Download A COMPARISON OF THE CYTOPLASMIC CHANGES INDUCED IN

A COMPARISON O F T H E CYTOPLASMIC CHANGES INDUCED I N
T H E WALKER RAT CARCINOMA 256 BY D I F F E R E N T
TYPES AND DOSAGES O F RADIATION
LLOYD C. FOGG
AND
SHIELDS WARREN
( F r o m the Laboratories of Pathology, iVpw England Deaconess Hospital and Collis P. Huntington
Mcntorial Hospital, and the Departr~ieizt of Pathology, Hurvard Medical School)
The changes in the cytoplasm of both normal and tumor cells induced by
radiation have been described by many workers, including Stenstrom, Alter,
Kimura, Dustin, Lucas, Spear, Whitman, Ludford, and others (1-8). The
present study is an attempt to compare the changes that occur in the Golgi
apparatus in the cells of the transplantable rat tumor, MTalker 256, after the
rat has been treated with radiation of different types and dosages. This
tumor was used because it has been fully described cytologically (Earle, 9 ;
Waldschmidt-Leitz, 10; Lewis, 11) and the growth characteristics have been
standardized in this laboratory by continued transplantation in our inbred
strain of Sloniker white rats. These rats withstand considerable amounts of
radiation, and the tunlor itself is fairly radioresistant, so that premature death
of animals or tumor did not occur in this series.
In evaluation of the effects of different types of radiation, test objects
have been nearly as varied as methods of irradiation, Ascaris ova, chick fibroblasts, salamander cornea, and many others being used. In the present study
we are attempting to follow not only the effect of radiation on various cytologic
structures in an animal tumor, but also the effect of radiation of different
wavelengths and in different dosages.
The cell of Walker carcinoma 256 as described by Lewis (11) and Earle
(9) is relatively large. The nucleus is large and varies in shape from nearly
round to elongate. There are usually present at least two prominent nucleoli
with different staining capacities, i.e. a karyosome and a plasmasome. This
was demonstrated in tissue culture by Lewis and confirmed in fixed material
by Earle. The cytoplasm appears to be more or less granular and dense, with
little evidence of vacuoles, metachromatic granules, or plastids. A centriole
has not as yet been identified as clearly distinguishable from other granules.
The nuclear membrane is clearly outlined. The above characteristics have
been confirmed in the present cytological study by supravital staining and in
fixed and stained preparations. Fig. 1 shows a characteristic lobular focus
of normal non-irradiated tumor. The cells reveal some evidence of polarity,
with the centrosomal area lying in the region between the nucleus and the
greater cytoplasmic space. I t is around this centrosomal area that the Golgi
apparatus is to be found.
1
Read in part before the American Society for Cancer Research, Chicago, March 24, 1937.
567
568
LLOYD C. FOGG AND SHIELDS WARREN
Rats bearing ten-day old tumor were used. For irradiation the rats
having uniformly growing, hard, non-necrotic tumors were selected. Only the
tumor and in~n~ediately
adjacent or underlying tissues were irradiated in the
radium and radon treated animals. In most of the x-ray experiments the
entire animal was irradiated. The following types of radiation and dosages
were studied.
180,000 volt x-ray. . . . . . .SO c ~ ndistance.
,
. .Filter Cu 0.5 mm.
+ Celluloitl 5 mni.
1600 r, 800 r, 400 r
400,000 volt x-ray. . . . . . .SO cm. distance.. .Filter Cu 2 m m . . . . . . . . . . . . . . . . . . ,1600 r , 400 r
1,000,000 volt x-ray. . . . . . .70 cm. clistance.. .Filter Pb 6 mm. . . . . . . . . . . . . . . . . . . . . . . . ,1600 r
Radon applicator, 450 mc.. . 1 cm. clistance.. .Filter Brass 2 mrn.
1 nrm. Steel
2 erythema closes
Radon bomb, 1020 mc.. . . . . 1 cni. tlistarice.. .Filter 1 mm. P b . . . . . . . . . . . . . M erythema (lose
Radium pack, 2 g r a m . . . . . . 6 cm. distance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
erythema close
Radium pack, 4 gram.. . . . . l 0 cm. distance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . >h erythema close
+
The rates of radiation in r per minute were as follows: for 180,000 volt
x-ray, 22.4; 400,000 volt x-ray, 2 7.5 ; 1,000,000 volt x-ray, approximately 50.
The Walker 256 tumor is known as a radioresistant growth. I t has been
shown (Warren, 1 2 ) that with a dose of radiation comparable to the larger
doses given in these experiments mitosis ceases within an hour and the cells
destined to recover do not show any consistent n~orphologicalevidences of
renewed mitotic activity until twelve to twenty-four hours have elapsed.
With the above in mind the experiments were designed to test the effect of
irradiation on the Golgi apparatus of the tumor cell within the twenty-fourhour period after the animals had been treated.
The following table lists the time intervals after irradiation before the
animals were killed :
180 kv. x-ray:
1600 r . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 3, 6, 9, 18, 19, 24, 30, and 48 hours
800 r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 6, 18, and 24 hours
400 r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 6 , 18, ant1 24 hours
400 kv. x-ray:
1600 r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 4 , 6, 9, 15, 16, 2-1, 30, 39, and 48 hours
400 r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 6, 18, ant1 24 hours
1,000,OUO volt x-ray, 1600 r . . . . . . . . . . . . . . 1 , 6, 18, ancl 24 hours
Iiadon applicator, 2 erythenlas . . . . . . . . . . . . . 1, 6, 18, and 24 hours
Radon bomb, %erythema. . . . . . . . . . . . . . . . 1, 4, and 24 hours
Two-gram racliunl pack, 55erythema. . . . . . . . 1, 18, and 24 hours
Four-gram radiuni pack, 54 erythema. . . . . . . 1, 18, and 24 hours
At each interval animals were killed by a blow to avoid the possible effect
of anesthesia on tissue. The tumor was excised and snlall fragments of
non-necrotic ( i . c . , pearly white, somewhat hard and opaque) tissue were placed
in fixative. Where possible, explants were used from areas nearest to the
point of irradiation, areas to one side, and areas a t the greatest distance.
Since there is a variability anlong tumors of the same type and strain, several
animals were used and the experiments repeated, where possible, for comparison for a given dose with one machine a t a stated interval. No study
of irradiated excised tissue was attempted.
The Nassanov, Cajal, Da Fano and modified Mann-Kopsch methods were
AND SPECIMENS
IRRADIATED
WITH 180 KV. X-RAY,1600 r
SPECIA~ENS
FIGS. 1-6. NON-IRRADIATED
1. Non-irradiated; Fleming's fixative. 2. Non-irradiated; Mann-Kopsch fixative. 3. Six
hours after irradiation. 4. Eighteen hours after irradiation. 5. Nineteen hours after irradiation.
6. Twenty-four hours after irradiation. Except as otherwise noted these and other illustrations
are of Mann-Kopsch preparations, X 1180.
used for the demonstrations of the Golgi apparatus. Tissues were fixed in
Fleming's strong and weak fluids as well. Only the modified Mann-Kopsch
method gave consistent results and the following description of changes is
based on that technic.
The Golgi apparatus is revealed as a net occupying a position next to the
nucleus in the greater cytoplasmic area. Fig. 2 shows three cells with typical
nets. The cytology of the Golgi apparatus follows the general description
for this component in cells of secretory type. In general, it is not demon-
570
LLOYD C. FOGG AND SHIELDS WARREN
strable in living cells, appears as a net only in the interkinetic phase, and
disappears when the cell is in mitosis.
For the purpose of clarity in description the changes that occur as the
result of irradiation will be described first for tissue taken from animals
treated by 180,000 volt x-ray and given a dose of 1600 r. I t should be noted
that 1600 r is a sufficiently large dose to kill most animals in a period of four
to five days. The tumor will by that time, however, show indications of
growth in some portions, although much of it is necrotic.
180,000-volt x-ray: Dose 1600 r
In the first few hours there is no apparent change in the Golgi apparatus
as compared to the controls.
Six Hours: Fig. 3 shows a central cell with Golgi apparatus revealed. A
study of many cells at this stage does not demonstrate any marked change
FIGS.7-8.
SPECIMENSIRRADIATED
WITH 400 KV. X-RAY,1600 r . LOWERMZGNIFICATION
7. Six hours after irradiation. 8. Sixteen hours after irradiation.
from the control. The net is in the same area, has not become fragmented
as a result of the radiation, has not increased in volume or shown any other
evidence of morphological change.
I t should be emphasized here that a difference was noted in the ease of
demonstration. The Golgi method is admittedly a somewhat capricious one,
resulting in some areas being stained and others not, an inconstancy which
extends to cells. However, with the same technic as far as possible, it is
more difficult to get well stained cells on tissue removed six hours after
irradiation than it is from non-irradiated tissue.
Eighteen Hours: By this time necrotic cells are abundant. In selected
areas, however, well delineated cells may be found. The studies are made
only from such clearly defined cells; the changes due to necrosis are disregarded.
The Golgi apparatus no longer has quite the sanle appearance. Selected
favorable cells show that the area occupied by the net is greater. The Golgi
rods forming the now somewhat disrupted net do not seem to be greater in
volume or diameter, but appear to have spread away somewhat from a central
area, the centrosomal area. Fig. 4 shows a cell, just at the left of center,
which gives an appearance almost like chromosomes. Just to the right is
another cell, cut at another level, which reveals the clear central area.
Nineteen Hours: At this time there appears to be a greater amount of
osmic-reducing substance throughout the tissue. As compared to tissue taken
from animals from one to six hours after irradiation, where impregnation was
difficult, here the tissues were frequently overstained and destaining with
turpentine was necessary.
The Golgi apparatus has seemingly lost its identity as a net. I t appears
as a blackened mass of substance which occupies a somewhat greater volume
than the typical net. Fig. 5 shows cells with this changed blackened area.
At a somewhat later stage the mass appears to break up into variable units.
Cells with normal nets were not found at this period after irradiation.
Twenty-four Hours: The general receptivity of the cytoplasm for osmic
acid is still apparent, but to a lesser degree than at sixteen to twenty hours.
I n those cells destined to recover, the fragmented Golgi units are re-collecting
as a net. The cell is again assuming the appearance of a non-irradiated typical tumor cell in so far as the Golgi apparatus is concerned. Fig. 6 shows
a cell nearly back to normal.
The series of changes described above provides an opportunity to compare the effect of the same dosage, 1600 r, transmitted by an x-ray machine
of greater power400,OOO volts--on the same type of tissue.
400,000-volt x-ray: Dose 1600 r
In the first few hours after irradiation no change in the Golgi apparatus
is observed. This is consistent with the results obtained with the 180 kv.
x-ray machine.
Six Hours: The same series of changes as described for tissue treated
with the 180 kv. x-ray machine is already under way. Fig. 7 shows cells in
different stages. T o the right of this center is a cell where the typical net,
clearly seen, is practically normal. Other cells can be identified which show
increased volume, massed blackened material, loss of a well defined net and
572
LLOYD C. FOGG AND SHIELDS WARREN
fragmentation. The most frequent condition is the appearance of the net.
The reason for the more advanced changes in some cells is problematic.
Sixteen Hours: No longer can a typical net be seen. The increased area
of osmic-impregnated material and the tendency of such material to avoid
the centrosomal area and begin to fragment are typical. Fig. 8 shows several
cells that are characteristic. The ease with which the cells may become
blackened with osmic acid at this time is marked.
A comparison of Fig. 8 with Fig. 5 gives no clue as to which machine was
used. This suggests that the series of changes described for the Golgi apparatus follow a definite pattern, irrespective of amount and type of irradiation.
Twenty-four Hours: Those cells destined to recover now show a lesser
capacity to become impregnated. The blackened mass has become more or
less fragmented and a net is re-forming. Fig. 9 shows a cell that is in the
process of reorganization.
WITH RADON
APPLICATOR,
TWO ERYTHE~LIA
DOSES
IRRADIATED
FIGS. 10-11. SPECIMI:NS
10. One hour after irradiation. 1 1 . Four hours after irradiation.
The excised tissue shows grossly large foci of necrosis. This tissue on the
fixed and stained slides uniformly shows the Golgi apparatus to be in a blackened mass roughly similar to that of the sixteen- to eighteen-hour specimen,
although more accentuated. This would suggest that the Golgi apparatus
must be reorganized into a typical net, after having been disorganized by irradiation, before the cell is capable of mitotic activity. Further work is necessary, however, before this point can be determined conclusively.
The experiments designed to test the comparative effects of different kinds
of radiation were continued with the use of radon. As nearly as possible a
dose equivalent to 1600 r was given.
Radon Applicator: 2345 mc. hrs.
The series of changes in the Golgi apparatus is the same, in so far as can
be determined, by this method of procedure as with x-ray irradiation. At
first there is little change, then an increase in osmic-impregnating substance,
a loss of the identity of the net, and finally a fragmentation of the mass followed by re-formation of the net. Figs. 10, 11, and 12 show the typical net,
the beginning of the mass formation, and the reorganization. The reorganization is notable in that it represents a stage where it is difficult to distinguish
between fragmented Golgi material and mitochondria. The mitochondria a t
this stage are numerous and receptive to stain.
Radium Pack: 1 / 2 Erythema Dose
By courtesy of Dr. Max Cutler of Chicago it was possible to test the effect
on the Golgi apparatus of radiation with the 2-gram and 4-gram radium packs.
The dose was equivalent to about one-half an erythema dose, an appreciably
smaller amount than that used in the experiments already described.
Two-gram Pack: The same cycle of changes takes place with this method
of treatment as with x-ray or radon irradiation. I t is suggestive that on histological examination of the tissue fewer cells are found to have been affected
by the treatment, which is in accord with the observation that the tumors in
general were more firm and showed less evidence of necrosis.
With the lesser dose and rate of irradiation the changes that do occur in
the Golgi apparatus are less marked (compare, for example, the eighteen-hour
picture in Fig. 13, with either of the x-ray method pictures for the same time
period). By twenty-four hours many mitoses are visible and normal appearing cells are abundant.
Four-gram Pack: That no visible change occurs in the first few hours is
again confirmed. Fig. 14 shows a cell with a typical net. This photograph
clearly demonstrates a cell in mitosis, showing graphically the complete lack
of anything like a Golgi apparatus.
Fig. 15 shows a cell selected to demonstrate the blackened mass that may
appear in one stage of the series of changes. I n this particular photograph
the mass is somewhat accentuated and the question arises as to whether the
cell would have recovered. In cells that do not recover this blackened mass
is rarely if ever as pronounced as here.
Fig. 16 shows two cells recovering at the twenty-four hour interval. The
lack of a definite net and the numerous mitochondria are typical. At this
time numerous cells in mitosis may be seen in other areas. Whether this
suggests a variability in cell susceptibility has not been determined. A com-
574
LLOYD C. FOGG A N D SHIELDS WARREN
parison of the illustrations of the material irradiated with the 2-gram pack
and with the 4-gram pack suggests that the latter has had somewhat more of
an effect. The changes that have occurred are the same, but seem more pronounced. The limited amount of material, however, does not permit emphasis
to be laid on this point.
FIGS.13-16. SPECIMENSIRRADIATED
WITH RADIUMPACK, % ERYTHEMA
DOSE
13. Eighteen hours after irradiation with 2-gram pack. 14. One hour after irradiation with
4-gram pack. 15. Eighteen hours after irradiation wtih 4-gram pack. 16. Twenty-four hours
after irradiation with 4-gram pack.
Other Sources of Radiation
The effect of lesser dosages, comparable to those of the radium packs, was
tested with the x-ray machine and with the radon bomb. The following doses
and types of radiation were used:
180,000 volt x-ray. . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 r and 800 r
400,000 volt x-ray. . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 r
Radon bomb. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.i erythema dose
In general the lesser dosages do not reveal any new development. More
cells show mitoses and the general effect is reduced. This result is reflected
in the Golgi apparatus. The significant point is that the changes do occur,
even though they are not so marked. In every case recovery was more rapid,
i.e. in less than twenty-four hours. Further, no morphological difference can
be detected in the appearance of the Golgi apparatus at any given stage after
irradiation with equivalent dosage.
The presence of a Golgi apparatus in malignant tissue has been demonstrated frequently and by different technics. By means of tissue culture
Vhzquez-L6pez (13) was able to show in the Jensen rat sarcoma that the surface area of the Golgi apparatus is equal to that of the nucleus. Turchini and
Broussy (14) maintain that during division some cells retain a reduced number
of Golgi vesicles. Ludford (8) demonstrated the Golgi apparatus in a number
of neoplasms, including the transplantable mouse adenocarcinoma 2 7 , sarcoma 3 7, and mammary carcinoma 63, and some human material. Guyer and
Claus (15) not only demonstrated the Golgi apparatus in the Jobling rat
carcinoma, but believe that increased activity of the Golgi apparatus bears
a relation to hypertrophy in the basophil cells in the anterior pituitary. Bartelmetz and Bensley (16) have reported that the volume of the Golgi apparatus
changes with the physiological state of the cell. In the present work a volume
change has been noted as the result of irradiation by x-rays, radium, and radon.
Nahm (17) pointed out that staining methods are inconstant in reference
to the Golgi apparatus, but suggested as a result of her studies that it represents unsaturated fatty acids. I t is in~possiblefrom this work to add any
information concerning its nature. The generally accepted conception is that
it is a fluid component of cytoplasm, probably ubiquitous, capable of reducing
osmic acid, is generally polar in secretory type cells, and probably composed
of both proteids and fats with the fats predominating.
Among others, Gatenby (18) has described the effect of radiation on the
Golgi apparatus in normal tissue. Ludford (8) has described changes as the
result of a constant dosage of radiation, using several tumors, both transplantable mouse and human neoplasms. In the transplantable mouse adenocarcinoma 2 7 he found that a dose of 58 mg. of radium element did not
produce regression, i.e. the tumor was radioresistant. His description of
changes that took place at different intervals is outlined below:
40 minutes after radiation. . . . . . . Golgi apparatus less clearly defined
7 hours
"
'
. . . . . . . Golgi apparatus like control
24 hours
"
'
. . . . . . . Cytoplasm stains less intensely. No statement concerning Golgi apparatus. No mitoses
50 hours
"
. . . . . . Golgi bodies surround secretion. Later
become scattered. Some normal cells, some
degenerate ones
3 days
"
"
. . . . . . . Mitoses
5days
"
'
. . . . . . . Golgi apparatus enlarged, tending to be
more granular and less sharply defined
6 days
"
"
. . . . . . . Tumor had grown. Golgi apparatus largcly
associated with secretion
7-11 days
"
"
. . . . . . . A period of decrease in size of tumor. Large
cells with enlarged Golgi apparatus
14-22 days
"
"
. . . . . . . Period of growth. Golgi apparatus like
control
Ludford summarizes the effects of irradiation by stating that it causes
cells to hypertrophy and this in turn may cause the Golgi apparatus to enlarge. The enlarged Golgi apparatus breaks up in degenerating cells. The
576
LLOYD C. FOGG AND SHIELDS WARREN
work of Ludford is comparable, therefore, to the present work in that he has
reported changes as a result of irradiation of tumor-bearing animals. The
enlargement of the Golgi apparatus is confirmed in this work on Walker 256.
'While Ludford has described the changes that occur after irradiation a t
intervals which include subsequent growth of the tumor, the present study has
been confined to the changes that occur from the cessation of most mitoses
until the resumption of nuclear activity, namely, for a period roughly equivalent to twenty-four hours. The degenerating cells have not been considered.
I t has been found that the Golgi apparatus does enlarge, then loses its identity
as a net, and subsequently becomes fragmented before reorganizing itself as a
net just previous to nuclear activity. This series of changes takes place regardless of the type of radiation if a sufficient dose is given. Lesser dosages
may not produce as marked changes, however.
A comparison of the 180 kv. x-ray with the 400 kv. x-ray when equivalent
doses in r units are given suggests that there is a direct relationship in time,
i.e. the series of changes occurs earlier with x-rays produced at the higher
voltages, but due to the lack of controlled quantitative studies this can be only
a suggestion.
I t is interesting to note that the Golgi apparatus disappears when mitosis
occurs. In this connection an attempt was made to ascertain if nuclear activity would begin previous to complete reorganization of the Golgi net subsequent to irradiation. No massed or enlarged Golgi apparatus was ever
found in what appeared to be normal cells capable of continued growth.
This would suggest that the disorganization caused by radiation in the cytoplasm must be remedied before normal activity can be resumed. This reorganization seems to precede normal mitotic activity. I t should be noted
that irradiation causes the cells to be in the resting stage previous to any
demonstrable change in the Golgi apparatus. Several hours may elapse
before spreading and massing are observed. The time at which this may
occur possibly bears some relationship to intensity.
1. The radioresistant transplantable rat adenocarcinoma Walker 256 was
used to test the effect of different types and intensities of radiation on the
Golgi apparatus.
2. A definite series of changes was noted, as follows:
(a) No change for a few hours.
( b ) The Golgi net spreads away from the centrosomal area.
( c ) I t becomes enlarged and massed, losing its identity as a net.
(d) Fragmentation occurs, followed by reorganization into a typical net.
3. This series of changes occurs within a period of twenty-four hours for
dosages up to 1600 r. This period is equivalent to the period required for
restoration of full mitotic activity in this tumor following similar dosage.
4. The amount of enlargement of the Golgi region bears some relationship
to the dosage of irradiation, the greater the dosage the more marked the enlargement and density of the blackened mass. The series of changes seems
to occur sooner with higher voltages.
5. The uniformity and ease with which this tissue may be impregnated
with osmic acid vary with time in the twenty-four hour interval. The greatest
ease of demonstration occurs in the interval from six to twenty-four hours.
6. Variation of wavelength within the range of radiation studied did not
alter the character of the cytoplasmic changes,.
NOTE:We are greatly indebted to Drs. R. C. Dresser, Jack Spencer, and H. L. Marks
of Boston for the use of the x-ray apparatus of the New England Deaconess and Collis P.
Huntington Hospitals, and to Dr. Max Cutler of Chicago for the privilege of using the twogram and four-gram radium packs at the Michael Reese Hospital.
BIBLIOGRAPHY
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
STENSTROM,
K. W.: J. Cancer Research 9 : 19@203, 1925.
ALTER,N. M.: J. M. Research 40: 241-264, 1919; 41: 439-456, 1920.
KIMURA,N.: J. Cancer Research 4 : 95-135, 1919.
DUSTIN,A. P.: Le cancer 7: 41-49, 1930.
LUCAS,F. F.: Proc. Nat. Acad. Sci. 16: 599-607, 1930.
SPEAR,F. G.: Brit. J. Radiol. 8: 68-86, 280-297, 1935.
WHITMAN,W. G.: Am. J. Cancer 17: 932-945, 1933.
LUDFORD,
R. J.: Tenth Scientific Report, Imperial Cancer Research Fund, 1932, pp.
125-168.
EARLE,W. R . : Am. J. Cancer 24: 566-612, 1935.
E . : Ztschr. f. physiol. Chem. 2 19. 115-127,
WALDSCHMIDT-LEITZ,
E., A N D MCDONALD,
1933.
LEWIS, M. R . : Abst. Anat. Record 45: 268-269, 1930.
WARREN,S. : Radiology (in press).
VAZQUEZ-LOPEZ,
E . : Arch espaii. de oncol. 3 : 395-420, 1933.
TURCHINI,J., AND BROUSSY,
J . : Compt. rend. Soc. de biol. 103: 1223-1224, 1930.
GUYER,M. F., A N D CLAUS,P. E.: Anat. Record 61: 57-69, 1934.
BARTELMETZ,
G. W., AND BENSLEY,C. M.: Human uterine gland cells, in Special Cytology, edited by E. V. Cowdry, Paul B. Hoeber, New York, 1932, vol. 3, pp. 15251564.
NAHM,L. J.: J. Morphol. 54: 259-301, 1933.
GATENBY,
J. B., MUKERJI,R. N., AND WIGODER,S. B.: Proc. Roy. Soc. ser. B, 105:
446-469, 1929.