Department of Physics, Molecular Life Science Center, Jacobs University, Campus Ring 1, 28759 Bremenm, Germany.
Chembiochem. 2011 Aug 16;12(12):1922-36. doi: 10.1002/cbic.201000653. Epub 2011 Jul 7.
Hsp70 is biologically relevant for its chaperon functions. The CX(-) and CX(+) sublines, which derive from the parental colon carcinoma CX2 cell line, are accordingly very similar. They have been reported to be specifically different only in Hsp70 membrane expression, which is associated with immunostimulatory effects. CX(-) /CX(+) have been phenotypically characterized by immunofluorescence studies and Raman spectroscopy combined with robust clustering and multivariate analysis. With the latter we address the potential of overall characterization for CX(-) /CX(+) discrimination and gain molecular insights into Hsp70 differential expression. Due to their strong resemblance, CX(-) and CX(+) show similar mean Raman spectra, which look indiscernible at first. Interestingly, their rather protein-dominated Raman spectra reveal, besides changes in protein and amino acids, very specific changes in DNA/RNA nucleotides involving pyrimidine ring Raman hypochromic effects. Therefore, discriminating CX(-) from CX(+) is ultimately achieved based on principal component scores. Because CX(-) /CX(+) are associated with the same lipid marker, changes in proteins support lipid interactions with regulatory proteins. More importantly, changes observed in nucleobases, which are indicative of DNA/RNA-protein binding interactions, suggest transcription deregulations as participating precursor onsets of different transport mechanisms that lead to Hsp70 differential expression and associated phenotypic variation. Besides immunofluorescence, we have used Raman spectroscopy combined with multivariate analysis within an autologous tumor system for label-free nondestructive cell-subline discrimination, and demonstrate, to our knowledge, the first overall phenotypic monitoring with insights into Hsp70 differential expression. This might well prove to be useful for Raman label-free cell-sorting of the CX(-) /CX(+) sublines.
热休克蛋白 70（Hsp70）因其伴侣功能而具有生物学相关性。源自亲本结肠癌细胞系 CX2 的 CX(-) 和 CX(+)亚系非常相似。据报道，它们仅在 Hsp70 膜表达方面存在特异性差异，这种差异与免疫刺激作用有关。CX(-) /CX(+) 通过免疫荧光研究和拉曼光谱学进行了表型特征分析，同时结合了强大的聚类和多变量分析。通过后者，我们解决了对 CX(-) /CX(+) 进行整体特征分析的潜力，并深入了解了 Hsp70 差异表达的分子机制。由于它们非常相似，CX(-) 和 CX(+) 的平均拉曼光谱看起来相似，乍一看难以区分。有趣的是，它们以蛋白质为主的拉曼光谱除了显示蛋白质和氨基酸的变化外，还显示出嘧啶环拉曼光谱减色效应等非常特定的 DNA/RNA 核苷酸变化。因此，最终基于主成分得分来区分 CX(-) 和 CX(+)。由于 CX(-) /CX(+) 与相同的脂质标记物相关，因此蛋白质的变化支持与调节蛋白的脂质相互作用。更重要的是，观察到的核苷碱基变化表明 DNA/RNA-蛋白质结合相互作用的转录调控异常，这是不同转运机制的起始点，导致 Hsp70 差异表达和相关的表型变化。除了免疫荧光，我们还在自体肿瘤系统中使用拉曼光谱学结合多变量分析进行无标记无损细胞亚系区分，并展示了对 Hsp70 差异表达的深入了解的首个整体表型监测。这对于 CX(-) /CX(+) 亚系的拉曼无标记细胞分选可能非常有用。
