Download overexpression of mcm protein potentially causes cancer

OVEREXPRESSION OF MCM PROTEIN POTENTIALLY CAUSES CANCER
Yujie Mu, Anthony Schwacha
Department of Bioengineering and Department of Biological Science, the University of Pittsburgh
INTRODUCTION
In the United States 2015, the total population of new
cancer cases reaches to 1.6 million, within which around 0.6
million deaths are projected to occur. [1] From data estimated by
International Agency for Research on Cancer, there are 14.1
million new cancer cases in 2012, and 8.1 million cancer deaths
occurred worldwide. [2] In recent years, cancer becomes one of
the most fatal diseases threatening human health. Targeted
therapy, one of the most common treatments for the cancer,
targets the small molecules in cancer cells that help them grow,
divide and spread. [3] Hence, small molecular inhibitor becomes
a new promising treatment to cancers under targeted therapy
category. In order to come out with a more effective and novel
molecular inhibitor to treat cancer, it is important to first
understand mechanisms of cancer development and how it is
affected by small molecular inhibitors.
Cancer results from a corruption of an organism’s
genetic code (genomic instability), and mistakes made during
DNA replication are a particularly prevalent cause of such
instability. The cause of genomic instability can be attributed to
defects in genes required for nucleotide repair, double-strand
DNA break recognition and repair, genetic recombination and
mismatch repair, which also associate with human cancer
syndrome. [4]
The previous study established the possibility that
alterations to one particular replication factor – the Mcm2-7
helicase – might contribute to genomic instability. Mcm2-7 is
formed from six different subunits (labeled 2 through 7) and
serves to unwind duplex DNA in advance of DNA polymerase
during DNA replication progress. [5] My lab and others have
shown that Mcm2-7 is also involved in various regulatory
processes that ensure accurate DNA replication. [6] Interestingly,
human tumors commonly contain mutations in at least one of the
Mcm subunits, and many overexpress individual subunits.
OBJECTIVE
The goal of my research is to test if Mcm subunit
overexpression can lead to genomic instability that potentially
triggers cancer, or if in contrast the observed Mcm mutations are
simply a result of cancer development.
HYPOTHESIS/SUCCESS CRETERIA
I hypothesize that there is at least one Mcm subunit
overexpressed causes significant genomic instability since
overexpression of Mcm protein will break up the balance in
reacting with other regulatory molecules.
We designed three success criteria aiming to achieve
more confidential results and set the standard cutoff analyzing
data. Firstly, we need to construct target Mcm subunit gene under
galactose promoter and insert appropriate selectable marker to
strains. Secondly, we set mcm2DENQ already known to cause
genomic instability as experimental positive control. The results
of 2DENQ are considered as statistical significant number, which
determines whether overexpression of Mcm subunit causes
genomic instability. Thirdly, we will perform each experiment
for three times to increase reliability and minimize statistical
errors.
METHOD
Fundamental replication processes are completely
conserved between yeast and humans; therefore yeast provides a
good human disease model, especially for cancer study. As
Mcm2-7 complex is highly conserved among all eukaryotes, we
will be conducting experiments using budding yeast as a highly
tractable model system. [7] Mcm subunits have around 50%
protein sequence identity between human and budding yeast by
testing genomic stability following conditional overexpression of
individual Mcm subunits. [5] Genomic instability can be
determined by whether it causes both DNA-level instability and
chromosome-level instability by examining the fraction of
single-strand and double-strand DNA breaks and the rate of
plasmid loss per generation respectively.
To overexpress individual Mcm subunits, an appropriate
genetic construction will be stably transformed into 6 strains that
will place each individual Mcm gene under the high-level
inducible expression of the galactose promoter; thus cells grown
on glucose will contain normal levels of Mcm proteins, while
cells grown on galactose will overexpress the target Mcm
subunit.
As a cytological assay for DNA instability, we will use
a reporter strain that contains an in-frame fusion between green
fluorescence protein (GFP) and RPA, which is a protein
specifically required to repair DNA single strand breaks. In this
assay, cells lacking DNA damage will contain a diffuse
fluorescence throughout the entire nucleus corresponding to
RPA-GFP, where as cells with DNA breaks will localize this
protein to break sites and form sharply defined point (foci).
Similar to procedures of RPA-GFP assay, Rad52-GFP assay is
designed to localize double-strand DNA breaks sites. The test
strains will be grown in the appropriate media, and the percent of
cells containing RPA-GFP or Rad52-GFP foci will be scored
using fluorescence microscopy.
Plasmid loss is described as an event where daughter
cell does not obtain one copy of plasmid from mother cell during
replication. [8] Normally, yeast will replicate and segregate
plasmids with good efficiency. Genetic alteration that reduces the
ability of cells to perform either DNA replication or chromosome
segregation will generate cells at a higher rate of plasmid loss.
We perform this assay by constructing ADE3, which is a color
indicator, into the circular DNA. Cells with plasmid and ADE3
gene translated show red color. Otherwise, cells shown white are
considered to lose plasmid. Then we follow the previous
established protocol to calculate the rate of plasmid loss by each
generation. Reaching to cutoff, overexpression of specific Mcm
subunit is counted to cause chromosome instability.
RESULTS
RPA-GFP assay and Rad52-GFP assay are performed
with three trials, and calculated values for plot comparison are
reported as the average across all subjects. The purple bar
indicates percent of foci induced by mcm2DENQ positive
control as significant cutoff value. The wildtype strains grown in
glucose are shown in blue bars, and overexpressed strains grown
in galactose are shown in red bars.
Figure 1. Mcm subunit overexpression caused single-strand DNA damage. The
indicated strains were scored for RPA-GFP foci following growth in either
glucose or galactose containing media. The numbers shown reflect the percent of
cells in the population containing 1 or more RPA-GFP foci with standard
deviation ±2~3%.
Figure 2. Mcm subunit overexpression caused double-strand DNA damage. The
indicated strains were scored for Rad52-GFP foci following growth in either
glucose or galactose containing media. The numbers shown reflect the percent of
cells in the population containing 1 or more Rad52-GFP foci with standard
deviation ±1~2%.
Plasmid loss assay was performed with two trials, and
all values are averaged in the calculation. There is no control
group for this assay. Rate of plasmid loss can be scored under
5% generally, with which significant cutoff can be considered as
three times of plasmid loss rate of wildtype.
Figure 3. Mcm subunit overexpression caused chromosome instability. The
indicated strains were scored for plasmid loss rate of each generation following
growth in either glucose or galactose containing media. The numbers shown
reflect the percent of plasmid loss rate with respect to wildtype plasmid loss rate
with standard deviation ±3~4%.
The results of experiments support my hypothesis.
Overexpression of Mcm3, 6 and 7 causes significant singlestrand DNA breaks (~55%, 26% and 51% respectively),
compared with RPA-GFP foci of 2DENQ (~25%).
Overexpression of Mcm3 and 7 causes significant double-strand
DNA breaks (~44% and 22% respectively), as 2DENQ shows
around 18% defect. Overexpression of Mcm5 (~17%) and Mcm7
(~14%) has comparatively significant plasmid loss rate than that
of wildtype (<5%).
DISCUSSION
My success criteria were meet, except for repetition of
plasmid loss assay. The results for single-strand and doublestrand DNA breaks are consistent between Mcm3 and Mcm7 that
these two subunits significantly cause DNA instability, while
Mcm6 is somehow consistent but smaller effect on double-strand
DNA breaks.
The limitation of my research is that counting foci and
cells under microscope is subjective that varies from person
having different counting rules, and there is no existing advanced
method to count cells in a more effective and accurate way.
In conclusion, the subunit Mcm7 is consistent in above
three assays that it causes both DNA instability and chromosome
instability. Mcm7 causes genomic instability that potentially
triggers cancer.
For future study, experiments are in progress to test if
over expression of either Mcm3 or Mcm7 causes other types of
DNA damage, as well as to test various hypotheses for why
overexpression of either of these subunits would result in DNA
damage.
ACKNOLEGMENT
I would like to thank Dr. Anthony Schwacha for
mentoring my research, and Jennifer Liberato, Elle Fernander,
and Julia Verbier for construction of some of my strains.
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