Download Figure 2 - York College of Pennsylvania

Cloning and expression of a novel S100P-mCherry fusion protein in
HT29 colon carcinoma cells
Joseph T. Orlando
Department of Biological Sciences, York College of Pennsylvania
Methods
Results
Chemotherapy Drug Treatment
•
Introduction
• S100P is a 95-amino acid protein that belongs to the
21 protein S100 family.
• The nomenclature S100P refers to the protein’s ability
to solubilize in 100% saturated ammonium sulfate
solution. The specific designation “P” is used because
it was first purified from placenta (Becker et al. 1995).
• The gene coding for S100P is located on chromosome
4p16 where some fatal disease-related genes are also
mapped including: Huntington disease, Crohn’s
disease and cervical cancer (Arumugam and Logsdon
2011).
• S100P interacts with its cellular targets via a Ca2+
conformational change (Becker et al. 1995; Figure 2).
• S100P regulates many intracellular and extracellular
activities including: protein phosphorylation, enzyme
activity, gene transcription, and cell proliferation and
differentiation (Donato 2001; Figure 3).
• Several studies revealed that S100P was
overexpressed in human cancer cells including: colon
(Bertram et al. 1998), breast, and prostate (Basu et al.
2008).
• Due to its overexpression and role in carcinogensis
and cell proliferation in cancer cells, S100P serves as
a potential molecular target for cancer treatment.
• Targeted therapies interfere with cell proliferation by
focusing on proteins involved in signal transduction.
• Doxorubicin (topoisomerase II inhibitor/free radical
generator), cisplatin (DNA crosslinker), and etoposide
(topoisomerase II inhibtor) act via different cellular
mechanisms.
HT29 cells were plated in 200µL medium
(DMEM, 10% FBS, and 1% pen/strep) in a 96-well
plate and incubated overnight.
• Medium was replaced with varying doses (1 -1000
µM) of either cisplatin, doxorubicin, or etoposide,
and incubated for 24 hours. Medium was removed
and cells were washed 3x with PBS.
• 50µL MTT (5mg/ml) added to each well and
incubated for 4 hours. 50µL DMSO added to
dissolve formed formazan crystals.
• Create a novel S100P-mCherry fusion protein using
the pMcherry-N1 mammalian expression vector.
• Transfect HT29 colon carcinoma cells with S100PpmCherry-N1 fusion plasmid.
• Compare chemotherapy drug effectiveness - treat wild
type, S100P overexpressed, and S100P silenced (via
siRNA) HT29 cells with cisplatin, doxorubicin, and
etoposide.
Hypothesis
Etoposide
Doxorubicin
Cisplatin
100
b.
50
Conclusions
0
• Cell viability was measured by using an MTT assay
(absorbance at 590nm measured).
1
• RT-PCR of human mammary epithelial cell RNA to
generate cDNA.
• Developed original primers to isolate S100P from
cDNA (BglII and EcoRI restriction sites).
5’-ggactcagatctgccgccaccatgacggaactagagacagccatgggc-3’
10
100
1000
L og D rug Concentration ( M)
Plasmid Construction
Figure 4. Mean (and SEM: n=3) percentage of cell viability of cisplatin,
doxorubicin, and etoposide treated HT29 colon cancer cells determined by
MTT assay. Cells were treated with chemotherapy drugs (1, 10, 50, 100 and
1000 µM) for 24 hours at 37°C followed by three PBS washes prior to addition
of MTT. Cells were incubated for 4 hours then absorbance at 590nm was
measured.
a. 100bp
Ladder
PCR
product
b
. 100bp 1
5’-catgcagaattcctttgagtcctgccttctcaaagtact-3’
Clone
2
3
4
ladder
• Digested PCR fragment with BglII and EcoRI to
generate sticky ends (Figure 5A).
• Ligated the PCR product into a linearized
pmCherry-N1 mammalian expression vector
(Clontech: Figure 7) with Instant Sticky-end Ligase
Master Mix® (New England Biolabs).
319bp
319bp
319bp
HT29 cell transfection
• Transformed pMcherry-N1 vector (Clontech)
containing S100P-mCherry fusion into E. coli.
• Isolated and purified plasmid from four transformed
E. coli colonies (selection with kanamycin).
Objectives
150
5x104
% Cell Viability
Figure 1. Ribbon structure of mCherry
Figure 7. Plasmid
map of pmCherryN1(right). S100P
(stop codon
excluded) inserted
50bp upstream of
mCherry gene. 15
AA linker sequence
inserted between
S100P and
mCherry gene.
MCS (below).
• S100P gene presence was verified by a restriction
digest (BglII and EcoRI) of the isolated plasmids
(Figure 5B).
Figure 5. Gel images A. Isolation of S100P
b. gene from cDNA. Digested
(BglII and EcoRI) S100P fragment was run on 2% agarose gel with 100 bp
ladder. B. Transformed E. coli colonies digested with BglII and EcoRI and
electrophoresed on a 2% agarose gel. Digestion was performed to verify
that isolated colonies contained the 319bp S100P gene.
a.
b.
• Sequenced isolated clone plasmids to verify correct
orientation of gene (PCMV IE primer).
• Transfected HT29 colon carcinoma cells using
FuGENE 6 (Promega) and analyzed cells via
fluorescent microscopy (Figure 6).
Future Studies
• Knockout S100P expression (via siRNA),
overexpress (via transfection) and treat cells –
compare cell viability.
• Treat transfected cells with CaCl2 to study Ca2+
interactions.
• Co-transfection of HT29 cells with H2B-GFP fusion
protein to confirm nuclear translocation.
• Observation of translocation with confocal
microscopy.
References
Arumugam, T., and Logsdon, C. D. 2011. S100P: a novel therapeutic target
for cancer. Amino Acids. 41:893-899.
A decreased expression level of S100P in HT29 cells will
significantly decrease the LD50 whereas an
overexpression of S100P will significantly increase the
LD50.
Becker, T., Gerke, V., Kube, E., and Weber, K. 1992. S100P a novel Ca2+ binding protein from human placenta cDNA cloning, recombinant protein
expression and Ca2+ binding properties. European Journal of Biochemistry
207:541-547.
Donato, R. 2001. S100: a multigenic family of calcium-modulated proteins of
the EF-hand type with intracellular and extracellular functional role.
International Journal of Biochemistry and Cell Biology. 33:637-668.
Figure 2.
Conformational
change of
S100P structure
caused by two
Ca2+ ions
binding to EFhand calcium
binding motifs
(Madej et al.
2012).
Ca2+
• HT29 cell treatment was unsuccessful with all three
chemotherapy drugs. Was not able to treat
overexpressed and siRNA knockout cells.
• S100P gene was isolated from cDNA, ligated into
pmCherry-N1 vector, and verified by sequencing.
• HT29 cells were successfully transfected with the
constructed mCherry-S100P fusion plasmid.
• The nuclear translocation of S100P-mCherry may
have been induced by UV light and/or heat during
fluorescence microscopy.
• Previous research has reported Ca2+ and binding
interaction induced translocation of S100P but
there has yet to be research reporting a UV or heat
induced translocation.
• Jiang et al. (2005) stated that S100P is associated
with enhanced survival in cells under stress, while
Al-Baker et al. (2004) reported that UV-light
induced DNA damage can cause a nuclear
translocation of proteins. In this case, S100P may
be translocating to the nucleus to serve as a
protective/repairing DNA agent.
c.
d.
Figure 6. Transfected HT29 cells viewed under fluorescent microscope
A) HT29 cells transfected with pmCherry-N1 control vector photographed
immediately after being brought into field of view B) Same group of cells
as photo A photographed 40 seconds later. C) HT29 cells transfected
with S100P-pmCherry-N1 vector photographed immediately after being
brought into field of view D) Same group of cells as photo C
photographed 40 seconds later.
Figure 3. The complexity of S100P signal transduction pathways and
regulatory molecules (Jiang et al. 2012).
Jiang, H., Hu, H., Tong, X., Jiang, Q., Zhu, H., and Zhang, S. 2012. Calciumbinding protein S100P and cancer: mechanisms and clinical relevance.
Journal of Cancer Research and Clinical Oncology. 138:1-9.
Madej, T., Addess, K. J., Fong, J. H., Geer, L. Y., Geer, R. C., Lanczycki, C.
J., Liu, C., Lu, S., Marchler-Bauer, A., Panchenko, A. R., Chen, J., Thiessen,
P. A., Wang, Y., Zhang, D., and Bryant, S. H. 2012. MMDB: 3D structures and
macromolecular interactions. Nucleic Acids Research. 40:461-464.
Acknowledgments
I would like to thank Dr. Ronald Kaltreider and Dr. Jeffrey Thompson for their
constant support and advice throughout the duration of this project.