Download A Factor from HeLa Cells Promoting Colonial

A Factor from HeLa Cells Promoting Colonial Growth
of Human Fibroblast-like Cells in Culture*
J. F. FOLEY,t B. J. KENNEDY,
(Department
of Medicine and Department
of Microbiology,
AND J. D. Ross
University of Minnesota,
Minneapolis,
Minnuota)
SUMMARY
Irradiated HeLa cells, lysed HeLa cells, and medium from irradiated HeLa cell cul
tures have been used as sources of a substance stimulating colonial growth of fibro
blast-like cells derived from human amnion cultures. Preliminary
characterization
suggested
that
the stimulating
substance
was not dialyzable,
was inactivated
by acid
or alkaline
hydrolysis,
was resistant
to degradationwith deoxyribonuclease
and ribo
nuclease, and was susceptible to tryptic digestion but resistant to boiling.
Interactions
between animal cells of diverse
cytologic type are of interest as regards the pos
sible relation to in vivo phenomena underlying the
organization of tissues and host cellular response
to malignant growth. A growth factor for human
fibroblast-like cells in culture elaborated by HeLa
cells was discovered in the course of attempts to
propagate human amnion cells by the use of irradi
ated HeLa “feeder―
layers (8). In several experi
ments,
irradiated
HeLa
cells
were
added
and 7.8. These solutions contained 0.1 mr@iglucose.
Salt solution was similar, except that calcium and
magnesium ions were 1 m@&,and phosphate and
phosphite
to amnion
cell monolayers : these cultures were overgrown by
fibroblast-like cells days to weeks earlier than were
control amnion cultures without HeLa cells. This
paper presents evidence for existence of a growth
stimulating substance released by HeLa cells and
describes some properties of the factor.
MATERIALS
AND METHODS
Media.—Solutions used as previously described
(10) included a culture rinsing solution and a cell
suspension-diluting
medium consisting of 1920mr@i
sodium chloride, 6 mr@ipotassium chloride, 0.1 nmi
magnesium and calcium ions (MgCl2' 6H20, CaCl2•
2H2O), and mixed phosphate (0.6 mM)-phosphite
(6 mM) buffer
S Supported
by
adjusted,
training
respectively,
grant
CRTY
5010
to pH
(C4)
of
the
7.3
Na
tional Cancer Institute,
U.S. Public Health Service, to Dr.
Jerome T. Syverton (deceased); an Institutional
Grant of the
American Cancer Society, IN 1SC; the Sabra Hamilton
Foundation; and in part by a Public Health Service fellowship,
number CSP 11,898, from the National Cancer Institute, Pub
lie Health Service.
t Special Fellowof the U.S. Public Health Service.
Received
for publication
August 13, 1962.
ions
92 and
20 m@i respectively.
For
basal medium the salt solution was supplemented
with the vitamins, amino acids, and glutamine of
Eagle's medium (3) as purchased from Microbio
logical Associates (Bethesda, Maryland). Penicil
un (100 mg/I), streptomycin
sulfate (100 mg/l),
and mycostatin (Squibb, 50 mg/l) were added to
complete growth medium. In early experiments
the energy source in basal medium was provided
by 10 mM fructose and 0.1 mr@iglucose supplement
ed with 0.1 mM pyruvate and 0.1 m@ oxalacetate.
This was replaced later by 10 m@ glucose without
effect on the system. Growth medium consisted of
basal medium supplemented with serum as noted.
Serum.—Calf serum was prepared from blood
obtained from an abattoir or purchased prepared
(Colorado Serum Company, Denver). Human
serum prepared aseptically was obtained locally.
All serum was filtered through Selas 03 filters,
pooled, and dispensed in small amounts
and
stored frozen at —18°C. until used.
Cells.—HeLa, line 2929of human cervical carci
noma (9), susceptible to polio virus, was obtained
from L. McLaren of the Department
of Microbi
ology. Stock cultures of HeLa cells were propagat
ed in basal medium supplemented with 20 per cent
calf serum.
Human fibroblast-like cells were obtained from
human amnion in primary culture. Membranes
from human placenta were obtained immediately
368
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1963 American Association for Cancer Research.
FOLEY et al.—Growth of Human
Fibroblast-like
Cells in Culture
369
after delivery and placed in culture rinsing solu
tion. Membranes were rinsed with culture rinsing
solution, the amniotic membrane was stripped
from the chorion and rinsed repeatedly with cul
ture rinsing solution to remove blood; then the
Wright's stain after 10—18days of incubation
colonies had achieved macroscopic size.
amniotic
HeLa cells.—Activity of a growth-stimulating
membrane
was stripped
free of clots with
forceps and placed in culture rinsing solution con
taming 0.2 per cent trypsin for 1 hour at 37°C.
The membrane was agitated for 3—5minutes to
free cells, and the freed cells were sedimented from
the supernatant
fluid at 60 X g for 15 minutes.
The cell pellet was redispersed in basal medium
supplemented
with S per cent calf serum and 15
per cent human adult serum, and large bottles
were inoculated with 15—30X l0@ cells per ml. of
medium. Cultures exhibiting prominent fibroblast
like growth after incubation were subcultured by
serial passage in basal medium supplemented with
20 per cent calf serum. Usually epithelial-like cells
were not seen after the first or second serial sub
culture. Fibroblast-like
cultures could be main
tamed by serial subculture from massive inocula
through ten or more passages over a 4- to 6-month
period. The term “fibroblast-like―cells refers only
to the morphologic state of the cells and is not
intended to denote a known mesothelial origin.
Cultural procedure.—Stock cultures of HeLa
cells and human fibroblast-like
cells of amniotic
origin were subcultured every 1—3weeks. Cultures
for passage were washed twice with culture rinsing
solution, treated for 20 minutes at 37°C. with
0.2 per cent trypsin, pipetted to disperse cells, di
luted in growth medium or in cell suspension-dilut
ing medium with 92per cent fetal calf serum (Hy
land Laboratories, Los Angeles), and inoculated in
appropriate
numbers into warmed Petri dishes
containing 5 ml. of medium. Cells were enumerat
ed with a hemocytometer
or a Coulter counter,
model B. All cultures were incubated at 37°C. ±
0.30 C. in a humidified,
continuously
changed
— 20°
C.
as
a
92 per
cent
(w/v)
solution
(10
X
tor
con
centrate). Ribonuclease
(Worthington
Biochemi
cal Corp., Friehold, New Jersey), 10 mg/mi in 0.9
per cent sodium chloride solution buffered with
0.02 M phosphate at pH 7.2, was stored at 4°C.
Deoxyribonuclease
(Worthington
Biochemical
Corporation,
type 1) was made up in 1 per cent
gelatin in balanced salt solution at pH 7.2. This
solution was stored at —70°C. and thawed imme
diately before use.
Observation of results.—Colonial cultures of
fibroblast-like
cells were stained with modified
for
human
fibroblast-like
HeLa cells was revealed
ments.
Culture
dishes
cells
factor from
fac
elaborated
by
by the following experi
were
each
seeded
with
2,000
human fibroblast-like cells from amniotic cultures.
Concomitantly,
test cultures were seeded with
varying numbers of IleLa cells which had received
an unfiltered dose of 2,600 roentgens in air deiiv
ered from a 220-ky. x-ray machine. In control
studies in which a total number of 37 X 106 cells
subjected to this dose of radiation were examined,
no HeLa cells were found to generate colonies of
cells. Although the number of seeded fibrobiast
like cells was constant, the number of fibroblast
like colonies with diameters greater than 1 mm.
afterincubation
for18dayswasalmost
propor
tional to the number of added irradiated HeLa
cells. The dishes in which 20,000; 40,000; and
80,000 radiated IleLa cells were added contained
eight, fourteen, and 32 such colonies. The fibro
blast-like cells in the control dishes without irradi
ated HeLa cells usually increased in size during
incubation. At the termination of the experiments,
the cells were very large in stretched area with
relatively small nuclei (Fig. a). In contrast, fibro
blast-like cells in colonies in the cultures contain
ing irradiated HeLa cells were much smaller and
fusiform in appearance (Fig. b). At termination of
the experiments,
the majority of the irradiated
HeLa cells had degenerated and had become de
tached from the dish surface. This stimulating
effect of radiated HeLa cells on human fibroblast
like cells was observed in eight separate experi
ments.
Time of release of HeLa factor.—A time study
at
mosphere of 92.5per cent carbon dioxide in air.
Enzymes.—Trypsin
(Difco 1 : 2.50) was dis
solved in culture rinsing solution and stored at
RESULTS
of growth-stimulating
Demonstration
when
was
done
to
see
when
HeLa
cells
elaborated
the
growth stimulant into cultural fluid. Five ml. of
basal medium containing 2 X 10@irradiated HeLa
cells was placed in each of a number of culture
bottles. Culture fluids were removed and replaced
after
2, 4, 6, 9,
12,
18,
and
28
days
of incubation.
Removed fluids were centrifuged to sediment cell
debris at 2,000 X g for 10 minutes, and the super
natant fluid was stored at —20@C. Portions of the
fluid were diluted equally with freshgrowth medium
(basalmediumwith 20percentcalfserum)for use
as
growth
medium
human
amniotic
ture
fibroblast-like
of
for
cultures
seeded
fibroblast-like
cells
after incubation exhibited
(Fig. a). Macroscopically
in
with
cells. Control
the
basal
2,000
cul
medium
only the enlarged cells
visible fibroblast-like
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1963 American Association for Cancer Research.
Cancer Research
870
Vol. 23, March
1963
colonies were generated in the presence of 10@
A portion of the HeLa culture supernate was
irradiated HeLa cells. Although fluid representa
placed in sterile washed dialyzing membrane in a
tive of the 4th day of HeLa cell culture produced
screw-cap
bottle
containing
an equal
volume
of
a few fibroblast-like
colonies,
appreciable
activity
fresh growth medium outside the membrane. After
of the growth stimulant as judged by colonies over
9 days at room temperature,
mediums inside and
1 mm. in size appeared first in the fluid representa
outside the membrane were tested for growth
tive of the 12th day of HeLa culture (Table 1).
stimulating
activity.
The medium
outside
the
HeLa cultures prepared and treated like those
membrane
contained
no activity,
whereas the
used for elaboration of growth stimulant were ana
medium inside the membrane promoted colonial
lyzed for number of glass-attached
cells and their
growth of the human fibroblast-like cells not seen
protein content. Protein of cells remaining at
in the control
fibroblast-like
cultures
in the usual
tached to glass was measured by the Oyama-Eagle
medium—i.e., colonies of fibroblast-like cells over
modification of the Folin-Ciocalteau
reaction (7). 1 mm. in diameter were 0, 23, and 0 in the control
Properties of these cultures (Table 1) suggested that
dish, the dish containing the dialyzed medium, and
elaboration of the growth stimulant occurred dur
the dish containing
the dialysate medium, re
ing the phase of culture when surviving cells were
spectively.
synthesizing protein, rather than during the early
Five-mi. portions of the HeLa culture supernate
phase when cells were most numerous. Lack of were adjusted, respectively, to pH 1.75 and 11.8
association
of factor
elaboration
with
active
HeLa
with 1 N hydrochloric acid and sodium hydroxide,
TABLE
1
RELATIONSHIPOF ELABORATIONOF GROWTHFACTORBYIRRADIATEDHELA CELLs
TO RELATIVE PROTEIN SYNTHESIS
Day:Q4691518*8CellcountXlO3
642
@gProtein/cell'
1,965
1,005
3,810
6,200
8,450
No. colonies greater than
01,488
lmm.insize1,676
S
Although
to indicate
electronically
determined
only relative
cell
protein content
counts
and
in
which
from
100
to
protein
500
experi
non-irradiated
HeLa cells and 2,000 amniotic fibrobiast-like cells
were mixed in dishes of medium consisting of basal
medium with 20 per cent calf serum. In two sepa
rateexperiments,
9Xl0@
HeLacellsfromvigor
ous
cultures
were
scraped
off
the
glass
surface
into
10 ml. of growth medium, then frozen and thawed
S times. Supernatant
fluid obtained by centrifuga
tion
stimulated
formation
of macroscopic
colonies
by amniotic fibroblast-like
cells, to suggest that
HeLa cells in sufficient number contain growth
stimulant.
Properties of the HeLa factor.—Some properties
of the factor elaborated by HeLa cells and stimu
lating colonial growth of the human amniotic
fibroblast-like
cells were determined by assay in
cultures seeded with 2,000 fibroblast-like cells. The
fluid containing marked activity from the previous
experiment
(that
obtained
933.4
045.6
determinations
are
accurate,
these
1835
values
should
be
considered
of cells.
cell growth was observed in three separate
ments
0472
5827
between
the
18th
and
28th day of incubation of the irradiated HeLa
cells) was used in the following experiments.
incubated at 37°C. for 60 minutes, and readjusted
to pH 7.0. Although the supernate at the usual pH
of 7.0 was active, the supernate treated with acid
or base was inactive. No colonies were found in the
testcultures,
but thecontrolculturewithun
treated HeLa factor contained fourteen macro
seopic colonies of fibroblast-like
cells. Control
growth medium subjected to the same increments
of chloride and hydrogen ions supported fibro
blast-like growth equally as well as untreated
growth medium.
A portion of the HeLa-culture supernate was
treated with trypsin (Difco 1 :2.50, 0.2 per cent
final concentration)
for 2 hours at 37°C., and
boiled for 15 minutes to inactivate the trypsin.
Examination
of fibroblast-like
colonies suggested
that the growth-stimulating
activity of the treated
fluid had resisted boiling and that its activity had
been
reduced
by tryptic
digestion
(Figs.
c, d, e).
Other portions of HeLa-culture
supernate were
treated,
respectively,
with deoxyribonuclease
(5 @g/mlfinal concentration)and ribonuclease
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1963 American Association for Cancer Research.
•_
.@.
0•
.*@.
0
0.•••.
a'
S
‘I
0―;'t@@
;@
A
@A
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1963 American Association for Cancer Research.
FIG. a.—Fibroblast-like
cell from a control
is very large in stretched
dish.
area with a relatively
The
cell
small nucleus.
Note its much greater area than a similar but multiplying
cell
in Figure b which is at the same magnification.
Wright's stain,
X30.
FIG.
Note
b.—Portion
the
of a colony
typical
of
fibroblast-like
fibroblasts
from
appearance.
Figure
Wright's
d.
stain,
X30.
FIGS.
C, d, e.—Effect
contained
of trypsin
2,000 human
on growth
fibroblast-like
factor.
Each
cells. The control
dish
dish
(c) contained
freshgrowthmedium
to whichtrypsinwas
added to a concentration of 0.@ per cent. The dishes of Figures
d and e contained growth fluid used to maintain radiated HeLa
cells. This fluid was known to contain the growth factor. To
one aliquot
of this fluid was added
trypsin
to a final concentra
tion of 0.2 per cent. Then the control fluid plus trypsin, the
growth factor fluid, and the growth factor fluid plus trypsin
were
incubated
at
37° C.
for
@Zhours.
Then
all
fluids
were
boiled for 15 minutes to inactivate
diluted equally with fresh growth
20 per cent calf serum) and used
contains the fresh growth medium
the trypsin. The fluids were
medium (basal medium plus
in the experiment. Figure c
plus trypsin. Figures d and
6 contain
and
the
growth
factor
fluid
plus trypsin, respectively. Cultures
Colonies were stained with Wright's
FIGS. f, g, h.—Effect
of RNase
the
growth
factor
fluid
were incubated 1@2days.
stain (shown X.6).
on growth
factor.
Each dish
contained 2,000 human fibroblast-like cells. To an aliquot of
medium used to maintain irradiated HeLa cells and known to
contain
the
growth
factor
was
added
ribonuclease
(100
@g/ml
final concentration).
The growth factor aliquot and the aliquot
containing the ribonuclease were incubated at 37°C. for 1 hour.
The fluids were then diluted equally with fresh growth medium
and used in the above experiment. Growth medium was basal
medium with 20 per cent calf serum. Figures e,f, and g contain
fresh growth medium, growth factor medium, and growth fac
tor medium plus RNase, respectively. Cultures were incubated
16 days. Colonies were stained with Wright's stain (shown X.6).
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1963 American Association for Cancer Research.
FOLEY et al..—Growth of Human
(100 ,@g/ml final concentration) for 1 hour at 370 C.
Growth-stimulating
activity for human amniotic
fibroblast-like
cells was not eliminated by this
treatment (Figs. f, g, h).
DISCUSSION
HeLa cells prevented from multiplying by x-radi
ation have been found to elaborate a factor into
culture fluid that stimulated fibroblast-like
cells
from human amniotic cultures to generate macro
scopically visible colonies. Active material was also
obtained from lysed HeLa cells, although IleLa
cells generating colonies in amniotic fibroblast-like
cultures did not produce or give off enough factor
to show effect. Since control fibroblast-like cells in
the absence of the factor did not degenerate but
survived in culture and enlarged and since activity
of the factor affected colony frequency more than
colony size, it appears that the factor in some man
ner promoted continuing cell division. The results
of preliminary characterization
suggested that the
factor was not dialyzable, was resistant to boiling
and treatment
with ribonuclease and deoxyribo
nuclease, and was susceptible to inactivation
by
trypsin. These properties would be exhibited by a
glycoprotein. Present results suggest that elabora
tion of the factor in quantity by HeLa cells may
not be associated with growth but with degenera
tion or protein synthesis without cell division.
Quantitative
biological and immunological assays
are needed to further resolve the origin and nature
of the factor. Initial observations suggest that fac
tor activity is not limited to the fibroblast-like cells
from amniotic cultures: stimulation of colony gen
eration has been observed with single specimens of
fibroblast-like
cells from human fetal lung and
breast cancer tissue.
Growth-stimulating
factors for animal cells in
culture reported by other workers have included
materials nondialyzable
as well as dialyzable. A
nondialyzable
proteinaceous
substance described
by Alfred and Pumper (1) was elaborated
by
mouse lung cells adapted to growth in a protein
free medium. This substance, stable at 56°C. for
45 minutes, could replace serum in medium for
growth of Chang liver cells in culture.
Low
Baron and
(2)reported that skim milk used as a serum
substitute
for cultural
maintenance
of cells con
Fibroblast-like
371
Cells in Culture
tamed a nondialyzabie
growth-stimulating
sub
stance withstanding boiling for 5 minutes or auto
claving for 15 minutes. An active nondialyzable
component of chick embryo extract was described
by Kutsky and Harris (6) as a protein extractable
together with nucleic acids. By ethanol precipita
tion of homogenates of human and animal tumors,
Kuru,
Kosaki,
Ito, Matusima,
and Matudo
(4, 5)
isolated a protein substance called “Oncotrephin.―
Like the substance reported here, this material was
nondialyzable,
withstood
boiling, and was de
stroyed by acid hydrolysis. Unlike our growth fac
tor, Oncotrephin
was not affected by trypsin,
although it was destroyed by the bacterial proteo
lytic enzyme, pronase. The mode of action of these
materials and of our stimulating
factor is un
known.
REFERENCES
1. ALFRED,L. J., and Puau'nn, R. W. Biological Synthesis of
a Growth Factor for Mammalian Cells in Tissue Culture.
Proc. Soc. Exp. BioL Med., 103:688-91, 1960.
2. BARON, S., and Low, R. J. New Maintenance
Medium
for
Cell Culture. Proc. Soc.Exp. Biol. Med., 128:89-90, 1958.
3. EAoi@n,
H. NutritionalNeeds of MammalianCellsin Tis
sue Culture.
Science, 122:561-4,
1955.
4. Kos@txx,G.; Iro, E.; M@usnt@t, T.; and K@mu,M. Fur
ther Analytical Studies on Oncotrephin, the Mitosis Pro
moting
Substance
in Malignant
Tumors;
Influences
of
Proteolytic Enzymes and Acid Hydrolysis upon Effective
Substances from Human Hepatoma Precipitated
by Etha
noL Gann, 51:409—13, 1960.
5. Kunu, M. ; KosAxI, G. ; M@nmo, K. ; and ITO, E. Electro
phoretic Analysis of Oncotrephin, the Mitosis Promoting
Substance in Malignant Tumors. Gann, 51:201—fl, 1960.
6. Ktrrsxy, R. J. Nucleoprotein Constituents Stimulating
Growth in Tissue Culture: Active Protein Fraction. Sci
ence, 129:1486—87, 1959.
7. OYAMA, V. I., and EAoui, H. Measurement
of Cell Growth
in Tissue Culture with a Phenol Reagent (Folin-Ciocal
teau).
8. Pucx,
Growth
tics of
Feeder
Proc. Soc. Exp. Biol.
Med.,
91:305—7, 1956.
T. T.; MARcUS, P. I.; and CIsx,iuL&, S. J. Clonal
of Mammalian Cells in Vitro. Growth Characteris
Colonies from Single HeLa Cells with and without
Layer. J. Exp. Med., 103:273—83, 1956.
9. ScHERER,W. F.; SYVERTON,
J. T.; and GEY, G. 0. Studies
on the Propagation in Vitro of Poliomyelitis Viruses. IV.
Viral Multiplication
in a Stable Strain of Human Malig
nant Epitheial
Cells (Strain HeLa) Derived from an Epi
dermoid Carcinoma of the Cervix. J. Exp. Med., 97:695709, 1957.
10. TREADwELL,P. E., and Ross, J. D. Effects of Human
Adult and Bovine Fetal Serum on Colonial Propagation
Rabbit Cells. J. Natl. Cancer Inst., 28:679—90, 1962.
of
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1963 American Association for Cancer Research.
A Factor from HeLa Cells Promoting Colonial Growth of Human
Fibroblast-like Cells in Culture
J. F. Foley, B. J. Kennedy and J. D. Ross
Cancer Res 1963;23:368-371.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/23/3/368
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1963 American Association for Cancer Research.