Download (Pierce, 2014). Figure 3. a gel after PCR reaction. L refers to which

Chemoprotective Effects of Overexpression of Milnesium
tardigradum Rad51 Gene in HUVEC Cells
Christopher Parker, Department of Biological Science, York College
http://apod.nasa.gov/apod/ap130306.html
Damage to DNA in humans from
environmental factors such as radiation
promotes tumorigenesis. Tardigrades
have a much higher resistance to DNA
damaging agents such as radiation
through their DNA repair and protection
mechanisms. One of the most significant
mechanisms is Rad51 which promotes
DNA
repair
through
homologous
recombination. Through isolation of
tardigrade RNA, a plasmid could be built
containing the tardigrade Rad51 gene
which could be then transfected into
human cells. In this study, tardigrade
RNA was isolated and the Rad51 gene
was
successfully
amplified.
The
tardigrade Rad51 gene if expressed in
HUVEC
cells
could
encourage
homologous recombination resulting in a
decrease of cell death in the presence of
reagents that cause double stranded
breaks in DNA such as doxorubicin.
Review of Literature (continued)
Rad51 was showed only to be effective in the
presence of reagents that cause DSB such as
cisplatin and mitomycin C. The study also
showed that the down regulation of Rad51
gave less resistance to these reagents
(Slupianek et al. 2001).
Mouse MmRad51 over and under expressed in
CHO cells negative for P53. The study showed
that CHO cells overexpressing the MmRad51
gene had a lower frequency of tumors and the
tumors grew slower compared with the CHO
cells not expressing MmRad51 (Bertrand et al.
2003).
Rad51 displaces RPA from ssDNA and
guides recombination mediators BRCA1
and BRCA2. The Rad51 protein is able to
catalyze strand exchange interactions
between sister chromatids in order to
repair DSB in DNA.
Radiation as well as cisplatin, miomycin
C, and doxorubicin all cause DSB in DNA.
DNA damage from these reagents can
promote tumorigenesis. Rad51 can
suppress the homologous recombination
repair system when it is mutated and
prone to make mistakes.
Humans have a homolog of the Rad51
gene although it does not provide the
same
amount
of
resistance.
The
expression of the tardigrade Rad51 gene
in human cells could provide a greater
resistance to reagents that cause DSB in
DNA.
Review of Literature
Study proposed Rad51 is one of the most
significant components in the tardigrade
DNA repair mechanisms. Sequenced
Rad51 gene and is where primer design
for this study was obtained (BeltránPardo et al. 2013).
100bp L1P1 L1P2 L1C L2P1 L2P2 L2C 500bp
Create cDNA library using Reverse Transcription kit
Amplify Rad51 by doing a PCR using primer sets
RadMT-70F/RadMT1135R and 366F/Rad-Ext-R
RadMT-70F: CACCTCTCGTGAGTTCTTCGCTTG, RadMT1135R: CCGGTAGACACGGAGAATCGTAG,
366F: AAGATGGTTCCGATGGGCTTC, Rad-Ext-R: CCTCATTAGCGATCGCGAACA
Transform E. coli and select for plasmid using
ampicillin
Tardigrades have been able to survive in
a wide array of hostile environments
including high radiation through DNA
protection and repair systems.
Preliminary Results
Isolate RNA from Milnesium tardigradum using
TRIzol
Inset Rad51 gene into G418 plasmid
Introduction
Rad51 is thought to be one of the most
significant
mechanisms
in
the
tardigrades ability to survive in radiation
because it repairs double stranded
breaks (DSB) through homologous
recombination.
Methods
Figure 3. a gel after PCR reaction. L refers to which library
I used and the P is for which primer set with RadMT being
1 and 366 being 2.The 100bp ladder is at the beginning
and the 500bp ladder is at the end. The two bands in lanes
4 and 7 are 18S control. The band in lane 6 is the Rad51
gene amplified using primers 336F and Rad-Ext-R. The
band’s size was expected to be around 756 bp. The PCR
reaction used 95˚C to denature,56˚C to anneal, and 72˚
to extend.
Expected Results
Figure1. shows the that the overexpression of
Rad51 had a lower percent of CHO cells with
tumors compared to those that had Rad51
Inhibited (Bertrand et al. 2003).
Hamster Rad51 was overexpressed in CHO
cells and exposed to increasing amounts of
radiation. The study showed that in the late S
and G2 phase the overexpressed cells had a
greater resistance to radiation exposure. This
was concluded to be caused by the lack of
formation of the sister chromatids in the
earlier phases (Vispe et al. 1998).
Soft tissue sarcoma cells showed a lower
resistance to doxorubicin, a reagent that
causes DBS in DNA, when anti Rad51 siRNA
was present (Hannay et al. 2007).
Isolate plasmid and transfect HUVEC cells using
Liptofectamine2000
R ad51
80
Culture transformed HUVEC cells and control HUVEC
cells in 0.1, 0.5, 1.0, and 2.0 µmol of doxorubicin
C o n tro l
60
40
20
0
0
Chemoprotective effects of Rad51 will be quantified
by an MTS assay every four hours for forty-eight
hours. The amount of viable cells will be compared
between groups of the same concentration.
4
8
12
16
20
24
28
32
36
40
44
48
T im e ( h o u r s )
Figure 4. Possible result for comparison of control and
Rad51 expressed HUVEC cells over the course of fourtyeight hours in 1.0 µmol of doxorubicin. Rad51 HUVEC
Cells would be expected to have greater resistance.
Literature Cited
Beltrán-Pardo, Eliana A., Ingemar Jönsson, Andrzej Wojcik, Siamak Haghdoost, Rosa
María Bermúdez Cruz, and Jaime E. Bernal Villegas."Sequence Analysis of the DNA-repair
Gene Rad51 in the Tardigrades Milnesium Cf. Tardigradum, Hypsibius Dujardini and
Macrobiotus Cf. Harmsworthi." Journal of Limnology 72.1s (2013): 80-91.
Hypotheses/Objectives
Bertrand, Pascale, Sarah Lambert, Christophe Joubert, and Bernard S. Lopez.
"Overexpression of Mammalian Rad51 Does Not Stimulate Tumorigenesis While a
Dominant-negative Rad51 Affects Centrosome Fragmentation, Ploidy and Stimulates
Tumorigenesis, in P53-defective CHO Cells." Oncogene 22.48 (2003): 7587-592.
There will be a significantly less amount
of dying HUVEC cells expressing Rad51
than the amount of dying wild type
HUVEC cells in the presence of higher
concentrations of doxorubicin.
HUVEC cells expressing the Rad51 gene
will be able to survive a significantly
greater dosage of doxorubicin than wild
type HUVEC cells.
100
% V ia b le C e lls
Project Summary
http://www.rcsb.org/pdb/explore.do?structureId=1SZP
Hannay, J. A.f., J. Liu, Q.-S. Zhu, S. V. Bolshakov, L. Li, P. W.t. Pisters, A. J.f. Lazar, D. Yu,
R. E. Pollock, and D. Lev. "Rad51 Overexpression Contributes to Chemoresistance in
Human Soft Tissue Sarcoma Cells: A Role for P53/activator Protein 2 Transcriptional
Regulation."Molecular Cancer Therapeutics 6.5 (2007): 1650-660.
Pierce, Andrew. "PGCGFP-G418 (Plasmid #31264)." Addgene: PGCGFP-G418. Addgene,
Slupianek, Artur, Christoph Schmutte, Gregory Tombline, Malgorzata NieborowskaSkorska, Grazyna Hoser, Michal O. Nowicki, Andrew J. Pierce, Richard Fishel, and Tomasz
Skorski. "BCR/ABL Regulates Mammalian RecA Homologs, Resulting in Drug
Resistance."Molecular Cell 8.4 (2001): 795-806.
Figure 2. G418 plasmid contains EGFP and ampicillin
Resistance (Pierce, 2014).
Vispe, S., C. Cazaux, C. Lesca, and M. Defais. "Overexpression of Rad51 Protein
Stimulates Homologous Recombination and Increases Resistance of Mammalian Cells to
Ionizing Radiation." Nucleic Acids Research26.12 (1998): 2859-864.
Acknowledgements
I would like to thank Dr. Ronald Kaltreider for being my mentor and aiding me
in the development of my thesis.