Identification and characterization of a novel Mdm2 splice variant acutely induced by the chemotherapeutic agents adriamycin and actinomycin D

Abstract

Mdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108 bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2(+108). Importantly, this insertion introduces in-frame nonsense codons, thus encoding a profoundly truncated mdm2 protein lacking the C-terminal RING finger domain and the E3 ubiquitin ligase activity. A wide range of pharmacological testing revealed that Mdm2(+108) is induced, in mouse and rat cells, in specific response to Adriamycin and actinomycin D, but not other modes of DNA damage. Meanwhile, antibodies against the N-terminal region of mdm2 reveal a marked reduction in detectable mdm2 protein upon Adriamycin treatment, while p53 accumulates to strikingly high levels. We thus conclude that this alternative spicing of Mdm2 may be an important mechanism to facilitate massive accumulation of p53 in response to genotoxic agents.

Figure 1

Discovery of a DNA damage-induced alternative mRNA form of Mdm2. (A) Asynchronous NIH3T3, p21^−/− 3T3 and p53^−/− 3T3 cells were treated for eight hours with 1 μg/mL Adriamycin, followed by RT-PCR analysis (as described in materials and methods) with primers for Actin and Mdm2 at indicated cycle numbers. (B) Asynchronous NIH3T3 cells were treated for eight hours with the indicated doses of Adriamycin (μg/mL) followed by RT-PCR analysis with primers for Mdm2. (C) Schematic of the M. musculus Mdm2 gene indicating primers used for the Mdm2 RT-PCR analysis in (A and B).

Figure 2

~100 bp of additional sequence appears between exon 10 and exon 11 in Mdm2 mRNA following Adr treament. (A–C) Asynchronous NIH3T3 (or p53^−/− 3T3 cells in (A) only) were treated for eight hours with 1 μg/mL Adriamycin, followed by RT-PCR analysis with primers corresponding to various regions within the Mdm2 mRNA. (x = exon; Two different exposures are shown for x9-12). (B) Higher-resolution picture. Labels indicate band sizes for the 1 kb DNA ladder.

Figure 3

Sequence of the intron 10-derived 108 bp insert in Mdm2 mRNA following Adr treatment. (A) Diagram of the exon 10–exon 11 region of the mdm2 mRNA before and after Adr treatment. (B) Partial genomic DNA sequence of the mdm2 gene: intron 10 (lower-case letters); exons 10 and 11 (capital letters); the region of intron 10 found in mature Mdm2 mRNA following Adr treatment (bold lower-case). Bars above the sequence indicate the apparent mRNA splicing events. (C) Complete sequence of the additional 108 bp found in mdm2 cDNA following Adr treatment. Bars above indicate putative nonsense codons. D) NIH3T3 cells were treated exactly as in Figure 4C, followed by Mdm2 RT-PCR using a 5′ primer (upper) or 3′ primer (lower) corresponding to sequences located within intron 10.

Figure 4

Pharmacological characterization of Mdm2⁺¹⁰⁸. Following the indicated treatment of NIH3T3 cells, mdm2 (x9-12) was analyzed by RT-PCR as previously. (A) Treatment with 1 μg/mL Adriamycin was for indicated lengths of time. [“ ⊘” indicates that all cells appeared dead or dying at time of harvest.] Pre-treatment, where indicated, was for 30 minutes with 25 μg/mL Cycloheximide, or 500 nM Actinomycin D. Actinmycin D alone was 450 nM for 8 hours; etoposide alone was 25 μM for 8 hours. (B–E) All drug treatments were for six hours. (B) Adr, 750 ng/mL, alone or with 30 minute pre-treatment with 4 mM Caffeine or 100 μM Cycloheximide; ionizing radiation (IR), 10 Gy (6 h recovery); UV, 220 nm, 100 J/m² (6 h recovery). (C) Adr, 750 ng/mL; IR, 25 Gy (6 h recovery); UV, 220 nm, 250 J/m² (6 h recovery), H₂O₂, 1 mM. (D) Adr, 750 ng/mL; HU, 2 mM; aphidicolin, 5 μg/mL; actinomycin D, 45 nM; Caffeine pre-treatment, where indicated was 4 mM for 30 minutes. (E) Adr, 750 ng/mL; cisplatin, 20 μM; actinomycin D, 450 nM.

Figure 5

Induction of Mdm2⁺¹⁰⁸ corresponds with ablation of mdm2 protein and accumulation of p53. NIH3T3 cells were treated for six hours with Adr (750 ng/mL), etoposide (25 μM), IR (10 Gy), UV (220 nM, 100 J/m²), actinomycin D (450 nM), with or without 30 minute pre-treatment with Caffeine (4 mM) or Cycloximide (100 μM). Cells were then lysed and probed for antibodies to mdm2 or p53 by western blotting. NS = nonspecific band.

Figure 6

Mdm2⁺¹⁰⁸ is induced by Adr treatment in C2C12 myoblasts. Asynchronous C2C12 mouse myoblasts were treated with Adr for six hours prior to RT-PCR with a panel of primers to amplify the indicated regions of the Mdm2 transcript.

Figure 7

Ablation of ATR has no effect on the induction of Mdm2⁺¹⁰⁸ by Adr. Rin1 cells were stably infected with retroviruses expressing pSuper-shRNAs directed against Atr or control retroviruses, followed by six hour Adr treatment and RT-PCR for Mdm2 (x9-12).