The early response to DNA damage can lead to activation of alternative splicing activity resulting in CD44 splice pattern changes.
Expression of the human papillomavirus 16 E6 oncogene interferes with several vital cellular processes, including the p53-dependent response to DNA damage. To assess the influence of E6 on the early response to DNA damage, we analyzed gene expression following mitomycin C-induced genotoxic stress in human E6-expressing U2OS cells (U2OSE64b) as well as in p53-expressing control cells (U2OSE6AS) by comparative global expression profiling. As expected, genes involved in p53-dependent pathways were activated in p53-expressing cells. In the U2OSE64b cells, however, a largely nonoverlapping group of genes was identified, including two splicing factors of the SR family. Immunoblot analysis revealed increased expression of several SR proteins during the early response to DNA damage, which was accompanied by activation of alternative splicing activity. Disruption of splicing activity by treatment with small interfering RNA directed against splicing factor SRp55 resulted in the increased viability of p53-deficient cells following DNA damage. To determine whether the transient activation of splicing activity was due to E6-mediated degradation of p53, or was due to some other activity of E6, we compared the early response of the p53 wild-type and p53-/- isogenic HCT116 cell lines, and found that the increase in splicing activity was observed only in the absence of p53. Finally, both the U2OSE64b and the p53-/- cells showed altered splicing patterns for the CD44 receptor. Together, these data show that cells lacking p53 can activate alternative splicing following DNA damage.
Filippov
Valery
V
Department of Biochemistry and Microbiology, Loma Linda University School of Medicine, Loma Linda, California 92354, USA.
Filippova
Maria
M
Duerksen-Hughes
Penelope J
PJ
D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001859:Bone Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D015536:Down-Regulation; D020869:Gene Expression Profiling; D020868:Gene Silencing; D045325:HCT116 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000235:genetics*; D016685:Mitomycin / Q000494:pharmacology; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D020033:Protein Isoforms; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D000068103:Serine-Arginine Splicing Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation
D000903:Antibiotics, Antineoplastic; C497483:CD44 protein, human; C068651:E6 protein, Human papillomavirus type 16; D018960:Hyaluronan Receptors; D009687:Nuclear Proteins; D009856:Oncogene Proteins, Viral; D010750:Phosphoproteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C489470:SRSF6 protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D000068103:Serine-Arginine Splicing Factors; D016685:Mitomycin