PNNL-13501 - Pacific Northwest National Laboratory

not DDM-PGE2 (Figure 1). The lack of correlation with a
TP receptor agonist suggests that the proliferative
response to 8-iso-PGF2α is unrelated to the endothelial
type TP receptor, and is consistent with evidence
suggesting smooth muscle cells may express a unique
isoprostane receptor. Of particular interest is the
proliferative response to 8-iso-PGF2α , which was
apparent at extremely low concentration (100 pM). In a
separate study, non-LDRD the proliferative response of
smooth muscle cells to 8-iso-PGE2 was investigated.
8-iso-PGE2 (0.1-100 nM) did not modulate smooth
muscle cells proliferation (data not shown).
Cell Proliferation
(% control)
200
180
160
B
140
120
8-iso-PGF2α
DDM-PGE2
100BJ
0
J
0.1
J
1
[nM]
J
10
Figure 1. Proliferative response of smooth muscle cells to
8-iso-PGF 2α, but not DDM-PGE 2
In the absence of enzymatic activity, 8-iso-PGF2α is the
dominant isoprostane generated by the oxygen free
radical catalyzed peroxidation of arachidonic acid.
However, experimental evidence suggests that 8-iso-PGE2
is the major isoprostane product formed in the liver,
indicating that prostanoid biosynthetic pathways may
influence the class of isoprostane generated in an organspecific
manner. This observation is relevant to the
response of primary hepatocytes to prototypical
isoprostanes. Hepatocytes were isolated from male
B6C3F1 mice using a standard collagenase digestion and
percoll gradient purification. Hepatocytes were incubated
in attachment medium for 4 hours, and subsequently
treated with epidermal growth factor (1 to 10 ng/mL),
10 nM 8-iso-PGF2α, 10 nM 8-iso-PGE2, or epidermal
growth factor + isoprostane for 48 hours in the presence
of 50 µM 5-bromo-2’-deoxyuridine in defined mitogen
restricted media. Cell proliferation was determined using
an anti-BrdU antibody, horse radish peroxidase
conjugated secondary antibody, and colorimetric
detection (and quantifying labeled nuclei). Epidermal
growth factor increased proliferation in a dose-dependent
fashion, while isoprostanes alone were without effect
(Figure 2; 8-iso-PGF2α not shown). However,
82 FY 2000 Laboratory Directed Research and Development Annual Report
B
B
cotreatment of cells with 1 ng/mL epidermal growth
factor and 10 nM 8-iso-PGE2 resulted in a proliferative
response comparable to a 10 ng/mL epidermal growth
factor concentration, despite the lack of effect of 8-iso-
PGE2 alone. In contrast, 8-iso-PGF2a as a cotreatment
was without effect. Therefore, cultured hepatocytes are
responsive to the major class of isoprostane (8-iso-PGE2)
observed in vivo, but not to the major class predicted in
the absence of enzymatic activity (8-iso-PGF2a). Further,
the lack of effect of 8-iso-PGE2 alone raises the
possibility that the cotreatment effect with epidermal
growth factor on cell proliferation may be synergistic.
Cell Proliferation
(BrdU Labeled Cells/dish)
200
160
120
80
40
0
Figure 2. 8-iso-PGE 2 modulates epidermal growth factordependent
proliferation in primary hepatocytes isolated
from male B6C3F1 mice
The effect of 8-iso-PGF2α on the growth of human breast
cancer cell line (MCF-7) was investigated. Cell
proliferation was determined using the picogreen assay.
Under anchorage-dependent conditions, 8-iso-PGF2α did
not modulate MCF-7 proliferation (data not shown). In
contrast, under anchorage-independent conditions (growth
on soft agar), 10 nM 8-iso-PGF2α increased MCF-7
proliferation as determined by an increase of DNA/dish
(Figure 3). Epidermal growth factor was included as a
positive control and to investigate cotreatment effects.
However, in contrast to the hepatocyte model,
cotreatment of cells with epidermal growth factor and
8-iso-PGF2α resulted in a proliferative response that was
additive.
We next investigated whether prototypical isoprostanes
modulated the proliferation of human renal epithelial cells
(HK-2). HK-2 cells were treated with 0.1 to 100 nM
8-iso-PGE2 and 8-iso-PGF2α for 3 days and cell
proliferation determined by quantifying DNA/dish.