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Ornithine decarboxylase activity correlates with the synthesis of DNA and not of rRNA in developing sea urchins
Cell Biology
International
Reports,
377
Vol. 7, No. 5, May 1983
ORNITHINE DECARBOXYLASE ACTIVITY CORRELATES WITH THE SYNTHESIS OF DNA AND NOT OF rRNA IN DEVELOPING SEA URCHINS A.M.
Pirrone^, R. Gambino+, S.A. G. Buttice^, P. Bellavia^, and G. Giudice^+
Istituto di Anatomia e Istituto di Biologia via Archirafi,20,22
Acute*,
Comparata^, UnivFrsita dello Sviluppo , CNR 90123 Palermo(Italy)
ABSTRACT Measurements of vity in oocytes and tive correlation of ty with cell cleavage ribosomal RNA (rRNA)
Ornithinedecarboxylase (ODC) actideveloping sea urchins show a posivariations of this enzymatic activiand DNA synthesis, but not with synthesis. INTRODUCTION
Ornithine decarboxylase (ODC) activity, leading to the production of polyamines, has been known to increase sharply in somatic cells undergoing replication(Russe1 and Snyder,1968; Fillingame et a1.,1975; Janne et al., 1977; Knutson and Morris,1978; Luk et a1.,1982; Russel and McVicker,l971). This increased activity has been correlated with the increased macromolecular syntheses of DNA and rKNA which preceed the division of somatic cells. Sea urchin embryonic cells, however, offer a special situation which permits the correlation of the increase in ODC activity with the synthesis of DNA or with that of rRNA or both to be distinguished. There are in fact embryonic stages in which rRNA synthesis is very active while DNA synthesis is silent, as for example during oogenesis, and there are stages in which, on the contrary, DNA synthesis is very active and rRNA synthesis is very low, as e.g. from fertilization to mesenchyme blastula ( see Giudice,l973 for a review). We therefore decided to measure ODC activity at different developmental stages in order to investigate to which of the two described syn0309-16511631050377-051$03.00/0
@
1983
Academic
Press
Inc.
(London)
Ltd.
378
teses, that ity correlates.
Cell Biology
of
DNA or
International
that
MATERIALS
of
Reports,
rRNA,
this
Vol. 7, No. 5, May
enzymatic
1983
activ-
AND METHODS
Embryonic cultures were carried out in sterile sea water with antibiotics as previously described(Giudice and Mutolo,1969). Oocytes were prepared following the procedure of Giudice et a1.(1972) as modified by Cognetti et a1.(1977). The enzyme source was prepfred+$s follows: oocytes or embryos washed twice in Ca -free sea water, were 943 homogenized in 50 mM Phosphate buffer pH 7.0, 2 mM Dithiothreitol( DTT ), 1 mM EDTA and the 30,000 xg supernatant dialyzed against 25 mM Phosphate buffer pH 7.0, 2 mM DTT and 0.1 mM EDTA. Ornithine decarboxylase activity was measured according to the method of Russel and Snyder(1968) modified as follows: incubation was carried out in Warburg flasks for 30’ at 37 "C in the presence of 25 mM Phosphate buffer pH 7.0, 0.1 mM PirydoxalphpJphate, 2 mM DTT, 2.1 mM LOrnithine, 0.03 mM D-L-(lC)Ornithine (58mCi/mMole) and 30,000 xg supernatant. The reaction was terminated by the addition of 0.3 ml of 4 M Cytric acid ( previously present in fQe lateral arm), and the radioactivity of the liberated CO2 exhaustively absorbed for a further 60' in the Ethanolaminemethoxyethanol solution ( contained in the central well) measured. Protein content was measured according to the Lowry procedure (1951). RESULTS
AND DISCIJSSION
The results of a typical experiment (selected from a series of three, all with the same trend) are reported in very low in the oocytes, Figure 1. The enzymatic activity, i.e. when rRNA synthesis is very active and DNA synthesis silent, shows a very sharp increase during the so-called cleavage period, i.e. when cell division and DNA synthesis are very active and rRNA synthesis very low. The curves of the mitotic index and of the DNA content per embryo(Parisi et al., 1978) are reported for comparison. A drop in ODC activity is observed coincident with a drop in the mitotic index and a plateau of the DNA content which occur around hatching. DNA synthesis is resumed during gastrulation, and here again a second increase in ODC activity is ob-
Cell Biology
International
Reports,
Vol. 7, No. 5, May 1983
379
1
I _’ I c
0 hours
Fig.1
after
fertilization
) Ornithine decarboxylase activity;((DNA content;(m-¤-¤)Mitotic index. These ter values are redrawn from Parisi et al.,
1 two lat1978
served, followed by a second drop in enzymatic activity when the DNA content, during the early prism stage, shows It may be important to recall here that a second plateau. this second plateau has been also statistically confirmed by our previous experiments( Giudice and Mutolo, 1970). These results, therefore, clearly show that the ODC activity correlates fairly well with the mitotic activity and the synthesis of the DNA, even in stages where rRNA synthesis is low, while it is low in developmental stages, when rRNA synthesis is very active, whether the latter is measured on a per nucleus basis as during oogenesis or on a per embryo basis( which in sea urchin is equivalent
380
Cell Biology
International
Reports,
Vol. 7, No. 5, May 1983
to a per milligram protein basis) as in postgastrular stages (Griffith et al., 1981; Sconzo et al., 1970). These results agree quite well with the cyclic changes in polyamine content related with the cleavage cycle reported by Kusunoki and Yasumasu (1976) in the first three cleavages of Hemicentrotus pulcherrimus and with the inhibitory effect of the alpha-hydrazineornithine on the first three cleavages of Hemicentrotus and Antocidaris eggs, observed by Kusunoki and Yasumasu (1978). Our results also reproduce the biphasic curve of the activity of an enzyme correlated with DNA synthesis like DNAase described by De Petrocellis and Vittorelli (1975). It is therefore reasonable to conclude that the variation in ODC activity positively correlates with cell division and DNA synthesis but not with rRNA synthesis, at least in sea urchin development. REFERENCES Cognetti, G., Platz, R.D., Meistrich, M.L. and Di Liegro 1.(1977) Studies on protein synthesis during sea urchin oogenesis I. Synthesis of Histone FZb. Cell Differentiation, 5, 283-291. M.L.(1975) Role of De Petrocellis, B. and Vittorelli, cell interactions in development and differentiation of the sea urchin Paracentrotus lividus. Experimental Cell Research, 94, 392-400. Fillingame, R.H., Jorstad, C.M., Morris, D.R.(1975) Increased cellular levels of Spermidine or Spermine are required for optimal DNA synthesis in limphocytes acof the National tivated by Concanavalin A. Proceedings Academy of Sciences USA, 72, 4042-4045. Giudice, G.(1973) Developmental Biology of the sea urchin embryo. Academic Press New York and London. V.(1969) Synthesis of ribosomal Giudice, G. and Mutolo, RNA during sea urchin development II. Electrophoretic analysis of nuclear and cytoplasmic RNA's, Biochimica et Biophysics Acta, 179, 341-347. G. and Mutolo, V.(l970) Reaggregation of dissoGiudice, ciated cells of sea urchin embryos. Advances in Morphogenesis, Academic Press, New York, 8, 115-158. Giudice, G., Sconzo, G., Bono, A. and Albanese, 1.(1972) Studies on sea urchin oocytes I. Purification and Experimental Cell Research, 72, cell fractionation.
Cell Biology
International
Reports,
Vol. 7, No. 5, May 1983
381
90-94. Griffith, B.B. and Humphreys, T.(1981) Griffith, J.K., Regulation of ribosomal RNA synthesis in sea urchin emDevelopmental Biology, 87, 220-228. bryos and oocytes. Janne, J., Paso, H. and Raina, A.(1977) Polyamines in Biochimica et Biophysics Acta, rapid growth and cancer. 473, 241-293. D.R.(1978) Cellular polyamine Knutson, J.C. and Morris, depletion reduced DNA sythesis in isolated lymphocyte Biochimica et Biophysics Acta, 520, 291-301. nuclei. Cyclic change in polyKusunoki, S. and Yasumasu, 1.(1976) amine concentration in sea urchin eggs related with cleavage cycle. Biochemical Biophysical Research Communications,68, 881-885. Kustinoki, S. and Yasumasu, 1.(1978) Inhibitory effect of alpha-hydrazinoornithine on egg cleavage in sea urchin eggs. Developmental Biology, 67, 336-345. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.(1951) Protein measurements with the Folin Phenol reagent. The Journal of Biological Chemistry, 193, 265-275.
Luk,
G.D., Civin, C.I., Weisman, R.M. and Baylin, S.B. (1982) Ornithine decarboxylase: essential in proliferation but not differentiation of human promyelocytic leukemia cells. Science, 216, 75-77. Parisi, E., Filosa, S., De Petrocellis, B. and Monroy, A. (1978) The pattern of cell division in the early development of the sea urchin Paracentrotus lividus. tievelopmental Biology, 65, 38-49. Russel, D.H. and McVicker, T.A.(1971) Polyamine metabolism in mouse liver after partial epatectomy. Biochimica et Biophysics Acta, 244, 85-93. Russel, D.H. and Snyder, S.H.(1968) Amine synthesis in rapidly growing tissues: Ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proceedings of the National Academy of Sciences USA, 60, 1420-1427. Sconzo, G., Pirrone, A.M., Mutolo, V. and Giudice, G.(1970) Synthesis of ribosomal RNA during sea urchin development III. Evidence for an activation of transcription. Biochimica et Biophysics Acta, 199, 435-440. Received:
15th March 1983.
Accepted: 21st March 1983
International
Reports,
377
Vol. 7, No. 5, May 1983
ORNITHINE DECARBOXYLASE ACTIVITY CORRELATES WITH THE SYNTHESIS OF DNA AND NOT OF rRNA IN DEVELOPING SEA URCHINS A.M.
Pirrone^, R. Gambino+, S.A. G. Buttice^, P. Bellavia^, and G. Giudice^+
Istituto di Anatomia e Istituto di Biologia via Archirafi,20,22
Acute*,
Comparata^, UnivFrsita dello Sviluppo , CNR 90123 Palermo(Italy)
ABSTRACT Measurements of vity in oocytes and tive correlation of ty with cell cleavage ribosomal RNA (rRNA)
Ornithinedecarboxylase (ODC) actideveloping sea urchins show a posivariations of this enzymatic activiand DNA synthesis, but not with synthesis. INTRODUCTION
Ornithine decarboxylase (ODC) activity, leading to the production of polyamines, has been known to increase sharply in somatic cells undergoing replication(Russe1 and Snyder,1968; Fillingame et a1.,1975; Janne et al., 1977; Knutson and Morris,1978; Luk et a1.,1982; Russel and McVicker,l971). This increased activity has been correlated with the increased macromolecular syntheses of DNA and rKNA which preceed the division of somatic cells. Sea urchin embryonic cells, however, offer a special situation which permits the correlation of the increase in ODC activity with the synthesis of DNA or with that of rRNA or both to be distinguished. There are in fact embryonic stages in which rRNA synthesis is very active while DNA synthesis is silent, as for example during oogenesis, and there are stages in which, on the contrary, DNA synthesis is very active and rRNA synthesis is very low, as e.g. from fertilization to mesenchyme blastula ( see Giudice,l973 for a review). We therefore decided to measure ODC activity at different developmental stages in order to investigate to which of the two described syn0309-16511631050377-051$03.00/0
@
1983
Academic
Press
Inc.
(London)
Ltd.
378
teses, that ity correlates.
Cell Biology
of
DNA or
International
that
MATERIALS
of
Reports,
rRNA,
this
Vol. 7, No. 5, May
enzymatic
1983
activ-
AND METHODS
Embryonic cultures were carried out in sterile sea water with antibiotics as previously described(Giudice and Mutolo,1969). Oocytes were prepared following the procedure of Giudice et a1.(1972) as modified by Cognetti et a1.(1977). The enzyme source was prepfred+$s follows: oocytes or embryos washed twice in Ca -free sea water, were 943 homogenized in 50 mM Phosphate buffer pH 7.0, 2 mM Dithiothreitol( DTT ), 1 mM EDTA and the 30,000 xg supernatant dialyzed against 25 mM Phosphate buffer pH 7.0, 2 mM DTT and 0.1 mM EDTA. Ornithine decarboxylase activity was measured according to the method of Russel and Snyder(1968) modified as follows: incubation was carried out in Warburg flasks for 30’ at 37 "C in the presence of 25 mM Phosphate buffer pH 7.0, 0.1 mM PirydoxalphpJphate, 2 mM DTT, 2.1 mM LOrnithine, 0.03 mM D-L-(lC)Ornithine (58mCi/mMole) and 30,000 xg supernatant. The reaction was terminated by the addition of 0.3 ml of 4 M Cytric acid ( previously present in fQe lateral arm), and the radioactivity of the liberated CO2 exhaustively absorbed for a further 60' in the Ethanolaminemethoxyethanol solution ( contained in the central well) measured. Protein content was measured according to the Lowry procedure (1951). RESULTS
AND DISCIJSSION
The results of a typical experiment (selected from a series of three, all with the same trend) are reported in very low in the oocytes, Figure 1. The enzymatic activity, i.e. when rRNA synthesis is very active and DNA synthesis silent, shows a very sharp increase during the so-called cleavage period, i.e. when cell division and DNA synthesis are very active and rRNA synthesis very low. The curves of the mitotic index and of the DNA content per embryo(Parisi et al., 1978) are reported for comparison. A drop in ODC activity is observed coincident with a drop in the mitotic index and a plateau of the DNA content which occur around hatching. DNA synthesis is resumed during gastrulation, and here again a second increase in ODC activity is ob-
Cell Biology
International
Reports,
Vol. 7, No. 5, May 1983
379
1
I _’ I c
0 hours
Fig.1
after
fertilization
) Ornithine decarboxylase activity;((DNA content;(m-¤-¤)Mitotic index. These ter values are redrawn from Parisi et al.,
1 two lat1978
served, followed by a second drop in enzymatic activity when the DNA content, during the early prism stage, shows It may be important to recall here that a second plateau. this second plateau has been also statistically confirmed by our previous experiments( Giudice and Mutolo, 1970). These results, therefore, clearly show that the ODC activity correlates fairly well with the mitotic activity and the synthesis of the DNA, even in stages where rRNA synthesis is low, while it is low in developmental stages, when rRNA synthesis is very active, whether the latter is measured on a per nucleus basis as during oogenesis or on a per embryo basis( which in sea urchin is equivalent
380
Cell Biology
International
Reports,
Vol. 7, No. 5, May 1983
to a per milligram protein basis) as in postgastrular stages (Griffith et al., 1981; Sconzo et al., 1970). These results agree quite well with the cyclic changes in polyamine content related with the cleavage cycle reported by Kusunoki and Yasumasu (1976) in the first three cleavages of Hemicentrotus pulcherrimus and with the inhibitory effect of the alpha-hydrazineornithine on the first three cleavages of Hemicentrotus and Antocidaris eggs, observed by Kusunoki and Yasumasu (1978). Our results also reproduce the biphasic curve of the activity of an enzyme correlated with DNA synthesis like DNAase described by De Petrocellis and Vittorelli (1975). It is therefore reasonable to conclude that the variation in ODC activity positively correlates with cell division and DNA synthesis but not with rRNA synthesis, at least in sea urchin development. REFERENCES Cognetti, G., Platz, R.D., Meistrich, M.L. and Di Liegro 1.(1977) Studies on protein synthesis during sea urchin oogenesis I. Synthesis of Histone FZb. Cell Differentiation, 5, 283-291. M.L.(1975) Role of De Petrocellis, B. and Vittorelli, cell interactions in development and differentiation of the sea urchin Paracentrotus lividus. Experimental Cell Research, 94, 392-400. Fillingame, R.H., Jorstad, C.M., Morris, D.R.(1975) Increased cellular levels of Spermidine or Spermine are required for optimal DNA synthesis in limphocytes acof the National tivated by Concanavalin A. Proceedings Academy of Sciences USA, 72, 4042-4045. Giudice, G.(1973) Developmental Biology of the sea urchin embryo. Academic Press New York and London. V.(1969) Synthesis of ribosomal Giudice, G. and Mutolo, RNA during sea urchin development II. Electrophoretic analysis of nuclear and cytoplasmic RNA's, Biochimica et Biophysics Acta, 179, 341-347. G. and Mutolo, V.(l970) Reaggregation of dissoGiudice, ciated cells of sea urchin embryos. Advances in Morphogenesis, Academic Press, New York, 8, 115-158. Giudice, G., Sconzo, G., Bono, A. and Albanese, 1.(1972) Studies on sea urchin oocytes I. Purification and Experimental Cell Research, 72, cell fractionation.
Cell Biology
International
Reports,
Vol. 7, No. 5, May 1983
381
90-94. Griffith, B.B. and Humphreys, T.(1981) Griffith, J.K., Regulation of ribosomal RNA synthesis in sea urchin emDevelopmental Biology, 87, 220-228. bryos and oocytes. Janne, J., Paso, H. and Raina, A.(1977) Polyamines in Biochimica et Biophysics Acta, rapid growth and cancer. 473, 241-293. D.R.(1978) Cellular polyamine Knutson, J.C. and Morris, depletion reduced DNA sythesis in isolated lymphocyte Biochimica et Biophysics Acta, 520, 291-301. nuclei. Cyclic change in polyKusunoki, S. and Yasumasu, 1.(1976) amine concentration in sea urchin eggs related with cleavage cycle. Biochemical Biophysical Research Communications,68, 881-885. Kustinoki, S. and Yasumasu, 1.(1978) Inhibitory effect of alpha-hydrazinoornithine on egg cleavage in sea urchin eggs. Developmental Biology, 67, 336-345. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.(1951) Protein measurements with the Folin Phenol reagent. The Journal of Biological Chemistry, 193, 265-275.
Luk,
G.D., Civin, C.I., Weisman, R.M. and Baylin, S.B. (1982) Ornithine decarboxylase: essential in proliferation but not differentiation of human promyelocytic leukemia cells. Science, 216, 75-77. Parisi, E., Filosa, S., De Petrocellis, B. and Monroy, A. (1978) The pattern of cell division in the early development of the sea urchin Paracentrotus lividus. tievelopmental Biology, 65, 38-49. Russel, D.H. and McVicker, T.A.(1971) Polyamine metabolism in mouse liver after partial epatectomy. Biochimica et Biophysics Acta, 244, 85-93. Russel, D.H. and Snyder, S.H.(1968) Amine synthesis in rapidly growing tissues: Ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proceedings of the National Academy of Sciences USA, 60, 1420-1427. Sconzo, G., Pirrone, A.M., Mutolo, V. and Giudice, G.(1970) Synthesis of ribosomal RNA during sea urchin development III. Evidence for an activation of transcription. Biochimica et Biophysics Acta, 199, 435-440. Received:
15th March 1983.
Accepted: 21st March 1983