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The Role of Synchronization Mediators in Circadian Rhythm in HT-29 Colon Cancer Cell Line

Year 2019, Volume: 9 Issue: 4, 2207 - 2215, 01.12.2019
https://doi.org/10.21597/jist.551238

Abstract

In mammals, the central circadian clock is located in the hypothalamus, coordinating peripheral clocks with neuronal and endocrine signals. It has been shown that circadian clock genes and proteins continue their rhythmic expression in peripheral cells and tissues independent of central clock. The rhythm continues even in the immortalized cell lines, but intercellular coordination is impaired. Cell lines are good mediators for studying cancer-related pathways and circadian rhythm relationships. However, it should first be shown that rhythmic gene expression continues. Some mediators are used to achieve intercellular synchronization. The aim of this study was to investigate the role of strong synchronization initiators, FBS, and dexamethasone, on the expression of the PER2 clock gene in the HT-29 colon cancer cell line. For this purpose; following FBS or dexamethasone administration, HT-29 cells were harvested at 6 different times to isolate RNA and the PER2 and ACTB gene expressions were quantified by real-time PCR assay. The results were evaluated by Linear Cosinor analysis. When the change in mRNA expression of the PER2 gene was compared to the ACTB reference gene, it was shown that rhythmic expression was achieved with both initiators. According to the Cosinor analysis, a significant rhythmic change was observed in both 18 and 24 hour periods. However, in the synchronization with dexamethasone, the confidence interval in the time Period was also significant in the 24 hour period. In this study, it was determined that the initiators may vary in the calculation of circadian Periods which was carried out with two different initiators in HT-29 cell line.

Project Number

Proje numarası:9.2016/9

References

  • Albrecht, U., B. Zheng, D. Larkin, Z. S. Sun and C. C. Lee (2001). "MPer1 and mper2 are essential for normal resetting of the circadian clock." J Biol Rhythms 16(2): 100-104.
  • Ballesta, A., S. Dulong, C. Abbara, B. Cohen, A. Okyar, J. Clairambault and F. Levi (2011). "A combined experimental and mathematical approach for molecular-based optimization of irinotecan circadian delivery." PLoS Comput Biol 7(9): e1002143.
  • Balsalobre, A., S. A. Brown, L. Marcacci, F. Tronche, C. Kellendonk, H. M. Reichardt, G. Schutz and U. Schibler (2000). "Resetting of circadian time in peripheral tissues by glucocorticoid signaling." Science 289(5488): 2344-2347.
  • Balsalobre, A., F. Damiola and U. Schibler (1998). "A serum shock induces circadian gene expression in mammalian tissue culture cells." Cell 93(6): 929-937.
  • Balsalobre, A., L. Marcacci and U. Schibler (2000). "Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts." Curr Biol 10(20): 1291-1294.
  • Cheon, S., N. Park, S. Cho and K. Kim (2013). "Glucocorticoid-mediated Periyot2 induction delays the phase of circadian rhythm." Nucleic Acids Res 41(12): 6161-6174.
  • Cornelissen, G. (2014). "Cosinor-based rhythmometry." Theor Biol Med Model 11: 16.
  • Dibner, C., U. Schibler and U. Albrecht (2010). "The mammalian circadian timing system: organization and coordination of central and peripheral clocks." Annu Rev Physiol 72: 517-549.
  • Feillet, C. A., J. A. Ripperger, M. C. Magnone, A. Dulloo, U. Albrecht and E. Challet (2006). "Lack of food anticipation in Per2 mutant mice." Curr Biol 16(20): 2016-2022.
  • Fourier, J. (1822). Théorie Analytique de la Chaleur. Paris, France.
  • Izumo, M., T. R. Sato, M. Straume and C. H. Johnson (2006). "Quantitative analyses of circadian gene expression in mammalian cell cultures." PLoS Comput Biol 2(10): e136.
  • Kassel, O. and P. Herrlich (2007). "Crosstalk between the glucocorticoid receptor and other transcription factors: molecular aspects." Mol Cell Endocrinol 275(1-2): 13-29.
  • Kiessling, S., L. Beaulieu-Laroche, I. D. Blum, D. Landgraf, D. K. Welsh, K. F. Storch, N. Labrecque and N. Cermakian (2017). "Enhancing circadian clock function in cancer cells inhibits tumor growth." BMC Biol 15(1): 13.
  • Kopp, C., U. Albrecht, B. Zheng and I. Tobler (2002). "Homeostatic sleep regulation is preserved in mPer1 and mPer2 mutant mice." Eur J Neurosci 16(6): 1099-1106.
  • Levi, F., A. Okyar, S. Dulong, P. F. Innominato and J. Clairambault (2010). "Circadian timing in cancer treatments." Annu Rev Pharmacol Toxicol 50: 377-421.
  • Livak, K. J. and T. D. Schmittgen (2001). "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method." Methods 25(4): 402-408.
  • Marquardt, D. W. (1963). "An Algorithm for Least-Squares Estimation of Nonlinear Parameters." Journal of the Society for Industrial and Applied Mathematics 11(2): 431–441.
  • Minami, Y., K. L. Ode and H. R. Ueda (2013). "Mammalian circadian clock: the roles of transcriptional repression and delay." Handb Exp Pharmacol(217): 359-377.
  • Nagoshi, E., C. Saini, C. Bauer, T. Laroche, F. Naef and U. Schibler (2004). "Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells." Cell 119(5): 693-705.
  • Nelson, W., Y. L. Tong, J. K. Lee and F. Halberg (1979). "Methods for cosinor-rhythmometry." Chronobiologia 6(4): 305-323.
  • Pardini, L., B. Kaeffer, A. Trubuil, A. Bourreille and J. P. Galmiche (2005). "Human intestinal circadian clock: expression of clock genes in colonocytes lining the crypt." Chronobiol Int 22(6): 951-961.
  • Pendergast, J. S., R. C. Friday and S. Yamazaki (2010). "Photic entrainment of Periyot mutant mice is predicted from their phase response curves." J Neurosci 30(36): 12179-12184.
  • Reddy, T. E., F. Pauli, R. O. Sprouse, N. F. Neff, K. M. Newberry, M. J. Garabedian and R. M. Myers (2009). "Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation." Genome Res 19(12): 2163-2171.
  • Refinetti, R., G. C. Lissen and F. Halberg (2007). "Procedures for numerical analysis of circadian rhythms." Biol Rhythm Res 38(4): 275-325.
  • Schibler, U. (2005). "The daily rhythms of genes, cells and organs. Biological clocks and circadian timing in cells." EMBO Rep 6 Spec No: S9-13.
  • Siegel, R. L., K. D. Miller, S. A. Fedewa, D. J. Ahnen, R. G. S. Meester, A. Barzi and A. Jemal (2017). "Colorectal cancer statistics, 2017." CA Cancer J Clin 67(3): 177-193.
  • So, A. Y., T. U. Bernal, M. L. Pillsbury, K. R. Yamamoto and B. J. Feldman (2009). "Glucocorticoid regulation of the circadian clock modulates glucose homeostasis." Proc Natl Acad Sci U S A 106(41): 17582-17587.
  • Tamai, T. K., Y. Nakane, W. Ota, A. Kobayashi, M. Ishiguro, N. Kadofusa, K. Ikegami, K. Yagita, Y. Shigeyoshi, M. Sudo, T. Nishiwaki-Ohkawa, A. Sato and T. Yoshimura (2018). "Identification of circadian clock modulators from existing drugs." EMBO Mol Med 10(5).
  • Toh, K. L., C. R. Jones, Y. He, E. J. Eide, W. A. Hinz, D. M. Virshup, L. J. Ptacek and Y. H. Fu (2001). "An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome." Science 291(5506): 1040-1043.
  • Van der Veen, D. R., J. Shao, Y. Xi, L. Li and G. E. Duffield (2012). "Cardiac atrial circadian rhythms in PERİYOT2::LUCIFERASE and per1:luc mice: amplitude and phase responses to glucocorticoid signaling and medium treatment." PLoS One 7(10): e47692.
  • Vanselow, K., J. T. Vanselow, P. O. Westermark, S. Reischl, B. Maier, T. Korte, A. Herrmann, H. Herzel, A. Schlosser and A. Kramer (2006). "Differential effects of PER2 phosphorylation: molecular basis for the human familial advanced sleep phase syndrome (FASPS)." Genes Dev 20(19): 2660-2672.
  • Xu, Y., K. L. Toh, C. R. Jones, J. Y. Shin, Y. H. Fu and L. J. Ptacek (2007). "Modeling of a human circadian mutation yields insights into clock regulation by PER2." Cell 128(1): 59-70.
  • Zheng, B., D. W. Larkin, U. Albrecht, Z. S. Sun, M. Sage, G. Eichele, C. C. Lee and A. Bradley (1999). "The mPer2 gene encodes a functional component of the mammalian circadian clock." Nature 400(6740): 169-173.
  • Zielinska, K. A., L. Van Moortel, G. Opdenakker, K. De Bosscher and P. E. Van den Steen (2016). "Endothelial Response to Glucocorticoids in Inflammatory Diseases." Front Immunol 7: 592.

HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü

Year 2019, Volume: 9 Issue: 4, 2207 - 2215, 01.12.2019
https://doi.org/10.21597/jist.551238

Abstract

Memelilerde merkezi sirkadiyen saat, hipotalamusta konumlanmıştır. Periferal saatleri nöronal ve endokrin sinyaller ile koordine etmektedir. Sirkadiyen saat gen ve proteinlerinin, merkezi saatten bağımsız olarak da periferal hücre ve dokularda ritmik ekspresyona devam ettiği yapılan çalışmalar ile gösterilmiştir. İmmortalize hücre hatlarında da ritim devam etmekte ancak hücreler arası koordinasyon bozulmaktadır. Hücre hatları kanser ile ilgili yolaklar ve sirkadiyen ritim ilişkisini çalışmak için en elverişli aracılardır. Ancak öncelikle ritmik gen ekspresyonunun devam ettiğinin gösterilmesi gerekmektedir. Hücreler arası senkronizasyonun sağlanması için bazı aracılar kullanılmaktadır. Bu çalışmadaki amacımız; güçlü senkronizasyon başlatıcılar olan fetal bovin serum ve deksametazonun HT-29 kolon kanseri hücre hattında PER2 saat geninin ekspresyonu üzerindeki rolünü incelemektir. Bu amaçla; fetal bovin serum veya deksametazon uygulamasını takiben HT-29 hücreleri altı farklı zamanda toplanarak RNA izole edilmiş ve PER2 ve ACTB gen ekspresyonları gerçek zamanlı PZR deneyi ile kantifiye edilmiştir. Sonuçlar Lineer Cosinor analizleri ile değerlendirilmiştir. PER2 genine ait mRNA ekspresyonlarındaki değişim ACTB referans geni ile kıyaslanarak değerlendirildiğinde, her iki başlatıcı ile de ritmik ekspresyonun sağlandığı gösterilmiştir. Her iki başlatıcı ile de Cosinor analizine göre 18 ve 24 saatlik periyot sürelerinde anlamlı ritmik değişim gösterilmiştir. Ek olarak deksametazon ile başlatılan senkronizasyonda, 24 saatlik periyot süresinde zaman dilimindeki güven aralığının anlamlı olduğu gözlenmiştir. HT-29 hücre hattında iki farklı başlatıcı ile yapılan bu çalışmada sirkadiyen periyot sürelerinin hesaplanmasında başlatıcıların değişkenlik gösterebildiği tespit edilmiştir.

Supporting Institution

Bezmialem Vakıf Üniversitesi

Project Number

Proje numarası:9.2016/9

Thanks

Çalışmada kullanılan HT-29 hücre hattı temini için Dr. Belma Z. Kurt’a (Bezmialem Vakıf Üniversitesi, Eczacılık Fakültesi, Farmasötik Kimya AD) teşekkür ederiz. Çalışmanın matematiksel modellemelerdeki yönlendirmeleri için Sadullah Göncü’ye (Fatih Dilek ÖZTÜRK ve Beyza GÖNCÜ 9(4): 2207-2215, 2019 HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü 2214 Sultan Mehmet Vakif Üniversitesi, Mühendislik Fakültesi, İnşaat Mühendisliği Bölümü) teşekkür ederiz. Bu çalışma Bezmialem Vakıf Üniversitesi Bilimsel Araştırmalar Proje Destekleme Ofisi tarafından desteklenmiştir (Proje numarası:9.2016/9).

References

  • Albrecht, U., B. Zheng, D. Larkin, Z. S. Sun and C. C. Lee (2001). "MPer1 and mper2 are essential for normal resetting of the circadian clock." J Biol Rhythms 16(2): 100-104.
  • Ballesta, A., S. Dulong, C. Abbara, B. Cohen, A. Okyar, J. Clairambault and F. Levi (2011). "A combined experimental and mathematical approach for molecular-based optimization of irinotecan circadian delivery." PLoS Comput Biol 7(9): e1002143.
  • Balsalobre, A., S. A. Brown, L. Marcacci, F. Tronche, C. Kellendonk, H. M. Reichardt, G. Schutz and U. Schibler (2000). "Resetting of circadian time in peripheral tissues by glucocorticoid signaling." Science 289(5488): 2344-2347.
  • Balsalobre, A., F. Damiola and U. Schibler (1998). "A serum shock induces circadian gene expression in mammalian tissue culture cells." Cell 93(6): 929-937.
  • Balsalobre, A., L. Marcacci and U. Schibler (2000). "Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts." Curr Biol 10(20): 1291-1294.
  • Cheon, S., N. Park, S. Cho and K. Kim (2013). "Glucocorticoid-mediated Periyot2 induction delays the phase of circadian rhythm." Nucleic Acids Res 41(12): 6161-6174.
  • Cornelissen, G. (2014). "Cosinor-based rhythmometry." Theor Biol Med Model 11: 16.
  • Dibner, C., U. Schibler and U. Albrecht (2010). "The mammalian circadian timing system: organization and coordination of central and peripheral clocks." Annu Rev Physiol 72: 517-549.
  • Feillet, C. A., J. A. Ripperger, M. C. Magnone, A. Dulloo, U. Albrecht and E. Challet (2006). "Lack of food anticipation in Per2 mutant mice." Curr Biol 16(20): 2016-2022.
  • Fourier, J. (1822). Théorie Analytique de la Chaleur. Paris, France.
  • Izumo, M., T. R. Sato, M. Straume and C. H. Johnson (2006). "Quantitative analyses of circadian gene expression in mammalian cell cultures." PLoS Comput Biol 2(10): e136.
  • Kassel, O. and P. Herrlich (2007). "Crosstalk between the glucocorticoid receptor and other transcription factors: molecular aspects." Mol Cell Endocrinol 275(1-2): 13-29.
  • Kiessling, S., L. Beaulieu-Laroche, I. D. Blum, D. Landgraf, D. K. Welsh, K. F. Storch, N. Labrecque and N. Cermakian (2017). "Enhancing circadian clock function in cancer cells inhibits tumor growth." BMC Biol 15(1): 13.
  • Kopp, C., U. Albrecht, B. Zheng and I. Tobler (2002). "Homeostatic sleep regulation is preserved in mPer1 and mPer2 mutant mice." Eur J Neurosci 16(6): 1099-1106.
  • Levi, F., A. Okyar, S. Dulong, P. F. Innominato and J. Clairambault (2010). "Circadian timing in cancer treatments." Annu Rev Pharmacol Toxicol 50: 377-421.
  • Livak, K. J. and T. D. Schmittgen (2001). "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method." Methods 25(4): 402-408.
  • Marquardt, D. W. (1963). "An Algorithm for Least-Squares Estimation of Nonlinear Parameters." Journal of the Society for Industrial and Applied Mathematics 11(2): 431–441.
  • Minami, Y., K. L. Ode and H. R. Ueda (2013). "Mammalian circadian clock: the roles of transcriptional repression and delay." Handb Exp Pharmacol(217): 359-377.
  • Nagoshi, E., C. Saini, C. Bauer, T. Laroche, F. Naef and U. Schibler (2004). "Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells." Cell 119(5): 693-705.
  • Nelson, W., Y. L. Tong, J. K. Lee and F. Halberg (1979). "Methods for cosinor-rhythmometry." Chronobiologia 6(4): 305-323.
  • Pardini, L., B. Kaeffer, A. Trubuil, A. Bourreille and J. P. Galmiche (2005). "Human intestinal circadian clock: expression of clock genes in colonocytes lining the crypt." Chronobiol Int 22(6): 951-961.
  • Pendergast, J. S., R. C. Friday and S. Yamazaki (2010). "Photic entrainment of Periyot mutant mice is predicted from their phase response curves." J Neurosci 30(36): 12179-12184.
  • Reddy, T. E., F. Pauli, R. O. Sprouse, N. F. Neff, K. M. Newberry, M. J. Garabedian and R. M. Myers (2009). "Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation." Genome Res 19(12): 2163-2171.
  • Refinetti, R., G. C. Lissen and F. Halberg (2007). "Procedures for numerical analysis of circadian rhythms." Biol Rhythm Res 38(4): 275-325.
  • Schibler, U. (2005). "The daily rhythms of genes, cells and organs. Biological clocks and circadian timing in cells." EMBO Rep 6 Spec No: S9-13.
  • Siegel, R. L., K. D. Miller, S. A. Fedewa, D. J. Ahnen, R. G. S. Meester, A. Barzi and A. Jemal (2017). "Colorectal cancer statistics, 2017." CA Cancer J Clin 67(3): 177-193.
  • So, A. Y., T. U. Bernal, M. L. Pillsbury, K. R. Yamamoto and B. J. Feldman (2009). "Glucocorticoid regulation of the circadian clock modulates glucose homeostasis." Proc Natl Acad Sci U S A 106(41): 17582-17587.
  • Tamai, T. K., Y. Nakane, W. Ota, A. Kobayashi, M. Ishiguro, N. Kadofusa, K. Ikegami, K. Yagita, Y. Shigeyoshi, M. Sudo, T. Nishiwaki-Ohkawa, A. Sato and T. Yoshimura (2018). "Identification of circadian clock modulators from existing drugs." EMBO Mol Med 10(5).
  • Toh, K. L., C. R. Jones, Y. He, E. J. Eide, W. A. Hinz, D. M. Virshup, L. J. Ptacek and Y. H. Fu (2001). "An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome." Science 291(5506): 1040-1043.
  • Van der Veen, D. R., J. Shao, Y. Xi, L. Li and G. E. Duffield (2012). "Cardiac atrial circadian rhythms in PERİYOT2::LUCIFERASE and per1:luc mice: amplitude and phase responses to glucocorticoid signaling and medium treatment." PLoS One 7(10): e47692.
  • Vanselow, K., J. T. Vanselow, P. O. Westermark, S. Reischl, B. Maier, T. Korte, A. Herrmann, H. Herzel, A. Schlosser and A. Kramer (2006). "Differential effects of PER2 phosphorylation: molecular basis for the human familial advanced sleep phase syndrome (FASPS)." Genes Dev 20(19): 2660-2672.
  • Xu, Y., K. L. Toh, C. R. Jones, J. Y. Shin, Y. H. Fu and L. J. Ptacek (2007). "Modeling of a human circadian mutation yields insights into clock regulation by PER2." Cell 128(1): 59-70.
  • Zheng, B., D. W. Larkin, U. Albrecht, Z. S. Sun, M. Sage, G. Eichele, C. C. Lee and A. Bradley (1999). "The mPer2 gene encodes a functional component of the mammalian circadian clock." Nature 400(6740): 169-173.
  • Zielinska, K. A., L. Van Moortel, G. Opdenakker, K. De Bosscher and P. E. Van den Steen (2016). "Endothelial Response to Glucocorticoids in Inflammatory Diseases." Front Immunol 7: 592.
There are 34 citations in total.

Details

Primary Language Turkish
Subjects Structural Biology
Journal Section Moleküler Biyoloji ve Genetik / Moleculer Biology and Genetic
Authors

Dilek Öztürk 0000-0003-2485-891X

Beyza Göncü 0000-0001-6026-8218

Project Number Proje numarası:9.2016/9
Publication Date December 1, 2019
Submission Date April 9, 2019
Acceptance Date July 18, 2019
Published in Issue Year 2019 Volume: 9 Issue: 4

Cite

APA Öztürk, D., & Göncü, B. (2019). HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü. Journal of the Institute of Science and Technology, 9(4), 2207-2215. https://doi.org/10.21597/jist.551238
AMA Öztürk D, Göncü B. HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü. J. Inst. Sci. and Tech. December 2019;9(4):2207-2215. doi:10.21597/jist.551238
Chicago Öztürk, Dilek, and Beyza Göncü. “HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü”. Journal of the Institute of Science and Technology 9, no. 4 (December 2019): 2207-15. https://doi.org/10.21597/jist.551238.
EndNote Öztürk D, Göncü B (December 1, 2019) HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü. Journal of the Institute of Science and Technology 9 4 2207–2215.
IEEE D. Öztürk and B. Göncü, “HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü”, J. Inst. Sci. and Tech., vol. 9, no. 4, pp. 2207–2215, 2019, doi: 10.21597/jist.551238.
ISNAD Öztürk, Dilek - Göncü, Beyza. “HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü”. Journal of the Institute of Science and Technology 9/4 (December 2019), 2207-2215. https://doi.org/10.21597/jist.551238.
JAMA Öztürk D, Göncü B. HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü. J. Inst. Sci. and Tech. 2019;9:2207–2215.
MLA Öztürk, Dilek and Beyza Göncü. “HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü”. Journal of the Institute of Science and Technology, vol. 9, no. 4, 2019, pp. 2207-15, doi:10.21597/jist.551238.
Vancouver Öztürk D, Göncü B. HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü. J. Inst. Sci. and Tech. 2019;9(4):2207-15.