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Title: Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. -more » Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [1] ;  [3]
  1. Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI (United States)
  2. Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI (United States)
  3. (United States)
  4. Division of Genetics, Department of Pathology, Brown University and Women and Infants Hospital, Providence, RI (United States)
Publication Date:
OSTI Identifier:
22462309
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 335; Journal Issue: 2; Other Information: Copyright (c) 2015 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; APOPTOSIS; BIOLOGICAL PATHWAYS; BLEOMYCIN; CELL CYCLE; CELL PROLIFERATION; DNA DAMAGES; DNA REPAIR; DNA REPLICATION; GENES; INFLAMMATION; LIVER; MASS SPECTROSCOPY; MESSENGER-RNA; PHOSPHORYLATION; POLYPLOIDY; PROTEINS; REDUCTION; SIGNALS; SPLICING