Stokke T, Holte H, Steen H B
Department of Biophysics, Norwegian Radium Hospital, Oslo.
Cancer Res. 1988 Dec 1;48(23):6708-14.
The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood B- and T-lymphocytes (G0 cells) bound low amounts of 7-AMD (7-AMD- phenotype), while G1 REH cells and purified B-cells stimulated with anti-mu + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD+ phenotype). 7-AMD binding increased up to threefold and the differences in binding between G0 and G1 cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1. 7-AMD binding increased in parallel with autofluorescence and approximately linearly with time during the G0-G1 transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor. In cell suspensions from lymph node biopsies, the 7-AMD+ phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9). The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD- cells. All tumors were diploid. The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (P less than 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity. Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (P = 0.07), while the light scattering (cell size) of G0/G1 cells was highly correlated to 7-AMD binding (P less than 0.001).
通过用DNA特异性荧光团7-氨基放线菌素D（7-AMD）对甲醛固定、透化处理的细胞进行染色，并通过流式细胞术测量单细胞荧光，研究了二倍体前体B细胞系（REH）、体外刺激的正常B淋巴细胞以及反应性和恶性淋巴结B淋巴细胞的染色质结构。静息外周血B淋巴细胞和T淋巴细胞（G0期细胞）结合少量的7-AMD（7-AMD阴性表型），而G1期REH细胞以及用抗μ抗体+B细胞生长因子刺激的纯化B细胞结合的7-AMD量几乎是前者的两倍（7-AMD阳性表型）。在固定前分离细胞核或用DNA酶1处理固定的全细胞时，7-AMD结合增加至三倍，G0期和G1期细胞之间的结合差异几乎消失。在体外刺激的B细胞从G0期向G1期转变过程中，7-AMD结合与自发荧光平行增加，并且与时间大致呈线性关系，这是通过同时测量7-AMD荧光、自发荧光或异硫氰酸荧光素标记的针对早期活化抗原4F2和转铁蛋白受体的抗体的荧光来确定的。在淋巴结活检的细胞悬液中，7-AMD阳性表型是高级别非霍奇金淋巴瘤（H-NHL， Kiel分类，5/5）患者肿瘤细胞的特征；仅在9个低级别非霍奇金淋巴瘤样本中的1个（L-NHL，1/9）中发现了具有这种表型的细胞。其他（8/9）L-NHL样本和反应性淋巴结仅含有7-AMD阴性细胞。所有肿瘤均为二倍体。在染色质中观察到的7-AMD结合与DNA对DNA酶1的敏感性之间的相关性（P小于0.001）表明，7-AMD结合是一般转录活性的标志物。令人惊讶的是，处于S期的肿瘤细胞百分比与7-AMD染色性无显著相关性（P = 0.07），而G0/G1期细胞的光散射（细胞大小）与7-AMD结合高度相关（P小于0.001）。
