Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats

AIM To evaluate the relationship between expression of ras, p53, bcl 2 gene products, and hepatocarcinogenesis since endotoxemia produced from lipopolysaccharide admi nistration and/or the hypophagocytic state of splenectomy significantly accelerated hepatocarcinogenesis induced by thioacetamide. 〖WTH4〗METHODS〓〖WTXFZ〗The hepatocarcinoma model was induced by oral intake of 0 03% thioacetamide for six months. During the induction of hepatocarcinoma model, rats were additionally treated with splenectomy and/or lipopolysaccharide administration. The techniques of flow cytometry, immunohistochemistry and immunoelectronmicroscopy were applied to quantitative analysis of the expression of oncogene proteins. RESULTS In this model system, overexpression of ras p21 protein mainly occurred on precancerous cell population or in early stage of hepatocyte transformation. And the levels of ras p21 declined when nuclear DNA aneuploid increased. Expression of bcl 2 protein slowly and steadily rose with more hepatocytes staying in S+G2M phases as the hepatocarcinoma became more malignant. P53 was moderately expressed during the hepatocarcinogenesis. There was no statistical correlation between endotoxemia levels and the changes of ras, p53 and bcl 2 gene products. CONCLUSION Over expression of oncogene ras p21 was likely to be a precursor of the premalignant hepatocytes and it might be responsible for the initiation of hepatocarcinogenesis. Bcl 2 protein expression is proportional to the severity of the malignancies. P53 may be a key pathway on the transformation and development of hepatocarcinoma. This study confirmed the hypothesis that there are multiple genes and multiple steps involved in hepatocarcinogenesis. Expressions of oncogene proteins reflected the properties of the premalignant and malignant cells, but not directly related to endotoxemia statistically.[JP]

APC and K-ras gene mutation in aberrant crypt foci of human colon

AIM:To study the genetic alteration in ACF and to define the possibility that ACF may be a very early morphological lesion with molecular changes,and to explore the relationship between ACF and colorectal adenoma even carcinoma. METHODS: DNA from 35 CRC, 15 adenomas, 34 ACF and 10 normal mucus was isolated by means of microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well as the mutation cluster region (MCR) of APC gene was performed. RESULTS: K-ras gene mutation frequency in ACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/15), and 14.3% (5/35) respectively, showing no difference (P 】 0.05) in K-ras gene mutation among three pathologic procedures. The K-ras gene mutation in adenoma, carcinoma and 4 ACF restricted in codon 12 (GGT GAT), but the other 2 mutations from ACF located in codon 13 (GGC GAC). K-ras gene mutation was found more frequently in older patients and patients with polypoid cancer. No mutation in codon 61 was found in the three tissue types. Mutation rate of APC gene in adenoma and carcinoma was 22.9% (8/35) and 26.7% (4/15), which was higher than ACF (2.9%) (P 【0.05). APC gene mutation in carcinoma was not correlated with age of patients, location, size and differentiation of tumor. CONCLUSION: ACF might be a very early morphological lesion in the tumorogenesis of colorectal tumor. The morphological feature and gene mutation status was different in ACF and adenoma. ACF is possibly putative microadenoma that might be the precursor of adenoma. In addition, the development of a subgroup of colorectal carcinomas might undergo a way of normal epithelium ACF carcinomas .

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury

Ras homolog enriched in brain(Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1(mTORC1).Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR.S6K1 and4E-BP1 are important downstream effectors of mTORC1.In this study,we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1in the protection of retinal ganglion cells.We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration.We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute(14 days) and chronic(21 and 42 days) stages of injury.We also found that either co-expression of the dominant-negative S6K1mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells.This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration.However,only S6K1 activation,but not 4E-BP1 knockdown,induced axon regeneration when applied alone.Furthermore,S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury,whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days postinjury.Ove rexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury.Likewise,co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury.These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rhe b/mTOR.Together,our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity.Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

腺病毒介导RA538基因转移及其诱导肿瘤细胞凋亡的研究

RA538是从维甲酸诱导终末分化的人食管癌细胞系中分离出的一个与分化相关的基因。通过重组体腺病毒将RA538基因成功地转导到人肺腺癌细胞系 (GLC - 82 )中 ,经X -gal染色证实转导效率达 10 0 % (MOI为 2 5) ,转导RA538基因后的GLC - 82肿瘤细胞生长受到明显的抑制 ,抑制率为 77 2 %。经流式细胞计数 (FCM)及TUNEL检测证实RA538可诱导肿瘤细胞发生凋亡 ,凋亡率为 59 4%。利用Westernblot法检测转导了RA538基因GLC - 82肿瘤细胞中癌基因蛋白发现 ,c -myc基因蛋白表达下调。结果提示 :RA538作为抑癌基因将有可能应用于肿瘤基因治疗。

Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

Detection of k-ras gene point mutation in fine needle aspiration and pancreatic juice by sequence special primer method and its clinical significance

INTRODUCTIONThe point mutation rate of k-ras gene at codon 12 inpancreatic adenocarcinoma is reported to be as highas 90%,and with no mutations in normalpancreas tissues or other pancreatic disorders.Wehave detected the presence of k-ras gene

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

腺病毒介导RA538基因转移及其对肿瘤的抑制作用

目的:RA538是从维甲酸诱导终末分化的人食管癌细胞系中分离出的一个与分化相关的基因,本研究探讨了其抗癌作用.方法:利用腺病毒介导基因转移观察对肿瘤细胞的抑制效应.结果:通过重组体腺病毒将RA538基因转导到人肺腺癌细胞系(GLC-82)和人结肠癌细胞系(HCY)中,获得较高的转导效率,显示出较明显的抑制肿瘤生长作用,抑制率分别为77.2%和77.9%,并能降低两细胞系体内肿瘤形成能力.流式细胞计数、DNA片段化分析及TUNEL检测证实RA538可诱导肿瘤细胞发生凋亡.Westem blot分析表明,RA538具有明显下调c-myc基因表达的作用,结论:研究结果提示,RA538是一个较广谱的抑癌基因,可以作为肿瘤基因治疗的一个新目的基因,具有良好的应用前景.

Preliminary Investigation on Regulating Effects of Different TCM Treatments on Transcription of the Correlated Genes of Liver Cancer in Rats

The regulating effects of TCM treatments including clearing away heat and toxic materials,promoting blood circulation and removing blood stasis,and strengthening the spleen and regulating qi on the oncogene transcription were observed in the liver cancer model rats.The preliminary results indicated that the mRNA levels of H-ras N-ras and K-ras,and signal molecules correlated with the ras/MAPK signal transduction pathway were down-regulated by the different TCM treatments in varying degrees.Also,the regulating effects of the treatments on differently-displayed genes were discrepant.It is suggested that the molecular mechanisms of the TCM treatments for liver cancer was complex with different target genes.

重组RA538腺病毒对人胃癌细胞的体内及体外分子治疗研究

目的：研究重组ＲＡ５３８腺病毒对胃癌细胞的体内外生物学作用。方法：采用ＬａｃＺ基因Ｘ－ｇａｌ染色、ＭＴＴ、克隆形成试验、ＤＮＡ梯度降解试验、原位末端标记、流式细胞仪、裸鼠致瘤性、裸鼠皮下移植瘤模型实验等方法，对ＲＡ５３８重组腺病毒在人胃癌ＳＧＣ７９０１细胞系中的作用进行体内外研究。结果：Ａｄ－ＲＡ５３８对ＳＧＣ７９０１细胞产生明显的生长抑制作用，ＭＴＴ显示生长抑制率为７６．３％。ＤＮＡ梯度降解试验、原位末端标记、流式细胞仪显示Ａｄ－ＲＡ５３８诱导了ＳＧＣ７９０１细胞凋亡。经Ａｄ－ＲＡ５３８处理的ＳＧＣ７９０１细胞裸鼠致瘤性消失。Ａｄ－ＡＳｃ－ｍｙｃ对裸鼠皮下移植瘤模型瘤内注射能有效降低肿瘤的生长速度，生长抑制率为６０．６６％。结论：Ａｄ－ＲＡ５３８对胃癌细胞具有显著的体内外生长抑制及凋亡诱导作用。

腺病毒介导的RA538基因转移及其对人结肠癌细胞HCT的抑瘤作用

目的 进一步研究ＲＡ５３８ 对人结肠癌细胞系的生物学效应，选用介导ＲＡ５３８ 重组体腺病毒，观察其对人结肠癌细胞系ＨＣＴ 的作用。方法 通过重组体腺病毒将ＲＡ５３８ 基因转导到人结肠癌细胞系ＨＣＴ 中，观察转染ＲＡ５３８ 基因后的肿瘤细胞生长情况。经流式细胞仪、ＤＮＡ 片段化分析及ＴＵＮＥＬ 检测分析抑瘤机制。结果 由重组体腺病毒ＡｄＲＡ５３８ 介导基因ＲＡ５３８ 转导在ＨＣＴ 获得较高的转导效率， ＭＯＩ 为１００ 时可使８０ ％ 细胞转染，并显示对肿瘤细胞生长有明显的抑制作用，抑制率为７４ ％ 。裸鼠皮下注射ＲＡ５３８ 处理的ＨＣＴ 细胞肿瘤形成能力为２０ ％ 。ＤＮＡ 片段化分析、ＴＵＮＥＬ 和流式细胞仪检测，结果证明ＲＡ５３８诱导肿瘤细胞发生凋亡。Ｗｅｓｔｅｒｎ ｂｌｏｔ 分析表明，ＲＡ５３８ 具有明显下调ｃｍ ｙｃ 基因表达的作用。结论 ＲＡ５３８ 对人结肠肿瘤细胞有抑制生长和诱导凋亡的作用，可以作为结肠肿瘤基因治疗的１ 种新的治疗基因。

Studies on the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer

AIM To study the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer.

Adenovirus-mediated transfer of RA538 gene and its antitumor effect

The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle Gl arrest/apoptosis of the tumor cells. The expression of c-myc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.

Expressions of myc and ras gene in human hepatocellular carcinoma by applying the double hybridization in situ

Objective: To establish a method of non-isotope double in situ hybridization in order to detect the expression of two kinds of oncogenes at single cell level simultaneously, and confirm the hypothesis of 'model of stepwise carcinogenesis'. Methods: The method of non-isotope double in situ hybridization was established with the digoxigenin (Dig) and biotin(Bio) labelled probes. The expression of two members of oncogenes of the myc and/or ras gene families (myc and N-ras, myc and K-ras, myc and H-ras, N-ras and K-ras , N-ras and H-ras, K-ras and H-ras) was further studied with the method in 10 cases of Chinese Hepatocellular Carcinomas (HCC). Results : Co-expression of two kinds of oncogenes was detected only in a few cases, reflected by coshowing two different hybridization signals ,i. e. , co-showing of Dig-myc and Bio-H-ras, Dig-myc and Bio-N-ras, Dig-myc and Bio-K-ras was observed in 2 cases, 2 cases and one case, respectively. The common characteristics were that positive cells of myc distributed diffusely, while positive cells of ras diatributed sporadically or locally among the positive cells of myc, and only a few cells exhibited co-showing of two oncogenes at single cell level. There were only 2 cases representing co-showing of two oncogenes in ras gene family (Dig-N-ras and bio-H-ras, Dig-N-ras and Bio-K-ras , respectively). The two kinds of positive cells of different ras gene represented mixed local and sporadical distribution, and co-showing of two signals was found in a few cells at single cell level. Conclusion: There are multiple oncogenes involving in tumorigenesis by their ordered activation. The activation of the ras gene family plays a role in promotion, while the activation of myc is an important event in late stage of tumorigenesis

Correlation between IL-1β,IL-1Ra gene polymorphism and occurrence of polycystic ovary syndrome infertility

Objective:To explore the relationship between IL-1β.IL-1Ra gene polymorphism and the occurrence of polycystic ovary syndrome(PCOS) infertility.Methods:A total of 59 PCOS infertility cases visiling the reproductive center of our hospital from Mar.2010 to Mar.2012 and 56 healthy women were selected.ELISA method was used lor the detection of IL-1β.IL-1Ra lewis,and the levels of serum supersensitivity C reaction protein(US-CRP).insulin(FINS),follieule-stimulating hormone(FSH) and fasting blood—glucose(FRG) were detected.PCR analysis technology was adopted to detect the gene polymorphism of the.511 site of IL-1βand the second introne of IL- 1Ra.Results:The levels of IL-1β.IL-1Ra.US-CRP.FINS and FBG in blood scrum of patients in PCOS group were significantly higher than those in control group(P【0.05 or P【0.01).The level of FSH in PCOS group was significantly lower than that in control group(P【0.05).The genotypic frequency of T/T.the 511 site of IL-1βin PCOS group was 42.37%.significantly higher than 1250%in control group 【P【0.01).The frequency of T allele was also significantly higher than that in control group(P【0.01).The genotypic frequency ofⅠ/Ⅴ.the second introne of IL-1Ra in PCOS group was 20.34%,signicianlly higher than 3.57%in control group(P【0.05).The frequency of V allele in PCOS group was significantly higher than that in control group(P【0.05).Conclusions: T allele of the 511 site of IL-1βgene and V allele of the second inlrone of IL-1Ra gene might be the genetic basis of the rising of IL-1β.IL-1Ra and US-CRP levels in blood serum of PCOS patients,and are associated with the infertility occurrence of PCOS patients.

The Role of Ras Gene Mutation in Gastric Cancer and Precancerous Lesions

Abnormality of ras gene family was studied in a total of 206 cases of gastric cancer and precancerous lesions by PCR-RFLP, PCR-SSCP and DNA sequencing. The results showed that mutation rate of H-ras 12 codon in metaplasia,atypical hyperplasia, early-stage cancer and advanced cancer was 16. 7%, 31. 2 %, 50. 0%, and 32. 2%, respectively. In the groups of superficial gastritis and normal controls, no mutation were detected in codon 12 of ras. Mutations of Hras 61 codon and N-ras 12 codon in various groups were the same as those in normal control. K-ras 12 codon mutation was detected in only 2 cases of gastric cancer by using PCR-SSCP, but it was not detected by DNA sequencing, which may be polymorphism. All H-ras 12 codon mutations were G→T mutation. There were significant difference between the groups of metaplasia, dysplasia, gastric carcinoma and normal control group (P<0.05, P<0.01, P<0.01,respectively). It was concluded that H-ras 12 codon mutation was an early event and may play an important role in gastric carcinogenesis. Although K-ras, N-ras mutation rates are high in colon cancer and leukemia, it seems to bear no relationship with gastric cancer.

In Vitro Anti-tumor Immune Response Induced by Dendritic Cells Transfected with Recombinant Adenovirus Carrying Mutant K-ras Genes

Summary： The specific anti-tumor immune response induced by mouse bone marrow dendritic cells （DCs） lransfected with recombinant adenovirus carrying mutant k-ras genes was investighted. DCs were generated from mouse bone marrow in the presence of rmGM-CSF （3.3 ng/mL） and rmIL-4 （1.3 ng/mL） and detected by FACS, and then transfecled with the recombinant adenovirus encoding mutant k ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The resuhs showed thai DCs had dendritic veiled morphology. BmDCs highly expressed B7-1（80%）, B7-2（77%）, MHC Ⅱ （70%）, CDllc （65%）, CD40 （70%） and CD54 （96%） with FACS, and no significant difference in the expression was observed before and after the transfection （P〈0.05）. The DCs transfeeled by mutant k-ras gene could significantly stimulate lymphoeytes proliferation as compared with those transfeeted by Ad e or non-modified DCs （P〈0.05）. DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific eytotoxicity against Lewis lung cancer in Ad-k-ras/12-transdueed DC group was signifieantly higher than those in the control, vector and non transfeeted DCs groups （P〈0.05）. It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.

Rapid Detection ofK-ras Gene Point Mutation at Codon 12 by PCR-SSPin Pancreatic Adenocarcinom a

To evaluate the feasibility and clinical significance of the PCR SSP technique in detecting K ras gene mutation at codon 12 in pancreatic adenocarcinoma tissues. 80 specimens of surgical resection or biopsy samples were tested at our hospital from January 1994 to September 1995. Three different special sequence primers (SSP) synthesized according to mutation styles of CGT, GTT, GAT were respectively prepared. Three amplification reactions were performed for each sample. The amplification products were analyzed by conventional polyacrylamide gel electrophoresis, stained with ethidium bromide and observed under UV transillumination. Results: All of the 34 pancreatic adenocarcinoma samples had positive PCR results with the mutation rate 100%. 7 cases were CGT mutation, 18 GGT and 17 GAT mutation, in which 2 types of mutation existed in 8 cases. No mutation appeared in 13 normal pancreatic tissues, 6 insulinomas, 6 chronic pancreatitis, 5 benign pancreatic cysts, 7 bile duct carcinoma, 5 ampulla carcinoma and 4 carcinomas of duodenal papilla. Conclusion: Pancreatic adenocarcinoma is one of the commonly encounted tumors and is still very difficult to diagnose at the early stage and to distinguish from other lesions preoperatively. Our study indicates that PCR SSP is an ideal assay in comparison with other methods to detect K ras gene mutation. It is simple, rapid, specific, sensitive and easily generalized for clinical application on preoperative diagnosis.