Vol.
183,
March
No.
2, 1992
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
16, 1992
Pages
DNA BINDING
FACTORS
Jorge
Alemany3,
' Centro '
AND
de
WHICH INTERACT PROMOTER ARE John
F.
Klement',
Investigaciones
Biologicas,
of Molecular National Institutes
and
Laboratory
3 Pharmagen,
Received
WITH TEE Spl DEVELOPMENTALLY
January
23,
OF TEE CHICKEN REGULATED
Teresa
Borrds3
CSIC,
Velazquez
Developmental of Health, Tres
SITE
and
Biology, Bethesda,
Cantos,
28160
61-CRYSTALLIN
Flora
144,
De Pablo'*
28006
National Maryland
Madrid,
659-665
Madrid,
Eye 20892
SPAIN
Institute,
Spain
1992
Transcription of the 61-crystallin gene is developmentally regulated in the embryonic chicken lens. Previous work defined a positive transcription regulatory element between positions -120 and -43 of the 61-crystallin promoter. This region contains a putative Spl binding site (-78 to -71), adjacent to a CAAT box (-70 to -67). Gel retardation assays using lens nuclear extracts revealed two protein-DNA complexes which involved the Spl site. The formation of the complexes increased from day 6 to day 11 of embryogenesis (period of lens organogenesis) peaked between days 11 and 15, then decreased in a non-parallel manner until hatching (day 21). A point mutation in the Spl binding site of the 61-crystallin promoter abolished formation of one of the complexes (complex 1, slower in mobility), while point mutations in the CAAT box had no effect on the formation of either complex. Studies using purified Spl protein and increasing amounts of embryonic chicken lens nuclear extracts showed cooperativity in the formation of both complexes, more remarkable with complex 1. a 199: Rcadrmlc Press. 1°C.
The
chicken
throughout highly the
6
undetectable growth
positive
the
CAAT
essential known, promoter
binding
site)
Adjacent
box
did
not
transcriptional however, are
* Corresponding
factor
how regulated
morphological cells
(IGF-I) the
was to
affect
promoter
trans-acting during
author.
659
increase to
stimulated
by
its
promoter box
of
-603
to
to
in -67).
regulate
61-
(6)
and one -70
viva
it
(10). the
of and
and
Mutations
that
promoter
and
The
-79
activity (-70
-120)
interaction between
suggesting which
the gene
(5). regulation
The
located
a CAAT
serum
61-crystallin
elements
Sl-crystallin
factor(s) development.
be
(7).
is and
mRNA
lens-specific
regulation, the
protein,
posthatching
The
region
for is
studied
differentiation
positions
stimulation GC-rich
be
differentiation
regulatory for
GC box in
decrease
(from
required
the
6-crystallin
(3,4).
negative
I
element the
with
with
of
can
fiber
essential
of
and
6-crystallin
can
soluble
stage
Levels
&-acting was
factors
(8,9).
of
contain
-43)
to
growth Spl
to
element
transcription
(1).
and
predominant the
embryogenesis
elongated
-120
its
cells of
embryogenesis
upon
Expression
shown
positive
(putative in
19
coincident
been
insulin-like
vitro
lens
into
(from
crystallin
in
of
factors, has
in
depending
day (2).
cells
promoter
more
and
levels
epithelial
for
expression age
day
early
6-crystallin,
in
developmental
and
develops
organogenesis. regulated
between
or
lens
is It
61-crystallin
not was
an not
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
The products of src and ablare non-receptorial tyrosine kinases associated with the inner layer of the membrane. 1111.fms and erbB encode growth factor receptors (respectively for Colony Stimulating
Factor-l
- CSF-1 - and Epidermal Growth
Factor -EGF- ),
which are permanently active and show increased tyrosine kinase activity. 11111.fosand myc code for nuclear proteins that interact with DNA (4, 5). Since each oncogene
transforms
cells by means of different
molecular
mechanisms, we hypothesized that the common feature responsible for radioresistance could
be found within
transformation.
those biochemical
Cell transformation
steps that are altered in oncogenic
by oncogenes that code for proteins involved in
mitogenic signalling has been associated with multifaceted alterations of intracellular second messengers. In particular, complex changes involving diacylglycerol (DAG) formation, inositol lipid metabolism, and phosphatidylcholine turnover have been reported (for rev. see 5). Therefore, we decided to study second messenger formation in cell lines transformed
by oncogenes that caused resistance, and whose transforming
products interfered with signal transduction. Our results allowed us to make a correlation between increased level of diacylglycerol
and phosphatidylcholine
metabolism,
and
resistance to ionizing radiations.
MATERIALS and METHODS. Normal NIW3T3 fibroblasts, and their counterpart transformed by the oncogenes IS, ra( src, abl, erbB, were previously described (6). The normal myeloid line 32D, and its counterparts either transformed by the oncogenes erbB, abl, src, or transfected with the genes coding for EGF and PDGF receptors, were described (7, 8). The study of mitogenic second messengers associated with transformation caused by different oncogenes, was carried out by prelabelling the cultures with radioactive precursors (New England Nuclear) for 48 h, in the presence of fetal calf serum. We measured: I. Phosphoinositide metabolism by labelling the cultures with 13Hlinositol (9). II. DAG metabolism by labelling the cultures with [3H]glycerol (10). III. Phosphatidylcholine metabolism by labelling the cultures with [3H]methyrcholine (11). Inositol phosphates, DAG and phosphatidylcholine metabolites were separated by ion exchange chromatography, and thin layer chromatography respectively (9-l 1). In order to compare the results obtained in each cell line, we normalized the obtained data for the incorporation of each radioactive precursor in the corresponding phospholipid. This procedure, repeatedly confirmed in different studies (9- 1 l), allowed reproducible normalization of data.
RESULTS.
Bearing in mind that many mitogenic
signals converge on the
metabolism of inositol lipids, we measured the basal level of inositol polyphosphates (IPs), and DAG in normal and transformed NIW3T3 Bbroblasts prelabelled with tritiated inositol and glycerol respectively. Normal NIW3T3 fibroblasts were chosen because they show a survival curve to ionizing radiations similar to that of several human tumor lines (2). Table 1 shows that cells transformed by the oncogenes ms, mf; srcand erbB had decreased level of inositol phosphates. These results, that are in agreement with those previously obtained in ms transformants, indicate that the turnover of inositol lipids was
Vol.
183,
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2, 1992
BIOCHEMICAL
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RESEARCH
COMMUNICATIONS
Table 1 Steady-state levels of phosphocholine, inositol phosphates, diacylglycerol, in NIH/3T3 cells transformed by ras, ra4 WC and erbB oncogenes
and
oncogene
IPS
DAG
p-cho
la.9
0.75
1.26
2.25
raf
0.45
1.84
2.06
sl-c
0.27
1.28
3.55
erbB
0.70
1.59
3.16
Cell cultures were labelled to equilibrium for 48 hours with methyl-[ 14C]choline, [3H]myoinositol, or [ 14C]glycerol in serum-containing medium. After radiolabelling, cultures were washed and maintained for 4 hours in serum-free medium, after which the medium was removed and cells incubated for 1 hour in 35-mm dishes containing 1 ml of serum-free medium. In experiments designed for measuring IPs formation, lithium chloride (20 mM) was present during the final incubation. Intracellular phosphocholine (p-cho), total inositol phosphates (IPs), and diacylglycerol (DAG) were extracted and measured as described. Values obtained for each metabolite were normalized for the amount of radioactivity associated with the precursor phospholipid. Data are expressed as fold increase over control (i.e. normal NW3T3 cells were taken as 1.OO), and are means of at least three determinations.
lower than in normal cells; this pattern was quite different from that expected if growth factor signalling was constitutively stimulated. Several explanations might account for these results. Increased incorporation of labelled inositol into phosphoinositides in transformed cells might result in an apparentdecreaseof inositol phosphates.However, we measuredthe pattern of labelling of phosphoinositidesin normal and transformed NW3T3 fibroblasts,and we found no significant difference betweenthe cell lines(Table 2). Therefore, the observed differences could not be ascribedto different metabolism and/or incorporation of precursorsin transformedcells. Another hypothesisconcernsthe
Incorporation
of [3H]myuinositol transformed
Table 2 into phosphoinositides NIW3T3 fibroblasts
[ 3 H]PIPdcell
oncogene
[3H]PIPs
NIW3T3
6843
618750
0.011
las
9179
849750
0.010
laf
11313
872250
0.012
SK
11801
906666
0.013
7288
548250
0.013
erbB
cell number
in normal
and number
Normal and transformed cultures were labelled as described in the legend to Table 1. [3H]phosphoinositides (PIPS) were separated by thin-layer chromatography; results are expressed as 3H-radioactivity (cpm) associated with phosphoinositides, and are means of at least three determinations. 654
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possibility that cells transformed by oncogenes interacting with signal transduction might show a pattern of receptor-phosphoinositide
de-sensitization analogous to that observed
during continuous stimulation with growth factors. Alternatively,
constitutive activation
of PKC by increased DAG production from sources other than phosphoinositides,
could
de-sensitize inositol lipid signalling, as it was observed with phorbol ester treatment (5). Whatever the case, transformation of NIW3T3 cells by oncogenes radioresistance was not associated with increased inositol lipid turnover.
that cause
DAG level was constitutively elevated in transformed cells, thus confirming an observation previously made in other NIW3T3 transformants (6- 11). The increase of DAG without corresponding increase of phosphoinositide turnover led us to hyphotesize that transformed cells could used alternative sources for DAG production. Beside the turnover of inositol lipids, DAG can be formed through the turnover of phosphatidylcholine (for rev. see 12). In order to assess whether phosphatidylcholine turnover was a general response in cells transformed by oncogenes causing resistance, we decided to study the intracellular accumulation of choline and phosphocholine, the decrease of phosphatidylcholine. increased
level
of phosphocholine
phosphatidylcholine), These
results
(p-cho;
i.e. the hydrolysis
product
accompanied by decreased level of phosphatidylcholine
suggest
phosphatidylcholine
and
Our experiments showed that transformed cells had
that an increase
of the hydrolysis
of
(Table 1).
and turnover
of
could be responsible for the high level of DAG.
Next, we decided to investigate whether the changes associated with oncogeneinduced
radioresistance
hematopoietic
were
cells transformed
chosen for the following
common
to other cell lines and we studied 32D
by the oncogenes erbB, srcand abl. These cells were
reasons: I. They are one of the most suitable model for the
study of normal hematopoiesis
and leukemogenesis
(7). II. Transformation
by the
oncogenes e&B, srcand abl caused an increase in radioresistance at 5 cG/min dose rate (3, 13). III. It is supposed that resistance to radiations could be responsible for the lack of therapeutical effect in patients subjected to radiotherapy for leukemia and lymphoma (13). At variance with NIW3T3
fibroblasts,
we observed an increase of DAG level
accompanied by an elevated turnover of phosphoinositides and of phosphatidylcholine (Fig. 1). These results, i.e. the presence of high turnover of phosphoinositides in 32D, led us to study the correlation between inositol lipid metabolism and radioresistance. To this end we used transfectants expressing the EGF (EGFR-32D) receptor. These transfectants exhibited normal phenotype, and stimulation by EGF was not accompanied by an increase of radioresistance (3, 13). 32D cells overexpressing (PDGFR-32D) metabolism
the PDGF receptor
were taken as control, since PDGF rapidly stimulates (8, 9). Fig. 2 shows that stimulation
inositol lipid
with EGF or PDGF triggered the
metabolism of inositol lipids and the formation of DAG, without any effect on the rapid turnover of phosphatidylcholine. These results seem to indicate that stimulation of the “classical” inositol lipid turnover was not directly associated with the radioresistant phenotype. It appears that the biochemical alterationz common to transformation by 655
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IPS
66-
01
32D
erbB abl cell line
-1 O2 O Coat
src
Figure I. Steady-state level of second messengers in normal and tmnsfonned 320 cells. 32D hemato3poietic cells were labelled to equilibrium with [3H]glycerol, [3H]myoinositol or methyl-[ HIcholine for 48 h in complete medium as described. Afterward, cultures were washed and maintained for 4 h in serum-free medium. The final incubation was for 1 h in serum-free medium. In those experiments designed to measure inositol phosphates, lithium chloride (20 mM) was present during the final incubation. Data are expressed as fold increase over control (i.e. normal 32D cells were taken as 1 .OO), and are means of at least three determinations. IPs: total inositol phosphates. p-cho: intracellular phosphocholine. DAG: diacylglycerol. Figure 2. Second messenger formation in response to different growth factors. 32D cells were labelled to equilibrium with [3H]glycerol, [3H]myoinositol or methyl[3H]choline as described. Wild-type 32D cells or transfectants expressing foreign growth factor receptors (for EGF and PDGF respectively) were then stimulated with the appropriate growth factor for 10 min. Final concentration of growth factors was: EGF, 500 rig/ml; PDGF 100 rig/ml. In those experiments where inositol phosphates were measured, lithium chloride (20 mM) was added during incubation with agonists. Results are expressed as fold increase over control (unstimulated) cells, and are means of at least 5 experiments, each performed in triplicate samples. Results were normalized for the total incorporation of radioactivity in parent phospholipids as described. DAG: diacylglycerol. IPs: total inositol phosphates. p-cho: intracellular phosphocholine.
oncogenes that cause resistance were the increase of DAG formation, and of phosphatidylcholinemetabolism.
DISCUSSION.
It hasbeen demonstratedthat someoncogenesconfer the ability to
metastatizeand acquire resistanceto ionizing radiations(2, 3). It could be hypothesized that somecommon mechanism(s)account for this phenomenon,since the products of theseoncogenesdiffer greatly in structureand function. We reasonedthat, sincemost of the genes causing resistance interfere, at different levels, with mitogenic signal transduction, the common mechanism(s)could be found in the cascadeof mitogenic secondmessengers whoseformation is triggered in responseto growth stimuli. The study of signal transduction in transformedNIH/3T3 fibroblasts and in 32D hematopoietic cells revealedthat elevated DAG and phosphatidylcholineturnover were common featuresof transformation causedby resistance-inducingoncogenes.However, the turnover of inositol lipids was differently altered in the two transformedcell types. At variance with transformed NIW3T3 fibroblasts, 32D cells showed a net increase of 656
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inositol
lipid metabolism.
immature
myeloid
proliferation
AND
BIOPHYSICAL
RESEARCH
These results were particularly
32D cell line is strictly
COMMUNICATIONS
intriguing;
the murine,
dependent on Interleukin-3
(IL3)
for
and survival (7, 9). Signalling through the IL3 receptor weakly stimulates
DAG production and phosphatidylcholine metabolism (14). Furthermore, stimulate inositol
turnover, without any effect on inositol lipid
32D cells are devoid of receptors for growth factors that
lipid turnover.
Therefore,
elevated phosphoinositide
turnover
in
transformed 32D cells could be attributed to a direct effect of the oncogenes, rather than to a “cross-talk”
between receptors and oncoproteins.
Whatever the case, our results
suggest that increased inositol lipid turnover in 32D cells was not associated with resistance to ionizing radiations: 32D made to express the receptor for EGF or PDGF, and stimulated with their cognate ligand, showed a strong phosphoinositide response without
acquiring
phosphatidylcholine
resistance. All gathered evidence points to DAG production
and
turnover as the biochemical passages commonly altered during
transformation by resistance-inducing oncogenes. As
far as alternative
phosphatidylcholine involved
for
DAG
turnover and synthesis
in transformation
transformed
sources
production
are
concerned,
de ROVO, are the two major pathways
(12, 15, 16). We demonstrated
that 3T3 fibroblasts
by a variety of oncogenes showed increased synthesis
(17). Thus, it appears that phosphatidylcholine
de novoof
DAG
turnover and synthesis de nova are both
operative during oncogene-induced transformation of 3T3 fibroblasts. It can be hypothesized that the two pathways are strictly interconnected, as DAG synthesized de ROVOcould contribute to enhanced phosphatidylcholine should be noted that de nova synthesized
turnover, and viceversa ( 12). It
DAG (9, 16), and DAG deriving
from
phosphatidylcholine (12) differ from DAG generated through inositol lipid turnover: chemically different DAGs could selectively stimulate diverse PKC isozymes, thus being responsible for different cellular responses. Concerning radioresistance, transformed showing
the molecular
mechanism
cells causes activation, down-regulation,
an effect that is superimposable
Transformation equivalent
through which
DAG
could favour
we propose the following hyphothesis. Constitutive increase of DAG in and redistribution
of PKC, thus
to that of tumor promoters
(5, 17, 18).
by oncogenes that increased DAG, might represents the endogenous
of prolonged
treatment with tumor promoters.
We demonstrated
that
constitutive DAG increase and PKC stimulation in transformed cells was associated with nuclear translocation of the kinase, and abnormal phosphorylation of nuclear proteins (18). It is proposable that this phenomenon could interfere with resistance to ionizing radiations in at least two different ways: I. Determining
a different
conformational
rearrangement of nuclear proteins that are complexed with DNA; thus, particular critical sytes, direct or indirect targets of radiation-induced damage, would be “hidden” and “protected”.
II. Inducing the transcription
of genes responsible for the synthesis
of
enzymes involved in the mechanism of repair (13). A recent report seems to support this hypothesis since PKC inhibitors were able to increase radiation sensitivity of human 657
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BIOCHEMICAL
tumor cell lines, and PKC-deficient
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cell clones were more radiation sensitive than wild-
type (19). Whatever the mechanism involved, our results demonstrate that elevated DAG correlates with resistance, independently of the oncogene expressed. These findings might be important in predicting the responsiveness
to irradiation;
in fact, it has been
demonstrated that several genetic hits are required for development of most spontaneous human tumors, thus making it diflicult to relate the presence of a single oncogene with the degree of malignancy. Further sudies will establish whether the analysis of intracellular DAG is clinically applicable to the prediction of the responses to radiation therapy. Acknowledgment: This work was partly Associazione Italiana per la Ricerca sul Cancro.
supported
by a grant
from
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
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Brock, W.A., Campbell, B., Goepfert H., Peters, L.J. (1987) Cancer Bull. 39: 98-102. McKenna, W.G., Weiss, M.C., Bakanauskas, V.J., Sandler, H., Kelstein, M.L., Bianglow, J., Tuttle, SW., Endlich, B., Ling, C.C. and Muschel, R.J. (1990) Int. J. Radiat. Oncol. Biol. Phis. 18: 849-859. Fitzgerald, T.J., Henault, S., Sakakeeny, M., Santucci, M.A., Pierce, J.H., Anklesaria, P., Kase, K., Das, I. and Greenberger, J.S. (1990) Radiat. Res. 122: 44-52. Chiarugi, V. P., Ruggiero, M., and Porciatti, F. (1987) Cancer Invest. 5: 215-230. Chiarugi, V.P., Basi, G., Quattrone, A., Micheletti, R. and Ruggiero, M. (1990) Second Mess. and Phosphoproteins 13: 69-85. Ruggiero, M., Srivastava, S.K., Fleming, T.P., Ron, D., and Eva, A. (1989) Oncogene 4: 767-77 1. Pierce, J.H. (1989) Biochim. Biophys. Acta 989: 179-208. Matsui, T., Pierce, J.H., Fleming, T.P., Greenberger, J.S., La Rochelle, W.J., Ruggiero, M. and Aaronson S.A. (1989) Proc. Natl. Acad. Sci. USA 86: 83 148318. Chiarugi, V.P., Magnelli, L., Pasquali, F., Basi, G. and Ruggiero, M. ( 1989) FEBS Lett. 252: 129-134. Pierce, J. H., Ruggiero, M., Fleming, T. P., Di Fiore, P. P., Greenberger, J. S., Vartikovski, L., Schlessinger, J., Rovem, G. and Aaronson, S. A.: (1988) Science 239: 628-63 1. Lacal, J.C., Moscat, J. and Aaronson, S.A. (1987) Nature 330: 269-272. Pelech, S.L. and Vance, D.E. (1989) Trends Biochem. Sci. 1: 28-30. Fitzgerald, T.J., Santucci, M.A., Das, I., Kase, K., Pierce, J.H. and Greenberger, J.S. (in the press). Duronio, V., Nip L. and Pelech, S.L. (1989) Biochem. Biophys. Res. Comm. 164: 804-808. Slivka, S.R., Meier, K.E. and Insel, P.A. (1988) J. Biol. Chem. 263: 1224212246. Peter-Riesch, B., Fathi, M., Sclegel, W. and Wolheim, C.B. (1987) J. Clin. Invest. 81: 1154-1161. Chiarugi, V., Bruni, P., Pasquali, F., Magnelli, L., Basi, G., Ruggiero, M. and Famararo, M. (1989) Biochem. Biophys. Res. Comm. 164: 8 16-823 . Chiarugi, V., Magnelli, L., Pasquali, F., Vannucchi, S., Bruni, P., Quattrone, A., Basi, G., Capaccioli, S. and Ruggiero, M. (1990) Biochem. Biophys. Res. Comm. 173: 528-533. Hallahan, D.E., Virudachalam, S., Grdina, D.J., Schwartz, J.L., Weichselbaum, R.R. (199 1) Radiat. Oncol. Biol. Phys. 2 l/l: 65. 658