Examining the Mechanisms behind the

G0/G1 Phase Turnover Rate in order to

Determine a Therapeutic Treatment for

Cellular Regeneration
Umang Gill, Neda Aminzadeh, Aidan Tan, Evan Chan,
Stefan Schoof, Sreyas Yeddula, Lukhjit Dhaliwal
 TABLE OF CONTENTS
Background &
01 Introduction 03 Specific Aim 2
Understand Role of
What are CDKs and how
CD4/6 on RB
do they regulate
regeneration and G1/G0
phase changes?

02 Specific Aim 1 04 Specific Aim 3

Introducing CDK 4/6 and Experiment to test the effects
RB of various proteins involved in
G0
Impact and
05 Significance
Why is our research
important?
 Background - What are Cyclin Dependent
Kinases (CDKs)?
● Protein kinase ● Studies focusing on CDK activity in relation to G1/G0
● Involved in cell cycle phases
○ Inhibition of CDK4 via p21 and p27 → more G0 (Gulappa, 2013)
● CDK/cyclin complex
○ Cyclin activates CDK for protein modification
○ Downregulation of calcineurin → more CDK 4 activity → more G1
(Baksh, 2000)
○ Regulate cell cycle checkpoints and transcriptional
events (Ding, 2020)
○ Fibroblasts: p21Sdi1 = CDK inhibitor → more G0 (Nakanishi, 1995)
● G0 phase
○ Special cases allow for cells to leave this phase ● Hepatocytes can switch to G1 in cases of injury, however
neural and cardiac cells rarely leave G0 phase (Frade, 2015;
● G0 to G1 Transition in Hepatocytes Li, 2014; Pasumarthi, 2002)

○ Damage to cell initiates process (Li, 2014) ● Cardiac Progenitor Cell Damage Repair

○ Upregulation of specific pathways in transcription ○ Temporary quiescence in hypoxic conditions (Bellio, 2017)

○ Decreased expression of c-Myc proto-oncogene

○ Can we apply this to adult neural cells?
 Gap in Knowledge & Hypothesis

● Gap in Knowledge
○ Not enough research focused on CDK & checkpoint bypass for cellular regeneration
○ Application of knowledge from research can be used to assess signal transduction pathways
for cyclins and CDKs for cells with lower regeneration rates

○ Can lead to discovery of therapy focused on increasing cellular regeneration in times of injury

● Hypothesis
○ We predict that modification of the signal transduction pathway that leads to increase in CDK
4/6 activity will result in greater regeneration rates of cells with lower to no turnover periods
 Aim 1 - Introducing CDK 4/6 and pRb
CDK 4/6 pRb
● CDK 4/6 effect on the cell cycle
● Sufficient for G0-arrested cells to reenter cycle
● Dependence on functional RB proteins
● E2F separation

Figure 1. Role of CDK 4/6 in RB pathway (Niu et al., 2019).

 Aim 2 - Understand Role of CD4/6
on RB
● Cyclin D-CDK4/6 promote progression of cell
cycle

○ Phosphorylation of RB

○ Positive feedback loop

● ↓ RB → abnormal migration & neuronal death

Figure 2. Graphical Representation of Positive
feedback loop (Topacio el al., 2019)
Aim 3 - The Experiment: Can adding additional cell
cycle proteins force cells out of G0?

Rationale Experimental Design

● Understanding G0 is key to ● Tests for whether or not neural cells
understanding how to cause cell can regenerate in damaged nerve
division and change its rate cells of a snail

● Three G0 states: quiescent, ● 6-hydroxydopamine (6-OHDA)

senescent, and differentiated.
● Differentiated cells perform their
main functions indefinitely without
dividing
○ Will be target of experiment:
nerve cells
injected on section of snail’s nervous
system to cause specific nerve
damage (Glinka et al., 1997)
● Snails then injected with serum
containing mixture of cdk4, cdk6,
and cdk3, E2F, cyclin, and pRB in
varying combinations
 Experimental Results and Future
Predicted Results
● E2F expected to increase the rate of
cellular division
● pRB may enhanced cellular growth
○ proliferated by the presence of
CDK4 and CDK6
● Absence of added Cyclin would likely
cause added CDKs not having an
effect on the cells
Future Directions
● Extrapolate the findings to be applied
to other cells
● Example: differences of healing due
to cell division rates regarding vivo
damage to hepatic tissue in mice
● Liver resection performed on mice
and their liver weight will be recorded
before and after the healing process
 Impact & Significance
Future Ideas
● Investigating the specific
regulatory mechanisms
involved during process from
G0 → G1
● Test possible down/up
regulation of certain proteins
involved in CDK activation
pathway for types of cells
Importance What can it lead to?
● Useful for future science ● Quicker regeneration of
and medical treatments tissue in times of injury, even
and discoveries in cases where tissue is
● Could change the way unable to regenerate on its
injuries are approached own
in a medical setting
 REFERENCES
Awang, N. A., Maan, N., & Sul’ain, D. (2018). Tumour-immune interaction model with cell cycle effects including G0 phase. MATEMATIKA, 34(3), 33–44. https://doi.org/10.11113/matematika.v34.n3.1137
Baksh, S., DeCaprio, J. A., & Burakoff, S. J. (2000). Calcineurin regulation of the mammalian G0/G1 checkpoint element, cyclin dependent kinase 4. Oncogene, 19(24), 2820–2827. https://doi.org/10.1038/sj.onc.1203585
Bellio, M.A., Pinto, M.T., Florea, V. et al. Hypoxic Stress Decreases c-Myc Protein Stability in Cardiac Progenitor Cells Inducing Quiescence and Compromising Their Proliferative and Vasculogenic Potential. Sci Rep 7, 9702 (2017).
https://doi.org/10.1038/s41598-017-09813
Ding, L., Cao, J., Lin, W., Chen, H., Xiong, X., Ao, H., Yu, M., Lin, J., & Cui, Q. (2020). The Roles of Cyclin-Dependent Kinases in Cell-Cycle Progression and Therapeutic Strategies in Human Breast Cancer. International journal of molecular
sciences, 21(6), 1960. https://doi.org/10.3390/ijms21061960
Frade, J. M., & Ovejero-Benito, M. C. (2015). Neuronal cell cycle: the neuron itself and its circumstances. Cell cycle (Georgetown, Tex.), 14(5), 712–720. https://doi.org/10.1080/15384101.2015.1004937
Gelbert, L.M., Cai, S., Lin, X. et al. (2014). Preclinical characterization of the CDK4/6 inhibitor
LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine. Invest New Drugs, 32, 825–837. https://doi.org/10.1007/s10637-014-0120-7
Giacinti, C., & Giordano, A. (2006). RB and cell cycle progression. Nature News, 25, 5220-5227. https://www.nature.com/articles/1209615
Glinka, Y., Gassen, M., & Youdim, M. B. (1997). Mechanism of 6-hydroxydopamine neurotoxicity. Advances in Research on Neurodegeneration, 55–66. https://doi.org/10.1007/978-3-7091-6842-4_7
Gulappa, T., Reddy, R. S., Suman, S., Nyakeriga, A. M., & Damodaran, C. (2013). Molecular interplay between cdk4 and p21 dictates G0/G1 cell cycle arrest in prostate cancer cells. Cancer letters, 337(2), 177–183.
https://doi.org/10.1016/j.canlet.2013.05.014
Lau, L. F., & Nathans, D. (1985). Identification of a set of genes expressed during the G0/G1
transition of cultured mouse cells. The EMBO journal, 4(12), 3145–3151.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554634/
Li, M., Zhou, X., Mei, J., Geng, X., Zhou, Y., Zhang, W., & Xu, C. (2014). Study on the activity of the signaling pathways regulating hepatocytes from G0 phase into G1 phase during rat liver regeneration. Cellular & molecular biology letters,
19(2), 181–200. https://doi.org/10.2478/s11658-014-0188-2
Linke, S. P., Clarkin, K. C., Di Leonardo, A., Tsou, A., & Wahl, G. M. (1996). A reversible,
p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the
absence of detectable DNA damage. Genes & development, 10(8), 934–947.
https://doi.org/10.1101/gad.10.8.934
Matsui, T., Nieto-Estévez, V., Kyrychenko, S., Schneider, J. W., & Hsieh, J. (2017).
Retinoblastoma protein controls growth, survival and neuronal migration in human cerebral organoids. Development (Cambridge, England), 144(6), 1025–1034. https://doi.org/10.1242/dev.143636
Nakanishi, M., Adami, G. R., Robetorye, R. S., Noda, A., Venable, S. F., Dimitrov, D., Pereira-Smith, O. M., & Smith, J. R. (1995). Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA. Proceedings of the
National Academy of Sciences of the United States of America, 92(10), 4352–4356. https://doi.org/10.1073/pnas.92.10.4352
Niu Y, Xu J, Sun T. Cyclin-Dependent Kinases 4/6 Inhibitors in Breast Cancer: Current Status, Resistance, and Combination Strategies. J Cancer 2019; 10(22):5504-5517. doi:10.7150/jca.32628.
Pasumarthi, K. B. S., & Field, L. J. (2002). Cardiomyocyte Cell Cycle Regulation. Circulation Research, 90(10), 1044–1054. https://doi.org/10.1161/01.res.0000020201.44772.67
Ren, S., & Rollins, B. J. (2004). Cyclin C/cdk3 promotes Rb-dependent G0 exit. Cell, 117(2),
239–251. https://doi.org/10.1016/s0092-8674(04)00300-9
Sage, J., Miller, A. L., Pérez-Mancera, P. A., Wysocki, J. M., & Jacks, T. (2003). Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature News, 424, 223–228.
https://www.nature.com/articles/nature01764
Tomashevski A, Webster DR, Grammas P, Gorospe M, Kruman II. Cyclin-C-dependent cell-cycle entry is required for activation of non-homologous end joining DNA repair in postmitotic neurons. Cell Death Differ. 2010 Jul;17(7):1189-98.
doi: 10.1038/cdd.2009.221. Epub 2010 Jan 29. PMID: 20111042; PMCID:
PMC2885568.
Tomura M, Sakaue-Sawano A, Mori Y, Takase-Utsugi M, Hata A, Ohtawa K, et al. (2013) Contrasting Quiescent G0 Phase with Mitotic Cell Cycling in the Mouse Immune System. PLoS ONE 8(9): e73801.
https://doi.org/10.1371/journal.pone.0073801
Topacio, B. R., Zatulovskiy, E., Cristea, S., Xie, S., Tambo, C. S., Rubin, S. M., Sage, J.,
Kõivomägi, M., & Skotheim, J. M. (2019). Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix. Molecular cell, 74(4), 758–770. e4.
https://doi.org/10.1016/j.molcel.2019.03.020
KAHOOT