mot-2-Mediated cross talk between nuclear factor-B and p53 is involved in arsenite-induced tumorigenesis of human embryo lung fibroblast cells

Abstract

Background: Inactivation of p53 is involved in arsenite-induced tumorigenesis; however, the molecular mechanisms remain poorly understood.

Objective: We investigated the molecular mechanisms underlying the inactivation of p53 and neoplastic transformation induced by arsenite in human embryo lung fibroblast (HELF) cells.

Methods: Anchorage-independent growth assays were performed, and tumorigenicity in intact animals was assessed to confirm arsenite-induced neoplastic transformation. We determined the levels and functions of p53, nuclear factor-kappa B (NF-B; a key transcriptional regulator), and mot-2 (a p53 inhibitor) and their relationships in arsenite-induced transformed HELF cells by two-dimensional electrophoresis, reverse-transcriptase polymerase chain reaction, Western blot, immunofluorescence, and co-immunoprecipitation assays.

Results: Exposure of HELF cells to low levels of arsenite increased their proliferation rate and anchorage-independent growth and disrupted normal contact inhibition. When introduced into nude mice, transformed cells were tumorigenic. We used proteomic analysis to identify proteins with altered expression between untreated and arsenite-exposed cells. We found decreased expression of NF-B repressing factor (NKRF; an inhibitor of NF-B-mediated gene transcription), increased expression of mot-2, and increased activation of NF-B. Changes in cells exposed to 1.0 microM arsenite were more marked than changes in cells exposed to 0.5 or 2.0 microM arsenite. Inactivation of NF-B prevented malignant transformation induced by 1.0 microM arsenite. Moreover, we also identified a mechanism whereby NF-B regulated p53. Specifically, activation of NF-B up-regulated mot-2 expression, which prevented nuclear translocation of p53 and switched the binding preference of the p53 and NF-B coactivator CBP [cyclic AMP-responsive element binding protein (CREB) binding protein] from p53 to NF-B.

Conclusions: mot-2-mediated cross talk between NF-B and p53 appears to be involved in arsenite-induced tumorigenesis of HELF cells.

Figure 1

Neoplastic transformation of HELF cells exposed to 0.0, 0.5, 1.0, or 2.0 μM arsenite (As) for about 15 weeks (30 passages). C, control (untreated) HELF cells. A549 carcinoma cells served as the positive control. Morphological images of cells (A) after culture with 10% FBS (bars = 500 μm) and (B) after culture with 1% FBS (bars = 50 μm). (C) Photomicrographs of cell colonies in soft agar; bars = 500 μm. (D) The number (mean ± SD) of cell colonies in soft agar (n = 3). (E) Representative pathological sections of tumors 4 weeks after cells were inoculated into nude/BALB/c mice; tumors induced by arsenite-transformed cells consisted of undifferentiated and spindle cells (bars = 100 μm). (F) Volume (mean ± SD) of tumors in nude/BALB/c mice (n = 6). *p < 0.01 compared with control group. **p < 0.05 compared with 0.5 or 2.0 μM arsenite groups. ^#p < 0.05 compared with mice implanted with cells exposed to 0.5 μM arsenite.

Figure 2

The levels of NKRF and mot-2 and the activation of NF-κB in HELF cells transformed by arsenite (As). β-Actin levels, measured in parallel, served as controls. (A) Levels of NKRF, p-RelA, RelA, and mot-2 in HELF cells exposed to 0.0, 0.5, 1.0, or 2.0 μM arsenite for about 15 weeks (30 passages) as detected by Western blots. (B) Relative protein levels (mean ± SD) of NKRF, p-RelA, and mot-2 (n = 3). (C) Western blot analyses of NKRF, mot-2, p-RelA, and RelA in HELF cells exposed to 0.0 μM (top) or 1.0 μM (bottom) arsenite for 1, 5, 10, 15, 20, 25, or 30 passages. (D) Relative protein levels (mean ± SD) of NKRF, p-RelA, and mot-2 in passage control cells (top) and in cells exposed to 1.0 μM arsenite (bottom); n = 3. *p < 0.01 compared with passage control cells. **p < 0.05 compared with cells exposed to 0.5 or 2.0 μM arsenite.

Figure 3

Effects of blocking NF-κB on mot-2 protein and mRNA levels and NF-κB activation in HELF cells exposed to arsenite (As). HELF cells were exposed to 10 μM Bay11-7082 (Bay) for 3 hr or to 20 nM control (Con) siRNA or RelA siRNA for 24 hr, then incubated with 0.0 or 1.0 μM arsenite for 3 hr (p-IκBα and p-RelA) or 24 hr (mot-2, IκBα, and RelA). β-Actin levels, measured in parallel, served as controls. (A) Western blot analyses of mot-2, p-IκBα, IκBα, p-RelA, and RelA (top), and RT-PCR analyses of mot-2 (bottom). (B) Relative protein or mRNA levels of mot-2 (mean ± SD; n = 3). ^##p < 0.01 compared with cells exposed to arsenite plus Bay11-7082 or arsenite plus RelA siRNA.

Figure 4

Involvement of NF-κB and mot-2 in the activation and translocation of p53 in HELF cells exposed to arsenite (As). HELF cells were exposed to 10 μM Bay11-7082 (Bay) for 3 hr or to 20 nM control (Con) siRNA, RelA siRNA, or mot-2 siRNA for 24 hr, then incubated with 0.0 or 1.0 μM arsenite for 3 hr (p-p53, p-IκBα, and p-RelA) or 24 hr (p53, mot-2, RelA, and IκBα); fluorescence intensities were analyzed with a multimode microplate reader. β-Actin levels, measured in parallel, served as controls. (A) Western blot analyses of p-p53, p53, mot-2, p-RelA, RelA, p-IκBα, and IκBα. (B) Relative protein levels (mean ± SD) of p-p53, p53, and mot-2 (n = 3). (C) Immunofluorescence staining for p53 localization in HELF cells: blue, DAPI staining for nuclear DNA; red, p-p53. Bar, 25 μm. (D) Relative intensity (mean ± SD) of nuclear p53 fluorescence (n = 3). ^##p < 0.05 compared with cells exposed to arsenite plus Bay11-7082, arsenite plus RelA siRNA, or arsenite plus mot-2 siRNA.

Figure 5

mot-2–mediated cross talk between NF-κB and p53 in HELF cells exposed to arsenite (As). (A) Western blot analyses of mot-2 and p53 after cell lysates were subjected to co-immunoprecipitation with p53 (IP) and mot-2 (IB) antibodies. HELF cells were exposed to 10 μM Bay11-7082 for 3 hr or to 20 nM control (Con) siRNA or RelA siRNA for 24 hr, then incubated with 0.0 or 1.0 μM arsenite for 24 hr. (B) Relative protein levels (mean ± SD) of mot-2 (n = 3). (C) Western blot analyses of RelA, p53, and CBP after cell lysates were subjected to co-immunoprecipitation with CBP (IP) and RelA or p53 (IB) antibodies. After mot-2–normal or mot-2–defective HELF cells were exposed to 10 μM Bay11-7082 for 3 hr, they were incubated with 0.0 or 1.0 μM arsenite for 24 hr. (D) Relative protein levels of RelA and p53 (mean ± SD; n = 3). ^##p < 0.05 compared with cells exposed to arsenite plus Bay11-7082 or arsenite plus RelA siRNA. ^†p < 0.05 compared with corresponding arsenite exposure in mot-2–knockdown cells. ^††p < 0.05 compared with corresponding arsenite and Bay11-7082 exposure in mot-2–knockdown cells.

Figure 6

Involvement of NF-κB in arsenite-induced tumorigenesis in HELF cells exposed for about 15 weeks (30 passages) to 0.0 or 1.0 μM arsenite (As), to 10 μM Bay11-7082 (Bay), or to arsenite plus Bay11-7082. A549 cells served as the positive control (Con). (A) Cell colonies in soft agar; bars = 500 μm. (B) Number of cell colonies in soft agar (mean ± SD; n = 3). (C) Representative pathological sections of tumors 4 weeks after cells were inoculated into nude/BALB/c mice. Tumors induced by arsenite-transformed cells were composed of undifferentiated and spindle cells; bars = 100 μm. (D) Volume (mean ± SD) of tumors in nude/BALB/c mice (n = 6). *p < 0.01 compared with control cells, Bay11-7082 cells, or arsenite plus Bay11-7082 cells.