Abstract
Aim
To investigate the impact of polyamine deprivation on the transcriptome of CHO cells
Results
Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3 days showed that cessation of growth, associated with increased G1/S transition and inhibition of M/G1 transition was accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi’s anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1).
Conclusions
Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.
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Acknowledgements
This work was conducted with the financial support of Scientific Foundation of Ireland (SFI) Grant No. [13/IA/1841] and Science Foundation Ireland co-funded by ERDF, Grant No. [12/RC/2275_P2].
Supporting information
Supplementary Table 1—Primers used for qRT-PCR validation.
Supplementary Table 2—List of the 50 most compelling up-regulated and down-regulated differentially expressed genes considering fold-changes.
Supplementary Figure 1—(a) RT-qPCR of DE genes chosen for validation: Skp2, Tfdp1, Mad2l1, Ddit3, Parcg, Ackr3, Tbc1d2, Tfrc, Thbs1, Extl1. Biological and technical triplicates were performed. The endogenous gene Gapdh was used. (b) Comparison between fold-changes (FC) from microarray data and relative quantification (RQ) data from RT-qPCR for DE genes validated. The y-axis represents the values for FC and RQ of DE genes. Fold-changes and relative quantification are from DE genes expressed in polyamine-deprived media relative to the genes expressed in polyamine-containing conditions.
Supplementary Figure 2—Western blot of eIF5A2 and alpha-tubulin in CHO-K1 cells in SFM-F10 supplemented with putrescine (+P) and without putrescine (NoP). Triplicates are represented. The human B lymphoblastoid cell line (Raji) was included as positive control.
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Capella Roca, B., Doolan, P., Barron, N. et al. Altered gene expression in CHO cells following polyamine starvation. Biotechnol Lett 42, 927–936 (2020). https://doi.org/10.1007/s10529-020-02841-0
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DOI: https://doi.org/10.1007/s10529-020-02841-0