Published May 1, 1956

ACTION OF X-RAYS ON MAMMALIAN CELLS*,

BY T H E O D O R E T. PUCK, PH.D., AND pHTLIP L M A R C U S
(From the Department of Biophysics, Florence R. Sabin Laboratories, Uni~rsity oJ
Colorado Medical Center, Denver)
(Received for publication, February 3, 1956)
Pxarr.s 26 AND 27
Study of the mechanisms of action of ionizing radiation in higher animals
has been impeded by lack of a precise method for measurement of reproductive

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potential in single mammalian cells comparable to that available for micro-
organisms, such as the plating technique of the bacteriologist (1) or the plaque
method of the virologist (2). While a number of quantitative and semiqnantita-
tire procedures for titmting animal cell reproductive capacity have been pro-
posed (3), these generally depend on complex interactions between members of
large populations, and their results are difficult to translate in terms of the fate
of the individual cell. A variety of different criteria have been used as an index
of cell "killing" by irradiation, and "cell lethal doses" have variously been
defined over a range extending from the neighborhood of 100 r to more than
100,000 r (3-5). The extent to which the metabolic state of the individual
animal cell can alter its radiation susceptibility is only poorly apprehended.
The degree to which the lethal radiation dose to an entire animal depends on
radiobiologic damage to reproductive processes within cells of specific types is
uncertain. And the rates of mammalian cell mutation have been very rarely
susceptible to experimental determination (6) because of lack of a reasonably
rapid method for cultivation of large populations from the single cell mutants
produced by irradiation, as is done in a routine manner with microorganisms,
(see reference 7, for example).
With the development of simple, rapid methods for growing single mam-
malian cells into macroscopic colonies with a plating efficiency of 100 per
cent (within the limits of sampling error) (8-9), it became possible to carry out
studies on such cells in precisely the same way as has been done for bacteria.
In this paper are presented experiments describing quantitatively the effects
of high energy irradiation on the reproductive capacity of single HeLa cells,
derived from a human cervical carcinoma (10).
* Contribution No. 39.
:~Aided by a research grant from the Division of Biology and Medicine of the Atomic
Energy Commission(Contract No. AT(11-1)-269) and by a grant from the Damon Runyon
Fund.
653
Published May 1, 1956

654 ACTION OF X-RAYS ON M P , ~ A . L I A N CELLS

Methods and Materials

The plating procedures used to obtain colonial growth from single HeLa cells on Petri
dishes have been described (9). Both the method employing a feeder layer and that involv-
ing more gentle tryps'mization which makes the feeder layer unnecessary, were observed
to yield similar results, so that after the first several experiments the use of feeders was dis-
continued. The clonal strain $3 was employed in most of these experiments, although a few
survival curve determinations carried out on the wild type HeLa cell and on another mu-
tant, $1, yielded essentially the same result.
Known aliquots of monodisperse HeLa cell suspensions were deposited on 60 ram. Petri
dishes containing 4 to 5 cc. of standard growth medium (9), or a modification thereof con-
taining 10 or 15 per cent porcine serum as the only serum component. (Use of porcine rather
than human serum permits a larger number of colonies on each plate because of the greater
compactness of the resulting growth (9).) In the standard procedure the plates were incu-
bated in 5 per cent CO2 in air at 37°C., for at least 5 hours and not more than 24 hours,
during which time the cells attach to the glass. Inspection of the plates just before irradia-

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tion confirmed that more than 90 per cent of the cells are indeed single at this point and in
interphase. The covered Petri plates were then exposed for measured time intervals to a
Westinghouse x-ray tube operating at 230 KVP 1 and 15 ma., filtered with 1 ram. of alumi-
num and 0.5 mm. of Cu. Dose rates of 100 to 200 r/minute were employed. These were
measured with a Victoreen r meter whose calibration had been confirmed by comparison
with meters standardized by the National Bureau of Standards. The meter was placed in
exactly the same position as the sample, and covered with a Petri dish lid to duplicate the
conditions of cell exposure. Readings were corrected to standard temperature and pressure.
After irradiation the plates were incubated in the COs chamber (9) for at least 9 days,
and sometimes as long as 17 days, as will be described. In some experiments, fresh growth
medium was substituted immediately after irradiation in case toxic materials should have
been produced in the suspending fluid, but experience soon demonstrated that this measure
had no appreciable influence on the experimental results for the dosages used in this study.
It was also necessary to determine whether toxic products arising from the irradiated cells
might inhibit growth of true survivors on the same plate. The following experiment showed
this effect to be negligible within the dose ranges here employed: Plates containing thou-
sands of cells were irradiated with 2000 to 4000r, a dose range sufficient to permit no sur-
vivors. Immediately afterward a known inoculum of normal cells was added, and the plates
incubated. In every case the colony count obtained closely approximated the number of
normal cells added to the heavily irradiated plate.

U n l r r a d i a t e d cells exhibit 100 per c e n t c o l o n y - f o r m i n g efficiency u n d e r t h e

conditions e m p l o y e d . H e n c e , it is r e a s o n a b l y certain t h a t the e x p e r i m e n t a l
p r o c e d u r e subjects these cells to no m a j o r stress o t h e r t h a n t h a t of t h e irradia-
tion. T h r o u g h o u t this p a p e r t h e words, " s u r v i v a l , " " v i a b l e , " a n d " k i l l i n g " are
used in t h e sense which has b e c o m e s t a n d a r d in m i c r o b i o l o g y ; i.e., referring
o n l y to t h e a b i l i t y of t h e i n d i v i d u a l cell to m u l t i p l y i n t o a m a c r o s c o p i c colony.
W e shall d e s i g n a t e Do as t h e n u m b e r of R o e n t g e n s n e e d e d to reduce a popula-
tion to the f r a c t i o n 1/e - 37 p e r cent, a n d shall use this as a m e a s u r e of the
slope of t h e s u r v i v a l c u r v e in regions w h e r e it is a l i n e a r l y l o g a r i t h m i c f u n c t i o n
of t h e dose.
1 KVP, kilovolt peak.
Published May 1, 1956

THEODORE T, PUCK AND PHILIP I. MARCUS 655

EXPERIME.NTAL RESULTS

Reduction in Growth Rate among the Surdvors, and tke Formation of Abortive
Colonies.-
Irradiation of a plate seeded with isolated HeLa cells kills part of the popula-
tion, the proportion depending on the dose. In addition, however, the survivors
may grow more slowly. These two effects are demonstrated in the plates of
Figs. 1 and 2, showing the colonies developing on two plates which have been
treated identically except that one was irradiated with 300 r. On the latter
fewer colonies have grown out and those which have developed are clearly
smaller than the colonies of the unirradiated control. This effect of progressive
slowing down of reproductive rate by increasing amounts of radiation is quanti-
tatively shown in the data of Text-fig. 1, in which the average number of

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cells per colony has been determined for a control plate and for the survivors
of a series of irradiations. The growth-delaying effect is also illustrated by the
fact that the largest colony produced by the survivors of a dose of 550 r
contained fewer cells than the average colony on the unirradiated plate.
The existence of this growth-delaying action adds some difficulty to the
scoring of survivors, since an incubation period perfectly adequate to produce
well developed colonies from all of the cells of a control plate may be insufficient
to permit identification of some slow growing survivors. The situation is further
complicated by the fact that cells lethally damaged by the irradiation do not
at once cease all activity but may go on multiplying for as much as five or six
generations before terminating their reproduction. In this respect, their be-
havior is exactly analogous to that of irradiated bacteria (11). Therefore, in
scoring survivors, it becomes necessary to distinguish clearly between the
abortive colonies and those of slow growing survivors. An unambiguous de-
termination of the survival of the capacity to reproduce as a function of radia-
tion dose can be obtained by increasing the time of incubation in the growth
medium. Ultimately even the slowest growing surviving colonies reach diame-
ters of 3 to 4 ram., while the abortive colonies attain a limiting size after which
they remain constant or even degenerate. These processes are clearly illustrated
in the plates of Figs. 3 and 4, in which the macroscopic appearance of colonies
on plates irradiated with 0 and 300 r, respectively, is shown after 17 days of
incubation, as contrasted with the 9 day old plates of Figs. 1 and 2. It is o b
vious that on long term incubation, many of the questionable survivors of
Fig. 2 have failed to maintain their growth, and that the plate of Fig. 4 makes
possible a count of the survival of the capacity to reproduce, which is as defini-
tive as that in a bacterial population.
The abortive colonies are also usually distinguishable from slow growing
but true survivors by the preponderance of giant cells in their constituency,
as will be discussed later. Experience has shown that an accurate count of
Published May 1, 1956

656 ACTION O~ X-RAYS ON MAMMALIANCELLS

1800

1600
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DOSE (R)
TI~T-FIO. I. Demonstration of growth-slowing action of x-irradiation on single HeLa
cells. Plates containing single cells deposited in the standard manner were x-irradiated, in-
cubated for the times ~hown, then fixed and stained. The cells per colony were counted by
microscopic examination, for all the colonies on each plate, or if these were too large in num-
ber, for a random sample of 10 to 20 colonies. In order to avoid weighting the data through
the effect of abortive colonies, only colonies with more than 50 cells were counted.

colony-forming survivors can be obtained after only 11 to 13, instead of 17,

d a y s of incubation if the criterion employed for survival is t h a t a t least 50
normal cells be present in the colony at the end of this period. T h e s t a n d a r d
practice has been a d o p t e d of scoring all the colonies which, to the unaided
eye, are obviously in active growth. Questionable colonies are then examined
microscopically using 50 or more normal cells as the index of retention of
Published May 1, 1956

THEODORE T. PUCK AND PHILIP I. MARCUS 657

multiplication potential. W i t h this system of scoring, the colony counts are

definitive and remain constant with continued incubation.

A typical example will illustrate the results obtained: A series of plates, each seeded with
200 $3 cells, was irradiated with 300 r, and incubated for 9, 12, and 17 days, respectively.
The plate stained after 9 days had a total of 117 aggregates visible to the unaided eye, rang-
ing in diameter from 0.1 to 1.0 ram. Estimation of the actual number of these which would
go on to further growth is difficult with the eye alone. This uncertainty was eliminated on
further incubation, for the 12 day and 17 day plates unequivocally differentiated between
cell aggregates which had remained less than 1 ram. in diameter, and those which now formed
large colonies of 2 to 5 ram. The number of these latter was 31 and 35 on the plates incu-
bated for 12 and 17 days respectively, values agreeing well within sampling uncertainty.
However, by employing the criterion that each colony with 50 or more normal cells is to be
counted as a survivor, the counts obtained on all 3 plates agreed, consisting in 35, 36, and
36, for the 9, 12, and 17 day incubations, respectively. It is impossible to avoid a cer-

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tain degree of arbitrariness in the criterion which will indicate loss of reproductive function.
We believe the operational definition employed in this study is biologically analogous to
that which has proved useful in microorganisms, in addition to furnishing a readily repro-
duceable end-point.

Shape of the Survival Curve.-

T h e survival curve for single cells of the $3 H e L a strain determined b y this
procedure is presented in Text-fig. 2. Survival determinations a t each dose
were carried out with duplicate or triplicate platings whose individual values
almost i n v a r i a b l y agreed within sampling uncertainty. All b u t one of the
points shown on the curve are the means of values determined in a series of
2 to 10 such separate experiments carried out over a period of several months.
A t each dose the s t a n d a r d deviations of individual survival values obtained
from such a series of independent experiments usually lay in the neighborhood
of 10 to 25 per cent of the mean. As is to be expected, the reproduceability of
individual points is less good a t doses greater t h a n 500 r, when inocula as
high as 104-105 cells are required on each plate to yield a countable n u m b e r of
survivors. S t u d y of survival after doses of 800 r or more necessitates such tre-
mendous cell crowding as to require changes in the experimental technique.
The existence of the shoulder in the survival curve of Text-Fig. 2 is unequivo-
cal and constitutes evidence for a multiple hit killing mechanism. This curve
furnishes strong contrast to the single hit curve obtained with haploid cells
like E. coli (12). T h e straight portion of the H e L a survival curve has a slope
corresponding to a value for Do = 96 r; i.e., in this linear region, an increase
in dose of 96 roentgens further reduces the survivors b y a factor of 37 per cent.
(See Discussion.)

Fate of the Cells Inactivated by Irradiation.--

T h e ceils which fail to form self-sustaining colonies suffer one of three fates.
T h e y m a y undergo a limited n u m b e r of divisions, forming microcolonies with
a cell n u m b e r t h a t almost never exceeds 50, even after 17 to 20 d a y s of incuba-
Published May 1, 1956

658 A C T I O N OF X - R A Y S O N M A M M A L I A N CELLS

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Published May 1, 1956

THEODORE T. P U C K AND pHILIP I. M A R C U S 659

tion; they may remain as single cells; or they may disappear, leaving no trace
on the plate which is fixed and stained. Concomitant with the first two processes
which we shall lump together under the name "abortive colony formation"
even when only a single cell remains, is the development of giant cells from
the non-reproducing cells which continue to grow, reaching diameters as great
as 0.8 ram.
At low doses almost every killed cell will still multiply several times, so the
non-survivors form microcolonies of 20 to 40 individuals corresponding to
4 to 5 divisions. Practically all these microcolonies have several giants at the
time of staining, and some consist only of giants. As the x-ray dose increases,
the number of divisions decreases and the abortive colonies become smaller in
cell number. At doses greater than 600 r the ability to divide even once is

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TABLE I
Illustration that with increasing x-ray dose, there occurs a decrease in number of ceils per
abortive colony, and an increase in the proportion of giant cells in such non-surviving popula-
tions, until at 800 to 1000 r, a population consisting practically entirely of single giant cells
is to be found on the plate.
Average cells/clone in abortive Proportion of giants in the cells of
Dose colonies the abortive colonies

r per cent
50 27. 6.7
200 23. 23.
4OO 13. 28.
600 1.7 100.
1000 1.0 100.
In unirradiated populations the frequency of giant cell occurrence is 0.19 per cent.

greatly reduced, and at doses equal to or greater than 800 r practically all the
killed cells remain single. In addition, the per cent of giant cells in this popula-
tion which fails to form self-sustaining colonies increases steadily with dose.
These relationships are illustrated in Table I. Thus, exposure of a plate con-
mining as many as 104 cells to a dose of 1000 r or more will, after incubation
and staining, yield a population consisting entirely of single, giant cells (Figs.
5 to 7).
The irradiation needed to stop the biosynthetic activities involved in giant
formation is enormously larger still. A dose as high as 10,000 r applied to a
population of single cells m a y yield 5 to 20 per cent of the original inoculum
as isolated, well developed giants.

Properties of the Giants Resultingfrom Radiation.--

These giant cells can be produced in very high yields, and in almost any
desired degree of freedom from admixture with normal cells. For example, a
Published May 1, 1956

660 A C T I O N O1~ X - R A Y S O N M A M M A L I A N C E L L S

population of single cells irradiated with 600 r will have approximately 20 per
cent of its population converted into abortive colonies. From Table I, it is
seen that the average number of giants developing in each of these is 1.7, so
that the number of giants recovered is 30 to 35 per cent of the initial cell popula-
tion. If smaller x-ray doses are employed, one can readily obtain more giants
than the initial number of normal cells employed, but these will be mixed with
a larger number of the latter which have survived and multiplied. With doses
greater than 1000 r, but less than 10,000 r, the giant yield falls to 10 to 15
per cent of the original inoculum, but extremely pure preparations can be
obtained. By adjustment of these parameters, it becomes readily possible to
prepare populations of giant cells of 106 or more, with any desired degree of
purity.
Though study of these bizarre forms has only begun, the following facts may

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be mentioned here : -
The glass-attached, stretched giant may occupy an irregular area roughly
circular but sometimes elliptical or elongated. They can readily be trypsinized,
and assume a spherical shape with diameter of about 50 #, indicating a cell
volume of approximately 6 X 1@/t 3, about 15 times as great as a normal HeLa
cell. The cytoplasm is often less densely staining than in normal cells, but the
nuclei stain intensely. The nucleus is always enlarged, and sometimes contains
several huge nucleoli. Occasionally normal, discrete, double nuclei are visible
but these are not particularly more frequent than in the normal cells. Nuclear
fragmentation, and bizarre rosette-shaped nuclear configurations are often
seen.
These cells metabolize at a high rate, changing the pH of standard growth
medium at a rate roughly comparable to that of an equal mass of actively
dividing cells. They can be maintained in the metabolically active state for
long periods if the medium is regularly replaced as it becomes exhausted. Large
numbers of these cells have been kept active at 37°C. in this fashion for 3
weeks.
The giants adhere tightly to glass as do normal cells. On removal by tryp-
sinization they can be resuspended in growth medium and will then reattach
to a new glass surface, with apparently the same configuration and properties
as before. Reattachment to glass after dispersal by trypsinization is presumably
an index of the ability to resecrete a new outer protein layer.
The "feeder" layer of irradiated cells which we have described earlier (8-9),
consists entirely of giants. These are able to promote colony formation of single
cells which have been traumatized by trypsinization procedures which cause
extensive leakage of the cell constituents and loss of ability to multiply in
isolation.
The ability to form large, deeply staining, nuclear bodies is direct evidence
of the retention by these cells of biosynthetic as well as catabolic activity. It
Published May 1, 1956

THEODORE T. PuCK AND PHILIP I. MARCUS 661

was of particular interest to determine whether the giant cells have indeed
lost the power to multiply. Populations of giant cells observed during 20 days
of incubation in complete growth medium which was regularly renewed every
3 to 4 days showed no increase in numbers, whereas normal cells multiply with
a generation time of 20 hours under these conditions. A more critical test was
performed by examination of large numbers of these cells for evidence of
mitosis. Populations of at least 10~ cells were examined for mitotic figures by
microscopic scanning. While occasionally giant cells were found with nuclear
configurations resembling those of early prophase, not a single case of late
prophase, metaphase, or telephase was found. Mitotic figures corresponding
to these latter stages occur in unirradiated cells in a frequency of 3 to 6 per
cent under these conditions. It may be concluded that the ability to multiply
in our standard conditions has been lost practically completely by the irradia-

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tion-produced giants.
Although they have lost the ability to multiply, these giant cells are highly
susceptible to destruction by virus. Addition of 200 particles of Newcastle's
disease virus to a plate containing 105 giants causes almost complete disinte-
gration of the cell layer within 5 days. Addition of this virus to a mixed popula-
tion of normal and radiation-produced giant HeLa cells selectively produces
greater destruction of the latter cell type, possibly because of its larger surface.
HeLa cells suspected of carrying a latent virus, were found to produce massive
areas of destruction when plated on these giants. Further details of these proc-
esses will be presented in another publication.

DISCUSSION

The data here presented distinguish four x-radiosensitive processes in the

isolated HeLa cell: (a) Loss of the ability of single cells to form a macroscopic
colony. This reproductive function possesses a shoulder in the neighborhood of
75 r, and thereafter exhibits a radiosensitivity such that the surviving popula-
tion is reduced by a factor of 37 per cent for each additional 96 r. Cells injured
mortally by this process in the dose range up to 600 r retain the ability to
multiply for 1 to 5 generations. After multiplication ceases, an appreciably large
proportion of the cells continue to grow, forming giants. (b) Slowing of growth
among the survivors. This effect is more difficult to quantitate, but is already
distinct with doses of 100 to 200 r. (c) With doses greater than 600 r, damage
to the reproductive apparatus itself appears, since cells so exposed rarely divide
even once. However, these cells still form giants, so that a considerable part of
their metabolic equipment is still functional. (d) With even greater doses, cells
disappear so that the yield of giants drops. This process is much less radio-
sensitive than any of the others, an appreciable fraction of the original popula-
tion still forming giants after exposure to 10,000 to 20,000 r.
The tremendous sensitivity of the HeLa cell to loss of reproductive potential
Published May 1, 1956

662 ACTION OF X-1LAYS O N MAMMALIAN C E L L S

as revealed b y these experiments demonstrates t h a t the reproductive a p p a r a t u s

of warm-blooded a n i m a l cells is enormously more radiation-sensitive t h a n t h a t
usually found in microorganisms. T h e Do value of 96 r for this cell m a y be
compared with its equivalent in round numbers for the following systems:
a b o u t 4000 r for E. coli (12); 40,000 r for T2 bacteriophage (13) and New-
castle's disease virus (14); 5000 to 18,000 r for haploid, diploid, triploid, or
tetraploid y e a s t (15 a, b); and more than 15,000 r for Ckilomonas paramecium
(16). A d m i t t e d l y the H e L a cell, which derives from a carcinoma rather than
from normal h u m a n tissue, m a y have an abnormal x-ray sensitivity. Similar
d e t e r m i n a t i o n s w i t h h u m a n cells from normal tissues are now in progress.
T h e following questions arise: Does the seat of this killing action in the H e L a
cell lie in: t h e genetic or physiological p a r t s of the cell? If genetic, does the
significant locus involve a single gene, or a larger p a r t of one or more chromo-

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somes?
T h e following considerations constitute good evidence (though not absolute
proof) t h a t lethal effect occurs in the genetic a p p a r a t u s , a t least for doses up
to 800 r:

(a) T h e ability of the killed cells still to reproduce for a limited number of times
appears to indicate that the mitotic mechanism is essentially intact. This phenomenon
of phenotypic lag is characteristic of lethal and other types of mutations in bacteria
(11, 17). I t is difficult to conceive of a damage not confined within the self-replicating
p a r t of the cell, which cannot be repaired by the cell, which delays its expression for
4 or 5 generations, and which then appears in every one of the progeny.
(b) The very minuteness of the value of 96 r indicates the existence of an exceed-
ingly sensitive structure whose inactivation can cause irreparable damage. This
amount of irradiation is equivalent to only 10-12 calories per cell. Even with 100 per
cent efficiency, this amount of energy could break only about 2 X 107 atomic bonds
per cell,2 and since these will be randomly distributed, the number of such bonds that
can be broken in any particular structure must be very small indeed. The sensitivity
of genetic structures to such exceedingly minute changes is well established. The ex-
istence of non-genetic cell constituents able to destroy reproductive capacity as a re-
sult of so small an amount of energy absorption has never been demonstrated.
(c) The multiple unit form of the survival curve qualitatively agrees with that ex-
pected from hits localized in the genetic structure of a polyploid cell and quantita-
tively yields the hit number of 2 as the following analysis s h o w s : -
If loss of a given function in an irradiated population occurs as a result of at least
one ionizing event in any of m different kinds of units, and if there are n units of each

A ~alue of 96 r is equivalent to 96 X 2 X 10-8 or 2 X 10-4 calories per gin. These cells

have a mass of about 4 X 10-9 gin. (9), so that the maximum energy per cell is
roughly 8 X 10-13 calories. Assuming an average only of 20,000 calories per gm. atom for
10-13
atomic binding energies, this represents a maximum of --82X
- - -X- - -10
- - T ~= 4 X 10-1~ gm. atomic
weights, or 6 X 10~3 X 4 X lff-1~ ~ 2 X 10~ atoms.
Published May 1, 1956

THEODORE T. PUCK AND PHILIP I. MARCUS 663

kind present, all of which must be inactivated before the function is lost, the fraction
of the population in which this function survives is given by S in the formula

S = [1 -- (I -- e-DJDo)'l" (I)

in which D is the x-ray dose, and Do is the 37 per cent lethal dose for an individual
site.This equation was originallyderived by Lurla (18) and its applicabilityhas bccn
examined in detail by Atwood and Norman (19). W e shall call n the hit number.
In genetic processes ~t = I for haploid, 2 for diploid,and 3 for triploid cells,etc.;wc
call m the unit number since it represents the number of different kinds of genetic
structures whose inactivation in all replicatespresent can lead to loss of the function
under consideration. For ~t = i, the survival curve is a linear logarithmic function of
the dose. For any value of n > 2, a plot of the log of the survivors against dose ex-
hibits an initialshoulder, after which it descends linearly,as in Text-fig. 2. Formula I
assumes all siteshave an equal radiation sensitivity.As the dose increases, contribu-

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tions to cellkillingmay be made by more radioresistantloci,and each time this hap-
pens, the survival curve will add an increment to its slope (19). By confining our at-
tention to the firstlinear region of the survival curve, we consider only the m loci
which arc maximally sensitive to the lethal effectsof radiation. The values of n, m,
and Do for such locimay be determined as follows: Extrapolation of the linearportion
of the curve to its intercept on the axis yieldsa value S ° == ~t% The slope of the linear
portion of the curve is -re~Do.
Application of these considerations to the survival curve of Text-fig. 2 yields the
following values: n = 2; m = I; and Do == 96 r. The equation
S = [1 - (I - e-D/°e) ~1

fits the cxperimental points well within experimental accuracy,a

These data make it most unlikely that the lethal action is due to a simple,
single gene mutation. The value of 96 r is completely incompatible with that
for any k n o w n single gene mutation. This figure leads to a sensitive volume
calculated by the method of Lea (20) in the neighborhood of I #s, a value more
than 105 times the size commonly accepted for a single gene (21). While the
theoretical uncertainties of volume calculations of this kind have been pointed
out m a n y times (19, 22) these almost never introduce an error greater than a
factor of 10 or at most I00. The enormous divergence in the present case appears
sufficient to rule out the single gene mechanism.
The site in question could well be chromosomal with the lethal action, a
reflection of chromosomal breakage without normal restitution.
The data can bc quantitatively explained if one chromosome pair in the cell
possesses a radioscnsitivity appreciably greater than the others; or if the killing
s While the extrapolationof Text-fig, 2 actually yields a value of 2.1, we consider this
to be identicalto 2 within the experimental precision,although the possibilityis real that a
process with a higher value of n is participatingto a very small extent in the observed kill-
ing process,yieldingan average n -= 2.1 (19).
Published May 1, 1956

664 A C T I O N OF X - R A Y S O N MAMMALIAN C E L L S

action is an expression of aberrations resulting from production of 2 or more

chromosome breaks, producing mitotic bridges, translocations, and genic im-
balance. Experiments testing these and other possibilities are in progress.
It is of interest that experimental behavior similar to that observed here was
obtained by Lucke and Sarachek (15 a) who found a 1 to 1 correspondence
between the values of n determined from survival curves and the true genetic
ploidy for haploid, diploid, triploid, and tetraploid yeast cells respectively.
They too observed a value of m equal to unity for all four types of cells. Zirkle
and Tobias (15 b) have studied diploid yeast cells with survival curves indica-
tive of high values of m.
The enormous radiosensitivity of the HeLa cell strongly suggests that the
killing action studied here may contribute materially to the processes which
determine the mean lethal dose for whole body irradiation of mammals. At 500

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r, the 50 per cent human lethal dose, 99 per cent of the ceils sharing the sensi-
tivity of the HeLa would be killed. The ability of such cells still to multiply
for several generations, or to persist as giants may contribute to the delay
before the full development of symptoms, which so strongly characterizes the
radiation syndrome. Studies are currently under way comparing the x-ray
susceptibility of various clonal human cells obtained from normal tissues,
and the effects thereon of changing the metabolic state of the cell population.
Experiments describing mutation occurrence among the cell survivors of various
doses of x-irradiation will be presented in a forthcoming report.
The parallelism between the giant cell formation here observed and the
development of huge snake-like forms on irradiation of E. coli (23) is striking.
Giant formation in mammalian cells has also been observed by Graham and
associates (24) who have carefully studied the appearance of these forms in
irradiated tumors, and have based an index of the tumor susceptibility on the
per cent of giants appearing in smears after various x-ray doses. The present
data are completely consistent with those of Graham and leave little doubt that
the processes observed in vitro are close counterparts to those seen in the body.
Experimental extension of the survival curve presented in Text-fig. 2 is now
in progress. Such data would provide a means of estimating the radiation
dosage needed to sterilize tumors of various sizes, provided all cells have a
uniform radiation sensitivity. The need for study of these relationships with a
variety of normal and malignant cells is obvious.
SUMMARY

The effects of x-irradiation have been quantitatively studied on single cells

of a human cervical carcinoma (HeLa) under conditions such that I00 per cent
of the unirradiated cells reproduce in isolation to form macroscopic colonies.
This technique eliminates complexities due to interactions of members of large
cell populations.
Published May 1, 1956

THEODORE T. PUCK AND PHILIP I. MARCUS 665

Survival of single ceils (defined as the ability to form a macroscopic colony

within 15 days) yields a typical 2 hit curve when plotted against x-ray dose.
The initial shoulder extends to about 75 r, after which a linear logarithmic
survival rate is obtained, in which the dose needed to reduce survivors to 37
per cent is 96 r. This radiation sensitivity is tens to hundreds of times greater
than that of any microorganism for which the equivalent function has been
studied.
Evidence, though not proof, is presented that the lethal effect is due to a
radiation-induced genetic defect which, however, cannot be a simple single
gene inactivation. The locus of the action could be chromosomal.
Beginning at doses of I00 r, or possibly earlier, growth-delaying effects of
radiation are visible.
Cells in which the ability to reproduce has been destroyed by doses below

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800 r, can still multiply several times. At higher doses even a single cell division
is precluded.
A large proportion of the ceils killed by radiation at any dose gives rise to
one or more giant ceils. These metabolize actively, grow to huge proportions
but never reproduce under the experimental conditions employed. Methods of
preparing large populations of giant cells are described. These giants are par-
ticularly susceptible to virus action.
Some of the irradiated cells disappear from the plate, presumably by disinte-
gration. This action of radiation is by far the least e~cient, since even after
I0,000 r, S to 10 per cent of the original cell inoculum is recoverable as giants.
BIBLIOGRAPHY
I. Wilson, G. S., and Miles, A. A., Topley and Wilson's Principles of Bacteriology
and Immunity, Baltimore, The Williams & Wilkins Co., 4th edition revised by
G. S. Wilson and A. A. Miles, 1955, 99.
2. Luria, S. E., Animal Virology, New York, John Wiley and Sons, 1953, 39.
3. Reviewed by Stroud, A. N., and Bruez, A. M., Texas Rep. Biol. and Med., 1954,
19., 931.
4. Halberstaedter, L., Goldhaber, G., and Doljanski, L., Cancer Research, 1941,2, 28.
5. Reid, T. R., and Gifford, M. P., J. Nat. Cancer Inst., 1952, 13, 431.
6. Russel, W. L., Cold Spring Harbor Syrup. Quant. Biol., 1952, 16, 327.
7. Demerec, M., and Latarjet, R., Cold Spring Harbor Syrup. Quant. Biol., 1946,
11, 38.
8. Puck, Theodore T., and Marcus, Philip I., Proc. Nat. Acad. Sc., 1955, 41, 432.
9. Puck, T. T., Marcus, P. I., and Cieciura, S., J. Exp. Med., 1956, 103,273.
10. Gey, G. O., Coffman, W. D., and Kubicek, M. T., Cancer Research, 19.52,12,264;
Scherer, W. F., Syverton, J. T., and Gey, G. 0., J. Exp. Meal. 1953, 97, 695.
11. Demerec, M., and Cahn, E., ]. Bact., 1952, 65, 27.
12. Lea, D. E., Actions of Radiations on Living Cells, Cambridge, Cambridge Uni-
versity Press, 1947, 318.
13. Watson, J. D., Y. Bact., 1950, 60, 701.
Published May 1, 1956

666 A C T I O N OF X - R A Y S O N M A M M A L I A N CELLS

14. Puck, T. T., Marcus, P. I., and Cieciura, S., data to be published.
15. (a) Lucke, W. H., and Saracher, A., Nature, 1953, 171, 1014. (b) Zirkle, R. E.,
and Tobias, C. A. Arck. Biockem. and Biopkysics, 1953, 47, 282.
16. Ducoff, H. S., Radiation Research, 1954, 1, 130, abstract 15.
17. Newcombe, H. G., Genetics, 1948, 33, 447; 1953, 38, 134.
18. Luria, S. E., Proc. Nat. Acad. Sc., 1947, 33, 253.
19. Atwood, K. C., and Norman, A., Proc. Nat. Acad. Sc., 1949, 38, 696.
20. Lea, D. E., Actions of Radiations on Living Cells, Cambridge, Cambridge Uni-
versity Press, 1947, 83.
21. Muller, H. J., Cold Spring ttarbor Syrup. Quant. Biol., 1941, 9, 151.
22. Puck, T. T., in Nickson, J. J., Symposium on Radiobiology, New York, John
Wiley and Sons, 1952, 385.
23. Lea, D. E., Actions of Radiations on Living Ceils, Cambridge, Cambridge Uni-
versity Press, 1947, 300.

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24. Graham, R. M., and Goldie, K. R., Cancer, 1955, 8, 71; Graham, R. M., and
Graham, J. B., Cancer, 1955, 8, 59.

EXPLANATION OF PLATES
PLATE 26
FIG. 1. Colonies developing On a plate seeded with 200 $3 HeLa cells and incubated
9 days in growth medium. Actual size.
FIG. 2. Colonies developing on a plate identical with that of Fig. 1 in every respect
except that the plate was irradiated with 300 r before incubation. Actual size.
FIG. 3. Plate identical with that of Fig. 1 (i.e., no irradiation), but which has in-
cubated 17 days instead of 9 days. Actual size.
FIG. 4. Plate identical with that of Fig. 2 (i.e., irradiated with 300 r), but which
has incubated 17 days instead of 9 days. Actual size.
FIG. 5. Plate on which 5700 cells were seeded, and which was then irradiated with
900 r. After 12 days of incubation the plate was fixed with Bouin's solution and stained
with hematoxylin. Only one actual co]ony developed on two such plates. Each of the
visible spots shown is a single giant cell. Actual size.
Published May 1, 1956

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. ]0,3 PLATE 26

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(Puck and Marcus: Action of x-rays on mammalian cells)

Published May 1, 1956

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PLATJg 27
FIG. 6. Single giant cell from plate like that in Fig. 5. (The scale is indicated.)
Phase contrast photomicrograph.
FIG. 7. Photomicrograph of a normal colony which has grown up amongst the
radiation-inactivated cells that have formed giants. × 25. The photograph demon-
strates that the giants achieve spread diameters that are 7 to 10 times that of the
normal cell.
Published May 1, 1956

THE .]OURNAL OF EXPERIMENTAL MEDICINE VOL. 103 PLATE 27

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(Puck and Marcus: Action of x-rays on m a m m a l i a n cells)