Cancer Letters, 67 (1992) 187- 192 Elsevier Scientific Publishers Ireland Ltd.
187
The induction of ornithine decarboxylase by ornithine takes place at post-transcriptional level in perifused Ehrlich carcinoma cells F. SAnchez-Jim&ez, Laboratory
J.L. Urdiales,
J.M. Mat& and I. Ntiiiez de Castro
ofBiochemistry and Molecular Biology, Facultad de C’iencias, hive&dad
(Received
28 August
1992)
(Accepted
9 October
1992)
Summary The increase in ODC activity during perifusion of Ehrlich carcinoma cells with 0.5 mM ornithine correlates with an increase in ‘de nouo’ synthetized ODC protein. ODC synthesis was followed by immunoprecipitation of equal quantities of 35S-labelled proteins after 10, 20 and 30 min of labelling. In addition, the rate of
‘de novo’ protein synthesis is very much elevated in cells perifused with saline buffer supplemented with 0.5 mM ornithine than in cells perifused with the saline buffer only. In spite of the higher specific ODC actiuify observed in cells perifused with saline buffer plus 0.5 mM ornithine respect to cells perifused with only saline buffer for 3.5 h, no elevation in ODC mRNA was observed when the cells were perifused in the presence of 0.5 mM ornithine.
Keywords: ornithine; ornithine lase; perifusion; polyamines
decarboxy-
Introduction Ornithine decarboxylase tant step for polyamine
(ODC) is the limi(putrescine, sper-
Correspondence to: F. Skhez-Jim6ne.q Laboratory of Biochemistry and Molecular Biology, Facultad de Ciencias, Universidad de Malaga, 29071 Mblaga, Spain.
0304-3&335/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
de M&laga, 29071 Mhlaga (Spain)
midine and spermine) biosynthesis [5]. ODC expression is induced by hormones, growth factors at both transcriptional and/or posttranscriptional levels [4]. Due to the complex regulation of the ODC and its extremely rapid turnover, the induction of the ODC activity is one of the primary events after triggering cell proliferation by growth stimulus [7]. In addition, ornithine-derived polyamines are essential for maintaining the macromolecular synthesis [9]. On the other hand, polyamines feedback regulate the ODC activity, the ODC protein level and the uptake of ornithine by the cells [6,11,15,21,22]. Neutral amino acids transported by the mammalian systems A and N are also described as ODC inducers for nontransformed and cancer cells: asparagine induces ODC synthesis in primary cultured rat hepatocytes [ES]; glutamine, asparagine or amino-isobutyric acid are essential for ODC induction caused by growth factors in confluent cultures of two murine cancer lines [18]. In 1989, Mat& et al. report that the cationic amino acids ornithine and arginine are also able to induce ODC activity in Ehrlich carcinoma cells [El. This effect can be clearly observed using cells confined in a perifusion chamber, since the accumulation of polyamines is prevented by the continuous renewal of the medium [14]. In this model, ornithine is a better ODC activity inducer than the neutral amino acids glutamine and asparagine [13]. The elevation of ODC activity caused by Ireland Ltd.
188
ornithine is avoided by cycloheximide, but is unchanged when actinomycin D is included in the perifusion medium [12]. In this work, ODC mRNAs detection by Northern blots and 35S incorporation experiments were carried out on perifused Ehrlich carcinoma cells. Results agree with those reported for the other ODC inducer amino acids, which elevate intracellular ODC protein synthesis with no effect on ODC mRNA levels. Materials
and Methods
Cells and perifusion system Ehrlich carcinoma cells (Lettre strain) were harvested from lo-day tumour bearing animals as described previously [ 161. At this time, tumour has reached the plateau phase of growth and ODC activity is the lowest observed ‘in vivo’ [lo]. Continuous incubation of cells have been reported elsewhere [l&13]. Briefly, a jacketed 10 ml perifusion chamber, which had been filled with the perifusion saline medium (6.16 mM KCl, 154 mM NaCl, 1.65 mM NaH2P04, 9.35 mM Na2HP04 pH 7.4) added or not with 0.5 mM ornithine, was used. Cellular density in the chamber was adjusted at 42 x lo6 cells/ml. The flow rate for renewal of perifusion medium was maintained at 0.18 ml/min and temperature at 37OC. ODC and protein synthesis After 2 h of perifusion with saline medium added or not with 0.5 mM ornithine, three 5 x lo6 cell aliquots from each incubation were collected from the perifusion chamber and resuspended in a final volume of 0.5 ml of the perifusion medium supplemented with 100 PCi of Tran35S-label TM (ICN, USA). Then, cells were further incubated for 10, 20 and 30 min at 37OC with gently shaking. Label incorporation was stopped by addition of an excess of cold methionine. Cells were again pelleted and resuspended in ODC buffer (50 mM Hepes, 10 mM dithiothreitoi, 1 mM EDTA, 50 PM PLP, pH 7.2). Cell suspensions were sonicated as described previously for ODC activity determinations [12]. The 35S incorpora-
tion into protein was determined on the supernatants of the centrifuged cell-free extracts (13 000 x g, 30 min) . Duplicates of 2 ~1 from each sample were precipitated using final 10% trichloroacetic acid and filtered as described by Bonifacino [2]. The acidinsoluble radioactivity was counted. In order to measure relative ODC synthesis, equal amounts of acid-insoluble radioactivity from the supernatants were incubated with an excess of polyclonal monospecific antibody raised against mouse ODC; the ODC-antibody complexes were collected by precipitation with insoluble Protein A [17,23]. Pelleted proteins were separated by SDS-polyacrylamide electrophoresis (SDS-PAGE) and visualized by fluorography [17,20,23]. The ODC subunit band (53 kDa) was identified by comparison between samples treated with immune and non-immune rabbit serum. Autoradiographs were digitalized in a Scanner Sharp, model 5X-450 (Japan) and processed using a Sun Microsystem Image Analyzer (USA). Northern blot analysis Total RNA was isolated as described by Chomczynski and Sacchi [3]. Ten micrograms from each sample was fractionated in formaldehyde-agarose gels and hybridized with 32P-labelled probes as described previously [19,20]. The insert of the clone pODC934 [l] was used as ODC mRNA probe. As an internal standard, mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected [20]. Results and Discussion When quiescent Ehrlich carcinoma cells are perifused with 0.5 mM ornithine for 1- 3.5 h, an important increase in ODC activity can be detected; however, ODC activity remains very similar to the initial value when cells are perifused with only saline buffer [12]. Neither asparagine nor other ornithine precursors are able to elicit such a ODC induction in Ehrlich cells, when added to the perifusion medium at the same concentration [13]. In ornithine-
189
labelling was stopped after 10, 20 and 30 min. Three different experiments were carried out and very similar results were obtained. A representative result is shown in Fig. 1. Slopes of label incorporation into proteins (counts per min/pl extract/min) in saline medium perifused cell extracts were the 41% * 4 of those calculated from ornithine-perifused cell extracts. No difference in the patterns of proteins synthetized ‘de novo’ could be observed by SDS-PAGE analysis of pulse-labelled extracts at different times (results not shown). Thus, a generalized increase in protein synthesis induced by ornithine during perifusion can be concluded. ODC synthesis rate can not be compared between saline medium and ornithine-
perifused cells, the increase in ODC activity relative to cell number is linear over the incubation time, but sigmoidal when the activity is expressed relative to cell protein, due to the progressive increase in the total protein content of the cell extracts detected after 1.5 h of perifusion with 0.5 mM ornithine [X2]. An elevation in protein content can be due to an increase of the total protein synthesis rate, an increase in the synthesis of a particular set of proteins, or a diminished proteolysis. In order to answer this question, total protein synthesis rates were studied by pulse-labelling experiments at different times. A =-SlabelledTM amino acid mixture was added to cells perifused for 2 h with saline medium only or saline medium plus 0.5 mM ornithine and
-c,
0 0
-
4190 r
-l-J Q, Cc
Y =
=
+
5864
X
0.9741
lo-
Y =
4300
+
2460
X
I
I
I
10
20
30
Lobelling
time
(min)
Fis. 1. Acid-insoluble radioactivity in cell-free extracts of Ehrlich carcinoma cells iabelled for different times after 2 h of perifusion with saline medium only (0) or saline medium supplemented with 0.5 mM ornithine ( V ).
190
perifused cells: firstly, the total protein synthesis rate is very low in cells perifused with only saline buffer; secondly, this rate is slower respect to the rate detected in ornithineperifused cells (Fig. 1). In addition, the extremely low levels of ODC in quiescent mammalian cells made ODC undetectable after immunoprecipitation experiments [8,17,22]. Even for cells in which ornithine has induced ODC activity, this is more than lo-fold lower than the ODC activity detected in cultured Ehrlich cells during exponential phase of growth when ODC represents less than the 5 x 10 -3% of the total ‘de novo’ protein synthesis [5,17,20]. ODC activity increases from 4 to 4.8 nmol Cop/h per mg protein between 2 and 2.5 h of perifusion with 0.5 mM ornithine [12]. In order to test a specific enrichment in ODC protein of ornithine perifused cells, equal quantities of radioactivity incorporated into protein at the different times were immunoprecipitated and analyzed by SDS-PAGE and fluorography. Results are shown in Fig. 2. Extracts were being enriched progressively in a peptide which had the reported M, for the mouse ODC subunit (53 kDa) and which could not be observed in samples immunoprecipitated with non immune rabbit serum [17,20]. Results confirm that ornithine induces an increase in ODC translation relative to total protein synthesis rate and they are coincident with the previous observation that ornithine induces a cycloheximide-sensitive increase in specific ODC activity [ 121. On the other hand, these results agree with those reported for other amino acids inducing mammalian ODC translation [4,8]. Moreover, the ornithine analog difluoromethylornithine (DFMO) which is a suicide ODC inhibitor, is also described as a short-term inducer of ODC translation without any effect on ODC mRNA levels [6,17,21]. Actually, as shown in Fig. 3, there is no elevation in relative ODC mRNA content induced by ornithine after 3.5 h of perifusion, when ODC activity is more t.han 30-fold higher in ornithine-perifused cells than in cells perifused with only saline medium [12,13].
A kD
66 -
ODC
45 -
36 -
z
C
75
CD .v) 0
50
0” F 25 ‘3 0 u c?L 0
10
Lobelling
20 time
30 (min)
Fi2. 2. Synthesis of 35S-labelled ODC protein in Ehrlich carcinoma cells after 2 h of perifusion with saline medium supplemented with 0.5 mM ornithine. Autoradiograph of samples from pulse-labelled cells for 10 (lane l), 20 (lane 2) and 30 min (lane 3) and immunoprecipitated with ODC antibodies. Numbers indicate the position of the M, standards (kDa) (A). Densitometric analysis of the autoradiograph shown in A; ODC signal intensity in lane 3 was considered as 100%
(B).
191
antibodies and to Dr. H. van Steeg (Utrecht, Netherlands) for providing us with the GAPDH probe. Thanks are due to R. Cameselle and Manuela Vega for technical assistance.
A
References 1
-
I3
1
ODC
2 GAPDH
Fig. 3.
Northern blot analysis of ODC mRNA (A) and GAPDH mRNA (B), after 3.5 h of perifusion with saline medium supplemented with 0.5 mM omithine (lane l), or saline medium only (lane 2). Both hybridizations (ODC and control mRNA) were carried out on the same filter.
Thus, as reported for other amino acids inducing mammalian ODC, we can conclude that the tumour ODC induction by ornithine also takes place at post transcriptional level. Acknowledgements This paper was supported by the Grant PB/88/0445 of the DGICYT. We are particularly indebted to Dr. P. Aller (CBM, Madrid, Spain) and Dr. F. Berger (Department of Biological Sciences, South Carolina) for the pODC934 clone, to Dr. L. Persson for his generous gift of polyclonal anti-mouse ODC
2
Berger, F.G., Szymanski, P., Read, E. and Watson G. (1984) Androgen-regulated ornithine decarboxylase mRNAs of mouse kidney. J. Biol. Chem., 259, 7941- 7946. Bonifacino, J.S. (1991) Purification of proteins by
precipitation. In: Current Protocols in Molecular Biology. Section VI, unit 10.16. Editors: F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl, Willey Interscience, New York. 3 Chomcynsky, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal. Biochem., 162, 156 - 159. 4 Hayashi, S. (1989) Multiple mechanisms for the regulation of mammalian ornithine decarboxylase. In: Qrnithine Decarboxylase: Biology, Enzymology and Molecular Genetics. pp. 35-45. Editor: S. Hayashi. Pergamon Press, New York. 5 Heby, 0. and Persson L. (1990) Molecular genetics of polyamine synthesis in eucaryotic cells. Trends Biol. Sci., 15, 153 - 158. 6 Helm, I., Persson, L., Stjernborg, L., Thorsson, L., Heby, 0. (1989) Feedback control of ornithine decarboxylase expression by polyamines. Biochem. J., 258, 343-350. 7 Kahana, C. (1989) Molecular genetics of mammalian ornithine decarboxylase. In: The Physiology of Polyamines, Vol. I, pp. 281-295. Editor: U. Bachrach and Y.M. Heimer. CRC Press, Boca Raton, Florida. 8 Kanamoto, R., Nishiyama, M., Matsufuji, S. and Hayashi. S. (1991) Translational control mechanism of ornithine decarboxylase by asparagine and putrescine in primary cultured hepatocytes. Arch. Biochem. Biophys., 291, 247 - 254. 9 Marton, L.J. and Morris, D.R. (1987) Molecular and cellular functions of the polyamines. In: Inhibition of polyamine metabolism, pp. 79 - 105. Editors: P.P. McCann, A.E. Pegg and A. Sjoerdsma. Academic Press, New York. 10 Mdrquez, J., Mates, J.M., Quesada, A-R., Medina, M.A., Ndiiez de Castro, I. and Sanchez-Jimenez, F. (1989) Altered ornithine metabolism in tumor-bearing mice. Life Sci., 45, 1877 - 1884. 11 Medina, M.A., Urdiales, J.L., Mates, J.M., Ntiiiez de Castro, I. and Sanchez Jimhnez, F. (1991) Diamines interfere with the transport of L-ornithine in Ehrlich cell plasma membrane vesicles. Biochem. J., 280, 825 - 827. F., Garcia-Caballero, M. 12 Mat&s, J.M., Sanchez-JimCnez, and NCltiez de Castro I. (1989) Histamine and serotonin inhibit the induction of ornithine decarboxylase in perifused Ehrlich ascites tumour cells. FEBS Len., 250, 257 - 261.
192
13
14
15
16
17
18
Mat&, J.M., Sbnchez-JimBnez, F., L6pez-Herrera, J. and NCltiezde Castro, 1. (1991) Regulation by 1,Cdiamines of the omithine decarboxylase activity induced by omithine in perifused tumor cells. Biochem. Pharmacol., 42, 1045 - 1052. Mat&, J.M., Garcia-Caballero, M., N6riez de Castro, I., Urdiales, J.L. and Sbnchez-JimBnez, F. (1992) Polyamine metabolism regulation by histamine and other biogenic amines in Ehrlich carcinoma cells. Agents and Actions, (Spec. Conf. Issue), C380 - C383. Murakami, Y., Nishiyama, M. and Hayashi, S. (1989) Involvement of antiime in stabilization of ornithine decarboxylase caused by inhibitors of polyamine synthesis. Eur. J. Biochem., 180, 181- 184. Quesada A.R., Medina, M.A., Mbrquez, J., SBnchezJimBnez, F. and Ndiiez de Castro, I. (1988) Contribution of host tissues to circulating glutamine in mice inoculated with Ehrlich ascites tumor cells. Cancer Res., 48, 1551- 1553. Persson, L., Helm, I. and Heby 0. (1988) Regulation of omithine decarboxylase mRNA translation by polyamines. J. Biol. Chem., 263, 3528-3533. Rinehart, C.A., Viceps-Madore, D., Fong, W.F., Ortiz, J.G. and Canellakis E.S. (1985) The effect of transport
19
20
21
22
23
system A and N amino acids and of nerve and epidermal growth factors on the induction of omithine decarboxylase activity. J. Cell. Physiol., 123, 435-441. Southard, J.N., S&nchez-Jimbnez, F., Campbell G.T. and Talamantes, F. (1991) Sequence and expression of hamster prolactin and growth hormone messenger RNAs. Endocrinology, 129, 2965 - 2971. Urdiales, J.L., Mat& J.M., N6fiez de Castro, I. and SBnchez-Jimhnez, F. (1992) Chlorpheniramine inhibits the ornithine decarboxylase induction of Ehrlich carcinoma growing “in viva”. FEBS Lett., 305, 260- 264. Van Daalen Weters, T., Macrae, M., Brabant, M., Sittler, A. and Coffino, P. (1989) Polyamine-mediated regulation of mouse omithine decarboxylase is post-translational. Mol. Cell. Biol., 9, 5484-5490. Van Steeg, H., Van Oostrom, C.T.M., Hodemaekers, H.M., I?e!eters,L., Thomas, A.A.M. (1991) The translation in vitro of rat omithine decarboxylase mRNA is blocked by its 5’ untranslated region in a polyamine-independent way. B&hem. J., 274, 521-526. Wallon, U.M., Persson, L. and Heby, 0. (1990) Superinduction of ornithine decarboxylase (ODC) by actinomycin D is due to stimulation of ODC mRNA translation. FEBS Lett., 268, 161- 164.
187
The induction of ornithine decarboxylase by ornithine takes place at post-transcriptional level in perifused Ehrlich carcinoma cells F. SAnchez-Jim&ez, Laboratory
J.L. Urdiales,
J.M. Mat& and I. Ntiiiez de Castro
ofBiochemistry and Molecular Biology, Facultad de C’iencias, hive&dad
(Received
28 August
1992)
(Accepted
9 October
1992)
Summary The increase in ODC activity during perifusion of Ehrlich carcinoma cells with 0.5 mM ornithine correlates with an increase in ‘de nouo’ synthetized ODC protein. ODC synthesis was followed by immunoprecipitation of equal quantities of 35S-labelled proteins after 10, 20 and 30 min of labelling. In addition, the rate of
‘de novo’ protein synthesis is very much elevated in cells perifused with saline buffer supplemented with 0.5 mM ornithine than in cells perifused with the saline buffer only. In spite of the higher specific ODC actiuify observed in cells perifused with saline buffer plus 0.5 mM ornithine respect to cells perifused with only saline buffer for 3.5 h, no elevation in ODC mRNA was observed when the cells were perifused in the presence of 0.5 mM ornithine.
Keywords: ornithine; ornithine lase; perifusion; polyamines
decarboxy-
Introduction Ornithine decarboxylase tant step for polyamine
(ODC) is the limi(putrescine, sper-
Correspondence to: F. Skhez-Jim6ne.q Laboratory of Biochemistry and Molecular Biology, Facultad de Ciencias, Universidad de Malaga, 29071 Mblaga, Spain.
0304-3&335/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
de M&laga, 29071 Mhlaga (Spain)
midine and spermine) biosynthesis [5]. ODC expression is induced by hormones, growth factors at both transcriptional and/or posttranscriptional levels [4]. Due to the complex regulation of the ODC and its extremely rapid turnover, the induction of the ODC activity is one of the primary events after triggering cell proliferation by growth stimulus [7]. In addition, ornithine-derived polyamines are essential for maintaining the macromolecular synthesis [9]. On the other hand, polyamines feedback regulate the ODC activity, the ODC protein level and the uptake of ornithine by the cells [6,11,15,21,22]. Neutral amino acids transported by the mammalian systems A and N are also described as ODC inducers for nontransformed and cancer cells: asparagine induces ODC synthesis in primary cultured rat hepatocytes [ES]; glutamine, asparagine or amino-isobutyric acid are essential for ODC induction caused by growth factors in confluent cultures of two murine cancer lines [18]. In 1989, Mat& et al. report that the cationic amino acids ornithine and arginine are also able to induce ODC activity in Ehrlich carcinoma cells [El. This effect can be clearly observed using cells confined in a perifusion chamber, since the accumulation of polyamines is prevented by the continuous renewal of the medium [14]. In this model, ornithine is a better ODC activity inducer than the neutral amino acids glutamine and asparagine [13]. The elevation of ODC activity caused by Ireland Ltd.
188
ornithine is avoided by cycloheximide, but is unchanged when actinomycin D is included in the perifusion medium [12]. In this work, ODC mRNAs detection by Northern blots and 35S incorporation experiments were carried out on perifused Ehrlich carcinoma cells. Results agree with those reported for the other ODC inducer amino acids, which elevate intracellular ODC protein synthesis with no effect on ODC mRNA levels. Materials
and Methods
Cells and perifusion system Ehrlich carcinoma cells (Lettre strain) were harvested from lo-day tumour bearing animals as described previously [ 161. At this time, tumour has reached the plateau phase of growth and ODC activity is the lowest observed ‘in vivo’ [lo]. Continuous incubation of cells have been reported elsewhere [l&13]. Briefly, a jacketed 10 ml perifusion chamber, which had been filled with the perifusion saline medium (6.16 mM KCl, 154 mM NaCl, 1.65 mM NaH2P04, 9.35 mM Na2HP04 pH 7.4) added or not with 0.5 mM ornithine, was used. Cellular density in the chamber was adjusted at 42 x lo6 cells/ml. The flow rate for renewal of perifusion medium was maintained at 0.18 ml/min and temperature at 37OC. ODC and protein synthesis After 2 h of perifusion with saline medium added or not with 0.5 mM ornithine, three 5 x lo6 cell aliquots from each incubation were collected from the perifusion chamber and resuspended in a final volume of 0.5 ml of the perifusion medium supplemented with 100 PCi of Tran35S-label TM (ICN, USA). Then, cells were further incubated for 10, 20 and 30 min at 37OC with gently shaking. Label incorporation was stopped by addition of an excess of cold methionine. Cells were again pelleted and resuspended in ODC buffer (50 mM Hepes, 10 mM dithiothreitoi, 1 mM EDTA, 50 PM PLP, pH 7.2). Cell suspensions were sonicated as described previously for ODC activity determinations [12]. The 35S incorpora-
tion into protein was determined on the supernatants of the centrifuged cell-free extracts (13 000 x g, 30 min) . Duplicates of 2 ~1 from each sample were precipitated using final 10% trichloroacetic acid and filtered as described by Bonifacino [2]. The acidinsoluble radioactivity was counted. In order to measure relative ODC synthesis, equal amounts of acid-insoluble radioactivity from the supernatants were incubated with an excess of polyclonal monospecific antibody raised against mouse ODC; the ODC-antibody complexes were collected by precipitation with insoluble Protein A [17,23]. Pelleted proteins were separated by SDS-polyacrylamide electrophoresis (SDS-PAGE) and visualized by fluorography [17,20,23]. The ODC subunit band (53 kDa) was identified by comparison between samples treated with immune and non-immune rabbit serum. Autoradiographs were digitalized in a Scanner Sharp, model 5X-450 (Japan) and processed using a Sun Microsystem Image Analyzer (USA). Northern blot analysis Total RNA was isolated as described by Chomczynski and Sacchi [3]. Ten micrograms from each sample was fractionated in formaldehyde-agarose gels and hybridized with 32P-labelled probes as described previously [19,20]. The insert of the clone pODC934 [l] was used as ODC mRNA probe. As an internal standard, mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected [20]. Results and Discussion When quiescent Ehrlich carcinoma cells are perifused with 0.5 mM ornithine for 1- 3.5 h, an important increase in ODC activity can be detected; however, ODC activity remains very similar to the initial value when cells are perifused with only saline buffer [12]. Neither asparagine nor other ornithine precursors are able to elicit such a ODC induction in Ehrlich cells, when added to the perifusion medium at the same concentration [13]. In ornithine-
189
labelling was stopped after 10, 20 and 30 min. Three different experiments were carried out and very similar results were obtained. A representative result is shown in Fig. 1. Slopes of label incorporation into proteins (counts per min/pl extract/min) in saline medium perifused cell extracts were the 41% * 4 of those calculated from ornithine-perifused cell extracts. No difference in the patterns of proteins synthetized ‘de novo’ could be observed by SDS-PAGE analysis of pulse-labelled extracts at different times (results not shown). Thus, a generalized increase in protein synthesis induced by ornithine during perifusion can be concluded. ODC synthesis rate can not be compared between saline medium and ornithine-
perifused cells, the increase in ODC activity relative to cell number is linear over the incubation time, but sigmoidal when the activity is expressed relative to cell protein, due to the progressive increase in the total protein content of the cell extracts detected after 1.5 h of perifusion with 0.5 mM ornithine [X2]. An elevation in protein content can be due to an increase of the total protein synthesis rate, an increase in the synthesis of a particular set of proteins, or a diminished proteolysis. In order to answer this question, total protein synthesis rates were studied by pulse-labelling experiments at different times. A =-SlabelledTM amino acid mixture was added to cells perifused for 2 h with saline medium only or saline medium plus 0.5 mM ornithine and
-c,
0 0
-
4190 r
-l-J Q, Cc
Y =
=
+
5864
X
0.9741
lo-
Y =
4300
+
2460
X
I
I
I
10
20
30
Lobelling
time
(min)
Fis. 1. Acid-insoluble radioactivity in cell-free extracts of Ehrlich carcinoma cells iabelled for different times after 2 h of perifusion with saline medium only (0) or saline medium supplemented with 0.5 mM ornithine ( V ).
190
perifused cells: firstly, the total protein synthesis rate is very low in cells perifused with only saline buffer; secondly, this rate is slower respect to the rate detected in ornithineperifused cells (Fig. 1). In addition, the extremely low levels of ODC in quiescent mammalian cells made ODC undetectable after immunoprecipitation experiments [8,17,22]. Even for cells in which ornithine has induced ODC activity, this is more than lo-fold lower than the ODC activity detected in cultured Ehrlich cells during exponential phase of growth when ODC represents less than the 5 x 10 -3% of the total ‘de novo’ protein synthesis [5,17,20]. ODC activity increases from 4 to 4.8 nmol Cop/h per mg protein between 2 and 2.5 h of perifusion with 0.5 mM ornithine [12]. In order to test a specific enrichment in ODC protein of ornithine perifused cells, equal quantities of radioactivity incorporated into protein at the different times were immunoprecipitated and analyzed by SDS-PAGE and fluorography. Results are shown in Fig. 2. Extracts were being enriched progressively in a peptide which had the reported M, for the mouse ODC subunit (53 kDa) and which could not be observed in samples immunoprecipitated with non immune rabbit serum [17,20]. Results confirm that ornithine induces an increase in ODC translation relative to total protein synthesis rate and they are coincident with the previous observation that ornithine induces a cycloheximide-sensitive increase in specific ODC activity [ 121. On the other hand, these results agree with those reported for other amino acids inducing mammalian ODC translation [4,8]. Moreover, the ornithine analog difluoromethylornithine (DFMO) which is a suicide ODC inhibitor, is also described as a short-term inducer of ODC translation without any effect on ODC mRNA levels [6,17,21]. Actually, as shown in Fig. 3, there is no elevation in relative ODC mRNA content induced by ornithine after 3.5 h of perifusion, when ODC activity is more t.han 30-fold higher in ornithine-perifused cells than in cells perifused with only saline medium [12,13].
A kD
66 -
ODC
45 -
36 -
z
C
75
CD .v) 0
50
0” F 25 ‘3 0 u c?L 0
10
Lobelling
20 time
30 (min)
Fi2. 2. Synthesis of 35S-labelled ODC protein in Ehrlich carcinoma cells after 2 h of perifusion with saline medium supplemented with 0.5 mM ornithine. Autoradiograph of samples from pulse-labelled cells for 10 (lane l), 20 (lane 2) and 30 min (lane 3) and immunoprecipitated with ODC antibodies. Numbers indicate the position of the M, standards (kDa) (A). Densitometric analysis of the autoradiograph shown in A; ODC signal intensity in lane 3 was considered as 100%
(B).
191
antibodies and to Dr. H. van Steeg (Utrecht, Netherlands) for providing us with the GAPDH probe. Thanks are due to R. Cameselle and Manuela Vega for technical assistance.
A
References 1
-
I3
1
ODC
2 GAPDH
Fig. 3.
Northern blot analysis of ODC mRNA (A) and GAPDH mRNA (B), after 3.5 h of perifusion with saline medium supplemented with 0.5 mM omithine (lane l), or saline medium only (lane 2). Both hybridizations (ODC and control mRNA) were carried out on the same filter.
Thus, as reported for other amino acids inducing mammalian ODC, we can conclude that the tumour ODC induction by ornithine also takes place at post transcriptional level. Acknowledgements This paper was supported by the Grant PB/88/0445 of the DGICYT. We are particularly indebted to Dr. P. Aller (CBM, Madrid, Spain) and Dr. F. Berger (Department of Biological Sciences, South Carolina) for the pODC934 clone, to Dr. L. Persson for his generous gift of polyclonal anti-mouse ODC
2
Berger, F.G., Szymanski, P., Read, E. and Watson G. (1984) Androgen-regulated ornithine decarboxylase mRNAs of mouse kidney. J. Biol. Chem., 259, 7941- 7946. Bonifacino, J.S. (1991) Purification of proteins by
precipitation. In: Current Protocols in Molecular Biology. Section VI, unit 10.16. Editors: F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl, Willey Interscience, New York. 3 Chomcynsky, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal. Biochem., 162, 156 - 159. 4 Hayashi, S. (1989) Multiple mechanisms for the regulation of mammalian ornithine decarboxylase. In: Qrnithine Decarboxylase: Biology, Enzymology and Molecular Genetics. pp. 35-45. Editor: S. Hayashi. Pergamon Press, New York. 5 Heby, 0. and Persson L. (1990) Molecular genetics of polyamine synthesis in eucaryotic cells. Trends Biol. Sci., 15, 153 - 158. 6 Helm, I., Persson, L., Stjernborg, L., Thorsson, L., Heby, 0. (1989) Feedback control of ornithine decarboxylase expression by polyamines. Biochem. J., 258, 343-350. 7 Kahana, C. (1989) Molecular genetics of mammalian ornithine decarboxylase. In: The Physiology of Polyamines, Vol. I, pp. 281-295. Editor: U. Bachrach and Y.M. Heimer. CRC Press, Boca Raton, Florida. 8 Kanamoto, R., Nishiyama, M., Matsufuji, S. and Hayashi. S. (1991) Translational control mechanism of ornithine decarboxylase by asparagine and putrescine in primary cultured hepatocytes. Arch. Biochem. Biophys., 291, 247 - 254. 9 Marton, L.J. and Morris, D.R. (1987) Molecular and cellular functions of the polyamines. In: Inhibition of polyamine metabolism, pp. 79 - 105. Editors: P.P. McCann, A.E. Pegg and A. Sjoerdsma. Academic Press, New York. 10 Mdrquez, J., Mates, J.M., Quesada, A-R., Medina, M.A., Ndiiez de Castro, I. and Sanchez-Jimenez, F. (1989) Altered ornithine metabolism in tumor-bearing mice. Life Sci., 45, 1877 - 1884. 11 Medina, M.A., Urdiales, J.L., Mates, J.M., Ntiiiez de Castro, I. and Sanchez Jimhnez, F. (1991) Diamines interfere with the transport of L-ornithine in Ehrlich cell plasma membrane vesicles. Biochem. J., 280, 825 - 827. F., Garcia-Caballero, M. 12 Mat&s, J.M., Sanchez-JimCnez, and NCltiez de Castro I. (1989) Histamine and serotonin inhibit the induction of ornithine decarboxylase in perifused Ehrlich ascites tumour cells. FEBS Len., 250, 257 - 261.
192
13
14
15
16
17
18
Mat&, J.M., Sbnchez-JimBnez, F., L6pez-Herrera, J. and NCltiezde Castro, 1. (1991) Regulation by 1,Cdiamines of the omithine decarboxylase activity induced by omithine in perifused tumor cells. Biochem. Pharmacol., 42, 1045 - 1052. Mat&, J.M., Garcia-Caballero, M., N6riez de Castro, I., Urdiales, J.L. and Sbnchez-JimBnez, F. (1992) Polyamine metabolism regulation by histamine and other biogenic amines in Ehrlich carcinoma cells. Agents and Actions, (Spec. Conf. Issue), C380 - C383. Murakami, Y., Nishiyama, M. and Hayashi, S. (1989) Involvement of antiime in stabilization of ornithine decarboxylase caused by inhibitors of polyamine synthesis. Eur. J. Biochem., 180, 181- 184. Quesada A.R., Medina, M.A., Mbrquez, J., SBnchezJimBnez, F. and Ndiiez de Castro, I. (1988) Contribution of host tissues to circulating glutamine in mice inoculated with Ehrlich ascites tumor cells. Cancer Res., 48, 1551- 1553. Persson, L., Helm, I. and Heby 0. (1988) Regulation of omithine decarboxylase mRNA translation by polyamines. J. Biol. Chem., 263, 3528-3533. Rinehart, C.A., Viceps-Madore, D., Fong, W.F., Ortiz, J.G. and Canellakis E.S. (1985) The effect of transport
19
20
21
22
23
system A and N amino acids and of nerve and epidermal growth factors on the induction of omithine decarboxylase activity. J. Cell. Physiol., 123, 435-441. Southard, J.N., S&nchez-Jimbnez, F., Campbell G.T. and Talamantes, F. (1991) Sequence and expression of hamster prolactin and growth hormone messenger RNAs. Endocrinology, 129, 2965 - 2971. Urdiales, J.L., Mat& J.M., N6fiez de Castro, I. and SBnchez-Jimhnez, F. (1992) Chlorpheniramine inhibits the ornithine decarboxylase induction of Ehrlich carcinoma growing “in viva”. FEBS Lett., 305, 260- 264. Van Daalen Weters, T., Macrae, M., Brabant, M., Sittler, A. and Coffino, P. (1989) Polyamine-mediated regulation of mouse omithine decarboxylase is post-translational. Mol. Cell. Biol., 9, 5484-5490. Van Steeg, H., Van Oostrom, C.T.M., Hodemaekers, H.M., I?e!eters,L., Thomas, A.A.M. (1991) The translation in vitro of rat omithine decarboxylase mRNA is blocked by its 5’ untranslated region in a polyamine-independent way. B&hem. J., 274, 521-526. Wallon, U.M., Persson, L. and Heby, 0. (1990) Superinduction of ornithine decarboxylase (ODC) by actinomycin D is due to stimulation of ODC mRNA translation. FEBS Lett., 268, 161- 164.
