Vol.

183,

March

No.

2, 1992

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

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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.

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2, 1992

BIOCHEMICAL

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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

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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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tumor cell lines, and PKC-deficient

AND

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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.

f ;: 13. 14. 15. 16. 17. 18. 19.

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