Kolset S O, Ivhed I, Overvatn A, Nilsson K
Institute of Medical Biology, University of Tromsø, Norway.
Cancer Res. 1988 Nov 1;48(21):6103-8.
A monoblastic cell line U-937 (clone 4), was induced to differentiate along the monocytoid lineage by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), and vitamin D3 (VD3). By immunochemical and morphological criteria the cells were found to differentiate into macrophage-like cells in the presence of all three inducers. The expression of proteoglycans was investigated in control cultures and in cells differentiated in the presence of both TPA, RA, and VD3. The cells were labeled with [35S]sulfate and cell and medium-associated 35S-macromolecules were either solubilized in sodium dodecyl sulfate or subjected to proteolytic digestion. By use of chondroitinase ABC digestions and deaminative cleavage at pH 1.5 it was demonstrated that all cell cultures incorporated [35S]sulfate exclusively into chondroitin sulfate proteoglycan (CSPG). The expression of CSPG was found to decrease with differentiation to 60% in the presence of TPA, 67% in RA, and 40% in VD3 of control cultures on a cellular basis. The CSPG synthesized was consistently recovered from the medium fractions, whereas free glycosaminoglycan (GAG) chains were found in the cell fraction in all the cell cultures. GAG chains from both control and TPA-, RA-, and VD3-induced cultures were found to be exclusively of the chondroitin 4-sulfate type. However, the CSPGs from RA- and VD3-treated cells were found to differ in molecular size from those of control and TPA-induced cultures, as judged by Sepharose CL-6B gel chromatography. This difference in macromolecular properties following the induced differentiation of the monoblastic cells into macrophage-like cells was found to reside in expression of CSPGs (in the presence of RA and VD3) with smaller GAG chains. Control cells and TPA-induced cells synthesized CSPGs with GAG chains of approximate Mr of 30,000, contrasted by approximate Mr of 17,000 and 16,000 in RA- and VD3-induced cells, respectively. Accordingly, all three agents used in this study were found to induce differentiation of the U-937-4 cells and a decrease in the expression of CSPG, but only RA and VD3 were found to influence the structure of the proteoglycans synthesized.
单核细胞系U - 937（克隆4），通过12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）、视黄酸（RA）和维生素D3（VD3）诱导其沿单核细胞谱系分化。根据免疫化学和形态学标准，发现细胞在所有三种诱导剂存在的情况下分化为巨噬细胞样细胞。研究了对照培养物以及在TPA、RA和VD3共同存在下分化的细胞中蛋白聚糖的表达。细胞用[35S]硫酸盐标记，细胞和培养基相关的35S - 大分子要么在十二烷基硫酸钠中溶解，要么进行蛋白水解消化。通过使用软骨素酶ABC消化和在pH 1.5下的脱氨基裂解，证明所有细胞培养物都仅将[35S]硫酸盐掺入硫酸软骨素蛋白聚糖（CSPG）中。发现在细胞基础上，CSPG的表达随着分化而降低，在TPA存在下降至对照培养物的60%，在RA存在下降至67%，在VD3存在下降至40%。合成的CSPG始终从培养基组分中回收，而在所有细胞培养物的细胞组分中发现游离糖胺聚糖（GAG）链。对照以及TPA、RA和VD3诱导培养物中的GAG链都仅为硫酸软骨素4 - 硫酸盐类型。然而，通过琼脂糖CL - 6B凝胶色谱法判断，RA和VD3处理细胞的CSPG在分子大小上与对照和TPA诱导培养物的不同。单核细胞系细胞诱导分化为巨噬细胞样细胞后，这种大分子性质的差异在于（在RA和VD3存在下）具有较小GAG链的CSPG的表达。对照细胞和TPA诱导的细胞合成的CSPG具有约30,000 Mr的GAG链，相比之下，RA和VD3诱导的细胞中分别约为17,000和16,000 Mr。因此，本研究中使用的所有三种试剂都能诱导U - 937 - 4细胞分化并降低CSPG的表达，但仅发现RA和VD3会影响合成的蛋白聚糖的结构。
