Inhibited neoplastic phenotype by the c-Ha-ras antisense RNA.

A 2.0 kb fragment DNA plasmid which expresses antisense to the upstream first exon of c-Ha-ras oncogene was transfected into Ha-ras transformed cell lines, GCM-3T3 and REF-4.3. The transfection leads to the inhibition of malignant behaviour, shown by decreasing of growth speed, colony forming ability on soft agar, tumorigenicity in nude mice and increasing of differentiation degree. In GCM-3T3 cells the lung metastasis frequency became much less (from 60% to 12.5%) after the transfection and the expression of ras oncogene product, and p21 protein was obviously decreased in the transfected cells. This work has first shown the inhibitory effect of antisense RNA on neoplastic behaviour in China.     

HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing

Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.     

The antisense c-Ha-ras oligodeoxynucleotides inhibit the proliferation of the gastrocarcinoma-DNA-transformed rat 3-3 cells and reduce the p21 expression of the cells

The Rat 3-3 is a secondary transformant of the rat fibroblast cell line (Rat-1) transfected with total DNA of a gastrocarcinoma cell line BGC-823. The cells over-express the c-Ha-ras oncogene which contains point mutation at the 12th codon. In order to determine how the activated c-Ha-ras oncogene expression governs the cell's transformation, two pentadecadeoxy-nucleotides AS-1 and AS-2 were synthesized. AS-1 was complementary to the single strand of the first three codons and the upstream sequence close to the ribosome binding site of c-Ha-ras mRNA. AS-2 was complementary to the 3' end of the first intron and the 5' end of the second exon of c-Ha-ras unripe RNA enclosed in the nucleus. The oligonucleotides could block either the translation of c-Ha-ras mRNA or the splicing of c-Ha-ras unripe RNA, thus inhibiting the expression of the activated c-Ha-ras oncogene and the proliferation of the transformed cells Rat 3-3. The inhibitory effect increased with a growing concentration of the antisense oligodeoxynucleotide (from 4-10 mumol/L) and reached its peak at 12 h after Rat 3-3 was treated with AS-1 and AS-2. This effect became weaker afterwards. The p21 level, product of c-Ha-ras, in the Rat 3-3, was examined by ELISA method. The result shows that the amount of p21 in the growth-inhibited cells is about 30% of that of the control cells.     

IDENTIFICATION OF A TRANSFORMING ras ONCOGENE IN AN ULTRAVIOLET RADIATION-INDUCED CORNEAL TUMOR OF Monodelphis domestica

Chronic exposure of the gray, short-tailed oppossum, Monodelphis domestica to ultraviolet radiation (UVR) induces mesenchymal tumors of the cornea. High molecular weight DNA samples from 6 UVR-induced corneal tumors were assayed for their ability to transform NIH 3T3 cells to tumorigenicity. NIH 3T3 cells transfected with DNA from 5 of the corneal tumors produced 14 tumors in nude mice. Cell lines were established from these tumors. DNA from 13 of 14 tumor cell lines contained repetitive opossum DNA sequences. Southern blot analysis revealed that DNA from 3 of 4 cell lines derived from tumorigenic NIH 3T3 cells transfected with DNA from a single oppossum tumor contained opossum Ki-ras oncogene sequences in addition to the murine Ki-ras gene. Northern blot analysis of mRNA from a mouse tumor cell line containing opossum Ki-ras gene sequences showed mRNA species identical in size to opossum Ki-ras mRNA, as well as murine Ki-ras mRNA species. These results suggest that an activated Ki-ras oncogene was present in one of the original opossum corneal tumors tested. Thus, activation of Ki-ras may play a role in the development of UVR-induced corneal tumors in Monodelphis domestica. Further characterization of ras oncogenes in these opossum tumors may provide information on the molecular mechanisms by which UVR induces corneal tumors in this species.     

Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays

The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.     

ENHANCEMENT OF ULTRAVIOLET-DNA REPAIR IN den V GENE TRANSFECTANTS and T4 ENDONUCLEASE V-LIPOSOME RECIPIENTS

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and into wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells.     

A cDNA CLONE WITH ANTIONCOGENE ACTIVITY

A cDNA clone, p14--6, which has an antioncogene activity on the v--Ki--Ras oncogene-transformed malignant cell line DT, was found. This clone was recovered from the revertantR14 cells, which had been isolated by transfections of DT cells with a normal human fibro-blast cDNA library cloned in pcD2, an Okayama-Berg vector. When transfected into DT cells,p14--6 clone gave rise to phenotypical flat reversion in 5--15% of DT transfectant colo-nies. The p14--6--transfected flat cell line, RR, was proven to be a true revertant with signif-icantly reduced malignancy by in ritro and in riro malignancy tests. All other clones recov-ered from R14 cells were unable to cause this reversion. Molecular hybridizations showedthat the p14--6 was inserted into RR genome as tandem repeats, and no structural changewas found in the D--Ki--Ras oncogene in RR genome. These facts suggest that the antioncogeneactivity of the p14--6 clone on the DT cells may be exerted through expression of thecDNA contained in this clone. Possib     

Monitoring liposome-mediated delivery and fate of an antisense drug in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used for evaluation of the effectiveness of delivery and fate of a model 25-mer DNA-based phosphorothiate antisense drug in cells. The antisense molecule was delivered to the cells through a simple incubation and by using a cationic liposome (Cytofectin GS 3815). The cationic lipid interacted with the negatively charged antisense to form a more positively charged complex. It was observed that uptake of the liposome-antisense complex by the cell was dependent on concentration of lipid, duration of transfection, and the cell type. The antisense drug interacted with intracellular components such as proteins and additional steps were needed to quantify the free antisense. Proteinase-K was able to release antisense from proteins. However, the addition of sodium dodecyl sulfate (SDS) to the sample or running buffer was more effective than Proteinase-K to release both naked and liposome-bound antisense from the cellular materials. Analysis of single HeLa cells for uptake of the unbound and liposome-complexed antisense revealed averages of 8.9x10(-19) moles and 4.9x10(-18) moles, respectively. The amount of uptake, however, varied greatly among individual cells and depended on the delivery method. With liposome-mediated delivery, the relative standard deviation (RSD) for the amount of antisense in individual cells was 130%, while the variation was much smaller (RSD = 45%) when the cells were incubated with the unbound antisense. These uptake variations agreed with those obtained from flow cytometry analysis.     

重组质粒pIRES-EGFP-BCL 11B电转染幼稚T细胞的可视化研究

将BCL 11B基因插入pIRES-EGFP构建重组质粒真核表达载体pIRES-EGFP-BCL 11B,采用电转染法将重组质粒转入人幼稚T细胞,转染24 h后,用原子力显微镜(AFM)观察转染前后细胞的表面形态以及生物物理性质的变化.转染72 h后,用CCK-8试剂检测幼稚T细胞的增殖情况.分别对空转的幼稚T细胞组、空载体转染组(pIRES-EGFP naked plasmid)、重组载体转染组(pIRES-EGFP-BCL 11B recombinant vector)、无电转无质粒的幼稚T细胞组进行实验.结果表明,4组幼稚T细胞的体积、高度、半宽度、粗糙度、表面颗粒大小等参数发生了变化,细胞杨氏模量以及细胞硬度也呈现很大变化,CCK-8结果显示,重组质粒pIRES-EGFP-BCL 11B电转染人幼稚T细胞后影响细胞的增殖.     

Synergistic effect of peroxiredoxin II antisense on cisplatin-induced cell death

Peroxiredoxin II (Prx II) is known not only to protect cells from oxidative damage caused by hydrogen peroxide (H202), but also to endow cancer cells with resistance to both H202 and cisplatin and to grant them radioresistance. In this study, we examined whether Prx II antisense could enhance cisplatin-induced cell death. When gastric cancer cells were transfected with various concentrations of Prx II antisense plasmid, pPrxII/AS, and then treated with the same concentrations of cisplatin, Prx II antisense enhanced cisplatin-induced cell death. The combination index (CI) at all doses of the combination was below 1, indicating that Prx II antisense sensitized cisplatin-induced cell death. This synergism was also observed in the cells transfected with a Prx II antisense oligomer. Our present results, therefore, suggest that Prx II antisense would be a very good sensitizer for cisplatin, and that Prx II as a target for chemosensitizers constitutes a promising avenue for future research.     

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.     

TNFa在血管内皮细胞ECV304中的靶向表达研究

在缺失了3'LTR U3区内病毒的启动子／增强子序列的逆转录病毒载体pLXSNd中,用血管内皮生长因子受体KDR的特异性启动子调控了TNFa在血管内细胞ECV304中的靶向表达。将构建的载体pLXSN-TNFa,pLXSNd-KDRp-TNFa和空载体pLXSN用PA317细胞包装后获得重组病毒,并用重组病毒分别感染NIH3T3细胞和ECV304细胞,培养物上清的ELISA结果证明,KDR启动子指导的TNFa在KDR阳性细胞ECV304中的表达量为在KDR阴性细胞NIH3T3中的表达量的8倍;而TR指导的TNFa在这两种细胞中的表达无明显差异,实现了TNFa在血管内皮细胞中的靶向表达,这可能为肿瘤基因治疗提供新途径。     

Transfer of foreign genes by electroporation and their expression in mammalian cells

Expression of foreign genes transferred into mammalian cells by electroporation has been studied. The pX1TK gene, pSV2Neo gene and pUCEJ oncogene have been introduced into MLTK- cells and NIH/3T3 cells, respectively. Stable transformation transient expression of TK gene by MLTK- cells as well as stable and malignant transformation of NIH/3T3 cells have been obtained. Transient expression frequency is about 80% and stable transformation frequency is about 10(-4). Integration of foreign genes into the cellular genome was verified with molecular hybridization. Tumor development was observed after inoculation of transformed cells into nude mice.     

TRANSFORMING GENES IN DUCK HEPATIC CARCINOMA——mht(raf) AND Ha-ras

The transfection of NIH 3T3 cells was performed with DNAs from 2 duck primary hepatic carcinomas (DHC 40K, 9K) and I tumor-adjacent liver tissue (TAL, 9N). Transfectants were found from 40K, 9K and 9N DNAs. The secondary transfectants were obtained after transfection of RAT-1 cells with DNAs from primary transfectants. After hybridization with Ha-ras, Ki-ras, N-ras and mht oncogenes, it was found that duck mht (5.2 and 3.2 kb EcoRI fragments) and duck Ha-ras (3.4 kb EcoRI fragments) were present in all these transformants.This is the first report on transforming genes in duck primary hepatic cancer as well as tumoradjacent liver tissue     

TRANSFER OF FOREIGN GENES BY ELECTROPORATION AND THEIR EXPRESSION IN MAMMALIAN CELLS

Expression of foreign genes transferred into mammalian cells by electroporation has been studied. The pX1TK gene, pSV2Neo gene and pUCEJ oncogene have been introduced into MLTK-cells and NIH/3T3 cells, respectively. Stable transformation transient expression of TK gene by MLTK-cells as well as stable and malignant transformation of NIH/3T3 cells have been obtained. Transient expression frequency is about 80% and stable transformation frequency is about 10~(-4). Integration of foreign genes into the cellular genome was verified with molecular hybridization. Tumor development was observed after inoculation of transformed celts into nude mice.     

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells

Transformation with viral oncogene extends the lifespan of normal cells beyond replicative senescence called M1, but most of them eventually succumb to second crisis called M2 when telomeres become critically short. To acquire an infinite growth capacity, these cells have to overcome M2 crisis, which is known to follow telomerase activation. We have investigated if telomerase expression is required for virus-transformed pre-M2 cells to avert M2 crisis. Human retinal pigment epithelial (RPE) cells were transformed with simian virus 40 large T antigen and a VR3 clone in pre-M2 stage was obtained. Then, VR3 cells were transfected with a telomerase-containing vector and two cell lines that expressed telomerase temporarily or continuously were cloned and designated as ST1 and ST2, respectively. Normal RPE cells went into senescence after 36 population doublings. Although the lifespan was extended in the VR3 clone about 20 times more, it eventually underwent second crisis. The telomere length of VR3 decreased compared to that of normal RPE cells and the decrease continued during subculture. However, the ST1 and ST2 clones that expressed both T antigen and telomerase could avert this crisis. The initial telomere length of ST1 and ST2 was longer than that of normal cells. The ST1 underwent growth arrest again as telomerase expression faded out and elongated telomere was shortened, but the ST2 that maintained telomerase activity and telomere length proliferated continuously. In conclusion, telomerase activation is definitely required to overcome M2 crisis and acquire an infinite lifespan in human somatic epithelial cells and this mechanism is independent from M1 crisis escape in cell immortalization.