A C O M P A R A T I V E S T U D Y OF T H E G R O W T H , N U T R I T I O N , A N D
M E T A B O L I S M OF T H E P R I M A R Y A N D T H E T R A N S F O R M E D
HUMAN CELLS IN VITRO*
BY R. SHIHMAN CHANG, M.D"
(From the Virus Laboratory, University of California, Berkeley, and the Department of
Microbiology, Harvard School of Public Health, Boston)
(Received for publication, September 22, 1960)
Materials and Methods
Nutrient Media.--These consisted, in general, of 10 per cent dialyzed human or horse
serum in Eagle's basal medium (22). The following modifications of Eagle's basal medium
were made: the omission of biotin, the addition of mesoinositol to about 10 microm01ar and
the use of Hanks's balanced salt solution. In all comparative experiments, this basal medium
was further supplemented by doubling the concentration of amino acids and amide, and adding glycine, alanine, sorine, proline, glutamic and aspartic acids, and asparaglne, all at a
final concentration of 0.2 mM. The collection, storage, and dialysis of serums have been described previously (23).
Human Cell Cultures.--The HeLa (1), conjtmctival (2), and FL cells (18) derived respectively from malignant cervical carcinoma, normal conjuncfiva, and amrdon were used as
representative ceils which have already undergone transformation. Since the differences
among these cells have not been consistent and were small in comparison to their difference
* Supported by research grants (SF131 and E553C6) from the United States Public
Health Service.
4O5
Downloaded from jem.rupress.org on July 31, 2017
Explanted h u m a n tissues, malignant or normal in origin, have been observed
b y numerous investigators to undergo alteration (1-21). This alteration enables
such cells to multiply autonomously and indefinitely under fairly well defined
conditions. The mechanism through which such alteration occurs, however,
remains obscure, as are the conditions optimal for the appearance of such
altered cells. T h e biological importance of this phenomenon, especially in its
relation to differentiation and carcinogenesis, prompts us to undertake a long
term study to elucidate the nature of this alteration. T o facilitate description,
this phenomenon is to be referred to in this manuscript as transformation, and
the altered cells, transformed cells (frequently referred to as established cell
lines in current literature).
This report is based upon a series of experiments designed to compare the
growth characteristics, nutritional requirements, and metabolic activities of the
transformed and freshly explanted h u m a n cells in vitro.
406
PRIMARY AND TRANSFORMED
HUMAN
CELLS
RESULTS
Growtk Ckaract~istics of the Primary Explants.--A total of 23 specimens
(11 amnion, 3 embryonic skin a n d muscle, 5 p o s t n a t a l kidney, 2 a d u l t conjunctivae, a n d 2 a d u l t skins) have been studied from 1956 to 1959. T h e results,
summarized in the hypothetical growth curve, (Fig. 1), are in general similar
to those reported previously (14, 19). T o facilitate description, the growth
curve was a r b i t r a r i l y divided into the following phases: glass a t t a c h m e n t ,
p r i m a r y growth, degeneration, transformation, and transformed growth. Owing
to the extreme v a r i a b i l i t y of results, the so called phases i n v a r i a b l y overlapped.
A decrease in cell n u m b e r was observed for the several days of the glass a t t a c h m e n t phase in most cultures, p r e s u m a b l y due to the elimination of cells incapable of adherence to glass. T h e extensive variation is illustrated b y the v e r y
limited a t t a c h m e n t of certain batches of epithelial-like cells contrasted with a
Downloaded from jem.rupress.org on July 31, 2017
from the primary explants, these cells are subsequently referred to as the transformed cells.
The preservation and propagation of stock cultures of the transformed cells have already
been described (23).
Primary explant cultures were prepared from 5 different sources: embryonic skin and
muscle, surgically removed postnatal kidney, the amniotic membrane obtained at full term
delivery, adult conjunctivae, and skin. Cell suspensions were prepared from these tissues by
the standard trypsinization procedure. Because of the availability of primary amnion, its
existence in a state of relative dormancy (resembling well differentiated tissue), and the ease
with which relatively pure morphologic cell type can be obtained, the metabolic and nutritional studies with primary tissues were performed chiefly with the primary amniotic cells.
Since it had been shown that in vitro aging of the primary amnion cell may alter its susceptibility to certain viruses (24), the preparation of amnion cells for metabolic study was standardized as follows: the cell suspension, prepared according to the method of Zitcer a al. (25),
was implanted in serum dilution bottles (200 mi. capacity) each containing 10 ml. of 10 per
cent human and 5 per cent calf serums in Eagle's basal medium; the medium was renewed
on the 4th and 7th day; on the 9th day, the medium was changed to that containing 10 per
cent dialyzed serum; on the l l t h day, subcultivation into culture tubes (18 X 150 ram.) was
made, and the cultures were used for different experiments in 2 to 3 days. The cultures from
certain placentae which failed to form sheets of polyhedral cells with clear or slightly granular
cytoplasm and showed no mitosis were discarded as unhealthy.
Compounds.--Commerdally available compounds of highest purity were used. The specific
activities of C1Mabelled compounds were 2.93, 2.2, 0.18, 4.18, 1.35 and 1.0 inc. per raM. of
ghicose-Ct% ribose-C1%xylose-l-C14,lactate-2, 3-C14,glycine-2-C1., and NaHCI~Os respectively.
Nutritional and Metabolic Study.--Roller cultures, revolving at the rate of 12 x.P.n, were
used exclusively; this continuous movement presumably facilitated equilibration of metaborites. In the CO2 depletion study, the method described previously was used (26). In experiments lasting more than 3 days, nutrient media were renewed every 48 hours. At the termination of each C" incorporation study, COs produced was first trapped; each culture rinsed
twice with 20 ml. of 0.85 per cent NaC1; the cells dissolved in 1 ml. of water containing 0.025
per cent sodium lauryl sulfate; materials from 5 to 10 replicate cultures pooled, and, 2 rag. of
bovine albumin, 1 mg. DNA, and 0.5 mg. RNA added per 10 mi. cell solution, which was then
fractionated according to the procedure of Schmidt-Tannhauser-Schneider (27). C" activities
in various fractions were measured as ultrathin specimens with a thin window Geiger tube
of about 15 per cent counting efficiency; the counting error was estimated at 5 per cent.
R. SHIH~.~.~T CHANG
407
multiplication of embryonic skin and muscle within 4 days after explantation.
The primary growth phase was also extremely variable. Fibroblast-like cells
usually maintained a high rate of multiplication, sometimes for several months.
On the other hand, the amnion cells remained chiefly in a relatively dormant
state, showing little primary growth (from slight decrease to about 2-fold
increase during the first 2 weeks). During the degenerative phase, cells became
granular and round so that they detached from the glass and were removed
during renewal of media. The relatively small number of cells remaining during
the transforming phase appeared thin, pale, and often bizarre in shape and of
very low metabolic activity. Degeneration proceeded very slowly to completion
i
2
t
5
I
4
I
i
5
Age of cutture
FIG. i. Hypothetical growth curve of primary explants. I, glass attachment phase; 2,
primary growth phase; 3, degenerating phase; 4, transforming phase; and 5, transformed
growth phase.
in most instances. In 1 experiment (kidney), 12 weeks after explanation, a few
cells appeared thicker and assumed polyhedral shape, mitosis soon followed,
and a few plaques of transformed cells appeared. These transformed cells could
then be subcultivated at irregular intervals. Subzero storage of these freshly
transformed cells failed to give rise to viable cultures in 2 trials. The long
period required for transformation to occur had been described by several
investigators: 51 days (7), about 50 days (8), about 3 months (18), 84 to 92
days (15), and 81 days (10). The established transformed cells are capable of
multiplying continually with a generation time of about 24 hours under ideal
conditions; this is too well known to require further elaboration.
Relative Incorporation of O*-LabeUed sugars.--The results of representative
experiments as presented in Table I, show that the total amount of glucose,
lactate, ribose, or xylose incorporated and converted to CO2 in 24 hours by the
transformed cells was at least 5 to 10 times more than in the primary amnion.
The largest difference in activities occurred in the nucleic acid fraction (about
Downloaded from jem.rupress.org on July 31, 2017
i
I
408
PRIMARY
AND
TRANSFORMED
HUMAN
CELLS
20 to 100 times more in the transformed cells); the least, in the acid-soluble
fraction. T h e utilization of xylose a n d ribose b y the p r i m a r y amnion cells
should be noted. I t should be emphasized t h a t the utilization of glucose through
glycolysis was not determined in this study. Since an extremely high rate of
glycolysis h a d been d e m o n s t r a t e d in the transformed cells (28) the actual
difference in glucose utilization should be much larger.
TABLE I
Relative Incorporation of CX4-Labdled Sugars
C ~4 activities (¢.p.m. per 106 cells)
in fractions
Exp.*
Cell~
Substratel[
286/285
509/327
glucose-C14
Primary
Transformed
266/277
302/257
glucose-C1.
Primary
Transformed
286/285
509/327
lactate-2,3-C14
Primary
Transformed
266/277
302/257
laetate-2,3-C1.
Primary
Transformed
286/285
509/327
ribose-C 14
Primary
Transformed
286/285
509/327
xylose-l-C14
....
"
"
"
"
"
"
"
"
"
"
Acidsoluble
93
1081
585
2521
220
752
34
947
7C
1414
193
1300
866
2123
170
914
46
890
11(3
1413
292
4306
1030
3555
317
7637
32
947
670
7886
307
1530
1199
3087
221
456
34
3052
690
983
46
213
832
3041
93
583
89
2710
51
1018
40
209
106
655
0
105
7
297
16
150
Lipid
Nucleic
acid
Protein
* Dialyzed horse serum used in Experiments 1, 3, 5, and 6; dialyzed human serum, in
2 and 4; duration of experiments, 24 hours.
:~Primary, primary amnion; transformed, Hela, conjunctival, or FL.
§ The denominator and numerator represented the actual number of ceils (in thousands)
in each culture at 0 and 24 hours.
[[ C14 substrate used at 1/zc. per ml. medium; respective carrier substrate also added to
final concentration of about 2.5 m~r.
R e l a t i v e I n c o r p o r a t i o n o f C1402.--A previous s t u d y (29) showed t h a t most of
the CO2 fixed b y the transformed cells was recovered in the nucleic acids a n d
their derivatives, and t h a t this fixation was essential for cell multiplication.
The p r i m a r y amnion cells showed v e r y much less fixation of CO2 in a comp a r a t i v e study. T h e results a p p e a r in Table I I ; again, the biggest difference
was found in t h e nucleic acids.
Relative Incorporation
of Glycine-2-C~*.--Table
I I I shows the results of 5
Downloaded from jem.rupress.org on July 31, 2017
Primary
Transformed
CO6
R. SHIHI~r~,N CHANG
409
experiments, I n contrast to the sugars a n d CO2, differences in the total a m o u n t
of glycine incorporated in 24 hours were only a b o u t 2-fold. Once again, the C I'
activities in the nucleic acid fractions of the transformed cells were about 12
TABLE II
Rdative Incorporation of 0~02 *
Exp.
CeU)
C14activities(e.P.M.per 106cell) in fraction:
Multiplication
of exp.
md~
Nucleic
AcidProteins
Lipid
add
soluble
Duration
]$r$.
65/61
66/61
240
180
27
31
111
9O
56
53
24
48
192/145
194/145
176
159
8
21
44
69
9
32
Primary
24
48
273/288
263/288
200
243
14
15
115
140
14
23
Transformed
24
48
112/81
187/81
1330
623
73
26
513
836
123
78
Transformed
24
48
115/76
162/76
1329
873
14
15
467
753
102
105
Primary
* 5/~c. of NaHCa~Os per 10 ml. nutrient medium.
:~See corresponding footnotes of table I.
TABLE HI
Rdative Incorporation of Glycine-g-C"
Exp.*
Cells
Primary
Primary
Primary
Transformed
Transformed
Multiplication
index$
185/155
113/93
132/131
181/125
165/116
C14 activities (c.P.•. per 106 cells) in fractions
Add-soluble
Lipids
Nudelc acid
Protein
974
2820
2047
3196
3022
304
422
242
1290
898
125
173
87
2166
867
775
21~
1277
344O
1440
* 1/zC. of glycine-2-CX4/per10 ml. medium; no carrier glycine added.
:~See corresponding footnotes in table I.
times treater t h a n those of the p r i m a r y amnion. Of equal interest was the
difference of less t h a n 2-fold in their protein fractions.
Incorporation of 0 4 Substrates at 8tk, 24th, and 48tk Hours. Results presented
thus far show very limited incorporation of C 14 substrates into the nucleic acid
Downloaded from jem.rupress.org on July 31, 2017
24
48
Primary
410
PRIMARY
AND
TRANSFORMED
HUMAN
CELLS
fractions of the p r i m a r y amnion cells. To exclude the possibility t h a t this low
a c t i v i t y was due to incomplete separation of the fractions, cell counts a n d C t4
activities were determined at intervals (8th, 24th, and 48th hours). Results of
TABLE IV
Incorporation of 0 4 Substrates at 8th, 24th, and 48th Hours by Primary Amnion Calls
C x'i activities(c.P.M.per culture)in fractions
Time
No. cell*
Substrate~
C~
[ Acidv______~* soluble
190, 207
245, 229
8
24
48
211, 208
223, 232
24
132
456
glucose-C 14
glycine-2-C 14
~c
~c
[ 58
[ 202
362
78
218
126
343
367
74
190
328
11
61
132
18
77
160
104
427
866
* Results of 2 replicate cultures, expressed as No. cell X I0a per culture. Zero hour count
174, 170.
:~Concentrations per 10 mt. media; 10/~c. glucose-C14 in 25 #M of carder glucose, and
1/~c. of glycine-2-C14without carder glycine.
TABLE V
Effect on Nutrient Depletion on Cell Multiplication
No. cell X 10z per culture on day
Exp.
Nutrient
depleted
Primary amnion*
7
None
Inositol
Glutamine
Glucose
None§
C02
37~4o6~
447, 465
378, 406 [ 487, 475
378, 406 I 388, 407
378, 406
73, 82
54, 50
54, 50
30, 31
32, 32
Transformed cells
14
0
7
14
585, 574
542, 527
597, 668
O, 0
133, 123
133, 123
133, 123
133, 123
729, 719
9, 4
57, 58
0, 0
1670, 1626
0,
0
43, 33
0,
0
18, 19
21, 21
43, 39
43, 39
225, 236
31, 29
217, 184
0,
0
* Cells from different placentae used in each experiment.
~tResults of replicate cultures.
§ Tds buffer used instead of NaHCOa; endogenous CO2 not depleted.
a representative experiment are shown in Table IV; there were small b u t consistent increases in cell counts as well as C 14 activities in the nucleic acids from
0 to 48 hours. These results suggested t h a t the low C 14 activities in the nucleic
acids represented synthesis rather than contamination.
Downloaded from jem.rupress.org on July 31, 2017
8
24
48
Nucleic [ Protein
Liplds
R. S H , H ~ A N
CHANG
411
FUDR, 5-fluorodeoxyuridlne.
Downloaded from jem.rupress.org on July 31, 2017
Effect o/Nutrient Depletion.--The effect of depletion of inositol, glutamine,
glucose, or endogenous CO2 is described in Table V. While depletion of any one
of these 4 compounds invariably led to rapid degeneration of the transformed
cells, only glucose depletion induced similar changes of the primary amnion
cells. For optimal cell multiplication, however, more regular results were obtained when an extraneous source of CO2 (such as NaHCO3 at about 5 raM) was
provided.
Virus Synthesis by C02-Depleted Primary Amnion CeUs.--Since the primary
amnion cell incorporated a very small quantity of C1K)2 (see Table II) and COz
depletion did not detectably affect its survival (see Table V), the question may
be raised as to whether CO2 participates in the synthesis of its nucleic acids.
The synthesis of Coxsackie virus, Group B, Type 1, in the primary amnion
cells depleted of CO2 was studied by the method described for the transformed
cells (30). As in the transformed cells, suppression of virus synthesis occurred
in COz-depleted cultures and this suppression was reversed by the addition of
CO, or the ribosides (uridine, cytidine, adenosine, and guanosine, all at concentrations of 1/~g. per ml.). This suggested that when induced by infection, the
primary amnion synthesized its purines and pyrimidines from C(h.
Effect of 5-Fluorodeoxyuridine (FUDR)I.--To corroborate the preceding evidence of a very low level of nucleic acid metabolism in the primary anmion,
the effect of FUDR was studied. FUDR had been shown to interfere specifically
with the synthesis of thymidine and to be incorporated into RNA following
degradation to 5-fluoro-uracil (31). As little as 0.01 #g. per ml. had been shown
to be toxic for a transformed cell (32). The results appear in Table VI. Within
48 hours after the addition of 10 #g. FUDR to the transformed amnion cells,
there was a very marked reduction of cells undergoing mitosis, followed by
degenerative changes involving all cells. At 0.1 #g. both the suppression of
mitosis and the appearance of degeneration occurred only after much longer
contact with FUDR; nevertheless, by the 14th day, only a few bizarre-shaped
cells were left. The reaction of primary amnion to FUDR was distinctly
different. In experiments in which the primary amnion showed no detectable
multiplication, FUDR, even at a concentration of 10 #g. per ml., exerted no
detectable effect after 7 days. By the 14th day, the majority of the primary
amnion cells exposed to 10 #g., but not those exposed to 1 t~g. of FUDR became
vacuolated. In those primary amnion cells which did multiply slowly for the
duration of the experiment, cell multiplication was suppressed within 7 days
even at 0.1 #g. per ml., while advanced degeneration appeared by the 14th day
only in those cultures containing 10 ~g. This slow appearance of degenerative
changes, which occurred only in cells exposed to 10 ~g. of FUDR, suggested
that the toxicity is the result of slow degradation of FUDR and the incorpor-
412
PRIMARY AND TRANSFORMED ttLrMAN CELLS
ation of its degraded product, 5-fluoro-uracil, into cell RNA giving rise to
defective RNA. It also indicated that RNA synthesis occurs even in cells
showing little evidence of multiplication.
TABLE VI
Effect of 5-Fluorodeoxyuridine (FUDR) on Cell Multiplication
No. cell X 10a per culture on day
Cell
Transformed
Primary
7
178, 116
1305, 1310~ (0)§
106, 8O (+)
6, 4 (-b-b-b)
O, 0 ( + + + )
177, 181
301, 292
14
196,
140,
163,
235,
156
130
188
180
(0)
(0)
(0)
(0)
468,
208,
180,
210,
488
221
190
232
(0)
(0)
(o)
(0)
1682, 1672 (0)
O,
0 (-b-b+)
O,
0 (-b-b-b)
110, 148 (0)
130, 148 (0)
128, 158
so,
80 (+)
570, 688 (0)
308, 280 (0)
258, 218 (0)
0,
0 (+++)
* Concentration expressed as micrograms per milliliter media.
:~ Results of replicate cultures.
§ (0), no evidence of degeneration; (-[-), moderate degeneration, (-J--b-b), advanced or
complete degeneration.
DISCUSSION
Comparative metabolic studies of primary explants and transformed cells
have been reported by several investigators. From a comparison of the amino
acids and amide requirements of the primary monkey kidney cells with the
transformed human and mouse cells, Eagle demonstrated that, in contrast to
the transformed cells, glycine was growth-stimulating and that glutamine may
be replaced by asparagine, glutamic and aspartic acids in the primary kidney
cells (33). Morgan and his associates, comparing primary chick embryonic
tissues with transformed human and mouse cells, found significant differences
in the uptake and release of certain amino acids (34). Lieberman and Ore (44)
found that primary cultures of rabbit tissues required folic acid at concentration
much higher than the transformed cells derived from human and from mouse
tissues. Riley, quoted by Moore (28), showed differences in the rates of anaerobic glycolysis between normal human fibroblasts and transformed epitheliallike cells.Other non-metabolic studies included the differences in cytopathology
Downloaded from jem.rupress.org on July 31, 2017
Primary
0
I~. SHIHI~I'.~II~T C]~ll~TC,-
413
Downloaded from jem.rupress.org on July 31, 2017
following polio infection (35), chromosome number (28, 36, 37), heterologous
transplantation (38), virus susceptibilities (36, 39, 40), multiplication of tubercle
bacilli (41), and effect of actinomycin D (42).
For the elucidation of the mechanism of transformation through comparative
studies, ideally the same cell before and after transformation should be used.
The technical obstacles to such a study have been described (43). The next
best approach would be the use of primary explant and transformed cells
derived from the same type of human tissues. This is the basis on which the
present experiments are designed; primary amnion cells obtained from over
15 placentae were compared with transformed cells derived from human
amnion, conjunctiva and cervical cancer.
The most consistent and striking of all the differences is the very low level
of nucleic acid synthesis in the primary amnion. A point of considerable importance is whether a few multiplying cells are synthesizing nucleic acids at
much higher rates among a large population of dormant cells with inactive
nucleic acid metabolism, or whether the majority of the cells are synthesizing
nucleic acids at very low rate. The latter possibility is supported by the vacuolization of most cells after prolonged contact with a high dose of FUDR, the
well known ability of most or all cells to synthesize viral nucleic acids, and the
random distribution of a few mitotic cells. The primary amnion cells are potentially capable of higher activities in their nucleic acid metabolism. This is
indicated by their well known ability to synthesize certain RNA and DNA
viruses; infectious virus (polio) appears several hours following inoculation
(35). A requirement for CO2 in the synthesis of nucleic acids, ordinarily difficult
to demonstrate, becomes readily demonstrable following infection with the
Coxsackie virus.
Differences in the utilization of glucose, lactate, ribose, and xylose deserve
consideration. The high glycolytic rates of rapidly growing cells as compared
to dormant cells are too well known to require further discussion. Our results
show that differences in the utilization of carbohydrate through the tricarboxylic acid cyde between the transformed and primary amion cells are also
great. Such differences cannot be attributed entirely to a better regulation of
available glucose in the primary amnion cell since, when lactate is supplied,
the conversion to COs occurs also at a much lower level. The ability of the
transformed cells to utilize ribose and xylose and the existence of mutants
capable of continuous multiplication with these pentoses as the sole carbohydrate source is well established (23). It is interesting that such dormant cells
as the amnion also utilized ribose and xylose. One may conclude that the
energy-generating mechanisms, glycolysis and respiration, are operating at
much higher rates in the transformed cells than in the primary amnion cells.
The failure to demonstrate specific requirements for inositol and glutamine
in the multiplying primary amnion cell deserves some emphasis (Table V). A
414
PRIMARY AND T R A N S F O R M E D H U M A N CELLS
S ~ R Y
The growth characteristics, nutritional requirements, and metabolic activities of primary explants (chiefly amnion) and transformed human cells
(derived from amnion, conjunctiva, and cervical cancer) were compared. Major
differences observed are as follows:
Downloaded from jem.rupress.org on July 31, 2017
deficiency in inositol has never been clearly demonstrated in man, although
Eagle and his associates, after some conflicting reports (45-47), concluded that
the transformed human cells did require an external source of inositol for
growth. A strain of transformed ceil (Hela) which multiplied without the
addition of inositol has also been reported (48). Eagle subsequently reported
that the transformed cell does synthesize inositol, presumably at a rate insufficient to meet the requirement of rapid growth (49). This information may
be integrated in the hypothesis that human cells are capable of synthesizing
sufficient inositol to meet their physiological growth requirement but that
an external source of inositol may become essential under conditions of rapid
and abnormal growth. The lack of a requirement for glutamine may be best
explained by the ability of the primary amnion to use asparagine and glutamic
acid in its place. It is obvious that the extrapolation of results obtained with
the transformed cell to those in vivo should be made with great caution.
The low level of nucleic acid metabolism in these slowly or nonmultiplying
primary amnion cells and the readiness with which it may be stimulated (such
as by viruses) support the hypothesis that abnormally rapid cell multiplication may be the consequence of the removal of suppressors of biosynthetic
enzymes or the deletion of catabolic enzymes which normally regulate cell
growth through a balanced nucleic acid metabolism (50). The 50 to 90 day
period required for transformation to take place among cell populations that
are apparently dormant and slowly disintegrating suggests a possible relation
between cell aging and abnormal growth. If various cell components and their
corresponding genetic counterparts degenerate at different paces randomly
in an aging cell population, and if the inhibitors of biosynthetic enzymes or
the catabolic enzymes are the first to disintegrate in a particular cell, it is not
too unreasonable to expect that this cell would resume multiplication. Based
on this hypothesis, it is not unfeasible that external agents, such as viruses
(.vet to be discovered for human cells), may enter a cell and initiate abnormal
growth through the suppression of hypothetical inhibitors and/or catabolic
enzymes.
Obviously, to substantiate this hypothesis, one would have to demonstrate
the existence of inhibitors for the biosynthesis, and/or catabolic enzymes for
the destruction of nucleic acids and their derivatives, and the removal or suppression of these inhibitors and/or catabolic enzymes. Such studies are currently in progress in this laboratory.
R, S H 1 H ' ~ T CHANG
415
The author is grateful to Dr. Wendell M. Stanley and Dr. John C. Snyder for their invaluable suggestions, and to Dr. M. J. Schiffronof Hoffmann-LaRoche, Inc., Chicago, for a
generous supply of FUDR.
BIBLIOGRAPHY
1. Gey, G. O., Coffman, W. D., and Kubicek, M. T., Tissue culture studies of proliferative capacity of cervical carcinoma and normal epithelium, Cancer Research, 1952, 12, 264.
2. Chang, R. S., Continuous subcultivation of epithelial-like cells from normal
human tissues, Proc. Soc. Exp. Biol. and Med., 1954, 87, 440.
3. Osgood, E. E., and Brooke, J. H., Continuous tissue culture of leucocytes from
human blood by application of gradient principles, Blood, 1955, 10, 1010.
4. Frisch, A. W., Jentoft, V., Berger, R., and Losli, E. J., A human epithelial-like
cell derived from an adenocarcinoma of lung, Am. J. Clin. Path., 1955, 9.§, 1107.
5. Moore, A. E., Sabachewsky, L. and Toolan, H. W., Culture characheristics of
4 permanent lines of human cancer cells, Cancer Research, 1955, 15, 598.
6. Eagle, H., Propagation in a fluid medium of a human epidermoid carcinoma,
strain KB, Proc. Soc. Exp. Biol. and Med., 1955, 89, 362.
7. Berman, L., Stulberg, C. S., and Ruddle, F. H., Long term culture of human
bone marrow, Blood, 1955, 10, 896.
8. Jordan, Jr., W. S., Human nasal cells in continuous culture, Proc. Soc. Exp. Biol.
and Med., 1956, 92, 867.
Downloaded from jem.rupress.org on July 31, 2017
1. Five types of human tissue when freshly explanted in vitro were found
to have varying but limited growth potential in contrast to the autonomous
and apparently unlimited growth of the transformed cell.
2. A requirement for an external source of inositol or glutamine could not
be demonstrated for the slowly multiplying primary amnlons in contrast to
the rapidly growing transformed cells which degenerated in the absence of
either metabolite.
3. Unlike the transformed cells, the primary amnion cells fixed an insignificant amount of COS; when infected by the Coxsackie virus, however, a requirement for COS in the formation of more virus became readily demoustrable.
4. The biggest difference in the incorporation of various C14 substrates occurred in the nucleic acid fractions.
5. The difference in the incorporation of glycine into the protein fraction
was very small.
6. The utilization of glucose, ribose, and x/lose as well as the oxidation of
lactic acid into COs by the primary amnlon cells occurred at a rate much
lower than that of the transformed cells.
7. The cytotoxic dose of 5-fluorodeoxyuridine for the primary amnlon was
at least 100 times that of the transformed cells although growth inhibition was
readily demonstrated at 0.1/~g. per ml. for both types of cells.
Based on these observations, several hypotheses are put forth for
consideration.
416
P~¥
AND TRANSFORMED HUM.AN CELLS
Downloaded from jem.rupress.org on July 31, 2017
9. Leighton, J., Kline, I., and Orr, H. C., Transformation of normal human fibroblasts into histologically malignant tissue in vitro, Science, 1956, 19.3, 502.
10. Bell, S. D., and Johnson, R. E., A strain of unclassified cells isolated from lymph
node of patient with reticulo-endotheliosis, Proc. Soc. Exp. Biol. and Med.,
1956, 9'2, 46.
11. Perry, V. P., Evans, V. J., Earle, W. R., Hyatt, G. W., and Bedell, W. C., Long
term tissue culture of human skin., Am. J. Hyg., 1956, 63, 52.
12. McCulloch, E. A., Continuous cultivation of cells derived from hemic cells of
man and pure strain of mice, Proc. Am. Assn. Cancer Research, 1956, 2, 132.
13. Baron, S., and Rabson, A. S., A culture strain of human epithelial-like cells from
an adenocarcinoma of the lung, Proc. Soe. Exp. Biol. and Meal., 1957, 96, 515.
14, Swim, H. E., and Parker, R. F., Culture characteristics of human fibroblasts
propagated serially, Am. J. Hyg., 1957, 66, 235.
15, Henle, G., and Deinhardt, F., The establishment of strains of human cells in
tissue culture, J. Immunol., 1957, 79, 54.
16. Girardi, A. F., Virus susceptibility of serially cultivated human heart tissue
cultures, Fed. Proc., 1957, 16, 414.
17. Syverton, J. T., and McLaren, L. C., Human cells in continuous culture. I.
Derivation of cell strains from esophagus, palate, liver and lung, Cancer Research, 1957, 17, 923.
18. Fogh, J., and Lund, R. O., Continuous cultivation of epithelial cell (strain FL)
from human amniotic membrane, Proc. Soc. Exp. Biol. and Med., 1957, 94,
532.
19. Zitcer, E. M., and Dunnebeck, T. H., Transformation of cells from normal human
amnion into established strains, Cancer Research, 1957, 17, 1047.
20. Fernandes, M. V., The development of a human amnion strain of cells, Texas
Rep. Biol. and Meal., 1958, 16, 48.
21. Hoch-Ligeti, C., and Hobbs, J. P., Behaviour of explants from human adult
bronchial epithelium in vitro, Proc. Soc. Exp. Biol. and Med., 1958, 97, 59.
22, Eagle, H., Nutritional needs of mammalian cells in tissue culture, Science, 1955,
129., 501.
23. Chang, R. S., Genetic study of human cells in vitro, J. Exp. Med., 1960, 111,235.
24. Frothingham, T. E., Effect of aging and rotation on human amnion cell response
to polio and sindbis viruses, Proc. Soc. Exp. Biol. and Med., 1959, 100, 505.
25. Zitcer, E. M., Fogh, J., and Dunnebacke, T. H., Human amnion cells for large
scale production of poliovirus, Science, 1955, 19.9., 30.
26. Geyer, R. P., and Chang, R. S., Bicarbonate as an essential for human cells in
vitro, Arch. Biochem. and Biophys., 1958, 73, 500.
27. Glick, D., Methods Biochem. Anal. 1957, 1,290.
28. Moore, A. E., Tumor formation by cultured cells derived from normal and cancerous tissues, Special Publ. New York Acad. So., 1957, 5, 321.
29. Chang, R. S., Liepens, H., and Margolish, M. Carbon dioxide requirement and
nucleic acid metabolism of human cells in vitro, Proc. Soc. Exp. Biol. and Med.,
in press.
30. Chang, R. S., Participation of bicarbonate in the synthesis of RNA and protein
as indicated by virus propagation in human cells, J. Exp. Meal., 1959, 109, 229.
31. Harbers, E., Chaudhuri, N. K., and Heidelberger, C, Studies on fluorinated
R. SHI~&N CHANG
32.
33.
34.
35.
36.
37.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
pyrivnidines. VIII. Further biochemical and metabolic investigations, J.
Biol. Chem., 1959, 234, 1255.
Rich, M. A., Bolafl~, J. L., Knoll, J. E., Cheong, L., and Edinoff, M. L., Growth
inhibition of a human tumor cell strain by 5-fluoro-uridine and 5-fluorodeoxyuridine-reversal studies, Cancer Research, 1958, 18, 730.
Eagle, H., Freeman, A. E., and Levy, M., The amino acid requirements of monkey
kidney cells in first passage, J. Exp. Meal., 1958, 1079 643.
Morgan, J. F., and Pasirka, A. E., Amino acid metabolism of normal and malignant cell cultures, Cannd. J. Biochem. and Physiol., 1960, 38, 399.
Dunnebacke, T. H., Correlation of the stage of cytopathic change with the release
of poliomyelitis virus, Virology, 1956, 2, 399.
Syverton, J. E., Comparative studies of normal and malignant human cells in
continuous culture, Special publication, New York Acad. So., 1957, 5, 331.
Hsu, T. C., and Moorhead, P. S., Mammalian chromosomes in vitro, J. Nat.
Cancer Inst., 1957, 18, 463.
Cofiell, L. L., McAllister, R. M., and Wagner, B. M., Criteria for determining
malignancy in tissue culture cell lines in the albino rat, Special Publ. New York
Acad. Sc., 1957, 5, 341.
Medearis, D. N., and Kibrick, S., An evaluation of various tissues in culture for
isolation of Eastern equine encephalitis virus, Proc. Soc. Exp. Biol. and Med.,
1958, 97, 152.
Quersin-Thiry, L., Nichoul, E., and Dekking, F., Echo virus type 9 as a cause of
epidemic meningitis, Science, 1957, 125, 744.
Shepard, C. C., A comparison of the growth of selected mycobacteria in Hela,
monkey kidney and human cells in tissue culture, J. Exp. Med., 1958, ].07, 237.
Cobb, J. P. and Walker, D. G., Effect of actinomycin D on tissue cultures of
normal and neoplastic cells, J. Nat. Cancer Inst., 1958, 2]., 263.
Chang, R. S., Indirect evidence of changes in nutrient requirements of human
epithelial-like cells in continuous culture, Special Publ. New York Aead. So.,
1957, 5, 315.
Lieberman, I., and Ove, P., Control of growth of mammalian cells in culture with
folic acid, thymidine, and purines, J. Biol. Chem. 1960, 235, 1119.
Eagle, H., The minimum vitamin requirements of the L and Hela cells in tissue
cultures, J. Exp. Med., 1955, ].02,595.
Eagle, H., Oyama, V. I., Levy, M., and Freeman, A., Myo-inositol as an essential
growth factor for normal and malignant human cells in tissue culture, Science,
1956, ].23, 845.
Eagle, H., Oyama, V. I., Levy, M., and Freeman, A. E., Myo-inositol as an
essential growth factor for normal and malignant human cells in tissue culture,
J. Biol. Chem., 1957, 9.26, 191.
Chang, R. S., Differences in inositol requirements of several strains of Hela,
conjunctival and amnion cells, Proc. Soc. Exp. Biol. and Meal., 1958, 99, 99.
Eagle, H., Agranoff, B. W. and Snell, E. E., The biosynthesis of mesoinositol by
cultured mammalian cells and the parabiofic growth of inositol-dependent and
inositol-independent strains, J. Biol. Chem., 1960, 235, 1891.
Potter, V. lZ., The biochemical approach to the cancer problem, Fed. Proc., 1958,
].7, 691.
Downloaded from jem.rupress.org on July 31, 2017
38.
417