Biochimica et Biophysica Acta, 1090 (1991) 188-194 © 1991 Elsevier Science Publishers B.V. All rights reserved 016%4781/91/$03.50 ADONIS 016747819100217T
188
BBAEXP 92297
On the translational control of ornithine decarboxylase expression by polyamines L o u i s e S1jernborg ~, Olle H e b y 2, I n g v a r H o l m 2 a n d L o P e r s s o n 3 I Department of Animal Physiology, Unirersity of Lund, Lund (Sweden). 2 Department of Zoophysiology, Unirersit) of Ume~, Umea (Sweden) and ~ Department of Physiology, Uniuersity of Lund, Lund (Sweden) (Received 13 March 1991)
Key words: Ornithine decarboxyla~; Polyamine; Translational control; Polysome profile
Ornithine decarboxylase (ODC, EC 4.1.1.17) expression is sabject to negative feedback regulation by the polyamines. The results of previous studies favor either translational or post-translational regulation. To facil;tate further analysis of the mechanism by which polyamines affect ODC expression we have used a cell line (L1210-DFMO r) that overproduces ODC. This cell line was isolated by selection for resistance to the antiproliferative effect of the ODC inhibitor w-difluoromethyiornithine (DFMO). These cells respond similarly to polyamine depletion and repletion as do their wild-type counterparts. When LI210-DFMO r cells were grown in the presence of 20 mM DFMO (i.e., when their polyamine content was reduced to an extent that still permitted a normal growth rate) ODC represented 4-5% of the soluble protein synthesized. After transfer of the cells to a medium lacking DFMO (i.e., when their polyamine pools were repleted), the rate of incorporation of [3SS]methionine into ODC was one order of magnitude lower. Since this difference in incorporation of radioactivity into ODE remained the same irrespective of the pulse-lahel time used (between 2 and 20 rain) it is likely to represent a true difference in ODC synthesis rate. Consequently, the pulse-inhel experiments cannot he explained by rapid degradation of the enzyme during the labeling period. The difference in ODE synthesis rate was not accompanied by a corresponding difference in the steady-stute level of ODE mRNA. Analyses of the distribution of ODE mRNA in polysome profiles did not demonstrate any major difference between cells grown in the absence or presence of DFTIO, even though the ODE synthesis rate differed by as much as 10-fold. However, the distribution of the ODE mRNA in the palysome profiles indicated that the message was poorly translated. Thus, most of the ODE mRNA was present in fractions containing ribosomal subunits or monosomes. Inhibition of elongation by cycioheximide treatment resulted in a shift of the ODC mRNA from the region of the gradient containing ribosomi subunits to that containing mono- and polysomes, indicating that most of the ODC mRNA was accessible to translation. Taken together these data lend support to a translational control mechanism which involves both initiation and elongation.
Introduction Cell growth and differentiation require optima! concentrations of the polyamines pucrescine, spermidine and spermine [1-5]. The initial step in polyamine synthesi~s is catalyzed by ornithine decarboxylase (ODC). Evidence is accumulating that this enzyme is feedback-regulated at the translational level by the polyamines [5-14]. Thus far, the mechanism involved is Abbreviations: ODC, ornithine decarboxylase; DFMO, a-difluoromethylornithine; SDS, sodium dodecyl sulfate. Correspondence: L. Persson, Department of Physiology, S61vegatan 19. S-223 62 Lund, Sweden.
largely unknown. Analyses of polysome profiles derived from Ehrlich ascites tumor ceils have revealed a surprisingly low number of ribosomes associated with the ODC mRNA [15]. Moreover, this number remained low and virtually constant even in a situation when ODC synthesis increased as a result of polyamine depletion, suggesting a coordinate regulation of initiation and elongation. At variance with our proposal of a translational control mechanism, Van Daalen Wetters et al. [16] suggested that the polyamine-mediated changes in ODC expression are mainly due to a posttranslational phenomenon. We have earlier isolated a cell line (L1210-DFMO r) with a very high expression of ODC [14]. The cause of this high expression is an amplification of the ODC
189 gene, with a concomitant increase in the steady-state level of the ODC mRNA. Although the synthesis of ODC in these cells is 100-fold higher than in the wild-type cells, ODC expression is still feedback-regulated by the polyamines [14]. L1210-DFMO r cells grown in the presence of the ODC inhibitor a-difluoromethylornithine (DFMO) synthesize ODC at a 10-fold higher rate (measured by pulse-labeling with [35S]methionine) than cells grown in the absence of the inhibitor. This difference in synthesis rate is not correlated with a change in the steady-state level of the ODC mRNA, or in the turnover of the enzyme, thus indicating a translational mechanism of control [14]. In the present study, we have used LI210-DFMO r cells to further investigate the mechanism by which polyamines affect O D C synthesis. In contrast to the results of Van Daalen Wetters et al. [16], the present results confirm a translational control mechanism, and suggest that this mechanism involves both initiation and elongation. Materials and Methods
Materials
Hybond-N membranes, [35S]methionine and [32p]. dCTP were purchased from Amersham. a-Difluoromethylornithine (DFMO) was generously provided by Merrell Dow Research Institute, Cincinnati, OH. The cDNAs, encoding mouse ODC [17] and the M2 subunit of mouse ribonucleotide reductase [18] were kind gifts from Dr. Chaim Kahana and Dr. Lars Thelande~., respectively.
Cells L1210-DFMO' cells [14] were routinely grown in RPMI 1640 medium containing 10% fetal calf serum, 50 ~ M ~-mercaptoethanol, antibiotics (50 units of penicillin/ml and 50 /tg of streptomyein/ml) and 20 mM DFMO. Cells to be used for experiments in the absence of D F M O had been grown without the inhibitor for at least five passages.
Polysome isolation an~. jer#ctionation For polysome analysis, plateau phase cells were seeded at a der.~;~" of 2.0.10 s cells/ml in the absence or presence of 20 mM D F M O and were harvested 24 h later. In some experiments a low concentration (0.75 /zg/ml) of cycloheximide was added 30 min prior to harvesting. Cells ((30-35). 106) were collected by centrifugation at room temperature and were then washed twice with ice-cold 20 mM Hepes buffer (pH 7.2) containing 0.15 M NaC! and 100 ~tg/ml cyclobeximide. After centrifugation, the cell volume was estimated and the cells were ~,vollen in 2 vol. of a hypotonic buffer (10 mM Hepes (pH 7.5), 10 mM KCI, 1.5 mM magnesium acetate, 7 mM ~-mercaptoethanol) and lyzed by the addition of 0.25% Nonidet P-40. Cytosolie
extracts, obtained by a 10 rain "entrifugation at 10000 x g (4°C) were layered onto 15-50% (w/v) linear sucrose gradients prepared in 20 mM Tris-HCI (pH 7.6), 0.1 M KCI and 3 mM magnesium aceta)e. After centrifugation at 40000 r.p.m, for 2 h at 4 ° C in a Beckman SW 41 rotor, the gradients were analyzed for absorbance at 254 nm and were then fractionated into 1 ml aliquots. Each fraction was treated with 0.1 m g / m l proteinase K and 0.5% sodium dodecyl sulfate (SDS) for 30 rain at 37 * C. 1hen the RNA was isolated by one extraction with phenol/chloroform and one with chloroform. The amount of RNA (5-10 ~g/fraction) was determined by measuring the absorbance at 260 rim.
Northern blot analysis The RNA was froctionated in a formaldehyde-containing 1% agarose gel. After transfe, to Hybond-N membrane, the R N A was hydridized t~ o!ther ODC eDNA or ribonucleotide reductase M2 eDNA labeled with the random oligonucleotide priming technique !19].
Determination of ODC synthesis The synthesis of ODC was determined by measuring the incorporation of [3SS]methionine into ODC. The cells (1.0. 106/ml) were preincubated for 5 rain in methionine-free RPM! 1640 medium. Then [35S]methionine (10 /zCi/ml) was added and the cells were incubated for 2, 5, 10, 20 or 25 min at 37 °C. Aliquots were collected and incorporation of radioactivity was discontinued by the addition of ice-cold medium conmining an excess of unlabeled methionine. The amount of radioactivity incorporated into ODC was determined by immunoprecipitation, SDS-PAGE and fluorography as described earlier [14]. Results The L1210-DFMO r cells were isolated for resistance to the antiproliferative effect of the ODC inhibitor D F M O [ 14]. The resistance was due to a more than 100-fold increase in ODC synthesis rate, corresponding to 4 - 5 % of soluble proteins synthesized [14]. The cells were routinely grown in a medium containing DFMO. However, when the cells were transferred to a medium without the inh~itor a marked decrease in ODC synthesis was observed (Fig. 1). The decrease in synthesis rate was most likely related to the increase in cellular polyamine levels occurring when the ODC molecules were no longer inactivated by DFMO (Table I). That the change in ODC synthesis rate is a translational phenomenon was indicated by the fact that the steady-state level of ODC m R N A as well as the turnover of the enzyme were largely the same whether the cells were grown in the presence or absence of D F M O [14].
19o
ODC
,'-
DFMO 4-Fig. I. Synthesis of ODC in DFMO-resistan!cells grown in the presence or absence of DFMO (20 raM). O13(2synthesiswas determined by incubation with [35S]methioninefor 25 rain followedby immunoprecipitation,SDS-PAGEand fluorography.Arrowhead indicates the migrationof pure mouse ODC (Mr = 53000) labeledwith [SH]DFMO.
Analyses of the distribution of ODC mRNA in polysome profiles from the DFMO-resistant cells grown in the presence of D F M O revealed that most of the message was present in fractions containing ribosomal subunits (especially 40S) or monosomes (Fig. 2). Only a very small fraction of the ODC m R N A was associated with large polysomes. No dramatic change in the distribution of ODC mRNA in the polysome profile was found when D F M O was omitted from the medium (Fig. 2). However, the fraction of ODC mRNA associated with polysomes appeared to be slightly smaller after omitting the inhibitor (Fig. 2). As a control of the technique, the distribution of n'bonucleotide reductase M2 m R N A was also determined in the polysome gradients. In contrast to ODC mRNA, this message was found to be mainly associated with larger polysomes (Fig. 2). To examine whether some of the ODC m R N A found in the untranslated region of the polysome profile was inaccessible to translation, we studied the effect of a low dose of cycioheximide. A low dose should specifically inhibit elongation, whereas the initiation should continue, resulting in larger and larger polysomes with time [20,21]. If some of the m R N A is not translated, i.e, not initiated, it should remain in the lighter fractions of the polysome profile. As shown in Fig. 3, a 30 rain treatment of the DFMO-resistant cells with 0 . 7 5 / t g / m l ¢ycioheximide resulted in a shift of the ODC mRNA from the lighter region of the polysome gradient to the region containing mono- and polysomes. Thus, it appears that none of the ODC mRNA was refractory to translation. This was true for the cells grown in the absence as well as those grown in the presence of D F M O (Fig. 3).
The evidence for a polyamine-mediated translational control of ODC expression is mainly based on experiments using the incorporation of [35S]methionine into the enzyme as a measurement of the synthesis rate [11-14]. In a recent publication by Van Daalen Wetters et al. [16] it was suggested that the polyaminemediated control of ODC is mainly, if not entirely, carried out by a post-trauslational mechanism and that the observed changes in incorporation of [35S]methionine are due to post-translational degradation of the cn~qne. To scrutinize this possibility, we have used shorter and shorter pulses labeling and then compared the incorporation of radioactivity into O D C of cells grown in the absence or presence of DFMO. If the difference seen in incorporation of [35S]methionine into ODC is due to degradation of the enzyme, the difference should diminish gradually with decreasing labeling time. However, as shown in Fig. 4 this was not the case. Even though pulses as short as 2 min were used, the incorporation of radioactivity into ODC was much less in cells grown in the absence than in the presence of DFMO, indicating a true difference in synthesis rate.
Discussion Our finding that most of the ODC m R N A in polysome prof'des was present in the region containing ribosomal subunits and monosomes indicates that this message is poorly translated in L1210-DFMO r cells. The condition is not restricted to the cells used in the present study, but appears to be a more general phenomenon. Thus, a similar distribution of ODC m R N A has been found in polysome gradients from Ehrlich ascites tumor cells [15] and $49 iymphoma cells [16]. Mammalian ODC m R N A contains an unusually long 5' untranslated leader which is GC-rich and may form stable secondary structures with a very high free energy of formation [22-26]. The theoretical value of the overall free energy of these s~condary structures is in the range of - 9 7 to - 126 kcal/mol, which should give rise to a substantial inhibition of the "-anslation of the
TABLE ! Polyamine coment o f LI210-DFMO" cells grown in the presence or absence o f D F M O "
Cells were grown in the presence or absenceof DFMO (20 JaM) for several passages. Cells were collected for polyamine analysis 24 h after seedinginto fresh medium. Mean:1:S.E., n = 4. Treatment +DFMO -DFMO
Putrescineb Spermidineb
188
BBAEXP 92297
On the translational control of ornithine decarboxylase expression by polyamines L o u i s e S1jernborg ~, Olle H e b y 2, I n g v a r H o l m 2 a n d L o P e r s s o n 3 I Department of Animal Physiology, Unirersity of Lund, Lund (Sweden). 2 Department of Zoophysiology, Unirersit) of Ume~, Umea (Sweden) and ~ Department of Physiology, Uniuersity of Lund, Lund (Sweden) (Received 13 March 1991)
Key words: Ornithine decarboxyla~; Polyamine; Translational control; Polysome profile
Ornithine decarboxylase (ODC, EC 4.1.1.17) expression is sabject to negative feedback regulation by the polyamines. The results of previous studies favor either translational or post-translational regulation. To facil;tate further analysis of the mechanism by which polyamines affect ODC expression we have used a cell line (L1210-DFMO r) that overproduces ODC. This cell line was isolated by selection for resistance to the antiproliferative effect of the ODC inhibitor w-difluoromethyiornithine (DFMO). These cells respond similarly to polyamine depletion and repletion as do their wild-type counterparts. When LI210-DFMO r cells were grown in the presence of 20 mM DFMO (i.e., when their polyamine content was reduced to an extent that still permitted a normal growth rate) ODC represented 4-5% of the soluble protein synthesized. After transfer of the cells to a medium lacking DFMO (i.e., when their polyamine pools were repleted), the rate of incorporation of [3SS]methionine into ODC was one order of magnitude lower. Since this difference in incorporation of radioactivity into ODE remained the same irrespective of the pulse-lahel time used (between 2 and 20 rain) it is likely to represent a true difference in ODC synthesis rate. Consequently, the pulse-inhel experiments cannot he explained by rapid degradation of the enzyme during the labeling period. The difference in ODE synthesis rate was not accompanied by a corresponding difference in the steady-stute level of ODE mRNA. Analyses of the distribution of ODE mRNA in polysome profiles did not demonstrate any major difference between cells grown in the absence or presence of DFTIO, even though the ODE synthesis rate differed by as much as 10-fold. However, the distribution of the ODE mRNA in the palysome profiles indicated that the message was poorly translated. Thus, most of the ODE mRNA was present in fractions containing ribosomal subunits or monosomes. Inhibition of elongation by cycioheximide treatment resulted in a shift of the ODC mRNA from the region of the gradient containing ribosomi subunits to that containing mono- and polysomes, indicating that most of the ODC mRNA was accessible to translation. Taken together these data lend support to a translational control mechanism which involves both initiation and elongation.
Introduction Cell growth and differentiation require optima! concentrations of the polyamines pucrescine, spermidine and spermine [1-5]. The initial step in polyamine synthesi~s is catalyzed by ornithine decarboxylase (ODC). Evidence is accumulating that this enzyme is feedback-regulated at the translational level by the polyamines [5-14]. Thus far, the mechanism involved is Abbreviations: ODC, ornithine decarboxylase; DFMO, a-difluoromethylornithine; SDS, sodium dodecyl sulfate. Correspondence: L. Persson, Department of Physiology, S61vegatan 19. S-223 62 Lund, Sweden.
largely unknown. Analyses of polysome profiles derived from Ehrlich ascites tumor ceils have revealed a surprisingly low number of ribosomes associated with the ODC mRNA [15]. Moreover, this number remained low and virtually constant even in a situation when ODC synthesis increased as a result of polyamine depletion, suggesting a coordinate regulation of initiation and elongation. At variance with our proposal of a translational control mechanism, Van Daalen Wetters et al. [16] suggested that the polyamine-mediated changes in ODC expression are mainly due to a posttranslational phenomenon. We have earlier isolated a cell line (L1210-DFMO r) with a very high expression of ODC [14]. The cause of this high expression is an amplification of the ODC
189 gene, with a concomitant increase in the steady-state level of the ODC mRNA. Although the synthesis of ODC in these cells is 100-fold higher than in the wild-type cells, ODC expression is still feedback-regulated by the polyamines [14]. L1210-DFMO r cells grown in the presence of the ODC inhibitor a-difluoromethylornithine (DFMO) synthesize ODC at a 10-fold higher rate (measured by pulse-labeling with [35S]methionine) than cells grown in the absence of the inhibitor. This difference in synthesis rate is not correlated with a change in the steady-state level of the ODC mRNA, or in the turnover of the enzyme, thus indicating a translational mechanism of control [14]. In the present study, we have used LI210-DFMO r cells to further investigate the mechanism by which polyamines affect O D C synthesis. In contrast to the results of Van Daalen Wetters et al. [16], the present results confirm a translational control mechanism, and suggest that this mechanism involves both initiation and elongation. Materials and Methods
Materials
Hybond-N membranes, [35S]methionine and [32p]. dCTP were purchased from Amersham. a-Difluoromethylornithine (DFMO) was generously provided by Merrell Dow Research Institute, Cincinnati, OH. The cDNAs, encoding mouse ODC [17] and the M2 subunit of mouse ribonucleotide reductase [18] were kind gifts from Dr. Chaim Kahana and Dr. Lars Thelande~., respectively.
Cells L1210-DFMO' cells [14] were routinely grown in RPMI 1640 medium containing 10% fetal calf serum, 50 ~ M ~-mercaptoethanol, antibiotics (50 units of penicillin/ml and 50 /tg of streptomyein/ml) and 20 mM DFMO. Cells to be used for experiments in the absence of D F M O had been grown without the inhibitor for at least five passages.
Polysome isolation an~. jer#ctionation For polysome analysis, plateau phase cells were seeded at a der.~;~" of 2.0.10 s cells/ml in the absence or presence of 20 mM D F M O and were harvested 24 h later. In some experiments a low concentration (0.75 /zg/ml) of cycloheximide was added 30 min prior to harvesting. Cells ((30-35). 106) were collected by centrifugation at room temperature and were then washed twice with ice-cold 20 mM Hepes buffer (pH 7.2) containing 0.15 M NaC! and 100 ~tg/ml cyclobeximide. After centrifugation, the cell volume was estimated and the cells were ~,vollen in 2 vol. of a hypotonic buffer (10 mM Hepes (pH 7.5), 10 mM KCI, 1.5 mM magnesium acetate, 7 mM ~-mercaptoethanol) and lyzed by the addition of 0.25% Nonidet P-40. Cytosolie
extracts, obtained by a 10 rain "entrifugation at 10000 x g (4°C) were layered onto 15-50% (w/v) linear sucrose gradients prepared in 20 mM Tris-HCI (pH 7.6), 0.1 M KCI and 3 mM magnesium aceta)e. After centrifugation at 40000 r.p.m, for 2 h at 4 ° C in a Beckman SW 41 rotor, the gradients were analyzed for absorbance at 254 nm and were then fractionated into 1 ml aliquots. Each fraction was treated with 0.1 m g / m l proteinase K and 0.5% sodium dodecyl sulfate (SDS) for 30 rain at 37 * C. 1hen the RNA was isolated by one extraction with phenol/chloroform and one with chloroform. The amount of RNA (5-10 ~g/fraction) was determined by measuring the absorbance at 260 rim.
Northern blot analysis The RNA was froctionated in a formaldehyde-containing 1% agarose gel. After transfe, to Hybond-N membrane, the R N A was hydridized t~ o!ther ODC eDNA or ribonucleotide reductase M2 eDNA labeled with the random oligonucleotide priming technique !19].
Determination of ODC synthesis The synthesis of ODC was determined by measuring the incorporation of [3SS]methionine into ODC. The cells (1.0. 106/ml) were preincubated for 5 rain in methionine-free RPM! 1640 medium. Then [35S]methionine (10 /zCi/ml) was added and the cells were incubated for 2, 5, 10, 20 or 25 min at 37 °C. Aliquots were collected and incorporation of radioactivity was discontinued by the addition of ice-cold medium conmining an excess of unlabeled methionine. The amount of radioactivity incorporated into ODC was determined by immunoprecipitation, SDS-PAGE and fluorography as described earlier [14]. Results The L1210-DFMO r cells were isolated for resistance to the antiproliferative effect of the ODC inhibitor D F M O [ 14]. The resistance was due to a more than 100-fold increase in ODC synthesis rate, corresponding to 4 - 5 % of soluble proteins synthesized [14]. The cells were routinely grown in a medium containing DFMO. However, when the cells were transferred to a medium without the inh~itor a marked decrease in ODC synthesis was observed (Fig. 1). The decrease in synthesis rate was most likely related to the increase in cellular polyamine levels occurring when the ODC molecules were no longer inactivated by DFMO (Table I). That the change in ODC synthesis rate is a translational phenomenon was indicated by the fact that the steady-state level of ODC m R N A as well as the turnover of the enzyme were largely the same whether the cells were grown in the presence or absence of D F M O [14].
19o
ODC
,'-
DFMO 4-Fig. I. Synthesis of ODC in DFMO-resistan!cells grown in the presence or absence of DFMO (20 raM). O13(2synthesiswas determined by incubation with [35S]methioninefor 25 rain followedby immunoprecipitation,SDS-PAGEand fluorography.Arrowhead indicates the migrationof pure mouse ODC (Mr = 53000) labeledwith [SH]DFMO.
Analyses of the distribution of ODC mRNA in polysome profiles from the DFMO-resistant cells grown in the presence of D F M O revealed that most of the message was present in fractions containing ribosomal subunits (especially 40S) or monosomes (Fig. 2). Only a very small fraction of the ODC m R N A was associated with large polysomes. No dramatic change in the distribution of ODC mRNA in the polysome profile was found when D F M O was omitted from the medium (Fig. 2). However, the fraction of ODC mRNA associated with polysomes appeared to be slightly smaller after omitting the inhibitor (Fig. 2). As a control of the technique, the distribution of n'bonucleotide reductase M2 m R N A was also determined in the polysome gradients. In contrast to ODC mRNA, this message was found to be mainly associated with larger polysomes (Fig. 2). To examine whether some of the ODC m R N A found in the untranslated region of the polysome profile was inaccessible to translation, we studied the effect of a low dose of cycioheximide. A low dose should specifically inhibit elongation, whereas the initiation should continue, resulting in larger and larger polysomes with time [20,21]. If some of the m R N A is not translated, i.e, not initiated, it should remain in the lighter fractions of the polysome profile. As shown in Fig. 3, a 30 rain treatment of the DFMO-resistant cells with 0 . 7 5 / t g / m l ¢ycioheximide resulted in a shift of the ODC mRNA from the lighter region of the polysome gradient to the region containing mono- and polysomes. Thus, it appears that none of the ODC mRNA was refractory to translation. This was true for the cells grown in the absence as well as those grown in the presence of D F M O (Fig. 3).
The evidence for a polyamine-mediated translational control of ODC expression is mainly based on experiments using the incorporation of [35S]methionine into the enzyme as a measurement of the synthesis rate [11-14]. In a recent publication by Van Daalen Wetters et al. [16] it was suggested that the polyaminemediated control of ODC is mainly, if not entirely, carried out by a post-trauslational mechanism and that the observed changes in incorporation of [35S]methionine are due to post-translational degradation of the cn~qne. To scrutinize this possibility, we have used shorter and shorter pulses labeling and then compared the incorporation of radioactivity into O D C of cells grown in the absence or presence of DFMO. If the difference seen in incorporation of [35S]methionine into ODC is due to degradation of the enzyme, the difference should diminish gradually with decreasing labeling time. However, as shown in Fig. 4 this was not the case. Even though pulses as short as 2 min were used, the incorporation of radioactivity into ODC was much less in cells grown in the absence than in the presence of DFMO, indicating a true difference in synthesis rate.
Discussion Our finding that most of the ODC m R N A in polysome prof'des was present in the region containing ribosomal subunits and monosomes indicates that this message is poorly translated in L1210-DFMO r cells. The condition is not restricted to the cells used in the present study, but appears to be a more general phenomenon. Thus, a similar distribution of ODC m R N A has been found in polysome gradients from Ehrlich ascites tumor cells [15] and $49 iymphoma cells [16]. Mammalian ODC m R N A contains an unusually long 5' untranslated leader which is GC-rich and may form stable secondary structures with a very high free energy of formation [22-26]. The theoretical value of the overall free energy of these s~condary structures is in the range of - 9 7 to - 126 kcal/mol, which should give rise to a substantial inhibition of the "-anslation of the
TABLE ! Polyamine coment o f LI210-DFMO" cells grown in the presence or absence o f D F M O "
Cells were grown in the presence or absenceof DFMO (20 JaM) for several passages. Cells were collected for polyamine analysis 24 h after seedinginto fresh medium. Mean:1:S.E., n = 4. Treatment +DFMO -DFMO
Putrescineb Spermidineb
