Animals ; Carcinoma, Hepatocellular ; genetics ; therapy ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms, Experimental ; genetics ; therapy ; Treatment Outcome

The outcome of current cancer therapy is far from satisfactory despite the considerable advancements have been achievedin life science and medicine.When looking for more potent drugs or technologies for cancer therapeutics,we shouht re-evaluate ourtraditional perception of caneer and have introspection whether we have some misunderstandings of cancer biology and the relationshipbetween cancer and host.In this commentary,the authors outlined their conceptual consideration of cancer,and proposed theirviewpoints about challenges and opportunities for cancer biotherapy.

The expression of cancer gene products of insuline-like growth factor Ⅱ (IGF-Ⅱ),IGF-Ⅱ receptors (IGF-Ⅱ-R),and colony-stimulating factor 1 receptors (CSF-1-R) /c-fms in 17 cases of human primary hepatic cancer,the non-cancerous liver tissue adjacent to the cancer,and normal liver tissue was studied with immunocytochemistry (ABC),Western blot and Northern blot techniques.It was found that the expression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-1-R was significantly higher in the cancers than in normal liver tissues,and the expression of IGF-Ⅱ and IGF-Ⅱ-R was higher in the non-cancerous liver tissues than in the cancers.It was noteworthy that the expression of IGF-Ⅱ in both the cancerous and non-cancerous hepatic tissues was characterized by its fetal type.However the expression of CSF-1-R was distinctly higher in the cancers than in the non-cancerous hepatic tissue.These findings,the authors believe,imply that the aberrant overexpression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-l-R might be related to the mechanism of auto-crine-stimulated growtth of the cells of human primary hepatic cancer.

Cationic polymers are being developed quickly as gene delivery vectors. For in vivo gene delivery, the cationic polymers are usually further modified by hydrophilic polymer grafting or ligand conjugation, which have been shown to increase the vector stability, gene delivery efficiency and specificity greatly. Some previous research had shown that modified hydrophilic polymer may partly shield the targeting ligand and result in poor delivery specificity. Developing a method to evaluate the influence of PEG modification on targeting delivery is particularly critical to cationic polymer design and gene therapy development. One of most commonly used cationic polymer polylysine (PLL) was chosen as a model. Targeting ligand epidermal growth factor(EGF)was conjugated with PLL to form PLL-EGF. Then hydrophilic polymer polyethylene glycol (PEG) with molecular mass 7 000 and 20 000 were used to modify PLL-EGF respectively to generate PEG7000-g-PLL-EGF and PEG20000-g-PLL-EGF. In BIAcore experiments, epidermal growth factor receptor (EGFR) was conjugated onto BIAcore chip and various PEG modified PLL-EGF solutions were flowed over the chip. By observing the change of RU value, the specific interaction of EGF to EGFR was compared. Compared with PLL-EGF, PEG modified PLL-EGF showed lower association rate and higher disassociation rate to EGFR. Furthermore, compared to PEG7000 modified PLL-EGF, PEG20000 modified PLL-EGF got lower association rate and higher disassociation rate to EGFR. The Scatchard analysis results showed that the interactions between EGFR and PLL-EGF or PEG-PLL-EGF are non-linear. It can be concluded that PEG modification indeed reduced the association rate and enhanced the dissociation rate of EGF to EGFR. The length of PEG chain was also a key factor to influence interaction between ligand and receptor. The results showed that it was critical important to evaluate the influence of PEG modification on delivery specificities. The BIAcore method developed in this paper can successfully evaluate the influence, which would be important for cationic polymer design and its application as potential non-viral gene delivery vectors.

Retroviral vectors are the most used gene delivery vehicle in human gene therapy.When the therapeutic gene is packagedfrom producer cell line, one has to determined whether thereplication-competent retrovirus(RCR) is being generated. In this study, two methods, namely the marker rescue assay and RT/PCR, were employed to detect RCR in the cells. While the marker rescue assay can detect RCR with a limit at 6?102CFU/ml, RT/PCR can be used to detect RCR at as low as lCFU/103ml. Combining the specificity of the marker rescue assay and the sensitivity of RT/PCR, both assays together should serve as an adequate test for detecting the generation of RCR in retroviral producer cell lines.

Objective To study the growth inhibition of human hepatoma cells tranfected with type Ⅱ insulin like growth factor receptor (IGF ⅡR) antisense gene. Methods We constructed the recombinant eucaryotic expression plasmids of IGF ⅡR sense and antisense genes, which were transfected into SMMC 7721 human hepatoma cell line using calcium phosphate coprecipitation, and selected in DMEM supplemented with 500 ?g/ml G418(Geneticin) for 2～3 weeks. Then the total number of colonies formed was counted. The effect of IGF ⅡR antisense gene transfection on endogenous IGF ⅡR mRNA levels was examined by Northern blot, and the growth rate and the ability of the transfected cells to form colonies in soft agar medium were also examined. Results The hepatoma cells transfected with IGF ⅡR antisense gene exhibited significant reduction in endogenous IGF ⅡR messenger RNA(mRNA) levels and loss of their ability for anchorage independent growth in soft agar. The IGF ⅡR antisense gene supressed the formation of colonies of SMMC 7721 cells resistant to G418 without alteration on cell growth curve. Conclusion The IGF ⅡR antisense gene expression effectively blocks IGF Ⅱ to IGF ⅡR autocrine and/or paracrine signal transduction pathway and reverses certain aspects of the cell malignant phenotype.

Objective: Purpose to investigate the different in vitro function of targetable non-viral vector containing poly-L-lysine or protamine. Methods: Using GV1 and GV2 targetable non-viral vectors, the influences of the poly-L-lysine and protamine on in vitro gene transfer efficiency and the course of gene expression were observed. Results: ?-galactosidase was expressed at intermediate level (50% ) in A375 cells using a complex containing either protamine or poly-L-lysine. Howerver, in case of ABAE cells, ?－galactosidase expression level was low (20% ) transferred with a comPlex containing protamine. On the contrary, ?－ galactosidase expression was at high level (70% ) provided that protamine was replaced with poly-L-lysine. In addition, ?-galac- tosidase activity reached the peak at the 6th day after transfection with the complex containing protamine. The expression was not altered with subsequent subcultures, at least for 3 passages. Using poly-L-lysine, the expression peak in A375 reached the peak at the 7th day after transfection, but the level declined along with subsequent passages of cells. Conclusion: The apllication of protamine in VEGF receptor mediated gene delivery system was limited.

0. 05). Conclusion: The HCAPl can be expressed in both normal peripheral white blood cells and the leukemic cells, and there is no difference in the protein levels between them.

Objective: To investigate the effects of novel targeted non-viral vector in gene therapy of human glioma. Methods: The EGF-R targeting gene delivery system GE7 was constructed. Human Glioma cell line U251 was transfected in vitro with ?－gal as reportor gene and p21 as therapeutic gene using this gene delivery system. By means of the assay of ?-galactosidase staining, Western blotting, in situ end labeling apoptosis cells and DNA ladder, the transferring of exogenous genes and the apoptosis of the tumor cells were examined.Results: It was showed that gene transfer efficiency is over 80%. When transfected with p21 gene, the growth of cells was inhibited significantly, and the apoptosis was detected in the transfected cell by the methods of in situ end labeling and DNA ladder. Conclusion: The GE7 gene delivery system has the ability to transfer exogenous gene to tumor cells and the expression of the therapeutic gene can inhibit the growth of the cells.

Overexpression of multidrug resistance gene, (MDR1) is responsible for the resistance of anticancer agents in cancer cells. In this study, we designed three pairs of DNA primers to clone MDR, cDNA from human normal liver by polymerase chain reaction. The sites of these primers in MDR cDNA are (I)-64-46 (5') and 1680-1698 (3'); (II) 1471-1489 (5') and 2905-2923 (3'); (III)2729-2747 (5') and 3845-3863 (3). The three reactions were underwent after the human normal liver mRNA was reverse transcripted into single strand DNA. The lengths of PCR products are 1762bp, 1452 bp and 1134bp, respectively. The former two products were subcloned in pBluscript SK, respectively and the latter product was subcloned in pGEM7Z (named as pMDR1.7, pMDR1.4 and pMDRl.l, respectively). Cloned genes were certificated as MDR1 cDNA by sequence analysis. Full-length MDR, cDNA was obtained after further subcloning. Full-length MDR1 cDNA we obtained will be a very important tool in molecular diagnosis and gene therapy of anticancer drug resistance.