Localization of Epidermal Growth Factor Receptor Enhancer Protein in A431 Epidermoid Carcinoma Cells by Southwestern Histochemistry
Localization of Epidermal Growth Factor Receptor Enhancer Protein in A431 Epidermoid Carcinoma Cells by Southwestern Histochemistry
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西南组织化学法定位 A431 表皮样癌细胞中表皮生长因子受体增强蛋白
DOI：
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
T. Koji
中科院分区：
文献类型：
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作者：
K. Komuta;T. Kanematsu;P. Nakane;T. Koji
由于表皮生长因子受体（EGFR）的异常表达常与肿瘤的发生发展相关，因此在转录水平和转录本水平上分析EGFR基因的表达有助于了解肿瘤生长的异常本质。在本研究中，我们试图定位EGFR转录因子，EGFR特异性转录因子（ETF）和GC因子（GCF），在A431人表皮样瘤移植到裸鼠的冷冻切片中，西南组织化学。合成了包含ETF和GCF调节元件的DNA序列（91个碱基对（bp））的（+）和（-）序列，退火后用地高辛配基（Dig）-11-dUTP进行末端脱氧核苷酸转移酶加尾。用辣根过氧化物酶标记的抗Dig进行酶免疫组织化学染色，观察Dig的位点。91 bp的探针有效地检测到一个单一的ETF带的分子量为120 kD的从A431肿瘤细胞提取的粗核馏分的South-western印迹，但不是GCF带。A431肿瘤冰冻切片经4%多聚甲醛固定后，与91 bp探针反应，可观察到核周区和核的斑点状染色，染色强度随探针浓度的增加而增加，在0.5-1μg/ml时达到稳定水平。此外，用探针进行的核染色依赖于盐浓度，并且在150 mM NaCl下信号/噪声比最大。过量的未标记的91 bp DNA或未标记的ETF反应元件DNA单独存在时，91 bp探针的染色被取消，但未标记的GCF DNA的染色不被取消，表明91 bp探针的核和核周染色反映了ETF的定位。因此，西南组织化学可以是一种新的工具来分析基因特异性转录调控因子的细胞表达。
Since abnormal expression of epidermal growth factor receptor (EGFR) is frequently associated with cancer development, the analysis of EGFR gene expression at the transcriptional level as well as the transcript level is helpful to understand the abnormal nature of cancer growth. In this study, we attempted to localize EGFR transcriptional factors, EGFR specific transcription factor (ETF) and GC factor (GCF), in the frozen sections of A431 human epidermoid tumor transplated into nude mice by southwestern histochemistry. As probes for southwestern histochemistry, (+) and (-) sequences of the DNA seqment (91 base pairs (bp)) including ETF and GCF regulatory element were synthesized, allowed to be annealed and then tailed by terminal deoxynucleotidyl transferase with digoxigenin (Dig) -11-dUTP. The sites of Dig were visualized enzyme-immunohistochemically with horseradish peroxidase-labeled anti-Dig. The 91 bp probe detected effectively a single ETF band with a molecular mass of 120 kD on a south-western blot of the crude nuclear fraction extracted from A431 tumor cells, but not a GCF band. When the frozen sections of A431 tumor were fixed with 4% paraformal-dehyde and reacted with the 91 bp probe, the staining of perinuclear area as well as nuclei in a speckled pattern were observed and the staining intensity was increased depending upon the concentrations of the probe and reached a plateu level at 0.5-1μg/ml. Moreover, the nuclear staining with the probe was dependent upon a salt concentration and the signal/noise ratio was a maximam at 150 mM NaCl. The staining with the 91 bp probe was abolished by the presence of an excess amount of unlabeled 91bp DNA or unlabeled ETF responsive element DNA alone, but not by that of unlabeled GCF DNA, indicating that the nuclear and perinuclear staining with the 91bp probe reflects the localization of ETF. Thus, southwestern histochemistry can be a novel tool to analyze cellular expression of gene-specific transcription regulatory factors.
微波稳定性增强了猕猴输卵管冷冻切片中雌激素受体的免疫细胞化学检测。
DOI：
10.1210/endo.136.9.7649110
发表时间：
1995
期刊：
Endocrinology.
影响因子：
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作者：
Slayden,OD;Koji,T;Brenner,RM
通讯作者：
Brenner,RM