The chemotherapeutic agent VP16 increases the stability of HB-EGF mRNA by a mechanism involving the 3'-UTR.
VP16 is a chemotherapeutic agent that introduces DNA damage. We demonstrate that cellular stress induced by VP16 in the human cervix cancer cell line HeLa increases the HB-EGF (heparin binding epidermal growth factor like growth factor) mRNA level dose dependently. Maximal induction (10-fold) was observed at 20-40 microM VP16. Increased HB-EGF peptide levels accompanied the increase in HB-EGF mRNA. We investigated the molecular mechanism involved in HB-EGF mRNA induction by VP16. Transcription was only slightly increased (60%) as determined by real-time PCR quantification of transcription from a reporter plasmid containing the HB-EGF promoter in front of the luciferase gene. In contrast, HB-EGF mRNA stability was increased significantly by VP16 as demonstrated by monitoring HB-EGF mRNA decay in cells treated with the transcriptional inhibitor actinomycin D. The 3'-UTR (3'-untranslated region) of HB-EGF was inserted at the 3'-end of LacZ mRNA. VP16 treatment of the cells caused a 5-fold increase in this chimeric mRNA, as compared to LacZ without this 3'-UTR. A 186 nucleotide region of HB-EGF contains five of the six AUUUA sequences found in the 1454 nucleotide 3'-UTR of HB-EGF and we demonstrate that this region caused an approximately 3-fold induction of LacZ mRNA when inserted at the 3'-end, as compared to LacZ without any insertion at the 3'-end, demonstrating that a significant proportion of the effect resides in this region. Induction of HB-EGF by VP16 has important implications as HB-EGF has been reported to prevent cell death, which might lower the efficacy of chemotherapy. We demonstrate that mRNA stability and in particular the HB-EGF 3'-UTR is involved in the HB-EGF mRNA induction.
Sorensen
Boe S
BS
Department of Clinical-Biochemistry, NBG, AS, Aarhus University Hospital, AS Norrebrogade 44, DK 8000 Aarhus C, Denmark. boess@as.aaa.dk
Ornskov
Dorthe
D
Nexo
Ebba
E
D020413:3' Untranslated Regions; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005047:Etoposide / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D066247:Receptor, ErbB-4; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; D000972:Antineoplastic Agents, Phytogenic; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D005047:Etoposide; C512478:EGFR protein, human; C587689:ERBB4 protein, human; D066246:ErbB Receptors; D066247:Receptor, ErbB-4