- Email: [email protected]
Radiomimetic effects of a nitrogen mustard on survival, growth, protein and nucleic acid synthesis of mammalian cells in vitro
Experimental
RADIOMIMETIC SURVIVAL,
EFFECTS GROWTH,
SYNTHESIS
OF A NITROGEN PROTEIN
OF MAMMALIAN
A. G. LEVIS, Institute
19
Cell Research 31, 19-30 (19G3)
of Zoology and Institute
L. SPAN10 of Pharmaceutical
AND CELLS
and A. DE Chemistry,
MUSTARD
NUCLEIC
ON
ACID
1N VITRO1 NADAI University
of Padua, Italy
Received July 27, 19622
NITROGES mustards are commonly referred to as “radiomimetic” substances, since their biological end efTects are similar to those produced by ionizing radiations. At the cellular level, mustards stop mitosis [W], produce chromosome aberrations [ 151, and induce mutations [2], as ionizing radiations do. At the chemical level, as Alexander and Stacey [ 1 ] have suggested on the basis of in vitro evidence, the biological effects both of alkylating agents and of ionizing radiations can probably be attributed to a direct action on the DSA molecule. Studies on “radiomimetic” substances of the alkylating group may therefore contribute to the understanding of the mechanism of radiation-induced biological effects, whenever a close comparison between the etTects of the two mutagenic agents is feasible. While accurate data are no\\- available for radiation-induced effects in mammalian cells, work on “radiomimetic” drugs is still lacking. We have thus undertaken a study of the action of nitrogen mustards on a guinea-pig cell strain cultivated in rdro. The results on survival, growth and synthesis of nucleic acids and proteins are here described.
MATERIAL
AND
METHODS
Nitrogen musfard.~Methyl-bis-(@-chloroethyl)amine hydrochloride (HN2) of the Ciba Chem. Ind. was used (Dichloren). This substance is prepared in phials containing 5 mg of white, crystalline powder, water-soluble and stable in an acid medium. At the moment of use, the content of a phial was dissolved in 5 ml of sterile Hanks’, so that a 1000 y/ml solution was obtained. Cells.-The RCP strain, derived from guinea-pig kidney cells by Gasparini et al. [lo], was used. The characteristics of this strain, grown adherent to glass in Melnick’s 1 This work was performed under contract n. 36 with the International (I.A.E.A.). 2 Revised version received November 5, 1962.
Atomic
Experimental
Energy Agency
Cell Research 31
20
A. G. Levis, L. Spanio and A. De Nadai
medium 1201 supplemented with 20 per cent whole calf serum, have been described in a preceding paper [5]. survival curues.-Samples from a single-cell suspension were plated in 60 mm Petri dishes with 5 ml of standard growth medium. Nitrogen mustard was added at the time of plating; 24 hr later, the HNB-containing medium was replaced with fresh growth medium. The plates were then incubated for 12 days at 37°C in a 5 per cent CO, supplemented incubator; after staining with acetic gentian violet, all the colonies visible by the naked eye were counted as survivors. Groats curves.---Equal samples from the same lnonodisperse cell suspension were inoculated in culture bottles, supplemented with 20 ml of standard medium. The bottles were divided into several lots: one was used as a control, and to the others nitrogen mustard at different concentrations was added, 24-36 hr after the cultures were started. The HN2-containing medium was replaced with fresh growth medium 24 hr later. At different times after treatment, the cells were harvested and counted in a Barker haemo~~tometer, while some of the bottles were used for quantitative biochemical analysis. Biochemical determinations.-Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were extracted with 10 per cent perchloric acid, according to the method indicated by Feinendegen et al. [9]. The RNA and DNA content of single bottles was determined by the spectrophotometer (Hilger-IJvispeck) at 260 nm for RNA and at 268 rnp for DNA. Protein determinations were made by the calorimetric method of Lowry as modified by Oyama and Eagle [22J, using a phenol reagent (Folin-Ciocalteau) for colour development. RESULTS Single-cell suspensions were treated with increasing doses of HN2, and surviving fractions, at any dose, were calculated as percentages of the controls, using the mean number of counted colonies [26]. In Fig. 1 the values obtained in two separate experiments are plotted in semi-logarithmic scale. The curve shows an initial shoulder which extends to about 0.3 y/ml, followed by a linear portion in which survival is an exponential function of dose. The average size of surviving colonies is somewhat smaller than that of the untreated series (Figs. 2, 3). microscopic examination revealed Besides colonies counted as “survivors”, the presence of “abortive” microcolonies (50 cells or less), usually including one or more giant cells, mono- or polynucleated (Figs. 4, 5, 6). Single giant cells characterized by cytoplasmic elongations (Fig. 7), by irregular-shaped or fragmented nuclei (Fig. S), and by large vacuoles (Fig. 9), were also observed. These giant cells may reach very large diameters. In Fig. 12 are reported the growth curves obtained by counting the total number of cells per bottle in treated and in untreated cultures. Cell-counts Experimentat
Cell Research 31
Radiomimefic effects of a nitrogen mustard on mammalian cells
21
were made every 12 hr, up to the 4th day after treatment. The number of cells in the control series increases at an exponential rate, that is, for the whole duration of the experiment the cell population was in a constant phase of growth (logarithmic phase). The doubling time is about 21 hr.
Fig. l.-Per cent survival to HN2 as a function of dose, obtained from two separate experiments. Survival values are based on the number of macroscopic colonies developed in three to five dishes per dose.
In the treated series, growth is inhibited to a variable extent: inhibition increases with dose. With 0.08 y/ml, the increase in cell number seems slightly reduced and the doubling time appears to be 24 hr. With 0.50 y/ml, the number of cells stays considerably below the values of the control series and the doubling time is extended to 66 hr. Finally with 0.75 y/ml, no significant increase in cell number is noticed: there seems to be here a complete inhibition of cell multiplication. These results are confirmed by a drop in the mitotic index, which was observed in cultures treated with the same doses of HN2. Particularly with 0.75 y/ml mitoses are immediately suppressed and no recovery was noticed until the 4th day after treatment [28]. Experimental
Ceil Research 31
A. G. Levis, L. Spanio and A. De Nadai
Figs. 2 and 3.-Effect x 1.5.
of Hi%;2 on colony
formation:
control
(I;ig. 2); 6.40 y/ml of HS2 (Fig. 3).
Bodenstein [cl] has shown that HN2 stops the mitotic activity of the ectodermal cells in embryos of Amblystomn punctntum. These cells are blocked in interphase but can still grow and become giant. A similar effect has been observed in cultures of Escherichirr coli treated with nitrogen and sulfur mustards [I 11. HN2-treatment appears to induce giant cell formation in our system as well (Figs. 10, 11). In order to investigate the changes in synthetic activity that accompany giant cell formation, the total number of cells and protein, RN4 and DNA content were determined in parallel cultures, up to the 24th hr after treatment with 0.75 y/ml of HN2. The results are reported in Fig. 13 together with the values obtained for the control series. The content of proteins, RNA and DN,4, unlike cell number, increases
Fig. 4.-An “abortive” microcolony with giant cells, from a culture treated with 1.00 y/ml of HN2, at the 12th day after treatment. x 240. Fig. 5.-As Fig. 4. x 240. Fig. B.-As Fig. 4. x 160. Fig. 7.-A single giant cell, from a culture treated with 0.75 y/ml of HN2, at the 12th day after treatment. x 480. Fig. 8.-As Fig. 7. x 480. Fig. 9.-As Fig. 7. x 480. Figs. 10 and ll.-Cell suspensions obtained from an untreated culture (Fig. 10) and from a culture at the third day after treatment with 0.75 y/ml of HN2 (Fig. 11). The suspensions were fixed in acetic alcohol (3: 1) and stained with Meyer’s haemalum. x 190. Experimental
Cell Research 31
Radiomimetic effects of a nitrogen mustard on mammalian cells
23
II)-
4
5 .
Experimental
Cell Research 31
24
A. G. Levis, L. Spanio and A. De Nadai
at a similar sate both in the treated and in the control series: the initial values double within 24 hr. From the above data, the average contents of proteins, RNA and DNA per cell have been calculated and are shown in Table I. In the control series TABLE
I.
content of proteins, RNA and DNA ment with 0.75 y/ml of HN2.
The average
The data are reported
Hr after HN2 treatment
Proteins y x 10-4/tell Contr.
0
7.9
6 12 18 24
6.8 7.7 7.0 8.0
graphically
treat-
in Fig. 13.
RNA y x 10-5/tell
DNA y x 10-5/tell
HN2
Contr.
HN2
Contr.
HN2
7.3 10.0 15.5 14.6
7.2 5.1 6.9 6.5 6.0
8.7 11.4 13.0 14.2
4.3 3.7 3.9 3.8 3.6
5.8 6.0 6.9 6.7
HOUR.5
Fig. 12.
per cell, after
AFTER
HN2-TREATMENT
Fig. 13.
Fig. 12.-Mean number of cells per bottle in cultures treated with different doses of HN2. Each point represents the average of three counts made on each of two different bottles. HN2 was added 24 hr after the cultures were started. Fig. 13.-Mean number of cells (A), and content of proteins (B), RNA (C), and DNA (D) per bottle, in cultures treated with 0.75 y/ml of HN2 (empty circles) and in untreated cultures (full circles). Each point represents the average of three determinations made on each of two separate bottles. The HN2 was added 36 hr after the cultures were started. Experimental
Cell Research 31
Radiomimetic effects of a nitrogen mustard on mammalian cells
25
DNA, RNA and protein content per cell does not show appreciable variations, indicating that throughout this 24 hr period the cell population was undergoing a fully exponential, balanced growth. In the treated series the amount of proteins and RNA per cell increases until it doubles the initial value; the amount of DNA per cell also increases, but to a lesser extent. In a subsequent experiment similar determinations were carried on, up to the 4th day after treatment. The results are reported in Fig. 14. The growth curves (Fig. 14A) are in good agreement with those shown in Fig. 12: the number of cells in the control series increases at an exponential rate, with a
0
12
24
48
72 HOURS
96 AFTER
0
12 24 HN2-TREATMENT
48
. ___---
_0
72
96
Fig. 14.-Mean number of cells (A), and content of proteins (B), RNA (C), and DNA (D) per bottle in cultures treated with 0.75 y/ml of HN2 (empty circles) and in untreated cultures (full circles). Each point represents the average of three determinations made on each of two separate bottles. The HN2 was added 24 hr after the cultures were started. Experimentnl
Cell Research 31
A. G. Levis, L. Spanio and A. De Nadai doubling time of about 21 hr. In the treated series cell multiplication is, on the contrary, practically stopped. During the first 24 hr the synthesis of proteins and RNA (Fig. 14 H, C) goes on at the same rate, both in the control and in the treated cultures: the initial values appear to be quadruplicated. This phase is followed, in the control series, by a less intense period of synthesis, during which the values double in 24-36 hr. In the treated cultures the synthesis of proteins and TABLE
II. The average
The data are reported
content of proteins, RLVA and DNA per cell, after merit with 0.75 y/ml of HN2.
graphically
in Fig. 14. Values (see text).
Proteins y X 10-4/cell Hr after HN2 treatment 0 6 12 18 24 48 72 96
RNA y X 10-5/tell
3.9 4.3 7.2 6.8 6.7 7.6 5.8 6.1
HN2
Giant cells
Contr.
6.5 12.6 10.8 15.4 17.7 25.6 20.7
4.7 6.6 7.3 6.9 7.2 6.0 6.1 4.4
6.1 11.5 10.0 13.7 15.6 21.G 17.0
indirectly
DNA y x lo-5/tell
r-h--Y
-_--A Contr.
for giant cells were calculated
trecrt-
,-----. HN2
Giant cells
Conk.
HN2
Giant cells
7.7 10.7 10.9 10.8 14.7 18.6 13.6
-7.9 11.6 11.8 11.7 16.9 21.x 16.7
2.6 3.5 3.2 3.1 3.1 2.4 2.4 1.9
3.9 4.4 4.2 3.6 4.6 5.0 4.1
4.0 4.8 4.5 3.7 5.2 5.6 4.8
RNA after the 24th hr, although not completely stopped, appears much more reduced. For DNA, also, two phases of synthesis can be observed (Fig. 14D). In the control series the initial value is practically triplicated during the first 24 hr, while in the following days the values double in about 36 hr. In the treated cultures, synthesis of DNA proceeds at the same rate as for the control only during the first 12-18 hr. Later, DNA synthesis is completely stopped. The average contents of proteins, RNA and DNA per cell, calculated from the above data, are reported in Table II. The variations in DNA, RNA and protein content per cell, observed in the control series, are a consequence of the cells probably being in stationary phase of growth when the experiment was started. Since a high number of cells was required, slightly overgrown cultures were used to set up the experiment. On the other hand, the decrease in protein, RNA and DNA content, that occurs in the control series after the 72nd hr, is likely due to the fact that cells are again entering the stationary Experimental
Cell Research 31
Radiomimetic effects of a nitrogen mustard on mammalian cells
27
phase. The variations of metabolic activity observed in the untreated cultures would be therefore in relation to different phases of growth, which follow on closely in heavy cultures. In the treated cultures the protein and RNA content per cell increases, reaching between the 12th and the 72nd hr values S-4 times greater than the ones of the control series, and then level off. The DNA per cell increases only to a value that is hardly t\vice as high as in the untreated cells.
Fig. 15.---Uistributions of cell diameters at the fourth day after treatment with 0.75 y/ml of HN2 (solid line) and in an untreated population (broken line). The diameters of 300 cells have been measured on smears of cell suspensions. The shaded area represents the fraction of cells in the treated population whose size does not exceed a “normal” value.
It must be pointed out that, from the 48th hr on, the average values per cell include both giant and normal-sized cells. With HS2, contrary to what happens after X-irradiation, pure populations of giant cells do not develop at any dose level. \\‘e have in fact observed that, with doses greater than 1.50 y/ml, the majority of cells disappear within a few hours after treatment, leaving no trace on the plates. At lower doses, such that will allow a proportion of cells to grow into giants, the true survivors will always represent a considerable fraction of the population. No mitotic activity, however, was observed until at least the 4th day after treatment with 0.75 y/ml [28;. At this time the number of “normal” cells, determined microscopically (Fig. 1.5), is in essential agreement with the value obtained on the survival curve. It would appear, therefore, that while damaged cells may grow into giants, the true survivors do not resume mitotic nor synthetic activity until after the 4th day. Experimental
Cell Research 31
28
A. G. Leuis, L. S~ff~io and A. L)e ~~~u~
In order to follow the biochemical activity of giant cells only, the values reported in Table II must be corrected by subtracting the contribution of the 20-25 per cent fraction of “normal” cells, which can be derived from the corresponding values obtained in the control series. The corrected values (Table II, columns III, VI, XI) are higher than those calculated previously, but also here both protein and RNA synthesis appear to level off after the 72nd hr, while DNA synthesis is inhibited somewhat earlier. DISCUSSION The survival curve of single cells, treated with increasing doses of HN2 is similar to those obtained after X-irradiation with other strains [8, 12, 18, 25, 321 and on the same RCP strain 1191. The extrapolation number is lower than the average values found in most of the X-rays survival curves for hyperploid mammalian cells [18, 261. It has been shown, however, that the extrapolation numbers obtained from different X-irradiation experiments may vary appreciably in relation to experimental conditions [17, 181. The low value of extrapolation number in the HN2 survival curve suggests nuclear damage such as the induction of lethal mutations, genie or more probably chromosomal. The data available on chromosome aberrations induced by nitrogen mustards in mammalian cell systems are, however, still too scanty to be quantitatively related with reproductive death. Microscopic examination of single-cell cultures, at the 12th day after plating, shows that the fate of cells treated with HN2 is roughly similar to that observed after X-irradiation. The treated cells, in fact, (1) may die and disappear leaving no trace on the plate, (2) may fail to divide at all, but microcolonies, (4) may retain grow into giant cells, (3) may form “abortive” the ability to form macroscopic colonies. Sub-lethal treatment seems to produce also a delay in cell multiplic.ation, since the surviving colonies are of reduced size as compared with those of l.he control series (Figs. 2, 3). The growth curves (Fig. 12) show evidence of an immediate inhibition of cell multiplication, which increases with increasing doses. After 0.75 y/ml treatment, the number of cells is constant at least until the 4th day, and observations on the mitotic index [28] seem to show that this is due to inhibition of cell division rather than to cell loss. The mitotic inhibition can be temporary; we have, in fact, emphasized that certain cells begin to divide only a few days after treatment. There are, however, cells that do not recover the capacity to multiply, but grow into giants. Giant cell formation represents one of the most characteristic efrects of ~x~erime~ful
Cell Research 31
Radiomimetic
effects of a nitrogen mustard on mammalian cells
29
X-irradiation on mammalian cells in vitro [14, 24, 253. The mechanism of giant cell formation is not clear, but it does reveal a specific damage to the process of cell division. In fact, it has been shown [B, 13, 21, 29, 331 that, following massive doses of radiation, the cells are no longer capable of division, but can still synthesize proteins and nucleic acids up to cellular levels highly abnormal, although at a reduced rate. DNA synthesis seems somewhat more sensitive than RNA synthesis [7, 12, 23, 27, 31, 341. However, any of the effects on synthetic processes, observed after low levels of irradiation, are likely to be secondary to the induced mitotic inhibition. From work on amphibian embryos [4] and on primary explants of chicken embryos [16], it has been shown that HN2 can stop DNA but not RNA synthesis. Our data also show that protein and RNA synthesis are less aflected than DNA synthesis, which ceases when the DNA content per cell is about double its normal value. However, there is an immediate inhibition of cell multiplication. Mustards, therefore, like X-radiation, seem to act directly on the division mechanism, while inhibition of synthetic processes would be secondary. It must be pointed out that some minor differences seem to exist between the effects of HN2 and X-radiation. First, HN’L-treated cells show a much longer mitotic delay than X-irradiated cells do. Second, the ratio between death must be lower for doses that cause “reproductive” and “metabolic” HN2 than it is for X-radiation, since a high yield of pure giant cells can never be obtained with HN2 treatment, as it can be with X-radiation. SUMMARY
The effects of different doses of a nitrogen mustard (HN2) on a guineapig cell strain grown in vitro, have been compared to the effects of X-rays on similar systems. 1. The survival curve is similar to those obtained after X-irradiation. The low extrapolation number suggests that, also in this case, the loss of reproductive capacity may be due to the induction of lethal mutations, probably chromosomal. The cells that have lost the ability to grow into macroscopic colonies can undergo a limited number of divisions and form “abortive” microcolonies, characterized by the presence of one or more giant cells. 2. The growth curves give evidence of the inhibition of cell multiplication. The inhibition is immediate and increases with increasing doses. Some of the inhibited cells, however, may eventually recover the capacity to multiply. When the inhibition is permanent, the cells grow into “giants”. Experimental
Cell Research 31
30
A. G. Levis, L. Spanio and A. De Nadai
3. Despite mitotic inhibition, the synthesis of proteins and RNA continues until highly abnormal cellular levels are reached. DNA synthesis is the most sensitive to HN2 treatment, and is stopped early, when the DNA content per cell doubles its normal value. The early inhibition of synthetic processes would appear to be secondary to the block of cell division, although a direct effect on DNA synthesis cannot be excluded. We wish to express our best thanks to Dr. G. Colombo, to Dr. G. Marin and to Dr. R. C. von Borstel for discussing the results of these experiments, and for their help during the preparation of the manuscript. REFERENCES ALEXANDER, P. and STACEY, K. A., Ann. N.Y. Acad. Sci. 68,1225 (1958). AUERBACH, C., Ann. S. Y. Acad. Sci. 68, 731 (1958). BODENSTEIN, D., J. Exptl. Zool. 104, 311 (1947). BODENSTEIN, D. and KONDRITZER, A. A., J. Exptl. Zool. 107, 109 (1948). COLOMBO, G. and MARIN, G., Exptl. Cell Res. 29, 268 (1963). DICKSON, M., PAUL, J. and DAVIDSON, J. N., Biochem. J. 70, 18 P (1958). DICKSOX, M. and PAUL, J., Intern. J. Radial. Biol. 3, 419 (1961). ELKIND, M. M. and SUTTON, H., Nature 184, 1293 (1957). FEINENDEGEN, L. E., BOND, V. P. and PAINTER, R. B., Exptl. Cell Res. 22, 381 (1961). GASPARINI, \‘., FARISANO, G. and GAMBA, F., Boll. I.S.M. 39, 132 (1960). HAROLD, F. M. and ZIPORIN, %. %., Biochim. Biophys. Acta 28, 482 (1958). HARRINGTON, H., Biochim. Biophys. Acta 41, 461 (1961). KLEIN, G. and FORSSBERG, A., Exptl. Cell Res. 6, 211 (1953). KOHN, H. J. and FOGH, J. E., .I. Natl. Cancer Inst. 23, 293 (1959). KOLLER, P. C., Ann. N. Y. Acad. Sci. 68, 783 (1958). KONIGSBERG, I. R., MCELVAIN, N., TOOTLE, M. and HERRMANN, H., J. Biophys. Biochem. Cytol. 8, 333 (1960). 17. I,OCKART, R. Z. and ELKIND, M. M., J. Satl. Cancer Inst. 27, 1393 (1961). 18 (suppl. 2), 227 (1960). 18. MARIS, G., Nuzzo, F. and DE CARLI, I,., Nuouo Cimento 19. MARIN, G. and COLOMBO, G., Atfi A.G. I. 6, 125 (1961). 20. MELNICK, .J. K., in Diagnostic procedure for virus and rickettsial diseases, 2nd ed., Am. Public Health Assoc., New York, 1956. 21. NIAS, A. H. W. and PAUL, J., Intern. J. Radiation Biol. 3, 431 (1961). 22. OYAMA, V. I. and EAGLE, H., Proc. Sot. Esptt. Biol. Med. 91, 305 (1956). 23. PAINTER, R. B., Radiation Res. 13, 726 (1960). 24. POMERAT, C. M., KENT, S. P. and LOGIE, I,. C., Z. Zellforsch. Mikroskop. Anat. 47, 158 (1957). 25. PUCK, T. T. and MARCUS, P. I., J. Exptt. Med. 103, 653 (1956). S. ,J., J. Exptl. Med. 103, 273 (1955). 26. PUCIC, T. T., MARCUS, P. I. and CIECIURA, M. R., DES ARMIER, R. M., SAGIK, B. P. and MAGEE, W. E., Exptl. Cell Res. 19, 549 27. SHEEK, (1960). 28. SPANIO, I,. and LEVIS, A. G., Caruoloqia. 15, 551 (1962). 29. TOLMACH, L. J. and MARCUS; P. <, &ptZ. Cell Res. 20,‘350 (1960). 30. TRUHAUT, R. and DEYSSON, G., Bull. Assoc. Franc. Etude Cancer 44, 222 (1957). 31. WHITFIELD, J. F. and RIXON, R. H., Exptl. Cell Res. 18, 126 (1958). 32. ~ ibid. 19, 531 (1960). G. F., TILL, J. E.. GWATKIN. R. B. L.. SIMINOVITCH. L. and GRAHAM. A. F.. 33. WHITMORE. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16.
Biochim. 34.
Bibphys.‘Acta
WHITMORE, G. F., STANNERS, 66 (1960).
Experimental
Cell Resenrch 31
i0, 583 (1958). C. P., GULY~S,
’ S. and TILL,
J. E., Biochim.
Biophys.
Acta 47,
RADIOMIMETIC SURVIVAL,
EFFECTS GROWTH,
SYNTHESIS
OF A NITROGEN PROTEIN
OF MAMMALIAN
A. G. LEVIS, Institute
19
Cell Research 31, 19-30 (19G3)
of Zoology and Institute
L. SPAN10 of Pharmaceutical
AND CELLS
and A. DE Chemistry,
MUSTARD
NUCLEIC
ON
ACID
1N VITRO1 NADAI University
of Padua, Italy
Received July 27, 19622
NITROGES mustards are commonly referred to as “radiomimetic” substances, since their biological end efTects are similar to those produced by ionizing radiations. At the cellular level, mustards stop mitosis [W], produce chromosome aberrations [ 151, and induce mutations [2], as ionizing radiations do. At the chemical level, as Alexander and Stacey [ 1 ] have suggested on the basis of in vitro evidence, the biological effects both of alkylating agents and of ionizing radiations can probably be attributed to a direct action on the DSA molecule. Studies on “radiomimetic” substances of the alkylating group may therefore contribute to the understanding of the mechanism of radiation-induced biological effects, whenever a close comparison between the etTects of the two mutagenic agents is feasible. While accurate data are no\\- available for radiation-induced effects in mammalian cells, work on “radiomimetic” drugs is still lacking. We have thus undertaken a study of the action of nitrogen mustards on a guinea-pig cell strain cultivated in rdro. The results on survival, growth and synthesis of nucleic acids and proteins are here described.
MATERIAL
AND
METHODS
Nitrogen musfard.~Methyl-bis-(@-chloroethyl)amine hydrochloride (HN2) of the Ciba Chem. Ind. was used (Dichloren). This substance is prepared in phials containing 5 mg of white, crystalline powder, water-soluble and stable in an acid medium. At the moment of use, the content of a phial was dissolved in 5 ml of sterile Hanks’, so that a 1000 y/ml solution was obtained. Cells.-The RCP strain, derived from guinea-pig kidney cells by Gasparini et al. [lo], was used. The characteristics of this strain, grown adherent to glass in Melnick’s 1 This work was performed under contract n. 36 with the International (I.A.E.A.). 2 Revised version received November 5, 1962.
Atomic
Experimental
Energy Agency
Cell Research 31
20
A. G. Levis, L. Spanio and A. De Nadai
medium 1201 supplemented with 20 per cent whole calf serum, have been described in a preceding paper [5]. survival curues.-Samples from a single-cell suspension were plated in 60 mm Petri dishes with 5 ml of standard growth medium. Nitrogen mustard was added at the time of plating; 24 hr later, the HNB-containing medium was replaced with fresh growth medium. The plates were then incubated for 12 days at 37°C in a 5 per cent CO, supplemented incubator; after staining with acetic gentian violet, all the colonies visible by the naked eye were counted as survivors. Groats curves.---Equal samples from the same lnonodisperse cell suspension were inoculated in culture bottles, supplemented with 20 ml of standard medium. The bottles were divided into several lots: one was used as a control, and to the others nitrogen mustard at different concentrations was added, 24-36 hr after the cultures were started. The HN2-containing medium was replaced with fresh growth medium 24 hr later. At different times after treatment, the cells were harvested and counted in a Barker haemo~~tometer, while some of the bottles were used for quantitative biochemical analysis. Biochemical determinations.-Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were extracted with 10 per cent perchloric acid, according to the method indicated by Feinendegen et al. [9]. The RNA and DNA content of single bottles was determined by the spectrophotometer (Hilger-IJvispeck) at 260 nm for RNA and at 268 rnp for DNA. Protein determinations were made by the calorimetric method of Lowry as modified by Oyama and Eagle [22J, using a phenol reagent (Folin-Ciocalteau) for colour development. RESULTS Single-cell suspensions were treated with increasing doses of HN2, and surviving fractions, at any dose, were calculated as percentages of the controls, using the mean number of counted colonies [26]. In Fig. 1 the values obtained in two separate experiments are plotted in semi-logarithmic scale. The curve shows an initial shoulder which extends to about 0.3 y/ml, followed by a linear portion in which survival is an exponential function of dose. The average size of surviving colonies is somewhat smaller than that of the untreated series (Figs. 2, 3). microscopic examination revealed Besides colonies counted as “survivors”, the presence of “abortive” microcolonies (50 cells or less), usually including one or more giant cells, mono- or polynucleated (Figs. 4, 5, 6). Single giant cells characterized by cytoplasmic elongations (Fig. 7), by irregular-shaped or fragmented nuclei (Fig. S), and by large vacuoles (Fig. 9), were also observed. These giant cells may reach very large diameters. In Fig. 12 are reported the growth curves obtained by counting the total number of cells per bottle in treated and in untreated cultures. Cell-counts Experimentat
Cell Research 31
Radiomimefic effects of a nitrogen mustard on mammalian cells
21
were made every 12 hr, up to the 4th day after treatment. The number of cells in the control series increases at an exponential rate, that is, for the whole duration of the experiment the cell population was in a constant phase of growth (logarithmic phase). The doubling time is about 21 hr.
Fig. l.-Per cent survival to HN2 as a function of dose, obtained from two separate experiments. Survival values are based on the number of macroscopic colonies developed in three to five dishes per dose.
In the treated series, growth is inhibited to a variable extent: inhibition increases with dose. With 0.08 y/ml, the increase in cell number seems slightly reduced and the doubling time appears to be 24 hr. With 0.50 y/ml, the number of cells stays considerably below the values of the control series and the doubling time is extended to 66 hr. Finally with 0.75 y/ml, no significant increase in cell number is noticed: there seems to be here a complete inhibition of cell multiplication. These results are confirmed by a drop in the mitotic index, which was observed in cultures treated with the same doses of HN2. Particularly with 0.75 y/ml mitoses are immediately suppressed and no recovery was noticed until the 4th day after treatment [28]. Experimental
Ceil Research 31
A. G. Levis, L. Spanio and A. De Nadai
Figs. 2 and 3.-Effect x 1.5.
of Hi%;2 on colony
formation:
control
(I;ig. 2); 6.40 y/ml of HS2 (Fig. 3).
Bodenstein [cl] has shown that HN2 stops the mitotic activity of the ectodermal cells in embryos of Amblystomn punctntum. These cells are blocked in interphase but can still grow and become giant. A similar effect has been observed in cultures of Escherichirr coli treated with nitrogen and sulfur mustards [I 11. HN2-treatment appears to induce giant cell formation in our system as well (Figs. 10, 11). In order to investigate the changes in synthetic activity that accompany giant cell formation, the total number of cells and protein, RN4 and DNA content were determined in parallel cultures, up to the 24th hr after treatment with 0.75 y/ml of HN2. The results are reported in Fig. 13 together with the values obtained for the control series. The content of proteins, RNA and DN,4, unlike cell number, increases
Fig. 4.-An “abortive” microcolony with giant cells, from a culture treated with 1.00 y/ml of HN2, at the 12th day after treatment. x 240. Fig. 5.-As Fig. 4. x 240. Fig. B.-As Fig. 4. x 160. Fig. 7.-A single giant cell, from a culture treated with 0.75 y/ml of HN2, at the 12th day after treatment. x 480. Fig. 8.-As Fig. 7. x 480. Fig. 9.-As Fig. 7. x 480. Figs. 10 and ll.-Cell suspensions obtained from an untreated culture (Fig. 10) and from a culture at the third day after treatment with 0.75 y/ml of HN2 (Fig. 11). The suspensions were fixed in acetic alcohol (3: 1) and stained with Meyer’s haemalum. x 190. Experimental
Cell Research 31
Radiomimetic effects of a nitrogen mustard on mammalian cells
23
II)-
4
5 .
Experimental
Cell Research 31
24
A. G. Levis, L. Spanio and A. De Nadai
at a similar sate both in the treated and in the control series: the initial values double within 24 hr. From the above data, the average contents of proteins, RNA and DNA per cell have been calculated and are shown in Table I. In the control series TABLE
I.
content of proteins, RNA and DNA ment with 0.75 y/ml of HN2.
The average
The data are reported
Hr after HN2 treatment
Proteins y x 10-4/tell Contr.
0
7.9
6 12 18 24
6.8 7.7 7.0 8.0
graphically
treat-
in Fig. 13.
RNA y x 10-5/tell
DNA y x 10-5/tell
HN2
Contr.
HN2
Contr.
HN2
7.3 10.0 15.5 14.6
7.2 5.1 6.9 6.5 6.0
8.7 11.4 13.0 14.2
4.3 3.7 3.9 3.8 3.6
5.8 6.0 6.9 6.7
HOUR.5
Fig. 12.
per cell, after
AFTER
HN2-TREATMENT
Fig. 13.
Fig. 12.-Mean number of cells per bottle in cultures treated with different doses of HN2. Each point represents the average of three counts made on each of two different bottles. HN2 was added 24 hr after the cultures were started. Fig. 13.-Mean number of cells (A), and content of proteins (B), RNA (C), and DNA (D) per bottle, in cultures treated with 0.75 y/ml of HN2 (empty circles) and in untreated cultures (full circles). Each point represents the average of three determinations made on each of two separate bottles. The HN2 was added 36 hr after the cultures were started. Experimental
Cell Research 31
Radiomimetic effects of a nitrogen mustard on mammalian cells
25
DNA, RNA and protein content per cell does not show appreciable variations, indicating that throughout this 24 hr period the cell population was undergoing a fully exponential, balanced growth. In the treated series the amount of proteins and RNA per cell increases until it doubles the initial value; the amount of DNA per cell also increases, but to a lesser extent. In a subsequent experiment similar determinations were carried on, up to the 4th day after treatment. The results are reported in Fig. 14. The growth curves (Fig. 14A) are in good agreement with those shown in Fig. 12: the number of cells in the control series increases at an exponential rate, with a
0
12
24
48
72 HOURS
96 AFTER
0
12 24 HN2-TREATMENT
48
. ___---
_0
72
96
Fig. 14.-Mean number of cells (A), and content of proteins (B), RNA (C), and DNA (D) per bottle in cultures treated with 0.75 y/ml of HN2 (empty circles) and in untreated cultures (full circles). Each point represents the average of three determinations made on each of two separate bottles. The HN2 was added 24 hr after the cultures were started. Experimentnl
Cell Research 31
A. G. Levis, L. Spanio and A. De Nadai doubling time of about 21 hr. In the treated series cell multiplication is, on the contrary, practically stopped. During the first 24 hr the synthesis of proteins and RNA (Fig. 14 H, C) goes on at the same rate, both in the control and in the treated cultures: the initial values appear to be quadruplicated. This phase is followed, in the control series, by a less intense period of synthesis, during which the values double in 24-36 hr. In the treated cultures the synthesis of proteins and TABLE
II. The average
The data are reported
content of proteins, RLVA and DNA per cell, after merit with 0.75 y/ml of HN2.
graphically
in Fig. 14. Values (see text).
Proteins y X 10-4/cell Hr after HN2 treatment 0 6 12 18 24 48 72 96
RNA y X 10-5/tell
3.9 4.3 7.2 6.8 6.7 7.6 5.8 6.1
HN2
Giant cells
Contr.
6.5 12.6 10.8 15.4 17.7 25.6 20.7
4.7 6.6 7.3 6.9 7.2 6.0 6.1 4.4
6.1 11.5 10.0 13.7 15.6 21.G 17.0
indirectly
DNA y x lo-5/tell
r-h--Y
-_--A Contr.
for giant cells were calculated
trecrt-
,-----. HN2
Giant cells
Conk.
HN2
Giant cells
7.7 10.7 10.9 10.8 14.7 18.6 13.6
-7.9 11.6 11.8 11.7 16.9 21.x 16.7
2.6 3.5 3.2 3.1 3.1 2.4 2.4 1.9
3.9 4.4 4.2 3.6 4.6 5.0 4.1
4.0 4.8 4.5 3.7 5.2 5.6 4.8
RNA after the 24th hr, although not completely stopped, appears much more reduced. For DNA, also, two phases of synthesis can be observed (Fig. 14D). In the control series the initial value is practically triplicated during the first 24 hr, while in the following days the values double in about 36 hr. In the treated cultures, synthesis of DNA proceeds at the same rate as for the control only during the first 12-18 hr. Later, DNA synthesis is completely stopped. The average contents of proteins, RNA and DNA per cell, calculated from the above data, are reported in Table II. The variations in DNA, RNA and protein content per cell, observed in the control series, are a consequence of the cells probably being in stationary phase of growth when the experiment was started. Since a high number of cells was required, slightly overgrown cultures were used to set up the experiment. On the other hand, the decrease in protein, RNA and DNA content, that occurs in the control series after the 72nd hr, is likely due to the fact that cells are again entering the stationary Experimental
Cell Research 31
Radiomimetic effects of a nitrogen mustard on mammalian cells
27
phase. The variations of metabolic activity observed in the untreated cultures would be therefore in relation to different phases of growth, which follow on closely in heavy cultures. In the treated cultures the protein and RNA content per cell increases, reaching between the 12th and the 72nd hr values S-4 times greater than the ones of the control series, and then level off. The DNA per cell increases only to a value that is hardly t\vice as high as in the untreated cells.
Fig. 15.---Uistributions of cell diameters at the fourth day after treatment with 0.75 y/ml of HN2 (solid line) and in an untreated population (broken line). The diameters of 300 cells have been measured on smears of cell suspensions. The shaded area represents the fraction of cells in the treated population whose size does not exceed a “normal” value.
It must be pointed out that, from the 48th hr on, the average values per cell include both giant and normal-sized cells. With HS2, contrary to what happens after X-irradiation, pure populations of giant cells do not develop at any dose level. \\‘e have in fact observed that, with doses greater than 1.50 y/ml, the majority of cells disappear within a few hours after treatment, leaving no trace on the plates. At lower doses, such that will allow a proportion of cells to grow into giants, the true survivors will always represent a considerable fraction of the population. No mitotic activity, however, was observed until at least the 4th day after treatment with 0.75 y/ml [28;. At this time the number of “normal” cells, determined microscopically (Fig. 1.5), is in essential agreement with the value obtained on the survival curve. It would appear, therefore, that while damaged cells may grow into giants, the true survivors do not resume mitotic nor synthetic activity until after the 4th day. Experimental
Cell Research 31
28
A. G. Leuis, L. S~ff~io and A. L)e ~~~u~
In order to follow the biochemical activity of giant cells only, the values reported in Table II must be corrected by subtracting the contribution of the 20-25 per cent fraction of “normal” cells, which can be derived from the corresponding values obtained in the control series. The corrected values (Table II, columns III, VI, XI) are higher than those calculated previously, but also here both protein and RNA synthesis appear to level off after the 72nd hr, while DNA synthesis is inhibited somewhat earlier. DISCUSSION The survival curve of single cells, treated with increasing doses of HN2 is similar to those obtained after X-irradiation with other strains [8, 12, 18, 25, 321 and on the same RCP strain 1191. The extrapolation number is lower than the average values found in most of the X-rays survival curves for hyperploid mammalian cells [18, 261. It has been shown, however, that the extrapolation numbers obtained from different X-irradiation experiments may vary appreciably in relation to experimental conditions [17, 181. The low value of extrapolation number in the HN2 survival curve suggests nuclear damage such as the induction of lethal mutations, genie or more probably chromosomal. The data available on chromosome aberrations induced by nitrogen mustards in mammalian cell systems are, however, still too scanty to be quantitatively related with reproductive death. Microscopic examination of single-cell cultures, at the 12th day after plating, shows that the fate of cells treated with HN2 is roughly similar to that observed after X-irradiation. The treated cells, in fact, (1) may die and disappear leaving no trace on the plate, (2) may fail to divide at all, but microcolonies, (4) may retain grow into giant cells, (3) may form “abortive” the ability to form macroscopic colonies. Sub-lethal treatment seems to produce also a delay in cell multiplic.ation, since the surviving colonies are of reduced size as compared with those of l.he control series (Figs. 2, 3). The growth curves (Fig. 12) show evidence of an immediate inhibition of cell multiplication, which increases with increasing doses. After 0.75 y/ml treatment, the number of cells is constant at least until the 4th day, and observations on the mitotic index [28] seem to show that this is due to inhibition of cell division rather than to cell loss. The mitotic inhibition can be temporary; we have, in fact, emphasized that certain cells begin to divide only a few days after treatment. There are, however, cells that do not recover the capacity to multiply, but grow into giants. Giant cell formation represents one of the most characteristic efrects of ~x~erime~ful
Cell Research 31
Radiomimetic
effects of a nitrogen mustard on mammalian cells
29
X-irradiation on mammalian cells in vitro [14, 24, 253. The mechanism of giant cell formation is not clear, but it does reveal a specific damage to the process of cell division. In fact, it has been shown [B, 13, 21, 29, 331 that, following massive doses of radiation, the cells are no longer capable of division, but can still synthesize proteins and nucleic acids up to cellular levels highly abnormal, although at a reduced rate. DNA synthesis seems somewhat more sensitive than RNA synthesis [7, 12, 23, 27, 31, 341. However, any of the effects on synthetic processes, observed after low levels of irradiation, are likely to be secondary to the induced mitotic inhibition. From work on amphibian embryos [4] and on primary explants of chicken embryos [16], it has been shown that HN2 can stop DNA but not RNA synthesis. Our data also show that protein and RNA synthesis are less aflected than DNA synthesis, which ceases when the DNA content per cell is about double its normal value. However, there is an immediate inhibition of cell multiplication. Mustards, therefore, like X-radiation, seem to act directly on the division mechanism, while inhibition of synthetic processes would be secondary. It must be pointed out that some minor differences seem to exist between the effects of HN2 and X-radiation. First, HN’L-treated cells show a much longer mitotic delay than X-irradiated cells do. Second, the ratio between death must be lower for doses that cause “reproductive” and “metabolic” HN2 than it is for X-radiation, since a high yield of pure giant cells can never be obtained with HN2 treatment, as it can be with X-radiation. SUMMARY
The effects of different doses of a nitrogen mustard (HN2) on a guineapig cell strain grown in vitro, have been compared to the effects of X-rays on similar systems. 1. The survival curve is similar to those obtained after X-irradiation. The low extrapolation number suggests that, also in this case, the loss of reproductive capacity may be due to the induction of lethal mutations, probably chromosomal. The cells that have lost the ability to grow into macroscopic colonies can undergo a limited number of divisions and form “abortive” microcolonies, characterized by the presence of one or more giant cells. 2. The growth curves give evidence of the inhibition of cell multiplication. The inhibition is immediate and increases with increasing doses. Some of the inhibited cells, however, may eventually recover the capacity to multiply. When the inhibition is permanent, the cells grow into “giants”. Experimental
Cell Research 31
30
A. G. Levis, L. Spanio and A. De Nadai
3. Despite mitotic inhibition, the synthesis of proteins and RNA continues until highly abnormal cellular levels are reached. DNA synthesis is the most sensitive to HN2 treatment, and is stopped early, when the DNA content per cell doubles its normal value. The early inhibition of synthetic processes would appear to be secondary to the block of cell division, although a direct effect on DNA synthesis cannot be excluded. We wish to express our best thanks to Dr. G. Colombo, to Dr. G. Marin and to Dr. R. C. von Borstel for discussing the results of these experiments, and for their help during the preparation of the manuscript. REFERENCES ALEXANDER, P. and STACEY, K. A., Ann. N.Y. Acad. Sci. 68,1225 (1958). AUERBACH, C., Ann. S. Y. Acad. Sci. 68, 731 (1958). BODENSTEIN, D., J. Exptl. Zool. 104, 311 (1947). BODENSTEIN, D. and KONDRITZER, A. A., J. Exptl. Zool. 107, 109 (1948). COLOMBO, G. and MARIN, G., Exptl. Cell Res. 29, 268 (1963). DICKSON, M., PAUL, J. and DAVIDSON, J. N., Biochem. J. 70, 18 P (1958). DICKSOX, M. and PAUL, J., Intern. J. Radial. Biol. 3, 419 (1961). ELKIND, M. M. and SUTTON, H., Nature 184, 1293 (1957). FEINENDEGEN, L. E., BOND, V. P. and PAINTER, R. B., Exptl. Cell Res. 22, 381 (1961). GASPARINI, \‘., FARISANO, G. and GAMBA, F., Boll. I.S.M. 39, 132 (1960). HAROLD, F. M. and ZIPORIN, %. %., Biochim. Biophys. Acta 28, 482 (1958). HARRINGTON, H., Biochim. Biophys. Acta 41, 461 (1961). KLEIN, G. and FORSSBERG, A., Exptl. Cell Res. 6, 211 (1953). KOHN, H. J. and FOGH, J. E., .I. Natl. Cancer Inst. 23, 293 (1959). KOLLER, P. C., Ann. N. Y. Acad. Sci. 68, 783 (1958). KONIGSBERG, I. R., MCELVAIN, N., TOOTLE, M. and HERRMANN, H., J. Biophys. Biochem. Cytol. 8, 333 (1960). 17. I,OCKART, R. Z. and ELKIND, M. M., J. Satl. Cancer Inst. 27, 1393 (1961). 18 (suppl. 2), 227 (1960). 18. MARIS, G., Nuzzo, F. and DE CARLI, I,., Nuouo Cimento 19. MARIN, G. and COLOMBO, G., Atfi A.G. I. 6, 125 (1961). 20. MELNICK, .J. K., in Diagnostic procedure for virus and rickettsial diseases, 2nd ed., Am. Public Health Assoc., New York, 1956. 21. NIAS, A. H. W. and PAUL, J., Intern. J. Radiation Biol. 3, 431 (1961). 22. OYAMA, V. I. and EAGLE, H., Proc. Sot. Esptt. Biol. Med. 91, 305 (1956). 23. PAINTER, R. B., Radiation Res. 13, 726 (1960). 24. POMERAT, C. M., KENT, S. P. and LOGIE, I,. C., Z. Zellforsch. Mikroskop. Anat. 47, 158 (1957). 25. PUCK, T. T. and MARCUS, P. I., J. Exptt. Med. 103, 653 (1956). S. ,J., J. Exptl. Med. 103, 273 (1955). 26. PUCIC, T. T., MARCUS, P. I. and CIECIURA, M. R., DES ARMIER, R. M., SAGIK, B. P. and MAGEE, W. E., Exptl. Cell Res. 19, 549 27. SHEEK, (1960). 28. SPANIO, I,. and LEVIS, A. G., Caruoloqia. 15, 551 (1962). 29. TOLMACH, L. J. and MARCUS; P. <, &ptZ. Cell Res. 20,‘350 (1960). 30. TRUHAUT, R. and DEYSSON, G., Bull. Assoc. Franc. Etude Cancer 44, 222 (1957). 31. WHITFIELD, J. F. and RIXON, R. H., Exptl. Cell Res. 18, 126 (1958). 32. ~ ibid. 19, 531 (1960). G. F., TILL, J. E.. GWATKIN. R. B. L.. SIMINOVITCH. L. and GRAHAM. A. F.. 33. WHITMORE. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16.
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