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.

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.

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).

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.

末端脱氧核苷酸转移酶相互作用因子1及其在癌症中的研究进展

末端脱氧核苷酸转移酶相互作用因子1(TdIF1)是一种直接与末端脱氧核苷酸转移酶结合的蛋白质。TdIF1在非小细胞肺癌、口腔癌、乳腺癌中高度表达,且这种高表达可促进癌细胞的生长。文章就TdIF1的结构、功能以及在癌症进展中的作用进行综述,指出TdIF1可能是一种潜在的癌症促进因子,为其成为癌症诊断的潜在标志物及靶向治疗的新靶标提供参考。

TdT阴性的淋巴母细胞淋巴瘤/急性淋巴细胞白血病临床特点、诊断及预后研究进展

末端脱氧核苷酸转移酶(terminal deoxynucleotide transferase,TdT)是一种特殊类型的DNA聚合酶,它可以在无DNA复制模板的条件下将单个脱氧核苷酸结合到DNA分子的3'-OH端。TdT是造血系统中不成熟淋巴细胞中特异性较强的细胞内标记,在淋巴源性肿瘤的诊断中具有重要意义。淋巴母细胞淋巴瘤/急性淋巴细胞白血病(lymphoblastic lymphoma/acute lymphoblastic leukemia,LBL/ALL)的诊断依赖免疫分型。TdT通常在LBL/ALL中呈阳性表达,是临床诊断LBL/ALL的重要依据。TdT阴性的LBL/ALL在临床中较为少见,鉴于该分子诊断特异性较高,其阴性表达会给诊断带来一定的困难。TdT阴性的原因有多种,在淋巴细胞不同分化阶段中TdT的表达情况不同,另因不同检测方法的灵敏度不同,也可导致偶见TdT的假阴性。此外,有文献指出TdT阴性与TdT阳性的LBL/ALL在临床特点及预后方面亦不相同。了解TdT的表达意义、阴性的原因,以及阴性或阳性表达的LBL/ALL的不同特点,有助于临床诊断、治疗选择及预后判断。该文对国内外相关文献进行了梳理,就TdT阴性的原因及其表达阴性的LBL/ALL的临床特点、诊断及预后等进行综述。

Single-stranded RNA as primers of terminal deoxynucleotidyl transferase for template-independent DNA polymerization

Terminal deoxynucleotidyl transferase(Td T) has been characterized as template-independent polymerase using single-stranded DNA(ss DNA) as primers to generate random oligonucleotides. However, the extension performance of Td T to single-stranded RNA(ss RNA) is vague. By systematically comparing and contrasting the performance of Td T-catalyzed ss DNA and ss RNA extension, it is indicated that the catalytic efficiency of ss RNA as primers was about 3 times lower than ss DNA as primers. Collectively, it is believed that understanding the catalytic performance of Td T will help to design the strategy to synthesize chimeric DNA on 3-OH of ss RNA, which becomes invaluable.

基于末端脱氧核苷酸转移酶协同G-四链体核酶的恩诺沙星检测方法

基于末端脱氧核苷酸转移酶(TdT)协同G-四链体核酶设计信号放大策略,建立了一种恩诺沙星电化学检测方法。目标物恩诺沙星与特异性核酸适体的结合触发TdT在电极表面的扩增反应,产生G-四链体核酶纳米线结构,进而发挥辣根过氧化物酶活性催化信号放大,实现恩诺沙星的高灵敏和高特异性检测。该方法对恩诺沙星的线性检测范围为0.5~50μg/L,检测限低至0.043μg/L。此外,该无标记电化学生物传感器简单快速,成本低,并成功应用于对实际食品样本的分析检测,显示出较好的应用潜能。

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。

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。

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。

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。