CELL BIOCHEMISTRY AND FUNCTION VOL.

11: 279-286 (1993)

Stimulation of HeLa Cell Growth by Physiological

Concentrations of 4-Hydroxynonenal
NEVEN ZARKOVICtS, ZORAN ILICt, MISLAV JURINt, R. JORG SCHAURS,
HERBERT PUHLS AND HERMANN ESTERBAUERS
tRudjer Boskovic Institute, Bijenicka 54, 41001 Zagreb, Croatia
$Institute of Biochemistry, University of Graz, Schubertstrasse I , A-8010 Graz, Austria

The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single
exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation
product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors,
particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of
the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days.
Fetal calf serum not only had a growth stimulating effect but also modulated the action of HNE. In neither respect
was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin-HNE
conjugate.
HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological
concentration (100 p ~ ) which
, was primarily cytotoxic and a physiological range (below 10 p ~ which ) showed
growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth,
which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared
to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of
lipid peroxidation, but a physiological growth regulating factor as well.
KEY wo~Ds-4-Hydroxynonenal (HNE); lipid peroxidation; cell proliferation; viability; human carcinoma (HeLa); protein
synthesis.

INTRODUCTION
4-Hydroxynonenal (HNE), a peroxidation product
of omega-6-polyunsaturated fatty acids, is thought
to be a ‘second toxic messenger’ of short-lived
oxygen free radicals.’Y2 Among its various bio-
logical effects the cytotoxic action and the
inhibition of growth have been described exten-
~ i v e l y . ~In, ~recent years it became evident that
HNE is a normal constituent of many cells and
tissues of animals and humans with a concentra-
tion range up to 1 p ~ The . ~question arises
whether these low concentrations are sufficient to
affect cellular growth. The aim of this study was
to analyze the growth response of mammalian
cells over an extended period of time to a single
exposure of different concentrations of HNE and
furthermore to examine the modulating effect of
serum factors, particularly albumin, on the growth
pattern. Hence, HeLa cells, a well characterized
established human carcinoma cell line, were cul-
tured under serum-free conditions in plain medium
or in medium supplemented with bovine serum
albumin (BSA), as well as in medium supplemented
with fetal calf serum (FCS). The results
show that HeLa cells cultured in FCS respond
to 0.1 to 1 ~ L MHNE with a transient inhibition
of growth followed by a strong stimulation of
proliferation.
MATERIALS AND METHODS

Cells and culture conditions

§Addressee for correspondence. On leave at the Institute of
Biochemistry, Graz. HeLa cells were seeded at the density of lo3 cells
Dedicated to Professor Dr Erwin Schauenstein on the occasion per culture into 96-well microcytoplates (Greiner,
of his 75th birthday. Frickenhausen, Germany) and cultured at 37°C
0263-6484/93/040279-08$09.00
0 1993 by John Wiley & Sons, Ltd.
280 N.ZARKOVIC ET AL.

in a humidified atmosphere of air with 5 per cent
CO,. The cells were incubated in RPMI 1640
medium, either under serum-free conditions
without any additional supplement, or under
serum-free conditions in medium supplemented
with 1-25mgml-' of bovine serum albumin
(BSA) (Sigma Chemical Co., St. Louis, U.S.A.),
as well as in medium supplemented with 5 per
cent of fetal calf serum (FCS) (Serva, Heidelberg,
Germany). Growth rate and cell viability, as
determined by the Trypan blue exclusion test,
were monitored daily for quadriplicates of the
cultures during the four consecutive days. The first
determination of the cell count and viability was
performed 3 h after the cells were seeded, i.e. just
before the addition of the aldehyde.
ruplicates was calculated. p values below 0.05
were regarded as indicating significant differences.
RESULTS
Cell Proliferation and Viability
As can be seen from Figure 1 the growth of HeLa
cells depended on the presence of serum in the
medium. If the medium was supplemented with
albumin instead of serum, no overall cell prolifera-
tion could be obtained during the four days of
m
100000 r Medium + 5% FCS

HNE Treatment
4-Hydroxynonenal was synthesized as
previously described5 and added to the cell culture
medium 3 h after the cells were seeded. The
concentrations of HNE used were: 100p ~ 10, p ~ ,
1 p~ and 0.1 p ~ There was no further addition
.
of HNE and the medium was not changed during
-
g loooor Medium + 1.25 mg ml-' BSA

the consecutive four days.

Incorporation of Radioactive Amino Acids

The incorporation of [3H]-amino acids
mixture (average sp. act. 45 Cimmol-l) 3 looo
(Amersham International plc, Buckinghamshire, 1 2 3 4
U.K.) was determined for quadriplicates of the
cultures. The labelled amino acids were added Medium alone
to the culture medium at an amount of 0.5pCi
per well, while the cells were harvested with
-2 loooo i
the flow harvester (PHD cell harvester,
Cambridge Technology, Inc., Watertown, U.S.A.)
after the 24h period, the amount of labelling
was measured in a @counter (Wallac 1209 -
Rackbeta liquid scintillation counter, Pharmacia, 6 1ooo 64
-
1 2 3
Sweden).
Days
--0- --A-- ...a*.* -.-o.-.
Statistics Control 0.1 pM HNE 1 pM HNE 10 pM HNE 100 pM HNE

The results obtained were analyzed using the
Mann-Whitney test, except for those obtained by
the Trypan blue exclusion test, because the cell
counts obtained for the serum-free cultures were
low. The mean values and the standard deviations
for the quadriplicates of cultures could not be
calculated; hence the proportion of the total
amount of the dead and live cells for quad-
Figure 1 . The influence of different concentrationsof HNE on
the growth rate of HeLa cells cultured under serum-free condi-
tions, in medium supplemented with 1.25mgml-' of BSA or
with 5 per cent of FCS. The values of the cell counts presented
for each group of cultures for particular days of incubation
represent the mean values obtained for quadriplicates of
cultures (SD 5.04-30.43per cent of the respective mean). *Sig-
nificant difference if compared with the control (p < 0.05,
Mann Whitney test).
EFFECTS OF H N E ON HeLa CELL GROWTH 28 1

incubation. However, an increase of the cell count further indicated the growth modifying effect of
was observed on the third day for the control the aldehyde, particularly for the cells cultured
serum-free cultures (twofold increase) and the with serum supplement. Thus, the decrease of the
control cultures supplemented with albumin cell count on the fourth day which was observed
(three-fold increase). Although in both cases the for the control serum-deprived cultures did not
increase of the cell count was significant if com- occur if the cells were cultured in the presence of
pared with the respective values obtained the day 0.1 p~ HNE, but only if albumin was used as a
before, (p = 0.014), it was followed by a decrease supplement.
of the cell count the day after. The lack of a decrease in the cell count on the
As expected, addition of 1 0 0 HNE ~ ~ inhibited fourth day was observed also if the cells were incu-
cell growth irrespective of the presence of serum bated in presence of 1p~ HNE, but in this case it
or albumin (significant difference if compared was irrespective of the addition of albumin.
with the control, p = 0.014, was obtained for the Moreover, 1 p~ and 0.1 p~ HNE showed the
serum-free and for albumin supplemented cultures same pattern of modifying the cell proliferation in
on the third day, while in case of serum supplemen- the presence of serum as did 1 0 p ~ indicating
, a
ted cultures the differences were always significant
except on the first day, p = 0.057). Medium + 5% FCS
Yet, it was apparent that the inhibition of cell
proliferation caused by 1 0 0 HNE ~ ~ was not s,80
only the result of the high concentration of the
aldehyde, because a gradual increase of the cell
count was observed for the serum supplemented
cultures. Thus, a four-fold increase of the cell
count was observed for serum supplemented cul-
tures between the second and the fourth day of
incubation after treatment with 100 p~ HNE
(p = 0.014). Therefore, it seems likely that the cells 100 Medium + 1.25mg mi-' BSA
which recovered from the toxic effect of the = WL
aldehyde retain the proliferating capacity, at least
if cultured in presence of serum.
A very similar finding was observed for the
cultures exposed to much lower concentrations of
HNE. Just like treatment with 100p~ HNE, expos-
ing the cells to 10~ L HNE
M prevented an increase of
the cell count on the third day of culturing, but
only if the culture medium was not supplemented
with serum or albumin. Thus a significant differ- 100 Medium alone
ence (p = 0.014) was obtained if the values of the - 9oF
cell count were compared with the control values
on the third day for the cells cultured in plain
medium. In the presence of serum this concentra-
tion of the aldehyde transiently inhibited cell prolif-
eration during the first two days of incubation
(both on the first and the second day the difference
was significant if compared with the control.

- E m =
282 N. ZARKOVIC ET AL

bifunctional growth-modifying activity of the alde- Medium + 5% FCS

hyde. Hence, for l p HNE ~ a significantly 25000
(p = 0.014) lower cell count was observed on the
second and a significantly higher on the third day
(p = 0.014) if compared with the untreated con-
trol, while in the case of 0-1 p~ there was only a sig-
nificantly higher cell count on the third day if
compared with the control (p = 0.014). Thus, a
transient inhibition of the cell growth after treat-
ment with HNE resulted in an increased rate of
cell proliferation if the cells were cultured under ,3000 r Medium + 1.25 mg m1-l BSA
optimal conditions, i.e. in presence of serum.
While the untreated control cells doubled in
number per culture between the second and the
third day of incubation, the number of the cells
treated with 1 0 , HNE ~ ~ increased at the same
time four-fold. The same increased rate of prolif-
eration was obtained for the cultures treated with __

0.1 p~ HNE, while the most striking finding was 0" -24 24 - 40 40 - 72 72 - 96
a nine-fold increase for 1 p~ HNE-treated cells
for the same 24 h period.
Furthermore, according to the Trypan blue
exclusion test, even the highest concentration of
HNE did not kill all the cells (Figure 2).
Particularly, it was not so in the case of serum-
supplemented cell cultures, since for the control
cultures and the cultures treated with lower concen-
trations of the aldehyde an increased incidence of
dead cells could be noticed as the cultures became
confluent. Due to the inhibiting effect of 1 0 0
HNE on cell proliferation, the cultures did not
become confluent and the proportion of dead cells
~ ~
- 0 -24

$
--
24-40

--A--
40 - 72
Incubation period (hours)
...
X..

Control 0.1 pM HNE 1 pM HNE 10 pM HNE 100 pM HNE

-. -0.
72 - 96

-.

was even lower than for the control cultures or the Figure 3. The influence of different concentrations of HNE on
cultures treated with lower concentrations of HNE. the degree of incorporation of [3H]-aminoacids into HeLa cells
In the case of serum-deprived cells incubated with cultured under serum-free conditions, in medium supplemented
1 0 0 , ~HNE,
~ a slightly increased incidence of with 1.25mgml-' of BSA or with 5 per cent of FCS. The CPM
values presented for each group of cultures for particular days of
dead cells could be observed in comparison to the incubation represent the mean values obtained for quadrupli-
untreated control cultures, but only on the second cates of cultures (SD 4.75-26.21 per cent of the respective
and the third day of incubation. A similar effect mean). *Significant difference if compared with the control
could also be observed for 1 0 , HNE ~ ~ cultures, (p < 0.05, Mann Whitney test).
but only if the cells were cultured in the presence
of albumin. Nevertheless, the lack of serum itself showed a daily increase of the amount of incor-
increased the proportion of dead cells in cultures poration of amino acids. A different pattern was
even for the control, and it seems that 1 0 p and ~obtained for the serum-free cultures. A sudden
100,~ HNE
~ caused a decay only in the serum- increase in the incorporation was observed during
deprived cell cultures. the second 24 h of incubation of the control cul-
tures (significant difference, if compared with the
control values obtained the day before,
Incorporation of Amino Acids p = 0.029), followed by a gradual daily decrease.
The pattern of change in the incorporation of In the presence of albumin, the degree of incor-
radioactive amino acids (Figure 3) showed an poration seems to be stabilized, since no particular
obvious similarity with the cell counts (Figure 1). daily change in the incorporation could be
In the presence of serum the control cultures detected.
EFFECTS OF HNE ON HeLa CELL GROWTH
The addition of 1 0 0 HNE ~ ~ resulted in a
constant inhibition of the incorporation, since the
differences obtained were irrespective of the condi-
tions used for cell culturing. They were significant
for all the intervals monitored if compared with
the control (except for the first 24h if the cells
were incubated in the absence of serum or
albumin, p = 0.057).
Although the inhibitory effect of 1 0 0 HNE ~ ~
did not seem to depend on the presence of serum
or albumin, the inhibitory effects of lower concen-
trations of the aldehyde on the incorporation of
amino acids showed the same pattern of interfer-
ence with serum as in the case of the cell count.
Hence, in the presence of serum, lower concentra-
tions of HNE (< I O ~ M )transiently inhibited the
degree of the incorporation, while afterwards the
cells exposed to the physiological range of HNE
concentrations showed an increase in the incor-
poration of amino acids. The inhibition of
incorporation was significant (p = 0.014) for all
HNE concentrations during the first and the
second 24 h of incubation, except for 0.1 p~ HNE
which did not show a significant effect during the
initial 24h (if compared with the control,
p = 0.1). A significant increase (if compared with
the control) in the incorporation was observed for
0.1 p~ (p = 0.014) and l p HNE ~ (p = 0.029)
for the third 24h, but in the case of cultures
exposed to l O p ~HNE the obtained difference
was not significant (p = 0.057).
If the cells were cultured in the presence of
albumin a moderate inhibition by HNE of the
rate of incorporation of amino acids could be
determined throughout the whole period. Thus,
for all concentrations of HNE used for each time
interval monitored, the degree of incorporation
was significantly lower (p = 0.014) if compared
with the control, except for 0 . 1 HNE ~ ~ for the
Table 1 . Growth promoting effect of HNE on HeLa cells cultured in presence of fetal calf serum.
HNE Cell count
added per well ( x lo3)
None 19.37 f 2.17
0.1 pM 39.75 f 3.80t
1I1M 52.15 f 554t
10 p M 25.89 f 377t
100 p M 2.1 1 f 0.55t
*Percentage of the mean value obtained the day before. tSignificant difference if compared with the control 0,< 0.05, Mann
Whitney test). The values presented were obtained on the 3rd day of in v i m incubationof the cells after a single exposure to different
concentrations of the aldehyde. The results are given as mean f SD for quadriplicates of cultures.
283
second 24-h interval (p = 0.243). Similar inhibi-
tion was observed for the cells cultured in the plain
medium. Although none of the HNE concentra-
tions used significantly inhibited incorporation of
amino acids during the first 24 h (p 2 0.057), for
all the concentrations of the aldehyde used signifi-
cant inhibition (p 5 0.029) was observed for the
consecutive 24-h intervals.
A comparison of the effects of HNE on the cell
count and the incorporation of amino acids for
the cells cultured in the presence of serum is pre-
sented in Table 1. At the particular time interval
between the second and the third day of incuba-
tion an obvious growth promoting activity of
lower concentrations of the aldehyde could be
observed. In the case of the toxic dose, i.e.
1 0 0 p ~ ,that period was also the time of the
gradual cell growth beginning after a period of
‘quiescence’, not yet accompanied by an increased
growth rate as seen for the pysiological
concentrations.
DISCUSSION
The results of this study indicate that HeLa cells
seem to respond to the addition of physiological
concentrations of HNE (0.1-1 p ~ by) changing
the pattern of proliferation, i.e. the initial inhibi-
tory effect of HNE on HeLa cell growth was
followed by a stimulation of cell growth (Table
2). Modification of the HeLa cell growth in the
presence of low concentrations of HNE seems to
be mediated through certain serum factors, but
not serum albumin. To our knowledge it is the first
time that a stimulatory effect of HNE on prolifera-
tion and incorporation of precursors into proteins
has been observed. In previous studies this phe-
nomenon was probably not recognized because
the duration of observation was too limited and/
Amino acids Amino acids
Cell count incorporation incorporation
increase* (CPM well-’ x lo3) increase*
229 18.04 f 4.72 119
449 2 8 . 0 4 f 1.71t 271
934 24.97 f 2.26 269
451 23.44 f 2.96 333
156 3.67 f 0.43t 173
284
Table 2. Summary of the effects of different concentrations of HNE on HeLa cell growth depending on the culture conditions.
Growth behaviour of the cells cultured in presence of
Medium + FCS'
Without HNE Optimal
0.1 p~ HNE Transient inhibition followed
by strong stimulation
1 p~ HNE Transient inhibition followed
by strong stimulation
1 O p HNE
~ Transient inhibition followed
by moderate stimulation
1 0 0 HNE
~ ~ Strong inhibition
~ ~~
*Medium supplemented with 5 per cent of fetal calf serum. tMedium supplemented with 1.25mgml-' of bovine serum albumin. The
effects on growth behaviour were summarized according to the effects of HNE on the daily change of the cell count, the vlability of
the cells (according to the Trypan blue exclusion test) and the degree of incorporation of [3H]-amino acids
or the ratio of HNE concentration to cell number
was lower. However, it is known that in vivo, a
moderate does of HNE (10mgkg-l) given
orally causes a significant increase of the total
body weight of young rats6 Thus, it seems
likely that, in vitro as well as in vivo, moderate
amounts of HNE could rather have growth
modifying effects. Whether this effect is a conse-
quence of the cellular, or in the case of the in vivo
model even systemic, reaction to moderate and
transient toxic effects of HNE remains an open
question.
The effect of HNE on HeLa cells was clearly
dose-dependent and a distinction could be made,
especially if the cells were cultured in the presence
of serum, between a high concentration (100 p ~
which was primarily cytotoxic and physiological
concentrations (below 10 p ~ which ) showed
growth modulatory effects. Physiological concen-
trations of HNE caused only a transient inhibition
of cell proliferation, which under optimal condi-
tions (in the presence of serum) was followed by a
period (3rd day) of increased proliferation as
compared with the untreated control.
It is possible that the addition of HNE to the cul-
ture medium caused a transient inhibition of cell
mitosis, thus the cells did not divide until they
had completely recovered from the moderately
toxic effect of HNE. In favour of this possibility
is the finding of a gradual increase of the cell num-
ber even for the cells treated with 1 0 0 HNE ~ ~ if
cultured in presence of serum.
The effect of HNE on cell growth was strongly
modified by the presence of fetal calf serum, but
not by albumin. Therefore the stimulatory effect
of serum cannot be explained by the formation of
a conjugate of HNE with albumin, but some
serum growth factor must be involved.
N. ZARKOVIC ET AL.
Medium + BSAt Medium alone
Moderate Moderate
Unchanged Unchanged
Unchanged Unchanged
Unchanged
Strong inhibition Strong inhibition
Recently, several studies have appeared
which showed that HNE may act as a
promoting factor for the expression of heat
shock genes, or in bacteria of the SOS-~peron.~-ll
These results indicate a certain gene-specific
effect of HNE influencing the cellular reaction
to the different unfavourable conditions.
Thus, it might be expected that the regulation of
cell proliferation might be also influenced by the
effects of HNE on particular genes as it is already
known that HNE at physiological concentration
can modify the transcription of genes (c-myc)
which in fact take part in the regulation of
proliferation. 12313 While toxic concentrations of
HNE 2 1 O p ~inhibited by c-myc expression, I-h
)treatment of K562 human erythroleukemic cells
with 1 p~ HNE resulted in a transient decrease of
c-myc mRNA levels during the first 4h. Later
higher levels of c-myc mRNA were found, and
the gene expression was normalized to the basal
level after four days.13
Such an effect of 1 p~ HNE did not influence the
cell viability or proliferation rate, but it was accom-
panied by an increased incorporation of [3H]-
leucine.l 3 Although the modified expression of
c-myc was observed for a different type of immor-
talized human malignant cells from those we
used, and the cells were cultured under serum-free
conditions, these results also point to the possible
involvement of physiological concentrations of
the aldehyde in the control of cell growth. In
favour of the possibility that HNE could modify
expression of the genes involved in the regulation
of cell behaviour is also a finding of the differentia-
tion of HL60 cells in vitro induced by continuous
exposure to 1 p~ HNE.14 However, it is an open
question if the effects of HNE in vitro depend
only on the concentration of the aldehyde and
EFFECTS OF HNE ON HeLa CELL GROWTH
the type of cells treated or also on the rate of
metabolism of the aldehyde in culture. Hence, it
could be speculated that even opposite effects
could be obtained by a single or by a continuous
exposure of the cells to a low amount of HNE. In
view of the finding by Dianzani’s group that 1 p~
HNE is sufficient to stimulate signal transduction
pathways especially via cyclic AMP,15 it can
be envisaged that target genes might exist which
are responsible for a promoting action of
HNE on cell growth. Furthermore, since the
growth-promoting activity was not observed for
the quiescent, serum-deprived, HeLa cell cultures,
but only for the cells cultured in presence of fetal
calf serum, it seems that the stimulated cell growth
was not a direct result of low, physiological
concentrations of HNE, but more likely of certain
serum growth factors (but not albumin) modified
by HNE. This possibility seems credible, since
UV light which generates an oxidative stress, with
consequential production of HNE, can enhance
the f o s expression, while the f o s promoter is
activated by binding of 62 and 67kDa serum
response proteins (SRPs) to a serum response
element.16.17 However, oxidative stress could
influence expression of numerous other genes,
and it is uncertain whether HNE or oxygen
radicals themselves could promote the growth of
the cells in vitro in the way described, by a
mechanism based on enhanced expression of
‘early-genes’ like myc or fos. More likely the
increased growth rate of the cells is based on the
cascade of events involving the modified
expression of the ‘effector-genes’ regulated by the
nuclear protein products of the early-genes.18 The
‘effector-genes’ and their protein products have
not yet been identified and their role in the control
of the normal and tumour cells is only theoretical.
However, HNE, as a ‘second toxic messenger’ of
the biological effects of oxygen free radicals, could
be considered not only as a toxic product of lipid
peroxidation, but as a physiological growth
regulating factor as well.
ACKNOWLEDGEMENTS
This study was supported by the Austrian
Science Foundation that granted the first
author the Lise Meitner fellowship research
grant and in a part by the AICR and the
Ministry of Science of Croatia.
The authors would also like to express their
sincere gratitude to Mrs Nevenka Hirsl and Mrs
285
Margareta Cvetkovski for excellent technical
assistance.
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Received (revised) 8 June 1993
Accepted 14 June 1993