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Amino acid requirements of normal and malignant human cells in tissue culture
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, 432-M
(1957)
Amino Acid Requirements of Normal and Malignant Human Cells in Tissue Culture Harry From
Eagle, Vance I. Oyama and Mina
the Section on Experimental Therapeutics, National Institute of Allergy and Infectious of Health,’ Bethesda,
Laboraiory
Levy
of Infectious Diseases, Institutes
Diseases, National Maryland
ReceivedAugust 10, 1956
It has been shown (1-3) that two mamalian cells in tissue culture, a human carcinoma of the cervix (st’rain HeLa) and a mouse fibroblast (st’rain L), required 13 amino acids for survival and growth. In the absence of any one of these, cytopathogenic changes developed which eventuated in the death of the cell. Since this cultured mouse fibroblast hasbeen found to produce tumors on injection intocortisone-treated mice (4), the possibility suggested itself that the anomalous requirement for 13 amino acids, as contrasted with the 8 and 9 required for nitrogen balance in adult’ man and rat, respectively (5)) might be peculiar to these two cell strains, and that other lines would show amino acid requirements more closely approximating those of the intact animal. Initial attempts to determine the specific amino acid requirements of other normal and malignant human cell lines were unsuccessful. Disappoint,ingly, t,he defined medium which, supplemented with serum protein, had supported t’he growth of the HeLa cell and the mousefibroblast, failed to permit the survival of any of a large number of cultured cell lines. This eventually proved to be due to t’he fact t’hat all of 20 human cell lines tested had a rigorous requirement for myo-(meso-)inositol (6). With inositol added, t’he medium now permitted the sustained propagation of every human tissue culture cell line examined; and it became possible to examine, both qualitatively and quant’itatively, the amino acid requirements of a variety of normal and malignant human cells. The results of these st,udiesare here reported. 1 Public
Health
Service,
U. S. Department
432
of Health,
Education,
and
Welfare.
;IMISO
ACID
REQCIREME?;TS
OF
CELLS
433
METHODS The methods used in growing stock cultures of the cells in l-l. Blake bottles, and of preparing replicate cultures in small T-15 flasks for individual experiments, have been described (l-3, 7). In previous studies the amount of cell growth had been determined by actual enumeration. In the present experiments, the protein content of the individual cultures was measured instead, using the Folin-Ciornltenu reagent in a modified Lowry procedure (8).
The composition of the basal medium, embodying only those components (amino acids, vitamins, salts, glucose, serum protein) demonst rahly essential for the growth and survival of the L fibroblast and the HeLa cell, is given in Table II of the preceding paper (66). The concentrations of the individual amino acids used in the present experiments were an arbitrary compromise between i he concentrations used for the cultivation of these two cell lines.
Cell Lines Five human cell lines were examined with respect to their amino acid requirements, in addition to the HeLa cell previously studied. Three lines derived from normal tissue: an embryonic intestinal epithelial cell cultured by 6. Henle and F. l>einhardt ,* and adult liver and conjunrtival cells cultured by Chang (9). The other two cell lines were cultures of human cancer: an epidermoid carcinom:l of the nasopharyns (strain KB) cultured directl>from the tumor into the medium here used (10). and a culture of the huffy coat from a case of human monocytic leltkemia, Osgood’s strain J-111 (I 1). EXPERIMENTAL
The I’niform Requirements for 13 Amino ;Icids by Humwn Culture, Whether Derived from Normal or lllaliynant
(‘ells in Tissue Tissue
,111five cell lines here studied xere found to require at least, 12 of the 13 amino acids previously shown to be essent,ial for the survival and growt’h of the HeLa cell and the mouse fibrohlast. In t’he case of tryptophan, with some of the cell lines there n-as a question as to whether it, was essential for survival, or merely growth-stimulat,ory (cf. below). With t’he other 12 amino acids, however, the omission of either arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methioninc, phenylalanine, threonine, tyrosine, or saline from the medium resulted in the death of the cell; and the early cytopathogenic effects produced b) such specific amino acid deficiencies are illustrated for a number of ~11 lines in Fig. 1, sew. 1, 1 and '7. 2 Personal
communication.
FIG.
1. The
7-day lines
Sec. Leucine Sec. 6. I,ysine Sec. 9n.U. 0. 3.
growth to
response individual
l-3:
01 reyr.esentat.ive amino acids.
Intestinal cell urld lcucine: 1. X0 lcurine. at 0.1 mM. 4-6: Conjunctival cell and lysine; 4. l;o lyaine. at 1 mM. 7-9: Liver cell and mcthionine: 7. No methionine. Methionirle nt 0.3 n&f. 331
human 2. Tencine 5.
Lyaim 8. Methionine
cell at
0.01
mM.
at
0.01
?m%f. at 0.03
AMINO
Illustrative
ACID
REQUIREMENTS
OF
435
CELLS
TABLE I Ezperimenk Showing the Growth Response of Various Human Cell Lines to Individual Essential Amino Acids
-
Concentration Cell line
Amino acid tested
of amino
acid in medium,
mill
CO?C?. COZKZL
3
j
1
( 0.3
( 0.1
Cellular
( 0.03 IO.01 jO.W3/O.Olll/
growth
0
permitting maximum growth
in s-7 days
pergttlY=
maxims growth (EDso)
--
ateatiie Znjunctiva WJ dver -nib a Culture b Culture
Argbliie Vdflle Lysine Metbionine TryPtoPlw
6.2 <0.5
24.0 10.3 7.9 1.7
24.8 9.4 7.0 10.2 4.4
16.7 10.2 6.1 10.8 9.2
8.2 6.8 2.65 8.9 7.7
8.1 3.9 1.1 6.2 6.9
1.9 1.9 0.6 2.2 6.3
1.3 3.4
1.3 0.9 0.5 0.8 1.0
mM
mhf
0.1-0.3 0.1 0.1 0.02 0.01-0.03
0.08-0.06 0.08 0.04 0.007 0.002
from epidermoid carcinoma of nanopharynr (10). from buffy coat of cane of mono&c leukemia (11).
As previously noted with the HeLa and L cell, with most of the amino acids the cytopathic effect of a single amino acid deficiency became evident within a few days, and the cells died off progressively thereafter (cf. Table I). With a few of the amino acids, however, there was initial growth, amounting to as much as a 2-3-fold increase over a period of 6-13 days; and only then did the cells begin to degenerate in consequence of the amino acid deficiency. In such casesthe death of the cells was greatly accelerated if the culture was subdivided after l-2 weeks’ incubation in the amino acid-deficient medium. In the case of tryptophan, all the cell lines here tested proved capable of initial slow growth in a tryptophan-deficient medium; but unlike the other amino acids, this slow growth usually continued in subculture for at least 34 passages.The determination of whether tryptophan is in fact essential for growth, or merely growth-stimulatory, is complicated by the extremely small amounts required even for maximum growth. These could easily be present as trace contaminants in someof the other amino acids, or they could be obtained by the limited utilization of the serum protein in the medium. Experiments on this point are now in progress. Quantitative Aspects of Amino Acid Requirement Illustrative experiments for various cell lines, showing the amount of growth as the concentration of a single essential amino acid in the medium was varied from 0 to 3 mM, are shown in Table I, and in
436
H. EAGLE,
V. I. OYAMA AND M. LEVY
Figs. 1 and 2. The concentrations of each of the 13 amino acids necessary to produce 50% of the maximal growth (EDsO) are listed for each cell line in Table II; and the concentrations permitting maximal growth are similarly listed in Table III. Previous results with the HeLa and L strains have been included for comparison. The range of variation in these values for the cell lines deriving from normal and from malignant tissues is shown in the last two columns of the tables. Within each group, there was usually a two- to threefold difference in the effective concentrations; but more significant is the fact that there was no regular difference in the amino acid requirements of the two types of cell. The EDso levels of cystine, glutamine, isoleucine, lysine, phenylalanine, and valine were higher for the malignant cells than for those deriving from normal tissue. Similar differences were, however, not observed with respect to the concentrations required for optimal growth. The Amino Acid Requirement of Cells in Tissue Culture as a Function of Population Density and Frequency of Feeding
In the preceding experiments the concentrations of the individual amino acids required for growth had been determined in cultures inoculated with relatively small numbers of cells. The experimental medium
r
1
0
aco1ooa3ca
CONCENTRATloN
003 Cf
AMINO
al ACID
03
I rnM IN MEMUM
FIO. 2. Illustrating the growth response of human cell lines to essential amino acids in tissue culture.
AMINO
ACID
REQUIREMENTS
OF
CELLS
437
had been added 24 hr. after planting, and had been replaced with fresh medium after 2,4,5,6, and 7 days. Experiments to determine the degree to which the amino acid requirement was affected by the number of cells and the frequency of feeding are summarized in Table IV. As there shown, the concentration of valine required for optimum growth of the KB cell was affected to only a minor degree by the density of the cell population and the frequency of feeding. However, these factors markedly affected the concentrations required to effect 50 % of optimum growth. With a relatively small inoculum (e.g. 49 pg. of protein per flask), and frequent (e.g., 12 hourly) changes of medium, 0.007 mM valine permitted 50% of the optimum growth; while with a fourfold larger inoculum, and infrequent (48 hourly) feedings, the EDso was 0.05 m&f. Quantitatively similar results were obtained in an experiment with threonine and the culture strain deriving from human liver. To avoid similar variation in the experiments of Table II, the inocula of all the cell lines were kept comparably low, and all the cultures were fed on the same schedule. Glutamine Requirement Under the conditions of the experiments so far described, the cell was under the necessity of making the nutritionally nonessential amino acids, purines, and pyrimidines from the amino acids and glucose of the medium, In consequence, relatively large amounts of glutamine (0.5-2 mM) were required for optimum growth, several times greater than the amounts required of any of the other amino acids. The EDso concentrations were similarly high, ranging from 0.15 to 0.3 mM. In the case of the HeLa cell and mouse fibroblast, it had previously been shown (3) that the addition of the non-essential amino acids, which relieved the cell of the necessity of making glutamic acid, proline, and aspartic acid, did not eliminate the need for this amino acid, but did have a glutamine-sparing effect. An additional minor glutamine-sparing effect was obtained on the addition to the medium of purines, pyrimidines, and NH4+. With three of the cell lines here studied (liver, intestine, and conjunctiva), the addition of the nutritionally non-essential amino acids, purines, pyrimidines, and NH*+ similarly reduced the glutamine requirement significantly (cf. Table V and Fig. 3). However, glutamine remained an essential component, and in its absence the cells eventually died. With all five human lines, normal or malignant, and again resembling the HeLa cell, glutamic acid at extremely high concentrations (lo-20
0.011
0.012
Iaoleucine
0.007
0.003 0.0023
0.904 0.003
Liver
0.25,0.25
)
Histidine
1 Intestine
0.2,0.3
acid tested EDro
0.01 0.01
0.005 0.003 0.003
0.2,0.150.6,
) cO~~~of amino
~.&a
0.3, 0.6, 0.6,0.7,0.
level
0.044
0.0120 0.01
)
0.018
0.04 0.02
0.009
0.6
0.04 0.03
0.0015
0.27
0.005 0.004
J-111
0.0025
T
/
0.03
n&f
KB
0.042 0.016
acid,
/
Cell line tested /
Mouse
0.025
0.004
0.12, 0.2, 0.14, 0.2,0.12,0.21
0.094
fibroblast
3
.
_-
c==z
-
Range
0 .007-0.012
0.018-0.04
0 .0023-0.0050.00154.009
0.27-0.7
0 .0008-0.0020.004-0.0064
0 .x-O.3
in
0.0164.05
of variation
Tissue Culture Lines
0 .1-0.2
P
Growth of 6 Human
0.0125
TABLE II of 18 Essential Amino Acids Permitting 60 Per Cent of Maximal
Glutamine
hino
I
The Concentrations
0.015 0.011
0.012 0.008
0.01 0.006
to.022 0.014
0.008 0.007
0.022 0.012
Lysine
Methionine
Phenylalanine
Threonine
Tyrosine
Valine
0.016
0.006
0.014 0.013
0.0048 0.004 0.0036
0.007 0.006
0.016 0.008 0.008
0.017 0.01 0.008
0.03
0.01 0.008
0.017 0.012
0.005
0.008
0.016 0.01
0.03
0.015
0.025
0.02
0.015 0.014
0.048 0.025
0.016 0.015
0.05
0.014 0.007
0.06
0.03
0.005
0.04
0.025
o Cells deriving from normal tissues (intestine, liver, conjunctiva, b Cells deriving from malignant tissues (KB, HeLa, J-111).
0.025
Leucine
fibroblast).
0.05
0.01 0.01
0.042 0.018 0.013
0.024
0.003
0.04
0.015
0.025
0.008
0.017
0.007
0.01
0.018
0.02
0.012-0.03
0.006-0.01
0.012-0.022
0.0036-0.01
0.008-0.016
0.0084.025
0.03-0.05
0.007-0.015
0.013-0.06
0.02-0.03
0.0250.04
0.015-0.025
0.1 0.03-0.1
0.1
Leucine
0.034.1
0.1
0.03 0.03
0.03 0.1
Isoleucine
0.03 0.03-0.1 0.03 I
I
0.03-0.1 0.1
0.1 0.03
0.03 0.03
0.01 0.01
0.5 li
P I
0.01 0.01
0.03 0.03
Lysine
Optimal
Amino
0.1 0.1 0.1 0.1 I I 0.0050.010.003-0.0: 0.01 0.01 0.01
Histidine
0.1 0.1-0.3 0.01 0.01 0.01-0.03
0.75 0.75
tested
of 12 Essential
1.0 l-2
acid
= I
Concentrations
Glutamine
Cystine
Arginine
Amino
The
TABLE
Cell
1
-
-
0.1
0.1
0.1 0.1
0.03 0.03
-
I.
0.1
0.1-0.3
0.1 0.1-0.3
0.03 0.03
l-2
1 1 2
mY
0.03 0.01-0.03
acid,
Maximal
0.1
of amino
- -
tested
III
0.03
0.1 0.1
line
Permitting
concentration
Acids
0.1
0.03
0.1-0.3 0.1
0.01
0.5
0.01 0.01
0.075i 0.1
Growth
0.05
0.05
0.1-0.2
0.03-0.1
0.03-0.1
0.03-0.1
0.01-0.03
0.01
Range
Culture
0.5-2
Tissue
0.2 0.2-0.5
of 6 Human in
Malignant human cells”
of variation
Lines
0.03 0.03
0.03-0.1 0.03
Tyrosine
Valine
from from
0.1 0.1
Threonine
deriving deriving
0.03 0.03
Phenylalanine
a Cells b Cells
0.03 0.03
Methionine
normal malignant
0.1 0.1
0.034.1 0.03
0.1 0.03
0.03
0.03
tissues (intestine, tissues (KB,
0.03-0.1
0.03-0.1 0.034.1
0.01-0.03 0.03 0.01-0.03
0.03 0.03
0.1
0.014.03 0.03
0.14.3
0.1-0.3
0.03
-~-- __-
liver, conjunctiva, HeLa, J-111).
0.1
0.03
0.1 0.1
0.03-0.1 0.1
0.03 0.03
-__
-
fibroblast).
_ .~
0.05
0.02-0.05
0.05
0.02
0.02-0.05
--
__ ~..-
0.03-0.1
0.034.1
0.03-0.1
0.01-0.03
IO.03
0.1
0.014.03
0.03-0.3
0.03-0.3
0.01-0.03
442
H.
EAGLE,
V.
I.
OYAMA
TABLE
AND
M.
LEVY
IV
The Growth Response of the KB Cell to r.-Valine as a Function of the Size of the Inoculum and the Frequency of Feeding Concentration
Growth
I
of valine
response
I
in medium,
in 6 days, referred
I
I
L5.318.515.515.613.911.0 19.619.317.912.1 11.010.6 9.5 5.3 14.715.213.1 9.1 14.312.714.710.9 9.4 8.9 8.3 4.7
I 7.4 3.1 6.7 5.8 2.7
to ino
- -
I 5.0 1.7 4.2 3.5 1.7
5.8 1.9 0.9 2.6 2.1 0.9
-
mN) could substitute for glutamine. the equilibrium in the reaction glutamate
+ NH*
+ adenosine
mnrole
-‘urn
3.5 1.F ) .B 1.4 1.3 0.5 - _-
I
-
as 1
.__ 2.6 0.9 0.4 1.1 0.7 0.4 .-
__ndd 1.10.0068 0.50.03 0.30.05 0.70.024 0.40.024 0.30.042
nrhf 0.05 0.1 0.1 0.1 0.1 0.1
This probably reflects the fact that
triphosphate adenosine
% glutamine diphosphate
+ + phosphate
is thereby shifted toward the right. It is of interest that the addition of those nutritionally non-essential factors which had a sparing effect on the glutamine requirement did not similarly reduce the effective concentration of glutamic acid. This suggests that the high concentrations of glutamic acid are not quantitatively related to the amount of glutamine required. The mouse fibroblast is the only cell so far examined wholly unable to utilize glutamic acid as a substitute for glutamine, no matter what the concentration added to the medium (3). DISCUSSION
One of the interesting aspects of the data here reported is the qualitative and quantitative similarity in the amino acid requirements of a wide variety of human cells in tissue culture, deriving from both normal and malignant tissues, and including both embryonic and adult cell lines (Tables II and III). If the embryonic intestine, adult liver, and adult conjunctival lines here studied are still “normal” cells (and there appears to be no presently available definitive criterion short of test in man), one must then conclude that malignant cells do not regularly differ from normal cells with respect to the exogenous amino acids needed for sur-
AMINO
ACID
REQUIREMENTS
OF
443
CELLS
TABLE V The Glutamine Requirement of Three Normal Human Cell Lines in Tissue Culture in Relation to the Composition of the Medium
Cell
strair
&al
mediuma
with
supplemented Degree
of cellula~ay~ltiplication
in 7
Approx. concn. needed A for ,ptimum I growth _nY
Liver
Intestine
Conjunctiva
mhf
0 14.816.212.611.3 4.9 1.80.t Non-essential b amine 16.015.716.116.010.4 5.51.: acids + NHit As above + purines 14.813.914.613.011.5 9.51.5 and pyrimidinesc
1.5-1.1 0 0.5
0.25 0.15
1.2-O.: 5
0.1
0 13.315.515.810.5 7.0 2.40.7 Non-essential amino 18.019.018.015.3 7.4 4.91.4 acids + NH,+ As above + purines 13.714.113.914.713.611.61.i and pyrimidines
1 1.0
0.2 0.25
1.1-o.: 2
0.1
11.610.6 9.7 8.0 4.9 2.30.7 12.312.212.510.8 8.0 4.51.4
1 0.5f
0.2 0.15
9.9 9.7 8.8 8.6 8.4 7.41.:
0.2
0.1
0 Non-essential amino acids + NH,+ As above + purines and pyrimidines
0 Essential amino acids, vitamins, salts, and serum protein. b Alanine, aspartic acid, glutamic acid, glycine, proline, and serine, each at 0.1 mmole; NH&l at 0.05 mmole. c Adenine, guanine, xanthine, and hypoxanthine, each at 0.025 mmole; thymine, uracil, cytosine and erotic acid, each at 0.05 mmole.
viva1 and growth, and the amounts required for maximum growth. Although all the cell lines here examined required the same amino acids for survival and growth, the possibility of minor differences in other cell strains is indicated by the additional requirement for serine by a rabbit fibroblast (12), and for asparagine by Walker carcinoma 256 (13). The requirement for 13 amino acids by every cell here tested,a whether normal or malignant, is to be contrasted with the requirement for only 8 8 Assuming that tryptophan was in fact essential for all the lines here studied, and not merely growth stimulatory.
444
H.
EAGLE,
V.
I.
OYAMA
AND
M.
LEVY
I=BASAL MEDIUM II=It NH,++ NON-ESSENTIAL AMINO ACIDS III=JIt PURlNESt WRIMIDINES I
I
I
V
0.1
0.2
-
CONCENTRATION
I
I
0.5
I
2mM
OF L-GLUTAMINE
3. Illustrating the glutsmine-sparing effect of nutritionally non-esaential amino acids, NHd’ purines and pyrimidines. The basal medium was the minimal medium, embodying only the essential amino acids, vitamins, salts, and serum protein. The growth response of the human intestine cell to varying concentrations of glutamine in this medium is shown in curve I. In curve II, the medium was supplemented with glutamic acid, proline, aspartic acid, glycine, serine, and alanine, each at 0.1 mM, and NH&l at 0.05 mM; and in curve III the medium was further supplemented with adenine, guanine, xanthine and hypoxanthine, each at 0.025 mM; thymine, uracil, erotic acid, and cytosine, each at 0.05 mM. In the latter two media, although there was limited initial growth in the absence of added glutamine, growth Boon stoppedand the cells eventually died. FIG.
amino acids for nitrogen balance in adult man. Several possible explanations are under present study: 1. The cells may have altered in the course of their propagation in tissue culture, one manifestation of that change being a new requirement for exogenous arginine, histidine, cystine, tyrosine, and glutamine, in addition to the eight amino acids normally considered “essential.” On this basis, it becomes necessary to assumethat modifications leading to precisely the same new requirements had occurred in culture in all the strains here tested. One interesting possibility along these lines is that all the cells deriving from normal tissuesmay have modified to assumesome of the metabolic properties of malignant cells. 2. The five additional amino acids may be necessary only in tissue
AMINO
ACID REQUIREMENTS
OF CELLS
445
culture, when the cells are growing at a much faster rate than in viva. All these cells may be able to make small quantities of arginine, histidine, cystine, tyrosine, and glutamine, sufficient for the relatively slow normal growth in vivo, but inadequate for the greatly accelerated pace of cellular growth and multiplication in tissue culture. 3. Most body cells may actually require the same 13 ammo acids here shown to be necessary in tissue culture. The fact that eight dietary amino acids suffice for nitrogen balance may mean only that the other five are provided for by some other mechanism, such as their overproduction in one or more organs capable of supplying the rest of the body’s minimum requirements. SUMMARY
Three tissue culture cell lines deriving from normal human tissue (liver, conjunctiva, and intestine) and two lines deriving from human cancer [KB (nasopharynx), and J-111 (monocytic leukemia)] have been examined with respect to their ammo acid requirements in comparison with those of a HeLa cell and a mouse fibroblast. With the possible exception of tryptophan, all seven cell lines required the same amino acids (arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, valine) ; and in the absence of any one of these 12, cytopathogenic changes developed which culminated in the death of the cell. The provision of non-essential amino acids, purines, pyrimidines, and NHd+ had a glutamine-sparing effect, but did not eliminate the need for this amino acid. Extremely high and nonphysiological concentrations of glutamic acid (20 mM) did, however, substitute for glutamine. It is not yet clear whether tryptophan is similarly essential for survival and growth, or whether it is merely growth stimulatory for some of the five cell lines here studied. The concentration of the individual amino acids necessary for optimum growth varied somewhat among the six strains; but there were not significant or consistent differences in this respect between the lines deriving from normal and from malignant tissues. REFERENCES 1. EAGLE, H., J. Biol. Chem. 214, 839 (1955). 2. EAGLE, H., J. Exptl. Med. 102, 37 (1965). 3. EAGLE, H., OYAMA, V. I., LEVY, M., HORTON, C. L., AND FLEISCHMAN, R., J. Biol. Chem. 218, 697 (1956).
446
II.
4. SANFORD,
EAGLE,
K. K., HOBBS,
V.
I. OYAMA
AND
G. L., AND EARLE,
M.
LEVY
W. R., Cancer Research 16, 162
(1956). 5. hlEIST?ZR, A., in “Biochemistry and Physiology of Nutrition” (Bourne, G. H., and Kidder, G. W., eds.) Vol. 1, p. 136. Academic Press, New York, 1953. 6~. EAGLE, Y., OYAMA, V. I., AND LEVY, M., Science 123, 845 (1956); 6. EAGLE, H., OYAMA, V. I., LEVY, M., AND FREEMAN, A., J. Biol. Chem. 1967, in press. 7. EAQLE, H., J. Ezptl. Med. 102, 595 (1955). 8. OYAMA, V. I., AND EAQLE, H., Proc. Sot. EzptE. Biol. Med. 91, 305 (1956). 9. CRANO, R. S., Trans. N. Y. Acad. Sci. Ser. II, No. ~$17, 534 (1955). 10. EAQLE, H., Proc. Sot. Exptl. Biol. Med. 89, 362 (1955). 11. OSOOOD, E. E., AND BROOKE, J. H., Blood, J. HematoEogy 10, 1010 (1955). 12. HAFF, R. F., AND SWIM, H. E., Federation Proc. 16, 591 (1956). 13. NEUMAN, R. E., AND MCCOY, T. A., Science 144,124 (1956).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
67, 432-M
(1957)
Amino Acid Requirements of Normal and Malignant Human Cells in Tissue Culture Harry From
Eagle, Vance I. Oyama and Mina
the Section on Experimental Therapeutics, National Institute of Allergy and Infectious of Health,’ Bethesda,
Laboraiory
Levy
of Infectious Diseases, Institutes
Diseases, National Maryland
ReceivedAugust 10, 1956
It has been shown (1-3) that two mamalian cells in tissue culture, a human carcinoma of the cervix (st’rain HeLa) and a mouse fibroblast (st’rain L), required 13 amino acids for survival and growth. In the absence of any one of these, cytopathogenic changes developed which eventuated in the death of the cell. Since this cultured mouse fibroblast hasbeen found to produce tumors on injection intocortisone-treated mice (4), the possibility suggested itself that the anomalous requirement for 13 amino acids, as contrasted with the 8 and 9 required for nitrogen balance in adult’ man and rat, respectively (5)) might be peculiar to these two cell strains, and that other lines would show amino acid requirements more closely approximating those of the intact animal. Initial attempts to determine the specific amino acid requirements of other normal and malignant human cell lines were unsuccessful. Disappoint,ingly, t,he defined medium which, supplemented with serum protein, had supported t’he growth of the HeLa cell and the mousefibroblast, failed to permit the survival of any of a large number of cultured cell lines. This eventually proved to be due to t’he fact t’hat all of 20 human cell lines tested had a rigorous requirement for myo-(meso-)inositol (6). With inositol added, t’he medium now permitted the sustained propagation of every human tissue culture cell line examined; and it became possible to examine, both qualitatively and quant’itatively, the amino acid requirements of a variety of normal and malignant human cells. The results of these st,udiesare here reported. 1 Public
Health
Service,
U. S. Department
432
of Health,
Education,
and
Welfare.
;IMISO
ACID
REQCIREME?;TS
OF
CELLS
433
METHODS The methods used in growing stock cultures of the cells in l-l. Blake bottles, and of preparing replicate cultures in small T-15 flasks for individual experiments, have been described (l-3, 7). In previous studies the amount of cell growth had been determined by actual enumeration. In the present experiments, the protein content of the individual cultures was measured instead, using the Folin-Ciornltenu reagent in a modified Lowry procedure (8).
The composition of the basal medium, embodying only those components (amino acids, vitamins, salts, glucose, serum protein) demonst rahly essential for the growth and survival of the L fibroblast and the HeLa cell, is given in Table II of the preceding paper (66). The concentrations of the individual amino acids used in the present experiments were an arbitrary compromise between i he concentrations used for the cultivation of these two cell lines.
Cell Lines Five human cell lines were examined with respect to their amino acid requirements, in addition to the HeLa cell previously studied. Three lines derived from normal tissue: an embryonic intestinal epithelial cell cultured by 6. Henle and F. l>einhardt ,* and adult liver and conjunrtival cells cultured by Chang (9). The other two cell lines were cultures of human cancer: an epidermoid carcinom:l of the nasopharyns (strain KB) cultured directl>from the tumor into the medium here used (10). and a culture of the huffy coat from a case of human monocytic leltkemia, Osgood’s strain J-111 (I 1). EXPERIMENTAL
The I’niform Requirements for 13 Amino ;Icids by Humwn Culture, Whether Derived from Normal or lllaliynant
(‘ells in Tissue Tissue
,111five cell lines here studied xere found to require at least, 12 of the 13 amino acids previously shown to be essent,ial for the survival and growt’h of the HeLa cell and the mouse fibrohlast. In t’he case of tryptophan, with some of the cell lines there n-as a question as to whether it, was essential for survival, or merely growth-stimulat,ory (cf. below). With t’he other 12 amino acids, however, the omission of either arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methioninc, phenylalanine, threonine, tyrosine, or saline from the medium resulted in the death of the cell; and the early cytopathogenic effects produced b) such specific amino acid deficiencies are illustrated for a number of ~11 lines in Fig. 1, sew. 1, 1 and '7. 2 Personal
communication.
FIG.
1. The
7-day lines
Sec. Leucine Sec. 6. I,ysine Sec. 9n.U. 0. 3.
growth to
response individual
l-3:
01 reyr.esentat.ive amino acids.
Intestinal cell urld lcucine: 1. X0 lcurine. at 0.1 mM. 4-6: Conjunctival cell and lysine; 4. l;o lyaine. at 1 mM. 7-9: Liver cell and mcthionine: 7. No methionine. Methionirle nt 0.3 n&f. 331
human 2. Tencine 5.
Lyaim 8. Methionine
cell at
0.01
mM.
at
0.01
?m%f. at 0.03
AMINO
Illustrative
ACID
REQUIREMENTS
OF
435
CELLS
TABLE I Ezperimenk Showing the Growth Response of Various Human Cell Lines to Individual Essential Amino Acids
-
Concentration Cell line
Amino acid tested
of amino
acid in medium,
mill
CO?C?. COZKZL
3
j
1
( 0.3
( 0.1
Cellular
( 0.03 IO.01 jO.W3/O.Olll/
growth
0
permitting maximum growth
in s-7 days
pergttlY=
maxims growth (EDso)
--
ateatiie Znjunctiva WJ dver -nib a Culture b Culture
Argbliie Vdflle Lysine Metbionine TryPtoPlw
6.2 <0.5
24.0 10.3 7.9 1.7
24.8 9.4 7.0 10.2 4.4
16.7 10.2 6.1 10.8 9.2
8.2 6.8 2.65 8.9 7.7
8.1 3.9 1.1 6.2 6.9
1.9 1.9 0.6 2.2 6.3
1.3 3.4
1.3 0.9 0.5 0.8 1.0
mM
mhf
0.1-0.3 0.1 0.1 0.02 0.01-0.03
0.08-0.06 0.08 0.04 0.007 0.002
from epidermoid carcinoma of nanopharynr (10). from buffy coat of cane of mono&c leukemia (11).
As previously noted with the HeLa and L cell, with most of the amino acids the cytopathic effect of a single amino acid deficiency became evident within a few days, and the cells died off progressively thereafter (cf. Table I). With a few of the amino acids, however, there was initial growth, amounting to as much as a 2-3-fold increase over a period of 6-13 days; and only then did the cells begin to degenerate in consequence of the amino acid deficiency. In such casesthe death of the cells was greatly accelerated if the culture was subdivided after l-2 weeks’ incubation in the amino acid-deficient medium. In the case of tryptophan, all the cell lines here tested proved capable of initial slow growth in a tryptophan-deficient medium; but unlike the other amino acids, this slow growth usually continued in subculture for at least 34 passages.The determination of whether tryptophan is in fact essential for growth, or merely growth-stimulatory, is complicated by the extremely small amounts required even for maximum growth. These could easily be present as trace contaminants in someof the other amino acids, or they could be obtained by the limited utilization of the serum protein in the medium. Experiments on this point are now in progress. Quantitative Aspects of Amino Acid Requirement Illustrative experiments for various cell lines, showing the amount of growth as the concentration of a single essential amino acid in the medium was varied from 0 to 3 mM, are shown in Table I, and in
436
H. EAGLE,
V. I. OYAMA AND M. LEVY
Figs. 1 and 2. The concentrations of each of the 13 amino acids necessary to produce 50% of the maximal growth (EDsO) are listed for each cell line in Table II; and the concentrations permitting maximal growth are similarly listed in Table III. Previous results with the HeLa and L strains have been included for comparison. The range of variation in these values for the cell lines deriving from normal and from malignant tissues is shown in the last two columns of the tables. Within each group, there was usually a two- to threefold difference in the effective concentrations; but more significant is the fact that there was no regular difference in the amino acid requirements of the two types of cell. The EDso levels of cystine, glutamine, isoleucine, lysine, phenylalanine, and valine were higher for the malignant cells than for those deriving from normal tissue. Similar differences were, however, not observed with respect to the concentrations required for optimal growth. The Amino Acid Requirement of Cells in Tissue Culture as a Function of Population Density and Frequency of Feeding
In the preceding experiments the concentrations of the individual amino acids required for growth had been determined in cultures inoculated with relatively small numbers of cells. The experimental medium
r
1
0
aco1ooa3ca
CONCENTRATloN
003 Cf
AMINO
al ACID
03
I rnM IN MEMUM
FIO. 2. Illustrating the growth response of human cell lines to essential amino acids in tissue culture.
AMINO
ACID
REQUIREMENTS
OF
CELLS
437
had been added 24 hr. after planting, and had been replaced with fresh medium after 2,4,5,6, and 7 days. Experiments to determine the degree to which the amino acid requirement was affected by the number of cells and the frequency of feeding are summarized in Table IV. As there shown, the concentration of valine required for optimum growth of the KB cell was affected to only a minor degree by the density of the cell population and the frequency of feeding. However, these factors markedly affected the concentrations required to effect 50 % of optimum growth. With a relatively small inoculum (e.g. 49 pg. of protein per flask), and frequent (e.g., 12 hourly) changes of medium, 0.007 mM valine permitted 50% of the optimum growth; while with a fourfold larger inoculum, and infrequent (48 hourly) feedings, the EDso was 0.05 m&f. Quantitatively similar results were obtained in an experiment with threonine and the culture strain deriving from human liver. To avoid similar variation in the experiments of Table II, the inocula of all the cell lines were kept comparably low, and all the cultures were fed on the same schedule. Glutamine Requirement Under the conditions of the experiments so far described, the cell was under the necessity of making the nutritionally nonessential amino acids, purines, and pyrimidines from the amino acids and glucose of the medium, In consequence, relatively large amounts of glutamine (0.5-2 mM) were required for optimum growth, several times greater than the amounts required of any of the other amino acids. The EDso concentrations were similarly high, ranging from 0.15 to 0.3 mM. In the case of the HeLa cell and mouse fibroblast, it had previously been shown (3) that the addition of the non-essential amino acids, which relieved the cell of the necessity of making glutamic acid, proline, and aspartic acid, did not eliminate the need for this amino acid, but did have a glutamine-sparing effect. An additional minor glutamine-sparing effect was obtained on the addition to the medium of purines, pyrimidines, and NH4+. With three of the cell lines here studied (liver, intestine, and conjunctiva), the addition of the nutritionally non-essential amino acids, purines, pyrimidines, and NH*+ similarly reduced the glutamine requirement significantly (cf. Table V and Fig. 3). However, glutamine remained an essential component, and in its absence the cells eventually died. With all five human lines, normal or malignant, and again resembling the HeLa cell, glutamic acid at extremely high concentrations (lo-20
0.011
0.012
Iaoleucine
0.007
0.003 0.0023
0.904 0.003
Liver
0.25,0.25
)
Histidine
1 Intestine
0.2,0.3
acid tested EDro
0.01 0.01
0.005 0.003 0.003
0.2,0.150.6,
) cO~~~of amino
~.&a
0.3, 0.6, 0.6,0.7,0.
level
0.044
0.0120 0.01
)
0.018
0.04 0.02
0.009
0.6
0.04 0.03
0.0015
0.27
0.005 0.004
J-111
0.0025
T
/
0.03
n&f
KB
0.042 0.016
acid,
/
Cell line tested /
Mouse
0.025
0.004
0.12, 0.2, 0.14, 0.2,0.12,0.21
0.094
fibroblast
3
.
_-
c==z
-
Range
0 .007-0.012
0.018-0.04
0 .0023-0.0050.00154.009
0.27-0.7
0 .0008-0.0020.004-0.0064
0 .x-O.3
in
0.0164.05
of variation
Tissue Culture Lines
0 .1-0.2
P
Growth of 6 Human
0.0125
TABLE II of 18 Essential Amino Acids Permitting 60 Per Cent of Maximal
Glutamine
hino
I
The Concentrations
0.015 0.011
0.012 0.008
0.01 0.006
to.022 0.014
0.008 0.007
0.022 0.012
Lysine
Methionine
Phenylalanine
Threonine
Tyrosine
Valine
0.016
0.006
0.014 0.013
0.0048 0.004 0.0036
0.007 0.006
0.016 0.008 0.008
0.017 0.01 0.008
0.03
0.01 0.008
0.017 0.012
0.005
0.008
0.016 0.01
0.03
0.015
0.025
0.02
0.015 0.014
0.048 0.025
0.016 0.015
0.05
0.014 0.007
0.06
0.03
0.005
0.04
0.025
o Cells deriving from normal tissues (intestine, liver, conjunctiva, b Cells deriving from malignant tissues (KB, HeLa, J-111).
0.025
Leucine
fibroblast).
0.05
0.01 0.01
0.042 0.018 0.013
0.024
0.003
0.04
0.015
0.025
0.008
0.017
0.007
0.01
0.018
0.02
0.012-0.03
0.006-0.01
0.012-0.022
0.0036-0.01
0.008-0.016
0.0084.025
0.03-0.05
0.007-0.015
0.013-0.06
0.02-0.03
0.0250.04
0.015-0.025
0.1 0.03-0.1
0.1
Leucine
0.034.1
0.1
0.03 0.03
0.03 0.1
Isoleucine
0.03 0.03-0.1 0.03 I
I
0.03-0.1 0.1
0.1 0.03
0.03 0.03
0.01 0.01
0.5 li
P I
0.01 0.01
0.03 0.03
Lysine
Optimal
Amino
0.1 0.1 0.1 0.1 I I 0.0050.010.003-0.0: 0.01 0.01 0.01
Histidine
0.1 0.1-0.3 0.01 0.01 0.01-0.03
0.75 0.75
tested
of 12 Essential
1.0 l-2
acid
= I
Concentrations
Glutamine
Cystine
Arginine
Amino
The
TABLE
Cell
1
-
-
0.1
0.1
0.1 0.1
0.03 0.03
-
I.
0.1
0.1-0.3
0.1 0.1-0.3
0.03 0.03
l-2
1 1 2
mY
0.03 0.01-0.03
acid,
Maximal
0.1
of amino
- -
tested
III
0.03
0.1 0.1
line
Permitting
concentration
Acids
0.1
0.03
0.1-0.3 0.1
0.01
0.5
0.01 0.01
0.075i 0.1
Growth
0.05
0.05
0.1-0.2
0.03-0.1
0.03-0.1
0.03-0.1
0.01-0.03
0.01
Range
Culture
0.5-2
Tissue
0.2 0.2-0.5
of 6 Human in
Malignant human cells”
of variation
Lines
0.03 0.03
0.03-0.1 0.03
Tyrosine
Valine
from from
0.1 0.1
Threonine
deriving deriving
0.03 0.03
Phenylalanine
a Cells b Cells
0.03 0.03
Methionine
normal malignant
0.1 0.1
0.034.1 0.03
0.1 0.03
0.03
0.03
tissues (intestine, tissues (KB,
0.03-0.1
0.03-0.1 0.034.1
0.01-0.03 0.03 0.01-0.03
0.03 0.03
0.1
0.014.03 0.03
0.14.3
0.1-0.3
0.03
-~-- __-
liver, conjunctiva, HeLa, J-111).
0.1
0.03
0.1 0.1
0.03-0.1 0.1
0.03 0.03
-__
-
fibroblast).
_ .~
0.05
0.02-0.05
0.05
0.02
0.02-0.05
--
__ ~..-
0.03-0.1
0.034.1
0.03-0.1
0.01-0.03
IO.03
0.1
0.014.03
0.03-0.3
0.03-0.3
0.01-0.03
442
H.
EAGLE,
V.
I.
OYAMA
TABLE
AND
M.
LEVY
IV
The Growth Response of the KB Cell to r.-Valine as a Function of the Size of the Inoculum and the Frequency of Feeding Concentration
Growth
I
of valine
response
I
in medium,
in 6 days, referred
I
I
L5.318.515.515.613.911.0 19.619.317.912.1 11.010.6 9.5 5.3 14.715.213.1 9.1 14.312.714.710.9 9.4 8.9 8.3 4.7
I 7.4 3.1 6.7 5.8 2.7
to ino
- -
I 5.0 1.7 4.2 3.5 1.7
5.8 1.9 0.9 2.6 2.1 0.9
-
mN) could substitute for glutamine. the equilibrium in the reaction glutamate
+ NH*
+ adenosine
mnrole
-‘urn
3.5 1.F ) .B 1.4 1.3 0.5 - _-
I
-
as 1
.__ 2.6 0.9 0.4 1.1 0.7 0.4 .-
__ndd 1.10.0068 0.50.03 0.30.05 0.70.024 0.40.024 0.30.042
nrhf 0.05 0.1 0.1 0.1 0.1 0.1
This probably reflects the fact that
triphosphate adenosine
% glutamine diphosphate
+ + phosphate
is thereby shifted toward the right. It is of interest that the addition of those nutritionally non-essential factors which had a sparing effect on the glutamine requirement did not similarly reduce the effective concentration of glutamic acid. This suggests that the high concentrations of glutamic acid are not quantitatively related to the amount of glutamine required. The mouse fibroblast is the only cell so far examined wholly unable to utilize glutamic acid as a substitute for glutamine, no matter what the concentration added to the medium (3). DISCUSSION
One of the interesting aspects of the data here reported is the qualitative and quantitative similarity in the amino acid requirements of a wide variety of human cells in tissue culture, deriving from both normal and malignant tissues, and including both embryonic and adult cell lines (Tables II and III). If the embryonic intestine, adult liver, and adult conjunctival lines here studied are still “normal” cells (and there appears to be no presently available definitive criterion short of test in man), one must then conclude that malignant cells do not regularly differ from normal cells with respect to the exogenous amino acids needed for sur-
AMINO
ACID
REQUIREMENTS
OF
443
CELLS
TABLE V The Glutamine Requirement of Three Normal Human Cell Lines in Tissue Culture in Relation to the Composition of the Medium
Cell
strair
&al
mediuma
with
supplemented Degree
of cellula~ay~ltiplication
in 7
Approx. concn. needed A for ,ptimum I growth _nY
Liver
Intestine
Conjunctiva
mhf
0 14.816.212.611.3 4.9 1.80.t Non-essential b amine 16.015.716.116.010.4 5.51.: acids + NHit As above + purines 14.813.914.613.011.5 9.51.5 and pyrimidinesc
1.5-1.1 0 0.5
0.25 0.15
1.2-O.: 5
0.1
0 13.315.515.810.5 7.0 2.40.7 Non-essential amino 18.019.018.015.3 7.4 4.91.4 acids + NH,+ As above + purines 13.714.113.914.713.611.61.i and pyrimidines
1 1.0
0.2 0.25
1.1-o.: 2
0.1
11.610.6 9.7 8.0 4.9 2.30.7 12.312.212.510.8 8.0 4.51.4
1 0.5f
0.2 0.15
9.9 9.7 8.8 8.6 8.4 7.41.:
0.2
0.1
0 Non-essential amino acids + NH,+ As above + purines and pyrimidines
0 Essential amino acids, vitamins, salts, and serum protein. b Alanine, aspartic acid, glutamic acid, glycine, proline, and serine, each at 0.1 mmole; NH&l at 0.05 mmole. c Adenine, guanine, xanthine, and hypoxanthine, each at 0.025 mmole; thymine, uracil, cytosine and erotic acid, each at 0.05 mmole.
viva1 and growth, and the amounts required for maximum growth. Although all the cell lines here examined required the same amino acids for survival and growth, the possibility of minor differences in other cell strains is indicated by the additional requirement for serine by a rabbit fibroblast (12), and for asparagine by Walker carcinoma 256 (13). The requirement for 13 amino acids by every cell here tested,a whether normal or malignant, is to be contrasted with the requirement for only 8 8 Assuming that tryptophan was in fact essential for all the lines here studied, and not merely growth stimulatory.
444
H.
EAGLE,
V.
I.
OYAMA
AND
M.
LEVY
I=BASAL MEDIUM II=It NH,++ NON-ESSENTIAL AMINO ACIDS III=JIt PURlNESt WRIMIDINES I
I
I
V
0.1
0.2
-
CONCENTRATION
I
I
0.5
I
2mM
OF L-GLUTAMINE
3. Illustrating the glutsmine-sparing effect of nutritionally non-esaential amino acids, NHd’ purines and pyrimidines. The basal medium was the minimal medium, embodying only the essential amino acids, vitamins, salts, and serum protein. The growth response of the human intestine cell to varying concentrations of glutamine in this medium is shown in curve I. In curve II, the medium was supplemented with glutamic acid, proline, aspartic acid, glycine, serine, and alanine, each at 0.1 mM, and NH&l at 0.05 mM; and in curve III the medium was further supplemented with adenine, guanine, xanthine and hypoxanthine, each at 0.025 mM; thymine, uracil, erotic acid, and cytosine, each at 0.05 mM. In the latter two media, although there was limited initial growth in the absence of added glutamine, growth Boon stoppedand the cells eventually died. FIG.
amino acids for nitrogen balance in adult man. Several possible explanations are under present study: 1. The cells may have altered in the course of their propagation in tissue culture, one manifestation of that change being a new requirement for exogenous arginine, histidine, cystine, tyrosine, and glutamine, in addition to the eight amino acids normally considered “essential.” On this basis, it becomes necessary to assumethat modifications leading to precisely the same new requirements had occurred in culture in all the strains here tested. One interesting possibility along these lines is that all the cells deriving from normal tissuesmay have modified to assumesome of the metabolic properties of malignant cells. 2. The five additional amino acids may be necessary only in tissue
AMINO
ACID REQUIREMENTS
OF CELLS
445
culture, when the cells are growing at a much faster rate than in viva. All these cells may be able to make small quantities of arginine, histidine, cystine, tyrosine, and glutamine, sufficient for the relatively slow normal growth in vivo, but inadequate for the greatly accelerated pace of cellular growth and multiplication in tissue culture. 3. Most body cells may actually require the same 13 ammo acids here shown to be necessary in tissue culture. The fact that eight dietary amino acids suffice for nitrogen balance may mean only that the other five are provided for by some other mechanism, such as their overproduction in one or more organs capable of supplying the rest of the body’s minimum requirements. SUMMARY
Three tissue culture cell lines deriving from normal human tissue (liver, conjunctiva, and intestine) and two lines deriving from human cancer [KB (nasopharynx), and J-111 (monocytic leukemia)] have been examined with respect to their ammo acid requirements in comparison with those of a HeLa cell and a mouse fibroblast. With the possible exception of tryptophan, all seven cell lines required the same amino acids (arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, valine) ; and in the absence of any one of these 12, cytopathogenic changes developed which culminated in the death of the cell. The provision of non-essential amino acids, purines, pyrimidines, and NHd+ had a glutamine-sparing effect, but did not eliminate the need for this amino acid. Extremely high and nonphysiological concentrations of glutamic acid (20 mM) did, however, substitute for glutamine. It is not yet clear whether tryptophan is similarly essential for survival and growth, or whether it is merely growth stimulatory for some of the five cell lines here studied. The concentration of the individual amino acids necessary for optimum growth varied somewhat among the six strains; but there were not significant or consistent differences in this respect between the lines deriving from normal and from malignant tissues. REFERENCES 1. EAGLE, H., J. Biol. Chem. 214, 839 (1955). 2. EAGLE, H., J. Exptl. Med. 102, 37 (1965). 3. EAGLE, H., OYAMA, V. I., LEVY, M., HORTON, C. L., AND FLEISCHMAN, R., J. Biol. Chem. 218, 697 (1956).
446
II.
4. SANFORD,
EAGLE,
K. K., HOBBS,
V.
I. OYAMA
AND
G. L., AND EARLE,
M.
LEVY
W. R., Cancer Research 16, 162
(1956). 5. hlEIST?ZR, A., in “Biochemistry and Physiology of Nutrition” (Bourne, G. H., and Kidder, G. W., eds.) Vol. 1, p. 136. Academic Press, New York, 1953. 6~. EAGLE, Y., OYAMA, V. I., AND LEVY, M., Science 123, 845 (1956); 6. EAGLE, H., OYAMA, V. I., LEVY, M., AND FREEMAN, A., J. Biol. Chem. 1967, in press. 7. EAQLE, H., J. Ezptl. Med. 102, 595 (1955). 8. OYAMA, V. I., AND EAQLE, H., Proc. Sot. EzptE. Biol. Med. 91, 305 (1956). 9. CRANO, R. S., Trans. N. Y. Acad. Sci. Ser. II, No. ~$17, 534 (1955). 10. EAQLE, H., Proc. Sot. Exptl. Biol. Med. 89, 362 (1955). 11. OSOOOD, E. E., AND BROOKE, J. H., Blood, J. HematoEogy 10, 1010 (1955). 12. HAFF, R. F., AND SWIM, H. E., Federation Proc. 16, 591 (1956). 13. NEUMAN, R. E., AND MCCOY, T. A., Science 144,124 (1956).