The Effects of Caffeine on the S-phase Checkpoint
in Saccharomyces cerevisiae
Mandi Pearsons, Stephanie Truc Nguyen, Justin Pham, and Dr. Wendy J. Dixon
D E PA R T M E N T
O F B I O L O G I C A L S C I E N C E,
CALI FO R N IA STAT E
RA TIONALE
AB STRACT
Cdc7/Dbf4 kinase triggers DNA replication in Saccharomyces cerevisiae
and also plays role in the S phase checkpoint. The S phase checkpoint prevents
incomplete DNA replication from proceeding to mitotic division. In
Schizosaccharomyces pombe, caffeine was shown to override the S phase
checkpoint. Previous students' research on Saccharomyces cerevisiae showed
that caffeine did not affect UV induced S phase delay in a statistically
significant manner. We investigated caffeine and its ability to override the S
phase checkpoint in Saccharomyces cerevisiae induced by hydroxyurea
treatment, exemplifying arrested cells through DNA replication inhibition. We
performed the experiment by adding caffeine to Saccharomyces cerevisiae in
the regular cell cycle or in a hydroxyurea induced S-phase arrest. The results
indicated that the addition of caffeine alone had no effect on the S phase
checkpoint, while the addition of caffeine to hydroxyurea treated
Saccharomyces cerevisiae showed an overridden S phase checkpoint. The
combination of hydroxyurea and 10mM caffeine showed an increase of cells in
G1 phase and cell death. Caffeine ultimately affects the cell cycle by triggering
irregular mitosis in hydroxyurea arrested yeast cells.
PO LYT E C H N I C
Studying the effects of caffeine on S. cerevisiae explores the pathways involved in
DNA replication and the targets, within these pathways, that can be manipulated to
treat disease and other afflictions. An example of a disease that can be treated using the
knowledge from this study is cancer. Anticancer drugs work to stop DNA replication in
cancer cells that are rapidly proliferating. By studying how caffeine is able to override
the checkpoint created by the cell, a novel drug that acts in the same way as caffeine
can be created and used in a synergistic way with the anticancer drug to destroy the
targeted cancer cells. Studying the pathways that govern the S-phase checkpoint can
also give insight to why certain anticancer drugs have a detrimental effect and lead to
the development of new anticancer drugs that circumvent these side effects. Cancer
may develop from checkpoints being bypassed. This approach allows for exploring
how that might occur and by what agents.
CA
KEY (FIGURE1, FIGURE 2)
G1 PHASE
S PHASE
G2 PHASE
FIG. 1 Yeast is treated with only
hydroxyurea, showing a
dominance of cell growth in the G2
phase.
FIG. 2 Yeast is treated with both
hydroxyurea and caffeine, showing
a dominance of cell growth still in
the G1 phase. This allows us to
observe caffeine’s affect on
overriding the S-phase.
METHODS
INTRODUCTION
5 ml
URA-/TRP/GLU
BJ5459
A B
pNCCDC7GP &
pRSDMC4
A
B
STARTER
CULTURE
10 ml
URA-/TRPA
B
/RAF
NEW TUBES
INCUBATE
12–16
HRS
A: CONTROL - CAFFEINE
B: +CAFFEINE
A
B
2 HRS
A
A: CONTROL - CAFFEINE
B: +CAFFEINE
B
INCUBATE
7 HRS
B
A
A
B
NEW TUBES
B
200𝜇l of 2M
STOCK
HYDROXYUREA
A: CONTROL - CAFFEINE
B: +CAFFEINE
FIG. 3 G1 Phase
FIG. 4 S-Phase, G2-Phase
FIG. 5 Cell apoptosis
CO NCLUSIO N
10 ml
A
FIG. 2 G1 PHASE
INCUBATE
INCUBATE
In different studies done in a related species, S. Pombe, it has been found
that Cid1 and Cid2 are essential genes in maintaining S phase arrest. It has been
shown that when targeted by caffeine, these genes lose their viability and are
unable to maintain the S phase checkpoint of that cell. This event induces
abnormal mitosis of the cell which ultimately ends up in cell death.
Using S. cerevisiae as our model organism has enabled us to study the
connection between proteins and genes, observing the biological properties that
similarly occur in both entities. Its complete genome sequence was published in
1996, allowing us to manipulate its genetic composure with ease under a short
period of time.
P O M O N A,
R E S U LTS
1 ml
The mitotic cell cycle is an intricate system that is primarily composed of 4
main phases of growth: G1, S, G2, and M. At each phase of growth, a
regulatory checkpoint is created by a complex pathway of proteins. The purpose
of these checkpoints are to prevent the cell from growing unnaturally. The Sphase is a significant step that ensures the correct duplication of genetic
material from accumulating the necessary proteins until completion.
U N I V E R S I T Y,
2.5
HRS
1.0 ml
20%
GALACTOSE
STOCK
A
B
Caffeine alone showed no effect on the Saccharomyces cerevisiae S phase
checkpoint as the 10mM concentration was used. On the other hand, with the
addition of caffeine to hydroxyurea treated Saccharomyces cerevisiae showed
an overridden S phase checkpoint. The combination of hydroxyurea and 10mM
caffeine showed an increase of cells in G1 phase and cell death. Caffeine
ultimately affects the cell cycle by triggering irregular mitosis in hydroxyurea
arrested yeast cells.
** OPTICAL DENSITY
(OD) IS CHECKED
EVERY STEP
FOLLOWING
INOCULATION Fan et al., 1995; Powell et al., 1995; Russell et al., 1995; Yao et al., 1996
REFERENCES
INCUBATE
3 HRS
0.61 ml OF 82
mM CAFFEINE
STOCK
A
A
B
B
MICROSCOPY
RESULTS
Ito, Keisuke, Tomonori Nakazato, Yoshitaka Miyakawa, Kenji Yamato, Yasuo Ikeda, and
Masahiro Kizaki.
"Caffeine
Induces G2/M Arrest and Apoptosis via a Novel P53-dependent Pathway in NB4 Promyelocytic
Leukemia Cells." Journal of Cellular Physiology 196.2 (2003): 276-83.
Lu, Pin-Zhen, Ching-Yu Lai, and Wen-Hsiung Chan. "Caffeine Induces Cell Death via Activation of Apoptotic Signal
and Inactivation of Survival Signal in Human Osteoblasts." International Journal of Molecular
Sciences 9.5
(2008): 698-718.
Wang, S., Norbury, C., Harris A. L., and Toda, T. (1999). Caffeine can Override the S-M
Checkpoint in Fission
Printed by
Yeast. Journal of Cell Science, 112, 927-937. Print.