人内源性逆转录病毒HERV-K Np9蛋白在人白血病中的表达及功能研究
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摘要
白血病是一种常见的严重危害人类健康的造血系统恶性肿瘤。据统计,全世界约有250,000人患上了白血病。不幸的是,仍有大部分白血病病人死于耐药和复发。然而,迄今为止,由于白血病的确切病因和发病机制仍未明确,使得研发能够有效能治愈该类疾病的药物和治疗方案成为当下一直未能攻克的难题。长久以来,在病毒、电离辐射、化学物质和遗传因素等病因学的研究中,由以病毒病因学的研究最为引入注目。多年来研究证明,逆转录病毒是一些动物白血病和成人T细胞白血病/淋巴瘤的直接病因。然而,逆转录病毒是否是引起人常见白血病如：人急/慢性髓细胞白血病、人急/慢性淋巴细胞白血病的病因却未见明确报道。
HERV-K是人内源性逆转录病毒家族成员中最可能具有生物学活性和致病性的一员。近年来陆续有研究报道HERV-K家族成员在许多人肿瘤中被再次激活。甚至有个别研究者在肿瘤病人中发现了HERV-K的病毒颗粒和抗HERV-K的抗体。人内源性逆转录病毒基因Np9是Ⅰ型HERV-Kenv基因编码和剪辑的产物。研究证明,Np9的转录本只在肿瘤细胞和转化的细胞中表达,而在正常的非转化的细胞中不表达。这高度提示着Np9转录的激活与肿瘤的形成密切相关。然而,现有的研究尚不明确Np9蛋白在肿瘤中的表达情况及功能。我们的前期研究已经证明,在急性髓细胞白血病病人的原代白血病细胞中存在典型的逆转录病毒颗粒。并且,现有的研究证实由Ⅰ型人T细胞白血病病毒编码的Tax蛋白与该病的发病相关。这为我们探究人内源性逆转录病毒HERV-K Np9蛋白在人白血病中的表达及功能提供了思路和基础。本论文将通过以下三部分重点探讨Np9蛋白在人白血病细胞系、病人中的表达情况、功能及具体的分子机制。这对揭示白血病病因和深入理解发病机制有重要意义,并有可能为人白血病的诊断和防治带来新的思路。
第一部分
Np9蛋白在人白血病中的表达研究
本部分旨在明确Np9蛋白在人白血病细胞系、白血病病人原代标本以及正常成年人中的表达情况。并在此基础上进一步了解与Np9相关的同由Ⅰ型HERV-Kenv基因编码的pol-env、env和transmembrane蛋白的表达情况。最后还将探讨激素、酒精、DNA去甲基化剂和病毒诱导剂等因素对诱导白血病细胞中Np9激活的影响。
我们收集了14种人恶性血液肿瘤细胞系和16种实体瘤细胞系,通过Western blot及抗Np9的特异性抗体检测各细胞中Np9蛋白的表达情况。结果显示：78.57%的人恶性血液肿瘤细胞系高表达Np9蛋白(11/14)。其中包括3种慢性髓细胞白血病细胞系：K562, K562/adr, KCL-22M;3种急性髓细胞白血病细胞系：HL-60, NB4, Kasumi-1;3种急性淋巴细胞白血病细胞系：Jurkat, Molt-4, H9;2种多发性骨髓瘤细胞系：8226,KM3。18.75%实体瘤细胞系(3/16)。其中包括：人恶性胶质瘤细胞系：U87；人肺癌细胞系：A549；人胰腺癌细胞系：PANC-1。
我们接着收集了50例白血病病人及22例正常成年人的外周血标本,通过淋巴细胞分离液分离单个核细胞,并用Western blot检测了Np9蛋白的表达情况。结果显示：56%原代白血病病人标本表达Np9蛋白(28/50)。相比之下,4.5%正常成年人弱表达Np9蛋白(1/22)。
综上结果：Np9蛋白在人白血病细胞系和白血病病人的原代细胞中普遍表达,而在正常人的血细胞中几乎不表达。
我们结合上一部分的实验结果及病人临床资料,对Np9蛋白水平与CD34+白血病干/祖细胞的相关性进行统计分析。结果发现,Np9蛋白水平可能与CD34+白血病干/组细胞正相关(P=0.003)。
我们进一步选择Np9蛋白阳性的白血病标本并应用Western blot技术检测与Np9相关的同由Ⅰ型HERV-Kenv基因编码的其它逆转录病毒蛋白pol-env、env和transmembrane蛋白。结果显示,Np9蛋白阳性的样本高表达白血病特异的pol-env、 env和transmembrane蛋白,而正常成年人外周血分离的单个核细胞却未见表达。
接着我们通过实时定量PCR技术测定了激素、酒精、DNA去甲基化剂和病毒诱导剂等因素对白血病细胞中Np9转录的影响。结果显示,随着Dexamethasone、酒精、IdUR浓度的增高,Np9的转录水平逐渐提高；5-Aza浓度的变化未见明显影响。
第二部分
Np9蛋白在体内和体外对白血病细胞生存与增殖的影响
本部分研究通过体外细胞系实验和体内动物模型的建立,旨在揭示Np9蛋白对白血病细胞生存和增殖的影响,明确其是否是白血病细胞生存和增殖的必须因素。
应用shRNA和慢病毒感染技术稳定下调了人慢性粒细胞白血病细胞系K562和人急性淋巴细胞白血病细胞系Jurkat中Np9蛋白的表达水平,并随后用MTS实验检测细胞增殖能力的变化。结果发现,两种细胞的生长都受到了明显的抑制。
相应地,利用慢病毒感染技术在Np9蛋白阴性的人B淋巴细胞白血病细胞系Raji稳定地过表达Np9蛋白,通过测定和绘制细胞的生长曲线发现过表达Np9蛋白的Raji细胞的增殖能力要明显高于对照组。
为了进一步评估Np9的表达对白血病细胞克隆形成能力的影响,应用上一步已建立的Raji-Np9和Raji-Ctr这两种细胞的克隆形成实验来观察所形成的克隆数量及大小的差异。结果显示,上调Np9蛋白的表达水平能显著增加Raji细胞的克隆形成数量和大小。
接着通过脂质体转染技术将p-EGFP-C1-Np9和p-EGFP-C1瞬时转入人慢性粒细胞白血病细胞系K562。应用激光共聚焦显微镜观察Np9蛋白的亚细胞定位,发现该蛋白主要分布细胞核内,但是仍有一小部分分散在胞浆。
为了明确Np9蛋白能否在体内促进白血病细胞的生长,我们将过表达Np9蛋白的Raji细胞经皮下注射到严重联合免疫缺陷的NOD-SCID鼠中,同时用转染空载体的Raji细胞作为阴性对照,Rec蛋白作为阳性对照,发现由Raji-Np9所形成的肿瘤生长速度要显著高于阴性对照组(*P<0.01)。25天以后,瘤重约为1.74g±0.42g,而相应的阴性对照组为0.62g±0.18g(*P<0.01)。实验组的瘤重约为阴性对照组的2.81倍。Real-time PCR分析结果显示,Np9蛋白在相应的细胞系和移植瘤中高表达。
综上结果我们认为Np9蛋白是白血病细胞生存的必须因素,并且能够显著促进白血病细胞的复制、增殖和产生子代的能力。
第三部分
Np9蛋白促进白血病细胞增殖的分子机制研究
本部分研究重点对白血病中常异常激活的Numb/Notch、Ras/ERK1/2、 c-myc/AKT和Wnt/β-catenin信号通路与Np9蛋白表达的相关性做出分析,并且结合Np9蛋白的氨基酸序列特征分析和功能域预测,以揭示Np9蛋白促进白血病细胞增殖的分子机制。
我们首先选取了高表达Np9蛋白的四种白血病细胞：K562、Kasumi、NB4和Jurkat,并同时通过Western blot技术检测了Numb/Notch、Ras/ERK1/2、c-myc/AKT和Wnt/β-catenin信号通路。结果显示,上述四条白血病相关的信号通路能够被同时激活。Np9的表达与共激活β-catenin、ERK、AKT和Notch1可能相关。
接着我们将正常成年人的外周血单个核细胞用上述同样地方法分析了Np9、 Numb、Cleaved Notchl、Grb2、Ras、ERK、pERK、c-myc、PLZF、pAKT和β-catenin等分子,发现在正常成年人的外周血单个核细胞不表达Np9,并且不能观察到白血病相关信号通路的激活。
同样地,我们在高表达Np9蛋白的白血病病人的原代标本中也观察到了β-catenin、cleaved Notch1、ERK和c-myc的共激活。
为了进一步明确是否是Np9的表达影响了上述关键信号分子的改变,我们建立了高表达Np9蛋白的Raji和低表达Np9蛋白的K562和Jurkat细胞系。利用Western blot技术对Np9蛋白和Numb/Notch、Ras/ERK1/2、c-myc/AKT和Wnt/β-catenin信号通路进行分析。结果发现,过表达Np9能显著上调β-catenin、 pERK、c-myc和Cleaved Notch1。而与此相应的是,Numb的表达水平降低(Numb和Cleaved Notchl互为抑制)。低表达Np9能显著下调了c-myc、pERK1、pAKT和Cleaved Notchl,而Numb的表达水平升高。
通过GeneBank和相关软件对Np9蛋白的氨基酸序列特征进行分析和功能域预测。我们在Np9蛋白的氨基酸序列中鉴定到典型的Grb2SH3(N)-typical motif (PxxPxR)。这与我们在本部分的实验结果中观察到了Np9蛋白的表达能直接影响phospho-ERK的水平相一致。
综上述三部分的实验结果,我们认为Np9蛋白在人白血病细胞系和白血病病人的原代细胞中普遍表达,而在正常人的血细胞中几乎不表达。Np9蛋白水平可能与CD34+白血病干／组细胞正相关。此外,Np9蛋白是白血病细胞生存和增殖的必须因素。该蛋白通过同时或者相继激活多条白血病相关的信号通路(Numb/Notch. Ras/ERK1/2、c-myc/AKT和Wnt/β-catenin)来调节白血病细胞甚至是白血病干／祖细胞的生存和增殖。
Leukemia is a common hematopoietic tumor of unknown etiology, which is estimated to affect250,000people worldwide. Unfortunately, the majority of leukemia patients will die due to drug resistance or relapse. Currently, the uncertain etiology and molecular mechanisms greatly hamper development of efficient drugs and regimens for curing this malignant disease. Among all the studies on etiology, like virus, ionizing radiation, chemicals and genetic factors, viral etiology has been long considered as the most significant and attractive topic. It has been demonstrated that the retroviruses have been etiologically linked to a number of animal leukemias, and a rare type of human leukemia:human adult T cell leukemia/lymphoma. However, whether retroviruses have been etiologically linked to human common leukemia such as acute and chronic myeloid leukemia, acute and chronic lymphoblastic leukemia remain elusive.
Although the majority of HERVs (human endogenous retrovirus sequences, HERVs) are dysfunctional due to the accumulation of multiple nonsense mutations, some are still active and may have a potential function, notably the human endogenous retrovirus type K (HERV-K) family. The viral Np9transcript, a small regulatory gene encoded by HERV-K type I, has been shown to be exclusively present in tumors and transformed cells. Our previous studies have demonstrated the presence of retrovirus particles in primary leukemia cells from patients with acute myeloid leukemia (AML). In addition, the viral Tax protein of human T-Cell leukemia virus type1(HTLV-1) has been implicated in leukemogenesis. However, Np9protein expression and its possible role in human common leukemia have not been studied. The aim of this study was to investigate whether the Np9protein is expressed in human leukemia cells and whether the viral Np9plays a role in cell growth of human common leukemia including myeloid and lymphoblastic leukemia. If so, we then further assess possible molecular mechanism by which Np9promotes the growth of leukemia cells. The results of this study may provide a potential novel therapeutic target and opens new perspectives to unravel the etiology of human leukemia.
Part Ⅰ The expression of Np9protein in human leukemias
We examined the expression of Np9in14various human hematopoietic malignant cell lines,16different solid tumor cell lines, primary leukemia cells from a cohort of50patients with various leukemia, and normal hematopoietic cell samples, including CD34+hematopoietic stem cells (HSCs), from22healthy adult donors.78.57%hematopoietic tumor cell lines showed high levels of Np9protein, including3chronic myeloid leukemia (CML)(K562, K562/adr, KCL-22M),3acute myeloid leukemia (AML)(HL-60, NB4, Kasumi-1),3acute lymphoblastic leukemia (ALL)(Jurkat, Molt-4,119), and2multiple myeloma (MM)(8226, KM3).18.75%tumor cell lines:U87(glioblastoma), A549(lung cancer), and PANC-1(pancreatic adenocarcinoma) also showed high levels of Np9protein. Significantly,56%primary leukemia samples showed expression of Np9protein. In contrast, Np9protein was rarely detected in normal hematopoietic cells from healthy individuals. These results indicate that Np9protein expression is universal in both human leukemia cell lines and primary leukemia samples, but rarely in normal blood cells.
We next examined the Np9protein levels in CD34+leukemia stem/progenitor cells in primary leukemia samples. Np9protein levels were positively correlated with CD34+leukemia stem/progenitor cells (P=0.003).
We next asked whether Np9-positive leukemia samples also express pol-env polyprotein, env and transmembrane proteins using a specific antibody against a defined domain of Env, the transmembrane (TM) domain. Leukemia cell lines also expressed leukemia-specific pol-env polyprotein, env protein and transmembrane proteins (TM). In contrast, these leukemia-specific viral proteins were not detected in normal blood cells from12normal individuals.
We then used real-time PCR to test whether Np9transcription could be up-regulated in K562by effect of different factors such as hormone, alcohol, demethylating substance and retroviral inducers. Dexamethasone,alcohol and IdUR up-regulated the expression of Np9as the concentration increased.5-Aza did not affect the transcription levels.
Part Ⅱ
Np9is essential for survival and growth of leukemia cells in vitro and in vivo
To determine whether Np9is essential for the survival of leukemia cells, we suppressed Np9expression by using shRNAs against the viral Np9gene. Recombinant lentiviruses transcribing short hairpin RNAs against Np9were generated and transduced to Np9protein-expressing human myeloid leukemia K562and lymphoblastic leukemia Jurkat cells. Proliferation of leukemia cells were measured by MTS assay. The results showed a marked growth inhibition of both K562and Jurkat cells by Np9-specific shRNAs.
To assess whether Np9promotes the growth of leukemia cells, we generated recombinant lentiviruses expressing Np9and transduced to Np9-negative human leukemia Raji cells and determined growth curves of Raji cells with over-expression of Np9and found that Np9significantly promoted the growth of Raji cells.
We next examined the effects of Np9expression on the colony-forming ability of tumor cells using colony forming assay. In contrast to control, Np9over-expression induced an increase of in the number and size of colonies.
We also evaluated the subcellular localization of the Np9protein, and found that viral Np9protein was predominantly localized in the nucleus of leukemia cells, but a small amount of viral Np9protein was also observed in the cytoplasm of tumor cells.
To confirm whether Np9promotes the growth of leukemia cells in vivo, Raji cells with Np9over-expression or control cells were injected subcutaneously into immunocompromized NOD-SCID mice following standard protocols. We observed that tumors in mice (n=6) that received cells with Np9grew much faster and tumor weight was1.74g±0.42g within25days. In contrast, tumors in mice that received empty vector as negative control grew slowly and tumor weight was0.62g±0.18g within25days. These data clearly indicate that Np9expression strongly supports and promotes the growth of leukemia cells in NOD-SCID mice.
Part Ⅲ The molecular mechanism by which Np9promotes the growth of leukemia cells
We examined whether Np9expression co-activates Wnt/β-catenin, Notch1, ras/ERK and c-myc/AKT signaling pathways, which are aberrantly activated in human leukemia, in4different leukemia cell lines (K562, Kasumi-1, NB4and Jurkat) with high levels of Np9expression. The results showed that these leukemia cell lines had a co-activation of multiple leukemia-associated signaling pathways. In contrast to leukemia cells, no obvious activation of these leukemia-related signaling pathways was observed in normal blood cells from healthy donors. To address whether Np9affects these signaling components, we transfected Np9-negative Raji cells using Np9expression plasmid and observed that over-expression of Np9led to a marked up-regulation of β-catenin, phospho-ERK (pERK), c-myc and cleaved Notch1with a concomitant decrease of Numb. To validate these results, we then silenced Np9gene expression in K562and Jurkat leukemia cells using shRNAs. The results revealed that Np9silencing caused significant decreases of expression levels of c-myc, pERKl, phospho-Akt (pAkt) and cleaved Notchl with a concomitant increase of Numb.
To characterize Np9protein sequences, we found that there were five intact ORFs for Np9protein. Alignment analysis showed that amino acid sequences were highly similar. We identified a Grb2SH3(N)-typical motif (PxxPxR) among these deduced Np9proteins which was absolutely conserved throughout all Np9proteins indicating a potential role in Np9-mediated leukemogenesis.
HERV-K是人内源性逆转录病毒家族成员中最可能具有生物学活性和致病性的一员。近年来陆续有研究报道HERV-K家族成员在许多人肿瘤中被再次激活。甚至有个别研究者在肿瘤病人中发现了HERV-K的病毒颗粒和抗HERV-K的抗体。人内源性逆转录病毒基因Np9是Ⅰ型HERV-Kenv基因编码和剪辑的产物。研究证明,Np9的转录本只在肿瘤细胞和转化的细胞中表达,而在正常的非转化的细胞中不表达。这高度提示着Np9转录的激活与肿瘤的形成密切相关。然而,现有的研究尚不明确Np9蛋白在肿瘤中的表达情况及功能。我们的前期研究已经证明,在急性髓细胞白血病病人的原代白血病细胞中存在典型的逆转录病毒颗粒。并且,现有的研究证实由Ⅰ型人T细胞白血病病毒编码的Tax蛋白与该病的发病相关。这为我们探究人内源性逆转录病毒HERV-K Np9蛋白在人白血病中的表达及功能提供了思路和基础。本论文将通过以下三部分重点探讨Np9蛋白在人白血病细胞系、病人中的表达情况、功能及具体的分子机制。这对揭示白血病病因和深入理解发病机制有重要意义,并有可能为人白血病的诊断和防治带来新的思路。
第一部分
Np9蛋白在人白血病中的表达研究
本部分旨在明确Np9蛋白在人白血病细胞系、白血病病人原代标本以及正常成年人中的表达情况。并在此基础上进一步了解与Np9相关的同由Ⅰ型HERV-Kenv基因编码的pol-env、env和transmembrane蛋白的表达情况。最后还将探讨激素、酒精、DNA去甲基化剂和病毒诱导剂等因素对诱导白血病细胞中Np9激活的影响。
我们收集了14种人恶性血液肿瘤细胞系和16种实体瘤细胞系,通过Western blot及抗Np9的特异性抗体检测各细胞中Np9蛋白的表达情况。结果显示：78.57%的人恶性血液肿瘤细胞系高表达Np9蛋白(11/14)。其中包括3种慢性髓细胞白血病细胞系：K562, K562/adr, KCL-22M;3种急性髓细胞白血病细胞系：HL-60, NB4, Kasumi-1;3种急性淋巴细胞白血病细胞系：Jurkat, Molt-4, H9;2种多发性骨髓瘤细胞系：8226,KM3。18.75%实体瘤细胞系(3/16)。其中包括：人恶性胶质瘤细胞系：U87；人肺癌细胞系：A549；人胰腺癌细胞系：PANC-1。
我们接着收集了50例白血病病人及22例正常成年人的外周血标本,通过淋巴细胞分离液分离单个核细胞,并用Western blot检测了Np9蛋白的表达情况。结果显示：56%原代白血病病人标本表达Np9蛋白(28/50)。相比之下,4.5%正常成年人弱表达Np9蛋白(1/22)。
综上结果：Np9蛋白在人白血病细胞系和白血病病人的原代细胞中普遍表达,而在正常人的血细胞中几乎不表达。
我们结合上一部分的实验结果及病人临床资料,对Np9蛋白水平与CD34+白血病干/祖细胞的相关性进行统计分析。结果发现,Np9蛋白水平可能与CD34+白血病干/组细胞正相关(P=0.003)。
我们进一步选择Np9蛋白阳性的白血病标本并应用Western blot技术检测与Np9相关的同由Ⅰ型HERV-Kenv基因编码的其它逆转录病毒蛋白pol-env、env和transmembrane蛋白。结果显示,Np9蛋白阳性的样本高表达白血病特异的pol-env、 env和transmembrane蛋白,而正常成年人外周血分离的单个核细胞却未见表达。
接着我们通过实时定量PCR技术测定了激素、酒精、DNA去甲基化剂和病毒诱导剂等因素对白血病细胞中Np9转录的影响。结果显示,随着Dexamethasone、酒精、IdUR浓度的增高,Np9的转录水平逐渐提高；5-Aza浓度的变化未见明显影响。
第二部分
Np9蛋白在体内和体外对白血病细胞生存与增殖的影响
本部分研究通过体外细胞系实验和体内动物模型的建立,旨在揭示Np9蛋白对白血病细胞生存和增殖的影响,明确其是否是白血病细胞生存和增殖的必须因素。
应用shRNA和慢病毒感染技术稳定下调了人慢性粒细胞白血病细胞系K562和人急性淋巴细胞白血病细胞系Jurkat中Np9蛋白的表达水平,并随后用MTS实验检测细胞增殖能力的变化。结果发现,两种细胞的生长都受到了明显的抑制。
相应地,利用慢病毒感染技术在Np9蛋白阴性的人B淋巴细胞白血病细胞系Raji稳定地过表达Np9蛋白,通过测定和绘制细胞的生长曲线发现过表达Np9蛋白的Raji细胞的增殖能力要明显高于对照组。
为了进一步评估Np9的表达对白血病细胞克隆形成能力的影响,应用上一步已建立的Raji-Np9和Raji-Ctr这两种细胞的克隆形成实验来观察所形成的克隆数量及大小的差异。结果显示,上调Np9蛋白的表达水平能显著增加Raji细胞的克隆形成数量和大小。
接着通过脂质体转染技术将p-EGFP-C1-Np9和p-EGFP-C1瞬时转入人慢性粒细胞白血病细胞系K562。应用激光共聚焦显微镜观察Np9蛋白的亚细胞定位,发现该蛋白主要分布细胞核内,但是仍有一小部分分散在胞浆。
为了明确Np9蛋白能否在体内促进白血病细胞的生长,我们将过表达Np9蛋白的Raji细胞经皮下注射到严重联合免疫缺陷的NOD-SCID鼠中,同时用转染空载体的Raji细胞作为阴性对照,Rec蛋白作为阳性对照,发现由Raji-Np9所形成的肿瘤生长速度要显著高于阴性对照组(*P<0.01)。25天以后,瘤重约为1.74g±0.42g,而相应的阴性对照组为0.62g±0.18g(*P<0.01)。实验组的瘤重约为阴性对照组的2.81倍。Real-time PCR分析结果显示,Np9蛋白在相应的细胞系和移植瘤中高表达。
综上结果我们认为Np9蛋白是白血病细胞生存的必须因素,并且能够显著促进白血病细胞的复制、增殖和产生子代的能力。
第三部分
Np9蛋白促进白血病细胞增殖的分子机制研究
本部分研究重点对白血病中常异常激活的Numb/Notch、Ras/ERK1/2、 c-myc/AKT和Wnt/β-catenin信号通路与Np9蛋白表达的相关性做出分析,并且结合Np9蛋白的氨基酸序列特征分析和功能域预测,以揭示Np9蛋白促进白血病细胞增殖的分子机制。
我们首先选取了高表达Np9蛋白的四种白血病细胞：K562、Kasumi、NB4和Jurkat,并同时通过Western blot技术检测了Numb/Notch、Ras/ERK1/2、c-myc/AKT和Wnt/β-catenin信号通路。结果显示,上述四条白血病相关的信号通路能够被同时激活。Np9的表达与共激活β-catenin、ERK、AKT和Notch1可能相关。
接着我们将正常成年人的外周血单个核细胞用上述同样地方法分析了Np9、 Numb、Cleaved Notchl、Grb2、Ras、ERK、pERK、c-myc、PLZF、pAKT和β-catenin等分子,发现在正常成年人的外周血单个核细胞不表达Np9,并且不能观察到白血病相关信号通路的激活。
同样地,我们在高表达Np9蛋白的白血病病人的原代标本中也观察到了β-catenin、cleaved Notch1、ERK和c-myc的共激活。
为了进一步明确是否是Np9的表达影响了上述关键信号分子的改变,我们建立了高表达Np9蛋白的Raji和低表达Np9蛋白的K562和Jurkat细胞系。利用Western blot技术对Np9蛋白和Numb/Notch、Ras/ERK1/2、c-myc/AKT和Wnt/β-catenin信号通路进行分析。结果发现,过表达Np9能显著上调β-catenin、 pERK、c-myc和Cleaved Notch1。而与此相应的是,Numb的表达水平降低(Numb和Cleaved Notchl互为抑制)。低表达Np9能显著下调了c-myc、pERK1、pAKT和Cleaved Notchl,而Numb的表达水平升高。
通过GeneBank和相关软件对Np9蛋白的氨基酸序列特征进行分析和功能域预测。我们在Np9蛋白的氨基酸序列中鉴定到典型的Grb2SH3(N)-typical motif (PxxPxR)。这与我们在本部分的实验结果中观察到了Np9蛋白的表达能直接影响phospho-ERK的水平相一致。
综上述三部分的实验结果,我们认为Np9蛋白在人白血病细胞系和白血病病人的原代细胞中普遍表达,而在正常人的血细胞中几乎不表达。Np9蛋白水平可能与CD34+白血病干／组细胞正相关。此外,Np9蛋白是白血病细胞生存和增殖的必须因素。该蛋白通过同时或者相继激活多条白血病相关的信号通路(Numb/Notch. Ras/ERK1/2、c-myc/AKT和Wnt/β-catenin)来调节白血病细胞甚至是白血病干／祖细胞的生存和增殖。
Leukemia is a common hematopoietic tumor of unknown etiology, which is estimated to affect250,000people worldwide. Unfortunately, the majority of leukemia patients will die due to drug resistance or relapse. Currently, the uncertain etiology and molecular mechanisms greatly hamper development of efficient drugs and regimens for curing this malignant disease. Among all the studies on etiology, like virus, ionizing radiation, chemicals and genetic factors, viral etiology has been long considered as the most significant and attractive topic. It has been demonstrated that the retroviruses have been etiologically linked to a number of animal leukemias, and a rare type of human leukemia:human adult T cell leukemia/lymphoma. However, whether retroviruses have been etiologically linked to human common leukemia such as acute and chronic myeloid leukemia, acute and chronic lymphoblastic leukemia remain elusive.
Although the majority of HERVs (human endogenous retrovirus sequences, HERVs) are dysfunctional due to the accumulation of multiple nonsense mutations, some are still active and may have a potential function, notably the human endogenous retrovirus type K (HERV-K) family. The viral Np9transcript, a small regulatory gene encoded by HERV-K type I, has been shown to be exclusively present in tumors and transformed cells. Our previous studies have demonstrated the presence of retrovirus particles in primary leukemia cells from patients with acute myeloid leukemia (AML). In addition, the viral Tax protein of human T-Cell leukemia virus type1(HTLV-1) has been implicated in leukemogenesis. However, Np9protein expression and its possible role in human common leukemia have not been studied. The aim of this study was to investigate whether the Np9protein is expressed in human leukemia cells and whether the viral Np9plays a role in cell growth of human common leukemia including myeloid and lymphoblastic leukemia. If so, we then further assess possible molecular mechanism by which Np9promotes the growth of leukemia cells. The results of this study may provide a potential novel therapeutic target and opens new perspectives to unravel the etiology of human leukemia.
Part Ⅰ The expression of Np9protein in human leukemias
We examined the expression of Np9in14various human hematopoietic malignant cell lines,16different solid tumor cell lines, primary leukemia cells from a cohort of50patients with various leukemia, and normal hematopoietic cell samples, including CD34+hematopoietic stem cells (HSCs), from22healthy adult donors.78.57%hematopoietic tumor cell lines showed high levels of Np9protein, including3chronic myeloid leukemia (CML)(K562, K562/adr, KCL-22M),3acute myeloid leukemia (AML)(HL-60, NB4, Kasumi-1),3acute lymphoblastic leukemia (ALL)(Jurkat, Molt-4,119), and2multiple myeloma (MM)(8226, KM3).18.75%tumor cell lines:U87(glioblastoma), A549(lung cancer), and PANC-1(pancreatic adenocarcinoma) also showed high levels of Np9protein. Significantly,56%primary leukemia samples showed expression of Np9protein. In contrast, Np9protein was rarely detected in normal hematopoietic cells from healthy individuals. These results indicate that Np9protein expression is universal in both human leukemia cell lines and primary leukemia samples, but rarely in normal blood cells.
We next examined the Np9protein levels in CD34+leukemia stem/progenitor cells in primary leukemia samples. Np9protein levels were positively correlated with CD34+leukemia stem/progenitor cells (P=0.003).
We next asked whether Np9-positive leukemia samples also express pol-env polyprotein, env and transmembrane proteins using a specific antibody against a defined domain of Env, the transmembrane (TM) domain. Leukemia cell lines also expressed leukemia-specific pol-env polyprotein, env protein and transmembrane proteins (TM). In contrast, these leukemia-specific viral proteins were not detected in normal blood cells from12normal individuals.
We then used real-time PCR to test whether Np9transcription could be up-regulated in K562by effect of different factors such as hormone, alcohol, demethylating substance and retroviral inducers. Dexamethasone,alcohol and IdUR up-regulated the expression of Np9as the concentration increased.5-Aza did not affect the transcription levels.
Part Ⅱ
Np9is essential for survival and growth of leukemia cells in vitro and in vivo
To determine whether Np9is essential for the survival of leukemia cells, we suppressed Np9expression by using shRNAs against the viral Np9gene. Recombinant lentiviruses transcribing short hairpin RNAs against Np9were generated and transduced to Np9protein-expressing human myeloid leukemia K562and lymphoblastic leukemia Jurkat cells. Proliferation of leukemia cells were measured by MTS assay. The results showed a marked growth inhibition of both K562and Jurkat cells by Np9-specific shRNAs.
To assess whether Np9promotes the growth of leukemia cells, we generated recombinant lentiviruses expressing Np9and transduced to Np9-negative human leukemia Raji cells and determined growth curves of Raji cells with over-expression of Np9and found that Np9significantly promoted the growth of Raji cells.
We next examined the effects of Np9expression on the colony-forming ability of tumor cells using colony forming assay. In contrast to control, Np9over-expression induced an increase of in the number and size of colonies.
We also evaluated the subcellular localization of the Np9protein, and found that viral Np9protein was predominantly localized in the nucleus of leukemia cells, but a small amount of viral Np9protein was also observed in the cytoplasm of tumor cells.
To confirm whether Np9promotes the growth of leukemia cells in vivo, Raji cells with Np9over-expression or control cells were injected subcutaneously into immunocompromized NOD-SCID mice following standard protocols. We observed that tumors in mice (n=6) that received cells with Np9grew much faster and tumor weight was1.74g±0.42g within25days. In contrast, tumors in mice that received empty vector as negative control grew slowly and tumor weight was0.62g±0.18g within25days. These data clearly indicate that Np9expression strongly supports and promotes the growth of leukemia cells in NOD-SCID mice.
Part Ⅲ The molecular mechanism by which Np9promotes the growth of leukemia cells
We examined whether Np9expression co-activates Wnt/β-catenin, Notch1, ras/ERK and c-myc/AKT signaling pathways, which are aberrantly activated in human leukemia, in4different leukemia cell lines (K562, Kasumi-1, NB4and Jurkat) with high levels of Np9expression. The results showed that these leukemia cell lines had a co-activation of multiple leukemia-associated signaling pathways. In contrast to leukemia cells, no obvious activation of these leukemia-related signaling pathways was observed in normal blood cells from healthy donors. To address whether Np9affects these signaling components, we transfected Np9-negative Raji cells using Np9expression plasmid and observed that over-expression of Np9led to a marked up-regulation of β-catenin, phospho-ERK (pERK), c-myc and cleaved Notch1with a concomitant decrease of Numb. To validate these results, we then silenced Np9gene expression in K562and Jurkat leukemia cells using shRNAs. The results revealed that Np9silencing caused significant decreases of expression levels of c-myc, pERKl, phospho-Akt (pAkt) and cleaved Notchl with a concomitant increase of Numb.
To characterize Np9protein sequences, we found that there were five intact ORFs for Np9protein. Alignment analysis showed that amino acid sequences were highly similar. We identified a Grb2SH3(N)-typical motif (PxxPxR) among these deduced Np9proteins which was absolutely conserved throughout all Np9proteins indicating a potential role in Np9-mediated leukemogenesis.
引文
1. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA.1980; 77:7415-7419.
2. Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuli Y, Shiraishi Y, et al. Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature.1981; 294:770-771.
3.郑葆芬白血病病毒、艾滋病病毒、癌基因.上海医科大学出版.1996；P1-2,55-59.
4. Lander ES, Linton LM, Birren B, Nubaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature.2001; 409:860-921.
5. Costas J. Characterization of the intragenomic spread of the human endogenous retrovirus family HERV-W. Mol Biol Evol.2002; 19:526-533.
6. Hughes JF, Coffin JM. Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution. Nat Genet.2001; 29:487-489.
7. Belshaw R, Kaltaourakis A, Paces J,et al. High copy number in human endogenous retrovirus families is associated with copying mechanism in addition to reinfection. Mol Biol Evol.2005,22:814-817
8. Dewannieux M, Harper F, Richaud A, Letzelter C, Ribet D, Pierron G, et al. Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements. Genome Res.2006; 16:1548-1556.
9. Tonjes RR, Lower R, Boller K, Denner J, Hasenmaier B, Kirsch H, et al. HERV-K: the biologically most active human endogenous retrovirus family. Acquir. Immune Defic. Syndr. Hum. Retrovirol.1996; 13:S261-267.
10. Kraus B, Boller K, Reuter A, Schnierle BS. Characterization of the human endogenous retrovirus K Gag protein:identification of protease cleavage sites. Retrovirology.2011; 8:21.
11. Barbulescu M, Turner G, Seaman MI, Deinard AS, Kidd KK, Lenz J. Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans. Curr. Biol. 1999; 9:861-868.
12. Hanke K, Kramer P, Seeher S, Beimforde N, Kurth R, Bannert N. Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain. J. Virol.2009; 83:12790-12800.
13. Boller K, Schonfeld K, Lischer S, Fischer N, Hofmann A, Kurth R, et al. Human endogenous retrovirus HERV-K113 is capable of producing intact viral particles. J. Gen. Virol.2008; 89:567-72.
14. Lee YN, Bieniasz PD. Reconstitution of an infectious human endogenous retrovirus. PLoS Pathog.2007; 3:e10.
15. Contreras-Galindo R, Almodovar-Camacho S, Gonzalez-Ramirez S, Lorenzo E, Yamamura Y. Comparative longitudinal studies of HERV-K and HIV-1 RNA titers in HIV-1-infected patients receiving successful versus unsuccessful highly active antiretroviral therapy. AIDS Res Hum Retroviruses.2007; 23:1083-1086.
16. Depil S, Roche C, Dussart P, Prin L. Expression of a human endogenous retrovirus, HERV-K, in the blood cells of leukemia patients. Leukemia 2002; 16:254-259.
17. Contreras-Galindo R, Kaplan MH, Leissner P, Verjat T, Ferlenghi I, Bagnoli F, et al. Human endogenous retrovirus K (HML-2) elements in the plasma of people with lymphoma and breast cancer. J. Virol.2008; 82:9329-9336.
18. Armbruester V, Sauter M, Krautkraemer E, Meese E, Kleiman A, Best B, et al. A novel gene from the human endogenous retrovirus K expressed in transformed cells. Clin. Cancer Res.2002; 8:1800-1807.
19. Wang-Johanning F, Rycaj K, Plummer JB, Li M, Yin B, Frerich K, et al. Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors. J Natl Cancer Inst.2012; 104:189-210.
20. Zhao J, Rycaj K, Geng S, Li M, Plummer JB, Yin B, Liu H, Xu X, Zhang Y, Yan Y et al:Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer. Genes Cancer 2011;2:914-922.
21. Buscher K, Trefzer U, Hofmann M, Sterry W, Kurth R, Denner J. Expression of human endogenous retrovirus K in melanomas and melanoma cell lines. Cancer Res.2005; 65:4172-4180.
22. Ishida T, Obata Y, Ohara N, Matsushita H, Sato S, Uenaka A, Saika T, Miyamura T, Chayama K, Nakamura Y et al: Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity. Cancer Immun 2008; 8:15
23. Goering W, Ribarska T, Schulz WA:Selective changes of retroelement expression in human prostate cancer. Carcinogenesis 2011,32(10):1484-1492.
24. Ruprecht K, Ferreira H, Flockerzi A, Wahl S, Sauter M, Mayer J, Mueller-Lantzsch N:Human endogenous retrovirus family HERV-K(HML-2) RNA transcripts are selectively packaged into retroviral particles produced by the human germ cell tumor line Tera-1 and originate mainly from a pro virus on chromosome 22q 11.21. J Virol 2008,82(20):10008-10016.
25. Lower R, Tonjes RR, Korbmacher C, Kurth R, Lower J:Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K. J Virol 1995;69:141-149.
26. Denne M, Sauter M, Armbruester V, Licht JD, Roemer K, Mueller-Lantzsch N: Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein. J Virol 2007; 81:5607-5616.
27. Xu R, Gao Q, Wang S, Kan H, Sheng L, Li C, et al. Human acute myeloid leukemias may be etiologically associated with new human retroviral infection. Leuk. Res.1996; 20:449-455.
28. Oteiza A, Mechti N. The human T-cell leukemia virus type 1 oncoprotein tax controls forkhead box 04 activity through degradation by the proteasome. J Virol. 2011; 85:6480-6491.
29. Gandillet A, Park S, Lassailly F, Griessinger E, Vargaftig J, Filby A, et al. Heterogeneous sensitivity of human acute myeloid leukemia to β-catenin down-modulation. Leukemia 2011; 25:770-780.
30. Yeung J, Esposito MT, Gandillet A, Zeisig BB, Griessinger E, Bonnet D, et al. β-Catenin mediates the establishment and drug resistance of MLL leukemic stem cells. Cancer Cell.2010; 18:606-618.
31. Golan M, Hizi A, Resau JH, Yaal-Hahoshen N, Reichman H, Keydar I, Tsarfaty I. Human endogenous retrovirus (HERV-K) reverse transcriptase as a breast cancer prognostic marker. Neoplasia.2008;10(6):521-33.
32.徐荣臻.人白血病相关逆转录病毒的克隆及鉴定.浙江大学.2001
33. Boese A, Sauter M, Galli U, Best B, Herbst H, Mayer J, et al. Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein. Oncogene.2000; 19:4328-4336.
34. Currer R, Van Duyne R, Jaworski E, Guendel I, Sampey G, Das R, Narayanan A, Kashanchi F. HTLV tax:a fascinating multifunctional co-regulator of viral and cellular pathways.Front Microbiol.2012; 3:406.
35. Magin C, Lower R, Lower J. cORF and RcRE, the Rev/Rex and RRE/RxRE homologues of the human endogenous retrovirus family HTDV/HERV-K. J Virol 1999; 73:9496-507.
36. Galli UM, Sauter M, Lecher B, et al. Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors. Oncogene 2005; 24:3223-8.
37.汤钊猷.现代肿瘤学.复旦大学出版社.2011
38. Stommel JM, Kimmelman AC, Ting H, Nabioullin R, Ponugoti AH, Wiedemeyer R, et al. Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science.2007; 318:287-290.
39. Torok S, Dome B.Possibilities for inhibiting tumor-induced angiogenesis:results with multi-target tyrosine kinase inhibitors. Magy Onkol.2012; 56:3-15.
40. Guruswamy S, Rao CV.Multi-Target Approaches in Colon Cancer Chemoprevention Based on Systems Biology of Tumor Cell-Signaling. Gene Regul Syst Bio.2008; 2:163-176.
41.王韫秀,张继红,顾兆伟.Wnt/β-catenin信号通路与恶性血液病.中国实验血液学杂志.2009；17：234-237
42.赵渊源,李春艳.Wnt与Notch信号通路的串话与肿瘤发生、发展的关系.国际病理科学与临床杂志.2010；30：255-259
43. Tatarek J, Cullion K, Ashworth T, Gerstein R, Aster JC, Kelliher MA. Notchl inhibition targets the leukemia-initiating cells in a Tall/Lmo2 mouse model of T-ALL. Blood.2011; 118:1579-1590.
44. Pece S, Confalonieri S, R Romano P, Di Fiore PP.NUMB-ing down cancer by more than just a NOTCH.Biochim Biophys Acta.2011; 1815:26-43.
45. Ito T, Kwon HY, Zimdahl B, Congdon KL, Blum J, Lento WE, et al. Regulation of myeloid leukaemia by the cell-fate determinant Musashi. Nature.2010; 466:765-768.
46. Wang J, Liu Y, Li Z, Du J, Ryu MJ, Taylor PR, et al. Endogenous oncogenic Nras mutation promotes aberrant GM-CSF signaling in granulocytic/monocytic precursors in a murine model of chronic myelomonocytic leukemia. Blood.2010; 116:5991-6002.
47. Steelman LS, Franklin RA, Abrams SL, Chappell W, Kempf CR, Basecke J, et al. Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy. Leukemia.2011; 25:1080-1094.
48. Gutierrez A, Grebliunaite R, Feng H, Kozakewich E, Zhu S, Guo F, et al. Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia. J. Exp. Med.2011; 208:1595-1603.
49. Polak R, Buitenhuis M. The PI3K/PKB signaling module as key regulator of hematopoiesis:implications for therapeutic strategies in leukemia. Blood.2011; 199:911-923.
50. Sykes SM, Lane SW, Bullinger L, Kalaitzidis D, Yusuf R, Saez B, et al. AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell.2011; 146:697-708.
51. Hu Y, Chen Y, Douglas L, Li S. Beta-Catenin is essential for survival of leukemic stem cells insensitive to kinase inhibition in mice with BCR-ABL-induced chronic myeloid leukemia. Leukemia.2009; 23:109-116.
52. Takebe N, Harris PJ, Warren RQ, Ivy SP.Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways.Nat Rev Clin Oncol.2011; 8:97-106.
53. Guo S, Liu M, Gonzalez-Perez RR.Role of Notch and its oncogenic signaling crosstalk in breast cancer.Biochim Biophys Acta.2011; 1815:197-213.
54. Wang Z, Ahmad A, Li Y, Azmi AS, Miele L, Sarkar FH.Targeting notch to eradicate pancreatic cancer stem cells for cancer therapy.Anticancer Res.2011; 31:1105-13.
55. Guilmeau S.Notch signaling and intestinal cancer.Adv Exp Med Biol.2012; 727:272-88.
56. Armbruester V, Sauter M, Roemer K, Best B, Hahn S, Nty A, et al. Np9 protein of human endogenous retrovirus K interacts with ligand of numb protein X. J. Virol. 2004; 78:10310-10319.
57. Nie J, McGill MA, Dermer M, Dho SE, Wolting CD, McGlade CJ. LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation. EMBO. J.2002; 21:93-102.
58. Denne M, Sauter M, Armbruester V, Licht JD, Roemer K, Mueller-Lantzsch N. Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein. J. Virol.2007; 81: 5607-5616.
59. Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, Wetzker R. Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase gamma.Science.1997; 275:394-397.
60. Modi H, Li L, Chu S, Rossi J, Yee JK, Bhatia R. Inhibition of Grb2 expression demonstrates an important role in BCR-ABL-mediated MAPK activation and transformation of primary human hematopoietic cells.Leukemia.2010; 25:305-312.
1. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W et al: Initial sequencing and analysis of the human genome. Nature 2001,409(6822):860-921.
2. Costas J:Characterization of the intragenomic spread of the human endogenous retrovirus family HERV-W. Mol Biol Evol 2002,19(4):526-533.
3. Hughes JF, Coffin JM:Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution. Nat Genet 2001, 29(4):487-489.
4. Lower R, Lower J, Kurth R:The viruses in all of us:characteristics and biological significance of human endogenous retrovirus sequences. Proc Nat1 Acad Sci USA 1996,93(11):5177-5184.
5. Bannert N, Kurth R:Retroelements and the human genome:new perspectives on an old relation. Proc Natl Acad Sci USA 2004,101 Suppl 2:14572-14579.
6. Singh S, Kaye S, Gore ME, McClure MO, Bunker CB:The role of human endogenous retroviruses in melanoma. Br J Dermatol 2009,161(6):1225-1231.
7. Blikstad V, Benachenhou F, Sperber GO, Blomberg J:Evolution of human endogenous retroviral sequences:a conceptual account. Cell Mol Life Sci 2008, 65(21):3348-3365.
8. Dewannieux M, Harper F, Richaud A, Letzelter C, Ribet D, Pierron G, Heidmann T:Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements. Genome Res 2006, 16(12):1548-1556.
9. Tonjes RR, Lower R, Boller K, Denner J, Hasenmaier B, Kirsch H, Konig H, Korbmacher C, Limbach C, Lugert R et al. HERV-K:the biologically most active human endogenous retrovirus family. J Acquir Immune Defic Syndr Hum Retrovirol 1996,13 Suppl 1:S261-267.
10. Kraus B, Boller K, Reuter A, Schnierle BS:Characterization of the human endogenous retrovirus K Gag protein:identification of protease cleavage sites. Retrovirology 2011,8:21.
11. Barbulescu M, Turner G, Seaman MI, Deinard AS, Kidd KK, Lenz J:Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans. Curr Biol 1999,9(16):861-868.
12. Hanke K, Kramer P, Seeher S, Beimforde N, Kurth R, Bannert N:Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain. J Virol 2009, 83(24):12790-12800.
13. Boller K, Schonfeld K, Lischer S, Fischer N, Hoffmann A, Kurth R, Tonjes RR: Human endogenous retrovirus HERV-K113 is capable of producing intact viral particles. J Gen Virol 2008,89(Pt 2):567-572.
14. Lee YN, Bieniasz PD:Reconstitution of an infectious human endogenous retrovirus. PLoS Pathog 2007,3(1):e10.
15. Lower R, Tonjes RR, Korbmacher C, Kurth R, Lower J:Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K. J Virol 1995,69(1):141-149.
16. Armbruester V, Sauter M, Krautkraemer E, Meese E, Kleiman A, Best B, Roemer K, Mueller-Lantzsch N:A novel gene from the human endogenous retrovirus K expressed in transformed cells. Clin Cancer Res 2002, 8(6):1800-1807.
17. Boese A, Sauter M, Galli U, Best B, Herbst H, Mayer J, Kremmer E, Roemer K, Mueller-Lantzsch N:Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein. Oncogene 2000,19(38):4328-4336.
18. Denne M, Sauter M, Armbruester V, Licht JD, Roemer K, Mueller-Lantzsch N: Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein. J Virol 2007, 81(11):5607-5616.
19. Depil S, Roche C, Dussart P, Prin L:Expression of a human endogenous retrovirus, HERV-K, in the blood cells of leukemia patients. Leukemia 2002, 16(2):254-259.
20. Contreras-Galindo R, Kaplan MH, Leissner P, Verjat T, Ferlenghi I, Bagnoli F, Giusti F, Dosik MH, Hayes DF, Gitlin SD et al. Human endogenous retrovirus K (HML-2) elements in the plasma of people with lymphoma and breast cancer. J Virol 2008,82(19):9329-9336.
21. Wang-Johanning F, Rycaj K, Plummer JB, Li M, Yin B, Frerich K, Garza JG, Shen J, Lin K, Yan P et al. Immunotherapeutic potential of anti-human endogenous retrovirus-K envelope protein antibodies in targeting breast tumors. JNatl Cancer Inst 2012,104(3):189-210.
22. Zhao J, Rycaj K, Geng S, Li M, Plummer JB, Yin B, Liu H, Xu X, Zhang Y, Yan Y et al. Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer. Genes Cancer 2011,2(9):914-922.
23. Buscher K, Trefzer U, Hofmann M, Sterry W, Kurth R, Denner J:Expression of human endogenous retrovirus K in melanomas and melanoma cell lines. Cancer Res 2005,65(10):4172-4180.
24. Schanab O, Humer J, Gleiss A, Mikula M, Sturlan S, Grunt S, Okamoto I, Muster T, Pehamberger H, Waltenberger A:Expression of human endogenous retrovirus K is stimulated by ultraviolet radiation in melanoma. Pigment Cell Melanoma Res 2011,24(4):656-665.
25. Li Z, Sheng T, Wan X, Liu T, Wu H, Dong J:Expression of HERV-K correlates with status of MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells. Cancer Invest 2010,28(10):1031-1037.
26. Ishida T, Obata Y, Ohara N, Matsushita H, Sato S, Uenaka A, Saika T, Miyamura T, Chayama K, Nakamura Y et al. Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity. Cancer Immun 2008,8:15.
27. Goering W, Ribarska T, Schulz WA:Selective changes of retroelement expression in human prostate cancer. Carcinogenesis 2011,32(10):1484-1492.
28. Ruprecht K, Ferreira H, Flockerzi A, Wahl S, Sauter M, Mayer J, Mueller-Lantzsch N:Human endogenous retrovirus family HERV-K(HML-2) RNA transcripts are selectively packaged into retroviral particles produced by the human germ cell tumor line Tera-1 and originate mainly from a pro virus on chromosome 22q11.21. J Virol 2008,82(20):10008-10016.
29. Blond JL, Beseme F, Duret L, Bouton O, Bedin F, Perron H, Mandrand B, Mallet F:Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family. J Virol 1999,73(2):1175-1185.
30. Yi JM, Kim HM, Kim HS:Expression of the human endogenous retrovirus HERV-W family in various human tissues and cancer cells. J Gen Virol 2004, 85(Pt5):1203-1210.
31. Kim HS, Ahn K, Kim DS:Quantitative expression of the HERV-W env gene in human tissues. Arch Virol 2008,153(8):1587-1591.
32. Mi S, Lee X, Li X, Veldman GM, Finnerty H, Racie L, LaVallie E, Tang XY, Edouard P, Howes S et al. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 2000,403(6771):785-789.
33. Holder BS, Tower CL, Abrahams VM, Aplin JD:Syncytin 1 in the human placenta. Placenta 2012,33(6):460-466.
34. Ruebner M, Langbein M, Strissel PL, Henke C, Schmidt D, Goecke TW, Faschingbauer F, Schild RL, Beckmann MW, Strick R:Regulation of the human endogenous retroviral Syncytin-1 and cell-cell fusion by the nuclear hormone receptors PPARgamma/RXRalpha in placentogenesis. J Cell Biochem 2012, 113(7):2383-2396.
35. Sun Y, Ouyang DY, Pang W, Tu YQ, Li YY, Shen XM, Tam SC, Yang HY, Zheng YT:Expression of syncytin in leukemia and lymphoma cells. Leuk Res 2010,34(9):1195-1202.
36. Larsson LI, Holck S, Christensen IJ:Prognostic role of syncytin expression in breast cancer. Hum Pathol 2007,38(5):726-731.
37. Larsen JM, Christensen IJ, Nielsen HJ, Hansen U, Bjerregaard B, Talts JF, Larsson LI:Syncytin immunoreactivity in colorectal cancer:potential prognostic impact. Cancer Lett 2009,280(1):44-49.
38. Jern P, Sperber GO, Blomberg J:Definition and variation of human endogenous retrovirus H. Virology 2004,327(1):93-110.
39. Santoni FA, Guerra J, Luban J:HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency. Retrovirology 2012,9(1):111.
40. Yi JM, Kim HM, Kim HS:Human endogenous retrovirus HERV-H family in human tissues and cancer cells:expression, identification, and phylogeny. Cancer Lett 2006,231(2):228-239.
41. Jern P, Sperber GO, Ahlsen G, Blomberg J:Sequence variability, gene structure, and expression of full-length human endogenous retrovirus H.J Virol 2005, 79(10):6325-6337.
42. Pichon JP, Bonnaud B, Cleuziat P, Mallet F:Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling. Nucleic Acids Res 2006,34(6):e46.
43. Liang QY, Xu ZF, Xu RZ, Zheng S, Ding JY:[Deletion of the env region in HERV-H-X gene and its expression in colon cancer]. Ai Zheng 2007, 26(9):952-956.
44. Liang Q, Xu Z, Xu R, Wu L, Zheng S:Expression patterns of non-coding spliced transcripts from human endogenous retrovirus HERV-H elements in colon cancer. PLoS One 2012,7(1):e29950.
45. Wentzensen N, Coy JF, Knaebel HP, Linnebacher M, Wilz B, Gebert J, von Knebel Doeberitz M:Expression of an endogenous retroviral sequence from the HERV-H group in gastrointestinal cancers. Int J Cancer 2007, 121(7):1417-1423.
46. Mullins CS, Linnebacher M:Endogenous retrovirus sequences as a novel class of tumor-specific antigens:an example of HERV-H env encoding strong CTL epitopes. Cancer Immunol Immunother 2012,61 (7):1093-1100.
2. Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuli Y, Shiraishi Y, et al. Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature.1981; 294:770-771.
3.郑葆芬白血病病毒、艾滋病病毒、癌基因.上海医科大学出版.1996；P1-2,55-59.
4. Lander ES, Linton LM, Birren B, Nubaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature.2001; 409:860-921.
5. Costas J. Characterization of the intragenomic spread of the human endogenous retrovirus family HERV-W. Mol Biol Evol.2002; 19:526-533.
6. Hughes JF, Coffin JM. Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution. Nat Genet.2001; 29:487-489.
7. Belshaw R, Kaltaourakis A, Paces J,et al. High copy number in human endogenous retrovirus families is associated with copying mechanism in addition to reinfection. Mol Biol Evol.2005,22:814-817
8. Dewannieux M, Harper F, Richaud A, Letzelter C, Ribet D, Pierron G, et al. Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements. Genome Res.2006; 16:1548-1556.
9. Tonjes RR, Lower R, Boller K, Denner J, Hasenmaier B, Kirsch H, et al. HERV-K: the biologically most active human endogenous retrovirus family. Acquir. Immune Defic. Syndr. Hum. Retrovirol.1996; 13:S261-267.
10. Kraus B, Boller K, Reuter A, Schnierle BS. Characterization of the human endogenous retrovirus K Gag protein:identification of protease cleavage sites. Retrovirology.2011; 8:21.
11. Barbulescu M, Turner G, Seaman MI, Deinard AS, Kidd KK, Lenz J. Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans. Curr. Biol. 1999; 9:861-868.
12. Hanke K, Kramer P, Seeher S, Beimforde N, Kurth R, Bannert N. Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain. J. Virol.2009; 83:12790-12800.
13. Boller K, Schonfeld K, Lischer S, Fischer N, Hofmann A, Kurth R, et al. Human endogenous retrovirus HERV-K113 is capable of producing intact viral particles. J. Gen. Virol.2008; 89:567-72.
14. Lee YN, Bieniasz PD. Reconstitution of an infectious human endogenous retrovirus. PLoS Pathog.2007; 3:e10.
15. Contreras-Galindo R, Almodovar-Camacho S, Gonzalez-Ramirez S, Lorenzo E, Yamamura Y. Comparative longitudinal studies of HERV-K and HIV-1 RNA titers in HIV-1-infected patients receiving successful versus unsuccessful highly active antiretroviral therapy. AIDS Res Hum Retroviruses.2007; 23:1083-1086.
16. Depil S, Roche C, Dussart P, Prin L. Expression of a human endogenous retrovirus, HERV-K, in the blood cells of leukemia patients. Leukemia 2002; 16:254-259.
17. Contreras-Galindo R, Kaplan MH, Leissner P, Verjat T, Ferlenghi I, Bagnoli F, et al. Human endogenous retrovirus K (HML-2) elements in the plasma of people with lymphoma and breast cancer. J. Virol.2008; 82:9329-9336.
18. Armbruester V, Sauter M, Krautkraemer E, Meese E, Kleiman A, Best B, et al. A novel gene from the human endogenous retrovirus K expressed in transformed cells. Clin. Cancer Res.2002; 8:1800-1807.
19. Wang-Johanning F, Rycaj K, Plummer JB, Li M, Yin B, Frerich K, et al. Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors. J Natl Cancer Inst.2012; 104:189-210.
20. Zhao J, Rycaj K, Geng S, Li M, Plummer JB, Yin B, Liu H, Xu X, Zhang Y, Yan Y et al:Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer. Genes Cancer 2011;2:914-922.
21. Buscher K, Trefzer U, Hofmann M, Sterry W, Kurth R, Denner J. Expression of human endogenous retrovirus K in melanomas and melanoma cell lines. Cancer Res.2005; 65:4172-4180.
22. Ishida T, Obata Y, Ohara N, Matsushita H, Sato S, Uenaka A, Saika T, Miyamura T, Chayama K, Nakamura Y et al: Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity. Cancer Immun 2008; 8:15
23. Goering W, Ribarska T, Schulz WA:Selective changes of retroelement expression in human prostate cancer. Carcinogenesis 2011,32(10):1484-1492.
24. Ruprecht K, Ferreira H, Flockerzi A, Wahl S, Sauter M, Mayer J, Mueller-Lantzsch N:Human endogenous retrovirus family HERV-K(HML-2) RNA transcripts are selectively packaged into retroviral particles produced by the human germ cell tumor line Tera-1 and originate mainly from a pro virus on chromosome 22q 11.21. J Virol 2008,82(20):10008-10016.
25. Lower R, Tonjes RR, Korbmacher C, Kurth R, Lower J:Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K. J Virol 1995;69:141-149.
26. Denne M, Sauter M, Armbruester V, Licht JD, Roemer K, Mueller-Lantzsch N: Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein. J Virol 2007; 81:5607-5616.
27. Xu R, Gao Q, Wang S, Kan H, Sheng L, Li C, et al. Human acute myeloid leukemias may be etiologically associated with new human retroviral infection. Leuk. Res.1996; 20:449-455.
28. Oteiza A, Mechti N. The human T-cell leukemia virus type 1 oncoprotein tax controls forkhead box 04 activity through degradation by the proteasome. J Virol. 2011; 85:6480-6491.
29. Gandillet A, Park S, Lassailly F, Griessinger E, Vargaftig J, Filby A, et al. Heterogeneous sensitivity of human acute myeloid leukemia to β-catenin down-modulation. Leukemia 2011; 25:770-780.
30. Yeung J, Esposito MT, Gandillet A, Zeisig BB, Griessinger E, Bonnet D, et al. β-Catenin mediates the establishment and drug resistance of MLL leukemic stem cells. Cancer Cell.2010; 18:606-618.
31. Golan M, Hizi A, Resau JH, Yaal-Hahoshen N, Reichman H, Keydar I, Tsarfaty I. Human endogenous retrovirus (HERV-K) reverse transcriptase as a breast cancer prognostic marker. Neoplasia.2008;10(6):521-33.
32.徐荣臻.人白血病相关逆转录病毒的克隆及鉴定.浙江大学.2001
33. Boese A, Sauter M, Galli U, Best B, Herbst H, Mayer J, et al. Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein. Oncogene.2000; 19:4328-4336.
34. Currer R, Van Duyne R, Jaworski E, Guendel I, Sampey G, Das R, Narayanan A, Kashanchi F. HTLV tax:a fascinating multifunctional co-regulator of viral and cellular pathways.Front Microbiol.2012; 3:406.
35. Magin C, Lower R, Lower J. cORF and RcRE, the Rev/Rex and RRE/RxRE homologues of the human endogenous retrovirus family HTDV/HERV-K. J Virol 1999; 73:9496-507.
36. Galli UM, Sauter M, Lecher B, et al. Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors. Oncogene 2005; 24:3223-8.
37.汤钊猷.现代肿瘤学.复旦大学出版社.2011
38. Stommel JM, Kimmelman AC, Ting H, Nabioullin R, Ponugoti AH, Wiedemeyer R, et al. Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science.2007; 318:287-290.
39. Torok S, Dome B.Possibilities for inhibiting tumor-induced angiogenesis:results with multi-target tyrosine kinase inhibitors. Magy Onkol.2012; 56:3-15.
40. Guruswamy S, Rao CV.Multi-Target Approaches in Colon Cancer Chemoprevention Based on Systems Biology of Tumor Cell-Signaling. Gene Regul Syst Bio.2008; 2:163-176.
41.王韫秀,张继红,顾兆伟.Wnt/β-catenin信号通路与恶性血液病.中国实验血液学杂志.2009；17：234-237
42.赵渊源,李春艳.Wnt与Notch信号通路的串话与肿瘤发生、发展的关系.国际病理科学与临床杂志.2010；30：255-259
43. Tatarek J, Cullion K, Ashworth T, Gerstein R, Aster JC, Kelliher MA. Notchl inhibition targets the leukemia-initiating cells in a Tall/Lmo2 mouse model of T-ALL. Blood.2011; 118:1579-1590.
44. Pece S, Confalonieri S, R Romano P, Di Fiore PP.NUMB-ing down cancer by more than just a NOTCH.Biochim Biophys Acta.2011; 1815:26-43.
45. Ito T, Kwon HY, Zimdahl B, Congdon KL, Blum J, Lento WE, et al. Regulation of myeloid leukaemia by the cell-fate determinant Musashi. Nature.2010; 466:765-768.
46. Wang J, Liu Y, Li Z, Du J, Ryu MJ, Taylor PR, et al. Endogenous oncogenic Nras mutation promotes aberrant GM-CSF signaling in granulocytic/monocytic precursors in a murine model of chronic myelomonocytic leukemia. Blood.2010; 116:5991-6002.
47. Steelman LS, Franklin RA, Abrams SL, Chappell W, Kempf CR, Basecke J, et al. Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy. Leukemia.2011; 25:1080-1094.
48. Gutierrez A, Grebliunaite R, Feng H, Kozakewich E, Zhu S, Guo F, et al. Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia. J. Exp. Med.2011; 208:1595-1603.
49. Polak R, Buitenhuis M. The PI3K/PKB signaling module as key regulator of hematopoiesis:implications for therapeutic strategies in leukemia. Blood.2011; 199:911-923.
50. Sykes SM, Lane SW, Bullinger L, Kalaitzidis D, Yusuf R, Saez B, et al. AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell.2011; 146:697-708.
51. Hu Y, Chen Y, Douglas L, Li S. Beta-Catenin is essential for survival of leukemic stem cells insensitive to kinase inhibition in mice with BCR-ABL-induced chronic myeloid leukemia. Leukemia.2009; 23:109-116.
52. Takebe N, Harris PJ, Warren RQ, Ivy SP.Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways.Nat Rev Clin Oncol.2011; 8:97-106.
53. Guo S, Liu M, Gonzalez-Perez RR.Role of Notch and its oncogenic signaling crosstalk in breast cancer.Biochim Biophys Acta.2011; 1815:197-213.
54. Wang Z, Ahmad A, Li Y, Azmi AS, Miele L, Sarkar FH.Targeting notch to eradicate pancreatic cancer stem cells for cancer therapy.Anticancer Res.2011; 31:1105-13.
55. Guilmeau S.Notch signaling and intestinal cancer.Adv Exp Med Biol.2012; 727:272-88.
56. Armbruester V, Sauter M, Roemer K, Best B, Hahn S, Nty A, et al. Np9 protein of human endogenous retrovirus K interacts with ligand of numb protein X. J. Virol. 2004; 78:10310-10319.
57. Nie J, McGill MA, Dermer M, Dho SE, Wolting CD, McGlade CJ. LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation. EMBO. J.2002; 21:93-102.
58. Denne M, Sauter M, Armbruester V, Licht JD, Roemer K, Mueller-Lantzsch N. Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein. J. Virol.2007; 81: 5607-5616.
59. Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, Wetzker R. Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase gamma.Science.1997; 275:394-397.
60. Modi H, Li L, Chu S, Rossi J, Yee JK, Bhatia R. Inhibition of Grb2 expression demonstrates an important role in BCR-ABL-mediated MAPK activation and transformation of primary human hematopoietic cells.Leukemia.2010; 25:305-312.
1. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W et al: Initial sequencing and analysis of the human genome. Nature 2001,409(6822):860-921.
2. Costas J:Characterization of the intragenomic spread of the human endogenous retrovirus family HERV-W. Mol Biol Evol 2002,19(4):526-533.
3. Hughes JF, Coffin JM:Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution. Nat Genet 2001, 29(4):487-489.
4. Lower R, Lower J, Kurth R:The viruses in all of us:characteristics and biological significance of human endogenous retrovirus sequences. Proc Nat1 Acad Sci USA 1996,93(11):5177-5184.
5. Bannert N, Kurth R:Retroelements and the human genome:new perspectives on an old relation. Proc Natl Acad Sci USA 2004,101 Suppl 2:14572-14579.
6. Singh S, Kaye S, Gore ME, McClure MO, Bunker CB:The role of human endogenous retroviruses in melanoma. Br J Dermatol 2009,161(6):1225-1231.
7. Blikstad V, Benachenhou F, Sperber GO, Blomberg J:Evolution of human endogenous retroviral sequences:a conceptual account. Cell Mol Life Sci 2008, 65(21):3348-3365.
8. Dewannieux M, Harper F, Richaud A, Letzelter C, Ribet D, Pierron G, Heidmann T:Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements. Genome Res 2006, 16(12):1548-1556.
9. Tonjes RR, Lower R, Boller K, Denner J, Hasenmaier B, Kirsch H, Konig H, Korbmacher C, Limbach C, Lugert R et al. HERV-K:the biologically most active human endogenous retrovirus family. J Acquir Immune Defic Syndr Hum Retrovirol 1996,13 Suppl 1:S261-267.
10. Kraus B, Boller K, Reuter A, Schnierle BS:Characterization of the human endogenous retrovirus K Gag protein:identification of protease cleavage sites. Retrovirology 2011,8:21.
11. Barbulescu M, Turner G, Seaman MI, Deinard AS, Kidd KK, Lenz J:Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans. Curr Biol 1999,9(16):861-868.
12. Hanke K, Kramer P, Seeher S, Beimforde N, Kurth R, Bannert N:Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain. J Virol 2009, 83(24):12790-12800.
13. Boller K, Schonfeld K, Lischer S, Fischer N, Hoffmann A, Kurth R, Tonjes RR: Human endogenous retrovirus HERV-K113 is capable of producing intact viral particles. J Gen Virol 2008,89(Pt 2):567-572.
14. Lee YN, Bieniasz PD:Reconstitution of an infectious human endogenous retrovirus. PLoS Pathog 2007,3(1):e10.
15. Lower R, Tonjes RR, Korbmacher C, Kurth R, Lower J:Identification of a Rev-related protein by analysis of spliced transcripts of the human endogenous retroviruses HTDV/HERV-K. J Virol 1995,69(1):141-149.
16. Armbruester V, Sauter M, Krautkraemer E, Meese E, Kleiman A, Best B, Roemer K, Mueller-Lantzsch N:A novel gene from the human endogenous retrovirus K expressed in transformed cells. Clin Cancer Res 2002, 8(6):1800-1807.
17. Boese A, Sauter M, Galli U, Best B, Herbst H, Mayer J, Kremmer E, Roemer K, Mueller-Lantzsch N:Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein. Oncogene 2000,19(38):4328-4336.
18. Denne M, Sauter M, Armbruester V, Licht JD, Roemer K, Mueller-Lantzsch N: Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein. J Virol 2007, 81(11):5607-5616.
19. Depil S, Roche C, Dussart P, Prin L:Expression of a human endogenous retrovirus, HERV-K, in the blood cells of leukemia patients. Leukemia 2002, 16(2):254-259.
20. Contreras-Galindo R, Kaplan MH, Leissner P, Verjat T, Ferlenghi I, Bagnoli F, Giusti F, Dosik MH, Hayes DF, Gitlin SD et al. Human endogenous retrovirus K (HML-2) elements in the plasma of people with lymphoma and breast cancer. J Virol 2008,82(19):9329-9336.
21. Wang-Johanning F, Rycaj K, Plummer JB, Li M, Yin B, Frerich K, Garza JG, Shen J, Lin K, Yan P et al. Immunotherapeutic potential of anti-human endogenous retrovirus-K envelope protein antibodies in targeting breast tumors. JNatl Cancer Inst 2012,104(3):189-210.
22. Zhao J, Rycaj K, Geng S, Li M, Plummer JB, Yin B, Liu H, Xu X, Zhang Y, Yan Y et al. Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer. Genes Cancer 2011,2(9):914-922.
23. Buscher K, Trefzer U, Hofmann M, Sterry W, Kurth R, Denner J:Expression of human endogenous retrovirus K in melanomas and melanoma cell lines. Cancer Res 2005,65(10):4172-4180.
24. Schanab O, Humer J, Gleiss A, Mikula M, Sturlan S, Grunt S, Okamoto I, Muster T, Pehamberger H, Waltenberger A:Expression of human endogenous retrovirus K is stimulated by ultraviolet radiation in melanoma. Pigment Cell Melanoma Res 2011,24(4):656-665.
25. Li Z, Sheng T, Wan X, Liu T, Wu H, Dong J:Expression of HERV-K correlates with status of MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells. Cancer Invest 2010,28(10):1031-1037.
26. Ishida T, Obata Y, Ohara N, Matsushita H, Sato S, Uenaka A, Saika T, Miyamura T, Chayama K, Nakamura Y et al. Identification of the HERV-K gag antigen in prostate cancer by SEREX using autologous patient serum and its immunogenicity. Cancer Immun 2008,8:15.
27. Goering W, Ribarska T, Schulz WA:Selective changes of retroelement expression in human prostate cancer. Carcinogenesis 2011,32(10):1484-1492.
28. Ruprecht K, Ferreira H, Flockerzi A, Wahl S, Sauter M, Mayer J, Mueller-Lantzsch N:Human endogenous retrovirus family HERV-K(HML-2) RNA transcripts are selectively packaged into retroviral particles produced by the human germ cell tumor line Tera-1 and originate mainly from a pro virus on chromosome 22q11.21. J Virol 2008,82(20):10008-10016.
29. Blond JL, Beseme F, Duret L, Bouton O, Bedin F, Perron H, Mandrand B, Mallet F:Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family. J Virol 1999,73(2):1175-1185.
30. Yi JM, Kim HM, Kim HS:Expression of the human endogenous retrovirus HERV-W family in various human tissues and cancer cells. J Gen Virol 2004, 85(Pt5):1203-1210.
31. Kim HS, Ahn K, Kim DS:Quantitative expression of the HERV-W env gene in human tissues. Arch Virol 2008,153(8):1587-1591.
32. Mi S, Lee X, Li X, Veldman GM, Finnerty H, Racie L, LaVallie E, Tang XY, Edouard P, Howes S et al. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 2000,403(6771):785-789.
33. Holder BS, Tower CL, Abrahams VM, Aplin JD:Syncytin 1 in the human placenta. Placenta 2012,33(6):460-466.
34. Ruebner M, Langbein M, Strissel PL, Henke C, Schmidt D, Goecke TW, Faschingbauer F, Schild RL, Beckmann MW, Strick R:Regulation of the human endogenous retroviral Syncytin-1 and cell-cell fusion by the nuclear hormone receptors PPARgamma/RXRalpha in placentogenesis. J Cell Biochem 2012, 113(7):2383-2396.
35. Sun Y, Ouyang DY, Pang W, Tu YQ, Li YY, Shen XM, Tam SC, Yang HY, Zheng YT:Expression of syncytin in leukemia and lymphoma cells. Leuk Res 2010,34(9):1195-1202.
36. Larsson LI, Holck S, Christensen IJ:Prognostic role of syncytin expression in breast cancer. Hum Pathol 2007,38(5):726-731.
37. Larsen JM, Christensen IJ, Nielsen HJ, Hansen U, Bjerregaard B, Talts JF, Larsson LI:Syncytin immunoreactivity in colorectal cancer:potential prognostic impact. Cancer Lett 2009,280(1):44-49.
38. Jern P, Sperber GO, Blomberg J:Definition and variation of human endogenous retrovirus H. Virology 2004,327(1):93-110.
39. Santoni FA, Guerra J, Luban J:HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency. Retrovirology 2012,9(1):111.
40. Yi JM, Kim HM, Kim HS:Human endogenous retrovirus HERV-H family in human tissues and cancer cells:expression, identification, and phylogeny. Cancer Lett 2006,231(2):228-239.
41. Jern P, Sperber GO, Ahlsen G, Blomberg J:Sequence variability, gene structure, and expression of full-length human endogenous retrovirus H.J Virol 2005, 79(10):6325-6337.
42. Pichon JP, Bonnaud B, Cleuziat P, Mallet F:Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling. Nucleic Acids Res 2006,34(6):e46.
43. Liang QY, Xu ZF, Xu RZ, Zheng S, Ding JY:[Deletion of the env region in HERV-H-X gene and its expression in colon cancer]. Ai Zheng 2007, 26(9):952-956.
44. Liang Q, Xu Z, Xu R, Wu L, Zheng S:Expression patterns of non-coding spliced transcripts from human endogenous retrovirus HERV-H elements in colon cancer. PLoS One 2012,7(1):e29950.
45. Wentzensen N, Coy JF, Knaebel HP, Linnebacher M, Wilz B, Gebert J, von Knebel Doeberitz M:Expression of an endogenous retroviral sequence from the HERV-H group in gastrointestinal cancers. Int J Cancer 2007, 121(7):1417-1423.
46. Mullins CS, Linnebacher M:Endogenous retrovirus sequences as a novel class of tumor-specific antigens:an example of HERV-H env encoding strong CTL epitopes. Cancer Immunol Immunother 2012,61 (7):1093-1100.