|Kim, Beum Jun - CORNELL UNIVERSITY|
|Shuler, Michael - CORNELL UNIVERSITY|
Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 5, 2005
Publication Date: March 31, 2005
Citation: Kim, B., Gibson, D.M., Shuler, M.L. 2005. Relationship of viability and apoptosis to taxol production in taxus sp. suspension cultures elicited with methyl jasmonate. Biotechnology Progress. 21:700-707. Interpretive Summary: Paclitaxel (Taxol) is a potent chemotherapeutic drug with proven utility against a range of cancers, including lung, breast, and ovarian cancer. The limited supply of this drug from the original tree source prompted the development of alternative sources of production, including the use of plant cell cultures. Taxol derived from plant cell cultures has been commercialized, but work is still needed to optimize production and understand the underlying mechanisms regulating production. This work describes our study to evaluate whether stimulation by methyl jasmonate, a known elicitor for taxol production in cell culture, causes loss of viability and cell death (apoptosis). We present evidence that while methyl jasmonate directly induces the production of the enzyme involved in the first committed step for taxol biosynthesis, it is not a direct factor in viability and growth reduction. This study will be useful to understanding the overall mechanisms involved in optimizing taxol production in plant cell cultures.
Technical Abstract: 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 the direct effect of methyl jasmonate. Upon subculture, the reduced viability continued in methyl jasmonate elicited cultures, but not in non-elicited 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 did not appear to be related to apoptosis based on DNA laddering analysis since it occurred very late 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. Compared to Taxol-producing cell lines, the viability of a non-producing 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 from both elicited and non-elicited T. cupsidata 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 are related to a high production level of Taxol (and related taxanes) upon methyl jasmonate elicitation, rather than to the direct effect of methyl jasmonate.