INCREASED EXPRESSION OF PDGF AND C-MYC GENES IN LUNGS AND PULMONARY ARTERIES OF PULMONARY HYPERTENSIVE RATS INDUCED BY HYPOXIA

The role of growth factors and proto-oncogene in pulmonary vascular structural remodelling is not well known.The present study examined gene expression of platelet-derived growth factor(PDGF)-A and -B chain and proto-oncogene,c-myc,in lung tissue and pulmonary artery of rats exposed to hypoxia and compared to those levels of gene expression in normal rats.Normal lungs and pulmonary artery expressed PDGF-A chain transcript of 1.7 kb and PDGF-B chain transcript of 3.5 Kb.The c-myc transcript of 2.2 kb was expressed as well. After hypoxic exposure for 7 and 14 days mRNA levels of PDGF-B chain and cmyc were elevated significantly compared with those of control rats.PDGF-A chain mRNA increased after hypoxia for 7 days,and then declined.These results suggest that activation of autocrine and/or paracrine is important in proliferation mechanism of pulmonary artery smooth muscle cells in hypoxic pulmonary hypertensive rats.

Gene therapy with antisense c-myc adenovirus for human gastric carcino-ma cell line in vitro and for implanted carcinoma in nude mice

Objective:To study the effects of recombinant antisense c-myc adenovirus (rAS-c-myc-Ad) on SGG 7901 human gastric carcinoma cell line in for and in nude mice. Methods:The effects of rAS-c-myc-Ad and LacZ-Ad on SGG 7901 gastric carcinoma cells were observed with X-galstaining, MTT, DNA gradient degradation test, TUNEL, flow cytometry, PCR and western blot. The therapeutic effects of rAS-c-myc-Ad on the implanted ax 7901 cells in nude mice were also ob served.Results: rAS-c-myc-Ad significantly inhibited the growth of SGG 7901 cells and induced their apoptosis. After the treatment of rAS-c-myc-Ad, the prolifetion rate of the cells was decreased by 44’ l% in de and SGC 7901 cells failed to form caxcinoma ther they were implanted into nude mice. Injection of rAS-c-myc-Ad into the carcinoma subcutaneously implanted to the nude mice significantly inhibited the growth of the implanted carcinoma with an inhibition rate of 68. 9%. Conclusion: rAS-c- myc- Ad significantly inhibits the growth of SGG 7901 human gastric carcinoma cells in vitro and in nude

INDUCTION OF C-MYC GENE AMPLIFICATION BY HYDROXYUREA AND ITS INHIBITION BY HOMOHARRINGTONINE

Induction of c-myc gene amplification in L1210 cells by hydroxyurea and its inhibition by homohar-ringtonine were investigated using the DNA-DNA molecular hybridization technique. When the cells were treated with hydroxyurea 1.0 mM for 16 hours, and incubated a further 16 hours in a drug-free medium, the c-myc gene amplified 23.5-fold. If homohar-ringtonine 50 μM was used at the same time as hydroxyurea, gene amplification did not occur. Cycloheximide, an inhibitor of protein biosynthesis, produced a similar effect. Our results indicated that a (or some) protein factor(s) might be involved in gene amplification. Detailed analysis showed that the synthesis of this protein factor(s) started 4 hours before the initiation of the S phase but did not continue in the S phase. It was also found that this protein factor(s) was very labile and began to degrade 2 hours after its appearance.

THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.

Deregulated c-myc expression in quiescent CHO cellsinduces target gene transcription and subsequent apoptotic phenotype

Human c-myc cDNA was fused with the hormonebinding domain (HBD) cDNA of murine estrogen receptorgene and the chimeric gene was introduced into the CHOcells. The fusion protein, c-MycER, becomes activatedwhen the synthetic steroid, 4-hydroxy-tamoxifen (OHT),binds HBD. Activated c-MycER, likely c-Myc, can inducequiescent CHO cells reentry into S phase and subsequentcell death under serum-free condition. In addition, theexpression of some proposed c-myc target genes such asODC, MrDb, cad, rcc1 and rc1 were found to increase uponOHT induction before S, phase entry and apoptosis, indicating that these target genes are involved in cell cycleregulation and/or apoptosis control. However, the mutantD106-143c-MycER protein does not have above activities.

Promoter hypermethylation of tissue specific tumor supressor genes and point mutation in K-ras, c-myc proto-oncogenes in urinary (transitional cell) bladder carcinoma

In a total of 83 UN specimens were investigated for proto-oncogene mutations, tumor supressor genes promoter methylation status and c-myc and Ki-67 expression. Point mutations in c-myc were detected in cases with high grade and proliferation index. Mutated K-ras proto-onco- gene profiles were detected in 17 (21%) tumoral spiecemens that examined. Tumor specimens were also showed hypermethylated promoter domain for the SFRP2, MGMT tumor supressor genes. These findings showed the combine effect of mutated c-myc and K-ras oncogene and epigenetic inactivation of tissue specific tumor supressor genes (TS) play a crucial role in tumor progression and recurrence in UN carcinogenesis.

Brg1及C-myc在宫颈癌中的表达及临床相关性研究

目的:探讨Brg1(brahma-related gene 1)和C-myc在正常宫颈、宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)及宫颈癌组织中的表达情况,分析两者的相互关系及临床意义。方法:应用免疫组织化学法检测正常宫颈、CIN和宫颈癌石蜡切片中Brg1和C-myc的表达。结果:Brg1蛋白在正常宫颈组织、CIN、宫颈癌中的阳性表达率分别为10.0%、32.5%、77.5%,差异均具有统计学意义(P<0.05)。C-myc蛋白在正常宫颈组织、CIN、宫颈癌中的阳性表达率分别为10%、25%、65%,差异有统计学意义(P<0.05)。并且宫颈癌中Brg1和C-myc的高表达与临床分期、淋巴结是否转移相关(P<0.05)。Pearson相关分析提示Brg1和C-myc在宫颈癌中的表达呈正相关(rs=0.716,P<0.05)。结论:在宫颈癌中Brg1、C-myc均呈高表达,二者在宫颈癌的发生、发展及转移方面可能存在促进作用。

INHIBITION OF C-MYC PROTEIN EXPRESSION AND CELL PROLIFERATION IN HL-60 CELLS BY ANTISENSE TRANSCRIPTS TO c-myc

The human promyelocytic cell line HL-60 overexpresses the c-myc protooncogene. Plasmid pDACx carrying antisense human c-myc DNA and neo gene was introduced into HL-60 cells with lipofectin reagent. Upon DNA entering the tar-geted celis and expression of antisense transcripts to c-myc, C-MYC protein level, cell proliferation and colony-forming potentiality were all definitely inhibited.

Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy

AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR).

EFFECT OF HYPOXIA ON DNA SYNTHESIS AND C－MYC GENE EXPRESSION OF PULMONARY ARTERY SMOOTH MUSCLE CELLS

The neonate is particularly susceptible to the development of hypoxic pulmonary hypertension. The present study was undertaken to observe the effect of hypoxia on DNA synthesis and c-myc gene expression between newborn calf and adult bovine PASMC in vilro. DNA synthesis measured by 3H- TdR incorporation was increased after hypoxic challenge for 24h. Hypoxia enhanced the increment in 3H-TdR incorporation induced by EGF. Northern blot analysis revealed that PASMC cultured in both normoxia and hypoxia expressed c- myc gene transcript of 2. 2Kb ,but there is a higher 2. 2Kb mRNA expression in hypoxic PASMC than that in normoxia. We speculate that newborn calf PASMC exhibited potential response to hypoxia than adult,which was augmented by EGF. Enhanced c-myc gene expression may lead to a great understanding of the mechanism of PASMC growth in the development of pulmonary hypertension.

EXPRESSION OF c－myc GENE AND BIOSYNTHESIS OF BIOLOGICAL MACROMOLECULES IN ANTISENSE TRANSFECTANT HL_（60）~R－9

The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientation in vitro, to make RNA probes for detection of c-myc mRNA and antisence RNA expression of tranfectant HL-9,which was obtained from HL60 cells transfected with inducible c-myc antisense RNA expression plasmid. The results from HL-9 cells induced by Cd2+ indicated that expression of c-myc antisense RNA increased with Cd2+ concentration and exposure time, while c-myc mRNA expression progressively reduced. Using immunohistochemical technique no c-myc P62 protein expression was detected. The incorporation of 3H-TdR, 3H-UR and 3H-Leu revealed significant suppression of DNA, RNA and protein biosynthesis. It is suggested that the reversion changes previously reported in malignant Phenotypes of HL-9 cells and the inhibition of macromolecular biosynthesis mentioned above were associated with the blockade of c-myc gene expression by its antisense RNA.

Effect of hammerhead ribozyme that specifically cleaves c-myc mRNA on proliferation of vascular smooth muscle cells

Ojective: To investigate the effect of hammerhead ribozyme that specifically cleaves c-myc mRNA on the proliferation of vascular smooth muscle cells (VSMCs ). Methods: Based on the computer analysis of the secondary structure of c-myc mRNA, nt 2029 in rat c-myc oncogene was selected as a cleaving site for hammerhead ribozyme and the ribozyme was designed. With automatic DNA synthesizer, the two complementary DNA strands of the ribozyme were synthesized. The ribozyme gene was cloned into pGEM3Zf ( + ) vector and subcloned into eukaryotic expression pcD-NA3 vector. The recombinant pcDNA-Rz was transfected into the cultured rat VSMCs by lipofectAMINE mediated DNA transfection protocol and individual cell clones were selected by G418. Results: The sequence of ribozyme gene inserted in pGEMSZf ( + ) vector was proved to be perfectly correct. In VSMCs transfected with recombinant pcDNA-Rz, flow cytometry analysis showed that the S phase and G2/M fractions were decreased significantly and cell proliferation stagnated in the G0/G1 phase. Conclusion: The results suggest that hammerhead ribozyme that specifically cleaves c-myc mRNA can significantly inhibit the proliferation of VSMCs.

Experimental study on siRNA expressing vector-based RNA interference targeting c-Myc in human hepatocellular carcinoma cell line BEL-7402

Objective: To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference (RNAi). Methods: Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results: Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion: The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.

Detection of Tumor Suppressor Gene and Oncogene in SO-Rb_(50) Human Retinoblastoma Cell Line

Retinoblastoma (Rb) is the most common malignant'cancer of eye.So-Rb_(50) is the first Rb cell line established in China in 1988.It has passed to the 387th passage now.We collected cells of the 327th passage of SO-Rb_(50),purified its genomic DNA and detected it with Rb and c-myc cDNA probes respectively(normal human white blood cells DNA was the control).We found the Rb gene was deleted while c-myc gene was amplified three times.This provides a basis for further study of the regulation of tumor development and tumor reversal with this cell line in vitro.Eye Science 1993;9:34-37.

Effects of rAAV-CD151 and rAAV-antiCD151 on the Migration of Human Tongue Squamous Carcinoma Cell Line Tca8113

Summary: This study was designed to determine the effects of the recombinant adeno-associated virus vector containing sense CD151 gene (rAAV-CD151) and antisense CD151 gene (rAAV-antiCD151) on the migration of Tca8113 cell. Functional fragment of CD151 gene was amplified by RT-PCR, and inserted into the vector pAAV in the sense direction and antisense direction, respectively. The rAAV-CD151 and rAAV-antiCD151 were produced and the titers were determined by dot blot. The CD151, at protein level, was detected by Western blot. The Transwell chamber was used to detect the effects of the rAAV-CD151 and rAAV-antiCD151 on the tumor cell migration. The titers of the rAAV-CD151 and rAAV-antiCD151 were 2×10~11 pfu/ml and 1.0×10~11 pfu/ml, respectively. The expression of CD151 was increased by 108 % in the cells transfected with rAAV-CD151 and decreased by 79 % in the cells transfected with rAAV-antiCD151, as compared with non-transfected cells, respectively. The number of the migrating cells was significantly increased in the cells transfected with rAAV-CD151 (93.56±11.59) and decreased in the cells transfected with rAAV-antiCD151 (24.00±4.36) as compared with non-transfected and rAAV-GFP transfected cells (53.00±6.56 and 46.00±7.00, P<0.05). It is an important molecular mechanism of the tumor metastasis that the overexpression of CD151 promotes the migration of the tumor cells. The rAAV-antiCD151 is a novel tool, which can reduce the expression of CD151 and inhibit the migration of the tumor cells, and brings us a new approach of anti-sene gene therapy targeted at CD151 in human carcinoma.

Detection of c-myc gene expression in nasopharyngeal carcinoma by nonradioactive in situ hybridization and immunohistochemistry

Objectives To investigate the c myc gene expression in nasopharyngeal carcinoma (NPC) at mRNA and protein levels, and to evaluate the relationship between the degree of expression and the clinicopathological data. Methods The expression of c myc specific mRNA and protein was detected using in situ hybridization with digoxigenin labeled probe and immunohistochemistry in 12 cases of normal nasopharyngeal epithelium (NE) and 48 cases of NPC. The intensity of staining for each sample was assessed as negative, weak, moderate and intense. Statistical analyses were performed using the Chi square test and the rank sum test. Results For normal NE, 41.7% (5/12) had weak (4/12) or moderate (1/12) staining for c myc protein and 41.7% (5/12) had weak staining for c myc mRNA. In contrast, 89.6% (43/48) of NPC showed some degree of c myc protein positive and 87.5% (42/48) showed some degree of c myc mRNA positive. In most samples, the expression of c myc protein was consistent with that of c myc mRNA. C myc protein and mRNA expression levels in NPC were significantly higher than those in normal NE, and correlated with early recurrence, but they were not significantly related to patient′s age, sex, EBVCA IgA titre, disease stage, lymph node status and metastasis. Conclusions C myc gene over expression may be involved in the pathogenesis and early recurrence of NPC. Further studies are needed to substantiate the preliminary findings.

Transformation of Chinese Cabbage(Brassica rapa L. ssp. pekinensis)by Agrobacterium Micro-Injection into Flower Bud

We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants(T0)with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One(DHZ-13-1)exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1)has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development.

Expression of telomerase hTERT in human non-small cell lung cancer and its correlation with c-myc gene

Objective To investigate the expression of human telomerase catalytic subunit,hTERT, in human non-small cell lung cancer (NSCLC) and its correlations to c-myc gene.Methods hTERT and c-myc mRNA expressions were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Statistical correlation analysis was made to estimate whether there was interrelation between them.Results Positive rate of hTERT expression in 51 surgically resected lung cancer specimens was 86.3%,significantly higher than that in adjacent non-neoplastic lung tissues and benign lesions,which were 14.3% and 27.3% respectively. No statistical significance was observed between the frequency of hTERT expression and histologic types,degree of differentiation,TNM stages,tumor size or lymph nodes metastases. Correlation analysis revealed that the expression of c-myc gene was significantly related to that of hTERT (correlation coefficient,r =0.633,P <0.001).Conclusions hTERT may be a useful tumor marker in diagnosing lung cancer. Significant correlation between the expression of hTERT and c-myc mRNA indicates that the activation and up-regulation of hTERT might be conferred by over-expression of c-myc gene.

Fusions of Dendritic Cells and C6 Cells Transfected with TGF-β1 Antisense in Treatment of Intracranial Gliomas

Objective： To investigate the immunotherapy efficacy of fusion cells （dendritic-C6anti-TGF-β1 cells） in the treatment of intraeranial gliomas. Methods： Dendritic cells were isolated from rat bone-marrow precursors stimulated in vitro with granuloeyte-macrophage colony-stimulating factor （GM-CSF） and Interleukin-4 （IL-4）. C6anti-TGF-β1 cells originally from C6 cell line of a rat glioblastoma were transfected with plasmid of TGF-β1 anti-sense gene. Fusions of dendritic cells and C6anti-TGF-β1 cells were prepared by polyethylene glycol （PEG）. The DC/C6anti-TGF-β1 fusion cells were observed and confirmed by fight microscopy and scanning electron microscopy. Experimental rats were divided into three groups at random： C6 cells （Ⅰ）, dendritic-C6anti-TGF-β1 fusion cells and C6 cells （Ⅱ） and IMDM medium only （Ⅲ）. The cells were injected into right parietal lobe region of the rat with stereotaxic technique. Histology, tumor necrosis and survival time were evaluated. Results： Compared with the rats that received C6 cells （survival median time was less than 20 days, tumor region was seen in all fields of observed）, the rats injected with dendritic-C6anti-TGF-β1 fusion cells and C6 cells got a more prolonged life span （more than 59 days）, as well as less tumor region （5.01%-6.2%）. There was no tumor necrosis, but some glias were seen in surroundings. All rats were survived and no necrosis was observed in negative control group. Statistical analysis showed that group Ⅱ had significant difference compared with group Ⅰ. Conclusions： Dendritic-C6anti-TGF-β1 fusion cells could prolong the life span of rats, providing a strategy to achieve an antitumor response against tumors in the central nervous system.

Reduction of Repair in c-myc Gene Within HL-60 Induced With DMSO

One of the important advancements in DNA repair research is the demonstration of preferential repair of transcriptionally active DNA in UV-irradiation cells. Experiment results have shown that repair in specific sequence in and around