Kim Beum Jun, Gibson Donna M, Shuler Michael L
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
Biotechnol Prog. 2005 May-Jun;21(3):700-7. doi: 10.1021/bp050016z.
Taxus cuspidata P991 in plant cell suspension culture is capable of producing the important anticancer agent Taxol (paclitaxel) and related taxanes. High-level production is obtained by elicitation with methyl jasmonate, but successful elicitation leads to loss of cell viability that cannot be recovered by subculture. Here, we test whether the loss of viability is due to a direct effect of methyl jasmonate. Upon subculture, the reduced viability continued in methyl jasmonate elicited cultures, but not in nonelicited control cultures. The growth reduction in elicited T. cuspidata P991 suspension cultures was evaluated by viability reduction measurements using phenosafranin and fluorescein diacetate. The viability reduction does not appear to be related to apoptosis based on DNA laddering analysis because it occurred very late (at day 35) in the culture period. DNA laddering was also found only after day 28 in T. canadensis C93AD (a Taxol-producing cell line) elicited with methyl jasmonate, implying that apoptosis is not the major death mechanism after elicitation. As compared to Taxol-producing cell lines, the viability of a nonproducing cell line, T. canadensis CO93D, was not severely affected by methyl jasmonate, indicating that methyl jasmonate itself is not the primary factor for viability reduction. Based on Northern analysis of taxadiene synthase mRNA from both elicited and nonelicited T. cuspidata P991, methyl jasmonate directly induces the production of this enzyme, which is the first committed step in the biosynthetic pathway for Taxol. As a result, both viability reduction and growth reduction appear related to a high production level of Taxol (and related taxanes) upon methyl jasmonate elicitation, rather than to the direct effect of methyl jasmonate.
东北红豆杉P991在植物细胞悬浮培养中能够产生重要的抗癌药物紫杉醇（泰素）及相关紫杉烷类化合物。通过茉莉酸甲酯诱导可实现高水平生产，但成功诱导会导致细胞活力丧失，且传代培养无法恢复。在此，我们测试活力丧失是否是由于茉莉酸甲酯的直接作用。传代培养后，茉莉酸甲酯诱导的培养物中活力持续降低，但未诱导的对照培养物中则没有。通过使用番红和荧光素二乙酸酯进行活力降低测量，评估了茉莉酸甲酯诱导的东北红豆杉P991悬浮培养物中的生长降低情况。基于DNA梯状分析，活力降低似乎与凋亡无关，因为它在培养期很晚（第35天）才出现。在用茉莉酸甲酯诱导的加拿大红豆杉C93AD（一种产生紫杉醇的细胞系）中，也仅在第28天后才发现DNA梯状现象，这意味着凋亡不是诱导后的主要死亡机制。与产生紫杉醇的细胞系相比，非产生细胞系加拿大红豆杉CO93D的活力未受到茉莉酸甲酯的严重影响，表明茉莉酸甲酯本身不是活力降低的主要因素。基于对诱导和未诱导的东北红豆杉P991中紫杉二烯合酶mRNA的Northern分析，茉莉酸甲酯直接诱导该酶的产生，这是紫杉醇生物合成途径中的第一个关键步骤。因此，活力降低和生长降低似乎都与茉莉酸甲酯诱导后紫杉醇（及相关紫杉烷类化合物）的高产量有关，而非与茉莉酸甲酯的直接作用有关。
