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Autophagy: Role of Ambra-1 in Cell Proliferation in Melanoma

Presentation for PBM 301
by

Rei Lee

on 17 May 2013

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Transcript of Autophagy: Role of Ambra-1 in Cell Proliferation in Melanoma

Autophagy Regulation in Cancer: Role of Ambra-1 in Melanoma Cell Proliferation by Analysis of Ki-67 Introduction Methods Yee Ling Kow and Dr. Jane Armstrong Faculty of Applied Sciences, University of Sunderland, UK Results Western blotting Immunocytochemistry Data Analysis Cell lysate were separated by SDS-PAGE and transferred to PVDF membranes for immunoblotting of Ki-67. Fixed with paraformaldehyde and incubated with anti-Ki67 and examined under a confocal microscope. The data generated from ImageJ were subjected to non-parametric and independent T-test on SPSS. Differences were considered significant when p< 0.05. References Autophagy Figure 1: Process and Regulation of Autophagy. The process is regulated by four subgroups of protein: the ATG1/ ULK complex and the Vps34 / Beclin1 complex which regulates the initiation of autophagy, LC3 and ATG12 which regulates autophagosome elongation and expansion ,and ATG9 and VMP1 which provides the membrane for the formation of autophagosomes. Adapted from Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev. 2011; 25: 1999-2010. Figure 1, The process and regulation of autophagy. Sample Preparation The A375 melanoma cells lines established with overexpression and knockdown of protein Ambra 1 by lentivector gene transfer prepared by cell lysis for Western blotting. Fluorescence data generated from confocal microscopy was analysed using ImageJ.
Manual cell count was also performed to determine the number of cells in specific stages of cycling based on the staining patterns of Ki-67. Figure 2. The PVDF image from Western blotting.
All samples are shown to be displaying two distinct bands. The bands are not of the right sizes for Ki-67, implying that protein might not have transferred through the gel. The red box highlights the position where Ki-67 is believed to be located. Figure 3. Immunocytochemical staining of Ki-67 by confocal microscopy.
The images illustrates the localization and intensity of Ki-67 fluorescence (right) and TOPRO nuclear staining (left) for the tested samples. Proliferating cells were characterized with distinct nucleoli-like formation within nucleus. Cells with dispersed speckles of Ki-67 staining were said to be at early G1 stage of the cell cycle. Figure 3b. Mean Ki-67 fluorescence intensity of sample melanoma cells by ImageJ analysis. r-Ambra cells were found to display lower intensity than its control which implied no significance in proliferation in the overexpressed system. This led to a cell count to assess proliferation based on staining patterns of Ki-67 within the nucleus. Data are expressed as mean±SD (n> 73) . ** denotes P> 0.01 Ki-67 TOPRO r-B-Gal r-Ambra sh-Ctrl sh-Ambra Table 1. Proliferation assessment with respect to Ki-67 staining patterns in the nucleus by cell count. 94% of the cells had the characteristic localization of Ki-67 for r-Ambra, suggesting cell cycling was increased as compared to r-B-galactosidase. Sh-Ambra cells were observed to have fewer cells that were undergoing cell cycling as compared to both the control sh-ctrl and r-Ambra. Discussion Conclusion Initial immunocytochemical analysis of the samples (Fig. 3b) showed lower fluorescence intensities for overexpression of Ambra1. Ki-67 was not well transferred onto the PVDF membrane under the automated protocol used for immunoblotting. Results implicate overexpression of Ambra1 increases proliferation while knockdown decreases proliferation of melanoma cells, supporting the hypothesis and is demonstrated by the results. Further work involving cell synchronization, use of proliferation assay kits and optimization of high molecular weight transfer in automated transferring are necessary to confirm the findings of this study. Acknowledgements The authors would like to thank Mr. Barry Thyne for his help in culturing the A375 melanoma cells for use in this study. 1. Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev. 2011; 25: 1999-2010.
2. Amaravadi RK, Thompson CB. The roles of therapy-induced autophagy and necrosis in cancer treatment. Clin Cancer Res 2007; 13: 7271-7279.
3. Fimia GM, Cozzari M, Antonioli M, Piacentini M. Ambra1 at the crossroad between autophagy and cell death. Oncogene 2012 doi: 10.1038/onc.2012.455
4. Ma XH, Piao S, Wang D et al. Measurement of Tumor Cell Autophagy Predict Invasiveness, Resistance to Chemotherapy, and Survival of Melanoma. Clin Cancer Res 2011; 17: 3478-3489
5. van Dierendonck JH, Keijzer R, Velde CJH et al. Nuclear Distribution of the Ki-67 Antigen during the Cell Cycle: Comparison with Growth Fraction in Human Breast Cancer Cells. A highly conserved process
Maintains homeostasis by removal of damaged proteins and organelles
Recycles them to basic building blocks
Promotes tumorigenesis [1], tumour survival and implicated to contribute to chemotherapy resistance. [2,3,4]
Loss of autophagy results in the loss of ability for tumour suppression and senescence
Is later reactivated due to cellular stress of the tumour such as hypoxia, starvation, and necrosis. [1,2] Ambra1 Novel protein recently found as an activator of autophagy and is a cofactor of Beclin1
Acts as an upstream regulator in the initiation of autophagy; binds only upon induction of autophagy. [3]
Implied to have role in tumour maintenance and proliferation. [4] Objective To investigate the possible role of Ambra1 in tumour proliferation by immunoblotting & immunocytochemical staining of Ki-67, a proliferation marker, in melanoma cells. Hypothesis Overexpression of Ambra1 would induce increased proliferation, reflected by the increase in Ki-67 concentrations & fluorescence intensity.
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