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Cytological analysis of colonies developed from mammalian cells irradiated in vitro with X-rays
Experimental
268
Cell Research 29, 26X-277 (1963)
CYTOLOGICAL ANALYSIS OF COLONIES MAMMALIAN CELLS IRRADIATED WITH X-RAYS1 G. COLOMBO Institute
DEVELOPED IN UZ’RO
FROM
and G. MARIN
of Comparative Anatomy, University of Perugia, and Institute and Comparative Anatomy, University of Padova, Italy
of Zoology
Received April 14, 1962
was first shown by Puck et nl. [ll, 121 that when mammalian cells are plated in vitro and irradiated with X-rays, only a fraction of the cells retain the ability to form macroscopic colonies. This fraction can be regarded as the proportion of the cell population whose reproductive functions have not been damaged by radiation. S-ray damage to reproductive functions, however, is expressed in a number of cytological features. Indeed an irradiated cell may fail to give rise to a macroscopic colony in several ways: (a) when it fails to divide at all and eventually dies; (b) when it fails to divide but grows into a “giant” cell; (c) when it undergoes a limited number of divisions and forms an “abortive” colony, i.e. a colony of less than 50 cells [ll]. In any case the end effect may be described as an inhibition of cell division which leads to a more or less delayed lethality. The proportion of cells dying through any of the above morphological pathways seems to vary at different doses. While extremely high doses are needed to induce immediate death, after a wide range of intermediate doses cell multiplication is entirely stopped and most of the survivors become giant. At low doses abortive division is the commonest form of reproductive death, although the incidence of giant cells is still high [ 111. It seemed useful to study quantitatively the occurrence in surviving colonies of these morphological features, i.e. abortive cell division and giant cell formation, in order to make a closer estimate of their relative lveight in overall lethality. Preliminary experiments showed that the fraction of colonies with one or more giant cells increases with time so that, four days after irradi-
IT
1 This work Agency. Experimental
was performed
Cell Research 29
under
contract
no. 36 with
the International
Atomic
Energy
Cytological analysis of colonies developed from mammalian cells
269
ation, the proportion of colonies without giant cells is close to the proportion of macroscopic colonies scored in standard survival experiments [6]. These results, however, could have been misleading, since the total number of colonies surviving at any time had not been recorded. In the present research the history of ?(-raved cell progenies has been traced for a longer period, namely eight days after irradiation, and the whole population of colonies arising from knovvn inocula of cells has been scored.
MATERIAL
AND
METHODS
Cells.-The RCP strain used in these experiments was derived from guinea pig kidney cells adapted to in vitro growth by Gasparini et al. [3] at the Institute of Hygiene of the University of Padova, Italy.’ The strain is heteroploid and polymorphic, having a variable proportion of tightly packed and loosely growing cells [5]. Doubling time in standard growth medium is about 24 hr [2]. Standard culture methods.--Standard medium consists of Hanks’ balanced salt solution containing 5 per cent lactalbumin hydrolysate (Nutritional Biochemicals Co.), 0.01 per cent yeast extract (Difco), 10 per cent undyalyzed calf serum heated for 1 hr at 56”C, 0.002 per cent phenol red (Merck), with 100 U/ml sodium penicillin and 0.1 mg/ml dihydrostreptomycin sulphate. When growth of isolated colonies is required the serum concentration is doubled (20 per cent): in such a “clonal medium” the plating efficiency averages 70 per cent or more. Stock cultures are grown in stoppered Jena culture bottles supplemented with standard medium and incubated at 37°C. For subculture the bottles are incubated for IO-15 min in 0.05 per cent Difco 200: 1 trypsin in Ca- and Mg-free Hanks’ solution, the cells are centrifuged at 1000 rpm for 3 min and dispersed in growth medium with a Pasteur pipette. Cell counts are made in a standard Btirker haemocytometer when the suspended cells appear to be at least 95 per cent single. Experimental cultures.-For routine survival experiments samples of irradiated and of control cell suspensions, at a known cell concentration, were plated in Petri dishes with “clonal medium” and kept for 12 days at 37°C in a CO, supplemented incubator. After staining, colony counts were made with the naked eye. Surviving fractions, at any dose, were calculated as percentages of the controls, using the mean number of counted colonies. For the cytological analysis of colony growth, cells were plated on 24 x 32 coverslip slides in 6 cm Petri dishes, by spreading on the coverslip 1 ml of a cell suspension in “clonal medium” at a final concentration of 250 cells per ml (500 cells per ml for the highest dose of X-rays). Care was taken to avoid any overflow of the medium so that all the cells settled down on the coverslip. Six hours later, when adherence of the cells to the glass was presumably complete, 4 ml of fresh medium were added to each dish. The plates were incubated at 37°C in a CO, supplemented incubator. At different times after plating the coverslips were fixed in Bouin-alcohol, stained 1 We are indebted
to Dr. V. Gasparini
for a sample of this cell line.
Experimental Cell Research 29
G. Colombo and G. Marin with Mayer’s haemallum and mounted. The total number of colonies developed on each coverslip was determined by microscopic examination. Any single isolated cell or cluster of cells, lying more or less together, was counted as a colony. In normal cultures spacing between colonies appeared wide enough to allow for an unambiguous identification of individual colonies, though of the loosely growing type. The progenies of irradiated cells often appeared more scattered and individual colonies were sometimes difficult to identify; however, the uncertainty introduced in the final values was negligible since errors in both directions were equally probable. Each colony was classed according to its size, i.e. number of cells per colony. In addition the colonies were classified according to whether giant cells were present or not. A cell was regarded as a “giant” when it showed a very large, often lobate nucleus, or when two or more nuclei were surrounded by a clearly single cell boundary. The term “giant” will be used with this meaning throughout this paper. Irradiation.--The cells, harvested from a log-phase culture and suspended in growth medium at a concentration of lo5 cells per ml, were spread over the bottom of a 50 ml beaker in a layer less than 1 mm thick. The beaker, covered by a 0.01 mm aluminium foil, was irradiated with an X-ray apparatus “Gilardoni Terapia 200/6”, operating at 180 kV and 6 mA, with inherent filtration equivalent to 3 mm of Al. Doses of 150, 300 and 450 roentgens were delivered at a dose rate of 150 r per min. Irradiation was always carried out at room temperature.
RESULTS The colonies developed from cells irradiated at different doses and plated in coverslips were examined at 2, 4, (i and 8 days after treatment. In the irradiated series giant cells appeared to be very frequent, both single or in colonies together with morphologicall?; normal cells (Figs. l-C,>. In Fig. 7 are reported the total numbers of colonies (solid circles) and the numbers of colonies with giant cells (empty circles) per slide, from normal and X-rayed inocula, at different days after plating. At least three slides were scored for each time and dose. The average values of total colony counts are joined by solid lines, whereas broken lines join the average numbers of colonies with giant cells. I’he difl’erence between the two sets of values gives the numbers of colonies zuithout giant cells, which will be referred to as “normal”. The zero-time values correspond to the size of the inocula (average number of cells plated on each slide), and to the mean number of colonies \vith giant cells found at the second day in the controls, which can be assumed to be an estimate of the average proportion of giant cells in the strain. From Fig. 7 it is apparent that in the controls there is no significant loss of colonies from the slides; about 20 per cent of the colonies contain giant cells and this proportion remains approximately constant during growth. In Experimenfal
Cell Research 29
Cytological analysis of colonies developed from mammalian cells
Fig. I.--Single Fig, 2.-Small Fig. 3.-“Normal” Fig. 4.-Large
giant cell colony,
8 days after irradiation
by 450 r ( x 400).
colony with 4 giant cells, 8 days after irradiation colony,
8 days after irradiation
271
by 450 r ( x 400).
by 450 r ( x 400).
colony with giant cells, 8 days after irradiation
by 450 r ( x 160). Experimenlal
Cell Research 29
G. Colombo and G. Marin
272
l:ig. 5.-Colony l:ig. G.-Large
from non-irradiated “normal”
colony
culture,
with giant cells, 8 days after plating
from non-irradiated
culture,
8 days after plating
( x 160). ( y 160)
the irradiated series, the total numbers of colonies per slide decrease as growth proceeds: the rate of colony loss is higher as higher doses are used. The numbers of colonies with giant cells increase, at the second day, to values almost proportional to dose; later they decrease at a rate roughly parallel to the total colony loss. Therefore the numbers of “normal” colonies appear to be fairly constant from the fourth day on, and loss affects mainly the class of colonies with giant cells. Since, after 150 and 300 r, the total numbers of colonies at the second day are about equal to the mean number of cells plated, we can assume that no significant cell loss occurs during the first two days after irradiation. During this period transformation of normal cells to giants takes place at a high rate. On the other hand, after the fourth day, when colonies have already begun to die off, few if any new giants appear, since the numbers of “normal” colonies remain fairly constant. An intermediate period, when the two phenomena may overlap to a variable extent, extends from the second to the fourth day. After irradiation with 450 r, a decrease in the total number of colonies is already apparent at the second day. It is possible that an early loss of Experimental
Cell Research 29
Cytological analysis of colonies developed from mammalian cells
273
“normal” colonies may have occurred here, even though loss of colonies with giant cells prevails later. The distribution of the numbers of cells per colony at different times after plating shows that an inhibition of colony growth occurs too (Fig. 8). It must be pointed out, however, that an apparent decrease in growth rate may result from cell mortality in individual colonies: small-sized colonies may be those in which some cells have died and become detached from the slide. 300 200 100
Control
. .
. I
-
8. . . . . . . . .--I-. . . . . .._.8.._.__-__-B
i
Fig. 7.-Total numbers -of colonies per slide (solid circles) and numbers of colonies with giant cells (empty circles), :in days following X-radiation.
b
i
b
i
DAYS
Anyhow the shift of the modal classes in following days indicates the overall rate of growth. In the irradiated series, by comparison with the controls, some kind of inhibition of growth appears to occur also among “normal” colonies, mainly in later-stages, and to a larger extent as larger doses are concerned. It seems, therefore, that colonies which appear morphologically normal have nevertheless been damaged to some extent by X-rays. However, growth appears to be more inhibited among colonies with giant cells, as may be expected. Although a small proportion of colonies with giant cells may attain a considerable size, the single giant cell class always prevails. 18 - 621806
Experimenlal
Cell Research 29
274
G. Colombo and G. Marin DISCUSSION
It is a well known fact that N-ray induced lethality displays a phenotypic lag in most living cells: it has been referred to as “reproductive death” since a specific damage to the reproductive system seems to be the reason why the cell eventually dies.
LOG,
NUMBERS
OF CELLS
PER
COLONY
Fig. S.-Per cent distribution of colonies of different size (i.e. number of cells per colony) in days following X-radiation. Abscissae in logarithmic scale, central values are log, integers, corresponding to doubling numbers of cells per colony. Shaded histograms = “normal” colonies; unshaded histograms = colonies with giant cells.
In agreement with this, the data here reported show that almost all the cells irradiated with doses ranging from 150 to 450 r do survive for some time, and most of them keep on dividing. Only the highest dose tested caused some cell to die before the second day following irradiation, that is, within the first or possibly the second division cycle. Part of the colonies developed from irradiated cells, however, are going to be lost, since a decrease in colony numbers will take place from the second day on. It appears, therefore, Experimental
Ceil Research 29
Cyfological
analysis
of colonies developed from mammalian
275
cells
that X-rays produce in the cell a transmissible lethal damage, which can be carried on by the cell’s progeny for some generations before showing up. Furthermore, our results confirm that one of the most striking morphological features induced by low-dose irradiation is the appearance of giant This efyect points out the specificity cells, either single or multinucleated. of X-ray damage on the mechanisms leading to cell division, and particularly to cytokinesis, since increase in mass and, to some extent, nuclear division I. Colonies of different size and morphology, surviving after irradiation (fractions of controls).
TABLE
0
150
300
450
at 8 days
1.00 (246.3 k 0.3ja
0.66 (162 2 5.7)
0.43 (105.3 2 4.3)
0.25 (62.0 i 5.3)
colonies at 8 days
1 .oo (220.0& 5.5)
0.24 (52.3 i 6.6)
0.10 (23.0 F 2.4) 0.06 (ll.lt1.9)
X-ray
Total
dose, r
colonies
“Normal”
at 8 and /cl days
Large colonies (2 32 cells) at 8 days
1.00 (177.6? 7.7)
0.42 (73.7 * 5.0)
0.17 (30.4 + 3.2)
1.00
0.45
0.15
Standard survival values, i. e. macroscopic colonies at 12 daysb a In brackets fractions. b Interpolated
0.53 (117.3i6.1)
are mean numbers
of colonies from three cultures,
values from a typical
survival
0.05
used to calculate
curve of the RCP strain (unpublished
surviving data).
are not affected. Giant cells may result from a primary block of division in the irradiated cell itself, or may appear in the irradiated cell’s progeny at different times, since the mean number of giant cells per colony was shown to increase in subsequent days [6]. Many authors have recorded the appearance of giant cells in cell populations irradiated in vitro [4, S-121, and biochemical studies have been carried out on these elements [7, 13, 141, but it has never been clearly established to what extent reproductive death is related to giant cell formation. Although both X-ray induced lethality and block of cell division followed by an increase of the cell’s size appear to be caused by some hereditable damage, the damage is not necessarily the same in both cases. Obviously the transformation of an irradiated cell into a giant equals reproductive death, but it is questionable whether reproductive death always and uniquely follows the appearance of giant cells in the irradiated cell’s progeny, Experimental
Cell Research 29
G. Colombo and G. Marin Puck and Marcus [ 1 l] report that practically all the abortive colonies scored at the time of colony counting (1 l-l 3 days) in X-ray survival experiments on HeLa cells contain giants. ,41so, our data seem to show that most of the colonies lost during early stages of growth (2-8 days) are colonies Avith, giant cells. Therefore, since giant cells appear only in lethal cell progenies, they should be looked upon as a phenotypic expression of a lethal hereditable damage. However, when the total numbers of “normal” colonies scored at 8 days are compared with standard survival values, i.e. macroscopic colonies at 12 days, the former appear to be somewhat higher. To approximate the standard survival values, only colonies with an average of 32 cells or mow must be taken into account (Table I). This means that a fraction of “normal” colonies may be regarded as abortive, since they will be lost or at least will not develop to macroscopic size within the twelfth day. The dill’erencc between the two sets of values represents a prospective colony loss due to a delayed lethal mechanism apparently unrelated to giant cell formation. Indirect evidence that more than one “target” is involved in X-ray induced lethality of mammalian cells comes also from the statistical analysis of survival curves. Bender and Gooch [l] showed that survival data for t\vo different strains of human cells grown in vitro fit better a “multi-hit” model than the “single-process” model which had been proposed by other authors. It seems, therefore, that although reproductive death may be primarily related, for a wide range of doses, to the same damage which causes the cell to become giant, other mechanisms appear to be involved in the induction of delayed lethality. SUMMARY
The relationship between X-ray induced reproductive death and blockage of cell division leading to giant cell formation, has been studied on a strain of colonies of mammalian cells grown in vitro. The size and morphology developed from single-cell platings were recorded from samples fixed and stained every second day, up to 8 days after irradiation with 150, 300 and 450 r. Early appearance of giant cells in the progenies of irradiated cells Since loss affected mainly the was followed by delayed loss of colonies. colonies with giant cells, the presence of giants turned out to be a morphological expression of some kind of lethal damage carried in the clones. Therefore giant cell formation and reproductive death could be ascribed to the same hereditable damage, although some minor lethality appeared to be related to a different mechanism. Experimental
Cell Research 29
Cytological analysis of colonies developed from mammalian cells
277
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
BENDER, M. A. and GOOCH, P. C., Inf. J. Rad. Biol. 5, 133 (1962). COLOMBO, G. and MARIN, G., Boll. Sot. If. Biol. Sper. 36, 1593 (1960). GASPARINI, V., FARISANO, G. and GAMBA, F., Boll. Ist. Sieroferapico Mifanese 39, 132 (1960). KOHS, H. J. and FOGH, J. E.. J. Nafl. Cancer Inst. 23, 293 (1959). MARIP~, G., Boll. di Zoof. 28, 727 (1962). MARIN, G. and COLOMBO, G., Affi Ass. Genefica lfafiana 6, 125 (1961). NIAS, A. H. UT. and PAUL, .J., Int. J. Radiation Biol. 3, 431 (1961). PATERSON, E., Brif. J. Radio!. 15, 257 (1942). Y. H. and KENT, S. I’., Z. Zellforsch. 48, POMERAT, C. M., FERNA~-DES, M. V., NAICAKISHI, 1 (1958). POMERAT, C. M., KENT, S. P. and LOGIE, L. C., ibid. 47, 158 (1957). PUCK, T. T. and MARCUS, P. I., .I. Expfl. Med. 103, 653 (1956). PUCK, T. T., MORKOVIN, D., MARCUS, P. I. and CIECIURA, S. J., ibid. 106, 485 (1957). SHEEK, M. R., DES ARMIER, R. M., SAGIK, B. P. and MAGEE, W. E., Espfl. Cell Research 19, 549 (1960). WHITX~ORE, G. T., TILL, J. E., GWATKIS, R. B. L., SIMISOVITCH, I,. and GRAHA~~, A. F., Biochim. Biophys. Acta 30, 583 (1958).
Experimental
Cell Research 29
268
Cell Research 29, 26X-277 (1963)
CYTOLOGICAL ANALYSIS OF COLONIES MAMMALIAN CELLS IRRADIATED WITH X-RAYS1 G. COLOMBO Institute
DEVELOPED IN UZ’RO
FROM
and G. MARIN
of Comparative Anatomy, University of Perugia, and Institute and Comparative Anatomy, University of Padova, Italy
of Zoology
Received April 14, 1962
was first shown by Puck et nl. [ll, 121 that when mammalian cells are plated in vitro and irradiated with X-rays, only a fraction of the cells retain the ability to form macroscopic colonies. This fraction can be regarded as the proportion of the cell population whose reproductive functions have not been damaged by radiation. S-ray damage to reproductive functions, however, is expressed in a number of cytological features. Indeed an irradiated cell may fail to give rise to a macroscopic colony in several ways: (a) when it fails to divide at all and eventually dies; (b) when it fails to divide but grows into a “giant” cell; (c) when it undergoes a limited number of divisions and forms an “abortive” colony, i.e. a colony of less than 50 cells [ll]. In any case the end effect may be described as an inhibition of cell division which leads to a more or less delayed lethality. The proportion of cells dying through any of the above morphological pathways seems to vary at different doses. While extremely high doses are needed to induce immediate death, after a wide range of intermediate doses cell multiplication is entirely stopped and most of the survivors become giant. At low doses abortive division is the commonest form of reproductive death, although the incidence of giant cells is still high [ 111. It seemed useful to study quantitatively the occurrence in surviving colonies of these morphological features, i.e. abortive cell division and giant cell formation, in order to make a closer estimate of their relative lveight in overall lethality. Preliminary experiments showed that the fraction of colonies with one or more giant cells increases with time so that, four days after irradi-
IT
1 This work Agency. Experimental
was performed
Cell Research 29
under
contract
no. 36 with
the International
Atomic
Energy
Cytological analysis of colonies developed from mammalian cells
269
ation, the proportion of colonies without giant cells is close to the proportion of macroscopic colonies scored in standard survival experiments [6]. These results, however, could have been misleading, since the total number of colonies surviving at any time had not been recorded. In the present research the history of ?(-raved cell progenies has been traced for a longer period, namely eight days after irradiation, and the whole population of colonies arising from knovvn inocula of cells has been scored.
MATERIAL
AND
METHODS
Cells.-The RCP strain used in these experiments was derived from guinea pig kidney cells adapted to in vitro growth by Gasparini et al. [3] at the Institute of Hygiene of the University of Padova, Italy.’ The strain is heteroploid and polymorphic, having a variable proportion of tightly packed and loosely growing cells [5]. Doubling time in standard growth medium is about 24 hr [2]. Standard culture methods.--Standard medium consists of Hanks’ balanced salt solution containing 5 per cent lactalbumin hydrolysate (Nutritional Biochemicals Co.), 0.01 per cent yeast extract (Difco), 10 per cent undyalyzed calf serum heated for 1 hr at 56”C, 0.002 per cent phenol red (Merck), with 100 U/ml sodium penicillin and 0.1 mg/ml dihydrostreptomycin sulphate. When growth of isolated colonies is required the serum concentration is doubled (20 per cent): in such a “clonal medium” the plating efficiency averages 70 per cent or more. Stock cultures are grown in stoppered Jena culture bottles supplemented with standard medium and incubated at 37°C. For subculture the bottles are incubated for IO-15 min in 0.05 per cent Difco 200: 1 trypsin in Ca- and Mg-free Hanks’ solution, the cells are centrifuged at 1000 rpm for 3 min and dispersed in growth medium with a Pasteur pipette. Cell counts are made in a standard Btirker haemocytometer when the suspended cells appear to be at least 95 per cent single. Experimental cultures.-For routine survival experiments samples of irradiated and of control cell suspensions, at a known cell concentration, were plated in Petri dishes with “clonal medium” and kept for 12 days at 37°C in a CO, supplemented incubator. After staining, colony counts were made with the naked eye. Surviving fractions, at any dose, were calculated as percentages of the controls, using the mean number of counted colonies. For the cytological analysis of colony growth, cells were plated on 24 x 32 coverslip slides in 6 cm Petri dishes, by spreading on the coverslip 1 ml of a cell suspension in “clonal medium” at a final concentration of 250 cells per ml (500 cells per ml for the highest dose of X-rays). Care was taken to avoid any overflow of the medium so that all the cells settled down on the coverslip. Six hours later, when adherence of the cells to the glass was presumably complete, 4 ml of fresh medium were added to each dish. The plates were incubated at 37°C in a CO, supplemented incubator. At different times after plating the coverslips were fixed in Bouin-alcohol, stained 1 We are indebted
to Dr. V. Gasparini
for a sample of this cell line.
Experimental Cell Research 29
G. Colombo and G. Marin with Mayer’s haemallum and mounted. The total number of colonies developed on each coverslip was determined by microscopic examination. Any single isolated cell or cluster of cells, lying more or less together, was counted as a colony. In normal cultures spacing between colonies appeared wide enough to allow for an unambiguous identification of individual colonies, though of the loosely growing type. The progenies of irradiated cells often appeared more scattered and individual colonies were sometimes difficult to identify; however, the uncertainty introduced in the final values was negligible since errors in both directions were equally probable. Each colony was classed according to its size, i.e. number of cells per colony. In addition the colonies were classified according to whether giant cells were present or not. A cell was regarded as a “giant” when it showed a very large, often lobate nucleus, or when two or more nuclei were surrounded by a clearly single cell boundary. The term “giant” will be used with this meaning throughout this paper. Irradiation.--The cells, harvested from a log-phase culture and suspended in growth medium at a concentration of lo5 cells per ml, were spread over the bottom of a 50 ml beaker in a layer less than 1 mm thick. The beaker, covered by a 0.01 mm aluminium foil, was irradiated with an X-ray apparatus “Gilardoni Terapia 200/6”, operating at 180 kV and 6 mA, with inherent filtration equivalent to 3 mm of Al. Doses of 150, 300 and 450 roentgens were delivered at a dose rate of 150 r per min. Irradiation was always carried out at room temperature.
RESULTS The colonies developed from cells irradiated at different doses and plated in coverslips were examined at 2, 4, (i and 8 days after treatment. In the irradiated series giant cells appeared to be very frequent, both single or in colonies together with morphologicall?; normal cells (Figs. l-C,>. In Fig. 7 are reported the total numbers of colonies (solid circles) and the numbers of colonies with giant cells (empty circles) per slide, from normal and X-rayed inocula, at different days after plating. At least three slides were scored for each time and dose. The average values of total colony counts are joined by solid lines, whereas broken lines join the average numbers of colonies with giant cells. I’he difl’erence between the two sets of values gives the numbers of colonies zuithout giant cells, which will be referred to as “normal”. The zero-time values correspond to the size of the inocula (average number of cells plated on each slide), and to the mean number of colonies \vith giant cells found at the second day in the controls, which can be assumed to be an estimate of the average proportion of giant cells in the strain. From Fig. 7 it is apparent that in the controls there is no significant loss of colonies from the slides; about 20 per cent of the colonies contain giant cells and this proportion remains approximately constant during growth. In Experimenfal
Cell Research 29
Cytological analysis of colonies developed from mammalian cells
Fig. I.--Single Fig, 2.-Small Fig. 3.-“Normal” Fig. 4.-Large
giant cell colony,
8 days after irradiation
by 450 r ( x 400).
colony with 4 giant cells, 8 days after irradiation colony,
8 days after irradiation
271
by 450 r ( x 400).
by 450 r ( x 400).
colony with giant cells, 8 days after irradiation
by 450 r ( x 160). Experimenlal
Cell Research 29
G. Colombo and G. Marin
272
l:ig. 5.-Colony l:ig. G.-Large
from non-irradiated “normal”
colony
culture,
with giant cells, 8 days after plating
from non-irradiated
culture,
8 days after plating
( x 160). ( y 160)
the irradiated series, the total numbers of colonies per slide decrease as growth proceeds: the rate of colony loss is higher as higher doses are used. The numbers of colonies with giant cells increase, at the second day, to values almost proportional to dose; later they decrease at a rate roughly parallel to the total colony loss. Therefore the numbers of “normal” colonies appear to be fairly constant from the fourth day on, and loss affects mainly the class of colonies with giant cells. Since, after 150 and 300 r, the total numbers of colonies at the second day are about equal to the mean number of cells plated, we can assume that no significant cell loss occurs during the first two days after irradiation. During this period transformation of normal cells to giants takes place at a high rate. On the other hand, after the fourth day, when colonies have already begun to die off, few if any new giants appear, since the numbers of “normal” colonies remain fairly constant. An intermediate period, when the two phenomena may overlap to a variable extent, extends from the second to the fourth day. After irradiation with 450 r, a decrease in the total number of colonies is already apparent at the second day. It is possible that an early loss of Experimental
Cell Research 29
Cytological analysis of colonies developed from mammalian cells
273
“normal” colonies may have occurred here, even though loss of colonies with giant cells prevails later. The distribution of the numbers of cells per colony at different times after plating shows that an inhibition of colony growth occurs too (Fig. 8). It must be pointed out, however, that an apparent decrease in growth rate may result from cell mortality in individual colonies: small-sized colonies may be those in which some cells have died and become detached from the slide. 300 200 100
Control
. .
. I
-
8. . . . . . . . .--I-. . . . . .._.8.._.__-__-B
i
Fig. 7.-Total numbers -of colonies per slide (solid circles) and numbers of colonies with giant cells (empty circles), :in days following X-radiation.
b
i
b
i
DAYS
Anyhow the shift of the modal classes in following days indicates the overall rate of growth. In the irradiated series, by comparison with the controls, some kind of inhibition of growth appears to occur also among “normal” colonies, mainly in later-stages, and to a larger extent as larger doses are concerned. It seems, therefore, that colonies which appear morphologically normal have nevertheless been damaged to some extent by X-rays. However, growth appears to be more inhibited among colonies with giant cells, as may be expected. Although a small proportion of colonies with giant cells may attain a considerable size, the single giant cell class always prevails. 18 - 621806
Experimenlal
Cell Research 29
274
G. Colombo and G. Marin DISCUSSION
It is a well known fact that N-ray induced lethality displays a phenotypic lag in most living cells: it has been referred to as “reproductive death” since a specific damage to the reproductive system seems to be the reason why the cell eventually dies.
LOG,
NUMBERS
OF CELLS
PER
COLONY
Fig. S.-Per cent distribution of colonies of different size (i.e. number of cells per colony) in days following X-radiation. Abscissae in logarithmic scale, central values are log, integers, corresponding to doubling numbers of cells per colony. Shaded histograms = “normal” colonies; unshaded histograms = colonies with giant cells.
In agreement with this, the data here reported show that almost all the cells irradiated with doses ranging from 150 to 450 r do survive for some time, and most of them keep on dividing. Only the highest dose tested caused some cell to die before the second day following irradiation, that is, within the first or possibly the second division cycle. Part of the colonies developed from irradiated cells, however, are going to be lost, since a decrease in colony numbers will take place from the second day on. It appears, therefore, Experimental
Ceil Research 29
Cyfological
analysis
of colonies developed from mammalian
275
cells
that X-rays produce in the cell a transmissible lethal damage, which can be carried on by the cell’s progeny for some generations before showing up. Furthermore, our results confirm that one of the most striking morphological features induced by low-dose irradiation is the appearance of giant This efyect points out the specificity cells, either single or multinucleated. of X-ray damage on the mechanisms leading to cell division, and particularly to cytokinesis, since increase in mass and, to some extent, nuclear division I. Colonies of different size and morphology, surviving after irradiation (fractions of controls).
TABLE
0
150
300
450
at 8 days
1.00 (246.3 k 0.3ja
0.66 (162 2 5.7)
0.43 (105.3 2 4.3)
0.25 (62.0 i 5.3)
colonies at 8 days
1 .oo (220.0& 5.5)
0.24 (52.3 i 6.6)
0.10 (23.0 F 2.4) 0.06 (ll.lt1.9)
X-ray
Total
dose, r
colonies
“Normal”
at 8 and /cl days
Large colonies (2 32 cells) at 8 days
1.00 (177.6? 7.7)
0.42 (73.7 * 5.0)
0.17 (30.4 + 3.2)
1.00
0.45
0.15
Standard survival values, i. e. macroscopic colonies at 12 daysb a In brackets fractions. b Interpolated
0.53 (117.3i6.1)
are mean numbers
of colonies from three cultures,
values from a typical
survival
0.05
used to calculate
curve of the RCP strain (unpublished
surviving data).
are not affected. Giant cells may result from a primary block of division in the irradiated cell itself, or may appear in the irradiated cell’s progeny at different times, since the mean number of giant cells per colony was shown to increase in subsequent days [6]. Many authors have recorded the appearance of giant cells in cell populations irradiated in vitro [4, S-121, and biochemical studies have been carried out on these elements [7, 13, 141, but it has never been clearly established to what extent reproductive death is related to giant cell formation. Although both X-ray induced lethality and block of cell division followed by an increase of the cell’s size appear to be caused by some hereditable damage, the damage is not necessarily the same in both cases. Obviously the transformation of an irradiated cell into a giant equals reproductive death, but it is questionable whether reproductive death always and uniquely follows the appearance of giant cells in the irradiated cell’s progeny, Experimental
Cell Research 29
G. Colombo and G. Marin Puck and Marcus [ 1 l] report that practically all the abortive colonies scored at the time of colony counting (1 l-l 3 days) in X-ray survival experiments on HeLa cells contain giants. ,41so, our data seem to show that most of the colonies lost during early stages of growth (2-8 days) are colonies Avith, giant cells. Therefore, since giant cells appear only in lethal cell progenies, they should be looked upon as a phenotypic expression of a lethal hereditable damage. However, when the total numbers of “normal” colonies scored at 8 days are compared with standard survival values, i.e. macroscopic colonies at 12 days, the former appear to be somewhat higher. To approximate the standard survival values, only colonies with an average of 32 cells or mow must be taken into account (Table I). This means that a fraction of “normal” colonies may be regarded as abortive, since they will be lost or at least will not develop to macroscopic size within the twelfth day. The dill’erencc between the two sets of values represents a prospective colony loss due to a delayed lethal mechanism apparently unrelated to giant cell formation. Indirect evidence that more than one “target” is involved in X-ray induced lethality of mammalian cells comes also from the statistical analysis of survival curves. Bender and Gooch [l] showed that survival data for t\vo different strains of human cells grown in vitro fit better a “multi-hit” model than the “single-process” model which had been proposed by other authors. It seems, therefore, that although reproductive death may be primarily related, for a wide range of doses, to the same damage which causes the cell to become giant, other mechanisms appear to be involved in the induction of delayed lethality. SUMMARY
The relationship between X-ray induced reproductive death and blockage of cell division leading to giant cell formation, has been studied on a strain of colonies of mammalian cells grown in vitro. The size and morphology developed from single-cell platings were recorded from samples fixed and stained every second day, up to 8 days after irradiation with 150, 300 and 450 r. Early appearance of giant cells in the progenies of irradiated cells Since loss affected mainly the was followed by delayed loss of colonies. colonies with giant cells, the presence of giants turned out to be a morphological expression of some kind of lethal damage carried in the clones. Therefore giant cell formation and reproductive death could be ascribed to the same hereditable damage, although some minor lethality appeared to be related to a different mechanism. Experimental
Cell Research 29
Cytological analysis of colonies developed from mammalian cells
277
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BENDER, M. A. and GOOCH, P. C., Inf. J. Rad. Biol. 5, 133 (1962). COLOMBO, G. and MARIN, G., Boll. Sot. If. Biol. Sper. 36, 1593 (1960). GASPARINI, V., FARISANO, G. and GAMBA, F., Boll. Ist. Sieroferapico Mifanese 39, 132 (1960). KOHS, H. J. and FOGH, J. E.. J. Nafl. Cancer Inst. 23, 293 (1959). MARIP~, G., Boll. di Zoof. 28, 727 (1962). MARIN, G. and COLOMBO, G., Affi Ass. Genefica lfafiana 6, 125 (1961). NIAS, A. H. UT. and PAUL, .J., Int. J. Radiation Biol. 3, 431 (1961). PATERSON, E., Brif. J. Radio!. 15, 257 (1942). Y. H. and KENT, S. I’., Z. Zellforsch. 48, POMERAT, C. M., FERNA~-DES, M. V., NAICAKISHI, 1 (1958). POMERAT, C. M., KENT, S. P. and LOGIE, L. C., ibid. 47, 158 (1957). PUCK, T. T. and MARCUS, P. I., .I. Expfl. Med. 103, 653 (1956). PUCK, T. T., MORKOVIN, D., MARCUS, P. I. and CIECIURA, S. J., ibid. 106, 485 (1957). SHEEK, M. R., DES ARMIER, R. M., SAGIK, B. P. and MAGEE, W. E., Espfl. Cell Research 19, 549 (1960). WHITX~ORE, G. T., TILL, J. E., GWATKIS, R. B. L., SIMISOVITCH, I,. and GRAHA~~, A. F., Biochim. Biophys. Acta 30, 583 (1958).
Experimental
Cell Research 29