Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice

The human breast adenocarcinoma cell line MDA-MB-231 has the triple-negative breast cancer (TNBC) phenotype, which is an aggressive subtype with no specific treatment. MDA-MB-231 cells express neurotensin receptor type 1 (NTSR1), which makes these cells an attractive target of therapeutic genes that are delivered by the neurotensin (NTS)-polyplex nanocarrier via the bloodstream. We addressed the relevance of this strategy for TNBC treatment using NTS-polyplex nanoparticles harboring the herpes simplex virus thymidine kinase (HSVtk) suicide gene and its complementary prodrug ganciclovir (GCV). The reporter gene encoding green fluorescent protein (GFP) was used as a control. NTS-polyplex successfully transfected both genes in cultured MDA-MB-231 cells. The transfection was demonstrated pharmacologically to be dependent on activation of NTSR1. The expression of HSVtk gene decreased cell viability by 49% (P<0.0001) and induced apoptosis in cultured MDA-MB-231 cells after complementary GCV treatment. In the MDA-MB-231 xenograft model, NTS-polyplex nanoparticles carrying either the HSVtk gene or GFP gene were injected into the tumors or via the bloodstream. Both routes of administration allowed the NTS-polyplex nanoparticles to reach and transfect tumorous cells. HSVtk expression and GCV led to apoptosis, as shown by the presence of cleaved caspase-3 and Apostain immunoreactivity, and significantly inhibited the tumor growth (55–60%) (P<0.001). At the end of the experiment, the weight of tumors transfected with the HSVtk gene was 55% less than that of control tumors (P<0.05). The intravenous transfection did not induce apoptosis in peripheral organs. Our results offer a promising gene therapy for TNBC using the NTS-polyplex nanocarrier.     

Tet-On调控HSVtk表达的重组腺相关病毒载体的构建和感染活性的检测

构建含有Tet基因调节系统及自杀基因HSVtk的重组腺相关病毒载体pAAV TRE HSVtk Tet_On ,并使用PCR技术和限制性内切酶消化进行鉴定。用构建好的重组质粒分别与辅助质粒pAAV_RC、pHelper以磷酸钙共沉淀法转染HEK2 93细胞,进行病毒包装后得到了AAV TRE HSVtk Tet_On重组腺相关病毒,以氯化铯密度梯度离心对包装好的病毒进行纯化。用纯化后的重组腺相关病毒感染乳腺癌细胞株MCF_7后,斑点杂交检测结果显示,HSVtk基因整合进入MCF_7细胞基因组中。有感染活性的重组腺相关病毒能将目的基因转移到宿主细胞中,在Dox诱导下,GCV对AAV感染的MCF_7细胞具有明显的杀伤作用。     

重组逆转录病毒载体GTK的构建及HyTK基因转移的研究

用逆转录病毒载体将单纯疱疹病毒胸苷激酶基因（ＨＳＶｔｋ）导入恶性肿瘤细胞,随后可应用药物９－（１,３－二羟基－丙氧基－甲基）鸟嘌呤（ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）选择性地杀死肿瘤细胞．将ＨｙＴＫ基因替换逆转录病毒载体ＧｌＮａ中的ｎｅｏ基因,构建成重组逆转录病毒载体ＧＴＫ,转染混合包装细胞（双噬性ＰＡ３１７细胞和单噬性ＧＰ＋Ｅ－８６细胞）,通过“乒乓效应”获得高滴度重组病毒．用该重组病毒转染小鼠恶性黑色素瘤细胞系Ｂ１６细胞,用ｈｙｇｒｏｍｙｃｉｎＢ筛选出阳性细胞克隆（ＨｙＴＫ＋）,经ＰＣＲ方法检测证明ＨｙＴＫ基因已成功地导入肿瘤细胞中,且不含可复制的辅助病毒．分别用不同浓度的ＧＣＶ作用于ＨｙＴＫ－及ＨｙＴＫ＋的Ｂ１６细胞,光镜下观察２４ｈ和４８ｈ后细胞形态及进行活细胞计数．结果表明,ＧＣＶ浓度大于０．１μｍｏｌ／Ｌ时即对Ｂ１６／ＨｙＴＫ＋细胞有显著的杀伤作用     

In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery

Transfer of the herpes simplex virus-thymidine kinase gene, followed by the administration of ganciclovir (HSV-tk/GCV), has been a major approach for cancer gene therapy. We investigated the antitumor effect of the HSV-tk/GCV strategy with the rat orthotopic hepatocellular carcinoma (HCC) model and the tumor-selective gene delivery by an adenovirus-mediated gene transfer through the hepatic artery. The complete antitumor effect was demonstrated, after the treatment with GCV in rat HCC established by the implantation of HSV-tk transferred rat HCC cells. The in vivo bystander effect was also observed. The marked infiltration of CD4+ and CD8+ T lymphocytes, macrophages and NK cells were found in the tumor area. After the injection of adenovirus carrying the LacZ gene into the hepatic artery, the selective expression of transgene in the tumor cell was achieved. These findings indicate that the HSV-tk/GCV strategy, using an adenoviral vector, could be a promising avenue for the treatment of hepatocellular carcinoma.     

Comparative analysis of DNA breakage,chromosomal aberrations and apoptosis induced by the anti-herpes purine nucleoside analogues aciclovir,ganciclovir and penciclovir

Nucleoside analogues have been used in antiviral therapy and suicide cancer gene therapy. Therefore, it is of importance to compare their potential cytotoxic and genotoxic action. Using metabolically competent CHO cells expressing the thymidine kinase gene of herpes simplex virus type 1 (CHO-HSVtk cells) as a model system, the induction of DNA breaks was compared with the induction of structural chromosomal aberrations and apoptosis/necrosis after exposure to the anti-herpes nucleoside analogues aciclovir (ACV), ganciclovir (GCV) and penciclovir (PCV). After continuous treatment of CHO-HSVtk cells with the drugs, LD(10) in a colony-forming assay was 50, 0.5 and 1 microM for ACV, GCV and PCV, respectively, with GCV to be the most potent agent as determined at a given dose level. There was a remarkable difference in the activity of the agents to kill HSVtk expressing and non-expressing cells: the difference in cellular sensitivity of HSVtk(+) versus HSVtk(-) cells at LD(10) level was 7-fold for ACV, 60-fold for GCV and 400-fold for PCV. The drugs were shown to be strong inducers of apoptosis that was analysed as to concentration- and time-dependence; they induced to only very low extent necrosis. The agents were also highly potent in the induction of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) (as measured by single cell gel electrophoresis (SCGE)) and chromosomal aberrations. Although PCV induced DNA DSBs with a kinetics and frequency similar to that of GCV, it caused mostly condensation defects instead of "typical" structural chromosomal aberrations. For the drugs used, the frequency of apoptotic cells and the induction of abnormal mitoses appear to be related indicating genotoxic effects induced by the agents to be involved in cell killing due to apoptosis.     

A promoter that drives gene expression preferentially in male transgenic rats

Gender-preferential gene expression is a widespread phenomenon in humans. It is important to study how gender differences influence the pathogenesis of various diseases and response to specific drugs. The aim of this study is to determine if the mouse albumin enhancer/promoter may serve as the promoter to introduce gender-preferential gene expression in transgenic animals. We created four independent transgenic rat lines in which the human C-reactive protein transgene was under the control of mouse albumin enhancer/promoter. Quantitative real time RT-PCR analysis showed that transgene expression in the liver of male rats was significantly higher than transgene expression in the female rats (P < 0.05).There was a 5.3-fold (male/female) difference in line-519, and a 12.2-fold (male/female) difference in line-488. Enzyme-linked immunosorbent assay showed that the serum of male transgenic rats had a 13- to 679-fold difference at the protein level on transgene production compared with female transgenic rats. The male-to-female difference in gene expression was 10- to 17-fold in the liver of transgenic rats. Orchiectomy dramatically reduced protein production from the transgene in the liver. Testosterone administration into female rats did not increase the transgene expression, but estrogen administration into the male rats reduced transgene expression. This study provides a valuable tool for investigating the pathological roles of genes that are expressed in a gender-preferential manner in human disease.     

Alrp, a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of 'Alrp homologous proteins' in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.     

Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.     

Alrp,a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats

Abstract

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of ‘Alrp homologous proteins’ in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.     

Green fluorescent protein-transgenic rat: a tool for organ transplantation research

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.     

Ribosomal protein S6 phosphorylation and function during late gestation liver development in the rat

The phosphorylation of ribosomal protein S6 is thought to be required for biosynthesis of the cell's translational apparatus, a critical component of cell growth and proliferation. We have studied the signal transduction pathways involved in hepatic S6 phosphorylation during late gestation in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of mitogenic signaling pathways that are operative in mature hepatocytes. Our initial studies demonstrated that there was low basal activity of two S6 kinases in liver, S6K1 and S6K2, on embryonic day 19 (2 days preterm). In addition, insulin- and growth factor-mediated S6K1 and S6K2 activation was markedly attenuated compared with that in adult liver. Nonetheless, two-dimensional gel electrophoresis demonstrated that fetal liver S6 itself was highly phosphorylated. To characterize the fetal hepatocyte pathway for S6 phosphorylation, we went on to study the sensitivity of hepatocyte proliferation to the S6 kinase inhibitor rapamycin. Unexpectedly, administration of rapamycin to embryonic day 19 fetuses in situ did not affect hepatocyte DNA synthesis. This resistance to the growth inhibitory effect of rapamycin occurred even though S6K1 and S6K2 were inhibited. Furthermore, fetal hepatocyte proliferation was sustained even though rapamycin administration resulted in the dephosphorylation of ribosomal protein S6. In contrast, rapamycin blocked hepatic DNA synthesis in adult rats following partial hepatectomy coincident with S6 dephosphorylation. We conclude that hepatocyte proliferation in the late gestation fetus is supported by a rapamycin-resistant mechanism that can function independently of ribosomal protein S6 phosphorylation.     

Gap junction-mediated bystander effect in primary cultures of human malignant gliomas with recombinant expression of the HSVtk gene

The ability of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-tk-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs via the transfer of phosphorylated GCV, the mechanism(s) of this bystander effect and the importance of gap junctions for the effect of prodrug/suicide gene therapy in primary human glioblastoma cells remains elusive. Surgical biopsies of malignant gliomas were used to establish explant primary cultures. Proliferating tumor cells were characterized immunohistochemically and found to express glial tumor markers including nestin, vimentin, glial fibrillary acidic protein (GFAP), S-100, and gap junction protein connexin 43 (Cx43). Western blot analysis revealed the presence of phosphorylated isoforms of Cx43 and Calcein/DiI fluorescent dye transfer showed evidence of efficient gap junction communication (GJC). In order to study the effect(s) of prodrug/suicide gene therapy in these cultures, human glioblastoma cell cultures were transfected with the HSVtk gene for transient or stable expression. Ganciclovir treatment of these cultures led to >90% of cells dead within 1 week. Eradication of cells could be inhibited by the addition of alpha-glycyrrhetinic acid (AGA), a GJC inhibitor. In parallel experiments, AGA decreased the immunodetection of phosphorylated Cx43 as analyzed by Western blot and inhibited fluorescent dye transfer. In conclusion, these observations are consistent with GJC as the mediator of the bystander effect in primary cultures of human glioblastoma cells by the transfer of phosphorylated GCV from HSVtk gene transfected cells to untransfected ones.     

In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats

To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.     

Transplanted reporter cells help in defining onset of hepatocyte proliferation during the life of F344 rats

Transplanted hepatocytes integrate in the liver parenchyma and exhibit gene expression patterns that are similar to adjacent host hepatocytes. To determine the fate of genetically marked hepatocytes in the context of hepatocellular proliferation throughout the rodent life span, we transplanted Fischer 344 (F344) rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. The proliferative activity in transplanted hepatocytes was studied in animals ranging in age from a few days to 2 yr. Transplanted hepatocytes proliferated during liver development between 1 and 6 wk of age, each dividing an estimated two to five times. DNA synthesis in occasional cells was demonstrated by localizing bromodeoxyuridine incorporation. There was no evidence for transplanted cell proliferation between 6 wk and 1 yr of age. Subsequently, transplanted cells proliferated again, with increased sizes of transplanted cell clusters at 18 and 24 mo of age. The proliferative activity of transplanted cells was greater in rats entering senescence compared with during postnatal liver development. In old rats, some liver lobules were composed entirely of transplanted cells. We conclude that hepatocyte proliferation in the livers of very young and old F344 rats is regulated in a temporally determined, biphasic manner. The findings will be relevant to mechanisms concerning liver development, senescence, and oncogenesis, as well as to cell and gene therapy.     

Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury

Overlapping cDNA clones for rat hepatocyte growth factor (rHGF) were isolated by cross-hybridization with the cloned cDNA for human hepatocyte growth factor (hHGF) and the nucleotide sequence of the cDNA was determined. The entire primary structure of rHGF was deduced from the sequence. Comparison of the amino acid sequences between rat and human HGFs revealed that the two sequences are highly conserved throughout the protein structures, suggesting that rat and human HGFs may be functionally similar. Responses of the rHGF mRNA during liver regeneration in rats were examined by Northern blot hybridization analysis with the aid of the cDNA probe for rHGF. The mRNA levels increased in the liver and spleen but not in the kidney after administration of carbon tetrachloride. At the maximum level of induction, the rHGF mRNA increased in the liver about 4.5-fold over its normal level. The mRNA levels also increased in the liver and spleen after administration of D-galactosamine. On the other hand, no obvious increase of the mRNA was observed in the liver and spleen after partial hepatectomy. These observations suggest that HGF may function as a regulator of liver regeneration following hepatic injury caused by hepatotoxins.     

Parabiosis model does not show presence of circulating osteoprogenitor cells

The goal of this study was to determine the presence of osteoprogenitor cells in the peripheral blood. Experiments were conducted with a parabiosis model in which osteoblast specific transgenic mice (Col2.3GFP or hOC‐GFP) were surgically joined with a transgenic mouse where herpes virus thymidine kinase gene is under the control of the collagen α1 promoter (Col2.3ΔTK). This method permits conditional ablation of osteoblasts by ganciclovir (GCV) treatment. In parabionts treated with GCV for 15 days or 1.5–2 months, GFP (hOC‐GFP or Col2.3GFP) expression was not detected in histological preparations or in marrow stromal cell cultures from the Col2.3ΔTK parabiont. Finally, Col2.3GFP/Col2.3ΔTK pairs were treated with GCV for 15 days and allowed to recover from GCV for 3 months. Again there was a failure to detect Col2.3GFP expressing cells in the Col2.3ΔTK parabiont. These observations, at least within the limits of this model system, allow the conclusion that osteoprogenitor cells do not readily circulate. genesis 48:171–182, 2010. © 2010 wiley‐Liss, Inc.     

CEA启动子控制HSV—TK基因表达对人结直肠癌细胞的杀伤作用

An expression plasmid pCEA-TK, in which HSV-TK gene was under the control of CEA promoter, was constructed. The human colorectal carcinoma cell line LoVo or the human uterine cervical cancer cell line HeLa was co-transfected with pSV2-neo and pCEATK, respectively. After G418 selection, both transgenic cell clones (LoVo/CEATK and HeLa/CEATK) were obtained. LoVo/CEATK cells were 1300 times more sensitive to the cytotoxicity of ganciclovir than LoVo cells. However, the elevation of GCV sensitivity induced by pCEATK gene in HeLa line was only 8 times. Injection of GCV resulted in significant regression of HSV-TK transfected LoVo tumor in nude mice. These data suggested that the expression of TK gene driven by CEA promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electromicroscopy and DNA fragmentation assay demonstrated that GCV could induce apoptosis in LoVo/CEATK cells. The possibility of the CEATK/GCV system in the treatment of human colorectal carcinoma was discussed.     

Sex-related differences in cadmium-induced lipid peroxidation in the rat

Male and female rats were dosed once a day for 2 days injection with 1.5 mg of Cd/kg as CdCl₂. 24 hr after administration of cadmium, lipid peroxidation determined by estimation of malondialdehyde (MDA) was greatly increased in male rat liver, but was not in female rats. Cadmium in a larger dose of 4.5 mg/kg, subcutaneous single injection, significantly increased content of MDA in female rat liver. These results suggest that sex-related differences exist in the ability of cadmium to induce MDA formation in rat liver, although administration of cadmium causes the enhancement of MDA formation in both male and female rats. The reason why sex-related differences exist in lipid peroxidation of rat liver is discussed.