Inhibition of NF2-negative and NF2-positive primary human meningioma cell proliferation by overexpression of merlin due to vector-mediated gene transfer.

OBJECT: The absence of in vitro models of neurofibromatosis Type 2 (NF2)-defective meningiomas has limited investigative efforts to study the biological effects of this gene in the pathogenesis of these tumors. The goals of this report are to show that gene transfer vectors can efficiently express the wild-type NF2 transgene into primary meningioma cells and to determine effects on cellular proliferation. METHODS: In this study, the authors have compared the transducing capacities of a retrovirus, an adenovirus, and a herpes simplex virus amplicon vector for use in primary human meningioma cells harvested from human tumors excised from patients with and without NF2. Transduction efficiencies with the latter vector approached 100% and it was selected to transfer the wild-type NF2 transgene into these cells. Western blot analysis confirmed that vector-mediated gene transfer mediated the expression of the NF2-encoded polypeptide merlin. Overexpression of merlin significantly inhibited the proliferation of both NF2-negative and NF2-positive human meningioma cells when compared to the proliferation of cells transduced with a control vector. CONCLUSIONS: This study demonstrates the feasibility of using vector-mediated gene transfer to study wild-type NF2 gene function in short-term cultures of primary human meningioma cells.     

腺病毒介导的基因治疗在骨创伤及骨病中的应用

目的 介绍腺病毒介导的基因治疗的特点及其在骨创伤和骨病中的应用。方法 广泛查阅有关重组腺病毒载体的制备原理、特点及应用研究进展等方面的文献，并总结腺病毒介导的基因治疗在促进骨愈合、治疗骨质疏松及类风湿性关节炎等骨病研究中的应用情况。结果 腺病毒载体具有高转染率、目的基因的高表达率及在宿主细胞内的表达有一定的时限等特点，能有效转染软骨细胞、成骨细胞、破骨细胞、骨髓基质细胞、关节滑膜细胞、椎间盘髓核细胞及成纤维细胞等，适用于骨折愈合、脊柱融合、骨质疏松及类风湿性关节炎等骨病的基因治疗。结论 腺病毒介导的基因治疗可望为骨创伤、骨病的治疗提供新的途径。     

Association between absence of epidermal growth factor receptor immunoreactivity and poor prognosis in patients with atypical meningioma

OBJECT: The clinical behavior of meningiomas is variable. Because multiple growth factor receptors have been identified in these tumors, the authors sought to assess the capacity of the expression patterns of a subset of these receptors to stratify meningioma cases. METHODS: Eighty-four meningiomas were analyzed, including 36 benign, 29 atypical, and 19 malignant lesions. Immunohistochemical staining was performed for epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR)-beta, basic fibroblast growth factor receptor (BFGFR), and MIB-1. Survival analyses were performed using follow-up data obtained in patients with newly diagnosed tumors. Immunoreactivity for EGFR was observed in 47% of benign, 48% of atypical, and 42% of malignant tumors. Staining for BFGFR was identified in 89% of benign, 97% of atypical, and 95% of malignant lesions. Immunostaining for PDGFR-beta was evident in all the lesions assessed. Mean MIB-I indices for benign, atypical, and malignant cases were 3.6 (range 0.5-15.3), 8.2 (range 1.5-23.1) and 18.3 (range 1.0-55.8), respectively. Overall mean follow-up duration was 9.0 years (range 5.1-18.8 years). Lack of EGFR immunoreactivity was identified as a strong predictor of shorter overall survival in patients with atypical meningioma (p = 0.003, log-rank test). This association was not evident in cases of benign or malignant meningiomas. CONCLUSIONS: There is a significant association between EGFR immunoreactivity and prolonged survival in patients with atypical meningioma. Given the variable behavior of atypical meningiomas, EGFR assessment could improve existing strategies for patient stratification and treatment.     

Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors

BACKGROUND: Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. HYPOTHESIS: Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. DESIGN: We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. RESULTS: In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44.8 +/- 3.4 vs 10.1 +/- 0.7 mU per milligram of protein; P<.001). Following heparin administration, the gene transfer efficiency achieved with AdZ.F(pK7) diminished slightly in a concentration-dependent manner. However, the transfer efficiency was still greater than with the unmodified virus (mean +/- SEM, 1342.3 +/- 101.8 vs 4.8 +/- 0.4 mU per milligram of protein; P<.001). In vivo, following injury to the pig iliac artery with a 4F Fogarty balloon catheter, we found that AdZ.F(pK7) transduced the artery approximately 35-fold more efficiently than AdZ.F and 3-fold more efficiently than AdZ.F(RGD) following the administration of intravenous heparin, 100 U/kg body weight, and heparinized saline irrigation. CONCLUSIONS: Modifications of the adenovirus that lead to receptor targeting resulted in significantly improved gene transfer efficiencies. These improvements in transfer efficiencies observed with the modified vectors decreased slightly in the presence of heparin. However, AdZ.F(pK7) was still superior to AdZ.F(RGD) and AdZ.F despite heparin administration. These data demonstrate that modifications of adenoviral vectors that enhance binding to heparan sulfate receptors significantly improve gene transfer efficiency even in the presence of heparin and suggest an approach to optimize gene transfer into blood vessels.     

Enhanced expression of the sis and c-myc oncogenes in human meningiomas

In 19 human meningiomas (14 primary and four recurrent tumors and one tumor transplanted into athymic nude mice), oncogene expression, amplification, and rearrangement, and loss of heterozygosity on chromosome 22 were examined. Compared to nontumor brain tissue, there was greater than a fivefold expression of the sis oncogene in six (40%) of 15 tumors studied and of the c-myc oncogene in 12 (63%) of the total 19 tumors. Expression of the sis gene was lower in the recurrent tumors than in the primary cases, and there was no detectable expression in anaplastic meningioma cells. Rearrangement of the sis gene was found in one meningioma. Loss of heterozygosity on chromosome 22 was detected in two of the five informative heterozygous cases. Expression of the c-myc gene was higher in cases with a loss of heterozygosity than in those without. These results suggest that the sis and c-myc oncogenes are associated with tumorigenicity and that c-myc may induce meningiomas through loss of the putative tumor suppressor gene.     

Epidermal growth factor receptor targeting of replication competent adenovirus enhances cytotoxicity in bladder cancer

PURPOSE: We evaluated the delivery and oncolytic potential of targeted replication competent adenoviruses in bladder cancer lines. MATERIALS AND METHODS: Seven established human bladder cancer tumor lines (5637, SW800, TCCsup, J82, Scaber, T24 and 253J) were studied for the expression of integrins alpha(v)beta3, alpha(v)beta5, Coxsackievirus and adenovirus receptor, epidermal growth factor receptor (EGF-R) and epithelial cell adhesion molecule antigens using flow cytometry analysis. Bispecific single chain Fv fragments were used to target replication deficient luciferase reporter adenovirus to EGF-R (425-s11) or to epithelial cell adhesion molecule (C28-s11) antigens. Moreover, a fiber modified adenovirus targeting alpha(v)-integrins was studied. Replication competent serotype-5 adenoviruses attenuated to replicate specifically in retinoblastoma pRb (Ad5-d24) or p53 deficient (Ad5-d55K) cells were tested in vitro for oncolytic properties. RESULTS: Low to absent Coxsackievirus and adenovirus receptor expression was found in 5 of the 7 tumor lines (SW800, J82, T24, 5637 and Scaber). EGF-R expression was found in all cell lines, whereas elevated epithelial cell adhesion molecule expression was seen in 3 (5637, Scaber and TCCsup), alpha(v)beta3-integrin was found in 1 (Scaber) and alpha(v)beta5-integrin was found in 3 (TCCsup, 253J and T24). EGF-R targeting using 425-s11 improved transgene expression in all cell lines from 2.1 to 12.5 times over nontargeted viruses. Epithelial cell adhesion molecule and integrin targeting was inferior to EGF-R targeting with a maximal increase in transgene expression of 2 times for epithelial cell adhesion molecule in 5637cells and 1.6 times for integrin targeting in T24 cells. Comparison of the wild-type replication competent virus with conditionally replicating adenoviruses (Ad5-d55K and Ad5-d24) showed superior oncolytic activity for the latter 2 in all lines. Furthermore, improved cytotoxicity (29% to 33%) was obtained in 4 of the 7 lines after pre-incubation of Ad5-d24 with 425-s11. CONCLUSIONS: EGF-R directed bispecific single chain antibodies enhance adenovirus mediated transgene expression and oncolysis in bladder cancer lines.     

Alpha(v)beta3 and alpha(v)beta5 integrin expression in meningiomas

OBJECTIVE: Integrins are emerging as alternative receptors capable of mediating several biological functions, such as cell-matrix and cell-cell adhesion, cell migration, signal transduction, and angiogenesis. Two alpha(v) integrins, i.e., alpha(v)beta3 and alpha(v)beta5, play critical roles in mediating these activities, particularly in tumors. No data are available on the expression of these integrins in meningiomas. METHODS: Using Western blot and immunohistochemical analyses with LM609 and PG32, two monoclonal antibodies capable of recognizing the functional integrin heterodimer, we evaluated the expression of alpha(v)beta3 and alpha(v)beta5 integrins in a series of 34 meningiomas of different histological subtypes and grades. We studied their expression in tumor cells and vasculature, as well as the expression of their related angiogenic factors (fibroblast growth factor 2 and vascular endothelial growth factor) and the alpha(v)beta3 ligand vitronectin. RESULTS: Alpha(v)beta3 and alpha(v)beta5 integrins were expressed by neoplastic vasculature and cells. Alpha(v)beta3 and alpha(v)beta5 expression was associated and correlated with that of their respective growth factors (fibroblast growth factor 2 and vascular endothelial growth factor) and microvessel counts and densities. Alpha(v)beta3 was more strongly expressed than alpha(v)beta5 in two cases of histologically benign meningiomas with aggressive clinical behavior. Alpha(v)beta3 expression was associated with that of its related ligand vitronectin and was also evident in small vessels of brain tissue closely surrounding meningiomas. CONCLUSION: Our data demonstrate the expression of alpha(v)beta3 and alpha(v)beta5 integrins in meningioma cells and vasculature. Our findings suggest a role for both of these integrins, and particularly alpha(v)beta3, in meningioma angiogenesis.     

Adenovirus‐mediated viral IL‐10 gene transfer prolongs survival of xenogeneic spheroidal aggregate‐cultured hepatocytes

Abstract Xenotransplantation of hepatocytes appears to be a novel promising therapy for some forms of liver disease, and may well overcome the problem of donor shortage. We have previously reported that hepatocytes with a spheroidal shape (spheroids) are ideal for cell transplantation. The application of gene transfer techniques to this hepatocyte transplantation could possibly regulate the xenogeneic rejection reaction and, therefore, result in prolongation of the survival of the transplanted hepatocytes. In this study, we chose the adenovirus as a vector and an immunosuppressive cytokine named viral IL‐10 (vIL‐10) for transfection. A series of experiments was performed to elucidate the efficacy of transfection to the spheroids with adenovirus vectors and the effect of transfected vIL‐10 on the survival of xenogeneic hepatocytes. We examined the cell survival quantitatively by evaluating β‐galactosidase (β‐gal) activity, which was transfected into the hepatocytes in the xenogeneic spleen, and semi‐quantitatively by the histological findings. The results of in‐vitro studies identified an efficient expression of the β‐gal gene within the spheroids infected with Ad‐CMVLacZ (LacZ‐encoding adenovirus vector with CMV promotor) and the presence of BCRF1 mRNA within the spheroids transfected with AdCMVvIL‐10 (vIL‐10‐expressing adenovirus vector with CMV promotor) under the condition of 1 MOI, for 1 h. Xenogeneic hepatocytes with a spheroidal shape showed comparable survival to syngeneic hepatocytes for up to 4 days after transplantation with co‐transplantation of the vIL‐10‐transfected hepatocytes. From this study, we concluded that adenovirus‐mediated vIL‐10 gene transfer prolongs the survival of xenogeneic hepatocyte spheroids. Furthermore, spheroids possess ideal properties for gene transfection, as well as cell transplantation.     

Intracranial injection of human meningioma cells in athymic mice: an orthotopic model for meningioma growth

OBJECT: Although human meningioma cells have been heterotopically implanted in nude mice, introducing these cells into intracranial locations seems more likely to reproduce normal patterns of tumor growth. To provide an orthotopic xenograft model of meningioma, the authors implanted a controlled quantity of meningioma cells at subdural and intracerebral sites in athymic mice. METHODS: Malignant (one tumor), atypical (two tumors), or benign (three tumors) meningiomas were placed into primary cell cultures. Cells (10(6)/10 microl) from these cultures and from an immortalized malignant meningioma cell line, IOMM-Lee, were injected with stereotactic guidance into the frontal white matter or subdural space of athymic mice. Survival curves were plotted for mice receiving tumor cells of each histological type and according to injection site. Other mice were killed at intervals and their heads were sectioned whole. Hematoxylin and eosin staining of these sections revealed the extent of tumor growth. CONCLUSIONS: The median length of survival for mice with malignant, atypical, or benign tumors was 19, 42, or longer than 84 days, respectively. Atypical and malignant tumors were invasive, but did not metastasize extracranially. Malignant tumors uniformly showed leptomeningeal dissemination and those implanted intracerebrally grew locally and spread noncontiguously to the ventricles, choroid plexus, convexities, and skull base. Tumors formed in only 50% of mice injected with benign meningioma cells, whereas injection of more aggressive cells was uniformly successful at tumor production. The three types of human meningiomas grown intracranially in athymic mice maintained their relative positions in the spectrum of malignancy. However, atypical meningiomas became more aggressive after xenografting and acquired malignant features, implying that there had been immune constraint in the original host. Tumor cells injected into brain parenchyma migrated to more optimal environments and grew best there. This model provides insights into the biology of meningiomas and may be useful for testing new therapies.     

Tissue-binding properties of a synthetic peptide DNA vector targeted to cell membrane integrins: a possible universal nonviral vector for organ and tissue transplantation

BACKGROUND: Gene delivery through a nonviral, receptor-mediated system widely expressed in transplanted tissue would have important advantages in transplantation, where gene delivery is performed ex vivo. Integrins are widely expressed cell surface receptors and can be targeted for gene delivery. METHODS: A synthetic 31 amino acid DNA vector (polylysine-molossin) comprising a 15-amino acid moiety for targeting cellular integrins (derived from the snake venom, molossin) and a 16-amino acid polylysine moiety for DNA-binding, has been evaluated. The 31-amino acid vector, as well as its separate 15-amino acid integrin-binding and (lys)16 components, were individually synthesized, and a monoclonal antibody was raised to the molossin peptide for these studies. Binding to cell lines and tissue sections and capacity for gene delivery were examined. RESULTS: Flow cytometric studies with the ECV304 cell line demonstrated that the binding of polylysine-molossin and polylysine-molossin/DNA complexes involved both electrostatic and integrin-mediated interactions with the cells, with the electrostatic binding being sufficient for maximal binding. However, binding to cellular integrins was essential for successful gene transfer. Binding studies on frozen tissue sections of the rat and pig demonstrated that the molossin peptide bound to many cell types of interest in transplantation, but not to all. Among the negative tissues were vascular endothelium and pancreatic islets. Small species differences in tissue binding were noted between the rat and pig. CONCLUSIONS: This study defines the cooperative nature of the binding of this vector system to target cells and establishes the cell types most likely to be effectively targeted for DNA transfer.     

Transduction of rabbit saphenous artery: a comparison of naked DNA, liposome complexes, and adenovirus vectors.

The methods and efficiency of gene transfer into rabbit saphenous artery were examined in this study. The purpose was to develop an animal model capable of evaluating the use of angiogenic gene therapy to revascularize necrotic bone more rapidly and completely than by surgical implantation of blood vessels alone. The success of transduction using adenovirus vectors, liposome/DNA complexes, and naked DNA was evaluated with delivery to both intra-luminal and adventitial sites. Intra-luminal and adventitial (extra-luminal) application was used for the viral and liposome methods. Naked DNA was evaluated only in the intra-luminal site, based upon previous reports. Relative transduction success was expressed as the percentage of total cells with beta-galactosidase activity. A 20-mm length of saphenous artery exposed surgically was targeted for lacZ gene transfer. Two days after transduction, the arteries were harvested and stained with X-gal for beta-galactosidase activity. The percentage of endothelial, media and adventitial cells with beta-galactosidase activity was determined. Intra-arterial injection of adenovirus vector transduced the largest amount of cells in all three areas of the vessel (endothelium, media and adventitia). The adenovirus vectors when applied to the adventitia only transduced adventitial cells. Following intra-arterial injection of liposome/DNA complexes transduction was detected only in endothelium. Extra-luminal liposome and intra-arterial naked DNA delivery resulted in no detectable gene transfer. Intra-arterial delivery of an adenovirus vector would likely provide optimal gene transfer for possible angiogenic gene therapy.     

A novel method for gene delivery and expression in esophageal epithelium with fibrin glues containing replication-deficient adenovirus vector

Background: Gene transfer to the esophageal epithelium holds the potential for the therapy of malignant as well as premalignant lesions in the upper gastrointestinal tract. Replication-deficient recombinant adenoviruses represent an efficient means of introducing genes in vivo into cells in a variety of organs. The majority of in vivo studies utilize direct submucosal injection for delivery of the viral vectors into the locoregional area of the gut; transferring genes into epithelial cells, however, is difficult because viruses are retained in the subepithelial space. To establish the efficient method for gene transfer into the epithelial cells, we have developed a multiluminal spray catheter that can be passed through the accessory channel of an endoscope, and we have evaluated the feasibility of fibrin glues as a vehicle of recombinant adenoviruses in a porcine model. Methods: The fibrinogen solution and the thrombion solution containing an E1/E3 deleted recombinant adenovirus expressing the bacterial lacZ gene (Ad-lacZ) were endoscopically sprayed on the porcine esophagus through the catheter attached to the dual-barrel syringe. Twenty-four hours after gene delivery, -galactosidase activity of the esophagus was determined under the microscope following X-gal staining. Results: The fibrin glue could be locally sprayed on the porcine esophagus by using the multichannel catheter through the endoscope. Attachment of the fibrin glue comtining Ad-lacZ caused strong -galactosidase staining on epithelial cells in the mucosal surface, but not in the basal cell layer. Conclusion: Endoscopic local delivery of recombinant adenoviruses in aerosolized fibrin glues through a multiluminal catheter could provide an optimal technique for gene transfer into epithelial cells in the mucosal surfece, which may have important implications for the treatment of human esophageal premalignant diseases.     

Enhanced transgene expression in androgen independent prostate cancer gene therapy by taxane chemotherapeutic agents.

PURPOSE: Chemotherapy is often used as a primary therapy for metastatic cancer because it kills cells en masse. However, high doses of chemotherapeutic drugs can cause toxicity in nontarget organs. Gene therapy may provide a better alternative to chemotherapy because its targeting of specific genes may reduce the undesirable toxicity associated with chemotherapy. We evaluated whether the chemotherapeutic agent docetaxel or paclitaxel may be combined with gene therapy to create a new therapeutic regimen for metastatic androgen independent prostate cancer. MATERIALS AND METHODS: The 2 androgen independent prostate cancer cell lines PC-3 and DU 145 were treated with docetaxel or paclitaxel. Three recombinant adenoviruses containing p21WAF-1/CIP1, p53 protein or beta-galactosidase complementary DNA under the control of cytomegalovirus promoter were used to determine transgene expression. They were evaluated by Western blot analysis, beta-galactosidase activity or in vitro growth assays. The [(3)H] labeled E1 deleted adenovirus dl312 was used to determine adenovirus uptake into cells. RESULTS: Docetaxel and paclitaxel enhanced adenovirus mediated transgene expression. Docetaxel appears to be a more potent growth inhibitor in vitro. Elevated transgene expression in virus infected cells induced by these 2 drugs was produced by increased cytomegalovirus promoter activity rather than increased virus uptake. CONCLUSIONS: The potential synergy of gene therapy with docetaxel and paclitaxel may be an important direction for future therapy for metastatic androgen independent prostate cancer.     

Adenoviral gene transfer in the peripheral nervous system

Background Viral vectors have gained widespread use as vehicles for somatic gene transfer, and the targeted expression of foreign proteins by these vectors offers advantages over the systemic administration of the drugs in some therapeutic situations. Selective virus-mediated gene transfer to the peripheral nervous system (PNS), however, remains to be established. There are no data showing efficiency of protein transduction in the PNS, which consists of a variety of cell types, many of which are postmitotic. Methods We prepared the first-generation replication-deficient recombinant adenovirus vectors engineered to express LacZ. Eight-week-old Wister rats were used in this study. Adenovirus vector (5 μl) containing the LacZ gene (5 × 10⁸ pfu) was injected into rat sciatic nerves or the dorsal root ganglia at the level of L5. The sciatic nerves, the dorsal root ganglia, and the spinal cords were obtained 7, 14, 21, and 28 days after injection. Expression of LacZ was assessed by X-gal histochemistry and β-gal immunohistochemistry. Results Following injection of the adenovirus carrying the LacZ gene into the sciatic nerve, LacZ expression was seen mainly in the Schwann cells and the small neurons in the dorsal root ganglion. In contrast, expression was observed in the primary nerve terminals of the spinal dorsal horn and the small to large dorsal root ganglion neurons and the Schwann cells after injection of the vectors into the L5 dorsal root ganglion. There were no side effects in rats with injection in the dorsal root ganglia or the sciatic nerve. Conclusions The present study shows efficient protein transduction by adenovirus vectors in the PNS. It is noted that injection of the virus into the dorsal root ganglia leads to extensive expression of LacZ in the spinal cord, the dorsal root ganglia, and the sciatic nerves.     

腺病毒载体介导反义Src基因治疗胶质瘤

目的 探讨腺病毒载体介导的反义Src基因对胶质瘤生长的作用及其机制.方法 应用携带反义Src基因的重组腺病毒体外感染C6胶质瘤细胞,观察其对细胞形态、生长曲线和克隆形成率的影响.建立大鼠胶质瘤动物模型,原位注射重组腺病毒.观察其治疗作用,Western blot检测肿瘤组织Src、Ras、MAPK蛋白的表达,实时逆转录-聚合酶链反应(Real-time RT-PCR)检测肿瘤组织Caspase-3、Caspase-8 mRNA的表达.结果 体外研究表明感染反义Src基因的C6胶质瘤细胞生长受到显著抑制.体内研究表明应用携带反义Src基因的腺病毒原位注射治疗大鼠胶质瘤能够抑制肿瘤生长,减少Src、Ras、MAPK蛋白的表达,增加Caspase-3、Caspase-8 mRNA的表达.结论 腺病毒载体介导的反义Src基因能够抑制大鼠胶质瘤细胞的生长.Src-Ras-MAPK信号通路参与胶质瘤的生长,抑制该信号通路可以增加凋亡通路关键蛋白Caspase-3、Caspase-8的表达.     

Multidrug resistance gene (MDR1) expression in human brain tumors.

Multidrug resistance for many types of cancer outside the central nervous system (CNS) has been found to be due to the overexpression of the multidrug resistance gene MDR1, of which the gene-product P-glycoprotein acts as a membrane-bound efflux pump for many anticancer drugs. To examine whether brain tumors overexpress the MDR1 gene, 25 brain-tumor specimens were subjected to Northern blot analysis: 10 gliomas, eight meningiomas, three schwannomas, one malignant lymphoma, and three tumors metastatic to the brain. Ten fresh-frozen autopsy specimens of various parts of normal brain were also analyzed. Blots were hybridized with 32P-labeled Chinese hamster complementary deoxyribonucleic acid (cDNA) and 32P-labeled human MDR1 cDNA. The MDR1 gene messenger ribonucleic acid (mRNA) was detected in two tumors using the Chinese hamster probe (one sphenoid wing meningioma and one metastatic prostate tumor) and in one CNS lymphoma using the human probe. Intact mRNA could not be extracted from the fresh-frozen autopsy specimens of normal brain. Seventeen tumors were examined for P-glycoprotein by immunohistochemical staining using murine monoclonal antibody C219: eight gliomas, eight meningiomas, and one craniopharyngioma. The neoplastic cells from two gliomas and three meningiomas and the blood vessels within six gliomas and two meningiomas stained positively for P-glycoprotein. Seven of 10 normal brain specimens stained positively for P-glycoprotein in blood vessels but no specimen demonstrated staining of parenchymal cells. This study demonstrates that the MDR1 gene can be detected in normal brain, and in malignant, benign, and metastatic lesions. P-glycoprotein can be present in tumor blood vessels even when it is not seen in neoplastic cells. Although the role of P-glycoprotein in tumor blood vessels needs to be further examined and more clearly defined, drug resistance in malignant primary brain tumors may result from characteristics not solely of neoplastic cells but also tumor vasculature.     

alpha1,3-Galactosyltransferase-gene knockout in cattle using a single targeting vector with loxP sequences and cre-expressing adenovirus

BACKGROUND: Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine alpha1,3-galactosyltransferase (alpha1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and alpha1,3GT-gene knockout cattle. METHODS: alpha1,3GT gene-disruption was accomplished using primary fetal fibroblasts with a single targeting vector, a promoter-less positive selection vector containing IRES (internal ribosome entry site)-antibiotic-resistance gene (neo) cassette and loxP sequences. At each step in establishing heterozygous and homozygous knockout cell lines, the antibiotic-resistance gene cassette in the targeted allele was removed by a Cre-loxP recombination system that utilizes an adenovirus with transient Cre recombinase expression. A nuclear transfer was performed using alpha1,3GT fetal fibroblasts, and one alpha1,3GT knockout calf was generated but died shortly after birth (day 287). RESULTS: Necropsy revealed normal morphology in all organs. The calf weighed 22.3 kg at birth and this value is within the normal range. CONCLUSION: The alpha1,3GT knockout- and antibiotic-resistance gene free (alpha1,3GT(-/-)neo-) cells could be cloned normally. Thus, cloned cattle from alpha1,3GT(-/-) neo- cells are potentially safer for human use. Additionally, our strategy is faster and more economical than backcrossing to produce homozygous knockouts. This method should be useful for future production of knockouts of multiple genes in livestock.     

Recombinant adenovirus mediated prostate-specific enzyme pro-drug gene therapy regulated by prostate-specific membrane antigen (PSMA) enhancer/promoter

Gene directed enzyme pro-drug therapy (GDEPT) is one of the adjuvant therapeutic regimens for advanced prostate adenocarcinoma, and this research intended to explore how to apply targeting therapy of prostate adenocarcinoma under the mediation of a promoter/enhancer of prostate-specific membrane antigen (PSMA(EP)) as a specific regulatory element. Recombinant adenoviruses (Ad-PSMA(E-P)-enhanced green fluorescent protein [EGFP], Ad-CMV-EGFP, Ad-PSMA(E-P)-CD, and Ad-CMV-CD) were constructed and could express cytosine deaminase (CD) or the EGFP reporter gene driven by a PSMA(EP) or cytomegalovirus (CMV) promoter. LNCaP, CL-1, MCF-7, and A549 were infected with CD-produced recombinant adenoviruses and treated with pro-drug 5-fluorocytosine (5-FC) in vivo and vitro; then, the growth inhibition of the cells and the cell cycle variation were assessed by an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay and flow cytometry. Growth suppression of the xenograft tumor was also adopted to evaluate the efficiency of the suicide system. Morphologic changes after treatment in vivo were assessed with hematoxylin and eosin staining. In the 4 examined cancer cell lines, PSMA-positive prostate cancer cells LNCap and CL-1 were exclusively sensitive to the Ad-PSMA(E-P)-CD/5-FC system. The S phase of cell cycle arrest was thought to be involved in the cytotoxicity of 5-fluorouracil (5-FU) converted from 5-FC by CD. CL-1 implanted Athymic BALB/c mice showed growth inhibition of tumors when they were treated with the Ad-PSMA(E-P)-CD/5-FC system without systemic conversion toxicity. The PSMA-based, CD-produced adenovirus, deserving further investigation in the future, might be a good candidate for targeting gene therapy of prostate adenocarcinoma.     

肝癌基因治疗的机遇和挑战

Gene therapy has emerged as an efficient modality to treat human diseases.This method is based on the transfer of genetic material to tissues to induce a curative effect.Gene therapy vectors are molecular constructs used to facilitate the penetration of genomic sequences inside the cells.Viral vectots have however several limitations when administered directly to the patient.They may cause significant toxicity by activating innate immunity or by eliciting an adaptive immune response against viral proteins.In addition,targeting the vector to the desired site is an issue when given systemically.The use of cells as vehicles for gene therapy vectors has many advantages.The combination of cell-viro-gene therapy has been thought as a new and promising strategy for therapy of cancer.The targeting vector to cancer stem cells will become a new direction in the field of gene therapy.In this article,we will introduce progressions,limitations and future directions of gene therapy of liver cancer.