Hamann K K, Cowles E A, Wang J L, Anderson R L
Department of Biochemistry, Michigan State University, East Lansing 48824.
Exp Cell Res. 1991 Sep;196(1):82-91. doi: 10.1016/0014-4827(91)90458-7.
We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to approximately 18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (pI8.7) protein, as well as the phosphorylated (pI8.2) derivative. In contrast, older (passage 27-35, 55-68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (pI 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.
我们比较了碳水化合物结合蛋白35（CBP35）在不同增殖能力的人SL66成纤维细胞中的表达和定位。当血清饥饿的静止年轻（第11代，相当于约18次累积群体倍增）SL66细胞用血清处理时，CBP35的表达受到明显刺激。这通过免疫荧光下抗CBP35阳性染色细胞百分比的增加、免疫印迹中蛋白质含量的增加以及Northern印迹中积累的mRNA水平的增加得以揭示。增殖细胞中CBP35表达的升高最明显地体现在细胞核部分，非磷酸化（pI8.7）蛋白以及磷酸化（pI8.2）衍生物的水平均有所升高。相比之下，较老的（第27 - 35代，55 - 68次累积群体倍增）SL66成纤维细胞培养物似乎已失去CBP35表达的正常增殖依赖性调节。在静止的高代细胞中CBP35水平较高，血清刺激后有所下降。此外，在高代SL66细胞的细胞核或细胞质中均未检测到凝集素的非磷酸化（pI 8.7）形式。最后，在高代细胞的静止培养物中CBP35的mRNA水平较高，但血清刺激17小时后无法检测到。这些结果表明，随着人成纤维细胞增殖能力的降低，CBP35的表达会发生改变。
