672
2.14. Western blot analysis.
To obtain total cell lysates, cells from supernatants were collected by
centrifugation at 2.000 x g for 5 min at 4ºC. Attached cells were scraped off in ice-
cold PBS, pelleted by centrifugation at 4.000 x g for 10 min at 4ºC and resuspended
in lysis buffer (25 mM HEPES, 2.5 nM EDTA, 0.1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride and 5 µg/ml leupeptin). Cellular lysates were
clarified by centrifugation at 12.000 x g for 10 min. After SDS-PAGE, gels were
transferred to Immobilon membranes (Millipore), blocked using 5% non-fat dried
milk or 3% bovine serum albumin (BSA) in 10 mM Tris-HCl, 150 mM NaCl pH 7.5
(TBS) and incubated overnight with antibodies as indicated in 0.05% Tween-20-
TBS. Immunoreactive bands were visualized using the ECL Western blotting
protocol (Millipore).
2.15. Statistical Analysis.
The data are expressed as means ± SD. The statistical significance was
estimated with Student's test for unpaired observation (p*<0.05; p**<0.01; and
p***<0.001).
3. RESULTS AND DISCUSSION
PTP1B expression is induced in human liver during APAP intoxication
.
Cell proliferation and cell death are governed by stimulatory and inhibitory
signals. Whereas trophic factors simultaneously stimulate mitosis and inhibit cell
death, negative growth signals regulate the opposite of these biological effects. In
the liver, trophic factors include endogenous growth factors such as EGF, bFGF,
TGF-alpha and IGFs that act through receptors belonging to the tyrosine kinase
superfamily (32). Thus, tyrosine phosphorylation may play a regulatory role in the
induction and execution of programmed cell death in the liver. On that basis, our
first goal in this work was to investigate whether an overdose of APAP induced the
expression of PTP1B, a negative modulator of survival-mediated signaling
pathways, in human liver biopsies obtained from individuals suffering from APAP
intoxication. In our previous work (33) we showed elevated PTP1B expression in
patients with APAP toxcicity that needed liver tranplantion. In agreement with
this, Figure 1 shows elevated PTP1B expression in another patient suffering from
APAP intoxication. Notably, immunostaining was detected in surviving
hepatocytes in the areas surrounding the central veins (right panel) as compared
to a normal liver (left panel).
PTP1B deficiency protects mouse hepatocytes against elevation of ROS.
The fact that PTP1B has been related to the induction of apoptosis in
hepatocytes under conditions of serum withdrawal (27) or activation of the Fas
death receptor (34) prompted us to investigate the involvement of PTP1B in the
