DTT-Induced Transcriptome Profiling Reveals Divergent ER Proteostasis and Stress-Adaptation Programs in CHO-S/HB8 and CHO-4BGD/DUL Producer Clones

Abstract

Chinese hamster ovary (CHO) producer cells differ in their capacity to accommodate secretory and endoplasmic reticulum (ER) stress, but how producer and host-cell backgrounds are associated with ER-proteostasis responses remains poorly understood. We compared 24 h dithiothreitol (DTT)-induced reductive ER stress responses in two clonal producers: CHO-S-derived HB8 secreting human chorionic gonadotropin and apoptosis-resistant CHO-4BGD-derived DUL secreting a GLP-1–Fc fusion protein with similar specific productivities. Responses were analyzed by strand-specific RNA-seq, Xbp1-splicing RT-PCR, immunoblotting, functional enrichment, and curated-module analysis. Analysis of the complete three-replicate dataset identified 299 DTT-responsive genes in HB8 and 877 in DUL, demonstrating a broader transcriptional response in the DUL clone. Analysis after exclusion of the atypical HB8#3 matched pair yielded 404 and 1019 differentially expressed genes, respectively, and was used for detailed sensitivity analysis. Both producers showed ISR/ATF4/CHOP-associated transcriptional activation and Xbp1 mRNA splicing, whereas changes in total Xbp1 and Hspa5/BiP transcript abundance were limited. BiP and CHOP accumulation was detected at the protein level, while phospho-eIF2α and ATF6 responses were variable. Baseline RNA-seq data comparison revealed extensive transcriptional divergence between HB8 and DUL. HB8 showed higher expression of several classical ER folding/redox factors, whereas DUL showed higher expression of selected ISR-, quality-control- and stress-survival-associated genes. DUL additionally displayed broader vesicle/endocytic and amino-acid/glutathione-related remodeling. Thus, the producers occupy distinct ER-proteostasis states, and DUL mounts a broader, but not uniformly stronger, canonical UPR response to reductive ER stress.