反义IGF-I基因对人7402肝癌细胞肿瘤免疫原性影响的研究

本研究利用基因治疗方法中的反义技术,通过脂质体包裹反义胰岛素样生长因子-I(IGF-I)基因导入人7402肝癌细胞株,研究肿瘤细胞表面MHC分子的表达和其对LAK细胞杀伤的敏感性。结果表明,反义IGF-I基因转染的人7402肝癌细胞,经潮霉素筛选后,Northern Blot分析显示反义IGF-I RNA表达阳性,MHC Ⅰ类和Ⅱ类抗原的表达水平高于亲本7402细胞。LAK细胞对反义IGF-I基因转染的人7402肝癌细胞的杀伤活性明显高于亲本7402细胞组(P<0.05)。说明反义IGF-I基因转移至该肝癌细胞,可提高肿瘤的MHC Ⅰ类和Ⅱ类分子的表达,显示肿瘤细胞的免疫原性增强,并可提高LAK细胞对其杀伤的敏感性。     

Treatment of hepatocellular carcinoma with adenoviral vector-mediated Flt3 ligand gene therapy

Fms-like tyrosine kinase 3 ligand (Flt3L) plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). It has been shown that administration of either tumor cells transfected in vitro with Flt3L vectors or soluble Flt3L fusion protein in a high dose can enhance host antitumor immunity in animal model systems. In this study, we developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter and investigated its biological efficacy in eliciting tumor-specific immune response against hepatocellular carcinoma in mouse hepatoma model. The constructed Ad-mFlt3L efficiently infected hepa 1-6 hepatoma cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCsNK cells and lymphocytes in local tumor tissues. Tumor cells infected with Ad-mFlt3L lost tumorigenicity and became more immunogenic in syngeniec animal models. Intratumoral injection of Ad-mFlt3L (10(9) expression-forming unit) x 3 significantly inhibited tumor growth with elicitation of long-lasting antitumor immunity, which is both preventive and curative. The tumor-specific immunity can be partially abrogated by depletion of either CD3+CD4+ T cells or NK cells and can be also re-established in na?ve animals by adoptive transfer of splenocytes from treated mice. The results suggest that adenovirus-mediated Flt3L gene therapy may provide a useful strategy for treatment of cancers.     

Induction in a murine tumor of immunogenic tumor variants by transfection with a foreign gene

Transfection of the undifferentiated murine colon carcinoma line CT-26 with the gene coding for the hemagglutination antigen (HA) of influenza virus resulted in the generation of highly immunogenic tumor cells. CT-26 cells transfected with HA not only failed to grow in syngeneic mice but also protected normal animals against a challenge with otherwise lethal doses of parental nontransfected cells. The immunogenicity of HA-transfected cells appeared to correlate with surface HA expression in that tumorigenic clones of HA-transfected CT-26 cells expressed little HA, while immunogenic clones were high expressers of HA. Irradiation of immunogenic HA clones did not abrogate their immunogenicity. These observations demonstrate that immune recognition of a poorly immunogenic tumor can be produced by immunization with tumor cells expressing a defined, foreign cell surface antigen.     

nm23H1对肝癌细胞增殖及体内肿瘤形成能力的影响

目的 研究ｎｍ２３Ｈ１对肝癌细胞增殖、体内肿瘤形成和转移的影响。方法 构建正反义ｎｍ２３Ｈ１ ｃＤＮＡ表达载体并转染肝癌细胞ＳＭＭＣ－７７２１,得到ｎｍ２３Ｈ１稳定最高和最低表达的两种细胞克隆,并进行细胞生长曲线测定、裸鼠皮下及脾包膜下移植试验。结果 转染反义表达载体后,肝癌细胞ｍＲＮＡ和蛋白表达下降,在体内、体外增殖加速;转染正义表达载体后,出现与之相反的结果。正义表达细胞接种组裸鼠肿瘤结节形成     

Antigen loss variants of a murine renal cell carcinoma: Implications for tumor vaccination

Vaccination with tumour cells genetically modified to support induction of an immune response either by production of cytokines or expression of co-stimulatory molecules provides a promising therapeutic approach. We have evaluated the efficiency of tumour vaccination using RENCA cells, a renal cell carcinoma of the BALB/c strain, which were stably transfected with MHC class II, B7.1 or both. Tumour growth after vaccination with MHC class II and/or B7.1 transfected RENCA cells was extremely variable, with protection close to 100% after vaccination with some clones and no effect of vaccination with others. To unravel the underlying mechanism, untransfected RENCA cells were cloned, and individual clones were tested for immunogenicity; that cloned RENCA cells varied considerably in immunogenicity. Whereas all clones displayed comparable growth rates in nude mice, some grew very slowly in immunocompetent syngenetic hosts. Vaccination with rapidly growing clones was ineffective and, importantly, this feature remained unaltered by vaccination with MHC class II and/or B7.1 transfected clones. Instead, 8 of 10 mice rejected the parental line after immunisation with a pool of MHC class II and B7.1 transfected clones. Finally, by cloning RENCA cells, we obtained one highly immunogenic clone (P2). Vaccination with this clone led to an individual-specific response, which indicates that during the cloning procedure a new strongly immunogenic entity must have arisen. Taken together, our results indicate that vaccination with MHC II and/or B7.1 transfected tumour cells induces an efficient immune response, but only if the tumour is weakly immunogenic. Since tumours may be composed of clones displaying different antigenicities, it is mandatory to use bulk cell populations for transfection and vaccination. Int. J. Cancer 77:114–122, 1998.© 1998 Wiley-Liss, Inc.     

Expression of co-stimulatory 4-1BB ligand induces significant tumor regression and protective immunity

Co-stimulatory molecules play an important role in initiating antitumor immune responses. Engineered tumor cells expressing co-stimulatory molecules have been used as cancer vaccines in both experimental tumor models and clinical trials. In this study, we cloned a cDNA gene coding for the mouse co-stimulatory molecule 4-1BBL by RTPCR. The expression vector pCI-4-1BBL was constructed by DNA recombinant technology and further transfected into a moderately immunogenic EL4 and a poorly immunogenic BL6-10 tumor cell line. Expression of the co-stimulatory molecule 4-1BBL is able to induce tumor regression of EL4/4-1BBL but not BL6-10/4-1BBL tumor cell line in syngeneic BALB/c mice. The tumor regression which is mainly mediated by CD8+ T cells further leads to protective immunity against the parental EL4 tumor. Our results thus indicate the potential utility of engineered tumor cells expressing co-stimulatory molecule 4-1BBL, especially in combination with other co-stimulatory molecules such as B7-1 in cancer vaccine.     

转染人CD80基因的黑色素瘤细胞生长特性及免疫原性探讨

】 ObjectivesTo investigate the effects of the CD80 costimulatory molecule expression on the immunogenicity and biological characteristics of poorly immunogenic B16 melanoma cells,explore the relationship between the immunogenicity of tumor cells and cell surface molecules. MethodsHuman CD80 cDNA was transfected into B16 melanoma cells by lipofectin-mediated gene transfer before the expansion of tumor cells were tested by MTT method in vitro;C57BL/6 mice were inculated subcutaneously either mock-transfected B16 cells (B16-neo)or CD80-transfected B16 cells (B16-CD80),then the latency,survival times and tumor mass growth were investigated.The lymphocytes were examined for both proliferation indices (PI) and specific cytotoxic activity by MTT method and improved LDH-releasing method,after syngenic Mixed Lymphocyte tumor cultures (MLTCs). ResultsIn the B16-CD80 cells,in which CD80 expression were detected by SABC method, demonstrated slower growth rate than B16-neo Clles in vivo (P<0.05),though they didn't in vitro. Compared with B16-wt and B16-neo cells,B16-CD80 cells more effectively induced the proliferation of effector lymphocytes and the generation of specific lytic activity against B16-wt cells. ConclusionsThe CD80 (B7-1) expression in poorly immunogenic tumor cells can increase their immunogenicity and decrease their tumorigenicity,the effects are associated with the expression of ICAM-1 and MHC molecules on tumor cells surface.     

Small cell lung carcinomas express shared and private tumor antigens presented by HLA-A1 or HLA-A2.

Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8+ CTLs. MHC-restricted CD8+ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8+ CTL responses are modulated by CD4+ helper T cells and by antigen-presenting cells. In this study, highly purified CD8+ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8+ T cells. Partial MHC class I matching of tumor and CD8+ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8+ CTLs. A1- and A2-restricted CD8+ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8+ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.     

碱性成纤维细胞生长因子反义寡核苷酸对肝癌细胞凋亡的影响

目的 检测碱性成纤维细胞生长因子(bFGF)反义寡核苷酸对肝癌细胞凋亡的影响,探讨bFGF作为肝癌治疗靶点的有效性.方法 以脂质体作为转染载体,转染bFGF反义寡核苷酸于HepG2细胞,用共聚焦显微镜和Western blot方法,检测转染bFGF反义寡核苷酸后肝癌细胞株HepG2中bFGF蛋白的表达,用流式细胞仪检测转染bFGF反义寡核苷酸后HepG2细胞的凋亡率.结果 与转染错义寡核苷酸组相比,bFGF反义寡核苷酸对HepG2细胞中bFGF蛋白表达有明显抑制作用,转染bFGF反义寡核苷酸可明显增加HepG2细胞的凋亡率(P＜0.01).结论 bFGF在肝癌细胞凋亡中有重要调节作用,可作为肝癌增敏治疗的有效靶点.     

Down-regulation of TGF-beta1 production restores immunogenicity in prostate cancer cells

The objective of this study is to determine if a non-immunogenic Dunning's rat prostate cancer cell line, MATLyLu, can become immunogenic by reducing the endogenous production of TGF-beta1. An expression construct containing a DNA sequence in an antisense orientation to TGF-beta1 (TGF-beta1 antisense) was stably transfected into MATLyLu cells. Following transfection, cellular content of TGF-beta1 reduced from 70 to 10 pg per 2x10(4) cells and the rate of in vitro 3H-thymidine incorporation increased 3-5-fold. After subcutaneous injection of tumour cells into syngeneic male hosts (Copenhagen rats), the tumour incidence was 100% (15/15) for the wild type MATLyLu cells and cells transfected with the control construct, but only 43% (9/21, P< or =0.05) for cells transfected with TGF-beta1 antisense. However, when cells were injected into immunodeficient hosts (athymic nude rats), the incidence of tumour development was 100% (10/10) for both the wild type MATLyLu cells and cells transfected with the control construct and 90% (9/10) for cells transfected with TGF-beta1 antisense. These observations support the concept that MATLyLu cells are immunogenic, when the endogenous production of TGF-beta1 is down-regulated.     

反义IGF—I基因转染人肝癌细胞分泌CEA和AFP水平的研究

本研究利用基因治疗方法中的反义RNA技术，通过脂质体包裹反义胰岛素样生长因子－I（IGF－I）基因导入人HepG2肝癌细胞株，NorthernBlot分析显示反义IGF－IRNA表达阳性。研究反义IGF－I基因转染的HePG2细胞分泌CEA和AFP的水平，放射性同位素的测定结果表明细胞培养上清中的CEA的水平显著地低于亲本细胞（P＜0．05），而分泌AFP的水平更显著地低于亲本细胞（P＜0．01）。预示着阳性克隆细胞的恶性程度低于亲本细胞，细胞内原有的生物学过程发生改变。     

Adenoviral transfer of xenogeneic MHC class I gene results in loss of tumorigenicity and inhibition of tumor growth

The immune system confers protection against a variety of pathogens and contributes to the destruction of neoplastic cells. Foreign major histocompatibility complex (MHC) protein serves as a potent stimulus to the immune system. In this report, a mouse H-2Kb gene was introduced into two poorly immunogenic tumor cell lines, a mouse colonic carcinoma cell line, MCA-26 (H-2Kd), and a rat mammalian carcinoma cell line, LN-4, in an effort to stimulate tumor rejection. Our results showed that the expression of xenogeneic MHC class I antigen completely abolished the LN-4 tumorigenicity in rats, whereas the expression of allogeneic MHC class I antigen only partially reduced the MCA-26 tumorigenicity in mice. Rats with tumor regression of LN-4/H-2Kb developed a T helper type 1-dominant response, whereas rats with LN-4 tumor growth developed a T helper type 2-dominant response. The immunized rats that experienced LN-4/H-2Kb tumor regression further developed protective immunity against a subsequent challenge of LN-4 cells. This protective immunity was mediated by the LN-4 tumor-specific cellular immune response against both the transduced and the parental LN-4 cells. Recombinant adenoviral vectors are highly efficient at in vitro and in vivo gene delivery. The LN4 cells transfected with the recombinant adenovirus AdV-H-2Kb in vitro expressed the cell surface H-2Kb molecule by fluorescence-activated cell sorter analysis. Adenovirus-mediated H-2Kb gene transfer in vivo can further significantly inhibit pre-established LN-4 tumors. Those rats with complete tumor regression further developed protective immunity against the subsequent challenge of a parental LN-4 tumor. Therefore, our study indicates that the adenovirus-mediated transfer of xenogeneic MHC class I gene may be an effective alternative to the current protocol of cancer gene therapy in which the allogeneic MHC class I gene is used.     

Treatment of rat hepatoma by locally injection of murine IL-12 retrovirus packaging cell

　Objective To investigate the therapeutic effects of the murine IL-12 (mIL-12) retrovirus packaging cell line on hepatoma injected locally. Methods The retrovirus vector encoding mIL-12 gene was constructed and transfected into packaging cell line PA317. The cells were then used to treat the rats with experimental orthotopic hepatoma at different time. The therapeutic effects, immune functions of the hosts, pathological and toxicological responses were documented. Results the results showed that the mIL-12 retrovirus packaging cell line could significantly inhibit the growth of the hepatoma cells injected locally to the hepatoma. The early treatment made the rats survive long, while the medium or late stage treatment could prolong the life time of the rats compared with the bland control group or bland vector control group, though the rats did not survive. The number of NK cells and T cells increased significantly in the treatment group. The effects of the early treatment were superior to those of the medium and late stage treatment. Moreover, the transfection of IL-12 gene locally in the hepatoma tissue could make the hepatoma disappear from other liver lobe. This phenomenon demonstrated that IL-12 could activate the immune cells of the host to kill the untransfected tumor cells. This is very important for IL-12 to be used in gene therapy clinically. Meanwhile, the hepatoma would not recur in the rats that had survived more than 2 months from the early treatment after being re-challenged with tumor cells. Conclusion the results showed that IL-12 gene injected locally in the hepatoma tissue could enhance the anti-tumor immunity of the host.     

抗胃癌生物活性肽对荷胃癌裸鼠酯酶同工酶代谢的影响

本文观察了协同刺激分子B7-1某因导入小鼠EL-4淋巴瘤细胞后在小鼠体内诱导的抗瘤效应.结果表明,逆转录病毒(PLXSN)载体重组的小鼠B7-1基因表达质粒导入小鼠EL-4淋巴瘤细胞,经有限稀释法克隆后获得高表达的B7-1~ EL-4细胞.B7-1~ 瘤细胞的形态,体外增殖能力及MHC Ⅰ类分子表达水平与野生型肿瘤细胞无显著差别,但致瘤性显著降低,用野生型肿瘤致死剂量接种C57BL/6小鼠完全排斥.同时免疫原性明显增强,以X-线灭活的B7-1~ 肿瘤细胞免疫后小鼠获得了对随后致死剂量野生型细胞攻击的免疫保护作用.以X-线灭活的肿瘤细胞作为瘤苗进行实验性免疫治疗,对早期(接种7天)形成的肿瘤有一定的治疗效果,但时晚期(接种14天)肿瘤.B7-1和B7-1~ 瘤细胞都未显示出明显的治疗效果.上述结果提示肿瘤细胞表达B7-1分子可有效激发机体的抗肿瘤免疫应答.     

The H-ras oncogene regulates expression of 70- and 45-kDa cell-surface molecules whose expression correlates with tumor-cell immunogenicity.

The effects of the H-ras oncogene on fibroblast cell tumorigenicity and immunogenicity was studied in transfectants of the BALB/c 3T3 clone A31 fibroblastoid cell-line. Cells that were transfected with MC29-LTR-H-ras (98/6) or MC29-LTR-v-myc + H-ras (98/4v) and were inoculated into syngeneic BALB/c mice were tumorigenic in 100% and 60% of animals respectively. By contrast, transfectants containing the pSV2neo plasmid alone (98/1) displayed normal characteristics both in vitro and in vivo. Inoculation of mice with mitomycin-C-treated 98/1 or 98/4v cells induced an effective protective immunity to a challenge of live 98/4v cells, and a partial immunity against 98/6 cells. Mitomycin-C-treated 98/6 cells failed to render immunity against a challenge of either 98/6 or 98/4v cells. To correlate immunogenicity and tumorigenicity of the different cell types with cell-surface-antigen expression, we prepared MAbs against 98/4v cells in syngeneic mice. Immunohistochemical and immunoblot analysis revealed that MAbs 102 and 104 recognized 2 protein band of 70 and 45 kDa respectively, which were expressed predominantly in 98/1 and 98/4v cells. A third immunoreactive protein band of 44 kDa that reacted with MAb 6 was expressed at a similar cell-surface density on all cell types. Cell-differentiation-inducing agents, such as DMSO, retinoic acid or sodium butyrate, were all found to induce 98/6 cell flattening and morphological changes toward a normal phenotype that were followed by up-regulation of the 70- and 45-kDa antigens. The results suggest that regulation of expression of the 70- and 45-kDa molecules is affected by H-ras, and that expression of these cell-surface molecules may be relevant to tumor cell immunogenicity.     

Adenovirus-mediated Gene Transfer of B7.1 Induces Immunological Anti-tumor Effects in a Murine Brain Tumor

The purpose of the present study was to determine if adenovirus-mediated transfection of a syngeneic mouse brain tumor with the gene encoding B7.1 enhances immunogenicity against tumor. Malignant astrocytoma cells were transfected with adenoviral vectors carrying the B7.1 gene (AdB7). Immunocytochemical analysis confirmed the expression of B7.1 in vitro and in vivo. To investigate the effects of B7.1 expression on tumorigenicity of the malignant astrocytoma, mice were implanted intracerebrally with B7.1-transfected glioma cells. There was no significant difference in proliferation between B7.1-transfected cells and controls in vitro. Nevertheless, mice implanted with B7.1-transfected cells survived significantly longer than those in the control groups. Immunocytochemical analysis of the tumors showed that there was infiltration of a number of CD8⁺ T-cells and CD25⁺ activated T-cells in the brain implanted with B7.1-transfected glioma cells. The results showed the possibility that adenovirus-mediated B7.1 gene transfection to a brain tumor induced activation of CD8⁺ cytotoxic T-lymphocytes.     

Immune response to somatic cell hybrids between ultraviolet radiation-induced regressor and spontaneous progressor C3H mouse tumor cells

We used somatic cell hybridization to determine whether the regressor phenotype exhibited by UV-induced murine tumors was dominant or recessive and whether this technique could confer immunogenic properties on nonimmunogenic syngeneic tumors. We transfected a highly antigenic UV-induced C3H mouse tumor cell line (UV-2240) with the plasmid pSV2-neo and selected G418-resistant clones. The resulting cell line was fused with a spontaneously transformed nonimmunogenic C3H progressor tumor cell line (SF-2T) that had been selected previously for resistance to 3.0 mM ouabain. These two cell lines were fused by a brief exposure to polyethylene glycol and heterokaryons isolated by growth in medium containing both G418 and ouabain. Hybrid cell lines established from individual colonies and from pools of colonies were tested for tumorigenicity in normal C3H and athymic nude mice. The results indicated that all the hybrid cell lines tested were highly antigenic in that they were completely rejected when transplanted into normal syngeneic mice but grew progressively in nude mice. Furthermore, immunization of C3H mice with the hybrid cell lines induced protective immunity against challenge with the immunizing tumor and generated cross-protective immunity against challenge with the regressor parental cell line but not against challenge with the progressor parental cell line. These results demonstrate that the regressor phenotype of the UV-2240 tumor is dominant in nature and that the immune response induced by somatic cell hybrids is uniquely directed against the dominant tumor-specific transplantation antigens expressed on the regressor tumor. This implies that introduction of tumor-specific transplantation antigens from an immunogenic tumor into a nonimmunogenic tumor, although sufficient to confer immunogenic properties to the hybrid, is insufficient to induce cross-protective transplantation immunity against the nonimmunogenic tumor.     

单纯疱疹病毒Ⅰ型tk基因的克隆及其真核表达载体的构建

目的:为观察携带小鼠白细胞介素12(mIL-12)逆转录病毒包装细胞株在肝癌局部注射后,对体内肝癌治疗效果.方法:构建携带mIL-12的逆转录病毒载体,将该载体转染包装细胞系PA317,应用该包装细胞对实验性原位肝癌大鼠分别在不同时间进行治疗,观察其抗肿瘤作用,免疫功能变化,病理及毒性反应.结果:携带mIL-12逆转录病毒包装细胞(PA317-mIL-12)在原位肝癌局部注射后能明显抑制肝癌细胞生长,其早期治疗可使大鼠长期生存,中晚期治疗,大鼠虽然不能长期生存,但较空白对照组及携带空载体对照组的生存期明显延长,(P<0.01).其治疗组NK细胞及T细胞明显增加,其早期治疗效果优于中期治疗,另外,本研究对肝癌局部进行IL-12基因转染,可使另一叶肝癌消退,这表明IL-12可通过激活机体免疫细胞,杀灭未转染的肿瘤细胞,这对于临床应用IL-12基因治疗非常重要.同时,生存期超过2个月大鼠,再次接种相当数量的肝癌细胞,该大鼠不能再次形成肝癌.结论:肝癌局部注射IL-12基因能激发机体抗肿瘤免疫反应.     

MiR-448 suppresses cell proliferation and glycolysis of hepatocellular carcinoma through inhibiting IGF-1R expression

BackgroundMicroribonucleic acids (miRNAs) have been shown to play important roles in hepatocellular carcinoma (HCC) progression. MiR-448 has frequently been shown to be a tumor suppressor, and is abnormally expressed in HCC tumor tissues. However, little is known about the role of miR-448 in HCC development. In this article, the regulatory role of miR-448 on insulin-like growth factor 1 receptor (IGF-1R) in modulating hepatoma cell viability and glycolysis was investigated.MethodsThe expression of miR-448 profiles in clinical tumor tissues and cell lines was examined using quantitative real-time polymerase chain reaction (qRT-PCR). HepG2 and Huh7 cells were transfected with miR-448 mimics, inhibitors, and scramble sequences. Cell viability and apoptosis were determined by a Cell Counting Kit-8 assay and a flow cytometry analysis. IGF-1R, a potential target of miR-448, was selected following a bioinformatic analysis, and the regulatory effects of miR-448 on IGF-1R expression was confirmed by luciferase reporter assay, qRT-PCR, and western blot. Glucose uptake, lactate production, and adenosine triphosphate (ATP) generation were detected by corresponding kits.ResultsDecreased miR-448 expression was observed in both HCC patients’ tumor tissues and hepatoma cells in vitro. The overexpression of miR-448 in HepG2 and Huh7 cells decreased cell viability and increased apoptosis. Additionally, the overexpression of miR-448 or the knockdown of IGF-1R lowered the level of glucose uptake, lactate production, and ATP generation, while the knockdown of miR-448 increased glycolysis. Further, aberrantly expressed miR-448 downregulated IGF-1R levels, while the inhibition of miR-448 resulted in the upregulation of IGF-1R in both HepG2 and Huh7 cells. In addition, miR-448 interacted with the wild-type 3''untranslated regions (3''UTRs) of IGF-1R, but had no effect on the mutant 3''UTRs. The expression of IGF-1R was increased in HCC patients’ tumor tissues and serum, and was inversely correlated with miR-448 expression.ConclusionsThe increased expression of miR-448 appears to downregulate the expression of IGF-1R by interacting with the 3’UTR in HCC progression. These findings highlight its role as a potential target for HCC therapy.     

Treatment of squamous carcinoma in mice with a vaccine enriched for cells that induce immunity to squamous carcinoma--a new vaccination strategy

We report a new vaccination strategy for squamous cell carcinoma (SCC). The vaccine was prepared by transfer of unfractionated DNA‐fragments (25 kb) from squamous carcinoma cells (KLN205, DBA/2 origin (H‐2^d)) into LM mouse fibroblasts (C3H/He origin; H‐2^k), a highly immunogenic cell line. To enhance their nonspecific immunogenic properties, the fibroblasts were modified before DNA transfer to secrete IL‐2 and to express additional allogeneic MHC class I determinants. As the transferred DNA integrates into the genome of the recipient cells, and is replicated as the cells divide, sufficient DNA to prepare the vaccine could be obtained from as few as 10⁷ squamous carcinoma cells (4 mm tumor). Since only a small proportion of the transfected cell‐population was expected to have incorporated genes specifying antigens associated with the squamous carcinoma cells (TAA), we devised a novel approach to enrich the vaccine for cells that induce immunity to the SCC. Aliquots of the transfected population were divided into 10 small pools (initial inoculums = 1 × 10³). We reasoned that if the starting inoculums were sufficiently small, then the distribution of highly immunogenic and weakly immunogenic cells in each pool would not be the same. Cells from individual pools were allowed to increase in number. A portion of the expanded cell populations were maintained frozen/viable for later recovery. The remaining portions were used to immunize naïve DBA/2 mice. Pools containing greater numbers of immunogenic cells were identified by 2 independent assays. Frozen aliquots of cells from the pool that stimulated immunity to the squamous carcinoma to the greatest extent were recovered and subdivided for additional rounds of immune selection. Enhanced immunity to squamous carcinoma mediated by CD8+ T cells was induced in tumor‐bearing mice treated solely by immunization with the enriched cell‐population. © 2006 Wiley‐Liss, Inc.