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Nature of the iron requirement for chick embryo cells cultured in the presence of horse serum
Cell Biology
International
Reports, Vol. 4, No. 5, May 1980
NATURE OF THE IRON REQUIREMENT FOR CHICK EMBRYO CELLS CULTURED IN THE PRESENCE OF HORSE SERUM
J. Tmbena;lAe.andC. Vagen
Institut
Groupe de Recherche No8 du C.N.R.S. Gustave Roussy 94800 Villejuif (France)
Abstract. Chick or bovine transferrin induces proliferation of chick embryo cells cultured in the presence of horse serum, which - cannot itself assure their multiplication. Cell growth can also be induced by iron salts and iron complexes such as hemoglobin or hemin, but also by biliverdin which has no iron atom in its molecule. Introduction. Many studies have shown that cell proliferation in cell culture requires the presence of iron (Messmer, 1973 ; Rudland et al., 1977 ; Fernandez-Pol et al., 1978). Chich embryo cells (CEC) do not proliferate at all in Eagle's minimal essential medium supplemented with horse serum alone (Verger and Imbenotte, 1977 ; Verger, 1979) * In the present study, we demonstrate that the absence of cell proliferation is due to the incapacity of horse transferrin to deliver its iron, since the addition of chick or bovine transferrin induces cell growth. Since we previously observed that iron complexes such as hemoglobin or hemin greatly stimulated CEC growth in the presence of horse serum, we studied the effects of derivatives of hemin with or without iron to try and define the role of the tetrapyrrole moiety of these compounds. MATERIAL AND METHODS Cell preparation, cultivation, transfer and counting. The methods employed at these different steps of experimentation have been previously described (Verger, 1979). Horse-and fetal bovine serum were obtained respectively from the Institut Pasteur and Sorga (France). Compounds tested. Horse and bovine transferrins were purchased from Koch-Light laboratories (iron percentage not specified). Chick transferrin (conalbumin) containing a maximal iron concentration of 0.03%, was obtained from Sigma. Other compounds were : iron as FeS047 Hz0 (Merck) ; bovine hemoglobin and hemin (Sigma) ; protoporphyrin IX as disodium salt (Calbiochem);biliverdin dihydrochloride (Sigma) ; chlorophyllin sodium-copper salt (Sigma). 0309-l
651/80/050447-06/$02.00/0
01980Academic
Press Inc. (London)
Ltd.
Cell Biology International
Eagle's minimal essential medium supplemented with Horse serum Horse serum Fetal bovineserum Horse serum 10 % + bovine transferrin Horse serum IO % + horse transferrin Horse serum 10 % + chick transferrin
No of Exp. 10 % 20 % 10 %
Reports, Vol. 4, No. 5, May 1980
Ratio
of cell in treated/control
3 3
1 (control) 1.23 k 0.43 30.84 k 5.67
10 UM
2
13.60 + 1.18
10 UM
2
1uM
2
0.1
density culture
(cytotoxic)
10.35 If 0.29
Table 1. Stimulation of chick embryo cell growth by various sera and transferrins in primary cultures at the 6th day. Cells were seeded at 6x104 per cm2 in Eagle's minimal essential medium plus IO%horse serum. This culture medium was removed on the second day and each compound tested was added with the renewed medium that was maintained during the entire experiment.
Stock solutions of hemin and biliverdin (ImM) were prepared in 0.02 N NaOH, then diluted in Eagle's minimal essential medium to the appropriate concentration. All the other compounds were solubilized in Eagle's medium. As specified by Calbiochem, protoporphyrin IX contained only 0.01% of iron. The contamination by iron in biliverdin and chlorophyllin was not specified. We thus determined the proportion of iron in these two compounds by a calorimetric reaction, (Harvey after mineralisation of compounds, with o-phenanthroline 1955). We found 0.042 u mole and 0.014 ~1mole of iron et al., per LI mole of biliverdin and chlorophyllin, respectively. RESULTS Inability of horse transferrin to induce growth. Table 1 shows that cells multiply in 10 % fetal bovineserum (FBS) but not in the even 20% of horse serum (HS). Nevertheless, induced in the presence of HS by the addition ferrin (1 MM) or bovine transferrin (10 uM). transferrin (10 crM) is cytotoxic.
chick embryo cell the presence of presence of 10 % or cell growth can be of chick transIn contrast, horse
Comparative effects on cell growth of FeSO4 or various compounds derived from heme in the presence of horse serum. Fig. I shows the kinetics of cell proliferation in primary
Cell Biology
3
A.
International
449
Reports, Vol. 4, No. 5, May 1980
3
Hemoglobin
C. FeSO4 1
i
x-xbx
DAYS
Fig. 1. Kinetics of hemoglobin, hemin, added at the second cultures seeded at of the mean of two bars).
cant rol
DAYS
cell proliferation under the effects of FeSOq and biliverdin. These compounds were day with the renewed medium in primary 6x104 cells per cm2. The standard deviation different counts is indicated (vertical
cultures over a period of six days, under the effect of iron, hemoglobin, hemin and biliverdin. It must be noted first that the maximal cell densities obtained with each compound were slightly different (the maximal rates of cell proliferation were observed with FeS04 (20 PM), hemoglobin (10 LIM), hemin (20 UM) or biliverdin (100 uM) and secondly that significant cell growth stimulations were obtained with a large rangeof concentrations (from I to 200 LAMfor FeS04, 3 to 50 uM ior hemoglobin, 4 to 40 UM for hemin and 20 to 100 uM for biliverdin). The kinetics of cell proliferation were also measured in primary cultures by the incorporation of tritiated thymidine, and it appeared that there was a good correlation between the results obtained by this method and those of cell counting (data not shown). Is stimulation of cell growth by biliverdin due to its contaminating iron ? The stimulating effect of hemoglobin and hemin can be attributed to their high iron content. The biliverdin used by us contained a small proportion of iron as a contaminant.
450
Cell Biology International
Reports, Vol. 4, No. 5, May 1980
Blliv. 20 yM
5 3 1
1
3
6
9
‘*
DAYS
Fig. 2. Comparison of the rates of cell growth obtained with various concentrations of biliverdin and their corresponding concentrations of contaminating iron, in secondary cultures (A) and in subcultures at the 4th cell transfer (B). Cells were seeded at 6~10~ per cm2 and the compounds were added simultaneously. In A, the cells came from primary cultures and in B, from subcultures propagated in the presence of HS plus hemoglobin (Verger, 1979). This raises the question whether biliverdin acted merely as an iron carrier or also as a molecule having a tetrapyrrole structure. To attempt to verify this, we tested FeS04 at concentrations approximately equivalent to the iron content found in the biliverdin concentrations utilized. This ex eriment was performed on subcultures seeded at 6x103 cells per cm!i at cell transfer I (secondary cultures) and 4. It appears clearly, in secondary cultures, that the level of cell growth stimulation is much more elevated with each concentration of biliverdin than the corresponding concentrations of FeS04 (Fig. 2A). In subculture 4, the superiority of biliverdin over its equivalent content of contaminating iron is evident only for biliverdin at 20 uM since the maximal rate of cell growth was already obtained with this concentration. In addition to biliverdin, we tested other compounds analogous to heme and devoid of iron atoms in their molecules. Protoporphyrin IX, which contains practically no iron, had no effect at concentrationsfrom IO to 100 nM and became cytotoxic above.
Cell Biology
International
Chlorophyllin, contaminating had no effect
451
Reports, Vol. 4, No. 5, May 1980
which contained about the same concentration of iron as did biliverdin (see material and methods}, on cell growth at concentrations from 20 to 100 uM.
DISCUSSION Chick embryo cells (CEC) are usually cultured in the presence of fetal bovine serum (FBS) but not horse serum (HS). The present work demonstrates that the absence of cell proliferation in the presence of HS is due to the fact that horse transferrin cannot give up its iron to CEC. The efficiency of transferrins is determined by their origin and not by their initial iron content, since there is enough iron in the culture medium to saturate the transferrins (Messmer, 1973 ; Young et al., 1979). It thus appears that HS contains all the growth-factors required by CEC in culture and that the growth inhibition is due to a functionally inefficient transferrin. Relatively high concentrations of FeS04 or compounds containing a high proportion of iron (hemoglobin or hemin) or a small proportion of iron (biliverdin) can be substituted for active transferrins to assure cell proliferation. A similar observation has been reported for hemoglobin in SV3T3 cells (Young et al., 1979). If one supposes that cell growth-stimulation is due to the iron proportion in each compound, the growth-promoting effect of biliverdin is surprising since it contains only a small proportion of contaminating iron. Moreover, it must be noted that chlorophyllin,which contains a comparable proportion of iron, did not stimulate cell growth at all. Concerning biliverdin, it can be envisioned either that it represents a particularly efficient iron carrier or eventually that the molecule itself, composed of a linear tetrapyrrole chain, might stimulate cell growth by an unknown mechanism. This stimulating effect of biliverdin suggests that perhaps porphyrins could stimulate cell growth. Such a possibility is supported by the fact that several metalloporphyrins stimulate protein synthesis in reticulocytes (Adamson et al., 1969), but also in nonerythroid cells (Beuzard et al., 1973). We tried to verify this hypothesis by adding protoporphyrin IX (heme molecule without central iron atom) in the culture medium. But the soluble compound we used (protoporphyrin IX disodium salt) appeared to be cytotoxic for CEC in culture, so that this experiment did not contribute any information concerning an eventual stimulating effect of porphyrins on cell growth. The possibility that iron stimulates cell growth partly via the synthesis of small amounts of porphyrins remains an open question but it seems that CEC cultured in the presence of HS could facilitate this study.
AKNOWLEDGBVENTSThis work was partly
supported
by I.N.S.E.R.M.
Cell Biology International
Reports, Vol. 4, No. 5, May 1980
REFERENCES Adamson, S.D., Herbert, E. and Kemp, S.F. (1969) Effects of hemin and other porphyrins on protein synthesis in a reticulocyte lysate cell-free system. Journal of Molecular Biology, 42, 247-258. Beuzard, Y., Rodvien, R. and London, I.M. (1973) Effect of hemin on the synthesis of hemoglobin and other proteins in mammalian cells. Proceedings of the National Academy of Science, U.S.A., 70, 1022-1026. Fernandez-Pol, J.A., Klos, D. and Donati, R.M. (1978) Iron transport in NRK cells synchronized in Gl by picolinic acid. Cell Biology International Reports, 2, 433-439. Harvey, A.E., Jr, Smart, J.A. and Amis, E.S. taneous spectrophotometric determination and total iron with I, IO-phenanthroline. Chemistry, 27, 26-29.
(1955) Simulof iron (II) Analytical
Messmer, T.O. (1973) Nature of the iron requirement for Chinese hamster V79 cells in tissue culture medium. perimental Cell Research, 77, 404-408.
Ex-
Rudland, P.S., Durbin, H., Clingan, D. and de Asua, L.J. (1977) Iron salts and transferrin are specifically required for cell division of cultured 3T6 cells. Biochemical and Biophysical Research Communications, 75, 556-562. Verger, C. and Imbenotte, J. (1977) L'temoglobine induit la multiplication et la differentiation de myoblastes de poulet cultives dans des conditions non optimales. Comptes Rendus de l'Acad&nie des Sciences, Paris, 285, 221-224. and morphology of chick emVerger, C., (1979) Proliferation bryo cells cultured in the presence of horse serum and hemoglobin. In Vitro, 15, 587-592. F.W., Chipman, S. and Hartman, S.C. Young, D.V., Cox III, (1979) The growth stimulation of SV3T3 cells by transferrin and its dependence on biotin. Experimental Cell Research, 118, 410-414. Received:
7th January
1980
Accepted:
12th March 1980
International
Reports, Vol. 4, No. 5, May 1980
NATURE OF THE IRON REQUIREMENT FOR CHICK EMBRYO CELLS CULTURED IN THE PRESENCE OF HORSE SERUM
J. Tmbena;lAe.andC. Vagen
Institut
Groupe de Recherche No8 du C.N.R.S. Gustave Roussy 94800 Villejuif (France)
Abstract. Chick or bovine transferrin induces proliferation of chick embryo cells cultured in the presence of horse serum, which - cannot itself assure their multiplication. Cell growth can also be induced by iron salts and iron complexes such as hemoglobin or hemin, but also by biliverdin which has no iron atom in its molecule. Introduction. Many studies have shown that cell proliferation in cell culture requires the presence of iron (Messmer, 1973 ; Rudland et al., 1977 ; Fernandez-Pol et al., 1978). Chich embryo cells (CEC) do not proliferate at all in Eagle's minimal essential medium supplemented with horse serum alone (Verger and Imbenotte, 1977 ; Verger, 1979) * In the present study, we demonstrate that the absence of cell proliferation is due to the incapacity of horse transferrin to deliver its iron, since the addition of chick or bovine transferrin induces cell growth. Since we previously observed that iron complexes such as hemoglobin or hemin greatly stimulated CEC growth in the presence of horse serum, we studied the effects of derivatives of hemin with or without iron to try and define the role of the tetrapyrrole moiety of these compounds. MATERIAL AND METHODS Cell preparation, cultivation, transfer and counting. The methods employed at these different steps of experimentation have been previously described (Verger, 1979). Horse-and fetal bovine serum were obtained respectively from the Institut Pasteur and Sorga (France). Compounds tested. Horse and bovine transferrins were purchased from Koch-Light laboratories (iron percentage not specified). Chick transferrin (conalbumin) containing a maximal iron concentration of 0.03%, was obtained from Sigma. Other compounds were : iron as FeS047 Hz0 (Merck) ; bovine hemoglobin and hemin (Sigma) ; protoporphyrin IX as disodium salt (Calbiochem);biliverdin dihydrochloride (Sigma) ; chlorophyllin sodium-copper salt (Sigma). 0309-l
651/80/050447-06/$02.00/0
01980Academic
Press Inc. (London)
Ltd.
Cell Biology International
Eagle's minimal essential medium supplemented with Horse serum Horse serum Fetal bovineserum Horse serum 10 % + bovine transferrin Horse serum IO % + horse transferrin Horse serum 10 % + chick transferrin
No of Exp. 10 % 20 % 10 %
Reports, Vol. 4, No. 5, May 1980
Ratio
of cell in treated/control
3 3
1 (control) 1.23 k 0.43 30.84 k 5.67
10 UM
2
13.60 + 1.18
10 UM
2
1uM
2
0.1
density culture
(cytotoxic)
10.35 If 0.29
Table 1. Stimulation of chick embryo cell growth by various sera and transferrins in primary cultures at the 6th day. Cells were seeded at 6x104 per cm2 in Eagle's minimal essential medium plus IO%horse serum. This culture medium was removed on the second day and each compound tested was added with the renewed medium that was maintained during the entire experiment.
Stock solutions of hemin and biliverdin (ImM) were prepared in 0.02 N NaOH, then diluted in Eagle's minimal essential medium to the appropriate concentration. All the other compounds were solubilized in Eagle's medium. As specified by Calbiochem, protoporphyrin IX contained only 0.01% of iron. The contamination by iron in biliverdin and chlorophyllin was not specified. We thus determined the proportion of iron in these two compounds by a calorimetric reaction, (Harvey after mineralisation of compounds, with o-phenanthroline 1955). We found 0.042 u mole and 0.014 ~1mole of iron et al., per LI mole of biliverdin and chlorophyllin, respectively. RESULTS Inability of horse transferrin to induce growth. Table 1 shows that cells multiply in 10 % fetal bovineserum (FBS) but not in the even 20% of horse serum (HS). Nevertheless, induced in the presence of HS by the addition ferrin (1 MM) or bovine transferrin (10 uM). transferrin (10 crM) is cytotoxic.
chick embryo cell the presence of presence of 10 % or cell growth can be of chick transIn contrast, horse
Comparative effects on cell growth of FeSO4 or various compounds derived from heme in the presence of horse serum. Fig. I shows the kinetics of cell proliferation in primary
Cell Biology
3
A.
International
449
Reports, Vol. 4, No. 5, May 1980
3
Hemoglobin
C. FeSO4 1
i
x-xbx
DAYS
Fig. 1. Kinetics of hemoglobin, hemin, added at the second cultures seeded at of the mean of two bars).
cant rol
DAYS
cell proliferation under the effects of FeSOq and biliverdin. These compounds were day with the renewed medium in primary 6x104 cells per cm2. The standard deviation different counts is indicated (vertical
cultures over a period of six days, under the effect of iron, hemoglobin, hemin and biliverdin. It must be noted first that the maximal cell densities obtained with each compound were slightly different (the maximal rates of cell proliferation were observed with FeS04 (20 PM), hemoglobin (10 LIM), hemin (20 UM) or biliverdin (100 uM) and secondly that significant cell growth stimulations were obtained with a large rangeof concentrations (from I to 200 LAMfor FeS04, 3 to 50 uM ior hemoglobin, 4 to 40 UM for hemin and 20 to 100 uM for biliverdin). The kinetics of cell proliferation were also measured in primary cultures by the incorporation of tritiated thymidine, and it appeared that there was a good correlation between the results obtained by this method and those of cell counting (data not shown). Is stimulation of cell growth by biliverdin due to its contaminating iron ? The stimulating effect of hemoglobin and hemin can be attributed to their high iron content. The biliverdin used by us contained a small proportion of iron as a contaminant.
450
Cell Biology International
Reports, Vol. 4, No. 5, May 1980
Blliv. 20 yM
5 3 1
1
3
6
9
‘*
DAYS
Fig. 2. Comparison of the rates of cell growth obtained with various concentrations of biliverdin and their corresponding concentrations of contaminating iron, in secondary cultures (A) and in subcultures at the 4th cell transfer (B). Cells were seeded at 6~10~ per cm2 and the compounds were added simultaneously. In A, the cells came from primary cultures and in B, from subcultures propagated in the presence of HS plus hemoglobin (Verger, 1979). This raises the question whether biliverdin acted merely as an iron carrier or also as a molecule having a tetrapyrrole structure. To attempt to verify this, we tested FeS04 at concentrations approximately equivalent to the iron content found in the biliverdin concentrations utilized. This ex eriment was performed on subcultures seeded at 6x103 cells per cm!i at cell transfer I (secondary cultures) and 4. It appears clearly, in secondary cultures, that the level of cell growth stimulation is much more elevated with each concentration of biliverdin than the corresponding concentrations of FeS04 (Fig. 2A). In subculture 4, the superiority of biliverdin over its equivalent content of contaminating iron is evident only for biliverdin at 20 uM since the maximal rate of cell growth was already obtained with this concentration. In addition to biliverdin, we tested other compounds analogous to heme and devoid of iron atoms in their molecules. Protoporphyrin IX, which contains practically no iron, had no effect at concentrationsfrom IO to 100 nM and became cytotoxic above.
Cell Biology
International
Chlorophyllin, contaminating had no effect
451
Reports, Vol. 4, No. 5, May 1980
which contained about the same concentration of iron as did biliverdin (see material and methods}, on cell growth at concentrations from 20 to 100 uM.
DISCUSSION Chick embryo cells (CEC) are usually cultured in the presence of fetal bovine serum (FBS) but not horse serum (HS). The present work demonstrates that the absence of cell proliferation in the presence of HS is due to the fact that horse transferrin cannot give up its iron to CEC. The efficiency of transferrins is determined by their origin and not by their initial iron content, since there is enough iron in the culture medium to saturate the transferrins (Messmer, 1973 ; Young et al., 1979). It thus appears that HS contains all the growth-factors required by CEC in culture and that the growth inhibition is due to a functionally inefficient transferrin. Relatively high concentrations of FeS04 or compounds containing a high proportion of iron (hemoglobin or hemin) or a small proportion of iron (biliverdin) can be substituted for active transferrins to assure cell proliferation. A similar observation has been reported for hemoglobin in SV3T3 cells (Young et al., 1979). If one supposes that cell growth-stimulation is due to the iron proportion in each compound, the growth-promoting effect of biliverdin is surprising since it contains only a small proportion of contaminating iron. Moreover, it must be noted that chlorophyllin,which contains a comparable proportion of iron, did not stimulate cell growth at all. Concerning biliverdin, it can be envisioned either that it represents a particularly efficient iron carrier or eventually that the molecule itself, composed of a linear tetrapyrrole chain, might stimulate cell growth by an unknown mechanism. This stimulating effect of biliverdin suggests that perhaps porphyrins could stimulate cell growth. Such a possibility is supported by the fact that several metalloporphyrins stimulate protein synthesis in reticulocytes (Adamson et al., 1969), but also in nonerythroid cells (Beuzard et al., 1973). We tried to verify this hypothesis by adding protoporphyrin IX (heme molecule without central iron atom) in the culture medium. But the soluble compound we used (protoporphyrin IX disodium salt) appeared to be cytotoxic for CEC in culture, so that this experiment did not contribute any information concerning an eventual stimulating effect of porphyrins on cell growth. The possibility that iron stimulates cell growth partly via the synthesis of small amounts of porphyrins remains an open question but it seems that CEC cultured in the presence of HS could facilitate this study.
AKNOWLEDGBVENTSThis work was partly
supported
by I.N.S.E.R.M.
Cell Biology International
Reports, Vol. 4, No. 5, May 1980
REFERENCES Adamson, S.D., Herbert, E. and Kemp, S.F. (1969) Effects of hemin and other porphyrins on protein synthesis in a reticulocyte lysate cell-free system. Journal of Molecular Biology, 42, 247-258. Beuzard, Y., Rodvien, R. and London, I.M. (1973) Effect of hemin on the synthesis of hemoglobin and other proteins in mammalian cells. Proceedings of the National Academy of Science, U.S.A., 70, 1022-1026. Fernandez-Pol, J.A., Klos, D. and Donati, R.M. (1978) Iron transport in NRK cells synchronized in Gl by picolinic acid. Cell Biology International Reports, 2, 433-439. Harvey, A.E., Jr, Smart, J.A. and Amis, E.S. taneous spectrophotometric determination and total iron with I, IO-phenanthroline. Chemistry, 27, 26-29.
(1955) Simulof iron (II) Analytical
Messmer, T.O. (1973) Nature of the iron requirement for Chinese hamster V79 cells in tissue culture medium. perimental Cell Research, 77, 404-408.
Ex-
Rudland, P.S., Durbin, H., Clingan, D. and de Asua, L.J. (1977) Iron salts and transferrin are specifically required for cell division of cultured 3T6 cells. Biochemical and Biophysical Research Communications, 75, 556-562. Verger, C. and Imbenotte, J. (1977) L'temoglobine induit la multiplication et la differentiation de myoblastes de poulet cultives dans des conditions non optimales. Comptes Rendus de l'Acad&nie des Sciences, Paris, 285, 221-224. and morphology of chick emVerger, C., (1979) Proliferation bryo cells cultured in the presence of horse serum and hemoglobin. In Vitro, 15, 587-592. F.W., Chipman, S. and Hartman, S.C. Young, D.V., Cox III, (1979) The growth stimulation of SV3T3 cells by transferrin and its dependence on biotin. Experimental Cell Research, 118, 410-414. Received:
7th January
1980
Accepted:
12th March 1980