Evans C A, Peterkofsky B
J Cell Physiol. 1976 Nov;89(3):355-67. doi: 10.1002/jcp.1040890302.
Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.
在接触抑制的小鼠成纤维细胞系Balb 3T3、柯斯顿病毒转化细胞系Ki-3T3以及经二丁酰环磷腺苷（dbcAMP）处理的Ki-3T3细胞的生长周期各阶段，对胶原蛋白合成、胶原蛋白中脯氨酸的羟化作用以及胶原蛋白分泌进行了研究。对数期和静止期的转化细胞产生的胶原蛋白水平均低于亲代细胞系，但即使在培养基中不添加抗坏血酸的情况下，胶原蛋白中理论上可能存在的羟脯氨酸残基仍有85% - 90%得以形成。此外，添加抗坏血酸盐后，转化细胞的胶原蛋白分泌仅增加约20%。这与亲代Balb 3T3细胞中脯氨酸最大程度羟化对抗坏血酸的需求以及抗坏血酸对胶原蛋白分泌2 - 3倍的刺激作用形成对比。尽管dbcAMP处理使Ki-3T3细胞呈现出更正常的形态，并使胶原蛋白合成的相对速率提高到与3T3细胞相似的水平，但这种处理并未恢复脯氨酸羟化或胶原蛋白分泌对抗坏血酸的需求。脯氨酰羟化酶的比活性也不受dbcAMP处理的影响，尽管这种处理增加了胶原蛋白的合成。此外，还发现抗坏血酸在对数期或静止期均无法有效激活来自Ki-3T3或经dbcAMP处理的Ki-3T3细胞培养物的脯氨酰羟化酶。Ki-3T3细胞培养物在细胞或培养基中均不积累抗坏血酸，细胞匀浆中也无法从前体14C - 葡萄糖醛酸合成抗坏血酸。结果表明，病毒转化的Balb 3T3细胞获得了合成脯氨酰羟化酶还原辅因子的能力，并且这种功能可能与这些细胞糖酵解代谢的增加有关，因为用dbcAMP处理既未改变细胞代谢，也未改变不依赖抗坏血酸的羟化作用。
