+ cells has been demonstrated an efficient purging method to reduce the number of tumour cells in the graft. We conducted a non randomized pilot study using CD34+ selected ASCT in refractory/relapsing HD patients and compared the clinical outcome to HD patients receiving unselected PBSC. Eleven patients received CD34+ selected ASCT (group A) and 11 patients received unmanipulated ASCT (group B). Patients were matched for age, sex, diagnosis, IPI and response to first-line therapy. Group A received a median number of 4.74 x 106/kg CD34+ selected cell while patients of B group of 8.45 x 106/kg unselected PBSC (p=0.23). No difference was observed between the two groups in any of the endpoints analyzed (haematological engraftment, infections, morbidity and mortality, progression, survival). These results show no advantages for patients receiving CD34+ selected ASCT compared to unselected PBPC transplant at least for refractory/relapsing HD.

Keywords: autologous stem cell transplantation, CD34+, cell immnuoselection, Hodgkin’s disease



27-36

Gene therapy for arthritis: defining novel gene targets

Author(s): Dr. Charles J. , Malemud,

Abstract: Rheumatoid arthritis (RA) and osteoarthritis (OA) are debilitating diseases of the musculoskeletal system. RA is characterized by immune dysfunction and the classical cellular and soluble mediators of inflammation, whereas during its early stages, OA is considered a non-inflammatory disorder. However, a common pathway inherent to both RA and OA is articular cartilage and subchondral bone destruction resulting in non-functional synovial joints. Over the past decade or so, many of the candidate pathophysiologic pathways including those regulated by cytokines, growth factors and transcription factors that promote RA and OA disease progression have been elucidated. Although medical therapies directed at neutralizing cytokine activity have emerged and are now employed in treating RA and OA, there is considerable debate as to how long these biologics can be employed to modify chronic disease progression without emergent serious adverse events. This has led to a significant increase in studies that have employed gene transfer strategies in RA and OA animal models directed at inhibiting inflammatory cytokines, immune-mediated inflammation, growth factors, angiogenesis factors, apoptosis and matrix metalloproteinases that play a prominent role in RA and OA pathology. The significant abrogation of inflammation in arthritis animal models, in particular, by interleukin-1 receptor antagonist (IL-1Ra) and soluble TNF receptor gene constructs makes them potentially useful for treating RA and OA.

Keywords: Adenoviral/Adenoviral-associated Vector, Gene Transfer, Inflammation, Osteoarthritis, Rheumatoid Arthritis



37-42

The PPAR-! Pro12Ala allele polymorphism of the Peroxisome Proliferator-Activated Receptor (!) Gene (PPARG2) is a risk factor with a self-identified obese Dutch population

Author(s): Dr. Kenneth, Blum,

Abstract: Obesity has been identified as a global epidemic and presents a significant increased risk factor for a variety of co- morbidities. Positive energy balance and resultant weight gain is largely attributed to a chronic mismatch between energy intake and energy expenditure. The PPAR gene (PPAR-! Polymorphism - Pro12Ala Allele) influences many biological factors including serving as the master regulator of fat-cell formation and influencing insulin resistance. Based on the literature and proposed associations of the PPAR gene and the Pro12Ala allele polymorphism as a fat regulator gene, we decided to determine its allelic presence in a self-identified (via a cross sectional survey questionnaire) obese Dutch population. In this preliminary observational study, a total of 1,058 subjects were genotyped for the PPARG2 polymorphism. The PPAR gene Ala allele polymorphic frequency was 25.05% of the study subjects (n=1,058) versus 14% of the literature controls (n=2,245). This difference was significant (Z=17.398, p= 0.001). Our results suggest that in a highly motivated (wanting to loose weight) group of self-identified obese subjects (n= 1,058) from the Netherlands, the Pro 12A1a polymorphism of the PPARG2 gene significantly associates with these individuals compared to non-obese controls (n=2,245) and may be considered a risk factor.

Keywords: Obesity, Genotrim, PPAR-! gene, fat regulation



43-50

Preliminary study on the recombinant endostatin engineering Lactococcus lactis

Author(s): Dr. Chongbi, Li,

Abstract: Endostatin is a specific inhibitor of endothelial proliferation and agiogenesis from the COOH-terminal portion of human collagen XVIII. In order to examine the effect on Lactococcus lactis (L. lactis) and endostatin for curing cancer, rat endostatin gene was isolated by RT-PCR from rat kidney and cloned into the plasmid of L lactis and expressed in Lactococcus lactis NZ9000. And the effects were observed by orally L .lactis and recombinant L lactis expressing endostatin for colorectal cancer-induced rats with 1, 2-dimethylhydrazine (DMH) through both the survival and histopathological examination of the rats. The results showed that recombinant endostatin L lactis had a significant effect on the Duke’s stage of the experimental rats (P<0.05). Furthermore, the mean survival of the rats taken orally with recombinant L lactis was longer than that of the rats treated with DMH alone. The study would lay a theoretical foundation for an application of L lactis and endostatin to the anti-tumor.

Keywords: Endostatin; cloning and expression, Lactococcus lactis, experimental rats, effects, Tumor



51-60

Epstein-Barr Virus associated gastric carcinoma

Author(s): Dr. Runjan, Chetty,

Abstract: Epstein-Barr Virus (EBV) is a ubiquitous human herpesvirus associated with a variety of human malignancies including lymphoma and so-called lymphoepithelial carcinoma seen in a variety of sites, including the stomach. EBV has been detected in 5-20% of gastric carcinomas worldwide. Evidence is presented which suggests that failure of EBV-specific immunity may play a role in the pathogenesis of EBV-associated malignancy. In this paper, we review the clinicopathologic features, molecular pathology, immunologic aspect, environmental factors in EBV- associated gastric carcinoma and lastly, EBV-targeted therapy.

Keywords: Epstein-Barr virus, gastric carcinoma, clinicopathologic features, molecular pathology, immunology, environmental factors, EBV-targeted therapy



61-74

Reviewing the role of putative candidate genes in “Neurobesigenics,” a clinical subtype of Reward Deficiency Syndrome (RDS)

Author(s): Dr. Kenneth, Blum,

Abstract: While there is a considerable body of literature correlating the role of dopaminergic genes and obesity, body mass index, body type, overeating, carbohydrate binging, energy expenditure and low dopamine D2 receptor (D2R) density, there is a paucity of research concerning the dopamine D2 receptor gene (DRD2) variants and percent body fat. We propose that the degree of obesity involving the interactive relationship of the brain’s reward circuitry and the body’s response to stress and caloric deprivation is represented by a new term Reward Deficiency Syndrome (RDS), which is subject to strong genetic influence. We report here the first potential association of DRD2 genotypes and the percent fat phenotype. We genotyped, at the DRD2 Taq1 A1 polymorphism, 122 obese Caucasian subjects and 135 non-obese controls. The first control group consisted of 30 non-obese Caucasians screened to exclude a wide range of addictive behaviors (Controls A). The second control group consisted of 105 non-obese Caucasians 61 Chen et al: Association of The Taq A1 Allele of The Dopamine Receptor Gene (DRD2) and Percent Body Fat in Humans screened to exclude substance abuse and psychiatric disorders (Controls B). Controls A were assessed for weight, body mass index (kg/m2) [BMI] and percent body fat using dual energy X-ray absorptiometry (DEXA). The controls B were assessed for weight and BMI. The sample was separated into two groups, those with the Taq1 A1 allele (A1/A1 or A1/A2) and those without the A1 allele (A2/A2). The controls A had a normal range of body fat (25- 31% for females and 18-25% for males), a mean % body fat of 28.4±3.4 % and a mean BMI of 22.4 ± 2.9 kg/m2. The controls B had a mean BMI of 21.9 ± 2.9 kg/m2 The obese subjects had a percent body fat value of over 32 % for females and over 25% for males, a mean % body fat of 42.1± 7.5%, and mean BMI of 29.3 ± 6.25 kg/m2. The DRD2 Taq1A1 allele was present in 67% of the obese subjects compared to 3.3 % of the well-screened controls A and 33.3 % for controls B. These differences were significant: Controls A vs. Obese subjects: c2 = 39.6 d.f. =1, p<0.0001, and Controls B vs. Obese subjects c2 = 25.9 d.f. 1, p <.0001. These results are consistent with a role of the DRD2 gene in obesity as measured by percent body fat as well as by BMI. Additionally, it is proposed that since fat distribution is under extensive genetic control (h2<0.80) one putative gene may be the DRD2.

Keywords: Obesity, percent body fat, dopamine, body mass index, DRD2gene, DEXA, screened controls, Reward Deficiency Syndrome (RDS)



75-78

Genetically modified stem cells for cellular therapy

Author(s): Dr. Philippe , Taupin,

Abstract: Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, to neurological diseases and injuries. Over the past decade, significant progresses have been made in stem cell research; the derivation of embryonic stem cells (ESCs) from human tissues, the development of somatic cell nuclear transfer (SCNT) technology, and the confirmation that neurogenesis occurs in the adult mammalian brain, including in human. Despite these advances, there may be decades before stem cell research translates into therapy. Beside the scientific and technical challenges, there are ethical and political constraints and debates over stem cell research, particularly on ESCs and SCNT. In this manuscript, I will discuss how gene therapy is applied to stem cell research, in an attempt to unlock some of the technical, ethical and political hurdles associated with stem cell research.

Keywords: embryonic stem cells, neural stem cells, somatic cell nuclear transfer, cellular therapy, gene therapy



79-92

Gene therapy trials for the treatment of high-grade gliomas

Author(s): Dr. Maciej S, Lesniak,

Abstract: protocols. Gene Therapy and Molecular Biology Vol 11, page 79 Summary High-grade gliomas remain relatively resistant to current therapy. Local recurrence is a common feature and the majority of patients progress despite conventional therapy. One modality-gene therapy-has shown a lot of promise in early preclinical and clinical studies aimed at advancing the treatment of this disease. In this review, we provide a comprehensive overview of clinical trials involving gene therapy in the field of neuro-oncology. The use of different delivery vehicles, including liposomes, cells, and viruses, as well genes, especially cytokines and suicide genes, are explored in detail. The unique features and advantages/disadvantages of the different vectors employed are compared based on results of human studies. We discuss both the limitations and successes encountered in these clinical trials, with an emphasis on the lessons learned and potential ways of improving current gene therapy

Keywords: Glioblastoma multiforme (GBM), brain tumor, glioma, clinical trial, gene therapy, virus



93-102

Preliminary association of both the Dopamine D2 Receptor (DRD2) [Taq1 A1 Allele] and the Dopamine Transporter (DAT1) [480 bp Allele] genes with pathological aggressive behavior, a clinical subtype of Reward Deficiency Syndrome (RDS) in adolescents

Author(s): Dr. Kenneth, Blum,

Abstract: Advances in our knowledge of the neurobiology of aggression have given rise to rational pharmacological treatments for these behaviors. The main biological systems which are known to be involved are certain reward neurotransmitters that include serotonin (5HT), opioid peptides (END), gamma-aminobutyric acid (!ABA), and the 93 Chen et al: DRD2 and DAT1genes as a clinical subtype of RDS in adolescents catecholamines (dopamine [DA] and Norepinephrine [NE]). We hypothesize that pathological aggression in adolescents may in part involve polymorphisms of genes linked to the dopaminergic system. It is our notion that pathological aggression is in part similar mechanistically to other forms of impulsive behaviors such as pathological gambling. By analogy to drug dependence, it has been speculated that the underlying pathology in pathological gambling is a reduction in the sensitivity of the reward system. The potential correlation of both the dopamine D2 receptor gene (DRD2) and the dopamine transporter gene (DAT1) polymorphisms with pathological aggression in adolescents was investigated in a total of 291 subjects. Only eleven Caucasian adolescent subjects were diagnosed to have impulsive-aggressive behavior or pathological aggression. For this study 30 super normal controls were screened to exclude a number of reward deficit behaviors including pathological aggression and were genotyped for the DRD2 gene only (the DAT gene genotyping was not similarly genotyped in the super control sample). In the “super normal control group” only one person out of the 30 individuals genotyped carried the A1/ A2 genotype (3. 3%). Additionally, 91 controls were screened to exclude only Attention Deficit Hyperactivity Disorder (ADHD), pathological aggression, alcohol, drug dependence and tobacco abuse. In the present study, 6 out of 11 of the pathological aggressive subjects had the DRD2 A1 allele (55%), 11 out of 11 of the subjects (100%) carried the DAT1 10 allele, whereas 7 out of 11 or 64% carried the 9 allele. When the DRD2 A1 allele (A1/A1 or A1/A2) genotype in these subjects was compared to the super controls (1/30 or 3. 3%) a significant association was observed (Fisher’s exact test p= 0.0006); a similar trend was found with DAT1 480 bp 10/10 genotype when compared to controls (Fisher’s exact test p=0.089). However, when the DAT1 9/10 and 10/10 geneotypes were compared with controls a significant association was observed (Fisher’s exact test p= 0.00006). Albeit the small number of subjects, this is the first report on DNA polymorphisms to suggest a role for both the DRD2 and DAT genes in pathological aggressive behavior and warrants further investigation.

Keywords: Dopaminergic genes, Polymorphisms, Pathological Aggression, Super controls, Dopamine D2 receptor, Dopamine Transporter, Aggression



103-112

IAP as a new diagnostic and effective therapeutic target molecule for prostate cancer

Author(s): Dr. Takeo, Nomura,

Abstract: Prostate cancer is the second most common cause of cancer-related death among men in the United States. The conversion from androgen-dependent to androgen-independent state constitutes an important event in prostate cancer progression and is the main obstacle to improving the survival and quality of life in patients with advanced prostate cancer. Considerable progress has been made in the understanding of the molecular basis of prostate cancer. Prostate cancer progression and the development of the androgen-independent characteristics have been largely related to genetic abnormalities that not only androgen receptor (AR) but also crucial molecules involved in the regulation of survival or apoptotic pathways. One of these molecules including p53 and the B-cell lymphoma-2 (Bcl-2) family, the antiapoptotic protein, the inhibitor of apoptosis proteins (IAPs) have been associated with the promotion of tumorigenesis and drug sensitivity in prostate cancer due to their overexpression in prostate cancer cells treated with androgen ablation or chemotherapeutic agents. Therefore, IAPs may be of great value in clinical and prognostic markers in patients with prostate cancer and therapies that target IAPs may have the potential to improve outcomes for patients. In this review, we focus on the experimental evidence that associates IAPs expression with prostate carcinogenesis and cancer progression, and summarize the roles of IAPs in chemotherapy to develop a new target for the diagnosis and treatment of prostate cancer.

Keywords: Prostate cancer, Inhibitor of apoptosis protein



113-116

Gene cloning of P43 surface protein of toxoplasma gondii tachyzoite and bradyzoite (SAG3)

Author(s): Dr. Bahram, Kazemi,

Abstract: Toxoplasma gondii is an obligate intracellular parasite which its sexual and asexual cycle respectively takes place in the intestinal epithelial of definitive host and tissue of intermediate hosts. Congenital toxoplasmosis is more important when the mother acquired the infection during pregnancy period for the first time. Having a specific antigen is an important element in prevention and detection of parasite. This study has designed and performed in the aim of cloning a specific toxoplasma antigen for further studies. We have amplified gene of P43 of toxoplasma tachyzoite and bradyzoite surface antigen. PCR product was cloned in pGEMEX1 expression vector (named pGEM43) and is ready to make the recombinant protein for using as antigen.

Keywords: Toxoplasma, tachyzoite, bradyzoite, surface antigen, cloning



117-132

Developing and applying a drug delivery model for liposomal and dendritic multifunctional nanoparticles

Author(s): Dr. Constantinos, M. Paleos,

Abstract: This account deals with a strategy for designing multifunctional liposomes and dendritic polymers. Such nanoparticles, although quite different in size and structure, both fulfill properties that drug carriers should exhibit, i.e. specificity or targeting ability, extended time of circulation in biological fluids and ability to be transported through cell membranes. Furthermore, having developed these multifunctional liposomal and dendritic carriers, a drug delivery model is presented that employs instead of cells multilamellar liposomes, which interact with the above mentioned multifunctional carriers. This interaction should primitively mimic the processes which occur in living cells when they interact with loaded or unloaded liposomal and dendritic nanoparticles. Multifunctionality and multivalency coupled with molecular recognition between the interacting pairs render the loaded nanoparticles effective drug delivery vehicles.

Keywords: Drug Delivery Systems, Liposomes, Dendrimers, Hyperbranched Polymers



133-142

In vitro anticarcinogenic effect of a nutrient mixture on human rhabdomyosarcoma cells

Author(s): Dr. Aleksandra , Niedzwiecki,

Abstract: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma of mesenchymal origin, has metastasized in ~25% of all patients at time of diagnosis. Though current treatment strategies have achieved some success, they are associated with severe adverse effects. We investigated the effect of a nutrient mixture (NM), which has shown antitumor effects on various cancer cell lines, on rhabdomyosarcoma cell growth, apoptosis, MMP secretion, and invasion. Human rhabdomyosarcoma cells, grown in DME, were treated at near confluence with NM at 0, 10, 50, 100, 500 and 1000 μg/ml in triplicate at each dose. MMP secretion was studied by zymography, viability by MTT assay, cell invasion through Matrigel, and morphology and apoptosis by H&E staining and live green caspase kit. Zymography demonstrated MMP-2 secretion and PMA-induced MMP-9 secretion. NM inhibited the secretion of both MMPs in a dose-dependent fashion, with virtual total inhibition at 500 μg/ml NM. Cell invasion through Matrigel was inhibited at 10, 50, 100 and 500 μg/ml by 75%, 80%, 92% and 100% (p=0.02) respectively. NM was slightly toxic at 1000 μg/ml (20% over control, p=0.016) to rhabdomyosarcoma cells. Cells exposed to NM showed dose-dependent apoptosis with 90% of cells in late apoptosis at 1000 μg/ml. These results suggest that NM has potential in the treatment of rhabdomyosarcoma by inducing cell apoptosis and inhibiting cell invasion and MMP secretion without toxic effects.

Keywords: rhabdomyosarcoma, apoptosis, MMPs, Matrigel invasion, nutrients, green tea extract, ascorbic acid, lysine



143-150

Transfection with glutathione-dependent dehydroascorbate reductase genes exerts cytoprotective effects against hydroperoxide- induced cell injury through vitamin C regeneration and oxidative-stress diminishment

Author(s): Dr. Nobuhiko , Miwa,

Abstract: To evaluate the potential for utilization of overexpression of glutathione (GSH)-dependent dehydroascorbate (DehAsc) reductase (DHAR) as a tool to alleviate deleterious effects induced by an oxidative stress, the effect of overexpressed DHAR on tert-butylhydroperoxide (t-BuOOH)-induced cell injury was examined using DHAR gene (dhar)-transfected Chinese hamster ovary (CHO) cells. The transfected dhar products-derived DHAR was shown to be expressed all over the cytoplasm rather than in the nucleus, and repressed t-BuOOH-induced cell death, DNA strand cleavages, and intracellular reactive oxygen species (ROS)-generation in cells pretreated with DehAsc plus GSH isopropylester, but not without such pretreatment in contrast to no repression in their vector-transfected counterparts with such pretreatment. Upon the similar pretreatment, the intracellular levels of both ascorbic acid (Asc) and total vitamin C (Asc plus DehAsc) were 1.4- to 1.7-fold higher in dhar-transfected cells than in the vector- transfectants. Thus, our results suggest that overexpressed DHAR exerts cytoprotective effects against hydroperoxide-induced cell injury, and that more abundant intracellular Asc accumulation induced by expression of exogenous dhar may be involved in the mechanism.

Keywords: glutathione-dependent dehydroascorbate reductase, dehydroascorbic acid, ascorbic acid, reactive oxygen species, oxidative stress, cell death



151-160

Adenovirus-mediated Expression of both Antisense Ornithine Decarboxylase (ODC) and S- adenosylmethionine Decarboxylase (AdoMetDC) inhibits human esophageal squamous carcinoma cell growth

Author(s): Dr. Hui , Tian,

Abstract: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad- ODC-AdoMetDCas). To evaluate the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ODC and S-adenosylmethionine decarboxylase (AdoMetDC), the human esophageal squamous carcinoma cell line Eca109, was infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess the properties of tumor growth and invasiveness. Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. Our study suggests that as a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of esophageal cancers.

Keywords:



151-160

Adenovirus-mediated Expression of both Antisense Ornithine Decarboxylase (ODC) and S- adenosylmethionine Decarboxylase (AdoMetDC) inhibits human esophageal squamous carcinoma cell growth

Author(s): Dr. Hui , Tian,

Abstract: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad- ODC-AdoMetDCas). To evaluate the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ODC and S-adenosylmethionine decarboxylase (AdoMetDC), the human esophageal squamous carcinoma cell line Eca109, was infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess the properties of tumor growth and invasiveness. Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. Our study suggests that as a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of esophageal cancers.

Keywords: Ornithine decarboxylase, S-adenosylmethionine decarboxylase, Polyamine, Esophageal neoplasms, Eca109 cell line, Gene therapy



161-170

Chromium Picolinate (CrP) a putative anti-obesity nutrient induces changes in body composition as a function of the Taq1 dopamine D2 receptor polymorphisms in a randomized double-blind placebo controlled study

Author(s): Dr. Kenneth, Blum,

Abstract: There is controversy regarding the effects and safety of chromium salts (picolinate and nicotinate) on body composition and weight loss in humans. Thus, we decided to test the hypothesis that typing the obese patients by genotyping the dopamine D2 receptor (DRD2) gene prior to treatment with Chromium Picolinate (CrP) would result in a differential treatment outcome. We genotyped obese subjects for the DRD2 gene utilizing standard PCR techniques. The subjects were assessed for scale weight and for percent body fat using dual energy X-ray absorptiometry (DEXAR). The subjects were divided into matched placebo and CrP groups (400 μg. per day) accordingly. The sample was separated into two independent groups. Those with either an A1/A1 or A1/A2 allele or those with only the A2/A2 allelic pattern. Each of these groups were tested separately for differences between placebo and treatment means for a variety of measures of weight change. The measures of the change in fat weight (p<0.041), change in body weight (p<0.017), the percent change in weight (p<0.044), and the body weight change in kilograms (p<0.012) were all significant for carriers of the DRD2 A2 genotype, whereas no significance was found for any parameter for those subjects possessing a DRD2 A1 allele. These results suggest that the dopaminergic system, specifically the density of the D2 receptors, confers a significant differential therapeutic effect of CrP in terms of weight loss and change in body fat, thereby strengthening the need for DNA testing.

Keywords: Chromium Picolinate, genotyping, body-composition, Genotrim®, and dopamine D2 receptor gene



171-176

Stringent control of NFATc1 nuclear occupancy is critical for maintaining balanced immune response

Author(s): Dr. Minggui , Pan,

Abstract: Many immune and inflammatory diseases still lack a clear mechanistic explanation. NFATc transcription factors are involved in immune homeostasis and response. NFATc1 is rapidly imported into the nucleus upon activation of lymphocytes that leads to its stimulation of a battery of cytokines responsible for immune response and is rapidly removed from the nucleus upon termination of the signaling. We have previously established a tetracycline- regulated transgenic mouse model with a subtle increased nuclear NFATc1 expression. The level of nuclear NFATc1 expression was only 1/7th of the total NFATc1 molecules of a wild type T cell. Here we show that this subtle increase of NFATc1 nuclear occupancy caused a severe disease with multi-organ failure characterized with infiltration of immune cells, elevated auto antibodies, leading to early animal death. Suppression of the transgene expression by doxycycline suppressed and reversed the disease. These results indicate that stringent control of NFATc1 nuclear occupancy is critical for maintaining balanced immune response and may have important clinical implications.

Keywords: NFATc1; Immune Response; Nuclear Occupancy; Autoimmunity



177-184

Assessment of cytogenetic effect of antiblastic therapy by means of micronucleus assay in exfoliated epithelial cells

Author(s): Dr. Armen K., Nersesyan,

Abstract: Literature data concerning possibility to use micronuclei (MN) level in exfoliated epithelial cells of patients under radio- and chemotherapy as a biomarker of cytogenetic effect are presented and discussed. The number of MN in buccal cells of patients under chemotherapy are very few and contradictory. Significant dose-dependent increment of MN in tumor and normal epithelial cells due to radiotherapy of cancer patients was shown by almost all investigators. Evaluation of MN induced by radiotherapy in exfoliated tumor cells can potentially identify radiosensitivity of tumors and the treatment outcome after the first fractions of irradiation. MN assay is almost completely non-invasive and easily done in accessible tumors (oral cavity and uterine cervix).

Keywords: micronucleus assay; exfoliated cells; chemotherapy; radiotherapy



185-202

Integration of human DNA fragments into the cell genomes of certain tissues from adult mice treated with cytostatic cyclophosphamide in combination with human DNA

Author(s): Dr. Sergei S., Bogachev,

Abstract: We demonstrate here that, when administered i.p. to adult mice in combination with the cytostatic cyclophosphamide, an inducer of cross-links in the DNA molecule, human exogenous DNA, having reached the nuclear space of liver, thymus, spleen cells, integrates into the mouse genome. The integration of foreign DNA produces change in blood counts and is lethal to the treated mice. It is suggested that the integration mechanism acts through the repair events induced by the formation of covalent interstrand cross-links resulting in double strand breaks during replication fork arrest.

Keywords: transgenic mice; human DNA; cyclophosphamide; Alu repeats; blood count (hemogram)



203-218

Gene expression profiling for adult human olfactory neuroepithelial-derived progenitors

Author(s): Dr. Abdelnaby , Khalyfa,

Abstract: The olfactory neuroepithelium (ONe) is, a specialized tissue that lines a region of the nasal cavity high in the nasal vault, characterized by its life-long regenerative capacity. The purpose of the study was to further characterize and to compare gene expression profiling in neurosphere-forming cells (NSFCs) derived from primary cultures of ONe using a genome-wide array approach. Total RNA was isolated from p14, p78, and p189 in vitro passages and hybridized to the human genome-wide CodeLink Bioarrays. Differentially expressed genes were identified in 9 arrays using statistical filters and bioinformatics analysis. Of 55,775 transcripts, 11,345 (in all passages) were detected and 7,115 were commonly expressed in 9 arrays. From the 7,115 transcripts, 2,690 transcripts were expressed in stem cells. Of these 2,690 transcripts, 1,117 genes containing RefSeq accession numbers were identified and further classified based on their functional similarities using Gene Ontology (GO) tools. The changes in expression levels revealed by microarray experiments were validated using real-time RT-PCR analysis. Biological pathways were constructed to better understand the biological significance of the differentially expressed genes. Each passage of NSFCs was characterized immunocytochemically and compared using antibodies to peripherin, β- tubulin III, and nestin. The array results indicate the presence of both neuronal and epithelial phenotypes within the population of NSFCs. The pattern of gene expression of the NSFCs was unique compared to other lines perhaps reflecting species related differences and or adult versus embryonic derived stem cells.

Keywords: a.khalyfa@louisville.edu



219-228

Monitoring green fluorescent protein for functional delivery of E. coli cytosine deaminase suicide gene and the effect of curcumin in vitro

Author(s): Dr. Siddhartha Sankar, Ghosh,

Abstract: Cytosine deaminase (CD) gene has been quite established as a suicide gene for cancer treatment, which converts nontoxic compound 5-flurocytosine (5-FC) to toxic chemotherapeutic agent 5-flurouracil (5-FU). However, besides choosing delivery system, the lack of suitable noninvasive probes to monitor quantitative gene transfer to the malignant tumor is another major concern that limits widespread application of suicide genes. In order to address this problem, we have constructed a dual expressing recombinant plasmid vector which carries CD and green fluorescent protein (GFP) genes. Herein, the GFP expression was used as a tool for monitoring functional CD gene transfer to investigate the mechanism of cell death by 5-FC/CD system in both cancer and non-cancer cells. The efficacy of CD transgene was enhanced when expressed under human ferritin promoter. Molecular analysis by RT- PCR established CD gene expression in the transfected cells, whereas mitochondrial activity (MTS) measurements showed the therapeutic effect of 5-FC/CD. Microscopic experiments, measurement of BrdU labeled DNA fragments release, characteristic laddering of chromosomal DNA and involvement of caspase-3 and Bcl-2 corroborated induction of apoptosis. Apoptosis was further synergized in presence of anticancer agent curcumin. Therefore, this noninvasive fluorescent technique was useful for easy monitoring of plasmid based 5-FC/CD system where a combinatorial effect of curcumin was shown to potentiate the therapeutic efficacy of CD gene.

Keywords: GFP, 5-flurocytosine (5-FC), cytosine deaminase (CD), apoptosis, suicide gene therapy



229-262

Genetic models of retinal degeneration and targets for gene therapy

Author(s): Dr. Stephen H. , Tsang,

Abstract: Studies utilizing animal models in combination with progress in the field of molecular genetics have improved our understanding of pathways leading to retinal degenerations. As a result, it has become clear that genes are involved in many processes that are responsible for the symptomatology seen in these conditions. However, it is still a mystery how certain genetic defects can cause a myriad of retinal degenerations while others defects, often in the same genes, lead to much more benign conditions such as stationary night blindness. As future research uncovers new details about specific genetic defects and the discovery of more accurate animal models, we can hopefully develop gene therapeutic strategies that will one day prevent, treat, and even cure these blinding and debilitating diseases.

Keywords: Animal models, congenital stationary night blindness, Models of Bardet-Biedl syndrome, Models of Best disease, Models of Norrie disease, Models of SFD, Models of Stargardt disease, Models of Stargardt-like macular dystrophy, Models of Usher syndrome, achr



263-268

Evaluation of cross immune response in DNA based vaccinated mice against HSV-1 and HSV-2

Author(s): Dr. Hoorieh , Soleimanjahi,

Abstract: Herpes simplex viruses are the most widespread human viral infections which are major targets of vaccine development. An effective vaccine for HSV must be stimulating both arms of immune system. DNA immunization with HSV-gD gene has been shown to induce both humoral and cellular immune responses against HSV infections. In the present study the cross immune responses of HSV-gD1 (gD1) or HSV-gD2 (gD2) against two HSV strains were evaluated. Mice were immunized with DNA vaccine containing gD1 or gD2 gene showed considerable responses against HSV-1 and HSV-2 respectively. While gD1 immunized mice showed significant cellular and humoral cross immune responses against HSV-2 strain but, gD2 did not. Due to important role of cellular immune responses against HSV infection, it can be concluded that vaccination with gD1 is more effective compared to gD2 when used as candidate vaccine against HSV infection.

Keywords: HSV-1, HSV-2, Glycoprotein D, DNA immunization, Cross immune response



269-274

Improving probiotic function using a patho- biotechnology approach

Author(s): Dr. Roy D. , Sleator,

Abstract: Although described for over a century, scientists and clinicians alike are only now beginning to realise the significant medical applications of probiotic cultures. Given the increasing commercial and clinical relevance of probiotics, improving their stress tolerance profile and ability to overcome the physiochemical defences of the host is an important biological goal. Patho-biotechnology describes the application of pathogen derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications as well as the development of novel vaccine and drug delivery platforms.

Keywords: Patho-biotechnology, probiotics, betaine, BetL, Listeria monocytogenes, Bifidobacterium, Lactobacillus



269-274

Improving probiotic function using a patho- biotechnology approach

Author(s): Dr. Roy D. , Sleator,

Abstract: Although described for over a century, scientists and clinicians alike are only now beginning to realise the significant medical applications of probiotic cultures. Given the increasing commercial and clinical relevance of probiotics, improving their stress tolerance profile and ability to overcome the physiochemical defences of the host is an important biological goal. Patho-biotechnology describes the application of pathogen derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications as well as the development of novel vaccine and drug delivery platforms.

Keywords: Patho-biotechnology, probiotics, betaine, BetL, Listeria monocytogenes, Bifidobacterium, Lactobacillus



275-288

Apoptosis prevention in neuronally differentiated PC12 cells, by bcl-2 gene transfection in the non- proliferative and differentiated state, with retention of neuron-specific proteins and blockage of mitocondrion-relayed apoptotic pathway

Author(s): Dr. Nobuhiko , Miwa,

Abstract: Transfected bcl-2 genes are known to inhibit apoptosis in proliferative or stable transfectants, but have been scarcely scrutinized in neuronally differentiated cells that are transiently transfected in the non-proliferative and differentiated state. In the present study, NGF-differentiated PC12 cells were transiently transfected with cDNA encoding human bcl-2 wrapped by HVJ virus-based liposomes. The resultant >50-fold Bcl-2 overexpression prevented NGF-removal-induced cell death, as shown by repression of either the diminished mitocondrial tetrazolium-reducing function or the augmented DNA-3’-OH cleavage terminals, through blockages of apoptosis- associated events such as lowered mitochodrial membrane potentials, release of cytochrome c and activation of caspase-3, together with transiently repressed intracellular ROS. Overexpressed Bcl-2 could also inhibit NGF removal-induced neurite-retraction, and the decreased levels of neurofilaments and neuron-specific enolase.

Keywords: bcl-2, NGF, PC12 cells, reactive oxygen species, apoptosis



289-298

Role of homologous recombination repair and non- homologous end joining in therapeutic resistance of BCR/ABL-expressing leukemia cells

Author(s): Dr. , Ireneusz Majsterek,

Abstract: The BCR/ABL oncogene resulting from chromosome translocation t(9;22) is the pathogenic principle of human chronic myelogenous leukemia. BCR/ABL-expressing cells may display drug and radiation resistance due to accelerated DNA repair stimulated by BCR/ABL. The aim of this study was determination of a role of homologous recombination (HRR) and non-homologous end joining repair (NHEJ) of double strand breaks (DSBs) in drug and radiation resistance of human BCR/ABL-expressing leukemic cells. K562 BCR/ABL-positive and CCRF-CEM BCR/ABL-negative human leukemia cells were used to examine the contribution of DSBs repair in their resistance to γ-radiation and idarubicin. Long-term viability and clonogenic test of the cell proliferation ability was used to estimate resistance against the DNA-damaging agents. The activity of BCR/ABL was inhibited by STI571 (Gleevec, Imatinib mesylate) as assayed by Western blotting. The kinetics of DNA repair after cell treatment with idarubicin at 0.5 and 1 μM or γ-radiation at 15 Gy and 25 Gy with or without STI571 pre-treatment were examined by the alkaline comet assay. It was found that DNA repair was more effective in K562 than in CCRF-CEM cells that correlated with drug and radiation resistance. The decrease of HRR pathway was 3.2-fold greater than NHEJ in K562 cells after STI571 treatment. Moreover, the induction of DSBs caused a 2.6-fold higher activation of HRR in K562 than in CCRF-CEM cells. We did not find significant changes in the activity of NHEJ pathway between both cell lines. Our results suggest that DSBs recovery by HRR-dependent pathway is critical for drug and radiation resistance of BCR/ABL-positive human leukemia cells.

Keywords: BCR/ABL; Leukemia; Drug resistance; DNA repair; STI571



299-304

Vitamin D receptor gene polymorphisms in patients with thyroid cancer

Author(s): Dr. , Seyed H. Ghaffari,

Abstract: The association between vitamin D receptor (VDR) gene polymorphisms and some diseases such as colorectal cancer, breast cancer, osteoporosis and psoriasis has been extensively investigated during the past few years. This research was performed not only because of the role of vitamin D as an anticancer agent, but also because of the suppressing action of vitamin D on TSH, the major regulator of thyroid cell growth. In this case-control study, comprising 71 thyroid cancer patients and 82 healthy population controls, we investigated the association between altered thyroid cancer risk with four polymorphisms located at the 3' end of the VDR gene detectable by ApaI, TaqI, Tru9 and BsmI restriction enzymes and with a start codon polymorphism located at the 5' end characterized by restriction enzyme FokI. The individual genetic pattern for VDR was evaluated by DNA extraction followed by PCR amplification of the VDR gene and the digestion with the restriction enzymes. For the effect of existence of the mentioned polymorphisms and thyroid cancer the odds ratio and 95% CI were calculated. All the odds ratios were within their CI, representing no relationship between these polymorphisms and risk of thyroid cancer. These observations suggest that VDR gene polymorphisms may not commonly contribute to the risk of thyroid cancer.

Keywords: vitamin D receptor; gene polymorphism; thyroid cancer



305-314

Exogenous DNA can be captured by stem cells and be involved in their rescue from death after lethal- dose γ-radiation

Author(s): Dr. , ,

Abstract:

Keywords:



305-314

Exogenous DNA can be captured by stem cells and be involved in their rescue from death after lethal- dose γ-radiation

Author(s): Dr. , Sergei S. Bogachev,

Abstract: Here we demonstrate that stem cells capture exogenous DNA internalized into the nuclear space. Injection of fragmented exogenous DNA to lethally radiated mice affords a very strong radioprotective effect: up to 90% of treated mice survived long after exposure to γ-rays. We also demonstrate that the high survival of mice after lethal- dose radiation is due to blood stem cell rescue whose offspring give rise to spleen colonies participating in recovery of the damaged immune system. It is suggested that the DNA radioprotective effect may result from involvement of exogenous DNA as substrate in homologous recombination during repair of double-strand break sites induced by high-dose radiation.

Keywords: gene therapy, radioprotection, stem cells, exogenous DNA, DNA uptake



315-320

Bioterrorism: warfare of the 21st century

Author(s): Dr. , Edit Nadasi,

Abstract: The terrorist attacks on and after September 11, 2001 have drawn attention to the fact that besides the traditional warfare, microorganism-based weapons are playing greater and greater role in military activities, especially in terror attacks. The ongoing war activities worldwide and the continuing international conflicts suggest that despite the international treaties, sooner or later germ warfare must be considered. The relatively easy preparation, storage and application of biologic weapons, despite the strictly regulated access, hold out major advantages when compared to traditional warfare, especially for terrorist activities. Germ warfare, divided into three different categories by the Center for Disease Control and Prevention USA, are based on use of bacteria, toxins and viruses. Besides the causative agents of the traditionally fearful contagious diseases (plague, smallpox, anthrax) other biologic agents (toxin of C. botulinum, hemorrhagic fever viruses) also play an important role in biologic warfare. Cognition of biologic weapons is very important because the infections originate from causative agents existing in the natural environment, but are generated artificially. These infections, whether air or food born, may generate numerous infections. Since the initial clinical features are usually not characteristic for the disease, it is crucial to be aware of the circumstances and clinical symptoms indicating biologic attack when produced en masse. When bio attack is recognized in time, suitable countermeasures may effectively reduce the severeness and expansion of the resulting infection.

Keywords: bio weapon, defence, prevention



321-328

Genotypic determination of Hepatitis C virus in Tehran using PCR-RFLP analysis

Author(s): Dr. , Bahram Kazemi,

Abstract: Hepatitis C virus (HCV) is the etiologic agent of most parentally transmitted hepatitis viruses and is responsible for more than 60% of chronic hepatitis cases that lead to liver transplantation. Based on its genetic variability, HCV is classified into at least six genotypes and a series of subtypes. HCV genotyping is important in order to select appropriate therapy. The aim of this study was to determine which HCV virus genotypes are most prevalent in Tehran. Serum HCV RNA was extracted by RNXplus buffer. RT-PCR and nested PCR were used on the 5’ untranslated region and a 250 bp fragment was amplified. 80 HCV positive sera samples were selectively chosen from 250 patients who were referred to the laboratory by their physicians. HCV genotypes were determined using restriction fragment length polymorphism (RFLP). The patients were grouped as follows: 32 cases (40%) had type 1a, 15 cases (18.75%) had type 3a, 7 cases (8.75%) had type 1b, 5 cases (6.25%) had type 3b, 3 cases (3.75%) had type 4, and 18 cases (22.5%) could not be typed because their sequences differed from any that were previously reported. Our results showed that types 1a and 3a were the most prevalent HCV genotypes in our Tehran samples, while types 2a, 2b, 5, and 6 were not observed.

Keywords: Hepatitis C virus; RFLP; Genotyping; Tehran




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