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. 2020 Apr 30;43(4):397-407.
doi: 10.14348/molcells.2020.2231.

DNAJB9 Inhibits p53-Dependent Oncogene-Induced Senescence and Induces Cell Transformation

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Free PMC article

DNAJB9 Inhibits p53-Dependent Oncogene-Induced Senescence and Induces Cell Transformation

Hyeon Ju Lee et al. Mol Cells. .
Free PMC article

Abstract

DNAJB9 is known to be a member of the molecular chaperone gene family, whose cellular function has not yet been fully characterized. Here, we investigated the cellular function of DNAJB9 under strong mitogenic signals. We found that DNAJB9 inhibits p53-dependent oncogene-induced senescence (OIS) and induces neoplastic transformation under oncogenic RAS activation in mouse primary fibroblasts. In addition, we observed that DNAJB9 interacts physically with p53 under oncogenic RAS activation and that the p53-interacting region of DNAJB9 is critical for the inhibition of p53-dependent OIS and induction of neoplastic transformation by DNAJB9. These results suggest that DNAJB9 induces cell transformation under strong mitogenic signals, which is attributable to the inhibition of p53-dependent OIS by physical interactions with p53. This study might contribute to our understanding of the cellular function of DNAJB9 and the molecular basis of cell transformation.

Keywords: DNAJB9; RAS; p53; senescence; transformation.

Conflict of interest statement

CONFLICT OF INTEREST

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. DNAJB9 inhibits H-RASV12-induced senescence in MEFs (A-D) and HDFs (E-H).
(A and E) Cells were infected with retroviruses as indicated (V, vector; JB9, DNAJB9; Ras, H-RASV12). Then, 1 × 104 cells were seeded and the number of cells was counted. Data are represent as mean ± SD (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001 vs V + V; n.s, not significant). (B and F) The ratios of SA-β-gal (+) cells were calculated. Data are represent as mean ± SD (n = 8; *P < 0.05 vs vector, # P < 0.05). (C and G) Representative SA-β-gal staining data were shown. Scale bars = 13.6 µm. (D and H) Western blot analysis was performed with cell lysates. β-Actin was used as a loading control.
Fig. 2
Fig. 2. DNAJB9 inhibits p53-induced senescence in MEFs (A-D) and Saos-2 cells (E and F).
(A and E) Cells were infected with retroviruses as indicated (V, vector; JB9, DNAJB9). Then, 1 × 104 cells were seeded and the number of cells was counted. Data are represent as mean ± SD for (A) and SEM for (E) (n = 5; *P < 0.05, **P < 0.01, ***P < 0.001 vs V + V; n.s, not significant). (B and F) The ratios of SA-β-gal (+) cells were calculated. Data are represent as means ± SD (n = 6 for Fig. 2B and n = 9 for Fig. 2F; *P < 0.05, ***P < 0.001 vs vector, # P < 0.05, ### P < 0.001). (C) Representative SA-β-gal staining data was shown. Scale bars = 13.6 µm. (D) Western blot analysis was performed with cell lysates. β-Actin was used as a loading control.
Fig. 3
Fig. 3. DNAJB9 induces neoplastic transformation with H-RASV12 in MEFs.
(A) Soft agar assay. MEFs were infected with retroviruses as indicated. Then, 2.5 × 104 cells were seeded on the soft agar and the total number of foci was counted. Data are represent as mean ± SD (n = 4; *P < 0.05 vs vector; n.s, not significant). (B) Representative data of soft agar assay was shown. Scale bars = 100 µm. (C-E) In vivo tumor formation assay. MEFs were infected with retroviruses and injected into nude mice. Total number of tumors per group (C), tumor volumes (D), and resected tumors (E) were shown. Data are represent as mean ± SEM (n = 8; *P < 0.05, **P < 0.01, ***P < 0.001 vs vector + vector; n.s, not significant).
Fig. 4
Fig. 4. DNAJB9 interacts physically with p53 under H-RASV12 expression.
MEFs were infected with retroviruses as indicated. (A) Cell lysates were subjected to immunoprecipitation using an anti-DNAJB9 antibody, followed by western blotting using an anti-p53 antibody (1c12) (upper panel) and the anti-DNAJB9 antibody (lower panel). (B) Confocal microscopy was performed using an anti-DNAJB9 antibody (red), an anti-p53 antibody (green), and DAPI (blue) as described in Materials and Methods section. Merged images of red and green channels were also shown. Scale bar = 50 µm. Magnification, 400×.
Fig. 5
Fig. 5. The DNAJB9-p53 interaction is involved in DNAJB9-mediated inhibition of p53- (B-D) and H-RASV12-induced senescence (E and F).
(A) A schematic representation of the structure of human DNAJB9 and its deletion mutants. (B and E) MEFs were infected with retroviruses as indicated (V, vector; Ras, H-RASV12). Then, 1 × 104 cells were seeded and the number of cells was counted. Data are represent as mean ± SEM (n = 15 for Fig. 5B and n = 10 for Fig. 5E; **P < 0.01, ***P < 0.001 vs V + V; n.s, not significant). (C and F) The ratios of SA-β-gal (+) cells were calculated. Data are represent as mean ± SD (n = 26 for Fig. 5C and n = 8 for Fig. 5F; **P < 0.01, ***P < 0.001 vs vector, ### P < 0.001; n.s, not significant). (D) Representative SA-β-gal staining data was shown. Scale bars = 13.6 µm.
Fig. 6
Fig. 6. The DNAJB9-p53 interaction is involved in DNAJB9-mediated neoplastic transformation.
(A) Soft agar assay. MEFs were infected with retroviruses as indicated. Cells were seeded on the soft agar and the total number of foci was counted. Data are represent as mean ± SD (n = 4; *P < 0.05 vs vector, # P < 0.05; n.s, not significant). (B) Representative data of soft agar assay was shown. Scale bars = 100 µm. (C-E) In vivo tumor formation assay. MEFs were infected with retroviruses and injected into nude mice. Total number of tumors per group (C), tumor volumes (D), and resected tumors (E) were shown. Data are represent as mean ± SD (n = 8; *P < 0.05, **P < 0.01, ***P < 0.001 vs vector + vector; n.s, not significant).

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