Clinical significance of telomerase and its associate genes expression in the maintenance of telomere length in squamous cell carcinoma of the esophagus

AIM: To observe the interaction between the expression of telomerase activity (TA) and its associate genes in regulation of the terminal restriction fragment length(TRFL) in esophageal squamous cell carcinoma (SCC).METHODS: Seventy-four specimens of esophageal SCC were examined. The TA was measured by telomeric repeat amplification protocol (TRAP) assay, and the associated genes [human telomerase-specific reverse transcriptase (hTERT), hTERC, TP1, c-Myc, TRF1,and TRF2] were detected using RT-PCR method. The TRFL was measured by Telomere Length Assay Kit and Southern blotting. The correlations between the expression of telomerase and its associated genes with the TRFL and survivals were examined.RESULTS: Expressions of the TA, hTERT, hTERC, TP1,c-Myc, TRF1, and TRF2 genes were observed in 85.1%,64.9%, 79.7%, 100.0%, 94.6%, 82.4%, and 91.9% of the tumor tissues, respectively. The TRFL of the tumor and normal esophageal tissues were 2.70±1.42 and 4.93±1.74 kb, respectively (P＜0.0001). The TRFL of the telomerase positive and telomerase negative tumor tissues were 2.72±1.44 and 2.58±1.32 kb, respectively (P = 0.767).The TRFL ratios (TRFLR) of the telomerase positive and telomerase negative tumor tissues were 0.55±0.22 and 0.59±0.41, respectively (P = 0.742). The expression rates of h-TERT (P = 0.0002), hTERC (P＜0.0001), and TRF1(P = 0.002) in the tumor tissues are higher than those of the normal paired tissues. Though TA is markedly activated in tumor tissues (P＜0.0001), its expression is not related to clinicopathological parameters including gender, tumor differentiation, and TNM stages. The cumulative 4-year survival rates of telomerase positive and telomerase negative cases were 35.86% and 31.2%,respectively (P = 0.8442). The cumulative 4-year survival rates of patients with their TRFLR ≤85% and ＞85%were 38.7% and 15.7%, respectively (P = 0.1307).CONCLUSION: Though telomerase expression is not related to tumor stages and prognosis, our data support that the TA increased as the TRFL decreased,probably under the control of hTERT, hTERC, and TRF1.When telomerase expression was activated, only TRF2overexpression persisted to stabilize T-loop formation.Furthermore, as the TRFLR decreased to 85%, a trend of better prognosis was observed. Cox model analysis indicates a higher t/n TRFLR and distant metastasis are independent poorer prognostic factors (P = 0.035 and P = 0.042, respectively).

The Cloning and Fluorescence In situ HybridizationAnalysis of Cotton Telomere Sequence

Telomeres form the ends of eukaryotic chromosomes and serve as protective caps that keep chromosomes structure independency and completeness. The first plant telomere DNA was isolated from Arabidopsis thaliana and was shown to have tandemly repeated sequence 5-TTTAGGG-3： The Arabidopsis-type telomere has been found in many plants, but several reports indicate that this sequence is absent in some plants. Up to now, no research has been conducted on the telomere of cotton. In this paper, the Arabidopsis-type telomere sequence was amplified and cloned using the primers designed based on the fragment containing telomere sequence in an Arabidopsis bacterial artificial chromosome （BAC）. Fluorescence in situ hybridization （FISH） with cotton metaphase chromosomes using the Arabidopsis-type telomere sequence as probes indicated that the signals were located at all chromosome ends of seven diploid and two tetraploid cotton species with different signal intensities among chromosome complements of different cotton species, even between long and short arms of the same chromosome. To identify the signals of FISH, the genome DNA of Xinhai 7, a cultivar of Gossypium barbadense, digested by BAL-31 nuclease was introduced in this study. The result of BAL-31 digestion indicated that the hybridization signals of FISH represent the outermost DNA sequence of each cotton chromosomes. So we first proved that the telomeric repeats of cotton cross-hybridize with that of Arabidopsis. The results of terminal restriction fragment （TRF） showed significant variation in telomere length among cotton species. The telomere length of cultivated cotton was close to 20 kb and was larger than those of wild cotton species whose telomere length rahged from 6 to 20 kb.

Transformation of marginal zone lymphoma into high-grade B-cell lymphoma expressing terminal deoxynucleotidyl transferase:A case report

BACKGROUND High-grade B-cell lymphoma(HGBL)is an unusual malignancy that includes myelocytomatosis viral oncogene(MYC),B-cell lymphoma-2(BCL-2),and/or BCL-6 rearrangements,termed double-hit or triple-hit lymphomas,and HGBL-not otherwise specific(HGBL-NOS),which are morphologically characteristic of HGBL but lack MYC,BCL-2,or BCL-6 rearrangements.HGBL is partially transformed by follicular lymphoma and other indolent lymphoma,with few cases of marginal zone lymphoma(MZL)transformation.HGBL often has a poor prognosis and intensive therapy is currently mainly advocated,but there is no good treatment for these patients who cannot tolerate chemotherapy.CASE SUMMARY We reported a case of MZL transformed into HGBL-NOS with TP53 mutation and terminal deoxynucleotidyl transferase expression.Gene analysis revealed the gene expression profile was identical in the pre-and post-transformed tissues,suggesting that the two diseases are homologous,not secondary tumors.The chemotherapy was ineffective and the side effect was severe,so we tried combination therapy including venetoclax and obinutuzumab.The patient tolerated treatment well,and reached partial response.The patient had recurrence of hepatocellular carcinoma and died of multifunctional organ failure.He survived for 12 months after diagnosis.CONCLUSION Venetoclax combined with obinutuzumab might improve the survival in some HGBL patients,who are unsuitable for chemotherapy.

STUDY ON ANTITUMOR DRUG-INDUCED APOPTOSIS IN HUMAN CANCER CELLS BY TERMINAL DEOXYNU-CLEOTIDYL TRANSFERASE ASSAY

Objective: Considerable evidence has showed that apoptosis is involved in both cancer development and inhibition. A new assay (terminal deoxynucleotidyl tansferase, TdT) was recently reported to have advan tages in the detection of apoptosis. In this study, this assay was used to investigate antitumor drug induced apoptosis in human cancer cells. Methods: TdT assay, DNA gel electrophoresis, electron and light microscopy were used to observe apoptosis. Results: Our results showed that cisplatin induced apoptosis in both HL 60 and SV40T transformed human bronchial epithelial cells was detected with a good dosage and time response. The occurrence of the apoptosis was preceded by the decrease of bcl 2 mRNA expression. With the TdT assay, apoptotic cells were observed in ovarian tumor of patients treated with carboplatin. Conclusion: TdT assay may be applicable to monitor apoptosis in human cancers induced by chemotherapy, and to evaluate tumor cell response during treatment.

The Possible Involvement of Apoptotic Decay of Terminal Deoxynucleotidyl Transferase-Positive Lymphocytes in the Reutilization of the Extracellular DNA Fragments by Surrounding Living Cells

The migrating TdT+ thymocytes can die in other tissues, promoting the surrounding cells’ renewing likes holocrine secretion does. To clarify the role of TdT-enzyme for this function of progenitor lymphocytes, their extracellular media with its components included by living cells analyzed in vitro before and after in vivo irradiation of donor rats. The nucleoid with DNase-sensitive (free) DNA and TdT activity discovered in extracellular media conditioned preliminary by spontaneous apoptotic death of a minor part of the thymocyte’s suspension in vitro. The penetration of labeled products of non-template synthesis with free DNA’ primers from media into cells by pinocytosis confirmed by exogenous polymeric DNA marked artificially. The DNA penetration into cells follows an increase of the cell’s viability and acceleration of spontaneous intracellular DNA-synthesis controlled with labeled thymidine uptake. Both phenomena are typical for either the lowest initial concentration of intact cells or their preliminary irradiation in vivo. The data point to possible involvement of apoptotic decay of TdT+ cells in the reutilization of the extracellular DNA fragments for reparation/regeneration of surrounding living cells.

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

链霉菌中一类同源非典型端粒的克隆及分析

采用自连接-PCR的方法克隆和测序了Streptomyces cattleya DSM46488、Streptomyces mobaraensisDSM40847及Streptomyces davawensisJCM4913的端粒,序列比对发现,这3种端粒具有较高的序列相似性,前60个核苷酸的序列相似性达到了80%以上;在二级结构水平,这些端粒含有多个回文序列,其中,包含相同的回文序列I和II,但并不能通过折返形成超级发夹结构;Streptomyces cattleya DSM46488、StreptomycesmobaraensisDSM40847及Streptomyces davawensis JCM4913的端粒与线性质粒pSHJG1端粒及Streptomyces albus J1074染色体端粒均具有较高的序列相似性;系统发育分析显示它们与典型端粒及其他非典型端粒处于不同的进化分支,S.cattleyaDSM46488、S.mobaraensis DSM40847、S.davawensisJCM4913、S.albus J1074及S.hydroscopicussubsp.Jinggangensis 5008都编码同源的潜在末端蛋白-端粒相关蛋白,暗示了这类同源的非典型端粒系统广泛存在于链霉菌中。

hTERT、c-fos、c-jun在成釉细胞瘤中的表达及意义

目的:探讨c-fos、c-jun及hTERTmRNA在人成釉细胞瘤(AB)中的表达及其临床意义。方法:用原位杂交法检测AB中c-fos、c-jun和hTERTmRNA的表达,同时选取正常口腔黏膜7例、牙源性角化囊性瘤(KCOT)16例作为对照。采用SPSS10.0统计软件包进行χ2检验和Fisher确切概率法及Kendall相关分析。结果:正常口腔黏膜无c-fos、c-junmRNA的表达,hTERTmRNA仅在1/7例正常上皮中表达。c-fos、c-junmRNA和hTERTmRNA在KCOT上皮阳性表达率分别为72.7%(8/11)、35.7%(5/14)和87.5%(14/16)。AB中,c-fosmRNA阳性表达率为91.5%(43/47)、c-junmRNA阳性表达率较低,为26.7%(12/45),而hTERTmRNA高达94.0%(47/50)。c-fos和hTERTmRNA在正常口腔黏膜、KCOT及AB3组间的表达差异显著(P<0.001),而c-jun的表达在各组间未见显著差异。Kendall相关分析表明,c-fos与hTERTmRNA呈正相关(rk=0.024,P<0.05)。结论:c-fos基因在AB的发生、发展及细胞的增殖中起重要作用,且可能为构成AP-1的一个主要亚基,参与AB中端粒酶活性的调节。

端粒酶RNA特异性核酶抑制卵巢癌细胞端粒酶的活性

目的 研究端粒酶 RNA特异性核酶对卵巢癌细胞活性的抑制作用 .方法 用脂质体法将构建好的核酶逆转录病毒载体转染至卵巢癌 HO- 8910细胞 ,以 MTT比色法检测细胞的生长 ,应用流式细胞仪分析细胞周期细胞 ,观察细胞的增殖情况 ,同时扩增端粒重复序列 ,结合杂交分析 ,检测细胞的端粒酶活性 .结果 MTT检测发现转染核酶后的细胞生长的潜伏期延长 (48→ 72 h) ,对数生长期减缓 (4→ 5 d) ,流式细胞仪结果显示其 G1期比例明显增高 (70 .0 %→ 80 .5 % ,P<0 .0 1) ,S期细胞比例明显降低 (19. 0 %→ 13. 7% ,P<0 .0 1) ,细胞增殖下降 ,细胞的端粒酶活性亦降低 .结论 端粒酶 RNA特异性核酶能抑制卵巢癌细胞端粒酶活性 ,抑制细胞增殖 .

二化性家蚕滞育过程中谷胱甘肽S-转移酶活性的变化

二化性家蚕卵以25℃和15℃催青,可分别诱导成虫产下子代滞育和非滞育卵,而即时浸酸和5℃左右的低温处理可以分别阻止和解除胚胎滞育。利用1-氯-2,4-二硝基苯(CDNB)显色法测定了经上述处理后的胚胎滞育过程中谷胱甘肽S-转移酶(glutathione S-transferase,GST)的活性,结果表明:胚胎发育后期的蚕卵经25℃催青,卵中的GST活性显著高于15℃催青蚕卵;蚕卵即时浸酸未显著改变胚胎滞育发动阶段蚕卵中的GST活性;5℃低温处理显著提高了滞育卵中的GST活性,但是未能显著改变即时浸酸卵的GST活性。上述结果表明,蚕卵中的GST活性变化与二化性家蚕胚胎滞育诱导和解除密切相关。

皮肤鳞状细胞癌皮损中hTERT mRNA的表达

目的探讨hTERT基因表达在皮肤鳞状细胞癌(鳞癌)进展过程中的作用。方法收集10例正常皮肤、11例假上皮瘤样增生(PEH)、66例皮肤鳞癌原发灶及12例淋巴结转移灶标本和临床、病理学资料,采用原位杂交法检测hTERT mRNA,结果以图像分析方法定量分析。结果hTERT mRNA在正常皮肤、PEH及鳞癌中的阳性系数(PU)分别为0.84±0.58、3.06±1.09和10.01±0.60(P<0.01),鳞癌与前两者间差异有非常显著性(P<0.01);高分化与低分化组PU值分别为8.52±0.63和13.42±1.02,差异有非常显著性(P<0.01);转移组与无转移组、已发生转移病例原发灶与转移灶间差异均无显著性(P>0.05)。结论与正常皮肤、PEH相比,鳞癌中hTERT基因表达明显增高,分化越低表达越强,提示端粒酶激活在鳞癌恶变过程中起了关键作用。

晚期糖基化终产物对心肌细胞细胞周期和凋亡的影响

目的探讨晚期糖基化终产物(advanced glycation end products,AGEs)对体外培养乳鼠心肌细胞细胞周期及凋亡的影响.方法体外培养3～5 d的乳鼠心肌细胞,用含1%或10%胎牛血清的DMEM培养基中继续培养24 h,应用流式细胞仪和原位末端标记技术观察不同浓度AGEs(50、100μg/ml)对心肌细胞刺激12、24、48 h后细胞周期分布、凋亡率及凋亡小体/凋亡细胞核分布的影响.结果AGEs对心肌细胞细胞周期分布无明显影响;在正常培养液培养时,心肌细胞的凋亡随着培养时间延长而增加,24、48 h与12h相比,凋亡小体/凋亡细胞核分别增加了0.55、1.23倍;AGEs可以明显增加心肌细胞的凋亡程度,并随着刺激时间、刺激浓度的增加而增加,50、100μg/ml AGEs组与对照组相比,凋亡小体/凋亡细胞核12、24、48 h分别增加了0.74和1.21、1.15和1.78、0.83和1.19倍.结论AGEs对心肌细胞细胞周期分布无影响,但可以通过增加心肌细胞凋亡率对心肌细胞造成损伤.

孕期酒精接触对子鼠视皮质神经元凋亡的影响

目的探讨孕期酒精接触对子鼠视皮质神经元凋亡的影响。方法从妊娠母鼠第5d酒精灌胃直至小鼠出生;采用激活的半胱氨酸天冬氨酸蛋白3(Caspase-3)免疫组织化学和原位末端标记(TUNEL)法观察P0、P7和P14小鼠视皮质神经元的凋亡。结果酒精实验组妊娠时间延长,出现死胎和畸形(小头畸形、无脑儿和脊柱脊髓裂等)。酒精实验组Caspase-3阳性率和凋亡指数明显高于对照组(P<0.001),高剂量酒精组明显高于低剂量组(P<0.01)。随着酒精剂量的增加,子鼠视皮质神经元的凋亡明显增加。结论孕期酒精接触可导致子鼠视皮质神经元凋亡,且有剂量依赖性和长时程效应。

尖锐湿疣组织中Survivin蛋白表达与角质形成细胞凋亡的相关性及意义

目的探讨Survivin蛋白在尖锐湿疣(CA)发生、发展中的作用。方法采用免疫组化和TUNEL技术检测56份CA组织(观察组)中Survivin蛋白表达和角质形成细胞凋亡指数,并与25份正常包皮组织(对照组)比较。结果观察组及对照组Survivin蛋白阳性率分别为64.29%(36/56)、4.00%(1/25),P<0.01;角质形成细胞凋亡指数分别为(9.73±3.51)%、(0.95±0.16)%,P<0.01;观察组Survivin蛋白表达阳性者及阴性者角质形成细胞凋亡指数分别为(6.16±2.58)%、(14.31±14)%,P<0.05。Survivin蛋白表达与凋亡指数呈负相关(r=-0.694,P<0.05)。结论 CA组织中存在Survivin蛋白过表达,可抑制角质形成细胞凋亡,促进CA的发生及发展。

TdT阴性的淋巴母细胞性白血病/淋巴瘤3例临床病理观察

目的探讨TdT阴性的淋巴母细胞性白血病/淋巴瘤(ALL/LBL)的临床病理特征、免疫表型、鉴别诊断及治疗预后。方法回顾性分析3例TdT阴性的ALL/LBL的临床及病理资料、随访,并进行相关文献复习。结果患者年龄分别为13岁、28岁和39岁,均为女性,临床首发症状分别为咽痛、腰背疼痛及多发皮下包块,镜下见小~中等大淋巴样细胞弥漫浸润性生长,胞质少,核染色质细腻,核仁不明显,核分裂象易见。瘤细胞CD99(3/3)、CD34(2/3)、CD43(1/2)、CD10(1/2)、PAX-5(2/3)、CD3(1/3)、CD7(1/3)(+),TdT、MPO、CD20均(-),1例诊断为T-ALL/LBL,2例为B-ALL/LBL。随访1年,2例死亡。结论 TdT是ALL/LBL的特异性标记物,TdT阴性时需结合临床、病理学形态及免疫表型综合分析明确诊断,避免误诊。

113例前列腺癌AR与凋亡相关因子表达的研究

目的探讨113例人前列腺癌AR与凋亡相关因子表达的相关性。方法应用免疫组化标记AR及细胞凋亡相关因子bcl-2、Bax及Tunel标记等,结合光镜、电镜观察及图像分析等方法,研究不同AR表达与PCa凋亡的相互关系。结果AR(-)组较AR(+)组bcl-2阳性表达明显增强,Bax表达增强,bcl-2/Bax比值增高,前列腺癌AR(-)组细胞凋亡作用较AR(+)组明显增强。结论AR表达缺失促进了bcl-2和Bax的表达,Bax表达上调促进细胞凋亡的发生,而bcl-2有癌基因的作用,其表达上调则促进了的细胞增殖,导致前列腺癌凋亡及增殖作用均增强。

激光辅助制动对精子核DNA的影响

目的:研究激光辅助制动精子是否引起精子核DNA损伤。方法:23例行体外受精(IVF)的男性精液标本,上游法处理后用0.45ms脉冲激光照射精子尾部制动精子。采用末端转移酶介导的dUTP末端标记法(TUNEL)和单细胞凝胶电泳法(SCGE)两种方法分别检测激光辅助制动处理前后阳性精子百分率。结果:TUNEL法检测激光辅助制动处理前后阳性精子的百分率分别为(1.32±0.61)%、(1.41±0.51)%,差异无显著性(P>0.05);SCGE法检测处理前后阳性精子百分率分别为(1.59±0.70)%、(1.83±0.68)%,差异亦无显著性(P>0.05)。结论:激光照射精子尾部辅助制动精子对精子核DNA无明显损伤。

端粒酶逆转录酶基因在口腔癌前病变及鳞癌的表达

目的 观察端粒酶催化蛋白基因 (h TRT)在口腔癌前病变及鳞癌中的表达 ,探讨其与口腔鳞癌(OSCC)发生发展及临床病理特征之间的关系。 方法 用原位杂交技术检测 5 4例标本 ,其中正常口腔粘膜 6例、上皮非典型增生 15例、口腔粘膜鳞癌 33例。 结果 正常口腔粘膜组织中 h TRT的 m RNA表达较弱 ,阳性信号仅局限于上皮基底层及副基底层间 ,阳性率 16 .6 7% (1/ 6 ) ;上皮非典型增生中 h TRT的 m RNA阳性表达见于多层上皮细胞 ,并随细胞异形性增高而表达增强 ,阳性率 6 0 % (9/ 15 ) ;口腔粘膜鳞癌组织中 h TRT的 m RNA有较强阳性表达 ,阳性率 87.88% (2 9/ 33) ;OSCC组织中 h TRT m RNA的表达与肿瘤临床病理分化无关 ,与淋巴结转移密切相关。 结论 端粒酶 h TRT基因的表达在

早期心肌缺血心肌细胞凋亡的实验研究

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