肺癌中14-3-3σ和hASH1表达关系及14-3-3σ表达与肺腺癌临床病理参数关系
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摘要
前言
14-3-3σ是14-3-3蛋白家族中的一员,14-3-3σ直接参与人类肿瘤的发生、发展,在多种人类恶性肿瘤中常有其蛋白或基因表达异常。hASH1基因是human achaete-scute homologue 1的简称,是bHLH(basic helix-loop-helix)基因家族成员之一,bHLH蛋白构成多个转录因子家族,这些转录因子参与胚胎的发育和肿瘤的形成。研究表明在肺神经内分泌癌中14-3-3σ表达明显减少。然而hASH1在肺神经内分泌肿瘤高表达,而在非神经内分泌肿瘤不表达或低表达。
14-3-3σ蛋白参与细胞周期G2期检测点,它的缺失会引起的G2期检测点缺陷,使细胞过早进入有丝分裂而导致肿瘤发生。14-3-3σ基因在乳腺癌、卵巢癌、子宫内膜癌、前列腺癌、皮肤癌和肝癌等多种类型肿瘤中因其启动子CpG甲基化而沉默或低表达。研究证实在神经内分泌肿瘤中同样存在14-3-3σ基因启动子高度的超甲基化从而引起其低表达,并且14-3-3σ表达下降对神经内分泌肿瘤形成可能是必需的。研究发现给予甲基转移酶抑制剂5-氮杂胞苷(5-Aza)或组蛋白脱乙酰基酶抑制剂便可逆转14-3-3σ的超甲基化。而hASH1是一种组蛋白甲基转移酶,可以使组蛋白去乙酰化和H3组蛋白4,9,20,27,36位赖氨酸甲基化,从而使基因沉默。同时Osada.H等研究发现：用RNAi方法抑制肺癌细胞系中的hASHl表达,可以引起该肿瘤细胞在G2-M期细胞周期停滞和细胞凋亡,从而达到治疗的目的,但是其分子机制不清。本研究旨在探讨在肺神经内分泌肿瘤中是否存在hASH1通过其甲基转移酶的作用使14-3-3σ启动子超甲基化,引起14-3-3σ沉默或低表达,造成G2-M期检测点缺失,细胞发生恶性转化,从而产生肿瘤,以及14-3-3σ表达与肺腺癌临床病理参数关系。
材料与方法
一、材料
47例新鲜肺腺癌标本(来自中国医科大学附属一院2008.5-2009.7),其中男性患者16例,女性患者31例；年龄34—73岁(平均52.5±10)；淋巴结转移阳性者23例,淋巴结转移阴性者24例；Ⅰ+Ⅱ共19例,Ⅲ+Ⅳ共28例(国际抗癌联盟2003年分期标准)。
五种细胞系：肺巨细胞癌细胞系BE-1；肺大细胞癌细胞系NCI-H460;肺腺癌细胞系LTEP-α-1,A549；肺小细胞癌细胞系NCI-H446。
二、主要试剂和来源
浓缩型羊抗人14-3-3σ蛋白多克隆抗体(SC-7683,Santa Cruz),
浓缩型兔抗人hAshl蛋白多克隆抗体(ab-38557,abCam),
Easy RT-PCR Kit(AE401,北京全式金公司),TRIzol Reagent (15595-026, invitrogen) hAsh1引物,14-3-3σ引物,β-actin引物
三、方法
(一)RT-PCR
取组织和对数生长期的细胞用TRIzol (invitrogen)提取总RNA,逆转录条件为30℃10min,42℃40min,99℃5min,5℃5min。PCR反应条件为：94℃5min,94℃30sec,55℃30sec,72℃40sec,72℃10min。取扩增产物5ul,2%琼脂糖凝胶电泳,用Image J软件进行表达强度分析。
(二)Western Blot
取对数生长期的细胞提取总蛋白,超声破碎2下,15000rpm离心15min,取上清约60u1,5%浓缩胶,15%分离胶电泳,电压浓缩胶60V,分离胶100V。40V电压转印约90min。在TBS/0.3%Tween-20/5%小牛血清中封闭2h。14-3-3σ和hASHl均一抗(1：500)4℃孵育过夜,二抗(1：5000)37℃孵育2h。中心实验室发光检测。以上实验相同条件重复3次。
四、统计学分析
采用SPSS13.0统计学分析软件,对14-3-3σ、hASH1的表达与临床病理因素的关系用X2检验；14-3-3σ和hASH1表达关系采用Spearman等级相关分析,其它采用t检验；P<0.05为有统计学意义。
结果
一、不同肺癌细胞系中14-3-3σ、hASHl的表达情况及两者关系
(一)RT-PCR结果
在五种肺癌细胞系中14-3-3σmRNA有不同程度的表达,其中小细胞癌细胞系446最高,其次是腺癌细胞系LTE和A549(正常肺组织不表达14-3-3σ)。hASH1在具有神经内分泌特征的肺小细胞癌细胞系446和肺大细胞癌细胞系460中高表达。但从mRNA水平来看两者之间似乎缺乏相关性
(二)Western Blot结果
在五种肺癌细胞系中14-3-3σ蛋白有不同程度的表达,其中小细胞癌细胞系446最高,其次是腺癌细胞系LTE和A549(正常肺组织不表达14-3-3σ)。hASH1在具有神经内分泌特征的肺小细胞癌细胞系446和肺大细胞癌细胞系460中高表达。以上结果与RT-PCR结果基本一致。从蛋白水平来看两者之间同样缺乏相关性
二、肺腺癌中14-3-3σ表达高低与肺腺癌的分化、临床病理分期和淋巴结转移相关
通过RT-PCR方法检测了47例肺腺癌切除标本和癌旁组织,结果显示14-3-3σmRNA在癌组织表达明显高于对应的正常肺组织,进一步结合临床病理参数统计发现14-3-3σmRNA在低分化的肺腺癌中表达明显高于高分化肺腺癌,即与肺腺癌的分化程度呈负相关。14-3-3σmRNA高表达患者与低表达患者相比进展更快(P=0.001),更容易发生淋巴结转移相关(P=0.004),而与患者年龄(P=0.496)和性别(P=0.765)无关。这些结果表明14-3-3σ可能参与了肺腺癌的分化、演进及淋巴结转移。
结论
14-3-3σ可能与肺腺癌的发生、发展、分化程度、临床病理分期和淋巴结转移有关。并且从总体上看在5种肺癌细胞系中14-3-3σ和hASH1两者表达之间缺乏明确的负相关性。
Introduction
14-3-3σwhich is one of 14-3-3 proteins series and has direct relation to human tumor always expresses abnormally in human carcinomas in respects with gene and protein. Its expression in different lung cancer is special,such as its expression in squamous cell caicinoma is higher than that in adenocarcinoma,but little or even no in lung neuroendocrine tumors. hASHl gene which is in short of human achaete-scute homologue 1 is one of bHLH gene series which play important roles in the development of embryo and tumors. In recent it has been identified that the expression of hASH1 is very high in lung neuroendocrine tumors but extremely low in lung noneuroendocrine tumors.
14-3-3a gene in breast cancer, ovarian cancer, endometrial cancer, prostate cancer, skin cancer and liver cancer is expressed lowly and even silenced which is induced by the methylation of promoter.The lack of 14-3-3σprotein which has relation to G2 checkpoint in cell cycle will induce malignance by promoting mitosis. The hypermethylation of promoter of 14-3-3a gene in lung neuroendocrine tumors is sspecial. hASH1 is a methyltransferase which can keep genes silenced by transporting methyl to the site of 4,9,20,27 and 36 of lysine of histone 3. Our investigation puts emphasis on the correlation between 14-3-3aand hASH1 in lung cancer cell lines and the relation of 14-3-3σwith clinical pathologic parameters of lung adenocarcinoma.
Materials and methods
一、Samples
Cell lines:BE-1(gaint cell carcinoma cell), NCI-H460 (large cell undifferentiated carcinoma cell), LTE and A549(adenocarcinoma cell),NCI-H446(small cell lung cancer cell)
47 fresh lung adenocarcinoma tissue samples and healthy tissue (obtained from patients who had surgery in the first Affiliated Hospital Of China Medcial University)
二、Reagent
The anti-human 14-3-3σgoat polyclonal antibody (SC-7683,Santa Cruz); The anti-human hASH1 rabbit polyclonal antibody (ab-38557,abCam); Easy RT-PCR Kit(AE401, TRANS); TRIzol Reagent (15595-026, invitrogen); hAshl primer; 14-3-3σprimer, p-actin primer(JINSITE BIOTECHNOLOGY)
三、Methods
(一)RT-PCR
Total RNA was isolated from cells in logarithmic growth phase and fresh lung adencarcinoma tissue samples using TRIZOL.The PCR primers were as follows:
14-3-3σforw 5'-TGCGAAGAGCGAAACCTGC-3'(19bp), 14-3-3σrev 5'-CTGTTGGCGATCTCGTAGTGG-3'(21bp) (the length of product is 446bp).
hASHl forw 5'-TCCCCCAACTACTCCAACGAC-3'(21bp), hASH1 rev 5'-CCCTCCCAACGCCACTG-3'(17bp) (the length of product is 233bp).
β-actin forw 5'-AAATCGTGCGTGACATTAA-3'(19bp),β-actin rev 5'-CTCGTCATACTCCTGCTTG-3'(19bp) (the length of product is 513bp).
The temperature for RT:30℃10min,42℃40min,99℃5min,5℃5min。
The temperature for PCR(14-3-3aand hASH1):94℃5min,94℃30sec,55℃30sec, 72℃40sec,72℃10min。
(二)Western Blot
Lung cancer cells were extracted with lysis buffer(150mMNacl,1%NP40, 0.1%SDS,2ug/ml aprotinin,1M PMSF)for 30 min at 4℃.The supernatants were centrifuged at 15000xg for 15 min at 4℃.Aliquots containing 100ug of protein were separated on 15% SDS-PAGE and transferred to PVDF membranes at 40v for 90 min at low temperature.The membranes were blocked with 5% skimmed milk.Then goat anti-human 14-3-3a polyclonal antibody(1:500 dilution), rabbit anti-human hASH1 polyclonal antibody(1:500 dilution) and anti-β-actin antibody(1:400) immunodetection was performed.The ECL Imaging System was used to catch up the bands.
四、Statistical analysis
ALL the data are analyzed with SPSS for Windows 13.0 software. We use x2 test to analyze the correlation between clinic pathologic parameters of lung cancer and expression of 14-3-3a and hASH1,We use the permutation test for the Spearman correlation coefficient to analyze the correlation between expression of 14-3-3σand hASH1. The statistical significance is defined as P＜0.05.
Result
一、The expression of 14-3-3σand hASHl in five lung cancer cells
(一)RT-PCR
the expression of 14-3-3σmRNA in five lung cancer cells is different and the highest is in 446 followed by LTE and A549(no expression in healthy lung tissue). The expression of hASHl mRNA is higher in 446 and 460 which are two kinds of lung neuroendocrine tumors.But perhaps there is no correlation between 14-3-3σand hASH 1 judging from their mRNA expression.
(二)Western Blot
the expression of 14-3-3σprotein in five lung cancer cells is different and the highest in 446 followed by LTE and A549 (no expression with healthy lung tissue). The expression of hASH1 protein is higher in 446 and 460 which are two kinds of lung neuroendocrine tumors.But perhaps there is no relation between 14-3-3aand hASH1 judging from their protein expression.
二、14-3-3σmRNA expression in 47 lung adenocarcinoma correlate with tumors differentiation,clinical stages and lymph node metastasis
The expression of 14-3-3amRNA is obviously higher in 47 lung adenocarcinoma than that in healthy lung tissue by RT-PCR detect. Furthermore, the expression of 14-3-3σmRNA in undifferentiated tumors is higher than that in differentiated tumors. As to say the relation between the expression of 14-3-3σmRNA and degree of differentiation of lung adenocarcinoma is negative.Also patients with high 14-3-3σmRNA expression were more likely than low expression patients have advanced disease (p=0.001) and lymph node metastasis(p=0.004). But there is no correlation between 14-3-3omRNA expression and patients age (p=0.496) and patients gender(p=0.765).Finally these results probably means 14-3-3σplays an important role in the development, differentiation and lymph node metastasis of lung adenocarcinoma.
Conclusions
14-3-3σprotein probably plays an important role in the development, differentiation and lymph node metastasis of lung adenocarcinoma.But the relation of higher hASH1 with lower 14-3-3σexisting in other lung neuroendocrine tumor cell lines does not exist in 446 cell line.
14-3-3σ是14-3-3蛋白家族中的一员,14-3-3σ直接参与人类肿瘤的发生、发展,在多种人类恶性肿瘤中常有其蛋白或基因表达异常。hASH1基因是human achaete-scute homologue 1的简称,是bHLH(basic helix-loop-helix)基因家族成员之一,bHLH蛋白构成多个转录因子家族,这些转录因子参与胚胎的发育和肿瘤的形成。研究表明在肺神经内分泌癌中14-3-3σ表达明显减少。然而hASH1在肺神经内分泌肿瘤高表达,而在非神经内分泌肿瘤不表达或低表达。
14-3-3σ蛋白参与细胞周期G2期检测点,它的缺失会引起的G2期检测点缺陷,使细胞过早进入有丝分裂而导致肿瘤发生。14-3-3σ基因在乳腺癌、卵巢癌、子宫内膜癌、前列腺癌、皮肤癌和肝癌等多种类型肿瘤中因其启动子CpG甲基化而沉默或低表达。研究证实在神经内分泌肿瘤中同样存在14-3-3σ基因启动子高度的超甲基化从而引起其低表达,并且14-3-3σ表达下降对神经内分泌肿瘤形成可能是必需的。研究发现给予甲基转移酶抑制剂5-氮杂胞苷(5-Aza)或组蛋白脱乙酰基酶抑制剂便可逆转14-3-3σ的超甲基化。而hASH1是一种组蛋白甲基转移酶,可以使组蛋白去乙酰化和H3组蛋白4,9,20,27,36位赖氨酸甲基化,从而使基因沉默。同时Osada.H等研究发现：用RNAi方法抑制肺癌细胞系中的hASHl表达,可以引起该肿瘤细胞在G2-M期细胞周期停滞和细胞凋亡,从而达到治疗的目的,但是其分子机制不清。本研究旨在探讨在肺神经内分泌肿瘤中是否存在hASH1通过其甲基转移酶的作用使14-3-3σ启动子超甲基化,引起14-3-3σ沉默或低表达,造成G2-M期检测点缺失,细胞发生恶性转化,从而产生肿瘤,以及14-3-3σ表达与肺腺癌临床病理参数关系。
材料与方法
一、材料
47例新鲜肺腺癌标本(来自中国医科大学附属一院2008.5-2009.7),其中男性患者16例,女性患者31例；年龄34—73岁(平均52.5±10)；淋巴结转移阳性者23例,淋巴结转移阴性者24例；Ⅰ+Ⅱ共19例,Ⅲ+Ⅳ共28例(国际抗癌联盟2003年分期标准)。
五种细胞系：肺巨细胞癌细胞系BE-1；肺大细胞癌细胞系NCI-H460;肺腺癌细胞系LTEP-α-1,A549；肺小细胞癌细胞系NCI-H446。
二、主要试剂和来源
浓缩型羊抗人14-3-3σ蛋白多克隆抗体(SC-7683,Santa Cruz),
浓缩型兔抗人hAshl蛋白多克隆抗体(ab-38557,abCam),
Easy RT-PCR Kit(AE401,北京全式金公司),TRIzol Reagent (15595-026, invitrogen) hAsh1引物,14-3-3σ引物,β-actin引物
三、方法
(一)RT-PCR
取组织和对数生长期的细胞用TRIzol (invitrogen)提取总RNA,逆转录条件为30℃10min,42℃40min,99℃5min,5℃5min。PCR反应条件为：94℃5min,94℃30sec,55℃30sec,72℃40sec,72℃10min。取扩增产物5ul,2%琼脂糖凝胶电泳,用Image J软件进行表达强度分析。
(二)Western Blot
取对数生长期的细胞提取总蛋白,超声破碎2下,15000rpm离心15min,取上清约60u1,5%浓缩胶,15%分离胶电泳,电压浓缩胶60V,分离胶100V。40V电压转印约90min。在TBS/0.3%Tween-20/5%小牛血清中封闭2h。14-3-3σ和hASHl均一抗(1：500)4℃孵育过夜,二抗(1：5000)37℃孵育2h。中心实验室发光检测。以上实验相同条件重复3次。
四、统计学分析
采用SPSS13.0统计学分析软件,对14-3-3σ、hASH1的表达与临床病理因素的关系用X2检验；14-3-3σ和hASH1表达关系采用Spearman等级相关分析,其它采用t检验；P<0.05为有统计学意义。
结果
一、不同肺癌细胞系中14-3-3σ、hASHl的表达情况及两者关系
(一)RT-PCR结果
在五种肺癌细胞系中14-3-3σmRNA有不同程度的表达,其中小细胞癌细胞系446最高,其次是腺癌细胞系LTE和A549(正常肺组织不表达14-3-3σ)。hASH1在具有神经内分泌特征的肺小细胞癌细胞系446和肺大细胞癌细胞系460中高表达。但从mRNA水平来看两者之间似乎缺乏相关性
(二)Western Blot结果
在五种肺癌细胞系中14-3-3σ蛋白有不同程度的表达,其中小细胞癌细胞系446最高,其次是腺癌细胞系LTE和A549(正常肺组织不表达14-3-3σ)。hASH1在具有神经内分泌特征的肺小细胞癌细胞系446和肺大细胞癌细胞系460中高表达。以上结果与RT-PCR结果基本一致。从蛋白水平来看两者之间同样缺乏相关性
二、肺腺癌中14-3-3σ表达高低与肺腺癌的分化、临床病理分期和淋巴结转移相关
通过RT-PCR方法检测了47例肺腺癌切除标本和癌旁组织,结果显示14-3-3σmRNA在癌组织表达明显高于对应的正常肺组织,进一步结合临床病理参数统计发现14-3-3σmRNA在低分化的肺腺癌中表达明显高于高分化肺腺癌,即与肺腺癌的分化程度呈负相关。14-3-3σmRNA高表达患者与低表达患者相比进展更快(P=0.001),更容易发生淋巴结转移相关(P=0.004),而与患者年龄(P=0.496)和性别(P=0.765)无关。这些结果表明14-3-3σ可能参与了肺腺癌的分化、演进及淋巴结转移。
结论
14-3-3σ可能与肺腺癌的发生、发展、分化程度、临床病理分期和淋巴结转移有关。并且从总体上看在5种肺癌细胞系中14-3-3σ和hASH1两者表达之间缺乏明确的负相关性。
Introduction
14-3-3σwhich is one of 14-3-3 proteins series and has direct relation to human tumor always expresses abnormally in human carcinomas in respects with gene and protein. Its expression in different lung cancer is special,such as its expression in squamous cell caicinoma is higher than that in adenocarcinoma,but little or even no in lung neuroendocrine tumors. hASHl gene which is in short of human achaete-scute homologue 1 is one of bHLH gene series which play important roles in the development of embryo and tumors. In recent it has been identified that the expression of hASH1 is very high in lung neuroendocrine tumors but extremely low in lung noneuroendocrine tumors.
14-3-3a gene in breast cancer, ovarian cancer, endometrial cancer, prostate cancer, skin cancer and liver cancer is expressed lowly and even silenced which is induced by the methylation of promoter.The lack of 14-3-3σprotein which has relation to G2 checkpoint in cell cycle will induce malignance by promoting mitosis. The hypermethylation of promoter of 14-3-3a gene in lung neuroendocrine tumors is sspecial. hASH1 is a methyltransferase which can keep genes silenced by transporting methyl to the site of 4,9,20,27 and 36 of lysine of histone 3. Our investigation puts emphasis on the correlation between 14-3-3aand hASH1 in lung cancer cell lines and the relation of 14-3-3σwith clinical pathologic parameters of lung adenocarcinoma.
Materials and methods
一、Samples
Cell lines:BE-1(gaint cell carcinoma cell), NCI-H460 (large cell undifferentiated carcinoma cell), LTE and A549(adenocarcinoma cell),NCI-H446(small cell lung cancer cell)
47 fresh lung adenocarcinoma tissue samples and healthy tissue (obtained from patients who had surgery in the first Affiliated Hospital Of China Medcial University)
二、Reagent
The anti-human 14-3-3σgoat polyclonal antibody (SC-7683,Santa Cruz); The anti-human hASH1 rabbit polyclonal antibody (ab-38557,abCam); Easy RT-PCR Kit(AE401, TRANS); TRIzol Reagent (15595-026, invitrogen); hAshl primer; 14-3-3σprimer, p-actin primer(JINSITE BIOTECHNOLOGY)
三、Methods
(一)RT-PCR
Total RNA was isolated from cells in logarithmic growth phase and fresh lung adencarcinoma tissue samples using TRIZOL.The PCR primers were as follows:
14-3-3σforw 5'-TGCGAAGAGCGAAACCTGC-3'(19bp), 14-3-3σrev 5'-CTGTTGGCGATCTCGTAGTGG-3'(21bp) (the length of product is 446bp).
hASHl forw 5'-TCCCCCAACTACTCCAACGAC-3'(21bp), hASH1 rev 5'-CCCTCCCAACGCCACTG-3'(17bp) (the length of product is 233bp).
β-actin forw 5'-AAATCGTGCGTGACATTAA-3'(19bp),β-actin rev 5'-CTCGTCATACTCCTGCTTG-3'(19bp) (the length of product is 513bp).
The temperature for RT:30℃10min,42℃40min,99℃5min,5℃5min。
The temperature for PCR(14-3-3aand hASH1):94℃5min,94℃30sec,55℃30sec, 72℃40sec,72℃10min。
(二)Western Blot
Lung cancer cells were extracted with lysis buffer(150mMNacl,1%NP40, 0.1%SDS,2ug/ml aprotinin,1M PMSF)for 30 min at 4℃.The supernatants were centrifuged at 15000xg for 15 min at 4℃.Aliquots containing 100ug of protein were separated on 15% SDS-PAGE and transferred to PVDF membranes at 40v for 90 min at low temperature.The membranes were blocked with 5% skimmed milk.Then goat anti-human 14-3-3a polyclonal antibody(1:500 dilution), rabbit anti-human hASH1 polyclonal antibody(1:500 dilution) and anti-β-actin antibody(1:400) immunodetection was performed.The ECL Imaging System was used to catch up the bands.
四、Statistical analysis
ALL the data are analyzed with SPSS for Windows 13.0 software. We use x2 test to analyze the correlation between clinic pathologic parameters of lung cancer and expression of 14-3-3a and hASH1,We use the permutation test for the Spearman correlation coefficient to analyze the correlation between expression of 14-3-3σand hASH1. The statistical significance is defined as P＜0.05.
Result
一、The expression of 14-3-3σand hASHl in five lung cancer cells
(一)RT-PCR
the expression of 14-3-3σmRNA in five lung cancer cells is different and the highest is in 446 followed by LTE and A549(no expression in healthy lung tissue). The expression of hASHl mRNA is higher in 446 and 460 which are two kinds of lung neuroendocrine tumors.But perhaps there is no correlation between 14-3-3σand hASH 1 judging from their mRNA expression.
(二)Western Blot
the expression of 14-3-3σprotein in five lung cancer cells is different and the highest in 446 followed by LTE and A549 (no expression with healthy lung tissue). The expression of hASH1 protein is higher in 446 and 460 which are two kinds of lung neuroendocrine tumors.But perhaps there is no relation between 14-3-3aand hASH1 judging from their protein expression.
二、14-3-3σmRNA expression in 47 lung adenocarcinoma correlate with tumors differentiation,clinical stages and lymph node metastasis
The expression of 14-3-3amRNA is obviously higher in 47 lung adenocarcinoma than that in healthy lung tissue by RT-PCR detect. Furthermore, the expression of 14-3-3σmRNA in undifferentiated tumors is higher than that in differentiated tumors. As to say the relation between the expression of 14-3-3σmRNA and degree of differentiation of lung adenocarcinoma is negative.Also patients with high 14-3-3σmRNA expression were more likely than low expression patients have advanced disease (p=0.001) and lymph node metastasis(p=0.004). But there is no correlation between 14-3-3omRNA expression and patients age (p=0.496) and patients gender(p=0.765).Finally these results probably means 14-3-3σplays an important role in the development, differentiation and lymph node metastasis of lung adenocarcinoma.
Conclusions
14-3-3σprotein probably plays an important role in the development, differentiation and lymph node metastasis of lung adenocarcinoma.But the relation of higher hASH1 with lower 14-3-3σexisting in other lung neuroendocrine tumor cell lines does not exist in 446 cell line.
引文
1 Mhawech P.14-3-3 proteins--an update. Cell Res,2005;15:228-236.
2 Pallas DC, Fu H, Haehnel L C, et al.Association of polyomavirus middle tumor antigen with 14-3-3 proteins[J]. Science,1994,265(5171):535-537.
3 Osada H, Tatematsu Y, Yatabe Y,et al. Frequent and histological type-specific inactivation of 14-3-3sigma in human lung cancers[J]. Oncogene,2002,21(15):2418-2424.
4 Nakajima T, Shimooka H, Weixa P, ET AL. Immunohistochemical demonstration of 14-3-3sigma protein in normal human tissues and lung cancers, and the preponderance of its strong expression in epithelial cells of squamous cell lineage[J]. Pathol Int,2003,53(6): 353-360.
5 Peng WX, Sano T, Oyama T, et al. Large cell neuroendocrine carcinoma of the lung:a comparison with large cell carcinoma with neuroendocrine morphology and small cell carcinoma[J]. Lung Cancer,2005,47(2):225-233.
6 Tanaka Y, Katagiri Z, Kawahashi K, et al. Trithorax-group protein ASH1 methylates histone H3 lysine 36[J]. Gene.2007,397(1-2):161-8.
7 Yatabe Y, Osada H, Tatematsu Y, et al. Decreased expression of 14-3-3sigma in neuroendocrine tumors is independent of origin and malignant potential[J]. Oncogene. 2002,;21(54):8310-9.
8 Osada H, Tomida S, Yatabe Y, et al. Roles of achaete-scute homologue 1 in DKK1 and E-cadherin repression and neuroendocrine differentiation in lung cancer[J]. Cancer Res.2008, 68(6):1647-55.
9 Duckworth BC, Weaver JS, Ruderman JV. G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A. Proc Natl Acad Sci USA,2002,99(26): 16794-16799.
10 Tanaka K, Hatada T,Kobayashi M, et al. The clinical implication of 14-3-3 sigma expression in primary gastrointestinal malignancy[J]. Int J Oncol,2004,25(6):1591-1597
11 Jiang S X,Kameya T,Asamura H,et al. hASHl expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors[J].Mod Pathol, 2004,17(2):222-229.
12 Kawabe T.G2 checkpoint abrogators as anticancer drug [J].Mol Cancer Ther,2004,3(4):513-519.
13 Osada H, Tatematsu Y, Yatabe Y,et al. ASH1 Gene Is a Specific Therapeutic Target for Lung Cancers with Neuroendocrine Features[J]. Cancer Res 2005,65(23):10680-5.
14 Kawabe T.G2 checkpoint abrogators as anticancer durgs[J]. Mol Cancer Ther,2004,3(4):513-519.
1 Ball D W, Azzoli C G, Baylin S B, et al.Idetification of a human achaete-scute homolog highly expressed in neuroendocrine tumors[J],Proc Natl Acad Sci USA,1993,90(12):5648-52.
2 Renault B, Lieman J, Ward D, et al.Localization of the human achaete-scute homolog gene (ASCL1)distal to phenylalanine hydroxylase (PAH) and proximal to tumor rejection antigen (TRal) on chromosome 12q22-q23[J].Genomics,1995,30(1):81-83.
3 Borges M, Linnoila RI, van de Velde HJ, et al. An achaete-scute homologue essential for neuroendocrine differentiation in the lung. Nature 1997;386:852-855.
4 Huber K, Bruhl B, Guillemot F, et al. Development of chromaffin cells depends on MASH1 function. Development 2002;129:4729-4738.
5 Lanigan TM, DeRaad SK, Russo AF. Requirement of the MASH-1 transcription factor for neuroendocrine differentiation of thyroid C cells. J Neurobiol 1998;34:126-134.
6 Kameda Y. Mashl is required for glomus cell formation in the mouse carotid body. Dev Biol 2005;283:128-139.
7 Linnoila RI. Functional facets of the pulmonary neuroendocrine system. Lab Invest 2006;86:425-444.
8 Ito T, Udaka N, Yazawa T, et al. Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development 2000;127:3913-3921.
9 Schuurmans C, Guillemot F. Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr Opin Neurobiol 2002;12:26-34.
10 Sommer L, Shah N, Rao M, et al. The cellular function of MASH 1 in autonomic neurogenesis. Neuron 1995;15:1245-1258.
11 Letinic K,Zoncu R, Rakic P.Origin of GABAergic neurons in the human neocortex[J].Nature,2002,417(6889)645-649.
12 Pattyn A.Simplicio N,van Doorninck J H,et al.Ascll/Mashl is requird for the development of central serotonergic neurons [J] Nature Neurosci,2004,7(6)589-595
13 Castro D S, Skowronska-Krawczyk D, Armant O, et al. Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif [J]. Dev Cell,2006,11(6):831-844
14 Battiste J,Helms A W, Kim E J,et al. Ascll defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord[J].Development,2007;134:285-293.
15 Kim EJ, Battiste J, Nakagawa Y,et al. Ascll (Mashl) lineage cells contribute to discrete cell populations in CNS architecture[J]. Mol Cell Neurosci.2008,38(4):595-606.
16 Hiroshi Kokubu, Toshiyuki Ohtsuka Ryoichiro Kageyama. Mashl is required for neuroendocrine cell development in the glandular stomach [J]. Genes to Cells 2008,13:41-51.
17 Ralston J, Chiriboga L, Nonaka D. MASH1:a useful marker in differentiating pulmonary small cell carcinoma from Merkel cell carcinoma[J]. Mod Pathol.2008,118:1357-1362.
18 Johnson JE, Birren SJ, Saito T, Anderson DJ. DNA binding and transcriptional regulatory activity of mammalian achaete-scute homologous (MASH) proteins revealed by interaction with a muscle-specific enhancer. Proc Natl Acad Sci USA 1992; 89:3596-600.
19 Tanaka Y, Katagiri Z, Kawahashi K, et al. Trithorax-group protein ASH1 methylates histone H3 lysine 36[J]. Gene.2007,397(1-2):161-8.
20 Linnoila RI, Zhao B, DeMayo JL, et al. Constitutive achaete-scute homologue-1 promotes airway dysplasia and lung neuroendocrine tumors in transgenic mice. Cancer Res 2000; 60:4005-9.
21 Jiang S X,Kameya T,Asamura H,et al. hASHl expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors[J].Mod Pathol, 2004,17(2):222-229.
22 Xiao-Yang Wang1, El Habib Dakir1, Xu Naizhen1,et al. Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium [J]. Laboratory Investigation 2007,87: 527-539.
23 Osada H, Tomida S, Yatabe Y, et al. Roles of achaete-scute homologue 1 in DKK1 and E-cadherin repression and neuroendocrine differentiation in lung cancer[J]. Cancer Res.2008, 68(6):1647-55
24 Rapa I, Ceppi P, Bollito E, et al. Human ASH1 expression in prostate cancer with neuroendocrine differentiation[J]. Mod Pathol.2008,39:700-707
25 Tanaka Y, Nakayama Y, Taniguchi M, et al. Regulation of early T cell development by the PHD finger of histone lysine methyltransferase ASH1 [J]. Biochem Biophys Res Commun. 2008,365(3):589-94.
26 Shida T, Furuya M, Kishimoto T, et al. The expression of NeuroD and mASH1 in the gastroenteropancreatic neuroendocrine tumors [J]. Mod Pathol.2008,121:1363-1370.
27 Gregory GD, Vakoc CR, Rozovskaia T, et al. Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes [J]. Mol Cell Biol. 2007,27(24):8466-79.
28 Struhl A, Adachi A.Nuclear access and action of notch in vivo[J].Cell,1998,93(4)649-660.
29 Chen H, Thiagalingam A, Chopra H, et al. Conservation of the Drosophila lateral inhibition pathway in human lung cancer:a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression. Proc Natl Acad Sci USA 1997; 94:5355-60.
30 Sriuranpong V, Borges MW, Strock CL, et al. Notch signaling induces rapid degradation of achaete-scute homolog 1. Mol Cell Biol 2002; 22:3129-39.
31 Elmi M, Faigle R, Yang W, et al. Mechanism of MASH1 induction by ASK1 and ATRA in adult neural progenitors[J]. Mol Cell Neurosci.2007,36(2):248-59.
32 Platta CS, Greenblatt DY, Kunnimalaiyaan M, et al. Valproic acid induces Notchl signaling in small cell lung cancer cells [J]. J Surg Res.2008,148(1):31-7.
33 Byrd KN, Shearn A. ASH 1, a Drosophila trithorax group protein, is required for methylation of lysine 4 residues on histone H3[J]. Proc Natl Acad Sci USA.2003,100(20):11535-40
2 Pallas DC, Fu H, Haehnel L C, et al.Association of polyomavirus middle tumor antigen with 14-3-3 proteins[J]. Science,1994,265(5171):535-537.
3 Osada H, Tatematsu Y, Yatabe Y,et al. Frequent and histological type-specific inactivation of 14-3-3sigma in human lung cancers[J]. Oncogene,2002,21(15):2418-2424.
4 Nakajima T, Shimooka H, Weixa P, ET AL. Immunohistochemical demonstration of 14-3-3sigma protein in normal human tissues and lung cancers, and the preponderance of its strong expression in epithelial cells of squamous cell lineage[J]. Pathol Int,2003,53(6): 353-360.
5 Peng WX, Sano T, Oyama T, et al. Large cell neuroendocrine carcinoma of the lung:a comparison with large cell carcinoma with neuroendocrine morphology and small cell carcinoma[J]. Lung Cancer,2005,47(2):225-233.
6 Tanaka Y, Katagiri Z, Kawahashi K, et al. Trithorax-group protein ASH1 methylates histone H3 lysine 36[J]. Gene.2007,397(1-2):161-8.
7 Yatabe Y, Osada H, Tatematsu Y, et al. Decreased expression of 14-3-3sigma in neuroendocrine tumors is independent of origin and malignant potential[J]. Oncogene. 2002,;21(54):8310-9.
8 Osada H, Tomida S, Yatabe Y, et al. Roles of achaete-scute homologue 1 in DKK1 and E-cadherin repression and neuroendocrine differentiation in lung cancer[J]. Cancer Res.2008, 68(6):1647-55.
9 Duckworth BC, Weaver JS, Ruderman JV. G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A. Proc Natl Acad Sci USA,2002,99(26): 16794-16799.
10 Tanaka K, Hatada T,Kobayashi M, et al. The clinical implication of 14-3-3 sigma expression in primary gastrointestinal malignancy[J]. Int J Oncol,2004,25(6):1591-1597
11 Jiang S X,Kameya T,Asamura H,et al. hASHl expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors[J].Mod Pathol, 2004,17(2):222-229.
12 Kawabe T.G2 checkpoint abrogators as anticancer drug [J].Mol Cancer Ther,2004,3(4):513-519.
13 Osada H, Tatematsu Y, Yatabe Y,et al. ASH1 Gene Is a Specific Therapeutic Target for Lung Cancers with Neuroendocrine Features[J]. Cancer Res 2005,65(23):10680-5.
14 Kawabe T.G2 checkpoint abrogators as anticancer durgs[J]. Mol Cancer Ther,2004,3(4):513-519.
1 Ball D W, Azzoli C G, Baylin S B, et al.Idetification of a human achaete-scute homolog highly expressed in neuroendocrine tumors[J],Proc Natl Acad Sci USA,1993,90(12):5648-52.
2 Renault B, Lieman J, Ward D, et al.Localization of the human achaete-scute homolog gene (ASCL1)distal to phenylalanine hydroxylase (PAH) and proximal to tumor rejection antigen (TRal) on chromosome 12q22-q23[J].Genomics,1995,30(1):81-83.
3 Borges M, Linnoila RI, van de Velde HJ, et al. An achaete-scute homologue essential for neuroendocrine differentiation in the lung. Nature 1997;386:852-855.
4 Huber K, Bruhl B, Guillemot F, et al. Development of chromaffin cells depends on MASH1 function. Development 2002;129:4729-4738.
5 Lanigan TM, DeRaad SK, Russo AF. Requirement of the MASH-1 transcription factor for neuroendocrine differentiation of thyroid C cells. J Neurobiol 1998;34:126-134.
6 Kameda Y. Mashl is required for glomus cell formation in the mouse carotid body. Dev Biol 2005;283:128-139.
7 Linnoila RI. Functional facets of the pulmonary neuroendocrine system. Lab Invest 2006;86:425-444.
8 Ito T, Udaka N, Yazawa T, et al. Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium. Development 2000;127:3913-3921.
9 Schuurmans C, Guillemot F. Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr Opin Neurobiol 2002;12:26-34.
10 Sommer L, Shah N, Rao M, et al. The cellular function of MASH 1 in autonomic neurogenesis. Neuron 1995;15:1245-1258.
11 Letinic K,Zoncu R, Rakic P.Origin of GABAergic neurons in the human neocortex[J].Nature,2002,417(6889)645-649.
12 Pattyn A.Simplicio N,van Doorninck J H,et al.Ascll/Mashl is requird for the development of central serotonergic neurons [J] Nature Neurosci,2004,7(6)589-595
13 Castro D S, Skowronska-Krawczyk D, Armant O, et al. Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif [J]. Dev Cell,2006,11(6):831-844
14 Battiste J,Helms A W, Kim E J,et al. Ascll defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord[J].Development,2007;134:285-293.
15 Kim EJ, Battiste J, Nakagawa Y,et al. Ascll (Mashl) lineage cells contribute to discrete cell populations in CNS architecture[J]. Mol Cell Neurosci.2008,38(4):595-606.
16 Hiroshi Kokubu, Toshiyuki Ohtsuka Ryoichiro Kageyama. Mashl is required for neuroendocrine cell development in the glandular stomach [J]. Genes to Cells 2008,13:41-51.
17 Ralston J, Chiriboga L, Nonaka D. MASH1:a useful marker in differentiating pulmonary small cell carcinoma from Merkel cell carcinoma[J]. Mod Pathol.2008,118:1357-1362.
18 Johnson JE, Birren SJ, Saito T, Anderson DJ. DNA binding and transcriptional regulatory activity of mammalian achaete-scute homologous (MASH) proteins revealed by interaction with a muscle-specific enhancer. Proc Natl Acad Sci USA 1992; 89:3596-600.
19 Tanaka Y, Katagiri Z, Kawahashi K, et al. Trithorax-group protein ASH1 methylates histone H3 lysine 36[J]. Gene.2007,397(1-2):161-8.
20 Linnoila RI, Zhao B, DeMayo JL, et al. Constitutive achaete-scute homologue-1 promotes airway dysplasia and lung neuroendocrine tumors in transgenic mice. Cancer Res 2000; 60:4005-9.
21 Jiang S X,Kameya T,Asamura H,et al. hASHl expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors[J].Mod Pathol, 2004,17(2):222-229.
22 Xiao-Yang Wang1, El Habib Dakir1, Xu Naizhen1,et al. Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium [J]. Laboratory Investigation 2007,87: 527-539.
23 Osada H, Tomida S, Yatabe Y, et al. Roles of achaete-scute homologue 1 in DKK1 and E-cadherin repression and neuroendocrine differentiation in lung cancer[J]. Cancer Res.2008, 68(6):1647-55
24 Rapa I, Ceppi P, Bollito E, et al. Human ASH1 expression in prostate cancer with neuroendocrine differentiation[J]. Mod Pathol.2008,39:700-707
25 Tanaka Y, Nakayama Y, Taniguchi M, et al. Regulation of early T cell development by the PHD finger of histone lysine methyltransferase ASH1 [J]. Biochem Biophys Res Commun. 2008,365(3):589-94.
26 Shida T, Furuya M, Kishimoto T, et al. The expression of NeuroD and mASH1 in the gastroenteropancreatic neuroendocrine tumors [J]. Mod Pathol.2008,121:1363-1370.
27 Gregory GD, Vakoc CR, Rozovskaia T, et al. Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes [J]. Mol Cell Biol. 2007,27(24):8466-79.
28 Struhl A, Adachi A.Nuclear access and action of notch in vivo[J].Cell,1998,93(4)649-660.
29 Chen H, Thiagalingam A, Chopra H, et al. Conservation of the Drosophila lateral inhibition pathway in human lung cancer:a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression. Proc Natl Acad Sci USA 1997; 94:5355-60.
30 Sriuranpong V, Borges MW, Strock CL, et al. Notch signaling induces rapid degradation of achaete-scute homolog 1. Mol Cell Biol 2002; 22:3129-39.
31 Elmi M, Faigle R, Yang W, et al. Mechanism of MASH1 induction by ASK1 and ATRA in adult neural progenitors[J]. Mol Cell Neurosci.2007,36(2):248-59.
32 Platta CS, Greenblatt DY, Kunnimalaiyaan M, et al. Valproic acid induces Notchl signaling in small cell lung cancer cells [J]. J Surg Res.2008,148(1):31-7.
33 Byrd KN, Shearn A. ASH 1, a Drosophila trithorax group protein, is required for methylation of lysine 4 residues on histone H3[J]. Proc Natl Acad Sci USA.2003,100(20):11535-40