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The roles of the nucleic acids and free nucleotides in chick embryonic extract on the growth of heart fibroblast
Experimental
502
Cell Research, 9, 502 ,509 (1055)
THE ROLES OF THE NUCLEIC ACIDS AND FREE NUCLEOTIDES IN CHICK EMBRYONIC EXTRACT ON THE GROWTH OF HEART FIBROBLASTl K. H. LU and T. WINNICK Department
of Biochemistry and the Radiation Research Laboratory, University of Iowa, Iowa City, lowa, U.S.A. Received February
College of Medicine.
26, 1955
1946 Fischer [tl] distinguished betlveen the high molecular \veight and the diffusible constituents of embryonic extract (EE). He found that dialyzed extracts failed to support growth of fibroblasts, but that this efrect of dialysis was partially reversed by the addition of a mixture of amino acids [3]. Subsequently, three groups of investigators [a, 13, 141 hart studied the role of ultrafiltrates, or of alcoholic extracts of EE, on proliferation of fibroblasts in vitro. These solutions were shown to contain a great variety of comamino acids, purines, pyrimidines, sugars, and coenpounds, including zymes. There appears to be general agreement that these lo\\ molecular compounds are essential for growth. However, the importance of the proteins and nucleic acids of El? has also been established. Fischer [4] described a nucleoprotein fraction, termed \vhich promoted growth of cultures; later work by Katsuta “embryonin”, and Takaoka [7] and Kutsky [tl] has emphasized the essentiality of nucleic acids in the nutrient media. Recent studies conducted in our laboratory have been concerned \vith the correlation of growth, as measured by increase in desoxpribose nucleic acid content, with the incorporation of labeled adeninc and thymidine into nucleic acids in roller tube tissue cultures [9]. The present paper is a continuation of these experiments, but with emphasis on the effect of fractionation of the EE nutrient medium on its grolvth-promoting properties. Various chemical analyses have been performed in order to characterize the major fractions.
IN
MATERIALS
The following Dialyzed
nutrient
embryonic
phane tube against
METHODS
fractions
were prepared: per cent EE [9] was dialyzed in a cellothree changes of Tgrode’s solution, for a total of 48 hours at ,5 . ex:tracf
(DEE).---50
1 Aided by a grant from the National Experimenlol
AND
Ceil Zlesearch 9
Institutes
of Health,
I’.S. Public Health
Service.
LYucleic acids and nucleotides Perchloric mid-soluble fraction (PS).-To 50 per cent ZYF:E was added an equal volume of cold 0.6 h’ perchloric acid. The precipitated protein was centrifuged and extracted again with one volume of cold 0.2 X HClO,. The combined supernatant fluids were carefully neutralized to pH 7.0 by the addition of 5 S KOH at 0°C. The resulting precipitate of KClO, was removed as completely as possible by centrifuging in the cold. The supernatant liquid was then evaporated to dryness in oacuo. The dry residue was dissolved in the minimal volume of water, aud the undissolvrtl KClO, was again removed by centrifugation. The clear solution was finally rcconstituted with water to the original volume of the 50 per cent EE, and sterilized b? filtration through UF-Pyrex sintered glass filters into “Plasma-Vat” bottles. In certain experiments, isotopically-labeled PS and DEE, containing purine-Cl4 nilcleotides and nucleic acids, were prepared from radioactive embryos (fertile eggs injected with adenine-8-C14) [lo].1 Heat-stable supernatant (HS).--50 per cent EE was heated at 100°C for 5 minutes. The coagulum was centrifuged, and the clear supernatant solution was decanted and retained. Ethanol fractionation.--One volume of HS was mixed with two volumes of 9.5 per cent ethanol, and kept at -20°C for 4 hours. The resulting alcohol-insoluble and the supernatant alcoholprecipitate (AZ) was separated by centrifugation, soluble phase (AS) was decanted. Each fraction was freed of alcohol by lyophilization. Then AS was reconstituted with water to the original volume of HS, while ;2Z was similarly reconstituted with Tyrode’s solution. Tissue culture procedures.-Previously described methods [O, lo] were used for explantation and cultivation of 13-day old chick heart fibroblasts, and for determining the nucleic acid content and radioactivity of the cultures. Besides the standard EE medium, combinations of the nutrient fractions discussed in the preceding section, were used. Changes in the composition of the media at successive stages of cultivation were determined by chemical analysis (described in the next section), and by Cl* measurements in those cases in which labeled adenine, nucleotides or nucleic acids were employed [lo]. Analysis of nutrient fractions.-The orcinol [ll] and diphenylamine [l] reactions were used for the quantitative determination of RNA and DNA, respectively. Likewise non-polynucleotide ribose compounds were determined by the orcinol method.2 Total nucleoprotein content [9] and the sum of amino acids plus peptides [ 151 were also measured.
RESULTS
AND
DISCUSSION
In Table I the main difference obserretl between the original EE and the ctialyzed preparation (DEE) was in the concentration of ribose compounds. These fell from 16 to 1.7 mg per 100 ml of 30 per cent EE. The levels ot nucleoprotein, RSA, and DNA &cl not tlecrease marketil~. Most probahl>I Allocated by the U.S. Atomic Energy Commission, Oak Ridge, Tennessee. T Desoxyribose nucleotides could not be measured accurately because of their low roncentrations and the insufficient sensitivity of the diphenplamine reagent. E.rperimenlol
Cell Resenrch 9
K. H. Lu and T. Winnick
504
TABLE I Characterization
of EE fractions.
Concentration in mg per 100 ml of 50 per cent EE Fraction
Nucleoprotein
DN.4
1360 1260 0 145
5.5 4.2 0 1.5
~
RNA
I Original EE . . . . . . . Dialyzed (DEE) . . . . . Perchloric acid soluble (PS) Heated supernatant (HS) . .4lcohol-soluble (AS) . . . Alcohol-insoluble (AI) . . .
. . . . . .
. . . . . . . . . . . ,
210‘,i’j
1Ez;i-&“l?;I;e I
55.8 41.1 0 9.0
16 1.7 16 14
i.5
7.6
the ribose compounds lost during dialysis were chiefly nucleotides (in the light of data to be presented later). Further confirmation of the extensive removal of purine and pyrimidine compounds was the shift in the ultraviolet absorption maximum from 258 to 270 mp, upon the dialysis of EE (Fig. 1). It may be seen (Table I) that the perchloric acid completely removed nucleoprotein, RNA, and DNA, while heat treatment of EE removed most, but not all of these substances. Ribose nucleotides were retained in both PS and HS. The latter fraction seems most nearly equivalent to the diffusible components of EE, in that the sums of DEE plus HS values for nucleoprotein, DNA, RNA, and ribose compounds were approximately equal to the AS plus AI the corresponding values for the original EE. Similarly, values approximeted those for HS, as was to be expected. It was found that the nucleoprotein and RNA remaining after heat treatment were precipitated by the addition of ethanol, while the residual DNA and most of the ribose compounds remained in solution. Fig. 2 shows that fractions PS, HS, and AS had almost identical absorption maxima (255 mp), and that these were only slightly displaced from that of the original EE. On the other hand, AZ had its peak at 263 rnk, and its curve resembled more nearly that of DEE. As reported in an earlier paper [9], the DNA content of chick tibroblasts, cultivated in 25 per cent EE, increases rapidly, after the initial fall associated with explantation. In Fig. 3 it is of interest that the combination of DEE and HS gave the same increase in tissue DNA at 6 days, as that obtained with the complete EE. -4 lesser response was obtained when PS replaced Experimental
Cell Research 9
.505
Nucleic kids und nucleofides TABLE It I.tilization
of protein
l:or each time interval
and nucleic
acid constituents
of EE during
cultivation
of heart tissue.
the changes in the EE were calculated in terms of the original medium, since the later was reuewed every three days. Concentration
I Period of cultivation
Nucleo1 protein
in mg per 100 ml of 25 per cent EE
DNA
RN.4
2.7 2.2 0.5 1.8 0.9 1.8 0.9
27.2 23.2 4.0 22.0 5.2 21.6 5.ti
Non-polynuAmino acids cleotide ribosel’and peptides’:
I Control EE (3 days tissue) ......... O-3 Days .......... Decrease ........ 3-6 Days .......... Decrease ........ (i-9 Days .......... Decrease ........
without 623 606 17 602 21 596 27
r Soluble in cold perchloric acid. e mM per 100 ml, determined with
trichloracetic
7.25 6.10 1.15 6.20 1.05 6.05 1.20
0.70 0.65 0.05 0.63 0.07 0.625 0.073
acid filtrates.
HS. The former fraction, unlike HS, was completely devoid of polynucleotides. Also, traces of perchlorate in PS may have been inhibitory, AS plus DEE was still less satisfactory. It is apparent that neither AS nor DEE alone, nor DEE plus AI, were able to cause an increase in the DNA content of the cultures. Kutsky [8] reported that the nucleoprotein fraction of EE exerts a stimulating effect in the production of heart Iibroblasts. Likewise, Nishioka and Ibuka [la] concluded that highly-polymerized RNA promotes the growth of cultures. In the present study, it was found that both HS and AS contain relatively large amounts of ribose compounds and amino acids. Thus the inability of these fractions to support tissue growth must be due in large measure to deficiencies in protein, RNA, and (probably) DNA. This conclusion agrees with that of other workers [4, 71. The utilization of both nucleic acid and protein components of the nutrient medium by the heart cultures is demonstrated in Table II. If the decreases in RNA and DNA in each of the time intervals are subtracted from the corresponding nucleoprotein values, the quantities of protein utilized can be seen to range from 12.5 to 20.5 mg. These values may be compared with the uptakes of the amino acid-peptide fraction, which range from about 8 to 11 mg (taking one mM as 150 mg). The extensive contriExperimental
Cell Research 9
K. I{. Lu and T. lb’innick
506 14 12 E IOI IO8 x i 06 t o 04 02
001 220
230
240 250 260 270 280 MU WAVE LENGTH
290
300
220
230
240 250 260 270 280 MU WAVE LENGTH
290 303
=o.o* Tl,.,t Of CULTIVATION(DAYS)
TIME
OF CULTIVATION (DAYS)
Fig. 1 (left). Ultraviolet absorption spectra of original EE and EE after dialysis. Fig. 2 (right). Ultraviolet absorption spectra of fractions derived from EE. Fig. 3 (upper left). Changes in DNA content during cultivation of heart tissue in different media. Equal proportions of the fractions were used in the composite media. When HS, AS, or SI were used alone, each was first diluted with an equal volume of Tyrode’s solution. Fig. 4 (lower left). Relative rates of incorporation of adenine-8-P into mixed nucleic acids of cultures, with different media. The labeled compound was employed at a 0.1 mM concentration in each case. Fig. 5 (right). Chromatography of a perchloric acid extract of radioactive chick embryos (13 g). h 20 x 0.9 cm Dowex 1 column was used. Elutions were performed with formic acid and ammonium formate [6], and 5 ml fractions were collected. The open symbols are optical densities of the eluted fractions, while the solid circles are the corresponding radioactivities. Experimental
Cell Research 9
Mucleic acids and nucleotides TABLE
Incorporation
of Cl* of labeled
I:qu:ll proporiions
fractions
of the constituents
III into
nwlcic
IIEE + labeled I’S rine per ml)’ . 1’S + labeled DEE rine per ml)B.
acids of heart
were employed in the nutrient
j-h.11 incorporated (:omposition
,5Oi
cultures mrdhl.
per gm of isolated
material
of medium
(0.020 . . (0.0105 . .
PM Cl&-pu- ’ . . . . . . p.11 Cl”-pu. . . . . ,
, 0.45 5.255
1 Contained in adenine nucleotides. 2 Representing W-adeninc and C14-guanine
~ I I
in both
1.7T
~
1.00
1 .!)l
1.17
~
O.&l
1.31
DSX
and RS.1.
butions of both protein and non-protein components of EE to the synthesis of tissue protein is thus apparent. Table II indicates that RNA was utilized some 5 to 8 times as extensively as 1)X,4. This is in accord with the known predominance of RNA in animal tissues (including embryonic heart). It is also noteworthy that about as much non-polynucleotide ribose as RNA ribosc was taken from the medium (considering that RSA contains about 30 per cent of this pentose). Since the degree of incorporation of isotopic adenine into tissue nucleic acids appears to be closely correlated with the rate of growth of tibroblasts 191, it was of interest to employ this criterion in studying the various nutrient fractions. The results in Fig. 4 are strikingly like those in Fig. 3 (in which DSA was measured). A combination of DEE and HS was fully equivalent whilt (actually superior) to EE, with respect to the uptake of adenine-8-C’*; DEE plus PS, or plus AS, was somewhat less effective at 6 days. Low rates of Cl* incorporation (with virtually no growth) resulted with HS, AS, AZ, OI DEE alone, or with DEE plus AI. Another approach was the use of labeled nucleotides and nucleic acids in studying the utilization of nutrient fractions (Table III). In the upper part of this table, the value of 0.45 for nucleoprotein represents the uptake of 2.5 per cent of the total Cl* available in adenine nucleotides of the medium, during a G-day cultivation period. The corresponding nucleoprotein value, 0.20, in the lower half of the table indicates a utilization of 2.0 per cent 01 the Cl4 in the nucleic acid purines. This approximately equal contribution of Cl4 bv the two phases of the medium agrees with the finding (Table 11) Experimenlnl
Cell Rraetrrch 9
508
K. H. Lu and T. \t’innick
that the nucleic acids of EE were utilized as estensiwly as the rihose nucleotides, for tissue nucleic acid synthesis. The higher Cl4 content of RNA, as compared \vith LISA, was due to the much greater proportion of RNA in the tissue cultures. In point of fact, the concentration of Cl4 was considerably higher in DNA, as compared to RS.4, in both experiments. In examining the role of the nucleotide constituents of EE, it \vas 01 interest to subject these to chromatographic analysis. Recause of the presence of interfering salts in the standard EE preparation, a direct perchloric acid extract of whole chick embryos was employed1 (Fig. 5). The nucleotides of the embryos had been biologically labeled with adenine-8-C14 (lo), so that radioactivity as well as nucleotide concentration could be measured in each chromatographic fraction. The nucleotide composition of each peak was ascertained from its ultraviolet absorption spectrum, and from E2,Jl&,, [G]. Ten acid-soluble nucleotides were identified in chick embryonic tissue: cytidine monophosphate, diphosphopyridine nucleotide, adenosine monophosphate, triphosphopyridine nucleotide, uridine monophosphate, adenosine diphosphate, uridine diphosphate, cytidine triphosphate, adenosine triphosphate, and uridine triphosphate. Guanosine di- and triphosphates arc tentatively indicated in Fig. 5. Thymine compounds could not be detected. The substance designated ADP-S was an unidentified adenine compound. ATP was by far the most abundant nucleotide, and contained the most C14. Lesser amounts of Cl4 occurred in DPN, AMP, TPK, ADP, and ADP-S. Ninety-two per cent of the total Cl4 was recovered in these six peaks. The apparent absence of labeled guanine compounds contrasts with the Cl* distribution observed in the nucleic acids of EE, in \vhich guanine accounted for about half of the purine-Cl4 [lo]. Apart from their multiple roles in enzymic processes, the nucleotides are generally believed to represent intermediate stages in the formation of polynucleotides. Hence it seems very likely that the nucleotides in EE were utilized directly for tissue nucleic acid synthesis. Obviously the EE contains a great variety of substances, in addition to protein and nucleic acid constituents, so that the elucidation of the complete nutritional requirements of living cells in uitro represents a difficult task. However the growth-promoting properties of the PS, and particularly the HS preparation in the present paper, suggest that further fractionation studies may be helpful. 1 This method, rather than heat coagulation, of nucleic acids.
Experimental Cell Research 9
was used because it resulted in complete
removal
Nucleic acids and nucleotides SUMMARY
Fractionation of chic.1~ embryonic extract has demonstrated that both the difTusible substances and the high molecular constituents were required for the in vitro growth of heart fibroblasts. Growth was evaluated by increase in tissue DNA content, and by incorporation of Cl*-labeled adenine. It was found that either the perchloric acid-soluble, or the heat-stable phase of embryonic estract could substitute fairly well for the components removed by dialysis. Direct chemical analysis of the nutrient medium during cultivation revealed the extensive uptake by tissue cells of nucleotides and nucleic Also the utilization of Cl*-labeled acids, as well as protein components. nucleotides and nucleic acids was demonstrated. .\nalysis of nucleic acid-free chick embryonic extract by Dowes column chromatography revealed a variety of different nucleotides, of which ten were identified. These compounds appear to have a dual role: in enzymic systems, and as building blocks of polynucleotide molecules. REFERENCES 1. DXSCHE, Z., Mikrochemie 8, 4 (1930). 2. FISCHER, A., Biology of Tissue Cells. Hafner, New-York, 1946. 3. -Biochem. J. 43, 491 (1948). 4. FISCHER, A., ASTRUP, T., EHRENSVXRD, G., and OEHLESSCX~LHGER, \‘., Proc. Sot. Expff. Biol. Med. 67, 40 (1948). 5. HARRIS, M., Growth 17, 147 (1953). 6. HURLBERT, R. B., SHMITZ, H., BRUMM, A. F., and POTTER, V. R., J. Hio[. Chem. 209,23 (1954). 7. KATSUTA, H. and TAKAOKA, T., Jap. J. Expfl. Med. 22, 163 (1952). 8. KUTSKY, R. J., Proc. Sot. Exptl. Biol. Med. 83, 390 (1953). 9. Lu, K. H. and WINNICK, T., Expfl. Cell Research 7, 238 (1954). 10. -ibid. 6, 345 (1954). 11. MEJBAUM, W., Z. Physiol. Chem. 258, 117 (1939). 12. NISHIOKA, K. and IBUKA, K., Jap. Expfl. Med. 22, 163 (1952). 13. ROSENBERG, S., ZIKEER, E., and KIRK, P. L., J. Gen. Physiol. 37, 231 (1953). 14. SASFORD, K. K., WALTZ, H. I<., SHANNON, J. E., EVANS, V. J., and EARLE, W. R., Paper presented at the March 1952 meeting of the American Tissue Culture Association, Providence, Rhode Island. 15. STEIS, W. H. and MOORE, S., J. Biol. Chem. 176, 367 (1948). 16. V~ISNICK, R. E. and WINNICK, T., J. Nat!. Cancer Inst. 14, 519 (1953).
32 - 553706
Experimenful
Cell Research 9
502
Cell Research, 9, 502 ,509 (1055)
THE ROLES OF THE NUCLEIC ACIDS AND FREE NUCLEOTIDES IN CHICK EMBRYONIC EXTRACT ON THE GROWTH OF HEART FIBROBLASTl K. H. LU and T. WINNICK Department
of Biochemistry and the Radiation Research Laboratory, University of Iowa, Iowa City, lowa, U.S.A. Received February
College of Medicine.
26, 1955
1946 Fischer [tl] distinguished betlveen the high molecular \veight and the diffusible constituents of embryonic extract (EE). He found that dialyzed extracts failed to support growth of fibroblasts, but that this efrect of dialysis was partially reversed by the addition of a mixture of amino acids [3]. Subsequently, three groups of investigators [a, 13, 141 hart studied the role of ultrafiltrates, or of alcoholic extracts of EE, on proliferation of fibroblasts in vitro. These solutions were shown to contain a great variety of comamino acids, purines, pyrimidines, sugars, and coenpounds, including zymes. There appears to be general agreement that these lo\\ molecular compounds are essential for growth. However, the importance of the proteins and nucleic acids of El? has also been established. Fischer [4] described a nucleoprotein fraction, termed \vhich promoted growth of cultures; later work by Katsuta “embryonin”, and Takaoka [7] and Kutsky [tl] has emphasized the essentiality of nucleic acids in the nutrient media. Recent studies conducted in our laboratory have been concerned \vith the correlation of growth, as measured by increase in desoxpribose nucleic acid content, with the incorporation of labeled adeninc and thymidine into nucleic acids in roller tube tissue cultures [9]. The present paper is a continuation of these experiments, but with emphasis on the effect of fractionation of the EE nutrient medium on its grolvth-promoting properties. Various chemical analyses have been performed in order to characterize the major fractions.
IN
MATERIALS
The following Dialyzed
nutrient
embryonic
phane tube against
METHODS
fractions
were prepared: per cent EE [9] was dialyzed in a cellothree changes of Tgrode’s solution, for a total of 48 hours at ,5 . ex:tracf
(DEE).---50
1 Aided by a grant from the National Experimenlol
AND
Ceil Zlesearch 9
Institutes
of Health,
I’.S. Public Health
Service.
LYucleic acids and nucleotides Perchloric mid-soluble fraction (PS).-To 50 per cent ZYF:E was added an equal volume of cold 0.6 h’ perchloric acid. The precipitated protein was centrifuged and extracted again with one volume of cold 0.2 X HClO,. The combined supernatant fluids were carefully neutralized to pH 7.0 by the addition of 5 S KOH at 0°C. The resulting precipitate of KClO, was removed as completely as possible by centrifuging in the cold. The supernatant liquid was then evaporated to dryness in oacuo. The dry residue was dissolved in the minimal volume of water, aud the undissolvrtl KClO, was again removed by centrifugation. The clear solution was finally rcconstituted with water to the original volume of the 50 per cent EE, and sterilized b? filtration through UF-Pyrex sintered glass filters into “Plasma-Vat” bottles. In certain experiments, isotopically-labeled PS and DEE, containing purine-Cl4 nilcleotides and nucleic acids, were prepared from radioactive embryos (fertile eggs injected with adenine-8-C14) [lo].1 Heat-stable supernatant (HS).--50 per cent EE was heated at 100°C for 5 minutes. The coagulum was centrifuged, and the clear supernatant solution was decanted and retained. Ethanol fractionation.--One volume of HS was mixed with two volumes of 9.5 per cent ethanol, and kept at -20°C for 4 hours. The resulting alcohol-insoluble and the supernatant alcoholprecipitate (AZ) was separated by centrifugation, soluble phase (AS) was decanted. Each fraction was freed of alcohol by lyophilization. Then AS was reconstituted with water to the original volume of HS, while ;2Z was similarly reconstituted with Tyrode’s solution. Tissue culture procedures.-Previously described methods [O, lo] were used for explantation and cultivation of 13-day old chick heart fibroblasts, and for determining the nucleic acid content and radioactivity of the cultures. Besides the standard EE medium, combinations of the nutrient fractions discussed in the preceding section, were used. Changes in the composition of the media at successive stages of cultivation were determined by chemical analysis (described in the next section), and by Cl* measurements in those cases in which labeled adenine, nucleotides or nucleic acids were employed [lo]. Analysis of nutrient fractions.-The orcinol [ll] and diphenylamine [l] reactions were used for the quantitative determination of RNA and DNA, respectively. Likewise non-polynucleotide ribose compounds were determined by the orcinol method.2 Total nucleoprotein content [9] and the sum of amino acids plus peptides [ 151 were also measured.
RESULTS
AND
DISCUSSION
In Table I the main difference obserretl between the original EE and the ctialyzed preparation (DEE) was in the concentration of ribose compounds. These fell from 16 to 1.7 mg per 100 ml of 30 per cent EE. The levels ot nucleoprotein, RSA, and DNA &cl not tlecrease marketil~. Most probahl>I Allocated by the U.S. Atomic Energy Commission, Oak Ridge, Tennessee. T Desoxyribose nucleotides could not be measured accurately because of their low roncentrations and the insufficient sensitivity of the diphenplamine reagent. E.rperimenlol
Cell Resenrch 9
K. H. Lu and T. Winnick
504
TABLE I Characterization
of EE fractions.
Concentration in mg per 100 ml of 50 per cent EE Fraction
Nucleoprotein
DN.4
1360 1260 0 145
5.5 4.2 0 1.5
~
RNA
I Original EE . . . . . . . Dialyzed (DEE) . . . . . Perchloric acid soluble (PS) Heated supernatant (HS) . .4lcohol-soluble (AS) . . . Alcohol-insoluble (AI) . . .
. . . . . .
. . . . . . . . . . . ,
210‘,i’j
1Ez;i-&“l?;I;e I
55.8 41.1 0 9.0
16 1.7 16 14
i.5
7.6
the ribose compounds lost during dialysis were chiefly nucleotides (in the light of data to be presented later). Further confirmation of the extensive removal of purine and pyrimidine compounds was the shift in the ultraviolet absorption maximum from 258 to 270 mp, upon the dialysis of EE (Fig. 1). It may be seen (Table I) that the perchloric acid completely removed nucleoprotein, RNA, and DNA, while heat treatment of EE removed most, but not all of these substances. Ribose nucleotides were retained in both PS and HS. The latter fraction seems most nearly equivalent to the diffusible components of EE, in that the sums of DEE plus HS values for nucleoprotein, DNA, RNA, and ribose compounds were approximately equal to the AS plus AI the corresponding values for the original EE. Similarly, values approximeted those for HS, as was to be expected. It was found that the nucleoprotein and RNA remaining after heat treatment were precipitated by the addition of ethanol, while the residual DNA and most of the ribose compounds remained in solution. Fig. 2 shows that fractions PS, HS, and AS had almost identical absorption maxima (255 mp), and that these were only slightly displaced from that of the original EE. On the other hand, AZ had its peak at 263 rnk, and its curve resembled more nearly that of DEE. As reported in an earlier paper [9], the DNA content of chick tibroblasts, cultivated in 25 per cent EE, increases rapidly, after the initial fall associated with explantation. In Fig. 3 it is of interest that the combination of DEE and HS gave the same increase in tissue DNA at 6 days, as that obtained with the complete EE. -4 lesser response was obtained when PS replaced Experimental
Cell Research 9
.505
Nucleic kids und nucleofides TABLE It I.tilization
of protein
l:or each time interval
and nucleic
acid constituents
of EE during
cultivation
of heart tissue.
the changes in the EE were calculated in terms of the original medium, since the later was reuewed every three days. Concentration
I Period of cultivation
Nucleo1 protein
in mg per 100 ml of 25 per cent EE
DNA
RN.4
2.7 2.2 0.5 1.8 0.9 1.8 0.9
27.2 23.2 4.0 22.0 5.2 21.6 5.ti
Non-polynuAmino acids cleotide ribosel’and peptides’:
I Control EE (3 days tissue) ......... O-3 Days .......... Decrease ........ 3-6 Days .......... Decrease ........ (i-9 Days .......... Decrease ........
without 623 606 17 602 21 596 27
r Soluble in cold perchloric acid. e mM per 100 ml, determined with
trichloracetic
7.25 6.10 1.15 6.20 1.05 6.05 1.20
0.70 0.65 0.05 0.63 0.07 0.625 0.073
acid filtrates.
HS. The former fraction, unlike HS, was completely devoid of polynucleotides. Also, traces of perchlorate in PS may have been inhibitory, AS plus DEE was still less satisfactory. It is apparent that neither AS nor DEE alone, nor DEE plus AI, were able to cause an increase in the DNA content of the cultures. Kutsky [8] reported that the nucleoprotein fraction of EE exerts a stimulating effect in the production of heart Iibroblasts. Likewise, Nishioka and Ibuka [la] concluded that highly-polymerized RNA promotes the growth of cultures. In the present study, it was found that both HS and AS contain relatively large amounts of ribose compounds and amino acids. Thus the inability of these fractions to support tissue growth must be due in large measure to deficiencies in protein, RNA, and (probably) DNA. This conclusion agrees with that of other workers [4, 71. The utilization of both nucleic acid and protein components of the nutrient medium by the heart cultures is demonstrated in Table II. If the decreases in RNA and DNA in each of the time intervals are subtracted from the corresponding nucleoprotein values, the quantities of protein utilized can be seen to range from 12.5 to 20.5 mg. These values may be compared with the uptakes of the amino acid-peptide fraction, which range from about 8 to 11 mg (taking one mM as 150 mg). The extensive contriExperimental
Cell Research 9
K. I{. Lu and T. lb’innick
506 14 12 E IOI IO8 x i 06 t o 04 02
001 220
230
240 250 260 270 280 MU WAVE LENGTH
290
300
220
230
240 250 260 270 280 MU WAVE LENGTH
290 303
=o.o* Tl,.,t Of CULTIVATION(DAYS)
TIME
OF CULTIVATION (DAYS)
Fig. 1 (left). Ultraviolet absorption spectra of original EE and EE after dialysis. Fig. 2 (right). Ultraviolet absorption spectra of fractions derived from EE. Fig. 3 (upper left). Changes in DNA content during cultivation of heart tissue in different media. Equal proportions of the fractions were used in the composite media. When HS, AS, or SI were used alone, each was first diluted with an equal volume of Tyrode’s solution. Fig. 4 (lower left). Relative rates of incorporation of adenine-8-P into mixed nucleic acids of cultures, with different media. The labeled compound was employed at a 0.1 mM concentration in each case. Fig. 5 (right). Chromatography of a perchloric acid extract of radioactive chick embryos (13 g). h 20 x 0.9 cm Dowex 1 column was used. Elutions were performed with formic acid and ammonium formate [6], and 5 ml fractions were collected. The open symbols are optical densities of the eluted fractions, while the solid circles are the corresponding radioactivities. Experimental
Cell Research 9
Mucleic acids and nucleotides TABLE
Incorporation
of Cl* of labeled
I:qu:ll proporiions
fractions
of the constituents
III into
nwlcic
IIEE + labeled I’S rine per ml)’ . 1’S + labeled DEE rine per ml)B.
acids of heart
were employed in the nutrient
j-h.11 incorporated (:omposition
,5Oi
cultures mrdhl.
per gm of isolated
material
of medium
(0.020 . . (0.0105 . .
PM Cl&-pu- ’ . . . . . . p.11 Cl”-pu. . . . . ,
, 0.45 5.255
1 Contained in adenine nucleotides. 2 Representing W-adeninc and C14-guanine
~ I I
in both
1.7T
~
1.00
1 .!)l
1.17
~
O.&l
1.31
DSX
and RS.1.
butions of both protein and non-protein components of EE to the synthesis of tissue protein is thus apparent. Table II indicates that RNA was utilized some 5 to 8 times as extensively as 1)X,4. This is in accord with the known predominance of RNA in animal tissues (including embryonic heart). It is also noteworthy that about as much non-polynucleotide ribose as RNA ribosc was taken from the medium (considering that RSA contains about 30 per cent of this pentose). Since the degree of incorporation of isotopic adenine into tissue nucleic acids appears to be closely correlated with the rate of growth of tibroblasts 191, it was of interest to employ this criterion in studying the various nutrient fractions. The results in Fig. 4 are strikingly like those in Fig. 3 (in which DSA was measured). A combination of DEE and HS was fully equivalent whilt (actually superior) to EE, with respect to the uptake of adenine-8-C’*; DEE plus PS, or plus AS, was somewhat less effective at 6 days. Low rates of Cl* incorporation (with virtually no growth) resulted with HS, AS, AZ, OI DEE alone, or with DEE plus AI. Another approach was the use of labeled nucleotides and nucleic acids in studying the utilization of nutrient fractions (Table III). In the upper part of this table, the value of 0.45 for nucleoprotein represents the uptake of 2.5 per cent of the total Cl* available in adenine nucleotides of the medium, during a G-day cultivation period. The corresponding nucleoprotein value, 0.20, in the lower half of the table indicates a utilization of 2.0 per cent 01 the Cl4 in the nucleic acid purines. This approximately equal contribution of Cl4 bv the two phases of the medium agrees with the finding (Table 11) Experimenlnl
Cell Rraetrrch 9
508
K. H. Lu and T. \t’innick
that the nucleic acids of EE were utilized as estensiwly as the rihose nucleotides, for tissue nucleic acid synthesis. The higher Cl4 content of RNA, as compared \vith LISA, was due to the much greater proportion of RNA in the tissue cultures. In point of fact, the concentration of Cl4 was considerably higher in DNA, as compared to RS.4, in both experiments. In examining the role of the nucleotide constituents of EE, it \vas 01 interest to subject these to chromatographic analysis. Recause of the presence of interfering salts in the standard EE preparation, a direct perchloric acid extract of whole chick embryos was employed1 (Fig. 5). The nucleotides of the embryos had been biologically labeled with adenine-8-C14 (lo), so that radioactivity as well as nucleotide concentration could be measured in each chromatographic fraction. The nucleotide composition of each peak was ascertained from its ultraviolet absorption spectrum, and from E2,Jl&,, [G]. Ten acid-soluble nucleotides were identified in chick embryonic tissue: cytidine monophosphate, diphosphopyridine nucleotide, adenosine monophosphate, triphosphopyridine nucleotide, uridine monophosphate, adenosine diphosphate, uridine diphosphate, cytidine triphosphate, adenosine triphosphate, and uridine triphosphate. Guanosine di- and triphosphates arc tentatively indicated in Fig. 5. Thymine compounds could not be detected. The substance designated ADP-S was an unidentified adenine compound. ATP was by far the most abundant nucleotide, and contained the most C14. Lesser amounts of Cl4 occurred in DPN, AMP, TPK, ADP, and ADP-S. Ninety-two per cent of the total Cl4 was recovered in these six peaks. The apparent absence of labeled guanine compounds contrasts with the Cl* distribution observed in the nucleic acids of EE, in \vhich guanine accounted for about half of the purine-Cl4 [lo]. Apart from their multiple roles in enzymic processes, the nucleotides are generally believed to represent intermediate stages in the formation of polynucleotides. Hence it seems very likely that the nucleotides in EE were utilized directly for tissue nucleic acid synthesis. Obviously the EE contains a great variety of substances, in addition to protein and nucleic acid constituents, so that the elucidation of the complete nutritional requirements of living cells in uitro represents a difficult task. However the growth-promoting properties of the PS, and particularly the HS preparation in the present paper, suggest that further fractionation studies may be helpful. 1 This method, rather than heat coagulation, of nucleic acids.
Experimental Cell Research 9
was used because it resulted in complete
removal
Nucleic acids and nucleotides SUMMARY
Fractionation of chic.1~ embryonic extract has demonstrated that both the difTusible substances and the high molecular constituents were required for the in vitro growth of heart fibroblasts. Growth was evaluated by increase in tissue DNA content, and by incorporation of Cl*-labeled adenine. It was found that either the perchloric acid-soluble, or the heat-stable phase of embryonic estract could substitute fairly well for the components removed by dialysis. Direct chemical analysis of the nutrient medium during cultivation revealed the extensive uptake by tissue cells of nucleotides and nucleic Also the utilization of Cl*-labeled acids, as well as protein components. nucleotides and nucleic acids was demonstrated. .\nalysis of nucleic acid-free chick embryonic extract by Dowes column chromatography revealed a variety of different nucleotides, of which ten were identified. These compounds appear to have a dual role: in enzymic systems, and as building blocks of polynucleotide molecules. REFERENCES 1. DXSCHE, Z., Mikrochemie 8, 4 (1930). 2. FISCHER, A., Biology of Tissue Cells. Hafner, New-York, 1946. 3. -Biochem. J. 43, 491 (1948). 4. FISCHER, A., ASTRUP, T., EHRENSVXRD, G., and OEHLESSCX~LHGER, \‘., Proc. Sot. Expff. Biol. Med. 67, 40 (1948). 5. HARRIS, M., Growth 17, 147 (1953). 6. HURLBERT, R. B., SHMITZ, H., BRUMM, A. F., and POTTER, V. R., J. Hio[. Chem. 209,23 (1954). 7. KATSUTA, H. and TAKAOKA, T., Jap. J. Expfl. Med. 22, 163 (1952). 8. KUTSKY, R. J., Proc. Sot. Exptl. Biol. Med. 83, 390 (1953). 9. Lu, K. H. and WINNICK, T., Expfl. Cell Research 7, 238 (1954). 10. -ibid. 6, 345 (1954). 11. MEJBAUM, W., Z. Physiol. Chem. 258, 117 (1939). 12. NISHIOKA, K. and IBUKA, K., Jap. Expfl. Med. 22, 163 (1952). 13. ROSENBERG, S., ZIKEER, E., and KIRK, P. L., J. Gen. Physiol. 37, 231 (1953). 14. SASFORD, K. K., WALTZ, H. I<., SHANNON, J. E., EVANS, V. J., and EARLE, W. R., Paper presented at the March 1952 meeting of the American Tissue Culture Association, Providence, Rhode Island. 15. STEIS, W. H. and MOORE, S., J. Biol. Chem. 176, 367 (1948). 16. V~ISNICK, R. E. and WINNICK, T., J. Nat!. Cancer Inst. 14, 519 (1953).
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Cell Research 9