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Investigation of The Role of Protein Phosphatase PPM1D/Wip1 in Basal and Genotoxic Stress Induced Autophagy
Turkey (TurkMSIC) - Adnan Menderes University, Aydin
Aydin Adnan Menderes University, ADU-BILTEM Center Research Lab. ADU Central Campus, Mesire Str., Aydin Adnan Mendere Universitesi Rd., Kepez Region, 09100, Aydin / TURKEY
Mehtap Kilic Eren, PhD
Mehtap Kilic Eren, PhD
Type of Research Project
- Basic science
What is the background of the project?
Please, provide the background of the project (Provide information about the project; min. 10 lines, max. 25 lines) PPM1D (Protein Phosphatase 1D) Wip1(Wild-type p53-induced phosphatase 1) is a Serine/threonine-specific protein kinase induced by p53(a tumor suppressor protein) in response to genotoxic stress in normal cells thus plays a central role in DNA damage response (DDR). In most of the human cancers, Wip1 is known to be impaired, amplified, and overexpressed. Overexpressed Wip1 negatively regulates the ATM/ATR (A-T mutated gene) mediated DNA damage response, DNA repair, cell cycle checkpoints, and global cellular stress responses such as apoptosis, and cellular senescence. Recently in mouse embryonic fibroblasts (MEFs) it was shown that genotoxic stress induced Wip1 dephosphorylates Ulk1 (Unc-51 like autophagy activating kinase 1) and accordingly triggers induction of autophagy.
What is the aim of the project?
If the project is successfully performed, the role of Wip1 in increased basal autophagic activity in mediating survival and maintenance of cancer cells, it’s role in genotoxic stress induced autophagy, third the relationship between autophagy and other cellular responses that may be important for anti-cancer therapies will be emerged. On the other hand, the answer to the question whether Wip1-mediated autophagy would be a therapeutic target in these tumors will be obtained. Thus, the proposed project is also of great importance in terms of generating valuable information that will potentially contribute to the identification of pharmacological and therapeutic targets and development of effective and advanced cancer therapy strategies.
What techniques and methods are used?
What techniques and methods are used? Mention the steps/stages in this project. (Provide examples and avoid abbreviations; which tools will be used in general in this project?) Hence utilizing Wip1 over-expressing cancer cell lines MCF-7(human breast adenocarcinoma cell line), IMR-32(human neuroblastoma cell line) and D283 cells and Flag-Wip1 overexpressing Hela cells; Wip1-Ulk1 dependent basal and genotoxic stress induced (etoposid) autophagy will be investigated by using LC3I-II(Microtubule-associated protein 1A/1B light chain 3 cytosolic form-phosphatidylethanolamine conjugated form) mobility shift, degradation of p62(nucleoporin p62), phosphorylation of Ulk1, Atg13(Autophagy related protein 13) LC3 proteins puncta, autophagic vacuole formation assays. The possible physical interaction between Wip1-Ulk1 genes will be investigated by co-IP(co immunoprecipitation) and co- localization analysis and confocal microscopy imaging methods. Furthermore, effect of Wip1- dependent autophagy on other cellular stress responses, will be investigated by using cell cycle analysis, Annexin, V/7AAD(7-amino-actinomycin D) and SA-β-gal (senescence-associated beta galactosidase) staining,H2K9 gene phosphorylation assay as well as Western Blot or qRT-PCR(Quantitative Real Time Polymerase Chain Reaction) analysis. Statistical Package for the Social Sciences (SPSS) 26 and Microsoft Excel will be used for the evaluation of the project results.
What is the role of the student?
- The student will mainly observe
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The following tasks will be expected to be accomplished by the student: -Observing the tissue culture preparation process -Examining the Western Blot method which is a laboratory method used to detect specific protein molecules from among a mixture of proteins. -Learning about the principles of Electrophoretic Mobility Shift Assay. -Analyzing the RT-qPCR (Real time Polymerase chain reaction) techniques steps which is a quantitative method in contrast to conventional PCR (Polymerase Chain Reaction), meaning that it enables the determination of exact amounts (relative or absolute) of amplified DNA in samples. -Being able to use Statistical Package for the Social Sciences (SPSS) 26 and Microsoft Excel at a fundamental level for the evaluation of the project results.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Will there be any theoretical teaching provided for the student (preliminary readings, lectures, courses, seminars)? Please describe how much teaching and by whom. There are many different subjects open for understanding methods. The subjects will be taught once per week, by the tutor Mehtap Kılıc Eren, Ph.D during the exchange depending on the availability: -Explaining Western Blot method which is a laboratory method used to detect specific protein molecules from among a mixture of proteins. - Explaining principles of Electrophoretic Mobility Shift Assay. - Describing RT-qPCR (Real time Polymerase chain reaction) techniques steps which is a quantitative method in contrast to conventional PCR (Polymerase Chain Reaction) -Learning about relations between medical genetics and medical biology.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
What skills are required of the student? The student should have knowledge regarding the following: - Laboratory safety and hygiene procedures - Essentials of medical genetics (could be looked up on a textbook like Molecular Biology of the Cell etc.) - The biochemical principles behind Polymerase Chain Reaction tests - Mechanisms and reasons/effects of cellular authophagy
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students
- Mercantepe; Tolga; Tumkaya; L.; Celiker; F.B.; Topal Suzan; Z.; Cinar; S.; Akyildiz; K.; Mercantepe; F.; Yilmaz; A.; 2018. Effects of gadolinium-based MRI contrast agents on liver tissue. J. Magn. Reson. Imaging 1–8. https://doi.org/10.1002/jmri.26031
- Mercantepe; T.; Unal; D.; Tumkaya; L.; Yazici; Z.A.; 2018. Protective effects of amifostine; curcumin and caffeic acid phenethyl ester against cisplatin-induced testis tissue damage in rats. Exp. Ther. Med. 15. https://doi.org/10.3892/etm.2018.5819
- Topcu; A.; Mercantepe; F.; Rakici; S.; Tumkaya; L.; Uydu; H.A.; Mercantepe; T.; 2019. An investigation of the effects of N-acetylcysteine on radiotherapy-induced testicular injury in rats. Naunyn. Schmiedebergs. Arch. Pharmacol. https://doi.org/10.1007/s00210-018-1581-6
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