Alterative Expression and Sequence of Human Elongation Factor-1δ during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.     

Role of cyclinD1 and CDK4 in the carcinogenesis induced by silica

To study the role of cyclinD 1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line （2BS） induced by silica. Methods Recombination vectors with sense and antisense pXJ41-cyclinD1 and pXJ41-CDK4 were constructed, and then transfected into the malignant transformed cells induced by silica, respectively. At the same time, pXJ41-neo was used as the control. Results During the progress of the malignant transformation of 2BS cells induced by silica, cyclinD 1 and CDK4 were overexpressed. Antisense RNA suppressed cyclinD 1 and CDK4 gene expression in the antisense pXJ41-cyclinD1 and pXJ41-CDK4 transfected cells. Antisense RNA led to cell cycle arrest, resulting in lengthened G1 phase （the percentages of cells in the G1 phase changed from 45.1% to 52.7% and 58.0% for cyclinD1 and CDK4 transfected cells, respectively）, and eventually attenuated the increase of the proliferation of malignant transformed cells induced by silica. Compared with malignant transformed cells induced by silica, cells transfected with antisense pXJ41-cyclinD1 and pXJ41-CDK4 showed obviously reduced growth rates. On the 8th day, the suppression rates were 58.69 and 77.43% （the growth rate of malignant transformed cells induced by silica was 100%）, doubling time changed from 21.0 h to 31.4 h and 21.0 h to 42.7 h, respectively, the growth capacities on soft agar of cells transfected by antisense pXJ41-cyclinD1 and pXJ41-CDK4 decreased obviously. Conclusion CyclinD 1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.     

Effects of Exogenous p16^ink4a Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16ink4a gene were investigated. Exogenous p16ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16ink4a gene was homozygously deleted. The expression of p16ink4a mRNA and protein was detected by RT-PCR and immunocyto-chemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16ink4a gene could be stably ex-pressed. The growth of A549 cells transfected with p16ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhib-ited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exoge-nous p16ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.     

Down-regulation of p110β expression increases chemosensitivity of colon cancer cell lines to oxaliplatin

This study examined the synergetic effect of class ?A Phosphoinositide 3-kinases cata-lytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-Ⅴ FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evalu-ated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G0-G1 phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by im-munoblotting in p110β knockdown group compared with normal control group and wild-type group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.     

Effects of anti- HPV16E6- ribozyme on phenotype and gene expression of a cervical cancer cell line

Objective To investigate the effects of anti- HPV16E6- ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line. Methods HRz was designed by computer programs.HRz’s activity was identified by cleavage experiments in vitro.HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi- R and CaSKi- P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot.The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot.Cell growth curves and soft agar forming ability were studied.The ability of each cell line to form tumors was assessed in nude mice.Apoptosis rates and expression of c- myc, bcl- 2, p53 and Fas were detected by flow cytometry (FCM).Antigens of tumor cells, HLA- 1, HLA- 2, B7- 1 and B7- 2 were also detected.NK, LAK, and CD3AK cells were induced.Their cytotoxicities were detected in CaSKi- R, CaSKi- P, and CaSKi cells. Results In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site- specific manner.HRz could be expressed stably in transfected CaSKi cells.Northern blot analysis showed that E6 mRNA levels were lower in CaSKi- R than in CaSKi.The growth rate of CaSKi- R was slower than those of CaSKi and CaSKi- P.The soft agar- forming rate of CaSKi- R was lower compared with those of CaSKi and CaSKi- P cells.The ability of CaSKi- R to form tumors in nude mice was also poor.The apoptosis rate of CaSKi- R cells was much higher than those of CaSKi and CaSKi- P.HRz could reduce the expression of E6, c- myc and bcl- 2 proteins, and increase the expression of p53 as well.HRz could increase the expression of HLA- 2, B7- 1 and B7- 2 antigens.The cytotoxicity of NK, LAK and CD3AK cells was much higher in CaSKi- R than in CaSKi- P and CaSKi cells.Conclusion HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells.     

Curcumin down-regulates PCNA, cyclin D1 and Bcl-XL expression in human keratinocyte cell lines

Objective：To evaluate the effects of curcumin on regulating the proliferation,cell cycle distribution,apoptosis and relevant mechanisms in keratinocyte cell lines.Methods：The human immortalized human keratinocyte lines（HaCaT cells） were treated with different doses of curcumin.The effects of curcumin on cell viability were measured by MTT assay,and the cell cycle distribution and apoptosis determined by flow cytometry.The mRNA expression changes of proliferating cell nuclear antigen（PCNA）,cyclin D1 and Bcl-xL were from real-time PCR analysis and the protein levels were detected by Western blotting.Results：Data obtained in the study showed that curcumin could cause significantly inhibitory effect on proliferation in HaCaT cells in a time- and dose-dependent manner.Cell arrest at G1/S phase and significant apoptosis were observed after being treated with curcumin for 24 h.In association with these,the expression of PCNA,cyclin D1 and Bcl-xL were decreased both at mRNA and protein levels for the same treatment.Conclusion：Curcumin can inhibit proliferation,induce cell arrest at G1/S phase and cause apoptosis in HaCaT cells.The decreased expression of PCNA,cyclin D1 and Bcl-xL induced by curcumin contributes to the above effects in vitro.     

镉和镍恶性转化16HBE上调基因eIF3 p36的克隆与序列

目的分析镉和镍恶性转化16HBE（人支气管上皮细胞）成瘤细胞株中翻译起始因子eIF3 p36基因序列的变化情况，探讨镉、镍重金属的致癌分子机制。方法取正常对照16HBE细胞、氯化镉（CAC12）和结晶型硫化镍（NiS）恶性转化的16HBE细胞，用有限稀释法分别建立单细胞克隆株，通过逆转录聚合酶链反应（RT—PCR）获得目的基因翻译起始因子eIF3 p36的eDNA，将目的基因产物连接到T载体中，转化、扩增重组质粒。提取大肠杆菌中的重组质粒进行DNA测序。将三组DNA测序所得到的基因序列进行对比分析。结果镉、镍转化组各细胞株（各10株）翻译起始因子的eIF3 p36 cDNA测序结果与正常对照细胞（10株）比较未发现基因的突变位点；所有镉、镍转化细胞和正常对照细胞株eIF3 p36 cDNA序列与Genbank数据库进行同源性分析，相似性比值均为100％。结论实验中镉、镍恶性转化16HBE细胞中翻译起始因子eIF3 p36上调未见基因序列改变，提示在镉、镍致癌中可能涉及到表遗传机制。     

BPDE诱发16HBE恶性转化过程中eIF3 p36的表达变化

目的探讨二羟环氧苯并芘（BPDE）诱发人支气管上皮细胞（16HBE）恶性转化过程中不同阶段真核生物蛋白翻译启始因子（elF3 p36 mRNA）表达水平的变化，为进一步阐明BPDE的分子致癌机理提供线索。方法应用RT—PCR和FQ—PCR方法，检测并分析BPDE诱发16HBE恶性转化不同阶段的eIF3p36mRNA表达量的变化。结果相对于非转化对照细胞，BPDE诱发恶性转化16HBE不同阶段细胞（转化细胞和成瘤细胞）的eIF3p36mR-NA基因表达水平均显著高于对照组（P〈0．01或P〈0．05），其中BPDE-转化细胞的elF3 p36平均表达量分别是对照细胞的2．3～5．1倍：而BPDE-转化细胞与BPDE-成瘤细胞的elF3 p36平均表达量相比，差别无显著性（P〉0．05），提示elF3 p36的异常表达量与BPDE诱发16HBE细胞恶变程度之间存在一定的正向关系。结论BPDE在诱发16HBE细胞恶变过程中，存在明显的elF3 p36的异常表达现象，其表达水平与细胞的恶变程度密切相关，这可能是BPDE诱发人细胞肿瘤的重要分子致癌机理之一。     

人成骨细胞株HOS TE85致瘤性转化的研究

目的建立人成骨细胞株HOS TE85致瘤性转化的模型, 作为骨肉瘤癌变过程细胞分子生物学研究的模型.方法通过克隆形成率实验确定N-甲基-N′-硝基-N-亚硝基胍(N-methyl-N′-nitro-N-nitrosoguanidine, MNNG)对HOS TE85细胞转化浓度后,以MNNG作启动剂,佛波酯(12-0-Te-tradecanoyl phorbol 13-acetate, TPA)作促进剂对其进行转化,66 d后通过细胞形态、软琼脂集落形成实验和裸鼠体内致瘤实验鉴定细胞转化程度.结果经MNNG和TPA协同处理HOS TE85细胞66 d后,细胞中出现形态异常的转化灶,转化灶细胞失去接触抑制;转化细胞凝集性增强;软琼脂克隆形成率明显增加;转化细胞在裸鼠皮下成瘤,病理组织学证实为低分化骨肉瘤.结论模拟人体细胞发生恶性转化的过程,建立了HOS TE85的恶性转化模型.     

Alterative Expression and Sequence of Human Elongation Factor-15 during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ(human EF-1δ p31) during malignant transformation of human bronchial epithelial cells induced by cadmium chloride (CdCI2) and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells (16HBE) induced by CdCI<.2> at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-1δ mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-1δ cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl<.2> was elevated (P<0.01 or P<0.05). Compared with their corresponding non-transformed cells, the overexpression level of EF-18 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed cells and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl<.2>. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl<.2>, but is not correlated with DNA mutations.     

Alterations of FHIT gene and P16 gene in nickel transformed human bronchial epithelial cells

INTRODUCTION Nickel is a widely distributed occupational and environmental pollutant. Epidemiological studies demonstrated that the incidence of respiratory tract cancer is correlated with worksite exposure to nickel. Nickel is also a potent carcinogen in laboratory animals. The International Agency for Research on Cancer (IARC) has classified nickel compounds as confirmed human carcinogens[1]. However, the mechanism of nickel carcinogenesis remains unknown. Insoluble nickel compounds…     

hOGG1基因在氯化镉恶性转化16HBE细胞系过程中的表达

目的探讨OGG1基因在氯化镉恶性转化16HBE细胞过程中mRNA的表达情况。方法用半定量反转录-聚合酶链反应(PT-PCR)技术检测16HBE细胞、氯化镉恶性转化16HBE细胞系不同阶段(第5、15、32代细胞及成瘤细胞)hOGG1基因mRNA的表达情况。结果与16HBE细胞比较,hOGG1基因mRNA在第32代细胞及成瘤细胞的表达明显低下(P<0.01)。结论氯化镉诱发16HBE细胞系恶性转化过程中hOGG1基因表达逐渐下降,hOGG1基因水平的改变可能是氯化镉的致癌机制之一。     

大鼠肠上皮细胞系IEC-6的恶性转化研究

目的建立N-甲基-N’-硝基-N-亚硝基胍（N-methyl-N’-nitro-N-nitrosoguanidine，MNNG）、佛波酯（12-O-tetradecanoylphorbol-13-acetate，PMA）对大鼠肠上皮细胞系IEC-6的恶性转化模型。方法通过克隆形成率实验确定MNNG、PMA对IEC-6细胞转化浓度，对IEC-6细胞进行分阶段多次干预。用软琼脂集落形成实验和裸鼠体内致瘤实验鉴定细胞恶性转化程度。结果经MNNG、PMA多次处理IEC-6细胞至24次后，细胞生长速度加快，排列紊乱，失去接触抑制，出现复层生长，并可在软琼脂上生长，且呈剂量反应关系，但在第24次之前的细胞则不能在软琼脂上生长。转化细胞在裸鼠体内形成瘤，病理组织学证实为低分化肠上皮细胞癌。结论MNNG、PMA具有使IEC-6细胞发生恶性转化的能力。     

人鼻咽癌DNA对Rat-Ⅰ细胞株的转化活性及其基因表达的研究

为了解EB病毒基因组阳性人鼻咽癌DNA在转染正常细胞后可能发生的生物学作用,作者将人鼻咽癌活检组织的DNA(EB病毒基因组阳性),用磷酸钙方法转染正常Rat-Ⅰ细胞,使其恶转;用软琼脂培养的转化细胞能贴壁生长,并形成克隆;转化细胞易与PHA发生凝集;克隆接种裸鼠形成纤维肉瘤;转化细胞和裸鼠肿瘤组织用抗C_3补体免疫荧光法检测EBNA呈阳性;染色体原位杂交显示在某些细胞中期染色体上有银粒出现。     

鼻咽癌癌基因研究：Ⅰ,鼻咽癌细胞株CNE—2DNA的转化活性

We have obtained foci of transformed NIH3T3 cells through transfection of human DNA from CNE-2 (an undifferentiated epithelial cell line of nasopharyngeal carcinoma). The DNA from primary foci can be used in subsequent cycles of transfection, resulting in secondary foci capable of forming clones on soft agar and producing tumors 30 days after innoculation into nude mice. The tumors were later proved histologically to be fibrosarcomas.     

鼻咽癌癌基因研究 I.鼻咽癌细胞株CNE-2DNA的转化活性

用低分化鼻咽癌细胞株CNE-2 DNA转染NIH3T3细胞,获得的转化细胞株经扩增进行二轮和三轮转染。转化细胞株含有人特异的DNA序列能在软琼脂上生长并在裸鼠体内形成肿块。