The quantity of nucleolar proteins nucleolin and protein B23 is related to cell doubling time in human cancer cells.
The quantity of nucleolar proteins nucleolin and protein B23 is related to cell doubling time in human cancer cells.
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发表时间：
1995-10
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通讯作者：
M. Derenzini;V. Sirri;D. Treré;R. Ochs
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作者：
M. Derenzini;V. Sirri;D. Treré;R. Ochs
背景在细胞组织学标本中原位测量的银染核仁蛋白（AgNOR蛋白）的数量与细胞增殖的速度有关。术语"AgNOR蛋白"包括几种蛋白。单个AgNOR蛋白量与细胞增殖活性之间的关系尚不清楚。我们研究了单个AgNOR蛋白的定量分布，特别注意到两个主要的AgNOR蛋白，核仁素和蛋白B23，在7个人癌细胞系，其特征在于不同的细胞倍增时间。设计通过定时计数非同步生长的癌细胞来测量癌细胞的倍增时间。在特定的一步染色程序后，通过计算机图像分析原位定量AgNOR蛋白。对于核仁素和蛋白B23的定量评价，遵循两种方法。通过SDS-PAGE分离后的核蛋白转移到硝酸纤维素膜上，并进行：1）通过AgNOR蛋白的银染色程序染色，或2）用抗核仁素和抗蛋白B23 mAb处理，然后与连接到过氧化物酶的二级抗体反应，并通过化学发光和放射自显影术显示。在这两种情况下，使用计算机化光密度分析进行单个AgNOR蛋白和核仁素以及蛋白B23量的测量。结果在所有细胞系中，105 kDa（核仁素）和38至39 kDa（蛋白B23）的银染条带的积分密度值占总银染条带值的60%以上。核仁素和B23蛋白的银染密度值与细胞增殖速度呈显著正相关（r = 0.85和r = 0.86，p <0.05）。用Western印迹法测得的核仁素和蛋白B23的值与细胞增殖速度密切相关（r分别为0.93和0.96，p <0.001）。最后，在原位细胞学制备物中定义的平均AgNOR蛋白面积值与银染硝酸纤维素膜（分别为r = 0.92和r = 0.90，p <0.01）和Western印迹（分别为r = 0.95和r = 0.94，p <0.001）中评估的核仁素和蛋白B23量之间观察到良好的相关性。结论原位细胞组织学标本中AgNOR蛋白的定量变化主要反映核仁素和B23蛋白的定量变化，并证明核仁素和B23蛋白的含量与人癌细胞倍增时间呈负相关。
BACKGROUND The quantity of the silver-stained nucleolar proteins (AgNOR proteins) measured in situ in cytohistologic preparations is related to the rapidity of cell proliferation. The term "AgNOR proteins" comprises several proteins. The relationship between the individual AgNOR protein amount and cell proliferating activity is not yet known. We studied the quantitative distribution of the individual AgNOR proteins, with specific attention to the two major AgNOR proteins, nucleolin and protein B23, in seven human cancer cell lines characterized by different cell doubling times. DESIGN The doubling time of cancer cells was measured by counting the asynchronously growing cells at regular time intervals. The AgNOR proteins were quantified in situ, after a specific one-step staining procedure, by computerized image analysis. For the quantitative evaluation of nucleolin and protein B23, two methods were followed. Nuclear proteins after separation by SDS-PAGE were transferred onto nitrocellulose membranes and were either: 1) stained by the silver staining procedure for AgNOR proteins or 2) treated with anti-nucleolin and anti-protein B23 mAb followed by reaction with secondary Ab linked to peroxidase and revealed by chemiluminescence and autoradiography. In both cases, measurement of individual AgNOR protein and nucleolin and protein B23 amount was carried out using computerized densitometric analysis. RESULTS Integrated density values of the silver-stained bands at 105 kDa (nucleolin) and 38 to 39 kDa (protein B23) represented, in all cell lines, more than 60% of the total silver-stained band value. A relationship was found between the densitometric values of silver-stained nucleolin and protein B23 and rapidity of cell proliferation (r = 0.85 and r = 0.86, respectively, p < 0.05). The values of nucleolin and protein B23 obtained using the Western blots were strictly related to the rapidity of cell proliferation (r = 0.93 and 0.96, respectively, p < 0.001). Finally, a good correlation was observed between the mean AgNOR protein area value, as defined in cytologic preparations in situ, and nucleolin and protein B23 amounts as evaluated in silver-stained nitrocellulose membranes (r = 0.92 and r = 0.90, respectively, p < 0.01) and in Western blots (r = 0.95 and r = 0.94, respectively, p < 0.001). CONCLUSIONS These data indicate that the quantitative changes of AgNOR proteins observed in cytohistologic preparations in situ mainly reflect the quantitative changes of nucleolin and protein B23 and demonstrate that nucleolin and protein B23 amounts are inversely related to cell doubling time in human cancer cells.