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Extracellular p53 fragment re-enters K-Ras mutated cells through the caveolin-1 dependent early endosomal system.

Lee SH, Woo TG, Lee SJ, Kim JS, Ha NC, Park BJ - Oncotarget (2013)

Bottom Line: The p53 DNA-binding domain directly associates with caveolin-1 and enters K-Ras mutated cells through early endosome-mediated endocytosis.In contrast, p53-Snail binding occurs at the 143-193 aa region.These results indicate that the p53 fragment might be useful as a specific delivery tool into K- Ras mutated cells as well as a diagnostic method.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, College of Natural Science, Pusan National University, Busan, Republic of Korea.

ABSTRACT
K-Ras mutation is detected in over 30% of human malignancies. In particular, 90% of human pancreatic cancers are initiated by K-Ras mutation. Thus, selective elimination of K-Ras mutated cells would be a plausible strategy to prevent or cure the malignancies. In our previous reports, it has been revealed that oncogenic K-Ras promotes the exocytosis of p53 with Snail. In this study, we have followed the final destination of extracellular p53, which is secreted by the Snail complex. Here we provide evidences that p53, exported from K-Ras-mutated cells, is specifically re-endocytosed by oncogenic K-Ras-containing cancer cells. The p53 DNA-binding domain directly associates with caveolin-1 and enters K-Ras mutated cells through early endosome-mediated endocytosis. Using a serial deletion approach, we revealed that a fragment of human p53 extending from 93-143 amino acids (AA) is responsible for binding with caveolin-1 and for endocytosis. In contrast, p53-Snail binding occurs at the 143-193 aa region. Finally, through in vivo study, we confirmed that injected recombinant p53 could be up-taken by tumor tissues, constructed by oncogenic K-Ras transformed MEF cells. In contrast, the tumors formed by H-Ras mutated MEF cells did not accumulate the injected p53 protein. These results indicate that the p53 fragment might be useful as a specific delivery tool into K- Ras mutated cells as well as a diagnostic method.

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Active internalization of p53 core domain occurs by K-Ras dependent manner(A-B) K-Ras dependent endocytosis was a p53-specific event. RP53 and recombinant Snail (Snail middle region; GST tag; 91-121 amino acids (AA)) were treated in a time-dependent manner for up to 12h in the K-Ras harboring A549 cells. The endocytosis levels were measured by western blot analysis using the indicated antibodies. (C) Internalization of RP53 was appeared through active mechanism. Cells were divided into two groups; one group was incubated with FITC-p53 first and then fixed with 2% PFA, while the other group was exposed to PFA before FITC-p53 treatment. Both groups were stained with PI (propidium iodide; Red) for nuclear staining. (D) Dynamin inhibitor remarkably reduced endocytosis of RP53 in HCT116 cells. After incubation with Dynasore (from 20 to 80 μM) for 3 h, cells were treated with FITC or FITC-p53 for 1 h as an indicated dose. After washing with PBS, cells were harvested and subjected to SDS-PAGE. Western blot analysis was performed with indicated antibodies. (E) Decreased internalization of RP53 in A549 was measured by FACS analysis. (F) Internalized RP53 was quantified by graph. Numbers of Y-axis showed the value of FITC-p53 positive portion/FITC positive portion. After incubation with Dynasore (80 μM) for 3 h, cells were treated with FITC or FITC-p53 (2 μg/ml) for 1 h.
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Figure 2: Active internalization of p53 core domain occurs by K-Ras dependent manner(A-B) K-Ras dependent endocytosis was a p53-specific event. RP53 and recombinant Snail (Snail middle region; GST tag; 91-121 amino acids (AA)) were treated in a time-dependent manner for up to 12h in the K-Ras harboring A549 cells. The endocytosis levels were measured by western blot analysis using the indicated antibodies. (C) Internalization of RP53 was appeared through active mechanism. Cells were divided into two groups; one group was incubated with FITC-p53 first and then fixed with 2% PFA, while the other group was exposed to PFA before FITC-p53 treatment. Both groups were stained with PI (propidium iodide; Red) for nuclear staining. (D) Dynamin inhibitor remarkably reduced endocytosis of RP53 in HCT116 cells. After incubation with Dynasore (from 20 to 80 μM) for 3 h, cells were treated with FITC or FITC-p53 for 1 h as an indicated dose. After washing with PBS, cells were harvested and subjected to SDS-PAGE. Western blot analysis was performed with indicated antibodies. (E) Decreased internalization of RP53 in A549 was measured by FACS analysis. (F) Internalized RP53 was quantified by graph. Numbers of Y-axis showed the value of FITC-p53 positive portion/FITC positive portion. After incubation with Dynasore (80 μM) for 3 h, cells were treated with FITC or FITC-p53 (2 μg/ml) for 1 h.

Mentions: To know that oncogenic K-Ras may promote random endocytosis in regardless of protein species, we compared the endocytosis of Snail and RP53 in K-Ras mutated cells, time-dependently. The intracellular RP53 could be detected after 10 min, and it reached its maximum level after 1 h (Figure 2A). However, the recombinant Snail was not detected in A549 cells (Figure 2B), indicating that A549 cells selectively uptake RP53. In addition, we could observe the decrease of extra- and intracellular RP53 after 4 h (Figure 2A). To test whether RP53 uptake occurs through an active mechanism, we treated FITC-p53 into A549 and MDA-MB-468 cells before and after PFA-fixation. Pre-fixation could completely block the RP53 endocytosis (Figure 2C). In addition, treatment of RP53 at 4°C also blocked the internalization (Figure S1E). Since large GTPase, dynamin is essential for vesicle formation during overall endocytosis [16], we monitored the internalization of RP53 as dynamin dependent endocytic pathway using dynamin inhibitor; dynasore [17]. Treatment of dynasore was remarkably reduced the RP53 endocytosis as dose-dependent manner (Figure 2D). To analyze the quantification of endocytosed RP53, we measured FITC positive cells through FACS analysis and found that entered FITC-p53 was obviously decreased by dynamin inhibitor (Figure 2E and F). These results suggest that internalization of p53 is not non-specific event and occurs via active endocytosis pathway.


Extracellular p53 fragment re-enters K-Ras mutated cells through the caveolin-1 dependent early endosomal system.

Lee SH, Woo TG, Lee SJ, Kim JS, Ha NC, Park BJ - Oncotarget (2013)

Active internalization of p53 core domain occurs by K-Ras dependent manner(A-B) K-Ras dependent endocytosis was a p53-specific event. RP53 and recombinant Snail (Snail middle region; GST tag; 91-121 amino acids (AA)) were treated in a time-dependent manner for up to 12h in the K-Ras harboring A549 cells. The endocytosis levels were measured by western blot analysis using the indicated antibodies. (C) Internalization of RP53 was appeared through active mechanism. Cells were divided into two groups; one group was incubated with FITC-p53 first and then fixed with 2% PFA, while the other group was exposed to PFA before FITC-p53 treatment. Both groups were stained with PI (propidium iodide; Red) for nuclear staining. (D) Dynamin inhibitor remarkably reduced endocytosis of RP53 in HCT116 cells. After incubation with Dynasore (from 20 to 80 μM) for 3 h, cells were treated with FITC or FITC-p53 for 1 h as an indicated dose. After washing with PBS, cells were harvested and subjected to SDS-PAGE. Western blot analysis was performed with indicated antibodies. (E) Decreased internalization of RP53 in A549 was measured by FACS analysis. (F) Internalized RP53 was quantified by graph. Numbers of Y-axis showed the value of FITC-p53 positive portion/FITC positive portion. After incubation with Dynasore (80 μM) for 3 h, cells were treated with FITC or FITC-p53 (2 μg/ml) for 1 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926846&req=5

Figure 2: Active internalization of p53 core domain occurs by K-Ras dependent manner(A-B) K-Ras dependent endocytosis was a p53-specific event. RP53 and recombinant Snail (Snail middle region; GST tag; 91-121 amino acids (AA)) were treated in a time-dependent manner for up to 12h in the K-Ras harboring A549 cells. The endocytosis levels were measured by western blot analysis using the indicated antibodies. (C) Internalization of RP53 was appeared through active mechanism. Cells were divided into two groups; one group was incubated with FITC-p53 first and then fixed with 2% PFA, while the other group was exposed to PFA before FITC-p53 treatment. Both groups were stained with PI (propidium iodide; Red) for nuclear staining. (D) Dynamin inhibitor remarkably reduced endocytosis of RP53 in HCT116 cells. After incubation with Dynasore (from 20 to 80 μM) for 3 h, cells were treated with FITC or FITC-p53 for 1 h as an indicated dose. After washing with PBS, cells were harvested and subjected to SDS-PAGE. Western blot analysis was performed with indicated antibodies. (E) Decreased internalization of RP53 in A549 was measured by FACS analysis. (F) Internalized RP53 was quantified by graph. Numbers of Y-axis showed the value of FITC-p53 positive portion/FITC positive portion. After incubation with Dynasore (80 μM) for 3 h, cells were treated with FITC or FITC-p53 (2 μg/ml) for 1 h.
Mentions: To know that oncogenic K-Ras may promote random endocytosis in regardless of protein species, we compared the endocytosis of Snail and RP53 in K-Ras mutated cells, time-dependently. The intracellular RP53 could be detected after 10 min, and it reached its maximum level after 1 h (Figure 2A). However, the recombinant Snail was not detected in A549 cells (Figure 2B), indicating that A549 cells selectively uptake RP53. In addition, we could observe the decrease of extra- and intracellular RP53 after 4 h (Figure 2A). To test whether RP53 uptake occurs through an active mechanism, we treated FITC-p53 into A549 and MDA-MB-468 cells before and after PFA-fixation. Pre-fixation could completely block the RP53 endocytosis (Figure 2C). In addition, treatment of RP53 at 4°C also blocked the internalization (Figure S1E). Since large GTPase, dynamin is essential for vesicle formation during overall endocytosis [16], we monitored the internalization of RP53 as dynamin dependent endocytic pathway using dynamin inhibitor; dynasore [17]. Treatment of dynasore was remarkably reduced the RP53 endocytosis as dose-dependent manner (Figure 2D). To analyze the quantification of endocytosed RP53, we measured FITC positive cells through FACS analysis and found that entered FITC-p53 was obviously decreased by dynamin inhibitor (Figure 2E and F). These results suggest that internalization of p53 is not non-specific event and occurs via active endocytosis pathway.

Bottom Line: The p53 DNA-binding domain directly associates with caveolin-1 and enters K-Ras mutated cells through early endosome-mediated endocytosis.In contrast, p53-Snail binding occurs at the 143-193 aa region.These results indicate that the p53 fragment might be useful as a specific delivery tool into K- Ras mutated cells as well as a diagnostic method.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, College of Natural Science, Pusan National University, Busan, Republic of Korea.

ABSTRACT
K-Ras mutation is detected in over 30% of human malignancies. In particular, 90% of human pancreatic cancers are initiated by K-Ras mutation. Thus, selective elimination of K-Ras mutated cells would be a plausible strategy to prevent or cure the malignancies. In our previous reports, it has been revealed that oncogenic K-Ras promotes the exocytosis of p53 with Snail. In this study, we have followed the final destination of extracellular p53, which is secreted by the Snail complex. Here we provide evidences that p53, exported from K-Ras-mutated cells, is specifically re-endocytosed by oncogenic K-Ras-containing cancer cells. The p53 DNA-binding domain directly associates with caveolin-1 and enters K-Ras mutated cells through early endosome-mediated endocytosis. Using a serial deletion approach, we revealed that a fragment of human p53 extending from 93-143 amino acids (AA) is responsible for binding with caveolin-1 and for endocytosis. In contrast, p53-Snail binding occurs at the 143-193 aa region. Finally, through in vivo study, we confirmed that injected recombinant p53 could be up-taken by tumor tissues, constructed by oncogenic K-Ras transformed MEF cells. In contrast, the tumors formed by H-Ras mutated MEF cells did not accumulate the injected p53 protein. These results indicate that the p53 fragment might be useful as a specific delivery tool into K- Ras mutated cells as well as a diagnostic method.

Show MeSH
Related in: MedlinePlus