反义RNA对人胃癌细胞生长抑制及恶性表型的阻断作用

ｒａｓ原癌基因的点突变是人胃癌发生发展的重要机理之一。利用能表达ｃ－Ｈ－ｒａｓ癌基因反义ＲＮＡ的质粒,导入人胃癌细胞系ＢＧＣ－８２３,研究了ｒａｓ癌基因反义ＲＮＡ对人胃癌细胞生长及恶性表型的作用,结果表明,ｃ－Ｈ－ｒａｓ反义ＲＮＡ可引起ＢＧＣ－８２３生长速率及形态的变化,在半固体培养基中细胞集落形成能力减弱,部分也抑制了ＢＧＣ－８２３在裸鼠体内的致瘤性。ｃ－Ｈ－ｒａｓ反义ＲＮＡ对其ＲＮＡ的过量表达     

毛喉萜对人胃癌细胞BGC－823中蛋白激酶C及其亚类的影响

以人胃癌细胞ＢＧＣ－８２３为模型,研究了毛喉萜（ｆｏｒｓｋｏｌｉｎ）对胃癌细胞中蛋白激酶Ｃ活性及其亚类基因表达的作用,同时也观察了毛喉萜对癌基因ｃ－ｊｕｎ及抑癌基因ｐ５３表达的影响．结果表明,２×１０~（－５）ｍｏｌ／Ｌ毛喉萜处理ＢＧＣ－８２３细胞７２ｈ,细胞质、膜和细胞核ＰＫＣ活性下降,ＰＫＣ亚类β,γ基因表达被抑制,癌基因ｃ－ｊｕｎ的表达也明显降低,而抑癌基因ｐ５３表达升高,上述变化可能是毛喉萜抑制胃癌细胞增殖等生理效应的重要分子事件。     

人肝细胞癌中N－ras、c－myc癌基因的表达──原位分子杂交和免疫组织化学研究

为了明确Ｎ－ｒａｓ、ｃ－ｍｙｃ癌基因在人肝细胞癌（ＨＣＣ）中的表达及其形态学分布,我们对３８例石蜡包埋的ＨＣＣ（其中３３例带有癌周肝组织）标本进行了原位核酸杂交和免疫组化研究。结果表明,ＨＣＣ及癌周肝组织中Ｎ－ｒａｓ表达阳性率分别为４２％和３９％;ｃ－ｍｙｃｍＲＮＡ阳性表达率分别为４７％和３６％;ｃ－ｍｙｃ蛋白阳性率分别为５３％和３９％。ＨＣＣ与癌周肝之间Ｎ－ｒａｓ、ｃ－ｍｙｃｍＲＮＡ及ｃ－ｍｙｃ蛋白表达水平无显著差别;ｃ－ｍｙｃｍＲＮＡ及蛋白阳性强度也无显著差别,其形态学分布基本一致,主要集中于癌细胞和癌周肝中的部分高度增生结节;Ｎ－ｒａｓ表达增强主要见于癌细胞、癌周肝中的高度增生结节、小细胞性不典型增生及少部分肝小多角细胞。它们与ＨＢｘＡｇ表达之间无明确的对应关系。上述结果显示,ＨＣＣ中Ｎ－ｒａｓ、ｃ－ｍｙｃ表达都显著增强,它们可能是人ＨＣＣ发生中较晚期的事件,而Ｎ－ｒａｓ表达增强早于ｃ－ｍｙｃ癌基因活化。     

毛喉萜抑制人胃癌细胞BGC－823增殖与ras基因表达相关性研究

毛喉萜（ｆｏｒｓｋｏｌｉｎ）对人胄癌细胞ＢＧＣ－８２３增殖有明显抑制作用,具药物剂量和作用时间之依赖性。剂量为２×１０~（－５）ｍｏｌ／Ｌ之毛喉萜使胃癌细胞在软琼脂中形成集落的能力显著降低;癌基因ｃ－Ｈａ－ｒａｓ之表达明显被抑制,细胞核中与ｒａｓ基因上游调控区２．５ｋｂ片段结合的三种蛋白结合能力下降。联系到以同样浓度药物处理胃癌细胞７２ｈ,细胞质、膜与细胞核中蛋白激酶Ｃ（ＰＫＣ）活性均下降的现象,可能ＰＫＣ活性下降与Ｈａ－ｒａｓ基因上游片段２．５ｋｂ结合蛋白之结合能力下降存在相关性,ＰＫＣ可能通过影响ＤＮＡ结合蛋白的磷酸化作用,导致了Ｈａ－ｒａｓ基因表达之被阻抑。而ｒａｓ基因表达下降可能是毛喉萜抑制胃癌细胞增殖的一个重要分子事件。     

新型生长抑肽对成纤维细胞的DNA合成及某些癌基因表达的影响

从细胞水平和基因水平研究了新型生长抑肽（ＥＰＰ）的生物学作用,研究表明：ＥＰＰ对ＮＣ３Ｈ１０及ＴＣ３Ｈ１０细胞的ＤＮＡ合成均有抑制作用,当ＥＰＰ与ｃＡＭＰ的位点选择性类似物８－Ｂｒ－ｃＡＭＰ共同作用时,其对ＮＣ３Ｈ１０细胞的ＤＮＡ合成的抑制作用消失,而对ＴＣ３Ｈ１０仍具有抑制作用;核酸杂交分析表明,ＥＰＰ可以抑制ｃ－ｆｏｓ、ｎｅｕ、ｋｉ－ｒａｓ三类癌基因在转化细胞中的表达。证明了ＥＰＰ对转化细胞的生长具有一定的抑制效应,且与８－Ｂｒ－ｃＡＭＰ联合使用时其效果更佳。     

重组RA—538及反义c—myc腺病毒对人胃癌,食管癌和bcl—2高表达细胞系的作用

本课题研究ＲＡ５３８、反义c-ymc重组腺病毒对人胃癌（ＳＧＣ７９０１）、食管癌（Ｅ Ｃ１０９、ＥＣ８７１２）、正常人胚肺２ＢＳ（２ＢＳ）及ｂｃｌ－２高表达细胞第的体仙外生物学作用及其分子机制。结果显示Ａｄ－ＲＡ５３８及Ａｄ－ＡＳｃ－ｍｙｃ对ＳＧＣ７９０１细胞体内外均具有明显的生长抑制及凋亡诱导作用,并能抑制其ｃ－ｍｙｃ、ｂｃｌ－２、ｃｙｃｌｉｎＤ１基因的表达及刺激ｂａｘ基因的表达。对ＥＣ１０９、ＥＣ８     

新型生长抑肽对成纤维细胞的DNA合成及某些癌基因表达的影响

从细胞水平和基国水平研究了新型生长抑肽（ＥＰＰ）的生物学作用，研究表明：ＥＰＰ对ＮＣ３Ｈ１０及ＴＣ３Ｈ１０细胞的ＤＮＡ合成均有抑制作用，当ＥＰＰ与ｃＡＭＰ的位点选择必类似物８－Ｂｒ－ｃＡＭＰ共同作用时，其对ＮＣ３Ｈ１０细胞的ＤＮＡ合成的抑制作用消失，而对ＴＣ３Ｈ１０仍具有抑制作用；核酸杂交分析表明，ＥＰＰ可以抑制ｃ－ｆｏｓ，ｎｅｕ，ｋｉ－ｒａｓ三类癌基因在转化中的表达，证明了ＥＰＰ对转化细胞的生长     

外源性神经节苷脂GM3影响人肝癌细胞生长的可能机制的初步研究

通过苔酚蓝染色细胞发现,外源性ＧＭ３能明显抑制人肝癌细胞株ＳＭＭＣ－７７２１细胞生长,在ＧＭ３处理３ｄ时,出现明显差异,通过ＮｏｒｔｈｅｒｎＢｌｏｔ分析发现,外源性ＧＭ３可明显影响人肝癌细胞株ＳＭＭＣ－７７２１细胞中ｃ－ｆｏｓ、ｃ－ｊｕｎ、ｃ－ｍｙｃ和Ｎ－ｒａｓ四种癌基因的ｍＲＮＡ表达,未经ＧＭ３处理的细胞中没有检测到ｃ－ｆｏｓｍＲＮＡ,但ｃ－ｊｕｎ微量表达,并有ｃ－ｍｙｃ和Ｎ－ｒａｓｍＲＮＡ的高     

稀土化合物氯化亚鈰对人肺癌细胞PG、人胃癌细胞BGC-823的作用

 以人肺癌细胞 Ｐ Ｇ 和人胃癌细胞 Ｂ Ｇ Ｃ ８２３ 作为研究对象,利用 Ｍ Ｔ Ｔ 测定、３ Ｈ Ｔｄ Ｒ 参入、流式细胞术、软琼脂培养、 Ｎｏｒｔｈｅｒｎ ｂｌｏｔ、 Ｗ ｓｔｅｒｎ ｂｌｏｔ 等实验方法,观察了稀土化合物氯化亚鈰（ Ｃｅ Ｃｌ３）抑癌作用．结果表明, Ｃｅ Ｃｌ３ 浓度为 ００５ ｍ ｍ ｏｌ／ Ｌ,０１ ｍ ｍ ｏｌ／ Ｌ,０５ ｍ ｍ ｏｌ／ Ｌ和 １ ｍ ｍ ｏｌ／ Ｌ可抑制 Ｐ Ｇ 细胞的增殖;浓度为 ０５ ｍ ｍ ｏｌ／ Ｌ和 １ ｍ ｍ ｏｌ／ Ｌ可抑制 Ｐ Ｇ 细胞 Ｄ Ｎ Ａ 的合成,其 Ｇ１ 期细胞比例增加而 Ｓ期细胞比例减少,在软琼脂中的生长能力降低,原癌基因 ｃ ｍ ｙｃ 和 ｃ ｒａｓ 表达降低,ｐ１６ 蛋白质表达降低．而同样浓度的 Ｃｅ Ｃｌ３ 对 Ｂ Ｇ Ｃ ８２３ 细胞和正常细胞 ２ Ｂ Ｓ未见影响．提示：稀土化合物抑制肺癌细胞 Ｐ Ｇ 的增殖以及降低其恶性度的作用机制可能与一些增殖相关的原癌基因的表达和细胞周期的调控有关,其确切的机理还需进一步的研究．     

反义癌基因c－jun对大鼠血管平滑肌细胞增殖的影响

构建可表达原癌基因ｃ－ｊｕｎ正义和反义ＲＮＡ的真核细胞表达载体,将其导入体外培养的大鼠血管平滑肌细胞（ＶＳＭＣ）后,经Ｎｏｒｔｈｅｒｎ分析证实,两种插入方向的ｃｊｕｎ基因均在ＶＳＭＣ中大量表达;￣３Ｈ－ＴｄＲ参入实验表明,ｃ－ｊｕｎ反义ＲＮＡ对ＶＳＭＣ的增殖具有显著抑制作用．提示用反义核酸抑制ｃ－ｊｕｎ表达对治疗某些由于ＶＳＭＣ增殖所引起的心血管病可能具有一定意义．     

Effects of PTX1 expression on growth and tumorigenicity of the prostate cancer cell line PC-3

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It encodes a nuclear protein that is downregulated in prostate carcinoma. Expression constructs containing PTX1 cDNA in both sense and antisense orientations were transfected into prostate tumor cell line, PC-3 cells. The effects of the expression of PTX1 and antisense PTX1 on PC-3 cells were examined using cell growth, proliferation, soft agar, invasion chamber, senescence-associated beta-galactosidase, and nude mice assays. Cells transfected with PTX1 construct in the sense orientation were growth-arrested. These cells displayed multiple morphological changes consistent with cellular senescence, including the expression of a senescence-associated beta-galactosidase. On the other hand, expression of antisense PTX1 RNA in PC-3 cells resulted in uncontrolled cell growth and increase of invasive potential. In nude mice, cells expressing antisense PTX1 grew sixfold faster than the control. These results suggest that PTX1 may play an important role in the growth and tumorigenicity of PC-3 cells.     

Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene.

Several lines of evidence have suggested that c-fos may act downstream from c-Ha-ras in a growth-regulatory signal transduction pathway. We used antisense RNA to inhibit c-fos gene expression and investigated the effects of diminished c-fos expression on the phenotypes induced by the EJ c-Ha-ras oncogene in NIH 3T3 cells. Immunofluorescent staining demonstrated that the antisense RNA caused a marked reduction in the amount of c-fos protein expressed following serum stimulation. EJ cells containing antisense-fos RNA continued to overexpress ras and remained capable of proliferating in vitro. However, the antisense-fos RNA caused a partial reversion of the major transformed phenotypes of EJ cells, including a restoration of both density-dependent growth arrest and the ability to be rendered quiescent by serum deprivation, a reversion to a flat morphology, inhibition of anchorage-independent growth, and inhibition of tumorigenicity in nude mice. Our results indicate that inhibition of c-fos expression, to a level still supporting in vitro proliferation, prevents the transforming effects of the ras oncogene; they thus provide additional evidence for the participation of c-fos in ras-regulated signal transduction pathways.     

抑制PKCα对乳腺癌细胞特性及cyclinE和CDK2基因表达的影响

Human breast cancer cell line Bcap-37 was stably transfected with the plasmid expressing antisense PKC alpha RNA, and cells, in which PKC alpha was inhibited due to antisense PKC alpha RNA, were isolated. Changes in serum-dependent growth in cell culture, cell clonogenicity in soft agar and growth in nude mice were tested, and the expressions of cyclin E and CDK2 were analyzed. After PKC alpha was inhibited, the cells showed that serum-dependent growth and anchorage-dependent growth enhanced, tumorigenicity in nude mice decreased. The results suggest that less aggressive breast cancer phenotypes may be induced by inhibition of PKC alpha. Levels of cyclin E and CDK2 mRNA in cells with antisense PKC alpha RNA were lower than those in control cell. These indicate that signal transduction system with PKC alpha is closely related to cell cycle control system with cyclin/CDK in the functions.     

Reversible tumorigenesis induced by deficiency of vasodilator-stimulated phosphoprotein

Random homozygous knockout (RHKO) is an antisense RNA strategy capable of identifying genes whose homozygous functional inactivation yields a selectable phenotype in cells growing in culture. Using this approach, we isolated NIH 3T3 fibroblast clones that showed the ability to form colonies on 0.5% agar and tumors in nude mice. The gene inactivated in one of these clones was found to encode VASP (vasodilator-stimulated phosphoprotein), a previously identified protein that binds to components of the cadherin-catenin junctional complex and has been implicated in cell-cell interactions, the formation of actin filaments, and the transmission of signals at the cytoskeleton-membrane interface. Fibroblasts made deficient in VASP by RHKO showed loss of contact inhibition, and consequently, continued cell division past confluence. Restoration of VASP function by reversal of RHKO yielded cells that had lost the neoplastic capabilities acquired during RHKO. Overproduction of VASP mRNA in the sense or antisense orientation from expression constructs introduced by transfection into naive NIH 3T3 fibroblasts also resulted in neoplastic transformation, implying that normal cell growth may require the maintenance of VASP expression within a narrow range. Our results implicate VASP in tumorigenesis and/or cancer progression.     

Matrix metalloproteinase 11 depletion inhibits cell proliferation in gastric cancer cells

Our previous study has shown that matrix metalloproteinase 11 (MMP11) is highly expressed in tumor cell lines and primary tumor of gastric cancer (GC). In order to reveal the correlation between expression of MMP11 and biological features of GC cell, we have constructed the recombinant plasmids producing hairpin small interfering RNA (siRNA) to target MMP11 mRNA using a vector-based RNA interference technology. Stable transfection of recombinants into GC cell line BGC823 specifically depleted the mRNA and protein of MMP11 as demonstrated by RT-PCR and Western blotting analysis. The siRNA-treated cells exhibited significantly decreased growth ability compared with mock transfectants and parental BGC823 cells. Furthermore, colony formation of MMP11 deficient cells was dramatically inhibited in soft agar and tumorigenicity was reduced in nude mice, respectively. These results provide new insights into the function of MMP11 and suggest that MMP11 may play an important role in the control of cell proliferation and tumor development in GC.     

Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.     

Neoplastic transformation and tumorigenesis associated with overexpression of IMUP-1 and IMUP-2 genes in cultured NIH/3T3 mouse fibroblasts

Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.     

人乳头瘤病毒16型E6和E7基因及其突变体转化活性的研究

为筛选出可用于研制HPV治疗性疫苗的HPV16型E6和E7基因突变体,故将HPV16型原型株(德国株)E6和E7基因及其各种突变体分别转染Balb/c3T3细胞,观察转染后的细胞在软琼脂培养中的集落形成能力和在裸鼠体内的成瘤能力.结果表明,单独转染和共转染HPV16野生型E6和E7基因的Balb/c3T3细胞系,在软琼脂中呈集落样生长,并在裸鼠体内成瘤;而转染E6基因突变体mE6(50G)、E7基因的两种突变体mE7-1(24G26G)和mE7-3(24G26G67R)以及共转染mE6和mE7-1的Balb/c3T3细胞,在软琼脂培养中极少形成集落,也不能在裸鼠体内成瘤.提示经结构改造后的HPV16 E6和E7基因已失去了对Balb/c3T3细胞的转化活性,而保留了免疫原性,可用于HPV16相关肿瘤治疗性疫苗的构建.     

Expression in rat fibroblasts of a human transforming growth factor-alpha cDNA results in transformation

Acquisition of the ability to produce and respond to a growth factor may result in increased cellular proliferation and could lead to malignant transformation. The fact that a large variety of tumor cells secrete transforming growth factor-alpha (TGF-alpha) suggests involvement of TGF-alpha in cellular transformation and provides supporting evidence for the autocrine stimulation model. In order to determine directly the role of TGF-alpha in tumorigenicity, we introduced a human TGF-alpha cDNA expression vector into established nontransformed Fischer rat fibroblast (Rat-1) cells. Synthesis and secretion of human TGF-alpha by these cells results in the loss of anchorage-dependent growth and induces tumor formation in nude mice. Anti-human TGF-alpha monoclonal antibodies prevent TGF-alpha expressing Rat-1 cells from forming colonies in soft agar.