1. Jak2 Protein Knockdown in BaF3 Cells Causes Differences in Proliferation Dependent on
the Growth Colony Stimulating Factor Receptor Isoforms
M Quinn, H Mehta, SJ Corey
Robert H. Lurie Comprehensive Cancer Center
Northwestern Feinberg School of Medicine
Loyola University Chicago
Abstract Methodology Discussion
Granulocyte colony stimulating factor (G-CSF) is a hormone that Our main method for studying Jak2 protein knockdown was Our findings with the original HGPRT control determined that
has been recognized to play a role in the differentiation and through the use of siRNA nucleofection. 300 nM concentration of siRNA was optimal – though it still did
proliferation of hematopoietic stem cells into neutrophils via the not reach expected knockdown. We expected greater than 90%
G-CSF Receptor. Of the known isoforms of this receptor, Class I is knockdown with this control, and we were only able to achieve
the most common in healthy individuals. However, the Class IV roughly 75% knockdown. However, it is sufficient for our
isoform, which is shorter in the internal cellular domain, shows experimental purposes, and we will continue to use 300 nM as
increased expression in individuals with severe congenital our standard nucleofection concentration.
neutropenia (SCN), acute myeloid leukemia (AML), as well as
individuals who respond poorly to chemotherapy. The Our Jak2 post-nucleofection western blot results lead us to
mechanism by which the Class IV isoform signaling differentiates visually conclude that siRNA duplexes #2 & #4 yield the best
from Class I is not yet known. Previous research has indicated a knockdown in BaF3 HA-tagged cells. For future
Jak2-dependence within the internal signaling of Class IV that is experiments, these are the two duplexes which we will use in
not present in Class I. Through the use of a Jak2 siRNA both GR-I and GR-IV cell types.
nucleofection knockdown, we determined Class IV proliferation
is dependent on the GCSF-Receptor binding to Jak2; without
which, the cells cease to thrive. Our results validate previous Creates temporary block of protein translation through mRNA
findings that Jak2 is essential for Class IV proliferation but has no cleavage. Future Directions
significant effect on Class I. We intend to further examine the
downstream signaling effects of Jak2 through this knockdown o Using both Baf3 HA-GRI and HA-GRIV cells, we nucleofected o Further experiments are necessary to test the effects of Jak2
technique in future studies. 2x106 cells/well using Thermo Scientific Dharmacon siRNA knockdown on cell proliferation in both GCSF-R I and GCSF-R
duplex #2 and #4 (specific to Jak2). IV BaF3 cells. We would like to do this using both cell viability
o After the nucleofection, we incubated the cells in 1.5 ml of as well as MTT assays.
BaF3 media +serum (including mIL3). o Additionally, experiments to validate the downstream effects
Introduction o We collected cell lysates at 48 hours post-nucleofection of Jak2 on Shp2 found in our previous research are desired.
to do western blot analyses.
o A concern of ours is the murine interleukin-3 (mIL-3) serum
The Growth Colony Stimulating Factor Receptor (GCSF-R)
dependency of our BaF3 cells. mIL-3 serum supplement is
influences hematopoietic stem cell differentiation, We used a mouse monoclonal antibody to blot for Jak2 required in BaF3 media, or high cell death will result.
proliferation, and survival. It has previously been determined protein presence, and a rabbit polyclonal Actin antibody for However, mIL-3 requires protein Jak2 in order to be
that there are several different isoforms of this receptor our control. integrated into the cell for effectiveness. Because we are
present in humans. Isoform I is the most prevalent type knocking down Jak2, there is the fear that those cells that are
found in healthy bodies, whereas the truncated Isoform IV successfully nucleofected with siRNA will not survive because
has been found to be present in higher ratios in patients they will be unable to incorporate mIL-3 into the cell. We are
Results
with Severe Congenital Neutropenia (SCN) and Acute currently troubleshooting this issue prior to further
Myeloid Leukemia (AML). In vitro studies have previously A qPCR of HGPRT as control was performed to determine experimentation.
shown that truncated GCSF-R IV causes increased nucleofection efficiency at different siRNA concentrations:
proliferation, with no effective differentiation in myeloid
precursor cells. References
o Mehta HM, Muneyoshi F, Glaubach T, Lee DW, Andolina
JR, Whichard Z, Quinn MP, Kao W, Sarkar CA, Maciejewski
JP, Corey SJ. Leukemia-associated loss of the GCSF receptor c-
terminus alters growth and differentiation properties and Jak-
Stat signaling. Blood (submitted 10/30/2012)
Differentiation o Weigert O, Lane A, Bird L, et al. Genetic resistance to JAK2
enzymatic inhibitors is overcome by HSP90 inhibition. J Exp
Neutrophil Med. 2012; 209(2):259-273. DOI 10.1084/jem.20111694.
Monocytes
Proliferation
Lymphocytes
Survival Acknowledgements
A western blot analysis was done after the original nucleofection
to determine whether the siRNA was effective in knocking down I would like to thank the Corey Lab at Northwestern Feinberg for
Jak2 at 300 nM concentration (and which ones are most giving me this opportunity.
effective):
The differences in internal signaling pathways between
isoforms I and IV are of critical importance, as they could be
potential drug targets in future treatments. siRNA 0 Scr #1 #2 #3 #4
Jak2
Our previous research has shown a differential response to
Actin
the protein Jak2, which has a dose-dependent downstream
effect on protein Shp-2 in GCSF-R IV but not GCSF-R I. For BaF3 HA-GRI 48 Hr Post-Nucleofection
this reason, knockdown studies of Jak2 in both cell-receptor
types could provide an important avenue for researching the
differences between these two receptors.