Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV⁺ than HPV⁻ cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV⁺ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.     

新疆哈萨克族食管癌组织中乳头瘤病毒16型E6E7基因的研究

为了解HPV16感染后E6E7基因变化在哈萨克族食管癌发病中的作用 ,以及HPV16E6E7基因与食管癌病理组织分级的关系 ,采用聚合酶链 (PCR)技术 ,检测 82例食管癌及癌旁正常食管粘膜组织中HPV 16E6E7基因差别。食管癌、癌旁正常粘膜组HPV16E6E7基因的检出率分别为 3 4. 15 % ( 14 /4 1)和 19. 5 1% ( 8/4 1) ,63 . 41% ( 2 6/4 1)和48. 78% ( 2 0 /4 1) ,差别均无显著性 (P >0 . 0 5 )。然而 ,在食管癌组织的高分化组、中低分化组中HPV16E6E7的检出率分别为 7. 69% ( 1/13 )和 46. 43 % ( 13 /2 8) ,3 8. 46% ( 5 /13 )和 75 % ( 2 1/2 8) ,差别均有统计学意义 (P <0 .0 5 )。提示HPV16E6E7基因与哈萨克族食管癌的发生及癌组织分级密切相关。     

35例维吾尔族妇女宫颈癌组织中HPV16型E7致癌基因突变分析

目的本研究旨在检测HPV16E7基因在新疆南部维吾尔妇女宫颈癌组织中的突变。方法从35份新疆南部地区维吾尔族妇女宫颈癌活检组织标本中提取组织DNA,作为模板,PCR扩增HPV16 E7全长基因,PCR产物直接测序或克隆后测序,分析新疆维吾尔族妇女宫颈癌组织HPV16E7基因的突变。结果PCR检测结果宫颈癌组织中HPV16E7阳性率为82.86%(29/35);27个E7分离片段的测序和序列分析表明,26个分离株E7基因与原型相同,1个分离株E7基因核苷酸发生2处突变,即647位(在HPV16基因组中的位置)的A→G,氨基酸由Asn变异为Ser;845位的T→C,氨基酸不变。结论新疆南部地区维吾尔妇女宫颈癌患者感染的HPV16中存在E7基因的变异株,该变异株可能是由人口流动传入。     

新疆妇女宫颈脱落细胞HPV16E6基因的PCR及荧光定量PCR检测

目的:了解新疆妇女人乳头瘤病毒16型(13PV16)的感染状况。方法:用PCR及荧光定量PCR(FQ—PCR)方法检测妇科门诊普通患宫颈脱落细胞及分泌物中人乳头瘤病毒16型E6基因(HPV16E6),以普通门诊患宫颈脱落细胞及分泌物DNA作为样本,以人乳头瘤病毒(HPV16E6)作为扩增的靶基因,同时以人β—actin基因片段作为细胞内参照,借助于两对引物,两个特异的荧光探针,用荧光定量PCR(FQ—PCR)方法对这两个片段进行扩增,得到单位细胞HPV16E6的相对含量;同时进行定性PCR检测。结果:宫颈脱落细胞标本159例中,β—actin阳性154例;HPV16E6阳性共12例,阳性率为7．8％。PCR与FQ—PCR结果基本一致,但FQ—PCR更敏感。结论:建立的FQ—PCR检测宫颈脱落细胞内：HPV16E6基因的方法,能反映单位细胞内病毒的复制情况,可用于性传播感染(STI)及宫颈癌的筛查。     

人乳头状瘤病毒16型E6E7基因与MCA、TPA协同诱导细胞恶性转化的实验研究

目的探讨人乳头状瘤病毒(HPV)16型E6E7基因与化学致癌物MCA、TPA对Balb/c 3T3细胞恶性转化的协同作用。方法构建含HPV16 E6E7基因的重组质粒,用其转染Balb/c 3T3细胞。采用RT-PCR和Westernblot技术检测HPV16 E6E7基因和蛋白表达;应用细胞转化实验研究由MCA和TPA诱导的细胞恶性转化;并检验转化细胞在软琼脂上形成集落的能力及对SCID小鼠的致瘤能力。结果转染HPV16 E6E7基因的细胞比未转染细胞形成更多的转化灶,转化灶个数增加4～25倍,且实验时间明显缩短;其转化细胞在软琼脂上形成集落的能力及对SCID小鼠的致瘤能力更强。结论 HPV16 E6E7基因与MCA、TPA可协同诱导Balb/c 3T3细胞恶性转化。     

Identification of High-Risk Human Papillomavirus DNA,p16, and E6/E7 Oncoproteins in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas

Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC.     

HPV16 E6/E7基因及蛋白表达与宫颈癌的相关性研究

目的研究人乳头状瘤病毒16型（HPV16）E6／E7基因及其蛋白表达在宫颈疾病及其癌变中的作用。方法运用PCR技术检测51例宫颈癌（癌症组）、20例富颈上皮瘤变（CIN）Ⅱ～Ⅲ级（CIN组）、20例宫颈炎（炎症组）患者病变组织中HPV16 E6／E7基因,并运用免疫组化SP法检测癌症组癌组织中HPV16E6、E7的表达情况。结果癌症组、CIN组、炎症组HPV16 E6检出率分别为5％、35％、45％．后两者明显高于前者（P〈0．05）,HPV16 E7检出率分别为65％、75％、68．6％,P均〉0．05;癌症组45例HPV16 E6和42例E7蛋白阳性表达（88．2％、82．3％）。HPV16 E6蛋白表达与临床分期、肿瘤分化程度和淋巴结有无转移均无相关性（P〉0．05）,HPV16 E7蛋白表达与临床分期和淋巴结有无转移相关（P〈0．05）,与肿瘤分化程度无相关性（P〉0．05）。结论HPV16 E6／E7基因与宫颈疾病及其癌变的关系密切。     

HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

Human papillomavirus type 16 (HPV16) causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large). Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43), a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G) Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.     

靶向乳头瘤病毒18型E6基因的shRNA表达载体的构建与鉴定

目的以乳头瘤病毒(human papillomavirus,HPV)18型E6基因为靶标,构建小发夹状RNA(small hairpinRNA,shRNA)表达载体。方法人工方法合成针对靶序列的两条寡核苷酸链,两端加入酶切位点。寡核苷酸链退火后与酶切的线性化表达载体连接。连接产物转化大肠埃希菌,卡那霉素筛选阳性克隆。结果重组质粒酶切片段约为400 bp,与插入片段大小一致。DNA序列分析证实插入片段序列与预期一致。结论成功构建HPV shRNA表达载体pHPV1、pHPV2。     

人类乳头瘤病毒16型E7C亚基因在耻垢分枝杆菌中的表达

目的在耻垢分枝杆菌Mycobaterium smegmatis mc2155中表达人类乳头瘤病毒16型E7C亚基因,为其重组BCG疫苗的研究奠定基础。方法采用电穿孔转化法将重组质粒pBCG-E7C导入耻垢分枝杆菌(M.smegmatismc2155)中,通过卡那霉素抗性筛选经PCR鉴定的重组M.smegmatis mc2155,培养于middlebrook7H9 broth(M7H9)培养基中,并添加10%M7H9 enrichment ADC和0.05%Tween 80,37℃培养48 h~72 h,42℃诱导表达5 h,表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot分析。结果成功构建pBCG3000-E7C质粒,SDS-PAGE显示表达产物的分子质量单位约为6.5 ku,与预期理论值相符,Western blot分析表达产物能被宫颈癌患者血清识别。结论人类乳头瘤病毒16型E7C基因在M.smegmatis mc2155中成功表达。     

HLA-A2限制性CTL表位HPV18E7_(7-15)分子修饰的实验研究

目的对人类白细胞抗原A2分子(HLA-A2)限制性细胞毒性T淋巴细胞(CTL)表位HPV18E77-15进行氨基酸置换修饰,并探讨修饰后多肽的免疫原性。方法根据量化模体方案,比较置换后的多肽与HLA-A2分子的结合系数,采用标准Fmoc方案合成并纯化多肽、细胞毒实验(51Cr释放法),观察多肽是否能够诱导特异性CTLs。结果修饰肽TLQDIVLHV符合HLA-A2分子限制性细胞毒性T细胞的表位要求,具有特异性细胞毒性T细胞诱导活性。结论修饰肽TLQDIVLHV具有更好的结合力和较强的抗原性,可以作为高危型人乳头瘤病毒(HPV)感染治疗性肽疫苗分子设计的候选表位。     

Detection and typing of human papillomavirus in anal epidermoid carcinomas: Sequence variation in the E7 gene of human papillomavirus type 16

INTRODUCTION: Human papillomavirus, particularly Type 16, plays a central role in the development of anogenital squamous-cell carcinomas. A common sequence variation of human papillomavirus Type 16 in cervical cancer cell lines and in cervical cancer tissues from Korean patients was recently reported. The present study was performed to determine the integration type of human papillomavirus DNA in anal epidermoid carcinoma and to identify the common sequence variations in the human papillomavirus Type 16 E7 gene that had been previously reported. METHODS: Twenty-one formalin-fixed, paraffin-embedded specimens collected from 29 patients with anal epidermoid carcinomas treated at the Seoul National University Hospital over a ten-year period (1989–1998) were investigated. Genomic DNA from the 21 specimens was extracted and analyzed using the polymerase chain reaction with a general primer and a type-specific primer for human papillomavirus Types 16 and 18. Direct sequencing was performed. As a control, 13 normal anal epithelia available from these patients were microdissected. As another control, 21 hemorrhoidal squamous epithelia obtained from a demographically adjusted group were also analyzed. RESULTS: Human papillomavirus Type 16 DNA was present in all 21 anal epidermoid carcinomas. All controls were negative for human papillomavirus DNA. Sequence analysis revealed that 57 percent (12/21) specimens showed two types of sequence variation in the E7 gene. One variant with a single nucleotide change at position 647 (amino acid 29AATAGT, asparagine to serine) was found in 38 percent (8/21) of the samples. This variant has been detected in cervical cancers from Korean patients: 19 (39 percent) of 49 cervical cancer tissues and 6 (50 percent) of 12 cervical cancer cell lines. Another single nucleotide change at position 645 (amino acid 28, TTATTC, leucine to phenylalanine) was found in 19 percent (4/21) of the samples. These two variants exhibit a change of amino acid affecting the critical sites for Rb binding. CONCLUSION: Human papillomavirus Type 16 was found to be present in all 21 anal epidermoid carcinomas. Furthermore, in the Korean population, the most common sequence variant found in cervical cancer is also the major one in anal epidermoid carcinoma.     

Human papillomavirus (HPV) type 6 and 16 DNA sequences in bronchial squamous cell carcinomas demonstrated by in situ DNA hybridization

A series of 131 routinely processed, paraffin-embedded biopsy specimens derived from the same number of patients with a bronchial squamous cell carcinoma were analyzed using in situ DNA-hybridization technique with a probe cocktail containing³⁵S-labeled human papillomavirus (HPV) DNA of types 6, 11, 16, 18, and 30. The 12 carcinomas shown to contain HPV DNA by the probe cocktail were subjected to in situ hybridization with the specific HPV DNA probes applied separately under high stringency conditions. HPV DNA could be found in 9 of these carcinomas; 2 cases contained HPV 6 DNA and 7 hybridized with HPV 16 DNA. The role of HPV in the development of bronchial squamous cell carcinoma is discussed in the light of the previously established morphologic evidence as well as the increasing number of reports on malignant transformation of the respiratory tract HPV lesions. The present findings of HPV DNA sequences provide further support to the concept of HPV as a potential causative agent of some bronchial squamous cell carcinomas, possibly acting synergistically with chemical or physical carcinogens. As in the genital tract, it seems clear that a respiratory tract infection by “low-risk” HPV types 6 and 11 by no means excludes the possibility of malignancy, so far ascribed almost exclusively to the “high-risk” type HPV 16.     

Inhibition of angiogenesis and tumor growth by murine 7E3, the parent antibodyof c7E3 Fab (abciximab; ReoProTM)

Angiogenesis plays an essential role in the growth and dissemination of solid tumor cancers. The expression of endothelial cell integrin αvβ3 has been shown to increase during vascular proliferation associated with human tumors. Selective antagonists of αvβ3 can block angiogenesis and tumor growth by inducing programmed cell death in proliferating endothelial cells. Monoclonal antibody 7E3, an antagonist of the human, but not murine, integrins αvβ3 and αIIbβ3 (GPIIb/IIIa), inhibits platelet aggregation. It is the parent antibody of a mouse/human chimeric antibody fragment approved for adjunctive therapy of patients undergoing percutaneous coronary interventions to prevent ischemic complications (c7E3Fab; abciximab; ReoPro). To evaluate the potential of 7E3 to inhibit human angiogenesis and tumor growth independent of its antiplatelet effects, we established integrin αvβ3-negative human melanoma tumors in full-thickness human skin grafted onto SCID mice. The resulting tumors induce a human angiogenic response as assessed by the immunoreactivity of vascular cells with monoclonal antibodies specific for human CD31. Administration of 7E3 prevented or significantly inhibited the growth of tumors, and this effect correlated with a significant reduction in the number of blood vessels supplying the tumors. These results support the previous findings that blockade of integrin αvβ3 inhibits angiogenesis and tumor growth and indicates that dual inhibitors of αvβ3 and αIIbβ3 are effective in blocking tumor growth and angiogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution of Human Papillomavirus (HPV) Genotypes in HIV-Negative and HIV-Positive Women with Cervical Intraepithelial Lesions in the Eastern Cape Province,South Africa

South African women have a high rate of cervical cancer cases, but there are limited data on human papillomavirus (HPV) genotypes in cervical intraepithelial neoplasia (CIN) in the Eastern Cape province, South Africa. A total of 193 cervical specimens with confirmed CIN from women aged 18 years or older, recruited from a referral hospital, were tested for HPV infection. The cervical specimens, smeared onto FTA cards, were screened for 36 HPV types using an HPV direct flow kit. HPV prevalence was 93.5% (43/46) in CIN2 and 96.6% (142/147) in CIN3. HIV-positive women had a significantly higher HPV prevalence than HIV-negative women (98.0% vs. 89.1%, p = 0.012). The prevalence of multiple types was significantly higher in HIV-positive than HIV-negative women (p = 0.034). The frequently detected genotypes were HPV35 (23.9%), HPV58 (23.9%), HPV45 (19.6%), and HPV16 (17.3%) in CIN2 cases, while in CIN3, HPV35 (22.5%), HPV16 (21.8%), HPV33 (15.6%), and HPV58 (14.3%) were the most common identified HPV types, independent of HIV status. The prevalence of HPV types targeted by the nonavalent HPV vaccine was 60.9% and 68.7% among women with CIN2 and CIN3, respectively, indicating that vaccination would have an impact both in HIV-negative and HIV-positive South African women, although it will not provide full protection in preventing HPV infection and cervical cancer lesions.     

吲哚美辛对胃癌SGC 7901细胞增殖及Cyclin D1蛋白表达的影响

目的:观察引哚美辛对胃癌SGC 7901细胞增殖及细胞周期调控蛋白Cyclin D1表达的影响,探讨吲哚美辛抑制细胞增殖的机制.方法:采用MTT法观察吲哚美辛对胃癌细胞SGC 7901增殖的影响,采用流式细胞仪观察细胞周期分布的变化,采用免疫细胞化学方法检测Cyclin D1蛋白的表达.结果:吲哚美辛呈时间、浓度依赖方式抑制胃癌SGC 7901细胞增殖,改变细胞周期的分布,增加G0/G1期细胞的比例,下调Cyclin D1蛋白的表达.结论:吲哚美辛可能通过下调Cyclin D1蛋白表达,影响细胞周期的分布来抑制胃癌SGC7901细胞增殖.     

血管紧张素-(1-7)在内皮素诱导血管平滑肌细胞增殖反应中的作用

目的　探讨血管紧张素 (1 7) [Ang (1 7) ]在内皮素 1(ET 1)诱导血管平滑肌细胞增殖反应中的作用。方法　在ET 1诱导培养的SD大鼠主动脉血管平滑肌细胞模型中 ,应用Ang (1 7) ,通过测定3 H 胸腺嘧啶 (3 H TdR)掺入的方法 ,观察血管平滑肌细胞增殖情况。结果　Ang (1 7)呈剂量性抑制ET 1诱导血管平滑肌细胞的DNA合成 ,其作用受体不是血管紧张素Ⅱ受体 1(AT1)或血管紧张素Ⅱ受体 2 (AT2 ) ,而是通过一种特殊受体介导。结论　Ang (1 7)能抑制ET 1诱导的血管平滑肌细胞增殖。     

The Retrovirus-mediated Antisense Human Telomerase RNA (hTR) Gene Limits the Growth of Hepatocellular Carcinoma Growth in Cell Culture and Animals

Aim To investigate the inhibitory effect of retrovirus-mediated antisense human telomerase RNA (hTR) gene therapy on hepatocelluar carcinoma. Methods We first constructed the sense and antisense hTR vectors and then transfected these into HepG2 cells. Telomerase activity, cell growth curves, proliferating cell nuclear antigen expression (PCNA), cell cycle distribution, and cell apoptosis were detected by the means of telemere repeat amplification protocol (TRAP), MTT assay, immunofluorescence, flow cytometric analysis, and transferase-mediated nick end labeling (TUNEL), respectively. In order to further confirm the therapeutic effect of this gene therapy, we developed an experimental line of HepG2 tumor-bearing nude mice by and directly injected these with retrovirus expressing the antisense hTR gene. Tumor growth was determined by tumor volume, and cell apoptosis was analyzed by TUNEL. Results The antisense hTR gene was shown to be successfully integrated into the target cells’ genome. HepG2 cells transfected with the antisense hTR gene showed down-regulated telomerase activity, inhibited cell growth, decreased PCNA expression, and increased apoptotic rate. Moreover, flow cytometry revealed a decrease of cells in the S phase with cell cycle arrest at the G2/M phase. In the antisense hTR-treated group, tumor growth was significantly reduced and showed an increase of apoptotic cells. Conclusion The results indicate that the specific inhibitor of the hTR template is likely to be a very efficient tool for hepatocellular carcinoma research and may possess potential therapeutic significance in the future clinical practice.