Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM

• Published in: Cancer research (Chicago, Ill.) Vol. 65; no. 5; pp. 1670 - 1677
• Main Authors: PENG, Yuanlin, WOODS, Rick G, BEAMISH, Heather, YE, Ruiqiong, LEES-MILLER, Susan P, LAVIN, Martin F, BEDFORD, Joel S
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 01.03.2005
• Subjects: Animals; Antigens, Surface; Antineoplastic agents; Ataxia Telangiectasia - metabolism; Ataxia Telangiectasia Mutated Proteins; Biological and medical sciences; Catalytic Domain; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; CHO Cells; Cricetinae; DNA - genetics; DNA - metabolism; DNA - radiation effects; DNA Damage - genetics; DNA Repair - genetics; DNA-Activated Protein Kinase; DNA-Binding Proteins - antagonists & inhibitors; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Down-Regulation; Fibroblasts - metabolism; Fibroblasts - pathology; Gene Expression Regulation; Glioma - metabolism; Glioma - pathology; Humans; Immunoenzyme Techniques; Medical sciences; Membrane Proteins - genetics; Membrane Proteins - metabolism; Nuclear Proteins; Pharmacology. Drug treatments; Protein Subunits; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Interfering - pharmacology; Transfection; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism; Catalytic subunit; Regulation(control); Enzyme; Protein kinase; Transferases; DNA; Deficiency
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-04-3451

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.