DRAM-1 encodes multiple isoforms that regulate autophagy

Abstract

Macro(autophagy) is a cellular mechanism which delivers cytoplasmic constituents to lysosomes for degradation. Due to its role in maintaining cellular integrity, autophagy protects against various diseases including cancer. p53 is a major tumor suppressor gene which can modulate autophagy both positively and negatively. p53 induces autophagy via transcriptional activation of Damage-Regulated Autophagy Modulator (DRAM-1). We report here that DRAM-1 encodes not just one mRNA, but a series of p53-inducible splice variants which are expressed at varying levels in multiple human and mouse cell lines. Two of these new splice variants, termed SV4 and SV5, result in mature mRNA species. Different to ‘full-length’ DRAM-1 (SV1), SV4 and SV5 do not localise to lysosomes or endosomes, but instead partially localise to peroxisomes and autophagosomes respectively. In addition, SV4 and SV5 can also be found co-localised with certain markers of the endoplasmic reticulum. Similar to SV1, SV4 and SV5 do not appear to be inducers of programmed cell death, but they do modulate autophagy. In summary, these findings identify new autophagy regulators that provide insight into the control of autophagy downstream of p53.

Keywords:

• DRAM-1
• mRNA splice variants
• p53
• cell death
• autophagy

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

We are grateful to Aviva Tolkovsky for kindly providing us with an adenovirus expressing GFP-LC3 and to Jim Norman, Beatson Institute and Nicholas Ktistakis, Babraham Institute for kindly provided mRFP-Rab7 and eGFP-DFCP1 respectively. We are also thankful to Tony McBryan for help with statistics and to members of the Tumour Cell Death Laboratory for critical reading of the manuscript. Work in the Tumour Cell Laboratory is supported by Cancer Research UK and the Association for International Cancer Research.

Figures and Tables

Figure 1 DRAM-1 splice variants are induced by p53. (A) Tet-on p53 Saos-2 cells were treated with 1 µg/ml of doxycycline for 24 h. RNA was then harvested and analyzed on a 3% agarose gel. (B) p53 induction upon doxycycline treatment was verified by western blotting. (C) Schematic representation of splice variants from (A) which harbor different internal exon deletions. Images shown are representative of what was observed in at least three separate experiments.

Figure 2 DRAM-1 splice variants are expressed in multiple human and mouse cells. (A) RNA from a panel of human cell lines was subjected to semiquantitative RT-PCR with primers from exon 1 and exon 7 of DRAM-1. Induction of p53 in p53-inducible Saos-2 cells was achieved with 1µg/ml of doxycycline for 24 h. (B) RNA from human p53-inducible Saos-2 cells and from MEFs that had been incubated in the absence or presence of Nutlin-3A (10 µM for 48 h) were subjected to semiquantitative RT-PCR with primers from exon 1 and exon 7 of human and mouse DRAM-1 respectively. Induction of p53 in p53-inducible Saos-2 cells was achieved with 1 µg/ml of doxycycline for 24 h. Images shown are representative of what was observed in at least three separate experiments.

Figure 3 DRAM-1 isoforms 1, 4 and 5 do not induce cell death. (A and B) Saos-2 cells were infected with either an empty adenoviral vector, DRAM-1 isoforms 1, 4 or 5, or p53 (positive control). After 48 h, both adherent and floating cells were harvested and the extent of cell death measured by flow cytometry for the percentage of cells with sub-G₁ DNA content. The data represented are from three independent experiments and error bars indicate standard deviation (s.d.) (B). Protein expression of p53 and DRAM-1 isoforms was validated by western blotting (A). Saos2 cells were infected with either an empty adenoviral vector, DRAM-1 isoforms 1, 4, 5 or p53. After 48 h, apoptosis was determined using annexinV-Alexa Fluor 488 and PI staining and analyzed by microscopy (C). Each histogram represents the mean percentage of either Annexin-V positive and PI negative or Annexin-V positive and PI positive cells. Results are the mean ± s.d. of seven determinations (D). Measurement of mitochondrial potential (ΔΨm) was assessed using MitoTracker Green FM which stains mitochondria regardless of ΔΨm and and TMRE which is sequestered only by active mitochondria. The staining was analyzed by microscopy. Each histogram represents the mean percentage of cells positive for both MitoTracker Green and TMRE. Results are the mean±s.d. of four determinations (E). Saos-2 cells were transfected with 10 µg of either: DRAM-1 SV1, SV4 or SV5, p53 (positive control) or an empty plasmid (pCMV; negative control). After 48 h, transfected cell populations were replated with fresh media containing G418 and assessed for clonogenic survival two weeks later (F). Images shown are representative of at least three independent experiments.

Figure 4 DRAM-1 isoforms 1, 4 or 5 cannot rescue the inhibition of oncogene-induced death caused by total loss of dram-1 expression. DRAM-1^fl/fl MEFs expressing the tamoxifen-inducible adenovirus onco-protein E1A-ER were infected with DRAM-1 isoforms 1,4 and 5 or the empty retrovirus vector. Expression of DRAM-1 SV1, 4 and 5 were validated by western blot (A). These panels of cells were then infected with the Cre retrovirus to induce recombination, and an empty vector where indicated (-Cre). DRAM-1 excision was validated at the message level by RT-PCR (B). MEFs were treated with 500 nM of 4-hydroxy tamoxifen for 8 h to activate E1a in this system. The cells were then treated with 0.2 µg/ml doxorubicin. After 24 h, both adherent and floating cells were harvested and the extent of cell death measured by flow cytometry for the percentage of cells with sub-G₁ DNA content. The data are representative of what was observed in three independent experiments and error bars indicate standard deviation (C). Tam, 4-hydroxytamoxifen.

Figure 5 DRAM-1 isoform 1, but not isoforms 4 and 5, locates to lysosomes, as well as early and late endosomes. Saos-2 cells were infected with equivalent amounts of DRAM-1 adenoviral lysates for 48 h. Colocalization (yellow) of myc-tagged DRAM-1 (red) with the respective lysosome and endosome proteins, cathepsin D (A), CD63 (B) EEA1 (C) (green), was assayed by confocal microscopy. The scale bar shown represents 20 µm.

Figure 6 DRAM-1 isoforms 1 and 5 are partially localized with markers of autophagosomes/autolysosomes. Saos-2 cells were infected with an adenovirus carrying mCherry-LC3 transgene (A), and either DRAM-1 isoforms 1, 4 or 5 for 48 h. Colocalization (yellow) of myc-tagged DRAM-1 (green) with LC3 (red) was assayed by confocal microscopy. (B) Saos-2 cells were transfected with pEGFP-DFCP1 (green) using X-fect (Clontech) 24 h prior to infection with DRAM-1 isoforms (red) for 48 h, then harvested for immunofluorescent studies. The scale bar shown represents 20 µm.

Figure 7 DRAM-1 isoforms 4 and 5 are partially localized in the endoplasmic reticulum. Saos-2 cells were infected with equivalent amounts of DRAM-1 adenoviral lysates for 48 h. Colocalization (yellow) of myc-tagged DRAM-1 (red) with the respective endoplasmic reticulum protein, Bip (A) and Calnexin (B) (green), was assayed by confocal microscopy. The scale bar shown represents 20 µm.

Figure 8 DRAM-1 isoform 4 is partially localized in peroxisomes. Saos-2 cells were infected with equivalent amounts of DRAM-1 adenoviral lysates for 48 h. Colocalization (yellow) of myc-tagged DRAM-1 (red) with the peroxisomal protein PMP70 (A) and peroxisome-GFP (B) (green), was assayed by confocal microscopy. The scale bar shown represents 20 µm.

Figure 9 DRAM-1 isoform expression induces autophagosome formation. (A) Saos-2 cells were co-infected with GFP-LC3 and either DRAM-1 isoforms or empty vector for 48 h. Protein expression of DRAM-1 was validated by western blotting. (B) Representative images of cells overexpressing DRAM-1 isoforms displaying autophagosome formation. The scale bar shown represents 20 µm. (C) Quantitation of autophagosome formation. Cells with eight or more GFP-LC3 puncta were scored as positive for cells with accumulated autophagosomes. In each case at least 200 cells were counted and the error bar indicates standard deviation. (D) Saos-2 cells were infected with adenoviruses expressing either DRAM-1 isoforms 1, 4, 5 or ‘empty’ virus as control. Cell lysates were analyzed by western blotting for LC3, DRAM (myc) and actin. The data shown are representative of what was observed in at least three separate experiments.