Biology Reference
In-Depth Information
stress in RPE may involve differential formation of
homodimeric and heterodimeric AP-1 proteins based
on the relative molar abundance of each protein. Thus,
an increasing pool of Jun:Fos htereodimers after oxida-
tive stress will have increased stability over decreasing
Jun:Jun pools and favor binding to AP-1 consensus
sequences (TGAGTCA). At a later time point following
oxidative stress, increasing pools of ATF proteins will
favor Jun:ATF heterodimers that will preferentially
bind to cAMP-reponsive elements (TGACNTCA) (105).
de novo protein synthesis. The next question the authors
addressed was whether transient increases in APE1
expression confer an adaptive response to genotoxic
agents. Clastogenic adaptation was observed following
APE-inducing pretreatment with NaOCl or H
2
O
2
but
not when cells were challenged with MMS or UV light
as measured by the number of chromosomal aberrations
in CHO cells. Thus, in response to oxidative stress, cells
specifically activate transcription of the APE1 gene, one
of only a few DNA repair genes that are induced in
response to genotoxic stress.
107
Induction of the APE1
gene in response to H
2
O
2
was subsequently shown to
require the cyclic AMP response element-binding
(CREB) site within the APE1 promoter. ATF2 and c-Jun
were shown to bind to the CRE element, and expression
of c-Jun was also enhanced in response to H
2
O
2
in these
CHO cells. Thus, induction of c-Jun in response to oxida-
tive stress is involved in a signaling pathway that results
in activation of the APE1 promoter.
108
More recently, APE1 has been shown to be induced in
response to treatment with arsenite, a human carcin-
ogen, with similar increases in mRNA, protein, and
enzymatic activity levels in mouse fibroblasts.
109
Expo-
sure to high levels of arsenic has been shown to cause
cancer.
110
Arsenic causes oxidative stress in cells due
to its ability to induce formation of oxyradicals and
H
2
O
2
.
111,112
In this study, sodium arsenite was found to
induce APE1 through transcriptional activation by
ATF4 and c-Jun through binding to a CRE site in
APE1's promoter, and knock-down of either ATF4 or
c-Jun by siRNA was found to eliminate the induction
of APE1. Arsenite toxicity resulted in mouse fibroblasts
and human lymphoblastoid cells when either APE1 or
ATF4 was suppressed, and mutagenesis induced by
arsenite in ATF4-depleted cells was offset by expression
of transgenic APE1. The authors concluded that ATF4-
mediated upregulation of APE1 and other genes plays
an important role in safeguarding cells from arsenite-
induced toxicity and mutagenesis.
109
APE1 is Induced in Response to Oxidative
Stress
In response to sublethal doses of ROS or ionizing
radiation but not other genotoxic agents such as alkylat-
ing agents or UV light, increased amounts of APE1
mRNA and protein were observed in HeLa S3 tumor
and WI 38 primary cell lines.
106
Increases observed
were greater than two-fold. APE1 was also shown to
translocate to the nucleus in response to ROS. Concom-
itant with increases in APE1 protein levels in response
to ROS, an increase in endonuclease activity was
observed consistent with the likelihood that increased
APE1 increases BER. To determine whether increased
levels of APE1 result in an adaptive response, cells
were treated with HOCl and, after 12 hours when
a maximum increase in APE1 was observed, were
then subjected to genotoxic agents including H
2
O
2
,
bleomycin, MMS,
g
-rays, UVC light, or HOCl. Signifi-
cant resistance in the HOCl-treated cells was observed
for subsequent challenge with H
2
O
2
, bleomycin, and
MMS but not for the other agents. Despite APE1's role
in processing alkylative damage in DNA, alkylating
agents did not induce APE1 rather the induction was
ROS-specific. ROS generated by bleomycin, H
2
O
2
,and
ionizing radiation all induced APE1. It is somewhat
surprising that APE1 is induced as it is a relatively
abundant protein. However, its low 3' DNA phosphodi-
esterase activity may make it limiting in the repair
pathway.
106
Induction of APE1 mRNA, protein, and activity in
CHO cells in response to oxidative stress was also
shown by Kaina and coworkers.
107
In this study,
a three-fold increase in APE1 mRNA levels and protein
levels in response to treatment with H
2
O
2
was observed.
Transcriptional activation of APE1 was also increased in
response to H
2
O
2
and NaOCl as measured using an
APE1-promoter CAT construct. However, enzyme
activity did not increase to the same extent as protein
levels. The authors concluded that post-translational
modification may play a role in regulating APE1's endo-
nuclease activity. Treatment with cycloheximide/aniso-
mycin prevented accumulation of APE1 mRNA,
suggesting that
Oxidative Stress Alters Base Excision Repair
Base excision repair is the first line of defense against
oxidative damage to DNA. Knockdown of APE1 by
RNA interference in human MCF7 [breast cancer] and
HCT116 [colon carcinoma] with a functional p53-
dependent DNA damage checkpoint stopped cell
proliferation and activated apoptosis.
6
These effects
were reversed by expression of the structurally unre-
lated yeast Apn1 AP endonuclease suggesting that it
is the AP endonuclease function of human APE1 rather
than protein
protein interactions or other activities of
APE1 that are vital to cell viability.
6
To determine the
impact of oxidative stress on the base excision repair
pathway, 2-nitropropane (2-NP) was used to induce
e
the induction was dependent upon
