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Studies on the uptake of deoxyribonucleic acid by synchronized mammalian cells in tissue culture

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Studies on the uptake of deoxyribonucleic acid by synchronized mammalian cells in tissue culture

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Content STUDIES ON THE UPTAKE OF DEOXYRIBONUCLEIC
ACID BY SYNCHRONIZED MAMMALIAN CELLS
IN TISSUE CULTURE
by
Joel E. Adams
A D issertation P resented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In P artial Fulfillm ent of the
R equirem ents for the D egree
DOCTOR O F PHILOSOPHY
(Biology)
June 1964
UNIVERSITY O F S O U T H E R N CALIFORNIA
T H E G R A D U A T E S C H O O L
U N I V E R S IT Y P A R K
L O S A N G E L E S . C A L I F O R N I A 9 0 0 0 7
This dissertation, written by
_____ J.Q.el_K.. A dam s....................
under the direction of his.....Dissertation C o m
mittee, and approved by all its m embers, has
been presented to and accepted by the Graduate
School, in partial fulfillment of requirements
for the degree of
D O C T O R O F P H I L O S O P H Y
Dr an
Date..................June,. 1964
DISSERTATION COMMITTEE
a. A
Chairman
ACKNOWLEDGMENTS
I would like to ex p ress my appreciation to the m em bers
of my com m ittee, Dr. W. E. M artin, Dr. C. M. Pom erat,
Dr. P. R. Saunders, Dr. A. S. Dunn, Dr. B. G. Monroe, and
M r. R. L. Z im m er for th eir help in the preparation of this
thesis. I want to extend special thanks to Dr. D. E. Rounds and
Dr. F. H. K asten of the Pasadena Foundation for M edical
R esearch for th e ir suggestions and c ritic a l evaluations during the
course of the work.
I am grateful to the m em bers of the technical and clerica l
staff of the Pasadena Foundation for M edical R esearch for th eir
willing and continued help.
My sincere gratitude to my wife for her patience and u nder
standing throughout the course of this work.
This work was supported in p art by U. S. Public Health Grant
CA-03546-06, adm inistered by Dr. W. E. M artin and in p art by
the National Science Foundation Grant GB-15, adm inistered by
Dr. C. M. P om erat.
TABLE OF CONTENTS
Page
INTRODUCTION....................... 1
H istorical Review . . . ..................... ............................. 1
Effects of DNA on developing em bryos .... 1
DNA m ediated tran sfo rm atio n s t u d i e s 3
Studies on the induction of tu m o rs with
D N A ........................... 8
Studies with naked v iral D N A ............................. 9
Other effects of DNA . . ........................................... 11
In vivo DNA uptake s t u d i e s .................. 13
In vitro DNA uptake studies . . . . . . . . . .......... 16
STATEMENT OF THE PROBLEM AND METHOD
OF A T T A C K ............................................. . 21
MATERIALS AND M ETH OD S.............................. 24
M a te r ia l s ........................................................ 24
Routine stock culture m e th o d s ....................... 24
P rep aratio n of conditioned cu ltu res . .. . . . 26
P rep aratio n of synchronized c u ltu re s .... 26
M easurem ent of the degree of synchrony . 27
P rep aratio n of highly polym erized
DNA s o lu tio n ................................................. 28
P rep aratio n of low polym er DNA
solution ....................... 29
Page
Fixation and sta in in g .................... ................... 29
P hotography.......................................................... 29
Procedure for scoring DNA uptake
by c e l l s ............. ................. .. o.... 30
Determination of the degree of synchrony . 31
Experim ental p la n . . . ...................... 31
Effect of low concentrations of DNA . . . „. 31
Effect of washing and exchanging
medium ...................................... 32
Effect of high concentration of D N A ....... 32
Observation of pinocytosis of DNA-
treated c e l l s ................. 33
R ESU LTS...................... 35
Experim ents A and B: Production of
synchronized cells and determ ination
of duration of interphase periods .... 35
Experiment III-1: Low concentration
of highly polymerized DNA .. ....... 36
Experiment IV-D: Low concentration
of low polymer DNA ............................. 36
Experiment III-J: Highly polymerized
DNA after washing and exchanging
medium ............................. 36
Experiment IV-E: Low polymer DNA
after washing and exchanging
medium ............... 37
iv
Page
E xperim ent IV-F: High concentration
of low polym er DNA . ........................ 38
E xperim ent IV-G: D em onstration of
pinocytosis in DNA-treated cells ...». 39
DISCUSSION . . . . . . . . . . . . . 51
SUMMARY . . . . .. . 60
LITERATURE CITED ............... 62
A P P E N D IX .. .................... 71
LIST OF TABLES
Table
1 HeLa S3 Cells Synchronized after Two
P assa g es in Medium Containing
A m ethopterin-A denosine and Thymidine
2 HeLa S3 Cells Synchronized after T hree
P a ssa g e s in Medium Containing
A m ethopteii n-Adenosine and Thymidine
3 Uptake Index of HeLa S3 Cells after T re a t
m ent with 20 jug/ml of Highly Polym erized
DNA
4 Uptake Index of HeLa S3 Cells after T re a t
m ent with 20 jug/ml of Low Polym er DNA
5 Uptake Index of HeLa S3 Cells after T re a t
m ent for Six Hours in New Medium
Containing 20 jug/ml of Highly Poly
m e riz ed DNA
6 Uptake Index of HeLa S3 C ells after T re a t
m ent for Six Hours in New Medium
Containing 20 jug/ml of Low Polym er DNA
7 Uptake Index of HeLa S3 C ells after T re a t
m ent for Six Hours in New Medium
Containing 500 jug/ml of Low Polym er
DNA
Page
40
43
46
47
48
49
50
LIST OF TEX T-FIG U R ES
D eterm ination of the d eg ree of
synchronization of HeLa S3
cells conditioned for two
p assag es, by am ethopterin-
adenosine and thym idine
D eterm ination of the degree of
synchronization of HeLa S3
cells, conditioned for th re e
p assag es, by am ethopterin-
adenosine and thym idine
INTRODUCTION
There have been num erous studies dealing with the effects of
exogenous deoxyribonucleic acid (DNA) on anim al cells bothjin vitro
and in vivo; however, there appear to be no reviews of the subject.
The purpose of this sum m ary is to give the read er as complete a
coverage as possible of the literatu re concerning work with DNA
from various sou rces on anim al cells. This section has been
divided into 7 m ajor categories in an effort to give some degree of
unity to the studies reported.
Evidence for DNA as a genetic m a te ria l received great
im petus with the report by Avery et al. (1944), of the successful
transform ation of a nonencapsulated b acterial type of DNA
extracted from a related but encapsulated type. Since then there
have been num erous rep o rts of bacterial transform ation and these
have been the subject of se v eral review s and will not be included
here.
H istorical Review
Effects of DNA on developing em bryos
M azia (1949), in an effort to determ ine if there was any
species specificity, studied fertilized frog (Rana pipiens) and
sta rfish (A ste ria s forbesii) eggs which w ere cultured in w ater
containing eith er homologous or heterologous DNA. He found that
frog eggs w ere a rre s te d at the late yolk plug stage of gastrulation
by homologous DNA but rem ained unaffected by a preparation from
calf thymus. The effect was rev ersib le if em bryos w ere not
blocked longer than 24 hours. Starfish em bryos w ere also
a rre s te d at the g astru la stage by homologous DNA but not by that
p rep ared from eith er the sea urchin or the sand dollar. However,
the sta rfish em bryos differed from those of the frog in that they
began to disintegrate after a very few hours in the gastrula stage
and the block was not rev ersib le by rem oval of DNA from the
culture medium.
U nfertilized sea urchin eggs w ere injected with homologous
and heterologous sp erm DNA by H orstadius et _al. (1954). They
rep o rted that fertilization m em branes w ere raise d and a surface
color change occurred in those eggs in which the DNA becam e
d isp ersed in the cytoplasm , but that they failed to develop. If the
injected m a terial rem ained undistributed, it eventually was
expelled as a drop from the surface and these eggs could then be
fertilized and develop norm ally. In a few c a ses the DNA-activated
eggs underwent an abortive cleavage before cytolysis occurred.
They w ere unable to detect any species specificity of the various
3
DNAfs employed in this study.
M artinovitch et ad. (1962) describ ed sev eral teratological
changes in and F 2 generations of White Leghorn chickens which
had been injected during the early em bryonic period with DNA
prepared from Rhode Island Red te s tis . Their DNA was dissolved
in tyrode salt solution which alone produced sim ila r but far le ss
m arked anom alies.
DNA mediated tran sfo rm atio n studies
In a se rie s of rep o rts beginning in 1957, Benoit et al. (1957a,
b, 1958, 1959, 1960a, b) described the effect of se ria l injections
of new ly-hatched Pekin ducks with DNA extracted from the te stis
or erythrocytes of Khaki Cam pbell ducks. They first observed an
altered pigmentation of the bill in tre a te d anim als which has been
described as a som atic transform ation. The F^ generation con
tinued to show som e of the pign ent changes observed in the
tre a te d parents and this was presented as evidence for a genetic
transform ation of the germ cells of injected p aren ts. The F 2
generation was reported to have pigm ent distribution sim ila r to
that observed in the F j anim als. In an effort to answ er questions
raise d as to the genetic purity of th e ir original anim als, they have
reviewed th eir early work and presented more detailed descriptions
of the morphology of the parents and progeny in th eir most recent
reports.
Induction of resistance to alanine nitrogen m ustard in sensitive
Yoshida sarcom a cells growing in ascites fluid of white rats,
following in vitro exposure to DNA extracted from resistant cells,
was claimed by Kurita et al. (1958). They failed to obtain
resistant cells if the DNA was first subjected to deoxyribonuclease
digestion or if the cells were exposed to native DNA for periods
less than 30 minutes. The treated cells were allowed a 3-day
growth period in the ascites fluid before the drug was adm inistered.
P erry and W alker (1958) failed to produce pigment in white
(Addis-Slonaker) rats or their Fj progeny following repeated
injections of DNA prepared from pigmented (Long-Evans) rat
tissues. They studied a total of 135 anim als, but indicated that
the failure to detect a change may have been the result of too sm all
a sample.
Employing DNA prepared from liver, spleen or testis of
August ra ts, Bearn and Kirby (1959) were unable to produce pig
mentation following injection of newborn W istar rats. They also
reported no effects on W istar em bryos which survived intra uterine
injection of August DNA at the blastocyst stage.
Schoffner et al. (1961) failed to detect any phenotypic changes
in White Rock chickens which w ere injected a s hatchlings with DNA
from ery th ro cy tes of Rhode Island Red chickens. They did not
observe any evidence of a genotypic change a fte r exam ination of
nearly 600 progeny. However, they pointed out that th e ir study
included only g ro ss m orphological observations and a change might
have been detected had a histological survey been m ade.
AKR m ice w ere tre a te d with DNA ex tracted from DBA m ouse
tissu e by Holoubek and H nilica (1961). They did not observe any
alteratio n of pigm entation or body configuration in e ith e r the
tre a te d anim als or their progeny which w ere followed for 3
generations. They also attem pted to d eterm in e if the tre a te d
anim als would accept skin g rafts from the DNA donor sp ecies, but
this proved unsuccessful. This indicated that th e re had been no
im m unological changes produced.
K raus (1961) employed tritiu m -la b e le d DNA from m arrow
cells of hum ans who w ere homozygous for type A hem oglobin to
tr e a t m arrow elem ents in tissu e culture from individuals
homozygous for type S hem oglobin. Labeling as detected by auto
radiography appeared o ver the nuclei of tre a te d ce lls afte r 16 to
48 hours. An electrophoretic an aly sis of c e lls exposed to DNA for
10 days showed both type S and type A hemoglobin.
Oregon K m ale D rosophila w ere injected with DNA extracted
from wild type tissu e s and the progeny of the treated m a le s and
untreated fem ales w ere examined by Fahm y and Fahm y (1961).
They found a twelvefold in crease in the num ber of ’’m inutes"
(reduced length and thickness of thoracic m acrochaetes) over the
control rate .
Szybalski et^aL (1962), with s im ila r techniques to those e m
ployed for b a c te ria l tran sfo rm atio n studies, w ere able to
dem o n strate the action of an enzym e not fo rm e rly p re se n t after
tre a tm e n t of a human bone m arrow cell line with DNA ex tracted
from an isologous cloned population which p o sse sse d the enzyme
activity. L ater these w orkers gave a m ore com plete d escrip tio n
of th e ir m ethods (Szybalska and Szybalski, 1962). They found
that a clone of the D etroit 98 cell line p o sse sse d inosinic acid
pyrophosphorylase (IMPPASE) activity as evidenced by the fact
that th ese cells w ere able to form colonies when plated in a
m edium which inhibits growth of ce lls lacking this enzym e.
S everal other clones isolated from the D etroit 98 line did not
fo rm colonies in the selective m edium . A fter trea tm en t of cells
lacking the enzyme with DNA from those showing activity for
sh o rt p erio d s, then plating on the selectiv e medium , they obtain
ed up to 4 tran sfo rm ed cells per one thousand treated. DNA
p re p a re d from HeLa, ra t, or m ouse cells did not produce
surviving cells detectable by th e ir techniques. This gave added
support to the conclusion that they had induced a specific bio
chem ical tran sfo rm atio n .
F lo ersh eim (1962) em ployed DNA ex tra cte d from CBA m ice
to tre a t hem opoietic cells of C s tra in m ice. He injected the
tre a te d cells into CBA m ice which had rece iv ed a lethal dose of
d im ethyl-m yleran (an alkylating agent producing pancytopenia)
in an effort to d eterm in e if histocom patability had been conveyed
to the C strain cells a s evidenced by su rv iv al of the m ice. None
of the anim als receiving homologous (C strain) cells survived,
but all of those tran sfu se d with isologous (CBA) cells lived. This
indicated the absence of tran sfo rm ed cells. However, he pointed
out that in terv al between DNA tre a tm e n t and the injection of cells
into m ice may not have been adequate fo r the developm ent of a
sufficiently la rg e population of tra n sfo rm e d cells to in su re s u r
vival of the anim als.
T ransform ation of 8 -azaguanine sen sitiv e cells with DNA
ex tra cte d from a re s is ta n t subline was re p o rte d by B radley et al.
(1962). They found th a t the D N A -treated ce lls, which w ere
capable of form ing colonies in a m edium containing 8 -azaguanine,
reta in ed their re s is ta n c e after se v e ra l p a ssa g e s in the absence
of the inhibitor.
8
Studies on the induction of tu m o rs with DNA
L euchtenberger et al. (1958) injected BA LB/C and CF^ m ice
with DNA p rep ared from liv e r, spleen or b re a s t tum or cells from
Agouti C 3 H m ice. In those anim als tr e a te d with tum or DNA, they
found a re a s of the liver with abnorm ally la rg e cells which had
nuclei of increased size, PAS positive nucleoli and d ecreased cyto
plasm ic glycogen s to r e s 0 They also found, by cytophotom etry,
cells containing in c re ase d am ounts of DNA u nrelated to norm al
polyploidy,, None of th ese changes w ere found in control m ice or
those tre a te d with spleen o r liver DNA0 They stated that th eir
re su lts w ere sim ila r to those found in m ic e which had received
Sarcom a 180 cells.
Newborn C3 H m ice w ere injected w ith com m ercially p re p a re d
h e rrin g -sp e rm DNA by Hewer and Meek (1958). The anim als w ere
autopsied, in a m oribund condition, 23 days after introduction of
the DNA and found to have m etastatic adenocarcinom as of the
duodenum. L itter m ate controls w ere healthy at 23 days and, when
sa crificed la ter, showed no evidence of tu m o rs. The sam e author^
M eek and Hewer (1959), stated that a s c ite s tum or cells produced
originally by injection of heterologous DNA into m ice had been
c a rrie d through se v eral p assag es in tis s u e culture.
B earn (1959) failed to produce tu m o rs in W istar ra ts injected
subcutaneously or intraperitoneally with DNA prep ared from ra t
sarcom a or hepatom a cells.
Several hundred newborn m ice w ere injected with DNA ex
trac ted from norm al or m alignant anim al tissu e or tissu e culture
cells by Dixon et al. (1961). The anim als w ere followed for n e a r
ly a y e a r, and at autopsy it was found that approxim ately 1 0 per
cent of those receiving DNA of m alignant origin had tu m o rs,
usually at the site of injection. None of the m ice which had been
trea ted with DNA of nonm alignant origin developed tu m o rs.
Studies with naked v iral DNA
Di M a y o rc a ^ t aL (1959) obtained a p rep aratio n by phenol
extraction of polyom a-infected tissu e culture cells which, when
incubated with mouse em bryo cells, produced cytopathogenic
effects identical to those seen following v ira l infection. They
w ere able to isolate whole v iru s from the tre a te d cells. Incuba
tion of the e x tra c t with deoxyribonuclease destroyed its activity,
but ribonuclease was ineffective. The fact that neither enzym e is
effective against whole v iru s was given as evidence that a subviral
agent was the active principle.
A phenolic ex tra ct of Shope papillom a v iru s was employed to
10
produce tu m o rs in do m estic rab b its by Ito (1960). He re p o rte d
that the p rep aratio n was inactivated by p re tre a tm e n t with deoxy
ribonuclease but rib o n u clease at the sa m e concentration w as not
effective. T reatm en t of the m a te ria l with specific a n tis e ra did
not dim inish its ability to produce tu m o rs in rab b its.
W eil (1961) re p o rted th a t a su b v iral agent, p re p a re d by
phenol ex tractio n from polyom a v iru s, produced plaques in tissu e
cu lture which w ere identical to those obtained with whole v iru s.
However, he found that the ex tra ct w as s e v e ra l o rd e rs of m agni
tude le s s effective than the v iru s 0 The phenol p rep aratio n was
se n sitiv e to deoxyribonuclease but not to ribonuclease.
Ito and Evans (1961) re p o rte d that tum or induction by a phenol
e x tra c t of polyom a v iru s or v ira l-in fe c te d tis s u e w as not inhibited
by incubating the m a te ria l with try p sin , chym otrypsin, specific
a n tis e ra or low doses of ribonuclease, but w as prevented by de
oxyribonuclease or high d oses of rib o n u clease. They concluded
that the active p rin cip le w as p ro te in -fre e v ira l DNA and attrib u ted
the effect with high concentrations of rib o n u clease to a non
specific binding of the enzym e to th e nucleic a c id 0 Heating of the
phenol e x tra c t to 100°C for 30 m inutes (Ito, 19 61a) did not reduce
its capacity to induce tu m o rs and, sin ce this is 10°C above the
m elting point of DNA, he suggested that ren atu ra tio n had o cc u rred .
11
P reparation of a tumorigenic extract from polyom a-infected
tissue of wild and domestic rabbits by various phenol techniques
was reported by Ito (1961b). He found no significant difference
in the activity of the several preparations.
Orth et al. (1962) described the characteristic nuclear
lesions and subsequent cytolysis following incubation of newborn
mouse kidney cells with DNA extracted from polyom a-infected
cells.
DNA prepared from SV^q virus was reported to produce
cytopathic changes in monkey kidney cells by G erber (1962). He
stated that the lesions obtained with the DNA w ere identical to
those produced by whole virus and that deoxyribonuclease,
but not ribonuclease, destroyed the activity of the extract.
Diderholm and W esslen (1963) produced lesions in cells non-
susceptible to whole virus with DNA extracted from polyoma and
SV4 0 viruses. With the polyoma extract, they obtained typical
lesions in kidney cells from monkey, swine, and calf, w hereas
with SV4 Q DNA they were able to infect mouse, swine and calf
cells.
Other effects of DNA
Dumont (1959) found that DNA, prepared from sev eral species,
12
enhanced fibroplasia during the healing of experim entally p r o
duced wounds in the rabbit ear. However, the effect w as m uch
g re a te r if the DNA was ad m in istered in conjunction with deoxy
ribonuclease. T herefore, he concluded that the phenom enon was
p rim a rily due to uptake of p re c u rs o rs resulting from enzym e
digestion of the nucleic acid.
Strain L fibroblasts w ere grown in the p re se n c e of DNA ex
tra c te d from m ouse sa rc o m a 180 or E hrlich ascites cells by
Smith and C ress (1961). They found that the L cells becam e
rounded and g ran u lar and failed to m ultiply, but w ere unable to
produce sim ila r d etrim ental effects with DNA p re p a re d fro m
sa rc o m a 180 cells grown in tissu e culture. The inhibition was
prevented if the DNA p rep aratio n s w ere firs t incubated with deoxy
ribonuclease.
Rounds (1961) re p o rted a slight rad ioprotective effect with
calf thym us DNA when it was added to cultures of am nion cells
for a 24-hour period preceding exposure to 700 r of gam m a ra d ia
tion from a cobalt-60 source. He obtained a g re a te r degree of
protection from ribonucleic acid (RNA) or o rotic acid, however,
and this finding, plus the necessity of incubating the cells with the
p rep aratio n for 24 hours p rio r to irra d iatio n in o rd e r to obtain
protection, led him to the conclusion that the p rim a ry effect was
13
due to p re c u rs o rs of RNA.
Djordjevic^et al. (1962) subjected s tra in L cells to u ltra
violet radiation and then tra n s fe rre d them to medium containing
isologous DNA and followed their growth ra te for 4 days. C ells
receiving DNA after irrad iatio n m ultiplied fa ste r than those i r
radiated but not grown in the p rese n ce of DNA; how ever, both
w ere far below the controls. They speculated that the enhance
m ent of growth of cells in the p re se n c e of DNA over those not
tre a te d was probably due to incorporation of p r e c u r s o r s since the
difference between the two groups was m o st m arked on the 4th day
p o stirrad ia tio n .
In vivo DNA uptake studies
Since 1959, num erous re p o rts of uptake of high m olecular
weight DNA have ap p eared in the lite ra tu re . Employing m ouse
lym phom a cells in the peritoneal cavity and a P ^ - l a b e l e d b a c te
ria l DNA p rep aratio n , Sirotnak and Hutchison (1959) found that
uptake reach ed a peak afte r 5 minutes* incubation, then leveled
off. They observed that absorption was g re a te s t when cells w ere
approaching the plateau of the growth phase,
H udnik-Plevnik et al, (1959) noted uptake of polym erized
P ^ - l a b e l e d homologous spleen DNA by elem ents of the ra t spleen,
14
following in tra p e rito n e a l injection. However, cells of the liv er,
te s tis and blood did not show in co rp o ratio n of the label.
W orking with le th a lly -irra d ia te d m ic e which received in je c
tions of homologous thym ocytes containing p32_iabeled DNA,
Hill and D ra sil (1960) found F eulgen-positive, labeled m a te ria l in
the cytoplasm and nuclei of c e lls from lymph nodes, spleen,
m arro w , kidney, gut and liver,, In an attem pt to show that the
DNA was in a p o ly m erized condition, th e se w o rk e rs com pared the
num ber of g rain s over the cells when labeled thym ocytes w ere in
jected, as contrasted w ith labeled DNA p r e c u r s o r s or inorganic
p32^ They found that the am ount of labeling of individual cells
w as se v e ra l o rd e rs of m agnitude g re a te r when thym ocytes w ere
injected than when e ith e r the labeled p r e c u r s o r s or inorganic P*^
w e re employed. In an extension of his e a rlie r work, Hill (19 61a)
found P ^ 2 concentrated in c e rtain a re a s of the m arrow cell
nucleus. F rom this he postulated that DNA entered at specific
points in the nuclear m em b ran e. Hill (1961b) re p o rted that g ran u
locytes of the m arrow showed the g re a te s t uptake of polym erized
O O
DNA from P -lab eled thym ocytes injected into m ice. None of the
m itotic figures from th e m arro w contained labeled m a terial.
T h erefo re , he concluded that thym ocyte DNA w as not incorporated
by cells during the synthetic p erio d of the interphase cycle.
T ritiu m -lab eled DNA, extracted from isologous lymphocytes
and sarco m a cells, was injected into ascites fluid of tum or-
bearing m ice by Rieke (1962). He found that only cells capable of
DNA synthesis contained labeled m aterial. In an effort to d e te r
m ine if the degree of polym erization influenced the uptake, he
com pared the amount of labeling of cells treated with high polym er
DNA with that found afte r incubation with DNA which had been p r e
viously subjected to deoxyribonuclease digestion. M oreover, the
high polym er DNA was m o re effective in producing labeling of the
cells. He concluded that the DNA was apparently ingested by pino-
cytosis ra th e r than phagocytosis.
In a recent study, Hill and Jokubickova (1962) rep o rted the
incorporation of p32_labeled thymocyte DNA into the cytoplasm
and nuclei of m arrow ce lls which w ere synthesizing DNA.
Employing homologous tritiu m -lab eled DNA injected in tra
venously in m ice, T su m ita and Iwanaga (1963) found that after
30 m inutes, approxim ately one per cent of the labeled DNA was in
cells of the liver. They repeated the experim ents with an acid-
hydrolyzed preparation and w ere unable to detect the label in
hepatic elem ents after the sam e tim e interval.
Fichtelius and G roth (1963) transplanted cells which had been
stained with trypan blue and also contained tritium -labeled DNA
16
subcutaneously into m ice. With autoradiographic techniques,
they found leukocytes, which invaded the tran sp lan t site, to con
tain both the label and trypan blue stained phagocytized m aterial.
However, they did not determ ine to what degree the labeled m ate
r ia l was polym erized.
In vitro DNA uptake studies
Using homologous tritiu m -la b e le d DNA with Henle cells in
tissu e culture, G artler (1959) rep o rted the uptake of about one per
cent of the label after incubation for one hour. He also added the
labeled DNA in the p rese n ce of unlabeled thymidine or thym idilate
and found no significant change in the amount of label incorporated.
However, when labeled DNA was added in the p resence of non
radioactive DNA, the degree of labeling was greatly reduced. On
the basis of this observation he concluded that the DNA was taken
up in an undegraded form .
The stra in L fib ro b lasts, grown in suspension culture and
tre a te d with tritiu m -la b e le d DNA extracted from mouse m elano-
b lasts or sa rc o m a 180 cells and then coated with gelatin, w ere
found by King and Bensch (1960) to contain Feulgen-positive
m a te ria l in the cytoplasm which was shown to be active with the
use of autoradiography.
17
Ehrlich ascites cells grown in flask cultures and treated with
tritium -labeled DNA for two hours by Chorazy et aj^ (1960) were
harvested and fractionated. These w orkers found activity only in
the DNA moiety out did not determ ine whether the DNA was of
nuclear or cytoplasmic origin.
14
E hrlich-L ettre cells were treated with autologous C -labeled
DNA for one hour bv Kay (1931). He then harvested the cells,
extracted and chrom atographed the DNA from the treated elements,
14
and compared their patterns with those prepared from the C
labeled DNA. From these observations he concluded that some
high molecular weight DNA had been tran sferred intact through
the cytoplasm and into the nucleus.
Uptake of DNA by He La cells in culture was first studied by
Borenfreund and Bendich (1S31). They labeled DNA from human
leukocytes and pneumococci, following the Wilzbach technique,
and treated cells for periods of 1 to 24 hours. Autoradiographs
showed that the cultures subjected to the laoeled m aterial for 8
hours contained approximately 35 per cent tagged cells, while
those exposed for 3 hours or less were virtually unlabeled.
Cultures in contact with the DNA for 24 hours were reported to
be alm ost 100 per cent labeled. When treated cells were
harvested and then the DNA extracted and chromatographed, they
18
found all four bases to be equally radioactive. M oreover, the
fact that most of the activity was rem oved by p retreatm en t with
deoxyribonuclease, but not with ribonuclease or mild hydrolysis
at low tem perature, led them to the conclusion that high m olecular
weight DNA was incorporated into the cells. They'proposed
pinocvtosis as the mechanism by means of which high polym er
DNA enters the cell.
Strain L fr moolasts w ere subjected to heterologous
m ammalian or bacterial DNA in suspension culture for periods of
2 hours :> v Fensch and King (1831). After treatm ent with the DNA
which had oei-n (1 ) coacervated by the addition of gelatin, or (2 )
oound with acridine orange, or (3) previously labeled with tritia te d
thymidine, they plated the cells and allowed them to form a m ono
layer. They found Feulgen-positive a re a s in the cytoplasm and
nucleus which correlated with the location of silv er grains in
autoradiogram s. In those cells which had received acridine
orange oound-DNA, they observed green fluorescence in the
cytoplasm..
Mathias and Fischer (1932) studied mouse leukem ia cells
treated with isologous tritium -labeled DNA. They m easured the
activity of the hot trichloroacetic acid ex tract of cells subjected to
the labeled m aterial for a 2-hour period. It was found that at
19
concentrations below 1 fig/ml the uptake was proportional to the
dose of DNA, but at higher levels the incorporation was not
greatly increased . They concluded from these re su lts that a
lim ited num ber of binding sites w ere available and that at doses
slightly above 1 jug/m l these w ere satu rated .
32
Cocito et al. (1932) tre a te d HeLa ce lls with P -labeled
autologous DNA or with the sam e labeled prep aratio n combined
with m ethylated bovine serum album in. They reported that uptake
of the la tte r was 40 tim es g re a te r than when DNA was employed
alone. However, the com bined protein-nucleic acid was not acted
upon in trac ellu larly by enzym es during the tim e req u ired for two
cell generations, nor was it digested in vitro by deoxyribonuclease.
This suggested that it probably was biologically inert.
14
E hrlich a sc ite s cells trea ted with homologous C -labeled
DNA w ere studied by Schimizu et aL (1962). They reported that
DNA was probably incorporated intact because c e lls contained
labeled m a te ria l when tre a te d in the p resen ce of u racil methyl
sulfone which inhibited the incorporation of labeled p re c u rso rs
resulting from the breakdown of labeled DNA. They reported
that at le a st som e DNA was incorporated into the nuclei of the
cells, since m ild deoxyribonuclease trea tm en t failed to rem ove
all activity. Koyama (1963), in an extension of these studies
20
employing the same test conditions, found that uptake, as
m easured by the degree of radioactivity in the DNA fraction from
treated cells, reached its peak after 2 0 minutes* incubation in the
labeled preparation. The amount of activity was reduced by about
90 per cent when living cells w ere subjected to deoxyribonuclease
treatm ent, after incubation in medium containing DNA, and this
led him to the conclusion that most of the incorporated m aterial
was in the cytoplasm, not in the nucleus.
Chorazy et al. (1963) employed tritium -labeled chrom osom es
from mouse leukemia cells to tre a t mouse macrophages, rat
embryo and He La cells. With autoradiographic techniques, they
found that incubation of the cells in the chrom osom e preparation
for 6 hours produced labeling alm ost exclusively in the cytoplasm,
while periods of 16 to 26 hours yielded some labeling in the nuclei.
The incorporation of labeled chrom osom es by macrophages far
exceeded that of either the fibroblasts or the He La cells, the
latter two of which only infrequently contained tagged m aterial.
They found that the addition of glucose and insulin to the incubating
medium, as agents to enhance pinocytosis, did stimulate uptake by
fibroblasts but was ineffective with m acrophages and HeLa cells.
However, they concluded that pinocytosis was the mechanism of
uptake because chrom osom es were frequently found in the cyto
plasmic vacuoles.
STA TEM EN T OF THE PRO BLEM AND
METHOD OF A TTA CK
As was indicated in an earlier section, the literature dealing
with the uptake of high molecular weight DNA is conflicting or, at
best, not clear in defining the conditions under which positive
results were obtained. In several of the studies, more than one
cell type was subjected to the treating m aterial sim ultaneously
and, therefore, it is difficult to be certain whether the uptake was
dependent upon the physiological state of the cells or if it was
prim arily dependent upon the cell type.
The purpose of this study was to determ ine if the uptake of
DNA by HeLa S3 cells was dependent upon (1) a specific interphase
period of the cell cycle, (2 ) the degree of polymerization of the
DNA employed, or (3) the concentration of the DNA, and to
dem onstrate a mechanism for the incorporation of high m olecular
weight DNA.
It was felt that the most significant manner of expressing data
for a study of this type would be to define the percentage of cells
showing uptake rather than the amount of DNA incorporated per
cell. Therefore, experiments were planned to allow a large
sample to be studied with a cytochemical technique rather than a
21
22
sm aller sample with labeled m aterial and autoradiographic
methods.
In order to determine whether or not uptake was dependent
upon a given phase of the cell cycle, a series of experiments
was planned whereby HeLa S3 cells, which had been partially
synchronized by suitable biochemical methods, were subjected to
DNA preparations. The cells were treated during two periods of
the interphase cycle and compared with nonsynchronized controls.
E arlier studies had employed numerous DNA preparations
which were obtained by various methods and, therefore, were of
different molecular weights. To determine if the degree of poly
merization was a limiting factor in the percentage of cells showing
uptake, parallel experiments were conducted employing DNA of
the same species origin but of different molecular weight. To
determine if uptake was dependent upon concentration, an
experiment was conducted employing high concentrations of DNA.
Several authors have proposed pinocytosis as the mechanism
of uptake of DNA, but none have presented direct evidence of its
taking place under their experimental conditions. It was, there
fore, decided that a tim e-lapse cine study of cells subjected to
DNA treatm ent would give definitive evidence as to whether this
could be the mechanism of entry of such substances into cells.
Experiments were carried out whereby conditioned HeLa S3 cells
were treated with DNA and photographed for varying periods in
an effort to visualize pinocytotic activity.
M ATERIALS AND METHODS
M aterials
All of the salts employed in the preparation of EagleT s medium
were of analytical grade. Pooled fetal bovine serum was obtained
from Microbiological Associates, Bethesda, Maryland. Anti
biotics were purchased from local pharm acies. High polymer DNA
was obtained as sodium salt from the California Corporation for
Biochemical Research, Los Angeles, California. Low polymer
DNA, also a sodium salt, came from Mann Research Laboratories,
New York City, New York. Acridine orange (Orange Dr Acridine
Brillant E. Z. ) was obtained from. Francolor, Paris, France.
Auramine G, certification number CAu 3, and azure B bromide,
CAb 1, w ere from Matheson, Coleman and Bell, Cincinnati,
Ohio.
Routine stock culture methods
All procedures involving the tra n sfe r of cells were carried
out in a glass-enclosed culture room used exclusively for this
purpose. Aseptic technique, as outlined by Paul (1960), was
followed during all operations. Sterile solutions were obtained
from supply houses or passed through a Seitz sterilizing filter.
24
Stock cultjres were maintained in E arle's T-60 flasks and
O
incuoated at 37 C. Natrient fluid consisted of Eagle's basal
medium (Eagle, 1955) supplemented with 10 per cent inactivated
fetal bovine serum, 100, 000 units per liter penicillin G, 125, 000
gamma per liter streptomycin, and 2. 5 ml of 0. 5 per cent phenol
red solution. Prior to use, the medium was adjusted to a pH of
7. 5 with 1 . 5 N sodium hydroxide or 1. 0 N hydrochloric acid. The
medium was replaced 48 hours after setup and the cells routinely
subcultured on the 4th day. The fluid was withdrawn into a
vacuum flask and 1 0 ml of 1:5000 versene in calcium and magne
sium free Gey’s balanced salt solution (BSS) were added. After
incubation for 5 to 10 minutes at 37°C, gentle aspiration several
times through a 15-gauge needle was sufficient to remove indi
vidual cells from the glass before centrifuging at 600 and 800 rpm.
The supernatant was withdrawn and the pellet resuspended in
i0 ml Eagle’s medium, again aspirated gently, and one-half of
the suspension added to each of two T-60 flasks containing medium
sufficient to yield a final volume of 20 ml. When possible, cells
were maintained as duplicate stocks which were handled on
succeeding days with different solutions to provide added
insurance against loss by contamination.
26
Preparation of conditioned cultures
In our experience, it was found, after several preliminary
experim ents with the methods described by Stubblefield and
M ueller (1962), that in order to obtain a high degree of synchrony
a conditioned population must be employed. This was developed
from the HeLa S3 stock after several passages in a nutrient
fluid consisting of EagleT s medium, as previously described, plus
- 6 - 5
the addition of 10 M amethopterin, 5x10 M adenosine and
2. 5 jug/ml thymidine. All experimental studies were conducted
with cells from stock cultures maintained under the conditions
which have been described.
Preparation of synchronized cultures
Cells grown in T-30 flasks were employed in prelim inary
experiments. All operations were performed in a 37°C constant
tem perature room built especially for these studies, since any
m arked tem perature fluctuation interferes with the achievement
of synchronized cultures (Swann, 1957). Experimental cultures
were housed in an incubator within the constant tem perature room,
except during handling, as a further precaution against
tem perature variation. Cells from stock flasks were harvested
with versene as in routine subculturing procedures and
27
resuspended in temperature equilibrated Eaglers medium without
amethopterin, adenosine and thymidine. A 5 ml aliquot was re
moved and diluted with 15 ml of 0.85 per cent saline and
enumerated with the aid of a Model B Coulter Counter. The
population density was then adjusted to yield a concentration of
50,000 to 100,000 cells per milliliter of medium. Five milliliter
aliquots of this suspension were seeded in T-30 flasks with an
automatic syringe, while the stock suspension was continuously
agitated to prevent cell settling. As a further precaution to
insure uniformity, the flasks were seeded in groups of 3 or 4,
and one flask from each group comprised a set for subsequent
treating and counting. Cells were allowed to settle and attach to
the glass during a period of 24 to 28 hours, after which 0. 1 ml
of prewarmed amethopterin adenosine, giving a final concentration
equal to that used in the conditioning medium, was added to all
flasks except those of the control group. Sixteen hours after the
addition of the blocking agent, 0 . 1 ml of thymidine yielding a
final concentration equal to that in the conditioning medium was
added to all flasks except the controls.
Measurement of the degree of synchrony
Sets of flasks to be counted were rinsed with 0.85 per cent
28
saline and 5 ml of 1:5000 versene added for 15 minutes at 37°C
to insure the removal of all cells in a monodisperse condition.
Exactly 15 ml of 0.85 per cent saline were added to the versene
suspension in each flask and the cells in the suspension counted on
a Model B. Coulter Counter. The average of 5 counts for each
flask was determined. The values presented in the section deal
ing with results represent the average of 2 or 3 flasks for each
interval. Counts were made immediately prior to the addition of
amethopterin, after 8 hours, 1 2 hours, and 16 hours in amethop
terin, and a c t 2- or 3-hour intervals after the addition of the
thymidine. Nonblocked controls were counted at the time of the
addition of the thymidine to treated flasks and 15 or 16 hours
later. Flasks which received the blocking agent but no thymidine
were counted at the same time as the second set of control flasks.
Preparation of highly polymerized DNA solution
Dried DNA was weighed and handled aseptically, but no
further precautions to sterilize the preparation were taken. The
material was added to either GeyT s BSS or EagleT s medium and
dissolved with slow stirring on a magnetic mixer at 4°C to prevent
possible enzymatic degradation of the DNA by nucleases present
in the constituents of the medium. The preparation was rapidly
29
o
warm ed to 37 C in a water bath immediately prior to use.
Preparation of low polymer DNA solution
This m aterial was obtained as a powder and was handled
the same as the highly polymerized fibrous m aterial except that
the powder did not require stirring to dissolve.
Fixation and staining
Cells were fixed with Carnoy7s 1:3 acetic alcohol. P re
parations for ordinary light microscopy were stained with Feulgen-
fast green or azure B bromide (Flax and Himes, 1952).
Fluorescence microscopy preparations (Plate I, figs. 1, 2) were
stained with 0. 01 per cent acridine orange at pH 4. 0 (Mayor, 1961)
or with auramine O - SOg (Kasten, 1959a, b).
Photography
Acridine orange and auramine O preparations were photo
graphed with the use of a Zeiss fluorescence microscope and an
Osram HBO 200 m ercury vapor lamp.
Tim e-lapse cine records were made with the aid of a 16 mm
Kodak Cine Special cam era and Zeiss or Wild phase contrast
optics, adapted for continuous recording at 37°C (Lefeber, 1963).
A cine abstracting device was employed for selecting and enlarg
30
ing of individual film frames.
Procedure for scoring DNA uptake by cells
All values were determined from a minimum of 500 or 1000
cells counted per slide and 2 or 3 slides were utilized for each
experimental condition. Since the auramine O fluorescent
Feulgen is the most sensitive and specific for DNA (Kasten,
personal communication), all uptake scores were determined from
these preparations. Counts were made with the use of oil
immersion optics by placing the objective at the edge of the pre
paration and moving the slide only in one axis to the opposite
margin then moving the other axis and repeating the procedure
until the reported number of cells had been observed. Cnly those
cells which fell completely within the optical field and were not
immediately adjacent to a degenerating cell were counted. Cells
were scored as positive if they contained DNA in the cytoplasm, but
not over the region of the nucleus. The uptake index was defined
as the number of DNA-positive cells divided by the total cells
counted with the quotient multiplied by 100, Examination of non
treated HeLa cells in a preliminary study revealed a mean uptake
index of 0. 65.
31
Determination of the degree of synchrony
In a se rie s of prelim inary studies the growth curves of
synchronized cultures w ere compared with those of random
populations and with blocked but not reversed cultures. E xperi
ments A and B were planned to determine the degree of synchrony
and the approximate duration of the periods of the interphase cycle.
The results of these studies form the basis for all further
experimental work.
Experimental plan
In experiments numbered in the III se ries the highly
polymerized DNA was employed while in those preceded by the
number IV the low polymer DNA was used. DNA was added to the
medium already present in experiments TIT-1 and IV-D. Fresh
medium was added at the time of DNA treatm ent in numbers III-J
and IV-E. High concentrations of DNA were added along with
fresh medium in experim ents IV-F and IV-G.
Effect of low concentrations of DNA
Experim ents III-1 and IV-D were conducted simultaneously
and under identical conditions with the single exception being the
degree of polymerization of the DNA. A 5 ml suspension of cells
32
was inoculated into 110 mm x 77 mm Yerganian tubes containing
25 x 75 mm coverslips. Synchronized cells were treated with the
DNA during the postmitotic growth phase (G-l) and the synthetic
phase (S); nonsynchronized sister cultures were treated and served
as controls. One ml of EagleT s medium containing sufficient DNA
to give a final concentration of 2 0 jug/ml was added to two cultures
for each experimental condition. The preparation was left in
contact with the cells for 6 hours. Before fixing, the cultures
were washed 3 times with Gey?s BSS at 37°C to remove extra
cellular DNA and debris. The G -l cultures were treated with the
DNA 12 hours after the addition of the blocking agent. The
synthetic phase cultures were treated two hours after the addition
of the thymidine and the nonsynchronized cells at a time between
the other two as a matter of convenience.
Effect of washing and exchanging medium
In experiments III-J and IV-E cultures were washed with BSS
and new medium containing the DNA added in an effort to enhance
pinocytosis. These studies were otherwise identical to numbers
III-1 and IV-D.
Effect of high concentration of DNA
Before these experiments were undertaken, a prelim inary
33
study, employing nonsynchronized cultures, was carried out in
which cells were left in contact with 500 /ig/ml of DNA for periods
up to 48 hours (approximately two cell generations) then fixed,
stained and examined for any morphological alterations. Cine
records were also made to determine any gross abnormalities
resulting from this treatment.
Experiment TV-F was designed to establish whether high
concentrations of DNA increased the per cent of cells showing
uptake or the quantity of DNA ingested by individual cells. The
procedure in this study was the same as that in IV-E except the
concentration was raised to 500 /ig/ml.
Observation of pinocytosis _in DNA-treated cells
Experiment IV-G was designed in an effort to visualize by
direct observation a mechanism for the entrance of macro-
molecular substances such as polymerized DNA. Nonsynchronized
HeLa S3 cells from conditioned stocks were seeded in Rose multi
purpose chambers and allowed to settle and attach to the glass for
periods of at least 24 hours. Medium was then withdrawn, cells
washed with warm GeyT s BSS. Cells were selected for photo
graphy which exhibited well spread cytoplasmic membranes but
were not undergoing active pinocytosis before treatment. New
34
medium containing 500 /ig/m l of low polymer DNA was then
added. Immediately after treatm ent, tim e-lapse cinematographs
were made at the rate of 4 fram es per minute for a 6 -hour period
with oil im m ersion optics.
RESULTS
Experiments A and B: Production of synchronized cells and
determination of duration of interphase periods
Tables 1 and 2 as well as text-figures 1 and 2 summarize
these preliminary studies. It was demonstrated that conditioned
HeLa S3 line could be virtually entirely blocked in the postmitotic
- 6 -5
growth phase by the addition of 10 M amethopterin and 5 x 10 M
adenosine. Eight to 12 hours after the drugs were administered,
cell multiplication, as detected by Coulter Counter techniques,
was almost entirely absent. When amethopterin and adenosine
were allowed to remain in the medium and no exogenous thymidine
added, cells failed to undergo DNA synthesis as indicated by the
nearly total absence of cell division for periods of 15 to 16 hours.
The addition of 2. 5 p.g/ml of thymidine 16 hours after the cells
received amethopterin and adenosine resulted in cell division
following an 8 - to 9-hour lag period. Cell multiplication
continued at a highly accelerated rate until approximately 90 per
cent of the control population size was reached. This increase
required about 7 hours as compared with approximately 20 hours
for a sim ilar increase in nonsynchronized populations.
35
36
Experim ent III-1: Low concentration of highly polymerized DNA
Cells w ere examined for Feulgen-positive m aterial in the
cytoplasm after treatm ent with highly polymerized salmon sperm
DNA for 6 -hour periods. Table 3 sum m arizes the findings of this
experiment. It shows that cells treated during the postmitotic
growth phase (G-l) had an average uptake index of 5. 5 with a
range of 5. 0 to 6 . 4 for the three slides counted. The figures for
cells treated during the synthetic phase (S) indicated an average
of 5. 9 and a range from 5. 7 to 6 . 6 . The nonsynchronized control
cells had an average uptake index of 4. 8 and a variation, for the
three slides counted, of 3.8 to 6.4.
Experiment IV-D: Low concentration of low polym er DNA
Data from this experiment a re presented in Table 4. The
postmitotic growth phase cells yielded an average of 5 . 9 and a
distribution of 5. 6 to 6 . 3. The range for the synthetic period was
4. 8 to 7. 3 and averaged 6 . 4. Control figures were 4. 6 to 6 . 4,
with a mean of 5. 5.
Experim ent III-J: Highly polymerized DNA after washing
and exchanging medium
Table 5 sum m arizes the results of this study. The averages
37
for three slides, with a total count of 1 0 0 0 cells per slide, were:
G -l, 5. 1; S phase, 6.4; and nonsynchronized controls, 4.5. The
variation between the three uptake indices calculated for each
experim ental condition was 4.8 to 5. 5 for the postmitotic growth
phase, 5. 6 to 7. 5 for the synthetic phase, and 3.7 to 5.4 for the
controls.
Experiment IV -E : Low poly m er DNA after washing
and exchanging medium
A total of 9000 cells were counted in this experiment and the
data presented in Table 6 sum m arize the findings. The range for
the three G -l slides was 4. 4 to 5. 1 and the average was 4.6.
The S phase showed an average of 7. 4, with a range of 7. 0 to 8 . 1.
The control averaged 5. 2 and the degree of variation was from
5. 0 to 5. 4.
The average figures from Experim ents III-1 and IV-D and E
showed a slight elevation in the average uptake indices for cells
in the synthetic phase; however, because of the degree of
variation between counts for the slides in the sam e experim ental
groups, it was felt that the differences fell within the range of the
experim ental e rro r.
38
Experiment IV-F: High concentration of low polymer DNA
Preliminary studies employing conditioned HeLa cells
indicated that 500 pg/m l DNA was not detrimental for periods up
to 48 hours. Since this is approximately the period required for
two cell generations, it was considered that DNA, at this concen
tration, was not toxic. Plate II shows HeLa cells after varying
periods in the low polymer salmon sperm DNA. Figures 5
through 8 were abstracted from motion picture records of living
cells and trace a mitotic event to its normal completion after the
cells had been in the DNA-containing medium for 4 hours. Plate I,
figure 3 shows living conditioned HeLa S3 cells photographed with
phase contrast optics 45 hours after the addition of 500 /ig/ml of
DNA. Figure 4 shows cells from the same culture fixed and
stained with acridine orange one hour later.
The uptake indices for cells subjected to 500 p g /m l of low
polymer DNA are presented in Table 7. The values for the G -l
phase ranged from 5.0 to 7.0, the synthetic phase values were
5. 2 to 7. 6 , and those for the controls were 4. 6 to 6 . 4. The
averages were 6 . 0, 6 . 5, and 5. 4. These figures fell within the
same order as those in experiments employing lower doses of
DNA. There was no detectable increase in the amount of DNA
found in the cytoplasm of positive cells.
39
Experiment IV-G: Demonstration of pinocytosis in
DNA-treated cells
Tim e-lapse cinematography was employed with the use of
oil im m ersion optics. Plate III shows conditioned HeLa S3 cells
in the presence of 500 jug/m 1 of low polymer DNA. Figures 9
through 14 trac e the appearance of pinocytotic vacuoles at the
margin of the cell and follow their course a short distance into
the cytoplasm. Time intervals following the addition of DNA-
containing medium a re indicated by the numbers in the lower left
corner of the figures. Pinocytosis was observed in cells treated
with DNA for 6 -hour periods; however, it was not a frequent
occurrence and this may relate to the relatively low uptake indices
recorded for the HeLa cell under the various conditions employed.
TABLE 1
HELA S3 CELLS SYNCHRONIZED AFTER TWO PASSAGES IN MEDIUM
CONTAINING AMETHOPTERIN-ADENOSINE AND THYMIDINE
Experimental Conditions Number of Flasks Average Cell Number
Per Cent of Control
at Termination
Set Up 2 264400
__a
Amethopterin added 3 298413 42
Amethopterin - 3 hours 3 348466 50
Amethopterin - 16 hours 3 394906 57
Control*3 3 527640 75
Thymidine - 4 hours 3 418826 59
Thymidine - 7 hours 2 420280 60
Thymidine - 10 hours 3 467480 67
Thymidine - 13 hours 3 559573 87
A
Thymidine - 16 hours 3 637973 90
TABLE 1--Continued
Experimental Conditions Number of Flasks Average Cell Number ^>erj_ ^L en^ °/ Control
at Termination
aNot calculated.
^Counted at time of thymidine addition to experimental flasks.
cCounted 16 hours after addition of thymidine to experimental flasks.
^Counted at time of second control.
Control 3 707906 100
Nonreversed 3 417346 59
Control
Text-Figure 1. Determination of the degree of syn
chronization of HeLa S3 cells, conditioned for
two passages, by amethopterin-adenosine and
thymidine.
oo
u o j j b u j u i j o j , i o j j u o o j o j u a o a a d
Number o f Hours
TABLE 2
HELA S3 CELLS SYNCHRONIZED AFTER THREE PASSAGES IN MEDIUM
CONTAINING AMETHOPTERIN-ADENOSINE AND THYMIDINE
Experimental Conditions
Number of Flasks Average Cell Number
Per Cent of Contro]
at Termination
Set up 2 293060
a
Amethopterin added 3 309893 47
Amethopterin - 12 hours 3 392013 59
Amethopterin - 16 hours 2 387640 58
Control^ 2 482580 73
Thymidine - 3 hours 2 373380 57
Thymidine - 5 hours 2 379200 57
Thymidine - 7 hours 3 396920 60
Thymidine - 10 hours 3 431693 64
I
Thymidine - 13 hours 2 504300 77
TABLE 2 --Continued
Experimental Conditions Number of Flasks Average Cell Number
Per Cent of Control
at Termination
Thymidine - 15 hours 3 560653 87
Control 3 664026 1 0 0
Nonreversedd 3 398946 60
aNot calculated.
^Counted at time of thymidine addition to experimental flasks.
cCounted 16 hours after addition of thymidine to experimental flasks.
^Counted at time of second control.
Text-Figure 2 . Determination of the degree of syn
chronization of HeLa S3 cells, conditioned for
three passages, by amethopterin-adenosine
and thymidine.
.c
s
o
in
o
C O
o
t—
o
00
u o T jttitu u a x iojjuoo jo ^ u a o ja j
TABLE 3
UPTA K E INDEX OF HELA S3 C ELLS A FT E R
TREA TM EN T WITH 20 M g/m l OF HIGHLY
POLY M ERIZED DNA
CeU Cycle Phase SUde Uptake Indexa
G -1 1 5.0
G -l 2 6.4
G -l 3 5.2
S 1 5.7 (1000)
S 2 6 . 6
S 3 5.5 (1000)
Nonsynchronized 1 3.8
Nonsynchronized 2 4.2
Nonsynchronized 3 6.4
a _ .
Figures based on 500 cells counted except
where otherwise indicated.
TABLE 4
UPTAKE INDEX OF HELA S3 C ELLS A FT E R
TREATM ENT W ITH 20 jxg/m l OF LOW
POLYM ER DNA
Cell Cycle Phase Slide Uptake Indexa
G -l 1 6.3
G -l 2 5.7
G -l 3 5.6 (500)
S
♦
1 7.3
s 2 4.8
s 3 7. 2
Nonsynchronized 1 6 . 4 (500)
Nonsynchronized 2 4.6
c i.
Figures based on 1000 cells counted except
where otherwise indicated.
TABLE 5
48
UPTAKE INDEX OF HELA S3 CELLS A FT E R TREATM ENT
FOR SIX HOURS IN NEW MEDIUM CONTAINING 20 jug/m l
OF HIGHLY POLYM ERIZED DNA
Cell Cycle Phase Slide Uptake Indexa
G-l 1 4.8
G-l 2 5.0
G-l 3 5.5
S 1 6.3
S 2 5. 6
S 3 7.5
Nonsynchronized 1 4.4
Nonsynchronized 2 3.7
Nonsynchronized 3 5.4
a
All fig u re s based on 1000 c e lls counted.
49
TABLE 6
UPTAKE INDEX OF HELA S3 CELLS AFTER TREATMENT
FOR SIX HOURS IN NEW MEDIUM CONTAINING 20 jLig/ml
OF LOW POLYMER DNA
Cell Cycle Phase Slide
3.
Uptake Index
G -l 1 5.1
G -l 2 4.9
G -l 3 4.4
S 1 7.1
S 2 8 . 1
s 3 7.0
Nonsynchronized 1 5. 0
Nonsynchronized 2 5. 1
Nonsynchronized 3 5.4
a
A ll fig u re s b ased on 1000 c e lls counted.
50
TABLE 7
UPTAKE INDEX OF HELA S3 CELLS AFTER TREATMENT
FOR SIX HOURS IN NEW MEDIUM CONTAINING 500 Mg/ml
OF LOW POLYMER DNA
Cell Cycle Phase Slide
a
Uptake Index
G -l 1 5. 0
G -l 2 7.0
S 1 7.6
s 2 5.2
s 3 6 . 8
Nonsynchronized 1 4.6
Nonsynchronized 2 5.2
Nonsynchronized 3 6.4
2 L
A ll fig u re s based on 500 c e lls counted.
DISCUSSION
In spite of obvious advantages, there have been very few
studies employing synchronized mammalian cells as an analytical
tool. There are several types of problems which can best be
studied when the experimenter knows what phase the cells are in
at the time of treatment. Ionizing radiation studies are an obvious
example since the results may vary markedly, depending on what
phase of the cycle cells are in at the time of treatment (Painter,
1962). Until very recently, the problem has been dealt with in
directly by usinc, log phase cultures and calculating the percentage
of cells at a given point from a knowledge of their generation time
and the approximate duration of the various phases. Another type
of experiment which lends itself to the use of synchronized cells in
culture is the study of DNA synthesis by individual chromosomes
during the S piiase (Stubblefield and Mueller, 1962).
Terasima and Tolmach (1963a) have recently developed a
method of obtaining partially synchronized populations by sub-
culturing only tho.se cells which are dividing. This technique de
pends upon the reduced adhesiveness exhibited by cells in mitosis
52
which allows them to be selectively removed from the glass and
transferred to another vessel. However, the number of cells ob
tained by this method is at best only about one per cent of the total
population. A further difficulty is that the degree of synchrony
tends to decay during the G -l phase (Sisken, 1963).
The use of amethopterin and thymidine to produce synchrony
offers several advantages over the techniques of Terasim a and
Tolmach (1963b). A much larger population can be obtained because
the method does not depend upon the collection of cells in a specific
phase of the cell cycle. Because the chemicals are applied sequen
tially, one can predict with confidence when a given phase begins.
Finally, since the progression from one phase to another is regu
lated by the experimenter, studies such as this one, where it is
necessary to subject cells to prolonged exposure to an experimental
variable, are possible because it is safe to assum e that the tre a t
ment did not extend beyond the lim its of the desired interphase
period.
There are technical difficulties involved in the use of drugs to
produce cell synchronization and these have perhaps tended to limit
the number of studies employing this technique. The necessity of
avoiding tem perature fluctuations, which tend to impose a certain
53
degree of synchrony, according to Newton and Wildy (1959),
requires that all work be done in a constant tem perature room.
Ruecker and Mueller (1960) emphasized the importance of d eter
mining the optimal population size in order to prevent "break
through” or incomplete blocking of the cells in the presynthetic
phase. The population must consist of cells which a re capable of
utilizing alternate biochemical pathways to insure the incorpora
tion of exogenous thymidine into the newly synthesized DNA.
The difficulties discussed have been largely overcome as
indicated by the data presented in experiments A and B. Consistent
results have been obtained on several occasions, two of which were
reported, when the cell density was between 50, 000 and 100, 000
per ml and the drug concentrations as stated in the section dealing
with m aterials.
The degree of synchrony which was obtained did not quite reach
that reported by Mueller et^ aL, (1962). Stubblefield (personal com
munication) has suggested that the discrepancy may have resulted
from the fact that Mueller et^al. (1962) used a highly purified
preparation of amethopterin, whereas in this study the com m ercial
ly available form of the drug was employed.
The data indicate that cells in both the postmitotic growth and
54
the synthetic phases are capable of taking up high m olecular weight
DNA. The fact that the uptake indices of the synchronized cells did
not differ significantly from those of the random population controls
would suggest that the addition of drugs to the cells did not affect
the uptake. The results of this study show that cytoplasm ic incor
poration of DNA is independent of the phase of the cell cycle in
contrast to reports by Sirotnak and Hutchison (1959) and Hill (1961).
However, in the earlier cases, nonsynchronized populations were
employed. Therefore, only indirect methods were available for
determining the phase of cells at the time of treatm ent. This could
easily account for the discrepancy with the present work.
The mean uptake index for treated cells is 6 . 0 ( 6 per cent) as
compared with a mean of 0 . 65 in nontreated cells. Statistical te s t
ing indicates that the difference between these figures is significant
beyond the 0.001 level. However, Borenfreund and Bendich (1961)
reported that approximately 35 per cent of their cells contained
cytoplasmic DNA after a 6 -hour treatm ent period. Three possible
explanations can be proposed for the difference in the results
obtained in the two studies. F irst, cells which contained very-
sm all amounts of incorporated nucleic acid may have been undetect
ed in the present study whereas, with autoradiographic techniques.
55
they would have been easily spotted by the silver grains overlying
the cell. Second, cells which may have contained DNA in the p o r
tion of cytoplasm over the nucleus could not be detected cyto-
chemically, but would have been scored positive with labeling
techniques. Finally, as may be deduced from a careful study of
their results, Borenfreund and Bendich may have inadvertently
included in their figure some cells which contained labeled m ate
rial other than high molecular weight DNA. Since they do not state
clearly what criteria they selected for scoring cells as positive, it
is impossible to determine the actual per cent of cells with DNA in
the cytoplasm. It is reasonable to assum e that the true value was
somewhere between 6 and 35 per cent.
It was shown that there was no difference in the number of
positive cells between the high and low polymer experiments. This
gives indirect evidence that the ingested m aterial was not attacked
by cytoplasmic enzymes such as those found in the lysosomes. Had
there been significant enzyme digestion, one would have expected
that the cells containing lower polymer m aterial would lose their
Feulgen-positive cytoplasmic inclusions sooner than those treated
with the highly polymerized DNA.
The addition of a high concentration of DNA in experiment IV-F
did not yield cells with increased quantities of Feulgen-positive
m aterial in their cytoplasm. A possible explanation for this has
been presented by Mathias and Fischer (1962). They postulated
that the number of binding sites were limited and that these w ere
saturated by doses of DNA slightly above 1 pg/m l. Therefore,
since 2 0 /ig/ml were employed for the other experiments, the addi
tion of still m ore DNA would be ineffective.
Rose (1957) found an increase in the number of HeLa cells
exhibiting pinocytosis following the addition of fresh nutrient fluid.
It was thought, therefore, that increased uptake might be observed
in experiments EJ-J and IV-E where the medium was exchanged at
the tim e DNA was added. The failure to detect an increased number
of cells with DNA in their cytoplasm was probably related to the
different type of cells employed in the two studies. Rose worked
with Gey's strain HeLa (wild type) which is the original cell line
obtained from a cervical carcinoma. The present study employed
HeLa S3, a clone derived from the Gey's strain, which tends to
grow in rather tightly packed colonies with minimal free cytoplas
mic borders (Puck and M arcus, 1955). In monolayer cultures of
the wild type, there are large single cells with expansive cytoplas
mic borders as well as the sm aller elements which grow in colonies
resembling those in the S3 cultures. Gropp (1963) has pointed out
57
that pinocytosis is m ore frequent in the large cells of HeLa and
H. Ep 2 cultures than in the sm aller, more compact ones. The
present study agrees with that of Chorazy et al. (1963) who were
unable to enhance pinocytosis in HeLa cells with glucose and
insulin, but did detect an increase with rat embryo fibroblasts. It
should be pointed out that, like the large cells of wild type HeLa,
rat embryo fibroblasts have extensive free cytoplasmic margins.
Pinocytosis must be the mechanism of uptake since the m olec
ular size of DNA precludes its entry into cells by simple diffusion.
In this study, pinocytosis was demonstrated in cells suejected to
DNA treatm ent. This provides experimental evidence that pino
cytosis is a mechanism for uptake.
On the basis that incorporation of high molecular weight DNA
is independent of the cell cycle phase and, as pointed out in the
review of the literature, it occurs to some degree in virtually all
cell types, it is interesting to speculate on tne different results
obtained in the various transformation studies. The first variable
which must be considered is the route by which the transform ing
m aterial leaches the susceptiole cell. It may come in contact with
digestive enzymes such as those in the serum or with special culls
whose function it is to capture and destroy foreign substances, out
which a re not transform ed themselves., If it is assumed that the
58
DNA reaches the cell surface in an active state and that it is in
corporated by the relatively small percentage of the population
which is undergoing pinocytosis at the time, it would be expected
that most of the material would be ingested by cells in the G -l
phase since this period normally occupies the greatest portion of
the cycle (Gelfant, 1962). However, if the DNA is added to cells
undergoing rapid multiplication such as in tissue culture or in the
early embryonic period in vivo, a relatively greater number would
ingest it during either the synthetic or premitotic growth phase
(G-2), since the G -l period becomes shortened under these
conditions (Sisken and Kinosita, 1962). If the cell is more sensitive
in one phase than in the others to exogenous DNA reaching its
chromosomal replicating centers, it might be possible to explain
the conflicting results obtained by different workers with the various
test systems employed. It is possible to te st this hypothesis with
the methods developed during the course of this work.
As a natural outgrowth of the results of this thesis, several
important directions for further work invite attention. Experi
ments employing labeled nucleic acid should be undertaken to
determine the fate of DNA which is ingested during the various
phases of the cycle. A study in which cells would be infected with
DNA viruses or the naked nucleic acid might show differential
59
sensitivity during a p articular period; a problem of great interest
in the light of current thinking in the field of cancer etiology.,
SUMMARY
1. HeLa S3 cells w ere synchronized by blocking them in
-6
the postmitotic growth phase with 10 M am ethopterin and then
with the addition of thymidine at 2. 5 p.g/ml allowing them to
proceed through synthetic and prem itotic growth phases.
2. Uptake of salmon sperm DNA (20 jug/ml) w as demon
strated by approximately 6 per cent of the cells in the G -l and S
phases. A sim ilar number of cells in a nonsynchronized control
population showed cytoplasmic incorporation of the DNA.
3. No difference in the number of cells showing uptake was
detected between cells treated with highly polymerized DNA and
those treated with low polymer m aterial.
4. Addition of new medium at the tim e of DNA treatm ent
did not enhance pinocytosis.
5. Pinocytosis was observed with the use of phase contrast,
tim e-lapse cinemicrography in cells treated with high concen
trations of the nucleic acid.
6 . Cells were not injured by growth in medium containing
500 p g /m l of the DNA for a 48-hour period. However, no increase
in the amount of m aterial incorporated was detected following a
6 -hour treatm ent period at this concentration.
60
61
7. Indirect evidence based on the results obtained with
high and low polymer DNA was presented to show that intracellular
digestion of the DNA did not occur during the 6 -hour treatment
period.
8 . A possible explanation for the conflicting results
obtained in DNA transformation studies is discussed on the basis
of conditions under which the experiments were conducted.
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APPENDIX
PLA TE I
HeLa S3 cells treated with 500 jug/ml of low
polym er DNA.
Figure 1. Cells treated with DNA 6 hours.
Fluorescent-Feulgen stain. Optics:
12. 5 x 90. Arrows indicate DNA in
cytoplasm.
Figure 2. Cells treated with DNA o hours.
Acridine orange stain. Optics: 10 x 40.
A rrow s indicate DNA in cytoplasm.
Figure 3. Cells treated with DNA 45 hours.
Living cells with phase contrast. Optic
1 0 x 2 0 .
Figure 4. Same cells as figure 3. Stained
with acridine orange. Optics: 10 x 20.
PLATE
74
PLA TE II
HeLa S3 cells treated with 500 ptg/ml of low
polymer DNA and photographed with tim e-
lapse cinemicrography.
Figure 5. Three hours after addition of
DNA. (Arrow indicates cell to be
followed through mitosis).
Figure 3. Three hours and thirty minutes
after addition of DNA.
Figure 7. Three hours and fifty minutes
after addition of DNA.
Figure 8 . Four hours and fifteen minutes
after addition of DNA. (Arrows indicate
daughter cells).
!! I , A T I' II
PLATE m
HeLa cell photographed with oil immersion
phase contrast optics after addition of
500 /i”'/ml of low polymer DNA. Arrows
indicate pinocytotic vacuoles. Time interval
(hours and minutes), after addition of DNA,
is shown in lower left corner of figures.
rz
t
t
I’ I.A I'I. Ill
T h is d i s s e r t a t i o n h a s b e e n 64—12,465
m i c r o f i l m e d e x a c tly as r e c e i v e d
ADAM S, J o e l E ., 1 9 3 6 -
STU D IES O N T H E U P T A K E O F D E O X Y R IB O N U C
L E IC ACID BY S Y N C H R O N IZ E D M A M M A LIA N
C E L L S IN TISSU E C U L T U R E .
U n iv e r s ity of S o u th e rn C a lif o r n ia , P h .D ., 1964
B io lo g y — G e n e tic s
University Microfilms, Inc., Ann Arbor, Michigan

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Adams, Joel E., 1936- (author)

Core Title Studies on the uptake of deoxyribonucleic acid by synchronized mammalian cells in tissue culture

School Graduate School

Degree Doctor of Philosophy

Degree Program Biology

Degree Conferral Date 1964-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, genetics,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Martin, Walter E. (committee chair), Dunn, Arnold S. (committee member), Monroe, Barbara G. (committee member), Pomerat, Charles M. (committee member), Saunders, Paul R. (committee member), Zimmer, Russel L. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-337456

Unique identifier UC11359168

Identifier 6412465.pdf (filename),usctheses-c18-337456 (legacy record id)

Legacy Identifier 6412465.pdf

Dmrecord 337456

Document Type Dissertation

Rights Adams, Joel E.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA