New York: John Wiley and Sons, 1988. 2. Romeo .... 1995. 24. Benech, P., Mory Y., Revel M., and Chebath J. Structure of two forms .... Bursch, W., Oberhammer,.
Vol. 8, 91-100,
January
1997
Cell Growth
Retinoic Acid and IFN Inhibition of Cell Proliferation Associated with Apoptosis in Squamous Carcinoma Cell Lines: Role of IRF-1 and TGase IIdependent Pathways1
and RA are able to increase activity in this cell line.
Valeria Gianna
Giandomenico, Francesca Lancillotti, Fiorucci, Zulema A. Percario, Roberto Rivabene, Walter Malomi, Elisabetta Affabris, and Giovanna Romeo2 Laboratory of Virology [V. G., F. L, G. F., Z. A. P., E. A., G. R.] and Department of Ultrastructures [R. A., W. M.], Istituto Supeniore di Sanit#{224}; Istituto Tecnologie Biomediche, Consiglio Nazionale delle Ricerche [G. F., G. R.j; and Department of Biology [E. A.], Universit#{224}di Roma Tre, Rome, Italy
Abstract
Both retinoids
and IFNs are known to inhibit of many normal and transformed cells and to have an in vivo antitumor effect against a variety of cancers, including squamous cell carcinoma. Because the combination of IFNs and all-trans retinoic acid (RA) could improve their antitumor effectiveness (depending on the histological origin and state of differentiation of the cells), we compared the activity of RA and/or IFNa2b with regard to the mechanism of growth inhibition of MEI8O and SiHa cell lines, derived from squamous cervix carcinoma at different stages of differentiation. We reported previously that, in the MEI8O cell line, the combined treatment significantly increased the growth inhibitory effect of the single agents. Here, we show that the SiHa cell line appears more sensitive to IFNa2b than the MEI8O cell line, and resistant to RA, which does not significantly inhibit SiHa cell growth. Induction of apoptotic cell death clearly occurs and correlates with the inhibition of cell proliferation in both cell lines. It is interesting that the induction of the proliferation
transcription
factor
IFN regulatory
factor
Received 5/16/96; revised 9/30/96; accepted 11/6/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1
This work
was supported
by grants
from
the Associazione
Italiana
91
Is
TGase
II expression
and
Introduction Both
IFNs
and
functions,
retinoids
including
are known
growth,
to regulate
differentiation,
basic and
cellular
immune
re-
activity (1-3). They exhibit an antiproliferative effect on many normal and transformed cells and have in vivo antitumor effects against a variety of cancers (4-6). A growing body of evidence from both laboratory and clinical research now supports the concept that simultaneous exposure to both cancer chemopreventive and therapeutic agents, retinoids and lFNs, can result in enhanced antiproliferative effects compared with either agent alone in a number of malignancies or malignant cell types. Recent clinical work in advanced squamous cell carcinoma reports major activity with this regimen
(7). New
nipulation
therapeutic
of normal
anisms,
such
as
paradigms
physiological terminal
involving
growth
cellular
the
regulatory
ma-
mech-
differentiation
or
pro-
grammed cell death, are being explored. In vitro studies have demonstrated that the combination of retinoids and IFNs resulted in additive or synergistic effects on the growth and differentiation of several SCC lines, which are thought to be the direct or indirect result of changes in gene expression (8-i 0). Strong evidence exists to support a major role for nuclear
retinoid
receptors,
which
transactivate
the
expres-
sion of target genes by binding to specific retinoic acid response elements (1 1), in mediating the effects of retinoids on gene expression and thereby altering the growth and differentiation of both normal and tumor cells. IFNs exert their
I correlates
with the subsequent induction of apoptosis, whereas TGase I and II expression does not. In particular, TGase I and II appear differentially expressed in the MEI8O and SiHa cell lines; i.e., TGase I is expressed in MEI8O and specifically inhibited by RA, whereas TGase II is expressed in SiHa. It is interesting that both IFN-a
& Differentiation
biological
of effector
of which and
activities
genes
is transiently
I3) or type
on target
(lSGs3; II (‘)
cells
by inducing
a number
Ref. 1 2), the transcriptional
increased IFNs
with
after the binding their
specific
activity
of type I
(a
cell-surface
receptors. The transcriptional stimulation is mediated by preexisting proteins that become activated and function as transcriptional factors that recognize an enhancer element (ISRE for type I IFN or y-activated sequence for type II IFN) within the regulatory sequence of target genes. Several studies have also been performed on the control of tumor
cell
retinoids. thelial
differentiation In particular,
cell
differentiation
pathways
by using
spontaneous have
and been
both
IFNs
drug-induced
associated
and epi-
with
the
per
Ia Ricerca sul Cancro, the Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica, and Consiglio Nazionale delle Ricerche No. 95.01697.CTO4 and Ministero Universit#{225}Ricerca Scientifica Tecnologica (40%). 2 To whom requests for reprints should be addressed, at Laboratory of Virology, Istituto Superiore di Sanit#{224},Viale Regina Elena, 299, 00161 Rome, Italy. Phone: (396) 49903231 ; Fax: (396) 49902082.
The abbreviations used are: ISG, IFN-stimulated gene; ISRE, IFN-stimulated response element; TGase, transglutaminase; RA, retinoic acid; SCC, squamous carcinoma cell; IRF-1 , IFN regulatory factor 1 ; 2-5A, 3
2’-S’oligoadenylate;
phate dehydrogenase.
RAR, RA receptor;
GAPDH,
glyceraldehyde-3-phos-
92
All-trans
RA and
IFN-a
Induce
Apoptosis
in SCCs
is7
is7
Ctr
‘RA 1O’U
‘IFNalOOIJ/ml
.
is5
2
2
4
3
3
5
4
7
6
Days
Days
Fig. 1. Effects on SiHa cell growth of RA, IFN-a2b, and their combination. Cells were treated with different concentrations of RA (106 and i0#{176} M) and IFN-a2b (100, 500, 1000, and 2000 lU/mi) and with the combination of RA (106 M) plus IFN-a2b at 100 lU/mI. Control and treated cells were counted daily. The results of a representative experiment are presented as mean values of viable cells counted in duplicate dishes.
activity
a group of Ca2-dependent
of
These
enzymes
cross-links
are
between
capable
enzymes,
of catalyzing
polypeptide
i.e., TGases.
the
chains
(13).
formation TGase
MEl 80 and of
I is in-
duced during squamous differentiation and plays a role in the formation of the cross-linked envelope (1 4), whereas the function of TGase II is less well established. This enzyme has been implicated in the activation of several cytokines (15, 1 6), and the expression of TGase II has been found in association
with
the active,
death
called
apoptosis
tion
of
ment,
cells
during
as well
genetically
controlled
(1 7-1 9), which morphogenesis
as in many
in
adult
process
occurs
tissues
in tumor
growth
(20). All-trans RA has been reported to induce TGase II expression and apoptosis in a variety of cell types (21 22). The role of IFN in inducing this phenomenon has yet to be ,
elucidated
The
(23).
present
mechanisms and their
of growth
their
combination
growth
(TGase involved synthetase
investigation
features,
was
inhibition
negative IRF-1).
exerted
to study
by IFN-a2b
in cultured
SCCs
expression
of differentiation
I and II), and IFN-induced in the and
designed
genes
via the
and RA of
markers
suggested
control of cell growth Two different cell lines
the
analysis
in their
SiHa,
belonging
to be
(i.e. , 2-5A were used,
to the
differentiative
stage.
same
histotype
In particular,
lished
from
a primary
noma of the cervix. that
both
optosis
agents,
in SCCs,
ing this
tissue
sample
Our results IFN and
IFN-a
phenomenon.
but
of a rapidly was estab-
of a squamous
provide
evidence
RA, are capable
dif-
MEl 80 was
derived originally from an omental metastasis spreading cervical carcinoma, whereas SiHa
expression, correlates tosis phenomenon.
develop-
embryonic
and
of cell
in the elimina-
fering
carci-
indicating
of inducing
ap-
being more active than RA in inducIRF-1 expression, but not TGase II with the RA- and IFN-induced apop-
Results Analyses Antiproliferative Growth RA
and
IFN-a2b
Effects.
We reported
inhibited
proliferation
previously of ME18O
(1 0) that cells
in a
dose- and time-dependent manner. A markedly increased growth inhibitory effect was observed when combination treatment was carried out. In all of the combination treatments
performed,
the single
agents
even
those
in which
did not significantly
low concentrations affect
growth,
a signif-
of
Cell Growth
& Differentiation
.
93
-..
4 Fig. 2. Fluorescence microscopy. Evaluation of apoptosis in SiHa cells collected from the supernatants of cultures exposed to RA (10_6 M; b), IFN-a2b (2000 IU/ ml) (c), and the combination of RA and IFN-a2b (d) for 48 h. Numerous mitotic figures are present in SiHa control cells (a). Apoptotic, fragmented nuclei are detectable from supernatants. In particular, chromatin clumps (arrows), as well as condensed nuclei with aggregated chromatin (0), are observable. Note that the number of detached cells undergoing apoptosis is remarkable in MEl 80 cells if compared with SiHa (d).
0
I
O
C II
:4!
a.
.?,
.
C icant
inhibition
(up
to
50%)
of the
proliferation
rate
Morphological
was
observed.
reported
Fig. 1 (left) shows SiHa
cell
proliferation,
IFN-a2b
hibitor. a dose-
and
hibitory
effect
lU/mI,
a significant
In contrast,
SiHa
by
(1 0).
was To
analyze
IFN-a2b growth analyses
and RA (right).
scribed
the
effect
induced
the
This
for TGase
by IFNs
effects
of apoptosis
or RA (21-23)
In fact, show
which
M,
different
treated
in
of in
not
growth
and have
inhibition
whether, effects performed
a
these
lines
cell
and
methods.
A different
adhering
the supernatants
both
also
SiHa
and
cells
by
cell
present
ME18O
in
cells
IFN. Both agents level of programmed
or
phenomenon
was
a different IFN. after
were capable cell death more
tumors
from
for the of viability.
morphological be
observed
, SiHa
2a and
untreated
showed
much
(i.e.
However,
the treatment with RA and massive apoptotic cell death
of
in fact
Figs.
ME18O
histotype
in spontaneously
means
detectable
lines.
the
out
cell lines
were
(a)
are related
be responsible
can
respectively;
untreated
were
carried
“baseline”
figures
results
underwent
IFN in terms
first
of the two
detect:
These of
could
to RA and were
the
to the same
features.
which
of to
substrate
characteristics
analyses
cells,
the
belong
cytological
are derived,
mitotic
out
(b) whether a different bethe two cell lines consid-
and
between
detached
of
de-
suggested
(1 8, 1 9). To examine
under our conditions, the observed antiproliferative correlated with the induction of apoptosis, we morphological and DNA fragmentation analyses.
shown).
have
carried
from
apoptosis;
susceptibility
untreated
consideration
be found
morphological
they
typical
incorporation
(data
the
In was
detached
cells
different
Separate
the
as effective
investigations in the
ered. to
analysis
by
would
at
resulted
was
death
but
cells
the
havior
of
we
Thymidine
cell
appeared
concentrations
combination alone.
in-
at 2000
whether
the growth of 106
treatment
Several
II in apoptosis
growth
in ME18O
effects,
suboptimal
observed
Analyses. involvement
growth
inhibitory of
as IFN-a2b
confirmed
cell
in-
cell line in
given
already
combination
growth
a combination
Apoptosis
role
whether
inhibition
was
of
a poor
A marked
IFN-a effect
in reducing
of the
with
was
RA was unable to inhibit than 20% at a concentration
effective
augmentation cells
more
when
inhibitory
RA
inhibitor
of the SiHa
manner.
observed
but
cells
proliferation
time-dependent was
was a potent all-trans
whereas
inhibited
100 lU/mI. which
that IFN-a2b
Studies.
above,
3a).
and
in
MEl
80
Numerous
in the supernatants few
apoptotic cells.
cells
Moreover,
susceptibility
to
ME18O cells underwent treatment with either RA of inducing a significant (Fig. 3, b and C). The extensive
when
the
two
94
All-trans
RA and lFN-
Induce
Apoptosis
in SCCs
Fig. 3. Fluorescence microscopy. Evaluation of apoptosis in ME18O cells collected from the supernatants of cultures exposed to RA (10 6 b), IFN-a2b (2000 lU/mI) (c), and the combination of RA and IFN-a2b (d) for 48 h. Apoptotic, fragmented nuclei are detectable from supernatants. In particular, chromatin clumps (arrows), as well as condensed nuclei with aggregated chromatin (asterisk), are observable. Note that the number of detached cells undergoing apoptosis is remarkable in ME18O cells if compared with SiHa (d).
drugs
were
cence
analyses
ence
of
given
together of
some
apoptotic
typical
of apoptosis
ments
(Fig.
terms
only
c and
2,
appeared
more
was
Methods” tween
agents
performed
pattern
of
dation,
a
two
cell
after
DNA
also
DNA
ladders
SiHa
cells,
with
RA
cell
whereas alone
analogous lines.
RA
induce
in
+
a general
RA
in
100
treat-
ME18O
rule,
agents,
at
least
A quantification
As
and the
was
in
treatment.
be-
80
0
0
and
detected
shown
in
used
formation
of
they
were
agreement
in Fig.
of The
evident
apoptosis, pattern
with
1 . The weights
of
combined
after not
Fig.
IFN-a
by the
also
molecular
.!
of these
Materials
“
endonucleolytic hallmark
increased
were
IFN
stated
pres-
clumping
C.)
60
.
0
a.
that to
h of
observed
inhibition
show
48
and
difference
internucleosomal
appears
fluoresthe
chromatin
these
Studies.
shows able
the
showed
types.
biochemical
cells
as
as
4). A marked
were
IFN
to
(Fig.
analysis
after Thus,
d).
DNA Fragmentation the
with
susceptibility.
then
the
cells
cells
responsive
of apoptotic
results
3d). Instead,
(Fig.
SiHa-detached
the
the
40 0
typical
degra-
in
ME18O
20
fragmented
treatment
pattern
(100-1000
(8
single
DNA
in cells
observed
0 a.
treatment.
IFN-a
observed
as
5 (!eft),
of
discrete
of treated growth
bands
Kb) in both
0
SIHA
ME18O
Fig. 4. Evaluation of apoptosis in SiHa and ME18O cultured cells. The high susceptibility to apoptosis of the latter is well evident. The average values of experiments performed in quadruplicate are shown. Variations between each experiment were
