高转移人肺癌表型去恶化研究─一细胞通讯、细胞骨架和癌基因、抑癌基因的相关变化

高转移人肺巨细胞癌ＰＧ细胞恶性表型明显。以人胚肺细胞为对照，荧光染料标记示踪法表明，ＰＧ的间隙连接通讯功能缺陷；免疫荧光染色显示微管解聚；荧光鬼笔环肽染色显示微丝网架破坏。分子杂交结果表明，ＰＧ细胞有ｃ－ｍｙｃＤＮＡ扩增和ｃ－Ｈａ－ｒａｓ、ｃ－ｍｙｃ基因高表达，抑癌基因Ｐ５３ｍＲＮＡ水平低于正常。用钙调蛋白拮抗剂ＣＤＺ（１００～２００ｎｍｏｌ／Ｌ）和中药Ｌ２（３～１３ｍｇ／ｍｌ）分别处理ＰＧ，细胞增殖受到抑制，表型向正常分化，细胞通讯功能再现，微管网架恢复。但只有Ｌ２能促进微丝骨架恢复，抑制软琼脂内的集落形成，并使ｃ－ｍｙｃ扩增减弱和ｍＲＮＡ水平下降，Ｐ５３表达升高。     

细胞间隙连接基因connexin32、connexin43及其蛋白在肝癌细胞系中的表达

为了探讨细胞间隙连接ｃｏｎｎｅｘｉｎ（ｃｘ）基因及其蛋白产物在肝癌细胞系的表达及意义,应用核酸分子原位杂交和流式细胞仪分析技术,研究肝癌细胞系ＨＨＣＣ、ＳＭＭＣ７７２１和正常肝细胞系ＱＺＧ中,间隙连接基因ｃｘ３２、ｃｘ４３ｍＲＮＡ及其蛋白产物的表达规律。原位杂交显示,ｃｘ３２、ｃｘ４３ｍＲＮＡ在ＨＨＣＣ、ＳＭＭＣ７７２１和ＱＺＧ中均呈强阳性。流式细胞仪分析,Ｃｘ３２蛋白在ＨＨＣＣ、ＳＭＭＣ７７２１和ＱＺＧ中阳性细胞计数率分别为１．９％、１．７％和９９．０％,Ｃｘ４３蛋白阳性细胞计数率分别为７．３％、２．３％和９９．１％。Ｃｘ３２、Ｃｘ４３蛋白在ＨＨＣＣ、ＳＭＭＣ７７２１中阳性细胞计数率与ＱＺＧ相比,有非常显著性差异（Ｐ＜０．０１）。ｃｘ３２、ｃｘ４３基因在转录后水平的调控异常所导致的蛋白表达降低与ＨＣＣ的发生密切相关。     

细胞间隙连接基因connexin32,connexin43及其蛋白在肝癌细胞？…

为了探讨细胞间隙连接ｃｏｎｎｅｘｉｎ（ｃｘ）基因及其蛋白产物在肝癌细胞系的表达及意义,应用核酸分子原位杂交和流式细胞仪分析技术,研究肝癌细胞系ＨＨＣＣ,ＳＭＭＣ－７７２１和正常肝细胞系ＱＺＧ中,间隙连接基因ｃｘ３２,ｃｘ４３ｍＲＮＡ及其蛋白产物的表达规律,原位杂交显示,ｃｘ３２,ｃｘ４３ｍＲＮＡ在ＨＨＣＣ,ＳＭＭＣ－７７２１和ＱＺＧ中均呈强阳性,流式细胞仪分析,Ｃｘ３２蛋白在ＨＨＣＣ,ＳＭＭＣ－７     

鸟氨酸脱羧酶反义RNA对人肺鳞癌细胞恶性表型的逆转

在已获两株有鸟氨酸脱羧酶（ＯＤＣ）反义序列稳定整合的细胞克隆的基础上，进一步研究发现，反义转染细胞的Ｇ０／Ｇ１期细胞数增多，Ｓ期细胞数相应减少；其软琼脂集落形成能力及裸鼠致瘤能力均显著下降。核酸杂交结果显示，在反义转染细胞中有ＯＤＣ反义ＲＮＡ表达，ＯＤＣｍＲＮＡ含量明显减少。结果表明，人肺鳞癌细胞恶性表型的逆转与ＯＤＣ反义ＲＮＡ有效控制多胺生物合成有关。     

c－ets－2、c－myc、N－ras三个癌基因联合反义RNA对肝癌细胞恶性表型的逆转

作者构建了一个能表达ｃ－ｅｔｓ－２、ｃ－ｍｙｃ及Ｎ－ｒａｓ三个癌基因联合反义ＲＮＡ的重组逆转录病毒载体，经病毒包装细胞ＰＡ３１７包装成假型逆转录病毒，利用此病毒成功地感染了人肝癌细胞株ＳＭＭＣ－７７２１，经Ｇ４１８筛选得到Ｇ４１８抗性细胞，基因组ＤＮＡ杂交结果表明重组病毒稳定地整合入７７２１细胞基因组中。ＲＮＡ杂交结果表明转化细胞中有较高水平的联合反义ＲＮＡ表达。初步结果表明，反义ＲＮＡ使７７２１细胞生长速率下降约７０％，软琼脂集落形成能力及裸鼠致瘤能力显著下降。这一结果表明，针对多个癌基因的联合反义ＲＮＡ可能给肿瘤基因治疗提供新的途径，有进一步探索的价值。     

逆转录病毒载体介导的IL-6大肠癌基因治疗实验研究

目的 探讨ＩＬ－６转基因表达对人肠癌细胞的体内外抑制作用。方法 参照分子克隆技术将重组建转录病毒载体ｐＺＩＰＩＬ－６ｃＤＮＡ转染ＰＡ３１７包装细胞,以Ｇ４１８筛选抗药性细胞人肠癌常规制备重组病毒液并感染大肠癌ＨＴ－２９细胞,采用ＮｏｒｔｈｅｒｎＢｌｏｔ分析基转录水平,ＥＬＩＳＡ法和ＭＴＴ显色法检测蛋白表达的量与细胞活性,以细胞生长曲线和集落形成实验以及裸鼠移植瘤实验,观察转导株的体内外抑瘤作用,结     

高转移人肺癌表型去恶化研究—细胞通讯,细胞骨架和癌基因…

高转移人肺巨细胞癌ＰＧ细胞恶性表型明显。以人胚肺细胞为对照，荧光染料标记示踪法表明，ＰＧ的间隙连接通讯功能缺陷；免疫荧光染色显示微管解聚；荧光鬼笔环肽染色显示微丝网架破坏。分子杂交结果表明，ＰＧ细胞有ｃ－ｍｙｃＤＮＡ扩增和ｃ－Ｈａ－ｒａｓ、ｃ－ｍｙｃ基因高表达，抑癌基因Ｐ＾５３ｍＲＮＡ水平低于正常。用钙调蛋白拮抗剂ＣＤＺ（１００～２００ｎｍｏｌ／Ｌ）和中药Ｌ２（３～１３ｍｇ／ｍｌ）分别处理ＰＧ，细     

HSP90β蛋白低调肿瘤细胞系的建立 *

目的：通过转染ＨＳＰ９０β反义核酸重组子ｐｃＤＮＡ－ＨＳＰ９０于４株人消化系肿瘤细胞系,以建立ＨＳＰ９０β蛋白低调肿瘤细胞系,并研究ＨＳＰ９０β蛋白低调后细胞的生长情况。方法：用Ｌｉｐｏｆｅｃｔａｍｉｎｅ介导将ｐｅＤＮＡ－ＨＳＰ９０转染人胃癌细胞系ＳＧＣ７９０１、人胃癌多药耐药细胞系ＳＧＣ７９０１／ＶＣＲ、人肝癌细胞系ＨＣＣ７４０２及人食道癌细胞系Ｅｃ１０９,用Ｇ４１８进行筛选,用ＲＮＡ酶保护分析法鉴定ＨＳＰ９０β反义核酸的表达,用Ｗｅｓｔｅｒｎ ｂｌｏｔ法检测ＨＳＰ９０β蛋白的表达。用细胞生长曲线研究基因转染细胞的生长情况。结果：ｐｃＤＮＡ－ＨＳＰ９０转染细胞ＡＨ－ＳＧＣ７９０１,ＡＨ－ＳＧＣ７９０１／ＶＣＲ,ＡＨ－ＨＣＣ７４０２及ＡＨ－Ｅｃ１０９有ＨＳＰ９０β反义ＲＮＡ表达,这些细胞ＨＳＰ９０β蛋白表达低调。研究还发现这些细胞的生长受到不同程度的抑制,其中ＡＨ－ＳＧＣ７９０１／ＶＣＲ及ＡＨ－Ｅｃ１０９细胞受抑程度更明显。结论：ｐｃＤＮＡ－ＨＳＰ９０转染细胞ＡＨ－ＳＧＣ７９０１,ＡＨ－ＳＧＣ７９０１／ＶＣＲ,ＡＨ－ＨＣＣ７４０２及ＡＨ－Ｅｃ１０９　ＨＳＰ９０β蛋白表达降低,细胞的生长受到不同程度的抑制。     

转化生长因子β1正反义基因对人肺巨细胞癌细胞体内外增殖的影响

探讨人转化生长因子β１（ＴＧＦβ１）正反义基因对人肺巨细胞癌细胞（ＰＬＡ－８０１Ｄ）增殖的影响及调控。方法通过基因转染方法,将ＴＧＦβ１正、反义基因分别导入ＰＬＡ－８０１Ｄ细胞,观察转染和未转染细胞体内外增殖变化、ｎｍ２３基因和ｃ－ｍｙｃ基因表达以及细胞凋亡特征。结果与未转染细胞比较,正义ＴＧＦβ１基因转染细胞培养上清中ＴＧＦβ１活性增加,体外生长速度减慢,软琼脂细胞培养集落形成能力降低,裸鼠体内成瘤率下降约４７％,肿瘤的转移受到抑制。其机制可能与正义ＴＧＦβ１基因转染细胞ｎｍ２３基因表达增加,ｃ－ｍｙｃ基因表达减少以及诱发细胞凋亡有关。而反义ＴＧＦβ１基因转染细胞则出现相反结果。结论ＴＧＦβ１对ＰＬＡ－８０１Ｄ细胞的体内外生长具有负调控作用     

鼻咽癌细胞系SUNE中EBV-LMP1基因对永生化人胚肾上皮细胞生长特性的影响

目的研究鼻咽癌细胞系ＳＵＮＥ中ＥＢ病毒潜伏膜蛋白１（ＬＭＰ１）基因对上皮细胞生长特性的影响，探索ＬＭＰ１在鼻咽癌发生中所起的作用。方法用ＬＭＰ１基因真核表达质粒转染人胚肾上皮细胞，检测ＬＭＰ１的表达，观察细胞的生长状态、生长速度、在软琼脂中的集落形成能力及对裸鼠的致瘤能力。结果被ＬＭＰ１基因转染的细胞生长旺盛，丧失接触抑制，生长速度增快，在软琼脂中能够形成多个集落，并能在裸鼠体内成瘤。结论ＬＭＰ１基因能明显改变上皮细胞的生物学行为，促进细胞的生长、增殖和转化，使转染的上皮细胞获得肿瘤细胞的生长特征。     

Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin43

The human lung carcinoma cell line PG is defective in gap junctional intercellular communication (GJIC). Connexin43 (Cx43) mRNA, which is expressed in normal human lung cells, is undetectable in these tumor cells. To explore if up-regulation of Cx43 gene expression will suppress malignancy of PG cells, Cx43 cDNA was co-transfected with pSV2neo cDNA into PG cells. Control cells were transfected with the blank vector plus neo cDNA. Several stable Cx43 transfectant clones, which acquired high levels of Cx43 expression and the capacity of GJIC, were compared with control clones and the parental cell line, both of which lacked Cx43 expression and GJIC. The control clones resembled the parental cells in exhibiting high cell growth rate, weak attachment to the substratum and a high frequency of colony formation in soft agar. In contrast to the control cells, Cx43 transfected clones showed reduced growth rate, enhanced attachment to the substratum and inhibition of colony formation in soft agar. In vivo results from nude mice experiments showed high tumorigenicity with control clones and inhibition of tumorigenicity in Cx43 transfected clones. The consistency between in vitro and in vivo results strongly suggests a tumor suppressing effect of the Cx43 gene in human lung carcinoma cells.      

Differential effect of subcellular localization of communication impairing gap junction protein connexin43 on tumor cell growth in vivo

There is a large body of evidence suggesting the connexin gap junction proteins appear to act as tumor suppressors, and their tumor inhibitory effect is usually attributed to their main function of cell coupling through gap junctions. However, some cancer cells (e.g. the rat bladder carcinoma BC31 cell line) are cell-cell communication proficient. Using specific site-directed mutagenesis in the third membrane-spanning (3M) domain of connexin43 (Cx43), we abolished the intrinsic gap junction intercellular communication (GJIC) in BC31 cells either by closing the gap junctional channels or by disruption of the transport of connexin complexes to the lateral membrane. Clones of BC31 cells transfected with a dominant negative Cx43 mutant giving rise to gap junctional channels, permeable only for a small tracer (neurobiotin), displayed accelerated growth rate in vivo, showing the critical role of selective gap junctional permeability in the regulation of cell growth in vivo. The use of other dominant-negative mutants of Cx43 also suggested that the effect of impaired communication on the tumorigenicity of cancer cells depends on the subcellular location of connexin. Inhibition of intrinsic GJIC in BC31 cells by sequestering of Cx protein inside the cytoplasm, due to expression of dominant-negative transport-deficient Cx43 mutants, did not significantly enhance the growth of transfectants in nude mice, but occasionally slightly retarded it. In contrast, augmentation of GJIC in BC31 cells by forced expression of wild-type Cx43, or a communication-silent mutant, fully suppressed tumorigenicity of these cells. Overall, these results show that cell coupling is a strong, but not the sole, mechanism by which Cx suppresses growth of tumorigenic cells in vivo; a GJIC-independent activity of Cx proteins should be considered as another strong tumor-suppressive factor.     

The effect of transfected Cx43 gene on the GJIC and proliferation of glioma cells

Cell to cell communication via gap junction plays an important role in the maintanence of normal cell growth. Its disruption may lead to aberrant cell growth and eventually development of tumors[1]. Gap junctions (GJ) are specialized intercellular channels between plasma membrane of two adjacent cells. Each GJ channel is composed of two connexons contributed by each of two communicating cells and each connexon is a hexameric assembly of protein subunits known as connexins (Cx). Cx genes fun…     

转染连接蛋白基因对人脑胶质瘤细胞间隙连接通讯及其生长变化的研究

目的 研究连接蛋白（Cx)基因对人脑胶质瘤细胞的细胞间隙连接通讯（GJIC）及其增殖的抑制作用,探索以Cx43基因治疗胶质瘤的可行性。方法 将含Cx43cDNA的质粒,以脂质体介导转染Cx43表达缺失的TJ905人胶质母细胞瘤细胞,通过Northern印染杂交、原位杂交及免疫组化染色检测Cx43mRNA及蛋白表达,划痕标记荧光染料示踪技术（SLDT）检测GJIC,MTT法测定细胞增殖率,核仁组成区嗜银蛋白（AgNOR)染色检测细胞增殖活性,TUNEL法检测细胞凋亡。结果 转染后TJ905细胞有不同程度的Cx43mRNA和蛋白表达及GJIC恢复。Cx43表达水平高的克隆细胞增殖明显下降,细胞凋亡并未增加。结论 Cx43基因及GJIC在恶性胶质瘤的发生发展过程中起重要作用,可能成为恶性胶质瘤基因治疗的优选靶的之一。     

Basic Investigations EXPRESSION OF GAP JUNCTION PROTEIN Cx43 IN CULTURED HUMAN NORMAL AND MALIGNANT LUNG CELLS

ＢａｓｉｃＩｎｖｅｓｔｉｇａｔｉｏｎｓＥＸＰＲＥＳＳＩＯＮＯＦＧＡＰＪＵＮＣＴＩＯＮＰＲＯＴＥＩＮＣｘ４３ＩＮＣＵＬＴＵＲＥＤＨＵＭＡＮＮＯＲＭＡＬＡＮＤＭＡＬＩＧＮＡＮＴＬＵＮＧＣＥＬＬＳＺｈａｎｇＺｈｉｑｉａｎ；张志谦；ＬｉｎＺｈｏｎｇｘｉａｎｇ...     

Inducible expression of the gap junction protein connexin43 decreases the neoplastic potential of HT-1080 human fibrosarcoma cells in vitro and in vivo

Numerous studies have demonstrated a correlation between dysregulation/loss of connexin expression or gap junction intercellular communication (GJIC) function and decreased growth control both in human tumors and tumor cell lines. Likewise, restoration of constitutive connexin expression/function is correlated with increased growth control/decreased tumorigenicity. Here, we show for the first time that inducible restoration of connexin43 (Cx43) expression and GJIC function in a human tumor line of mesenchymal origin (HT-1080, fibrosarcoma) resulted in a lowered neoplastic potential. Specifically, HT-1080 cells induced to express Cx43 demonstrated diminished foci formation when in co-culture with normal fibroblasts, decreased colony formation under anchorage-independent conditions, and reduced tumor growth when injected into immunodeficient mice. These results, obtained utilizing an inducible system that helps address issues of clonal heterogeneity, strongly implicate Cx43 as a tumor suppressor in human tissue of mesenchymal origin and GJIC as a regulatory mechanism for cellular growth control both in vitro and in vivo. This study also further supports the hypothesis that loss of Cx43/GJIC in human tumors may play an important role in the dysregulation of normal growth control.     

Gap junctional intercellular communication and connexin43 expression in human ovarian surface epithelial cells and ovarian carcinomas in vivo and in vitro.

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) are frequently decreased in neoplastic cells and have been increased by cAMP and retinoids. GJIC and connexin expression were investigated in early passage normal human ovarian surface epithelial (HOSE) cells, human ovarian adenocarcinoma cell lines (CaOV-3, NIH:OVCAR-3, SK-OV-3 and SW626) and surgical specimens of human serous cystadenocarcinomas. We hypothesized that GJIC and connexin expression would be decreased in neoplastic cells and would be increased by cAMP and retinoic acid. Cultured HOSE cells exhibited extensive fluorescent dye-coupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate and all-trans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.     

Aberrant expression and localization of connexin43 and connexin30 in a rat glioma cell line

Gap junctions are cellular structures which permit direct exchanges of small molecules from cytoplasm to cytoplasm in most of the cells of metazoan organisms. For four decades, it has been observed that the inhibition of this type of intercellular communication is often associated with tumorigenesis. The assumption that loss of homeostasis which characterizes tumor growth could be a consequence of a lack of gap junctional intercellular communication (GJIC) has been reinforced by strategies able to reinduce both GJIC and normalization of the phenotype. So far, no molecular data may explain clearly how gap junctions can regulate cell proliferation. It has been argued that the gap-junction tumor suppressive effect may depend specifically on the connexin type which is expressed. For instance, the transfection of connexin30 (Cx30), a gap junction protein, has been previously associated with a slower growth of rat glioma cells (9L cells). Here, we show that these cells do communicate less compared to the Cx43-expressing parental cells even if the Cx30-transfected cells do express more Cx43. This result was related to the cytoplasmic distribution of Cx43 and a nuclear localization of both the Cx30 and a 20-kDa fragment corresponding to a Cx43 signal. According to these data, it seems that cell growth regulation may depend more on the behavior of connexins than the simple establishment of GJIC.     

Gap junction proteins connexin32 and connexin43 partially acquire growth-suppressive function in HeLa cells by deletion of their C-terminal tails.

Our laboratory has previously reported that transfection of a connexin26 (Cx26) gene, but not connexin40 nor connexin43 (Cx43), into HeLa cells expressing no detectable level of connexins suppressed the tumorigenic phenotype of the HeLa cells both in vitro and in vivo, although all of these connexins induced gap junctional intercellular communication in HeLa cells to a similar extent. The most remarkable structural difference between connexin proteins is the length of the C-terminal cytoplasmic tail, Cx26 having almost no tail, while Cx43 and connexin32 (Cx32) have long and intermediate ones, respectively. When Cx32 and Cx43 lose their C-terminal tails, they seem to resemble Cx26 in structure. To examine whether such truncated connexins become tumor suppressive in HeLa cells, we introduced a stop codon into each of the Cx32 and Cx43 cDNAs to remove their C-terminal tails and transfected these constructs (DeltaCx) into HeLa cells. Both DeltaCx cDNAs induced GJIC as efficiently as the wild-type counterparts. Although none of the truncated connexins affected proliferation rate, the truncated Cx32 and Cx43 proteins suppressed anchorage-independent cell growth in soft agar. Furthermore, when the transfectants were injected into the backs of nude mice, tumor appearance was delayed by 7 days in animals given cells expressing truncated connexins, i.e. tumors became detectable on days 11 and 18 after injection of vector and DeltaCx transfectants, respectively. Although throughout these experiments the truncated connexins did not completely eliminate the tumorigenicity of HeLa cells, as Cx26 did, it was evident that deletion of the C-terminal tails gave both Cx32 and Cx43 a capacity for negative growth control, suggesting that the C-terminal tails of these two connexins function as a regulatory region for connexin-mediated growth control in HeLa cells.