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1.
Cells ; 12(22)2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37998396

RESUMO

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS-/- cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients.


Assuntos
Cricetulus , Cricetinae , Animais , Humanos , Células CHO , Bromodesoxiuridina/metabolismo , Transporte de Elétrons , Proteínas Recombinantes/metabolismo
2.
Mol Cancer Ther ; 16(12): 2881-2891, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28904132

RESUMO

Uterine serous cancer (USC) is aggressive, and the majority of recurrent cases are chemoresistant. Because the receptor tyrosine kinase AXL promotes invasion and metastasis of USC and is implicated in chemoresistance in other cancers, we assessed the role of AXL in paclitaxel resistance in USC, determined the mechanism of action, and sought to restore chemosensitivity by inhibiting AXL in vitro and in vivo We used short hairpin RNAs and BGB324 to knock down and inhibit AXL. We assessed sensitivity of USC cell lines to paclitaxel and measured paclitaxel intracellular accumulation in vitro in the presence or absence of AXL. We also examined the role of the epithelial-mesenchymal transition (EMT) in AXL-mediated paclitaxel resistance. Finally, we treated USC xenografts with paclitaxel, BGB324, or paclitaxel plus BGB324 and monitored tumor burden. AXL expression was higher in chemoresistant USC patient tumors and cell lines than in chemosensitive tumors and cell lines. Knockdown or inhibition of AXL increased sensitivity of USC cell lines to paclitaxel in vitro and increased cellular accumulation of paclitaxel. AXL promoted chemoresistance even in cells that underwent the EMT in vitro Finally, in vivo studies of combination treatment with BGB324 and paclitaxel showed a greater than 51% decrease in tumor volume after 2 weeks of treatment when compared with no treatment or single-agent treatments (P < 0.001). Our results show that AXL expression mediates chemoresistance independent of EMT and prevents accumulation of paclitaxel. This study supports the continued investigation of AXL as a clinical target, particularly in chemoresistant USC. Mol Cancer Ther; 16(12); 2881-91. ©2017 AACR.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzocicloeptenos/farmacologia , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Triazóis/farmacologia , Neoplasias Uterinas/tratamento farmacológico , Animais , Benzocicloeptenos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Paclitaxel/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Triazóis/administração & dosagem , Neoplasias Uterinas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase Axl
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