0020-7I I X/92 $5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd
1~. J. Biochem.Vol. 24, No. 9, pp. 1475-1480,1992 Printed in Great Britain. All rightsreserved
SUPERINDUCTION BY HALOGENATED FLAVIO FLAMIGNI,
OF ORNITHINE DECARBOXYLASE RIBOFURANOSYLBENZIMIDAZOLES
PAOLA PALADINI, CLAUDIO
Dipartimento
di Biochimica,
CLAUDIO
STBFANELLI, CARLO GUARNIERI
and
M. CALDARERA
Universita’ di Bologna, Via Irnerio [Tel. 51-351201; Fax 51-3512241
48, 40126 Bologna,
Italy
(Received 20 December 1991) Abstract-l. The effect of dichlororibofuranosylbenzimidazole (DiCl-RB), an inhibitor of hnRNA synthesis and casein kinase-2 activity, on ornithine decarboxylase (ODC) was investigated in a difluoromethylornithine (DFMO) resistant, ODC overproducing cell line. 2. In cells growing in the absence of DFMO, DiCl-RB provoked a marked, but transient increase in ODC activity and immunoreactive ODC content. 3. The ODC response to DiCl-RB was prevented by cycloheximide and was not due to stabilization of the enzyme. 4. The dibromo derivative analogue (DiBr-RB) exerted similar effects on ODC, but was effective at lower concentrations. 5. The halogenated ribofuranosylbenzimidazoles were ineffective in cells growing in the presence of DFMO and containing higher levels of ODC protein.
INTRODUCTION
The nucleoside analogue 5,6-dichloro-l-b-D-ribofuranosylbenzimidazole (DiCI-RB) is widely used to inhibit RNA polymerase II-specific transcription in vivo and in vitro (Tamm and Sehgal, 1978; Zandomeni et al., 1982). The mode of action of DiCI-RB entails selective inhibition of protein phosphorylation (Egyhazi et al., 1984; Zandomeni and Weinmann, 1984) and in particular DiCI-RB is a powerful inhibitor of casein kinase-2 (CK-2) acting competitively with respect to the phosphate donors ATP and GTP (Zandomeni et al., 1986; Meggio et al., 1990). The 5,6 dibromoderivative (DiBr-RB) is a more effective inhibitor of mRNA transcription and CK-2 activity than DiCl-RB, whereas the nonhalogenated l-b-D-ribofuranosylbenzimidazole is ineffective (Tamm and Sehgal, 1978; Zandomeni et al., 1986; Meggio et al., 1990). CK-2 is a muhifunctional protein kinase (Pinna, 1990), which is not dependent on second messengers, such as cyclic nucleotides, calcium or inositol phosphates; however its activity may be modulated by the naturally occurring polyamines, spermidine and spermine. Interestingly both CK-2 activity and polyamine biosynthesis are enhanced after stimulation to cell growth and proliferation (Pinna, 1990; Tabor and Tabor, 1984; Pegg, 1986). Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is characterized by a very short half-life, which allows rapid and marked changes in its concentration and activity. ODC can be finely regulated at transcriptional, translational and posttranslational level by hormonal and growth
stimuli or by the end-products polyamines (Tabor and Tabor, 1984; Pegg, 1986; Heby and Persson, 1990). We have shown that CK-2 can efficiently phosphorylate ODC in vitro at a serine residue, Ser 303, located within a perfect consensus sequence for CK-2 (Meggio et al., 1984, 1987). This consensus sequence is fully conserved in ODCs of vertebrates (Flamigni et al., 1988; Bassez et al., 1990) and is contained in a “PEST region”, typical of rapidly degraded proteins (Flamigni et al., 1988). Phosphorylation of ODC in intact cells has also been shown (Flamigni et al., 1990b) and occurs at the same site phosphorylated by CK-2 in vitro (RosenbergHasson et al., 1991). In order to obtain some information about the physiological meaning of this phosphorylation, as yet unknown, we have investigated the effect of halogenated ribofuranosylbenzimidazoles on ODC activity and turnover in intact cells. We took advantage of the use of an ODC overproducing cell line, L1210-DFMO’ (Persson et al., 1988), which has been selected for resistance to the ODC inhibitor cc-difluoromethylornithine (DFMO). The results show that halogenated ribofuranosylbenzimidazoles can cause a transient increase of ODC activity in the cells, but it does not result from modifications of the catalytic activity or the turnover of the enzyme. MATERIALS
AND METHODS
Materials L-[I-‘4C]Ornithine
chased
1475
from
and DuPont-New
DL-[3,b3H]DFMO
England
Nuclear.
were DFMO
purwas
FLAVIO FLAMIGNI et al.
1476 Table
I. Effect of halogenated ribofuranosylbenzimidazoles activity in LIZI~DFMO’
Treatment Quiescent cells Cells harvested at day I after seeding (control) DiCI-RB at seeding DiBr-RB at seeding DiCI-RB, 80 min before harvesting DiBr-RB, 80 min before harvesting
RESULTS
on ODC
cells ODC activity (units/mg protein) 2.1 f 0.5 (3) 131 i 27 (9) 5.5 + 2.1 (3) 4.5 & 1.4 (3) 661 f 122 (7) 639 + 103 (3)
LIZIl&DFMO’ cells were grown in the absence of DFMO for at least IO days. Quiescent cells were diluted in fresh medium and collected about 24hr later. DiCI-RB (60pM) or DiBr-RB (24 PM) were added to the cell medium at seeding or 80 min before harvesting. The results represent mean ri SD of(n) separate determinations.
generously provided by the Merrell Dow Research Institute (Strasbourg, France). LIZIO-DFMO’ cells (Persson ef al., 1988) and antibodies against mouse ODC (Persson, 1982) were a generous gift from Dr Lo Persson, University of Lund, Sweden. DiBr-RB (Meggio et al., 1990) was generously provided by Dr D. Shugar. University of Warsaw, Poland and Dr F. Meggio, University of Padova, Italy. DiCI-RB, actinomycin D, spermidine and cycloheximide were purchased from Sigma Chemical Co. Cell culture Mouse LIZlO-DFMO’ cells were routinely grown in the presence of 20 mM DFMO in RPM1 1640 medium containing 10% fetal calf serum, antibiotics and fi-mercaptoethanol as described by Persson et al. (1988). For most experiments, cells were shifted to and maintained in a DFMO-free medium for lo-40 days. In order to induce ODC, quiescent cells grown either in presence or absence of DFMO, were seeded at a density of 0.2 x 106/ml and harvested about 24 hr later. Friend murine erythroleukemia cells were cultivated as previously described (Flamigni et al., 1989a). DiCl-RB and DiBr-RB were dissolved in 95% ethanol before addition to the cell medium (2 pi/ml). Control cells received equal volumes of the vehicle.
Effect
of halogenated
ribofuranosylbenzimidazoles
ODC
in cells growing
in the absence
on
of DFMO
In order to induce ODC, L1210-DFMO’ cells grown for some weeks without DFMO were diluted in fresh medium and collected after about 24 hr. Under these conditions, the cells contain high levels of ODC activity, putrescine and spermidine (Persson et al., 1988). Table 1 shows the effect of halogenated ribofuranosylbenzimidazoles on ODC activity. When DiCl-RB or DiBr-RB were added to the cell medium at seeding time, ODC induction was almost completely prevented, in agreement with the notion that the induction of this enzyme in growing cells requires transcription of ODC mRNA (Pegg, 1986; Kahana and Nathans, 1984). However, a marked increase in ODC activity was observed if the treatment with these chemicals was limited to the last 80 min before harvesting the cells, when high levels of ODC mRNA are present (Persson, 1982). The average increase of ODC activity was Sfold, ranging from 3- to 8-fold in separate experiments. ODC activity rose rapidly following DiCI-RB or DiBr-RB addition, peaked between 1 and 2 hr and then declined fast to values below the control 4 hr after the beginning of the treatment (Fig. I). Dose-response curves are shown in Fig. 2. Half maximal response was achieved at 6 PM DiCI-RB, and at a slightly lower value, about 2nM, in the case of DiBr-RB. The effect of benzimidazole derivatives on ODC activity was not due to changes in K,,, for the substrate, nor to release of active ODC from ODC-antizyme complex (results not shown). Rather, the increase in ODC activity was
Analytical procedures ODC was extracted from cells as previously described (Flamigni et al., 1989b). ODC activity was assayed by measuring the release of 14C0, from t_-[l-‘4C]ornithine (Flamigni et al., 1989b). ODC activity is expressed as units/mg protein, where I unit is defined as I nmol CO,/hr incubation. Control assays showed that DiCI-RB did not affect ODC activity when added directly to the cell extract in the assay mixture. Attempts to detect ODC-antizyme complex were carried out as described in (Flamigni et al., 1990a). The content of immunoreactive ODC protein was determined by radioimmunoassay by using [‘HIDFMOlabeled ODC, essentially as described by Seely and Pegg (1983). As for preparation of [‘HIDFMO-labeled ODC, the enzyme was extracted from kidney of testosterone-treated mice and partially purified by ammonium sulfate fractionation (Seely et al., 1982). Then ODC was labeled with DL-[3,4-‘H]DFMO (Seely and Pegg, 1983) and further purified by Bio-Gel TSK DEAE-5 PW chromatography (Bio-Rad). This step also removed unbound [‘HIDFMO. Protein content was determined according to Bradford (1976). Immunoprecipitation of ODC followed by SDS polyacrylamide gel electrophoresis was carried out as previously indicated (Flamigni et al., 1990b).
0
1
3
2 Time
4
(hr)
Fig. 1. Time+ourse of ODC stimulation by halogenated ribofuranosylbenzimidazoles. At day 1 after seeding, L I2 IODFMO’ cells grown in absence of DFMO were treated as follows: (0) control; (a) DiCI-RB, 60 PM; (0) DiBr-RB, I2 p M. Cell samples were taken at the times indicated after treatment and assayed for ODC activity.
Dichlororibofuranosylbenzinidazole
0
I
I
20
30
I
10
0
DiCI-RB
1411
and omithine decarboxylase
I
40
(PM)
4020
40
Time
60
80
(min)
Fig. 4. Effect of DiCI-RB on the ODC turnover in LiZlODFMO’ cells. At day 1 after seeding, half of the cells were supplemented with 60 PM DiCI-RB. After 40 min, 0.2 mM cycloheximide was added and cell samples were removed at 20min intervals. (0) Control cells; (A) DiCl-RB treated cells.
This effect was not the result of stabilization of the enzyme, since ODC decayed at a similar rate following cycloheximide in control cells and in cells pretreated with DiCl-RB for 40min (Fig. 4). Table 2 shows that cycloheximide was able to inhibit the DiCl-RB-induced stimulation of ODC completely, when added to the cells just before DiBr-RB. This indicates that new protein synthesis is required for the stimulation. Besides, actinomycin D (10 pg/ml) was able to provoke a significant increase of ODC activity (Table 2). The effects of DiCl-RB and actinomy~n D were not additive, and actually ODC activity resulted lower (P < 0.01) in cells treated with both the transcriptional inhibitors than in cells receiving DiCl-RB only. It should be noted that actinomycin D, differently from DiCl-RB, has been reported to decrease the half-life of ODC (Wallon et al., 1990). molecules.
0
4
0
DiBr-RB
12
16
(gM)
Fig. 2. Dose-response curves of ODC stimulation by halogenated ribofuranosylbenzimidazoles. At day I after seeding, Ll210-DFMO’ cells grown in absence of DFMO were treated with increasing concentrations of DiCl-RB (A) or DiBr-RB (0). After 80 min, cells were harvested and assayed for ODC activity.
by a roughly proportional increase in the content of immunoreactive ODC protein (Fig. 3), indicating that the stimulation of ODC resulted essentially from an augmented number of ODC accompanied
=
._
000
al
z a.
pl 600 ,E v1 .?z 5
400
,x ..z t; m
200
ODC activity
ODC protein
Fig. 3. Effect of DiCI-RB on ODC activity and ODC protein content. At day I after seeding, LIZlO-DFMOr cells grown in absence of DFMO were treated for 80min as follows: (0) control; ) DiCI-RB, 60 pM. Each bar represents the mean value & SD of 3 separate experiments. 8C24/9-H
1478
FLAVIO FLAMIGNIet
Table 2. Effect of cycloheximide and actinomycin D on DiCI-RB stimulated ODC activity ODC activity (units/mg protein) Treatment Controi Cycloheximide Actinomyci” D
- DiCl-RB
+ DiCl-RB
1702 17 3.7 i 0.5 591 * 67
709A48 3.6 i 0.3 564 + 20
+/4.2 1.0
1.o
Ll2lGDFMO’ cells were treated with or without DiCI-RB as described in the legend on Fig. 3. Cycloheximide (0.2 mM) or actinomycin D (lO~~g/ml) were added just before DiCI-RB, 80 min before harvesting. Each value represents the mea” + SD of 3 separate experiments.
Effect of halogenated ribofuranosylbenzimidazoles on ODC in cells growing in the presence of DFMO
L1210-DFMO’ cells can grow in the presence of 20 mM DFMO as well as in its absence (Persson et al., 1988). Ceiis routinely grown with DFMO synthetize ODC at a rate at least IO-fold higher than cells maintained without DFMO. Thus, ODC accumulates greatly and, although most of the enzyme is inactivated by the inhibitor, some residual activity is detectable. In cells grown in the presence of DFMO, the response of ODC to DiCl-RB was inhibited (Table 3,a). This conclusion can be drawn either from the measurement of the residual ODC activity or the immunoreactive protein. No significant difference in the content of ODC was found even in an experiment in which ODC extracted from control or DiBr-RB treated cells was analyzed by SDS-polyacryiamide gel electrophoresis (not shown). The presence of DFMO in the cells was not directly responsible for the lack of effect of benzimidazole derivatives. In fact, cells routinely grown without DFMO and shifted to a DFMO containing medium 30 min before treatment with DiCl-RB, showed the usual response to the chemical (Table 3,d). Since cells grown in the presence of DFMO contain lower levels of spermidine (Persson et al., 1988), the ODC response to DiCl-RB was studied in cells treated with exogenous spermidine. Table 3 shows that treatment with 0.1 mM spermidine slightly increased the response of ODC to DiCl-RB.
al.
Other conditions affecting the response of ODC to halogenated ribofuranosylbenzimidazoles
During the course of this study we found that some experimental conditions affect the response of ODC to these chemicals in cells growing without DFMO. If cells at day 1 after seeding were collected and resuspended in fresh medium and immediately treated with DiCl-RB, the ODC response was reduced to a 2-3-fold increase (Table 4). Under these conditions, control cells showed a higher ODC activity, possibly as a consequence of increased ODC synthesis, whereas treated cells did not present a proportional increase. The same phenomenon, even more accentuated, was observed after dilution of cell medium by addition of distilled water (Table 4). ODC activity is known to be strongly enhanced in mammalian cells by a sudden reduction in medium osmolarity (Poulin and Pegg, 1990). Since it has been reported that cells growing in high concentrations of serum are less retarded in the cell cycle by DiCI-RB than cells growing in low serum concentrations (Tamm et al., 1982) the effect of serum was also studied. Omission of the serum during the treatment resulted in a decrease of the ODC response by about 50% (not shown). Finally superinduction of ODC by the benzimidazole derivatives was observed also in murine erythroleukemia cells, indicating that this effect was not limited to a particular cell line overproducing ODC (data not shown).
DISCUSSION
This research shows that halogenated ribofuranosylbenzimidazoles, known inhibitors of mRNA transcription, can paradoxically provoke marked, but transient increases of ODC activity in growing mammalian cells (“superinduction”). The concentrations effective are comparable to those reported to inhibit CK-2 in vitro (Zandomeni and Weinmann, 1984; Zandomeni et al., 1986; Meggio et al., 1990) and, actually, the incorporation rate of 32P into ODC was reduced by DiBr-RB treatment in intact cells (Meggio F., Flamigni F. and Pinna L. A., unpublished data).
Table 3. Effect of DiCI-RB on ODC activity and eonteent in LIZlO-DFMO’ cells in the presence of DFMO
Treatment Control (b) Spermidine, just before DiCI-RB (c) Spetmidine, at seeding (d) DFMO, 30mi” before WCI-RB (a)
- DiCI-RB 8.4
ODC activity fu”its/mg protein) -_ + DiCl-RB +/II
1.3
4.1
7.5
1.8
0.9
1.3
1.4
9.0
43
4.7
ODC protein (ng/mg protein) +/-
-.-.. (nl
(n)
- DiCl-RB
+ DiCI-RB
(5)
7875
7867
1.0
(3)
(2)
3544
3699
1.6
(2)
(2)
342
498
1.5
(2)
(2)
63
258
4.1
(2)
Ll210-DFMO’ cells were grow” in the presence of 20 mM DFMO (except for Treatment d) for several weeks. Then cells were treated at day I after seeding with or without DiCI-RB as described in Fig. 3. Spennidine (0.1 mM) was added just before DiCI-RB treatment, i.e. 80 min before harvesting the cells (Treatment b), or at cell seeding (Treatment c). In Treatment d, cells grow” in the absence of DFMO were shifted to a DFMO containing medium 30 min before DiCI-RB treatment. The results represent means of fnf separate experiments.
Dichlororibofuranosylbenzimidazole and omithine decarboxylase Table 4. Effect of distilled water and change of medium on DiCI-RB stimulated ODC activity ODC activity Treatment Control Change of medium Distilled water
(units/mg
- DiCI-RB
+DiCI-RB
142 334 649
647 821 III6
protein) +/4.6 2.4 I.7
cells were treated with or without DiCI-RB as the legend of Fig. 3. A portion of the cells was centrifuged and resuspended in fresh medium immediately before treatment with DiCI-RB. Another portion was treated with distilled water (up to 20%. v/v). The results are means of 2 separate determinations.
1479
condition characterized by a strong activation of translation of ODC (Poulin and Pegg, 1990). Analysis of the content of ODC mRNA and its translability in in vitro systems may prove useful to clarify the mechanism of this superinduction.
LIZI~DFMO’
SUMMARY
described in
However the increases in ODC activity by benzimidazole derivatives do not appear to result from a posttranslational control of the enzyme affecting the catalytic activity or the turnover. Interestingly, during the progress of this work, a study was reported by Rosenberg-Hasson et al. (1991), who failed to show a role for phosphorylation in the control of ODC activity or turnover, by using site-directed mutagenesis. Thus, the meaning of ODC phosphorylation by CK-2 remains unknown. DiCl-RB has been reported to cause superinduction of interferon (Tamm and Sehgal, 1978; Sehgal et al., 1978) a phenomenon that has been attributed mainly to the repression of a labile factor involved in the control of the stability of interferon mRNA. Actinomycin D, another inhibitor of transcription not structurally related to benzimidazole, can elicit superinduction of ODC in a variety of tissues and cell lines (Wallon et al., 1990; Clark, 1974; Goldstein et al., 1976; Endo, 1984). Wallon et al. (1990) have recently demonstrated a 3-fold increase in ODC synthesis in ELD cells, which occurred in absence of any accumulation of ODC mRNA, indicating an action of actinomycin D at level of translation. In the present investigation, superinduction by actinomycin D was observed under the same conditions favoring superinduction by DiCl-RB and the fact that the effects of actinomycin D and DiCI-RB were not additive may be consistent with the hypothesis of a same mechanism. Olson and Spizz (1986) have utilized DiCl-RB to measure the half-life of ODC mRNA in BC,Hl muscle cells and no increase in the amount of the messenger was reported. Actually, the rapid superinduction by halogenated ribofuranosylbenzimidazoles, leading to an increase of at least 3-fold within 1 hr, appears to be more consistent with a control of ODC synthesis at level of translation. This hypothesis seems also supported by the lack of ODC response to benzimidazole derivatives in L1210DFMO’ cells growing in the presence of DFMO. Under these conditions, ODC synthesis is greatly increased at level of translation, while the content of ODC mRNA remains unchanged and ODC stability increases only slightly (Persson et al., 1988). Furthermore, attenuation of the ODC response to DiCI-RB was also observed after hipoosmotic stress, another
Dichlororibofuranosylbenzimidazole (DiCl-RB) is a powerful inhibitor of RNA polymerase II-directed transcription and casein kinase-2 activity. The effect of DiCl-RB on the activity and turnover of omithine decarboxylase (ODC) was investigated in an ODC overproducing cell line, which can grow in the presence as well as in the absence of the ODC inhibitor difluoromethylornithine (DFMO). In cells growing in the absence of DFMO, DiCI-RB added at seeding time was able to prevent the induction of ODC elicited by dilution of the cells in fresh medium. On the contrary, DiCI-RB provoked a marked, but transient increase in ODC activity when added at day 1 after seeding to cells already containing high levels of ODC (“superinduction”). The enhancement of ODC activity was accompanied by an approximately proportional increase in the content of immunoreactive ODC protein, whereas the ODC turnover did not change significantly. Half-maximal stimulation was achieved at a DiCI-RB concentration of 6 PM. The dibromoderivative analog (DiBr-RB) exerted similar effects on ODC, but was effective at lower concentrations. Under the same conditions, actinomycin D was also able to provoke superinduction of ODC. However, the halogenated ribofuranosylbenzimidazoles were ineffective in cells growing in the presence of DFMO and containing higher levels of ODC protein. In conclusion, the increases in ODC activity provoked by halogenated ribofuranosylbenzimidazoles do not appear to be caused by a posttranslational modification of ODC affecting the catalytic activity or the turnover of the enzyme. Acknowledgements-We would like to thank Lo Persson for his generous gift of ODC antiserum and LIZlO-DFMO’ cells, and David Shugar and Flavio Meggio for kindly providing DiBr-RB. We also thank Lorenzo A. Pinna for many helpful suggestions during early stages of this investigation. The careful secretarial work of MS Angela Zarri is acknowledged. This research was supported by grants from M.U.R.S.T., Italy.
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