Development of an Agrobacterium-mediated transformation method for Taxus suspension cultures
Taxus plant cell culture is used industrially to supply the valuable anti-cancer compound paclitaxel, a natural specialized metabolite from the yew tree. This production system could be optimized through metabolic engineering efforts to improve and stabilize paclitaxel yields. However, robust techniques for transforming Taxuscell culture are currently lacking. Previously developed Agrobacterium-mediated transformation protocols result in in low transfection rates and are not translatable to other Taxusspecies and cultures. The method presented here expanded upon past studies to improve transfection rate and decrease time required to create a stably transformed cell line. This paper explores the effects of critical parameters including several Agrobacteriumtumefaciens strains and
varying concentrations of cefotaxime and hygromycin to improve plant transgenic cell selection. An anti-necrotic cocktail comprised of silver nitrate, cysteine, and ascorbic acid was supplemented to the media to reduce browning and enhance culture recovery. Optimization of the method resulted in establishment of multiple transgenic cultures, determined by homogenous GUS expression evident in the blue-staining phenotype, which remained stable over a 5-year period through continual application of selective pressure by hygromycin-enriched media. Genomic integration was confirmed through PCR amplification of a region of the GUS gene in transgenic cultures after 5 years. Not only did this method expedite the process and increase yields from previous Agrobacterium-mediated transformation methods for Taxus, it promoted stable transformation over a period of years. This technique can be used to genetically engineer Taxusplant cell culture through redirecting metabolic flux to deliver consistent and high paclitaxel yields.
Sarah A. Wilson, Patricia Keen, Michelle C. McKee, Nicole Raia, Joyce Van Eck, Susan C. Roberts. Development of an Agrobacterium-mediated transformation method for Taxus suspension cultures. In Vitro Cellular & Developmental Biology-Plant, 54:36-44, 2018.
Preclinical evaluation of the Aurora kinase inhibitors AMG 900, AZD1152-HQPA, and MK-5108 on SW-872 and 93T449 human liposarcoma cells
Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequently diagnosed soft tissue sarcomas in humans. There is great interest in identifying novel chemotherapeutic options for treating liposarcoma based upon molecular alterations in the cancer cells. The Aurora kinases have been identified as promising chemotherapeutic targets based on their altered expression in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhibitor (AMG 900) on undifferentiated SW-872 and well-differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0-1000 nM), AZD1152-HQPA (0-1000 nM), and AMG 900 (0-1000 nM) for 72 h resulted in a dose-dependent decrease in the total viable cell number. Based upon the EC50 values, the potency of the three Aurora kinase inhibitors in the SW-872 cells was as follows: AMG 900 (EC50 = 3.7 nM) > AZD1152-HQPA (EC50 = 43.4 nM) > MK-5108 (EC50 = 309.0 nM), while the potency in the 93T449 cells was as follows: AMG 900 (EC50 = 6.5 nM) > AZD1152-HQPA (EC50 = 74.5 nM) > MK-5108 (EC50 = 283.6 nM). The percentage of polyploidy after 72 h of drug treatment (0-1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant increase in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line.
The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipocytes and human mesenchymal stem cells. Western blot analysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immunoreactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differentiated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expression of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.
Sandhya Noronha, Lauren A.C. Alt, Taylor Scimeca, Omran Zarou, Justyna Obrzut, Brain Zanotti, Elizabeth A. Hayward, Akhil Pillai, Shubha Mathur, Joseph Rojas, Ribhi Salamah, Nalini Chandar, Michael J. Fay. Preclinical evaluation of the Aurora kinase inhibitors AMG 900, AZD1152-HQPA, and MK-5108 on SW-872 and 93T449 human liposarcoma cells. In Vitro Cellular & Developmental Biology-Animal, 54:71-84, 2018.