Improvement of a method to reproducibly immortalize human T cells by oncogene transfection

The method to immortalize human T cells efficiently and reproduciblyby oncogene transfection was improved. T cells were first grown selectively from peripheralblood lymphocytes population of healthy donors andatopic asthma patients, and from lymph nodelymphocytes population of lung cancer patients byactivating with mitogens (phytohemagglutinin andconcanavalin A) and recombinant human interleukin-2(rhIL-2) for five days. Plasmids expressingoncogenes, such as c-Ha-ras, c-myc,c-fos, v-myb and v-jun under the controlof human cytomegalovirus promoter, were then introducedinto these stimulated lymphocytes either separately orin various combinations by electropolation. Afterculturing these transfected lymphocytes for recoveryfor 1 day, they were fed every 3–4 days. Although all the control cells died within one month,oncogene-transfected lymphocytes continued toproliferate actively even for more than severalmonths, indicating that oncogene-transfectedlymphocytes were successfully immortalized. Flowcytometric analyses revealed that most of theimmortalized lymphocytes were T cells expressingCD3⁺ surface antigen. The ratios of CD4⁺and CD8⁺ subpopulations in immortalized T cellsderived from healthy donors varied, depending onthe kinds of oncogenes used. However, CD8⁺subpopulation in immortalized T cells derived fromcancer patients and atopic asthma patients weredominant, independent of the kinds of oncogenes. These immortalized T cells showed differentproliferative responses in the presence or absence ofexogenous human rhIL-2, depending on their origin ofdonors. Furthermore, immortalized T cells derivedfrom healthy donors showed stronger cytotoxicityagainst K562 cells, suggesting that MHC-nonrestrictedkiller T cells in T cell population were alsoimmortalized. Immortalized T cell lines, whichproliferate continuously without stimulation of amitogen or antigen in medium containing a lowconcentration of rhIL-2, have been maintained for morethan 2 years without any growth rate decrease.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

端粒酶在细胞永生化和癌变中的作用

重点讨论了端粒酶在肿瘤细胞和永生化细胞中的作用和功能,以及它与细胞衰老和永生化的关系.多数真核细胞的端粒酶能将单一重复序列加到端粒DNA的3′末端.端粒酶主要由模板RNA和端粒酶蛋白催化亚基组成,后者以前者为模板起逆转录酶的作用.端粒酶活性存在于肿瘤细胞中,而在良性肿瘤、体细胞中未发现端粒酶.     

Interactions of intergenic microRNAs with mRNAs of genes involved in carcinogenesis

miRNAs regulate gene expression by binding with mRNAs of many genes. Studying their effects on genes involved in oncogenesis is important in cancer diagnostics and therapeutics. The RNAHybrid 2.1 program was used to predict the strong miRNA binding sites (p < 0.0005) in target mRNAs. The program Finder 2.2 was created to verify 784 intergenic miRNAs (ig-miRNA) origin. Among 54 considered oncogenes and tumor suppressor genes, 47 genes are the best targets for ig-miRNAs. Accordingly, these genes are strongly regulated by 111 ig-miRNAs. Some miRNAs bind several mRNAs, and some mRNAs have several binding sites for miRNAs. Of the 54 mRNAs, 21.8%, 43.0%, and 35.2% of the miRNA binding sites are present in the 5'UTRs, CDSes, and 3'UTRs, respectively. The average density of the binding sites for miRNAs in the 5'UTR was 4.4 times and 4.1 times greater than in the CDS and the 3'UTR, respectively. Three types of interactions between miRNAs and mRNAs were identified, which differ according to the region of the miRNA bound to the mRNA: 1) binding occurs predominantly via the 3'-region of the miRNA; 2) binding occurs predominantly through the central region of the miRNA; and 3) binding occurs predominantly via the 5'-region of the miRNA. Several miRNAs effectively regulate only one gene, and this information could be useful in molecular medicine to modulate translation of the target mRNA. We recommend described new sites for validation by experimental investigation.     

Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.     

人乳头瘤病毒16L1-减毒志贺氏杆菌为载体疫苗的构建及免疫效应初步鉴定

为预防高危型人乳头瘤病毒16型(HPV16)诱发宫颈癌,制备以减毒志贺氏杆菌为载体的HPV16预防疫苗,以期载体可介导机体产生粘膜免疫反应,达到预防HPVl6感染的目的。为此构建了以HPV16L1为免疫原的减毒志贺氏杆菌苗,并初步鉴定候选疫苗的减毒特性和免疫效果。利用基于志贺氏杆菌virG／icsA基因的表达载体(pHS3199),将HPV16L1基因插入后构成pHS3199-hpv16L1质粒,电穿孔法将其转入减毒志贺氏杆菌sh42,经筛选获得重组减毒sh42-HPV16L1工程菌。用免疫印迹法检测HPV16L1蛋白表达,连续传代法确定其传代和目的蛋白表达的稳定性;豚鼠角膜巩膜炎症试验检测细菌的毒力和菌苗的免疫效果;小鼠红细胞凝集抑制试验检测免疫血清对病毒样颗粒(VLP)的中和活性。免疫印迹检测证实,重组菌株sh42-HPV16L1可稳定表达HPV16L1;豚鼠角膜巩膜炎症试验证实,该候选菌苗无致病性。减毒sh42-HPV16L1经结膜囊途径免疫豚鼠,可以产生特异性体液免疫应答,免疫动物体内的血清、肠道、阴道分泌物中抗HPV16L1 VLPIgG、IgA含量显著高于对照组,并且sh42-HPV16L1免疫动物血清可明显抑制HPV16L1 VLP引起的小鼠红细胞凝集。因而sh42-HPV16L1将是一种潜在的HPV16候选预防疫苗。     

表达人乳头瘤病毒16型E6/E7融合蛋白的非复制型重组痘苗病毒的构建及免疫效果观察

采用基因工程方法将HPV16E6、E7基因融合后插入痘苗病毒载体,通过同源重组构建表达人乳头瘤病毒16型E6/E7融合蛋白的非复制型重组痘苗病毒疫苗,用C57BL/6小鼠观察其免疫原性和抗肿瘤移植情况。测序结果表明融合的HPV16E6、E7基因序列与设计相符;构建的非复制型重组痘苗病毒经Dot blot鉴定,显示有E6、E7融合基因的插入;Western blot检测表明该重组病毒在鸡胚成纤维细胞中能表达HPV16型E6/E7融合蛋白。动物免疫试验表明,该重组病毒在小鼠体内可诱发E6、E7特异性抗体;被免疫小鼠能抵抗TC-1肿瘤细胞的攻击。此结果为将来进一步研制HPV16、18型联合疫苗打下了基础。     

腺伴随病毒载体介导HPV16 E6E7基因转化人胎食管上皮细胞

为了证实人乳头瘤病毒16型（HPV16）感染与食管鳞状细胞癌发生的关系,构建了包含HPV16E6E7基因的重组腺伴随病毒载体并包装重组病毒,重组病毒感染人胎食管粘膜组织,注射SCID小鼠皮下,在TPA协同下12周左右诱发肿瘤。PCR及打点杂交检测到瘤组织中HPV16E6E7基因的存在,HE染色表明为恶性鳞状上皮癌,培养形态及透射电镜观察证实了瘤组织的上皮来源。以上结果对于阐明食管癌发生的病毒病因、食管癌发生的分子机制以及为食管癌防治提供了理论和实践依据。     

How do mutated oncogenes and tumor suppressor genes cause cancer?

In recent decades we have been given insight into the process that transforms a normal cell into a malignant cancer cell. It has been recognised that malignant transformation occurs through successive mutations in specific cellular genes, leading to the activation of oncogenes and inactivation of tumor suppressor genes. The further study of these genes has generated much of its excitement from the convergence of experiments addressing the genetic basis of cancer, together with cellular pathways that normally control important cellular regulatory programmes. In the present review the context in which oncogenes such as proliferation, cell death/apoptosis, differentiation and senescence will be described, as well as how these cellular programmes become deregulated in cancer due to mutations.     

新疆维吾尔族妇女宫颈癌组织中乳头瘤病毒16型E6基因的克隆和序列分析

为了分析新疆南部地区维吾尔族妇女宫颈癌组织中HPV16型E6基因结构特点,从中国新疆南部地区维吾尔族妇女宫颈癌活检组织标本中提取DNA,以宫颈癌活检组织标本DNA为模板进行PCR扩增,获得HPV16 E6基因,将其克隆到pUCm-T载体上,并对其进行基因全序列分析.PCR检测结果显示宫颈癌组织中HPV16 E6阳性率为82.35%（14/17）;测序结果显示,新疆株HPV16 E6基因全长456 bp,大小与德国标准株一致.E6基因的第247位碱基发生T→G突变,并由此引起所编码的氨基酸亦发生改变.上述结果表明,中国新疆南部地区维吾尔族妇女宫颈癌患者组织中HPV16 E6的基因结构与德国标准株HPV16 E6基因之间存在差异.     

Sprouty1 induces a senescence-associated secretory phenotype by regulating NFκB activity: implications for tumorigenesis

Genes of the Sprouty family (Spry1–4) are feedback inhibitors of receptor tyrosine kinase (RTK) signaling. As such, they restrain proliferation of many cell types and have been proposed as tumor-suppressor genes. Although their most widely accepted target is the Extracellular-regulated kinases (ERK) pathway, the mechanisms by which Spry proteins inhibit RTK signaling are poorly understood. In the present work, we describe a novel mechanism by which Spry1 restricts proliferation, independently of the ERK pathway. In vivo analysis of thyroid glands from Spry1 knockout mice reveals that Spry1 induces a senescence-associated secretory phenotype via activation of the NFκB pathway. Consistently, thyroids from Spry1 knockout mice are bigger and exhibit decreased markers of senescence including Ki67 labeling and senescence-associated β-galactosidase. Although such ‘escape'' from senescence is not sufficient to promote thyroid tumorigenesis in adult mice up to 5 months, the onset of Phosphatase and tensin homolog (Pten)-induced tumor formation is accelerated when Spry1 is concomitantly eliminated. Accordingly, we observe a reduction of SPRY1 levels in human thyroid malignancies when compared with non-tumoral tissue. We propose that Spry1 acts as a sensor of mitogenic activity that not only attenuates RTK signaling but also induces a cellular senescence response to avoid uncontrolled proliferation.