Transformation of rat glioma cells with the M-CSF gene inhibits tumorigenesis in vivo

Macrophage colony-stimulating factor (M-CSF) is a potent stimulator of the effector cells such as monocytes and macrophages. To evaluate the effect of M-CSF on malignant gliomas, we transfected the rat gliosarcoma cell line (9L) with human M-CSF expression vector (pCEF-MCSF) by a liposome method. Transfectants were selected using G418-containing medium. As a control, 9L cells transfected with pRc/CMV and selected by G418 were used. The effects of M-CSF gene transfection on tumor cell proliferation in vitro and in vivo were examined. All growth rate did not change in vitro. While the control 9L cells formed progressively enlarging masses, 9L cells transfected with the M-CSF gene did not develop into tumors after the injection into rats. On the other hand, in rats receiving anti-asialo GM1 antibody, 9L cells transfected with M-CSF gene developed into tumors, though at a slower rate than control 9L cells. Histologic examination after transplantation of 9L cells transfected with M-CSF gene disclosed intense infiltration of macrophages in the tumor. Thus M-CSF gene transfection into glioma cells stimulates an antitumor effect.     

Transformation of rat glioma cells with the M-CSF gene inhibits tumorigenesis in vivo

Macrophage colony-stimulating factor (M-CSF) is a potent stimulator of the effector cells such as monocytes and macrophages. To evaluate the effect of M-CSF on malignant gliomas, we transfected the rat gliosarcoma cell line (9L) with human M-CSF expression vector (pCEF-MCSF) by a liposome method. Transfectants were selected using G418-containing medium. As a control, 9L cells transfected with pRc/CMV and selected by G418 were used. The effects of M-CSF gene transfection on tumor cell proliferation in vitro and in vivo were examined. All growth rate did not change in vitro. While the control 9L cells formed progressively enlarging masses, 9L cells transfected with the M-CSF gene did not develop into tumors after the injection into rats. On the other hand, in rats receiving anti-asialo GM1 antibody, 9L cells transfected with M-CSF gene developed into tumors, though at a slower rate than control 9L cells. Histologic examination after transplantation of 9L cells transfected with M-CSF gene disclosed intense infiltration of macrophages in the tumor. Thus M-CSF gene transfection into glioma cells stimulates an antitumor effect.     

A human bronchial epithelial cell strain with unusual in vitro growth potential which undergoes neoplastic transformation after SV40 T antigen gene transfection.

Bronchial epithelial cells were cultured from an individual with no evidence of malignant disease. These cells, designated HB56B, had a greatly extended in vitro life-span, being able to undergo 50 passages and 200 population doublings in contrast to the usual 3 to 4 passages and 20 to 30 population doublings characteristic of normal human bronchial epithelial cells. HB56B cells had karyotypic evidence of an amplified region on the short arm of chromosome II. Unlike normal bronchial epithelial cells, which undergo terminal squamous differentiation in vitro in response to fetal bovine serum, HB56B cells were only minimally affected by serum. These cells were readily established as an immortalized cell line, HB56B/5T, following transfection with a plasmid containing SV40 early region DNA. HB56B cells were non-tumorigenic in athymic nude mice, but HB56B/5T cells within a few passages of transfection with the SV40 plasmid formed tumors of which 28/37 regressed. HB56B cells may offer an experimental system for the study of proliferation, differentiation, and senescence control in human bronchial epithelial cells.     

外源p53对人肺癌细胞生长的抑制

目的　观察外源性野生型p53对有p53基因突变的人肺癌细胞系生长的影响。方法　用PCR SSCP及DNA测序 ,选择p53突变的人肺巨细胞癌系 80 1 D。构建野生型p53表达质粒PZiP p53。用基因枪介导外源基因。经G41 8筛选得到转染细胞系 80 1 D p53。用PCR检测外源基因 ,观察转染细胞恶性生长的变化。结果　转染细胞系 80 1 D p53体外长期传代有外源性p53基因存在 ,转染细胞生长明显受到抑制 ,集落形成抑制率达 96% ,裸鼠异种移植致瘤性降低 ,肿瘤生长明显缓慢。结论　外源性野生型p53经基因枪导入有p53基因突变的人肺癌细胞后可长期存在于转染细胞中 ,且明显抑制所转染的癌细胞的恶性生长。     

Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa

We examined the ability of human chromosome 11 derived from normal fibroblast cells to suppress the tumorigenicity of SiHa cells, a human cervical tumor cell line. Using DNA transfection, the human chromosome was tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic G418), transferred to mouse A9 cells by cell hybridization and microcell transfer techniques, and then transferred to SiHa cells by microcell transfer. These procedures resulted in the appearance of 15 independent, G418-resistant clones, 5 of which had one or two extra copies of an intact human chromosome 11. In situ chromosomal hybridization of these clones with the pSV2neo plasmid revealed the presence of a neo-tagged human chromosome 11 in all of the five SiHa-microcell hybrids. Two SiHa-microcell hybrids that contained a single copy of neo-tagged human chromosome 12 were also isolated by the same methods. The tumorigenicities of SiHa clones with one or two extra copies of chromosome 11 (SiHa-11) were suppressed; four of the five SiHa-11 clones formed no tumors in nude mice, whereas both parental SiHa cells and SiHa cells with an extra chromosome 12 formed tumors within 30 d. One SiHa-11 cell clone formed a single tumor 90 d after injection. This rare tumor had lost one copy of chromosome 11 and rapidly formed tumors when reinjected. These results indicate that the introduction of a single copy of normal human chromosome 11, but not chromosome 12, suppresses the tumorigenicity of SiHa cells, indicating the presence on human chromosome 11 of a putative tumor-suppressor gene (or genes) for human cervical tumors.     

突变型CD59蛋白对卵巢癌细胞的抑瘤效应

背景与目的：国外报道已在多种实体肿瘤细胞中发现有CD59分子的过表达,并与肿瘤的失控性生长和恶性转化密切相关。本研究探讨突变型CD59在卵巢癌细胞A2780表面的抗补体活性以及与LPS联合抑制A2780细胞增殖的活性。方法：取突变型CD59质粒、野生型CD59质粒分别转染A2780细胞．G418筛选稳定表达细胞克隆,并从基因水平和蛋白水平鉴定CD59突变基因和野生型基因的转染情况。MTT法观察野生型CD59与突变型CD59在A2780细胞表面的抗补体活性,以及突变型CD59在LPS存在时对细胞的抑瘤效应。结果：通过荧光免疫检测、流式细胞术分析、RT—PCR鉴定证明建立了稳定转染野生型和突变型CD59的A2780细胞。MTT结果显示,与野生型CD59相比,突变型CD59失去对补体的抑制功能,与对照组未转染的A2780细胞相比无显著性差异。MTT检测5μg／mLLPS作用30min．对转染野生型CD59、突变型CD59及未转染A2780细胞的增殖抑制率分别为（26．9±2．95）％、（36.3±4．87）％、（29．6±3．16）％,差异有统计学意义（P〈0．05）。结论：CD59的W40位点对其功能具有重要作用,封闭该位点能够提高补体溶细胞作用,并有助于LPS发挥抑制瘤细胞增殖活性．有望应用于肿瘤治疗。     

Comparative studies of human malignant mesothelioma in vivo, in xenografts in nude mice, and in vitro. Cell origin of malignant mesothelioma

Several decades ago, it was reported that normal and malignant mesothelial cells were transformed into distinct cell types (epithelial to fibrous and fibrous to epithelial) when transferred to in vitro conditions. Those tissue culture data are still cited as evidence supporting that the mesothelial cell has multipotentiality of differentiation and the mesothelial cell is a sole precursor of malignant mesothelioma cells. Six cell lines of heterotransplanted human malignant mesothelioma in nude mice and one cell line of subcultured human malignant mesothelioma in vitro have been established. To establish the validity of the classic concept of multipotentiality of malignant mesothelioma, we studied malignant mesothelioma cells of the seven cases using in vivo cultures, xenografts in nude mice and an in vitro tissue culture, utilizing histology, histochemistry, immunocytochemistry and electron microscopy. The nature of the original malignant mesothelioma cells was clearly shown to be well preserved in both the heterotransplanted and subcultured cells. Data did not support earlier hypotheses that mesothelial cells are capable of differentiating into distinct cell lines. The mesothelial cell and the submesothelial connective tissue cells are the precursors of the neoplastic cells in malignant mesothelioma.     

DNA transformation activity of a human gastrocarcinoma cell line

DNAs of three cell lines of human gastrocarcinoma (MGC-803, BGC-823 and PACM-82) and two fresh solid tumors of human stomach cancer were used to transfect NIH3T3 and Rat-1 cells. The transformed cells were selected with high concentration of glucose and low concentration of serum, or with medium containing Geneticin (G418) after co-transfection of pSVneo and DNAs of stomach cancer cell line or primary transformants. From the second round transfection, we had obtained transformants which could grow with high colony forming efficiency in soft agarose and were tumorigenic in nude mice. The southern blot analysis showed that the cellular DNA of the transformants contained human Alu repeat sequence and the transformed gene from stomach cancer cell line (BGC-823) and was homologous to proto-oncogene c-Ha-ras. The transforming gene is able to induce neoplastic transformation of NIH3T3 and Rat-1 cells.     

四环素调控的真核表达体系Hep3B Tet-On细胞株的建立

目的　建立可受四环素诱导调控的高表达外源基因的真核表达体系Hep3BTet On细胞株 ,用于基因功能的研究。方法　将pTet On质粒用脂质体介导法转染Hep3B细胞 ,经G4 18筛选后的克隆用有限稀释法进行单克隆化。单克隆分别扩增后 ,瞬时转染pTRE luc(编码荧光素酶蛋白 )质粒 ,Dox诱导表达后 ,检测荧光素酶活性 ,逐一筛选四环素调控高表达外源基因的Hep3BTet On细胞株。结果　成功构建了一株受Dox调控的高表达低背景的Hep3BTet On细胞株。结论　Hep3BTet On细胞株可用于外源基因的真核调控高表达 ,为研究真核基因功能提供了一种有力的实验手段。     

Humanized anti-CD26 monoclonal antibody as a treatment for malignant mesothelioma tumors.

PURPOSE: CD26 is a 110-kDa cell surface antigen with a role in tumor development. In this report, we show that CD26 is highly expressed on the cell surface of malignant mesothelioma and that a newly developed humanized anti-CD26 monoclonal antibody (mAb) has an inhibitory effect on malignant mesothelioma cells in both in vitro and in vivo experiments. EXPERIMENTAL DESIGN: Using immunohistochemistry, 12 patients' surgical specimens consisting of seven malignant mesothelioma, three reactive mesothelial cells, and two adenomatoid tumors were evaluated for expression of CD26. The effects of CD26 on malignant mesothelioma cells were assessed in the presence of transfection of CD26-expressing plasmid, humanized anti-CD26 mAb, or small interfering RNA against CD26. The in vivo growth inhibitory effect of humanized anti-CD26 mAb was assessed in human malignant mesothelioma cell mouse xenograft models. RESULTS: In surgical specimens, CD26 is highly expressed in malignant mesothelioma but not in benign mesothelial tissues. Depletion of CD26 by small interfering RNA results in the loss of adhesive property, suggesting that CD26 is a binding protein to the extracellular matrix. Moreover, our in vitro data indicate that humanized anti-CD26 mAb induces cell lysis of malignant mesothelioma cells via antibody-dependent cell-mediated cytotoxicity in addition to its direct anti-tumor effect via p27(kip1) accumulation. In vivo experiments with mouse xenograft models involving human malignant mesothelioma cells show that humanized anti-CD26 mAb treatment drastically inhibits tumor growth in tumor-bearing mice, resulting in enhanced survival. CONCLUSIONS: Our data strongly suggest that humanized anti-CD26 mAb treatment may have potential clinical use as a novel cancer therapeutic agent in CD26-positive malignant mesothelioma.     

Immunodiagnosis of mesothelioma: use of antimesothelial cell serum in an indirect immunofluorescence assay.

Cells isolated from human serous effusions were cultured in vitro. Monolayers of large multipolar cells were established. Antisera to the cultured cells were prepared in rabbits and rats. The antisera were absorbed with human red cells, liver powder and MOLT-4F cell line lymphocytes. Specificity of the absorbed antisera for human mesothelial cells were demonstrated in an indirect immunofluorescence assay. The antisera were used to confirm the diagnosis of mesothelioma in two cases. In both the patients, the morphologically identifiable malignant cell populations in the effusions stained positively with the antimesothelial cell serum thus establishing their mesothelial origin. Normal nonmesothelial tissue and known nonmesothelial tumors failed to react with the antisera thus confirming the specificity of the antisera.     

Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras

Activated ras oncogenes have previously been implicated in the pathogenesis of human lung carcinomas. A v-Ha-ras-containing retrovirus, Zip-ras, was generated by inserting the coding region of the v-Ha-ras oncogene into the Zip-NeoSV(X) [Cepko et al., Cell 37:1053-1062, 1984] retroviral vector. Amphotrophic Zip-ras retrovirus was used to infect an SV40 large T antigen-positive immortalized cell line, BEAS-2B, derived from normal bronchial epithelial cells, the predominant progenitor cells of human lung carcinomas. Zip-ras-infected BEAS-2B cells selected for G418 resistance formed anaplastic carcinomas in 12 of 15 athymic nude mice (latency 3 wk), whereas Zip-NeoSV(X)-infected BEAS-2B control cultures inoculated into 12 nude mice formed no tumors after a minimum of 7 mo. Tumor cell lines were established and demonstrated to be of human epithelial origin and to express v-Ha-ras p21 protein. A common feature of the tumor cell lines was an increase in ploidy. The increased efficiency of neoplastic transformation by v-Ha-ras of cell lines as compared with our previous results with normal bronchial epithelial cells [Yoakum et al., Science 227:1174-1179, 1985] is consistent with the hypothesis that the "immortalization" step is rate-limiting in in vitro human epithelial cell carcinogenesis.     

A BALB/c 3T3-transformed cell line suitable for transfection assay of metastasis-inducing genes

A clonal cell line, 1-1ras1000, transformed by the activated c-Ha-ras oncogene, does not form metastases after i.v. injection into mice (experimental metastasis assay). Here, we show that this cell line is useful as a recipient to detect metastasis-inducing genes, using a transfection assay. Cells (1-1ras1000) were susceptible to metastasis induction by transfection with either v-src or genomic DNA from a v-src- and v-fos-transferred highly metastatic rat cell line (SR202). The susceptibility of 1-1ras1000 cells for lung metastasis induction was suitable for a genomic transfection assay to detect a metastasis-inducing gene in the transfected cells which had incorporated genomic DNA from donor metastatic tumor cells. When DNAs extracted from 7 human tumors were tested for metastasis induction, 2 DNAs from nonmalignant tumor (non-tumorigenic tumors in athymic nude mice) (2/2) were negative and 4 DNAs from malignant tumors (4/5) were positive in 1-1ras1000 cells for primary transfection. In one of the resulting metastases, the ability to metastasize was also transferred in the second and third cycles of genomic DNA transfection at high frequencies. All of the resulting metastases carried the human repetitive A/u sequence. Neither re-arrangements of the endogenous c-Haras nor changes of protein amounts were detected. Recipient 1-1ras1000 cells had a negligible rate of spontaneously metastatic conversion during in vitro cultivation and transfection processes. The resulting metastasized cells were easily isolated from the lung after culturing in selection medium containing G418 (geneticin). Isolated cells stably retained the ability to form metastatic lung nodules when re-injected into mice. Thus, 1-1ras1000 cells appear to be a useful system for the isolation of metastasis-inducing genes from human metastatic tumors. Int. J. Cancer, 71:88–93, 1997. © 1997 Wiley-Liss, Inc.     

c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy

Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.     

Construction of Mouse A9 Clones Containing a Single Human Chromosome Tagged with Neomycin-resistance Gene via Microcell Fusion

Normal human fibroblasts (MRC-5 or NTI-4) were transfected with pSV2-neo plasmid DNA. Fifty G418-resistant fibroblast clones were isolated and independently fused to mouse A9 cells. The cell hybrids were selected and isolated in the medium containing G418 plus ouabaln. Since micronuclei were more efficiently induced in these hybrids compared to parental human fibroblasts by colcemid treatment, the transfer of neo-tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell fusion. Two hundred A9 microcell hybrids were isolated and karyotyped. Among them, thirteen microcell clones, each containing a single human chromosome 1, 2, 5, 6, 7, 8, 10,11,12,15,18,19 or 20 were established. Isozyme analyses confirmed the presence of each human chromosome in these A9 microcell clones. The results of Southern blot and chromosomal in situ hybridization analyses indicate that the human chromosomes in these clones were tagged with pSV2- neo plasmid DNA.     

外源人gp96基因在肿瘤细胞中的高效表达

背景与目的：从肿瘤细胞中分离出的热休克蛋白gp96制备物免疫小鼠可产生抗相应肿瘤的特异性保护性细胞毒性T细胞免疫应答,为肿瘤免疫治疗开辟了一条新的可行性途径,但gp96害细胞中的表达水平较低,从有限的细胞或组织中获得的gp96制备物难以满足研究的需要。因此,本研究拟为制备高质量和充足的gp96而建立高表达gp96的单克隆细胞株。方法：构建人gp96cDNA的重组表达质粒pcDNA-hgp96并将其转染HeLa细胞,G418筛选稳定转染的阳性细胞克隆,然后用Western印迹分析单克隆细胞株的gp96表达时。结果：成功构建了人gp96的重组表达质粒,建立了稳定转染的单克隆细胞株,并筛选出一株高表达gp96的单克隆细胞。结论：成功建立了高表达gp96的单克隆细胞株,为gp96抗肿瘤免疫的研究奠定了基础。     

绿色荧光蛋白基因标记的胃癌细胞系的建立

目的 建立能连续传代稳定高表达绿色荧光蛋白（GFP）的胃癌细胞株,探讨胃癌的生长与转移特征。方法 利用脂质体将携带GFP-cDNA的真核表达载体质粒转染胃癌细胞株GC9811-P,经过G418筛选和克隆化培养,用荧光显微镜及流式细胞仪检测转染的EGFP基因在癌细胞体内外的表达,MTT比色法观察细胞生长,Western blot检测转染细胞和未转染细胞肿瘤相关抗原的表达。结果 携带EGFP的真核表达载体能有效地转染胃癌细胞GC9811-P,转染的细胞能稳定、高效和持久地表达EGFP,与未转染细胞比较,他们的生物学特性未改变（P〉0. 05）。在裸鼠皮下肿瘤中,EGFP也能稳定、高效的表达。结论 胃癌GC9811P-GFP的建立为研究肿瘤侵袭和转移的发生机制提供了理想的细胞株。     

Morphological transformation, focus formation, and anchorage independence induced in diploid human fibroblasts by expression of a transfected H-ras oncogene

In an attempt to determine how normal human fibroblasts respond to high expression of the T24 H-ras oncogene, we tranfected such cells with the plasmid vector pHO6T1 (D. A. Spandidos and N. M. Wilkie, Nature (Lond.), 310:469-475, 1984), containing the T24 H-ras oncogene with 5' and 3' enhancer sequences, and the aminoglycoside phosphotransferase gene which confers resistance to the drug, G418. Approximately 1.5% of the G418-resistant colonies obtained after transfection and selection consisted of cells exhibiting obvious morphological transformation; i.e., they were highly refractile and more rounded than normal fibroblasts. DNA hybridization analysis showed that the morphologically transformed cells contained the transfected T24 H-ras oncogene, and radioimmunoprecipitation analysis showed that they were expressing the T24 H-ras protein product, M, 21,000 protein. Morphologically transformed cells formed colonies in soft agar at a frequency at least 60 times higher than that of cells that had been transfected with the control plasmid containing the normal cellular H-ras gene. Cells transfected with plasmid pHO6T1 could also be identified by their ability to form distinct foci when grown to confluence in nonselective medium following transfection. This study demonstrates that normal diploid human fibroblasts in culture can be transformed by transfection with a H-ras oncogene, and that such transformation correlates with expression of the mutant Mr 21,000 protein.     

Implantation of genetically manipulated fibroblasts into mice as antitumor alpha-interferon therapy

Long-term parenteral administration of human alpha-interferon (HuIFN-alpha) is effective in the treatment of several malignancies, including chronic myelocytic leukemia. In the present study, a model for fibroblast-mediated HuIFN-alpha gene therapy for the treatment of chronic myelocytic leukemia is described. Human IFN-alpha 5 complementary DNA was inserted into a bovine papilloma virus plasmid vector (BMGNeo) containing a neomycin resistance gene. The recombinant plasmid (BMGNeo-IFN) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and stably transformed cells were isolated by G418 selection. A fibroblast clone secreting a large amount of HuIFN into the culture supernatant was selected by radioimmunoassay using anti-HuIFN-alpha monoclonal antibodies. Southern blot analysis revealed that the transformed cells contained approximately ten copies of the BMGNeo-IFN plasmid per cell, and Northern blot analysis demonstrated high expression of HuIFN-alpha mRNA in the cells. This fibroblast clone strongly suppressed proliferation of a HuIFN-alpha-sensitive chronic myelocytic leukemia cell line (KU812) during cocultivation in vitro. When the HuIFN-alpha-producing fibroblasts were implanted into nude mice bearing KU812 tumors by the subcutaneous diffusion chamber method, tumor growth in vivo was also significantly suppressed. This study suggests the clinical potential of fibroblast-mediated gene therapy in the future.