Mitochondrial ribosomal protein L41 suppresses cell growth in association with p53 and p27Kip1 - PubMed (original) (raw)

Mitochondrial ribosomal protein L41 suppresses cell growth in association with p53 and p27Kip1

Young A Yoo et al. Mol Cell Biol. 2005 Aug.

Abstract

The p53 protein arrests the cell cycle at the G1 phase when stabilized by the interaction between ribosomal proteins and HDM2 under growth-inhibitory conditions. Meanwhile, p53, when translocated to the mitochondria in response to cell death signals, induces apoptosis via transcription-independent mechanisms. In this report, we demonstrate that the mitochondrial ribosomal protein L41 (MRPL41) enhances p53 stability and contributes to p53-induced apoptosis in response to growth-inhibitory conditions such as actinomycin D treatment and serum starvation. An analysis of MRPL41 expression in paired normal and tumor tissues revealed lower expression in tumor tissue. Ectopic MRPL41 expression resulted in inhibition of the growth of cancer cells in tissue culture and tumor growth in nude mice. We discovered that MRPL41 protein is localized in the mitochondria, stabilizes the p53 protein, and enhances its translocation to the mitochondria, thereby inducing apoptosis. Interestingly, in the absence of p53, MRPL41 stabilizes the p27(Kip1) protein and arrests the cell cycle at the G1 phase. These results suggest that MRPL41 plays an important role in p53-induced mitochondrion-dependent apoptosis and MRPL41 exerts a tumor-suppressive effect in association with p53 and p27 (Kip1).

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Figures

FIG. 1.

MRPL41 expression in various cells. (A) Physical and transcriptional mapping of the MRPL41 gene. MRPL41 is located within a 135-Mb region between the genetic markers D9S905 and D9S1838 on chromosome 9q34.3. Genetic markers are marked at the top. The intron-exon organization of the MRPL41 gene is depicted as solid lines. The boxes represent two exons, and a black box represents the open reading frame. Vertical arrows under exon 2 indicate the predicted translational start and stop sites. (B) Amino acid sequence of the predicted protein encoded by MRPL41. The open box indicates the predicted mitochondrial leader sequence. (C) The analysis of MRPL41 expression in human normal and tumor tissues. Northern blot analyses were performed to determine MRPL41 expression in various normal and tumor tissues. Normal human 12-lane multiple tissue Northern blot (left panel) and human tumor MTN Northern blot (right panel) were hybridized with 146-bp 32P-labeled HindIII fragments of MRPL41 or a human β_-actin_-specific probe (Clontech). (D) Comparison of the expression of MRPL41 in premalignant kidney cells and kidney tumor cells (left panel) and the expression of MRPL41 in various cancer cell lines (right panel). Northern blot containing total RNA (10 μg) was performed with a 146-bp 32P-labeled HindIII fragment of MRPL41 and GAPDH probe. Cell lines are as follows: NCI-H211, small-cell lung cancer; SNU-638, gastric carcinoma; SW-620, colon cancer; MCF-7, breast carcinoma; HNSCC-PCI50, head and neck squamous cell carcinoma. (E) Comparison of the expression of MRPL41 in non-small-cell lung cancer (NSCLC) cell lines and small-cell lung cancer (SCLC) cell lines. The 146-bp 32P-labeled HindIII fragments of MRPL41 and GAPDH probes were used. BEAS2B, premalignant lung cells.

FIG. 1.

FIG. 2.

The localization of MRPL41. H1299 cells were transfected with _MRPL41_-GFP or a control vector, fixed, and analyzed via confocal microscopy 48 h later. Mitochondria were stained with MitoTracker Red CMXRos dye. (A) GFP-tagged _MRPL41_-transfected cells. (B) Cells stained with MitoTracker Red dye. (C) Merged image demonstrating the colocalization of MRPL41 and mitochondria. (D) DAPI staining. (E) Western blot analysis with 10 μg mitochondrial and cytosolic extracts of NCI-H211 cells stably transfected with Myc-tagged MRPL41 or the Myc control vector (pcDNA3.1/myc-His). Ten micrograms of the fraction was separated, transferred onto the membranes, and incubated with anti-Myc antibody. The membrane was then reblotted with either COXIV, the mitochondria marker (Clontech), or the cytoplasm marker anti-Hsp70 antibody. C, cytosolic fraction; M, mitochondrial fraction.

FIG. 3.

Growth inhibition of tumor cells by MRPL41 in vitro and in vivo. (A) The enumeration of ACHN colonies. ACHN cells were transfected with MRPL41, cultured in suspension with G418, and stained with methylene blue, and colonies were counted. Double asterisk, P < 0.001 versus the vector (Student's t test). (B) NCI-H211 cells were transfected with MRPL41, cultured in soft agar with G418, and stained with nitroblue tetrazolium, and colonies were counted. The data are expressed as the means ± standard deviations (SD) from three independent experiments. Vector, pcDNA3; _MRPL41_-S, pcDNA3-MRPL41. (C) Northern blot of NCI-H211. The ectopic expression of MRPL41 in stable transfectant cells was confirmed by Northern blotting. The expression levels of endogenous (endo) and exogenous (exo) MRPL41 are shown. (D) The enumeration of NCI-H211 colonies. NCI-H211 cells were stably transfected with MRPL41 or control vector, cultured for 5 weeks in soft agar in the presence of G418, and stained with nitroblue tetrazolium, and colonies were counted. The data represent the means of three independent experiments ± SD. (E) The BrdU labeling assay. NCI-H211 cells stably transfected with MRPL41 or control vector were labeled with BrdU as described in Materials and Methods. Incorporated BrdU was measured by reading optical densities at 405 nm against the reference of 490 nm. The results are presented as the means of three independent experiments ± SD. (F) The effect of MRPL41 on the formation of tumors in vivo. Five animals were injected with NCI-H211 cells stably transfected with MRPL41 or control vector, as described in Materials and Methods. Tumors were measured every 3 days for 45 days. The data are expressed as the means of three independent experiments ± SD. Asterisk, P < 0.05 versus the vector (Student's t test). (G) The expression of introduced MRPL41 in nude mice was confirmed via Northern blot.

FIG. 3.

FIG. 4.

MRPL41-induced cell cycle arrest at G1. Flow cytometry of NCI-H211 stably transfected with MRPL41 or control vector. Cells were harvested, stained with propidium iodide, and analyzed by FACScan flow cytometry. Over 10,000 cells were acquired for analysis. The data are expressed as the means of three independent experiments ± standard deviations.

FIG. 5.

MRPL41 stabilized p53. (A) Western blot analysis. Forty micrograms of cell lysates of NCI-H211 cells stably transfected with _MRPL41_-Myc or control vector was resolved via SDS-PAGE, and Western blotting with anti-p53, anti-MDM2, or anti-β-actin antibodies was carried out. Increasing amounts of _MRPL41_-Myc (0.1 μg, 0.3 μg, 0.5 μg, and 1 μg) were transfected into NCI-H211 (B) and HEK-293T cells (C), and Western blotting with anti-p53, anti-Myc, or anti-β-actin antibodies was carried out. (D) Northern blot analysis of p53 in stable transfectant NCI-H211 cells. (E) Western blot analysis of NCI-H1299 cells cotransfected with equal amounts of p53 and increasing amounts of _MRPL41_-Myc. The lysates of cells cotransfected with 0.1 μg of p53 or increasing amounts of _MRPL41_-Myc (0.1 μg, 0.3 μg, 0.5 μg, and 1 μg) were resolved by SDS-PAGE and stained with anti-p53, anti-Myc, or anti-β-actin antibodies. (F) Effect of cycloheximide on p53-protein stability. NCI-H211 cells stably transfected with MRPL41 or control vector were treated with cycloheximide (50 μg/ml). In NCI-H1299 cells, cells were transfected either with p53 alone or with MRPL41 and treated with cycloheximide 48 h later. Extracts were subjected to SDS-PAGE, and p53 stability was evaluated via Western blot with anti-p53 antibody. (G) In vivo ubiquitination assay. H1299 cells were transfected with 0.1 μg of p53 and increasing amounts of _MRPL41_-Myc (0.3 μg and 1 μg). After 24 h, 10 μM of MG132 was treated for 6 h and then harvested. The lysates of cells were immunoprecipitated with anti-p53 antibody, followed by Western blot by anti-Ub antibody.

FIG. 6.

MRPL41 stabilized p27Kip1 and induced cell cycle arrest at the G1 phase in the p53-null NCI-H1299 cells. (A) Western blot analysis. Forty micrograms of cell lysates of NCI-H211 cells stably transfected with MRPL41 or control vector was resolved by SDS-PAGE, and Western blotting with anti-p27 Kip1 or anti-β-actin antibodies was carried out. (B) Forty micrograms of cell lysate of NCI-H211 cells transiently transfected with MRPL41 or control vector was resolved by SDS-PAGE, and Western blotting with anti-p27 Kip1 or anti-β-actin antibodies was performed. (C) Forty micrograms of cell lysates of NCI-H1299 cells transiently transfected with MRPL41 or control vector was resolved by SDS-PAGE, and Western blotting with anti-p27Kip1, anti-Myc, or anti-β-actin antibodies was performed. (D) Northern blot analysis of p27 Kip1 in stable transfectant NCI-H211 cells. (E) Effects of cycloheximide on p27 Kip1 protein stability. NCI-H1299 cells were transfected with p53 alone or with MRPL41 and then treated with cycloheximide 48 h later. Extracts were subjected to SDS-PAGE, and p27 Kip1 stability was assessed by Western blotting with anti-p27 Kip1 antibody. (F) NCI-H1299 cells were transfected with GFP or GFP-tagged MRPL41 and stained with propidium iodide 48 h later. The DNA content of GFP-expressing cells was quantitated by two-color fluorescence-activated cell sorting. Over 5,000 cells were used for this analysis. (G) One hundred nanomolar of p27Kip1 siRNA or a control RNA duplex was transiently transfected into NCI-H211 cells expressing MRPL41 for 24 h. After 24 h, 40 μg cell lysates was resolved by SDS-PAGE, and Western blotting with anti-p27Kip1, anti-Myc, or anti-β-actin antibodies was performed. (H) For flow-cytometric analysis, cells were harvested, stained with propidium iodide, and analyzed by FACScan flow cytometry. Over 10,000 cells were acquired for analysis.

FIG. 7.

MRPL41 induced p53-dependent apoptosis. (A) Effects of MRPL41 on p53-mediated apoptosis in H1299 cells. p53, GFP, and _MRPL41_-GFP were transfected into H1299 cells as indicated. Twenty-four hours after transfection, cells were stained with propidium iodide, and the DNA content of GFP-expressing cells was quantitated. Over 5,000 cells were used in this analysis. As a positive control, p53 was transfected into H1299 cells. Twenty-four hours after transfection, cells were treated with 5 μM camptothecin for 6 h. (B) MRPL41 induces cytochrome c release in cells cotransfected with p53 and MRPL41. H1299 cells cotransfected with equal amounts of p53 and increasing amounts of _MRPL41_-Myc (0.1 μg, 0.3 μg, and 0.5 μg), and mitochondrial (pellet) and cytosolic fractions (supernatant) were prepared from transfected cells. Ten micrograms of fraction was loaded, and Western blot analysis of cytochrome c, p53, COXIV, and PCNA was performed. wce, whole-cell extract.

FIG. 8.

MRPL41 induced the mitochondrial localization of p53. (A) The translocation of p53 induced by MRPL41. H1299 cells were transfected with either p53 alone or with _MRPL41_-Myc. After 24 h, cells were fixed and stained with anti-p53 antibody (green) in order to visualize the localization of p53. The location of p53 was detected by green fluorescence and was observed under confocal microscopy. Nuclei were stained with DAPI dye. As a positive control, p53 was transfected into H1299 cells. At 24 h after transfection, cells were treated with 5 μM camptothecin for 6 h. (B) H1299 cells were cotransfected with equal amounts of p53 and increasing amounts of _MRPL41_-GFP (0.1 μg, 0.3 μg, 0.5 μg, and 1 μg). Graphs show the percentage of cells exhibiting mitochondrial p53 staining for MRPL41-GFP staining. A total of 300 cells were scored to ascertain the location of p53 under each experimental condition. Data shown represent three independent experiments. (C) The subcellular localization of p53 and MRPL41-GFP was confirmed by confocal microscopy.

FIG. 8.

FIG. 9.

MRPL41 is essential for p53 and p27 activation on growth-inhibitory conditions. (A) NCI-H211 cells stably expressing MRPL41-Myc were transfected with MRPL41 RNAi or with a control RNA duplex and were harvested 24 h later. Forty micrograms of cell lysates was resolved by SDS-PAGE, and Western blotting with anti-p53, anti-p27Kip1, anti-Myc, or anti-β-actin antibodies was performed. (B) HEK-293T and NCI-H211 cells were treated with actinomycin D (5 nM) or serum starved and then harvested 24 h later. Forty micrograms of cell lysates was resolved by SDS-PAGE, and Western blotting with anti-p53, anti-p27Kip1, anti-MRPL41, or anti-β-actin antibodies was performed. (C) HEK-293T and NCI-H211 cells were transfected with MRPL41 RNAi or with a control scrambled RNA duplex for 24 h, followed by actinomycin D treatment (5 nM) for an additional 24 h. Forty micrograms of cell lysates was resolved by SDS-PAGE, and Western blotting with anti-p53, anti-p27Kip1, anti-MRPL41, or anti-β-actin antibodies was performed.

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Mitochondrial ribosomal protein L41 suppresses cell growth in association with p53 and p27Kip1 - PubMed (original) (raw)