Biology Reference
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28.3.4
Knockdown of TauT by RNAi Increases
Anticancer Drug Sensitivity
The above results demonstrated that knockdown of
TauT
in 293 cells could
significantly inhibit the growth of tumor cells. To determine whether inhibition of
TauT
could increase sensitivity to an anticancer drug in 293 cells, both the control
and TauT-deficient cells were treated with doxorubicin. Fig.
28.4
illustrates that
doxorubicin (200 ng/ml) induced cell death in both cell lines in a time-dependent
manner. The number of apoptotic cells was significantly higher in TauT-deficient
cells than in control cells, suggesting that knockdown of
TauT
increased the sensi-
tivity of doxorubicin-induced apoptosis in 293 cells.
To further determine if knockdown of
TauT
increased the sensitivity of doxoru-
bicin-induced apoptosis, both the control and shRNA-TauT 293 cell groups were
treated with doxorubicin (200 ng/ml) for 24 h, and DNA fragmentation was ana-
lyzed by DNA ladder in accordance with directions provided by the manufacturer
(R&D systems, MN). As shown in Fig.
28.5
, the 180-200 bp DNA laddering was
detected in cells treated with doxorubicin (lane 4) but not in the 200 ng/ml doxoru-
bicin-treated control cells (lane 2). These results indicate that the apoptotic effects
of doxorubicin are significantly enhanced in TauT-deficient 293 cells.
28.3.5
Knockdown of TauT Causes Cell Cycle G2 Arrest
To examine if inhibition of
TauT
alters the cell cycle, control and TauT-deficient 293
cells treated with or without cisplatin were analyzed by flow cytometry. As shown
in Fig.
28.6
, inhibition of
TauT
significantly increased the cell numbers in the G2
phase (10.2%) as compared to that in control cells (4.0%).
TauT
de fi ciency also
sensitized the 293 renal cells to cisplatin-induced apoptosis.
28.3.6
Effect of Inhibition of TauT on Signal Pathways
To further explore the impact of
TauT
deficiency on the signal pathways, microar-
ray analysis of gene expressions was performed using RNAs from control or TauT-
deficient 293 cells. As expected, inhibition of
TauT
shows both positive (>1.50-fold
upregulated genes;
p
< 0.01; 2,599 genes) and negative (>1.50-fold downregulated
genes;
p
< 0.01; 2,345 genes) effects on genes in various signal pathways that are
involved in the process of downregulation of cell proliferation and organic nutri-
ents transporters. Surprisingly, inhibition of
TauT
dramatically decreases (87-fold)
the expression of Slc2A3 (glucose transporter 3, GLUT3) in TauT-deficient 293
cells as compared to control cells. GLUT3, also known as solute carrier family 2,
facilitates the transport of glucose across the plasma membranes of mammalian
cells. GLUT3 is most known for its specific expression in neurons (Maher and
Simpson
1994
) .
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