Establishment of a Tumor-bearing Mouse Model Stably Expressing Human Tumor Antigens Survivin and MUC1 VNTRs

The eukaryotic vectors VR1012 expressing survivin or 33 tandem repeats of human mucin 1(MUC1)(VNTRs),namely,VR1012-S and VR1012-VNTR(VNTR=variable number of tandem repeat),were constructed by cloning s...     

Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases

Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to gamma-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases activities.     

Co-administration of Interleukin-2 Enhances Cellular and Humoral Immune Responses to HIV Vaccine DNA Prime／MVA Boost Regime

Interleukine-2 (IL-2) is a growth factor for antigen-stimulated T lymphocytes and is responsible for T-cell clonal expansion after antigen recognition. It has been demonstrated that DNA vaccine-elicited immune responses in mice could be augmented substantially by using either an IL-2 protein or a plasmid expressing IL-2. Twenty mice, divided into four experimental groups, were immunized with: (1) sham plasmid; (2) HIV-1 DNA vaccine alone; (3) HIV-1 DNA vaccine and IL-2 protein; or (4) HIV-1 DNA vaccine and IL-2 plasmid, separately. All the groups were immunized 3 times at a 2-week interval. Fourteen days after the last DNA vaccine injection, recombinant MVA was injected into all the mice except those in group 1. ELISA and ELISPOT were employed to investigate the effect of IL-2 on DNA vaccine immune responses. The obtained results strongly indicate that the efficacy of HIV vaccine can be enhanced by co-administration of a plasmid encoding IL-2.     

Antitumor immunity induced by tumor cells engineered to express a membrane-bound form of IL-2

Transduction of cytokine gene into tumor cells is a promising method of tumor therapy, but the value is limited by accompanying side effects. To focus antitumor immune response to tumor antigen-specific CTL, we developed an antitumor vaccine by transfecting modified IL-2 gene in a membrane-bound form (mbIL-2) into B16F10 melanoma cells. The mbIL-2 clone showed reduced tumorigenicity and metastatic ability, and inhibited metastasis and prolonged the survival of mice against B16F10 cells. The inhibition of B16F10 metastasis by mbIL-2 was accompanied by the increment of CD8(+) T cells. The metastasis of mbIL-2 clone was significantly increased in the CD8(+) T cell-depleted mice, but not in CD4(+) T cell depleted mice. Spleen cells immunized with the mbIL-2 clone showed higher CTL activity towards B16F10 cells than those immunized with control cells. The size of CD8(+) T cell population in the lung of mice injected with the mbIL-2 clone was markedly greater than that of mice injected with B16F10 cells, but there was no detectible change in CD4(+) and CD8(+) T cell populations of lymph nodes and spleen. These results suggest that when the mbIL-2 clone is introduced into the blood stream, it migrates mainly to lung and activates CD8(+) T cells in situ, possibly by direct priming. Such a tumor vaccine may ameliorate the toxic side effects encountered with conventional cytokine gene therapy.     

Gene expression profiling of tumor-associated macrophages after exposure to single-dose irradiation

Radiotherapy (RT) is a common cancer treatment approach that accounts for nearly 50% of patient treatment; however, tumor relapse after radiotherapy is still a major issue. To study the crucial role of tumor-associated macrophages (TAMs) in the regulation of tumor progression post-RT, microarray experiments comparing TAM gene expression profiles between unirradiated and irradiated tumors were conducted to discover possible roles of TAMs in initiation or contribution to tumor recurrence following RT, taking into account the relationships among gene expression, tumor microenvironment, and immunology. A single dose of 25 Gy was given to TRAMP C-1 prostate tumors established in C57/B6 mice. CD11b-positive macrophages were extracted from the tumors at one, two and three weeks post-RT. Gene ontology (GO) term analysis using the DAVID database revealed that genes that were differentially expressed at one and two weeks after irradiation were associated with biological processes such as morphogenesis of a branching structure, tube development, and cell proliferation. Analysis using Short Time-Series Expression Miner (STEM) revealed the temporal gene expression profiles and identified 13 significant patterns in four main groups of profiles. The genes in the upregulated temporal profile have diverse functions involved in the intracellular signaling cascade, cell proliferation, and cytokine-mediated signaling pathway. We show that tumor irradiation with a single 25-Gy dose can initiate a time-series of differentially expressed genes in TAMs, which are associated with the immune response, DNA repair, cell cycle arrest, and apoptosis. Our study helps to improve our understanding of the function of the group of genes whose expression changes temporally in an irradiated tumor microenvironment.     

Click-Chemistry-Mediated Cell Membrane Glycopolymer Engineering to Potentiate Dendritic Cell Vaccines

Dendritic cell vaccine (DCV) holds great potential in tumor immunotherapy owing to its potent ability in eliciting tumor-specific immune responses. Aiming at engineering enhanced DCV, we report the first effort to construct a glycopolymer-engineered DC vaccine (G-DCV) via metabolicglycoengineering and copper-free click-chemistry. Model G-DCV was prepared by firstly delivering tumor antigens, ovalbumin (OVA) into dendritic cells (DC) with fluoroalkane-grafted polyethyleneimines, followed by conjugating glycopolymers with a terminal group of dibenzocyclooctyne (DBCO) onto dendritic cells. Compared to unmodified DCV, our G-DCV could induce stronger T cell activation due to the enhanced adhesion between DCs and T cells. Notably, such G-DCV could more effectively inhibit the growth of the mouse B16-OVA (expressing OVA antigen) tumor model after adoptive transfer. Moreover, by combination with an immune checkpoint inhibitor, G-DCV showed further increased anti-tumor effects in treating different tumor models. Thus, our work provides a novel strategy to enhance the therapeutic effectiveness of DC vaccines.     

Functional Mechanism of Resveratrol in Inhabiting Growth of Cells ls174t and Its Mechanism in Subcutaneously Transplanted Tumor of Nude Mice

To explore the functional mechanism of Resveratrol against colon cancer cells Is174t and the growth of colon cancer tissue of tumor-bearing mice,MTT method was used to observe the functions of resveratrol for inhibition against cells ls174t in vitro.Transmission electron microscope was used to observe the cell apoptosis.FCM assay was performed to measure the change of the cell apoptosis rate and of cell cycle,RT-PCR method was used to detect the expressions of bc1-2 and bax mRNA.Western blot method was used to detect the expressions of bc1-2 and bax protein.Cells isi74t were transplanted subcutaneously to nude mice to observe the effect of resveratrol on the growth of subcutaneously transplanted tumor.RT-PCR method was used to detect the expressions of bc1-2 and bax mRNA in the tumor tissue.Western blot method was used to detect the expressions of bc1-2 and bax protein in the tumor tissue.Resveratrol has an effect of inhibiting proliferation of cells ls174t in vitro(P<0.01).It is able to induce the apoptosis of cells Is174t,causing the decrease in the expression of bc1-2 and the increase in the expression of bax.Resveratrol could inhibit the growth of subcutaneously transplanted tumor of nude mice(P<0.05),causing the decrease in the expression of bc1-2 and the increase in the expression of bax.Resveratrol can inhibit the growth of cells 174t and the growth of subcutaneously transplanted tumor.The mechanism is possibly related to the induction of the cell apoptosis and the regulation of bc1-2/bax expression.     

Functional Mechanism of Resveratrol in Inhabiting Growth of Cells Is174t and Its Mechanism in Subcutaneously Transplanted Tumor of Nude Mice

To explore the functional mechanism of Resveratrol against colon cancer cells ls174t and the growth of colon cancer tissue of tumor-bearing mice, MTT method was used to observe the functions of resveratrol for inhibition against cells ls174t in vitro. Transmission electron microscope was used to observe the cell apoptosis. FCM assay was performed to measure the change of the cell apoptosis rate and of cell cycle. RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA. Western blot method was used to detect the expressions of bcl-2 and bax protein. Ceils ls174t were transplanted subcutaneously to nude mice to observe the effect of resveratrol on the growth of subcutaneously transplanted tumor, RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA in the tumor tissue. Western blot method was used to detect the expressions of bcl-2 and bax protein in the tumor tissue. Resveratrol has an effect of inhibiting proliferation of cells ls174t in vitro（P〈0.01）. It is able to induce the apoptosis of cells ls174t, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol could inhibit the growth of subcutaneously transplanted tumor of nude mice（P〈0.05）, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol can inhibit the growth of cells 174t and the growth of subcutaneously transplanted tumor. The mechanism is possibly related to the induction of the cell apoptosis and the regulation of bcl-2/bax expression.     

In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.     

Effect of delivery system on the pharmacokinetic and phototherapeutic properties of bis(methyloxyethyleneoxy)silicon-phthalocyanine in tumor-bearing mice

A Si(IV)-phthalocyanine bearing two methoxyethyleneglycol axial ligands bound to the central metal ion (SiPc) has been prepared by chemical synthesis and analyzed for its phototherapeutic activity after administration in a Cremophor or liposome formulation to C57B1/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma (LLC). The maximum drug accumulation in the tumor is found at 24 h after intraperitoneal injection, independent of the delivery system. However, the tumor concentration of SiPc in the Cremophor formulation is about two-fold higher, while the drug concentration in liver and skin shows similar trends with the two delivery systems. The drug accumulation and retention in the brain is much larger when using Cremophor emulsion. Photodynamic therapy (672 nm, 370 mW m⁻², 360 J cm⁻²) at 24 h after the injection of Cremophor emulsion- or DPPC liposome-formulated SiPc causes a very efficient and similar response for the LLC (8 versus 22 mm mean tumor diameter for the control groups at 21 days after phototreatment). These very promising effects, obtained both at higher and lower tumor drug concentrations, clearly demonstrate the potential phototherapeutical activity of the newly synthesized SiPc.     

Sulfamide-substituted-BODIPY based fluorescence drugs: Synthesis,spectral characteristics,molecular docking,and bioactivity

BODIPY derivatives have attracted much attention in the field of biological probes, but probes with a single imaging function are no longer innovative. In this paper, two multifunctional sulfonamide-BODIPY derivatives were designed and synthesized. Photophysical properties, cytotoxicity, in vitro and in vivo imaging, apoptosis, cell cycle, and molecular docking simulation were studied. The results showed that the compound had low cytotoxicity to normal cells, but had strong inhibitory effect on tumor cells. The IC₅₀ value of compound 3 on HCT-116 cells was 58.61 ± 4.91 μmol/L, and 4 on HeLa cells was 52.29 ± 10.93 μmol/L. Cell imaging and mice experiments demonstrated that the probe had excellent biocompatibility and potential tumor targeting; and in vivo imaging of mice at different time periods showed that the fluorescence intensity of probes in subcutaneous lung tumor gradually increased with the extension of time. In addition, the flow cytometry analysis of 3 showed that the G1 phase of HCT-116 cells was inhibited and apoptosis of tumor cells was promoted. In molecular docking simulation, sulfonamide-BODIPY derivatives had high affinity scores with CDK2: −8.0 and −8.4 kcal·mol⁻¹, and the multiple interactions with receptors provided conditions for the probes to recognize tumor cells.     

ENHANCED GROWTH AND EXPERIMENTAL METASTASIS OF CHEMICALLY INDUCED TUMOR IN ULTRAVIOLET IRRADIATED SYNGENEIC MICE

Abstract— Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV-B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV-B irradiation were banks of 6 Westinghouse FS-40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30-min dorsal UV-B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 × 10⁵ J m^-2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16- to 35-fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.     

Distribution of metal-free sulfonated phthalocyanine in subcutaneously transplanted murine tumors

Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H(2)PcS(2.4)) was recently proved to be an efficient photosensitizer for the photodynamic therapy. Fluorescence spectral imaging microscopy was applied here to study localization and relative concentration of H(2)PcS(2.4) with micron-scale resolution in subcutaneously transplanted murine tumors: Ehrlich mammary gland carcinoma (EC), Lewis lung carcinoma (LLC), P388 lymphoid leukemia (P388) and B16 melanoma (B16). The study of cryogenic tissue sections prepared 24 h after H(2)PcS(2.4) intravenous injection revealed that H(2)PcS(2.4) was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H(2)PcS(2.4) was documented in tumor cells and adjacent non-tumor tissues (skin structures, fatty tissue, connective tissue enriched in fibrous component and infiltrated with fibroblasts and macrophages) for all the studied tumor models. P388 and B16 were stained with H(2)PcS(2.4) less than adjacent skin structures, whereas EC and LLC accumulated H(2)PcS(2.4) alike or higher than particular skin structures. Staining of EC and LLC was similar and ca. 1.4 and 2 times higher than that of B16 and P388, respectively, thus revealing the differences in ability of particular tumor strains to H(2)PcS(2.4) accumulation. The H(2)PcS(2.4) concentration in remote healthy tissues (skin, muscles and connective tissue) was 2-3 times lower as compared with the analogous tissue structures from the tumor area, whereas subcutaneous fatty tissue staining did not depend on the tissue-to-tumor distance. The tissue distribution of H(2)PcS(2.4) predefines the combined action of two photodynamic damage mechanisms: eradication of tumor due to the direct tumor cell destruction and suppression of tumor growth due to the injury of growth supporting system.     

Stage-specific alterations of cyclin expression during UVB-induced murine skin tumor development

We have evaluated the in vivo correlation between the expression of cell cycle markers and skin tumor development in SKH-1 hairless mice in a complete photocarcinogenesis protocol. Irradiated mice developed an average of 16 tumors per animal by week 23 with the average number of carcinomas per mouse being 2.1. The expression of p53 and cyclins A and D1 was confined initially to sporadic single cells and gradually developed into foci of patchy intense staining in the basal and granular layers of UVB-exposed epidermis. p53 was expressed in all the papilloma sections examined, whereas cyclins D1 and A were expressed in 68 and 71% of these lesions, respectively. In UVB-induced squamous cell carcinomas (SCC), p53 was expressed in >90% of the tumors, whereas cyclin D1 was detected in 55% of the lesions, and cyclin A staining was limited to 27%. These immunohistochemical observations were confirmed by Western blotting and protein kinase assays. We observed an early wave of cyclin A overexpression and cyclin A protein kinase activity preceding the appearance of detectable tumors. Cyclin D1 and p53 overexpression were coupled with the development of tumors, and these changes are likely to be relevant to the pathogenesis of these lesions.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

Tumor-associated antigenic differences between the primary and the descendant metastatic tumor cell populations.

The existence of antigenic differences between cell populations in the local growth of the 3LL tumor (L-3LL) and its lung metastases (M-3LL) was studied. Normal C57BL/6 spleen cells sensitized in vitro for 5 days against L-3LL monolayers lysed preferentially L-3LL targets but not M-3LL tumor cell targets. Conversely, anti-M-366-sensitized lymphocytes killed M-3LL targets more efficiently than they killed L-3LL targets. Furthermore, spleen cells from mice bearing subcutaneous L-3LL tumors were significantly more cytotoxic to L-3LL targets than to M-3LL targets and vice versa. M-3LL cells were found also to be more resistant in vitro and in vivo to natural killer cells than were L-3LL tumor cells. M-3LL cells were more resistant than L-3LL cells to hybrid resistant mechanisms when they were inoculated into F1 (C3Heb x C57BL/6) or F1 (BALB/c x C57BL/6) mice. Anti-M-3LL lymphocytes generated both in vitro and in vivo, but not anti-L-3LL lymphocytes, admixed with L-3LL or M-3LL tumor cells and inoculated into footpads of syngeneic recipients suppressed the development of lung metastases. These results suggest that metastatic cells are indeed phenotypic variants of the local growing tumor cell populations. Presumably, these variants are selected for their capacity to home to and grow in the lungs, and for their resistance to specific immune effects initially evoked against the local tumor and to nonspecific natural killer cells. These data may prove to be of importance with respect to any rational approach to the problem of immunotherapy.     

Tetrandrine Suppresses Human Brain Glioblastoma GBM 8401/luc2 Cell-Xenografted Subcutaneous Tumors in Nude Mice In Vivo

Tetrandrine (TET), a bisbenzylisoquinoline (BBI) alkaloid, is isolated from the plant Stephania tetrandra S. Moore and has a wide range of biological activity, including anticancer properties in vitro and in vivo. At first, we established a luciferase-expressing stable clone that was named GBM 8401/luc2 cells. Herein, the primary results indicated that TET reduced the total cell viability and induced cell apoptosis in GBM 8401/luc2 human glioblastoma cells. However, there is no available information showing that TET suppresses glioblastoma cells in vivo. Thus, we investigated the effects and mechanisms of TET on a GBM 8401/luc2 cell-generated tumor in vivo. After the tumor volume reached 100–120 mm³ in subcutaneously xenografted nude mice, all of the mice were randomly divided into three groups: Group I was treated with phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 25 mg/kg of TET, and Group III with 50 mg/kg of TET. All mice were given the oral treatment of PBS or TET by gavage for 21 days, and the body weight and tumor volumes were recorded every 5 days. After treatment, individual tumors, kidneys, livers, and spleens were isolated from each group. The results showed that TET did not affect the body weights, but it significantly decreased the tumor volumes. The TET treatment at 50 mg/kg had a two-fold decrease in tumor volumes than that at 25 mg/kg when compared to the control. TET decreased the total photon flux, and treatment with TET at 50 mg/kg had a lower total photon flux than that at 25 mg/kg, as measured by a Xenogen IVIS imaging system. Moreover, the higher TET treatment had lower tumor volumes and weights than those of the lower dose. The apoptosis-associated protein expression in the tumor section was examined by immunohistochemical analysis, and the results showed that TET treatment reduced the levels of c-FLIP, MCL-1, and XIAP but increased the signals of cleaved-caspase-3, -8, and -9. Furthermore, the hematoxylin and eosin (H & E) staining of kidney, liver, and spleen tissues showed no significant difference between the TET-treated and control groups. Overall, these observations demonstrated that TET suppressed subcutaneous tumor growth in a nude-mice model via the induction of cell apoptosis.     

Parkin Induces Upregulation of 40S Ribosomal Protein SA and Posttranslational Modification of Cytokeratins 8 and 18 in Human Cervical Cancer Cells

Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18.     

基于单克隆抗体G250修饰的肿瘤细胞靶向基因载体研究

用宫颈癌细胞Hela表面高表达G250抗原的单克隆抗体G250修饰非病毒基因载体, 获得肿瘤靶向基因载体. 通过注射G250杂交瘤细胞于小鼠腹腔, 制备富含G250mAb的腹水, 用正辛酸-硫酸铵沉淀法和Protein A Agarose分离纯化, 获得高纯度的G250mAb. 通过二硫键将PEI与G250mAb偶联, 得到修饰的基因载体G250mAb-PEI, 研究其转基因靶向性. 结果表明, G250mAb-PEI对Hela细胞的基因转染具有显著的靶向性, 对Hela细胞的转基因效率是肝癌细胞HepG2(G250阴性)的2倍; 而对正常血管平滑肌细胞(SMC)的基因转染效率比Hela低近20倍, G250mAb修饰与否对SMC没有靶向性; 对3T3细胞的毒性显著低于未修饰的PEI, 表明G250mAb-PEI是一种高效、低毒和具有靶向性的基因载体.     

Stable Cell Line of Human SH-SY5Y Uniformly Expressing TWIK-Related Acid-Sensitive Potassium Channel and eGFP Fusion

TWIK-related acid-sensitive potassium channels (TASK3) are pharmacological targets of CNS inflammation induced by acidification. They function as molecular switches between survival and death of neurons. In this report, TASK3 cloned from human brain cDNA was tagged with enhanced green fluorescent protein (eGFP), and the fusion gene was transiently expressed in human neuroblastoma SH-SY5Y cells. A cell line stably expressing TASK-eGFP fusion proteins was generated from transient expression cells by using fluorescence-activated cell sorting followed by antibiotic selection. The uniform expression of TASK3 fusion proteins was further confirmed by flow cytometry. Moreover, the localization of TASK3 tagged with eGFP was checked by confocal microcopy. TASK3-eGFP fusion proteins are observed on the SH-SY5Y cell membrane. The strategies using eGFP as a fusion tag facilitate the monitoring of the TASK3 expression and enable the successful employment of FACS for screening and construction of cell lines stably expressing TASK3. The TASK3 overexpression cell line will lay a fundamental for the in vitro evaluation of TASK3 function during hypoxic/ischemic injury.