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Some special effects of ionizing radiation
Experimental
Cell Research, Stcppl. 9, 563-568 (1963)
SOME SPECIAL
EFFECTS
563
OF IONIZING
RADIATION
P. C. KOLLER Chester Beatty Research Institute,
London, England
UPTON [S] has reviewed some of the most prominent facets of cell response to ionizing radiation, and showed the lack of our knowledge concerning the mechanism underlying radiation induced mitotic delay, chromosome injuries and their significance in relation to cell death. He drew attention, also, to the importance of interaction which occurs between irradiated malignant cells and the host, the nature of which is still obscure. My contribution will deal with three aspects of the radiation induced effects on the cancer cell. (1) The first concerns the importance of chromosome injuries. In a variety of human tumors, the type and frequency of cell injuries after irradiation, were studied; it was found that the amount of chromosome damage shown by individual cells and the number of cells having chromosome injuries, is dependent on the radiation dose; the higher the dose, the greater the damage per cell and the greater is the number of cells affected. The most important discovery, however, was the fact, that in different tumors which were exposed to the same dose of radiation, the number of injured cells differed. Table I shows the frequencies of chromosome injuries and their distribution per cell in three squamous cell carcinomas which had been irradiated with 300 rads 24 hr previously. The tumors were comparable in histological structure and size. It can be seen, that in spite of the same radiation treatment the percentage of injured cells varies between tumors when observed at 24 hr. Such variation TABLE
I. Radiation
induced cellular
Percentage Site Cheek Cheek Forehead
of injured cells 13.5 17.5 8.5’
damage in three tumors.
Percentageof cells with chromosome fragments 012345678 86.5 83.0 90.5
0.5 1.5 2.5
9-11 3.0 1.0 2.0
a 8 hr later the percentage increased to 12.0. [In each case 200 dividing cells were analysed
3.5 2.0 3.5
4.5 2.5 0.5
1.4 4.5 -
24 hr after treatment Experimental
0.5 2.0 -
1.5 -
0.5 -
1.0-1.5 -
with 30.0 rads.] Cell Research, Suppl. 9
P. C. Keller in the reaction of tumor cells to identical radiation doses indicates differences in growth rate or in cell sensitivity which may be an inherent property of individual tumors; it is also possible that the variation may only be the result of environmental factors. The tumor in the third patient illustrates the difference in response to irradiation due to growth rate. The percentage of analysed cells containing fragments was only 8.5 in the first sample (24 hr after irradiation) and had risen to 12 per cent in the second biopsy specimen removed eight hours later. Thus only at 32 hr was the re.sponse of this tumor similar to the response of the two other tumors at 24 hr. This example shows that more study is required to understand the cause of variation; this knowledge is necessary if we want to employ our radiotherapy methods effectively [3]. (2) The second point is the possible relationship which may exist between ploidy and radiation response. If there is a relationship between chromosome number and radiation sensitivity, it would be an important argument in favor of the view that genetic damage is the only underlying cause of cell death. There is some evidence that in natural polyploid plant species, in which the chromosome number is doubled, cells are more resistant to radiation than cells of corresponding diploid varieties [l]. In experimental transplanted tumors, and as recent studies indicate, in primary tumors also, heteroploid cells are common, i.e. cells in which the chromosome number deviate from the diploid number. Revesz and Norman [4] who studied the response to radiation in various sublines of Ehrlich ascites tumors, found that the hypertetraploid variant was less radiosensitive than the near diploid clone. During the analysis of radiation response in human epidermoid carcinomas, the present author came across a tumor in which heteroploid cells were present; heteroploidy was indicated by enlarged nuclear size of cells at interphase and by chromosome number in dividing cells. This particular tumor was treated by radiation and showed good initial response but eight months later the growth recurred. The new growth was completely composed of heteroploid cells. The inference is that cells with higher chromosome number than the diploid number, survived radiation and became the source of the new tumor which was found to be resistant to further treatment by radiation. It seems therefore that selection of polyploid cells.may contribute to the development of radioresistance in irradiated tumors. By comparing the DNA content of tumor cells in human uterine tumors before, during and after treatment by radium, Richards and Atkin [5] found evidence which favors the view that selection of polyploid cell variants by radiation, may occur. Experimental
Cell Research, Suppl. 9
FUC~OFS affecting
565
radiosensitivrty
(3) The third
point concerns the influence of host environment on tumor cells which are exposed to radiation. I am particularly interested in the effect of changes induced by radiation in the tissues surrounding the tumor; which in the literature, is referred to as “indirect effect” in relation to the tumor
I
2 DISTANCE
5
+ IN
5
CM.
Fig. l.-Graph illustrating the percentage of abnormal cells due to “direct” and “indiiect” effect of radiation at varying distances from the radiation source in two carcinomas of the cervix uteri. (The incidence of abnormal cells showing direct effects, that is, chromosome injuries, was the same in both cases, hence only that of case 1 is given.) Case No. l.-anaplastic adeno-carcinoma. Dose: 2 X 25 mg/24 hr. Time: 10 days. Case No. 2.-squamous cardnoma. Dose: 2 x 25 mg/24 hr. Time: 16 days.
cells. Cell death due to such an “indirect radiation effect” can be identified by the characteristic morphology of the degenerating cells. A comparison was made of the frequency of tumor cells showing chromosome injuries, indicating direct effect of radiation with that of tumor cells whose nuclear morphology indicated “indirect” radiation effects in two uterine cancers treated by radium. Fig. 1 shows the data obtained. It can be seen that the frequency of cells with chromosome injuries follows closely the estimated radiation dose, it decreases with the distance from the radium source, while the number of cells with pyknotic nuclei (indirect effect) increases with the distance. The fact that 80,000-150,000 rads are required to inhibit the growth of Experimental Cell Research, Suppl. 9
P. C. Keller tumor explant in culture [Z], while in vivo the growth of tumors can be inhibited by doses hundred times less, demonstrate the importance of interaction between tumor cells and the host environment. REFERENCES 1. EVANS, H. J. and SPARROW, A. H., Brookhauen Symposia in Biology, No. 14 (1961). 2. GOLDFEDER, A., Arm. N. Y. Acad. Sci. 95, 13 (1961). 3. KOLLER, P. C., Biological Basis of Radiotherapy in Cancer. P. RAVEN (ed.) Butterworths,
1959. 4. RbvBsz, L. and NORMAN, U., J. natl. Cancer Inst., 25, 5 (1960). 5. RICHARDS, B. M. and ATKIN, N. B., &it. J. Cancer 13, 788 (1959). 6. UPTON, A. C., Eqtl. Cell Res. Suppl. 9, 538 (1963).
Experimental
Cell Research, Suppl. 9
Cell Research, Stcppl. 9, 563-568 (1963)
SOME SPECIAL
EFFECTS
563
OF IONIZING
RADIATION
P. C. KOLLER Chester Beatty Research Institute,
London, England
UPTON [S] has reviewed some of the most prominent facets of cell response to ionizing radiation, and showed the lack of our knowledge concerning the mechanism underlying radiation induced mitotic delay, chromosome injuries and their significance in relation to cell death. He drew attention, also, to the importance of interaction which occurs between irradiated malignant cells and the host, the nature of which is still obscure. My contribution will deal with three aspects of the radiation induced effects on the cancer cell. (1) The first concerns the importance of chromosome injuries. In a variety of human tumors, the type and frequency of cell injuries after irradiation, were studied; it was found that the amount of chromosome damage shown by individual cells and the number of cells having chromosome injuries, is dependent on the radiation dose; the higher the dose, the greater the damage per cell and the greater is the number of cells affected. The most important discovery, however, was the fact, that in different tumors which were exposed to the same dose of radiation, the number of injured cells differed. Table I shows the frequencies of chromosome injuries and their distribution per cell in three squamous cell carcinomas which had been irradiated with 300 rads 24 hr previously. The tumors were comparable in histological structure and size. It can be seen, that in spite of the same radiation treatment the percentage of injured cells varies between tumors when observed at 24 hr. Such variation TABLE
I. Radiation
induced cellular
Percentage Site Cheek Cheek Forehead
of injured cells 13.5 17.5 8.5’
damage in three tumors.
Percentageof cells with chromosome fragments 012345678 86.5 83.0 90.5
0.5 1.5 2.5
9-11 3.0 1.0 2.0
a 8 hr later the percentage increased to 12.0. [In each case 200 dividing cells were analysed
3.5 2.0 3.5
4.5 2.5 0.5
1.4 4.5 -
24 hr after treatment Experimental
0.5 2.0 -
1.5 -
0.5 -
1.0-1.5 -
with 30.0 rads.] Cell Research, Suppl. 9
P. C. Keller in the reaction of tumor cells to identical radiation doses indicates differences in growth rate or in cell sensitivity which may be an inherent property of individual tumors; it is also possible that the variation may only be the result of environmental factors. The tumor in the third patient illustrates the difference in response to irradiation due to growth rate. The percentage of analysed cells containing fragments was only 8.5 in the first sample (24 hr after irradiation) and had risen to 12 per cent in the second biopsy specimen removed eight hours later. Thus only at 32 hr was the re.sponse of this tumor similar to the response of the two other tumors at 24 hr. This example shows that more study is required to understand the cause of variation; this knowledge is necessary if we want to employ our radiotherapy methods effectively [3]. (2) The second point is the possible relationship which may exist between ploidy and radiation response. If there is a relationship between chromosome number and radiation sensitivity, it would be an important argument in favor of the view that genetic damage is the only underlying cause of cell death. There is some evidence that in natural polyploid plant species, in which the chromosome number is doubled, cells are more resistant to radiation than cells of corresponding diploid varieties [l]. In experimental transplanted tumors, and as recent studies indicate, in primary tumors also, heteroploid cells are common, i.e. cells in which the chromosome number deviate from the diploid number. Revesz and Norman [4] who studied the response to radiation in various sublines of Ehrlich ascites tumors, found that the hypertetraploid variant was less radiosensitive than the near diploid clone. During the analysis of radiation response in human epidermoid carcinomas, the present author came across a tumor in which heteroploid cells were present; heteroploidy was indicated by enlarged nuclear size of cells at interphase and by chromosome number in dividing cells. This particular tumor was treated by radiation and showed good initial response but eight months later the growth recurred. The new growth was completely composed of heteroploid cells. The inference is that cells with higher chromosome number than the diploid number, survived radiation and became the source of the new tumor which was found to be resistant to further treatment by radiation. It seems therefore that selection of polyploid cells.may contribute to the development of radioresistance in irradiated tumors. By comparing the DNA content of tumor cells in human uterine tumors before, during and after treatment by radium, Richards and Atkin [5] found evidence which favors the view that selection of polyploid cell variants by radiation, may occur. Experimental
Cell Research, Suppl. 9
FUC~OFS affecting
565
radiosensitivrty
(3) The third
point concerns the influence of host environment on tumor cells which are exposed to radiation. I am particularly interested in the effect of changes induced by radiation in the tissues surrounding the tumor; which in the literature, is referred to as “indirect effect” in relation to the tumor
I
2 DISTANCE
5
+ IN
5
CM.
Fig. l.-Graph illustrating the percentage of abnormal cells due to “direct” and “indiiect” effect of radiation at varying distances from the radiation source in two carcinomas of the cervix uteri. (The incidence of abnormal cells showing direct effects, that is, chromosome injuries, was the same in both cases, hence only that of case 1 is given.) Case No. l.-anaplastic adeno-carcinoma. Dose: 2 X 25 mg/24 hr. Time: 10 days. Case No. 2.-squamous cardnoma. Dose: 2 x 25 mg/24 hr. Time: 16 days.
cells. Cell death due to such an “indirect radiation effect” can be identified by the characteristic morphology of the degenerating cells. A comparison was made of the frequency of tumor cells showing chromosome injuries, indicating direct effect of radiation with that of tumor cells whose nuclear morphology indicated “indirect” radiation effects in two uterine cancers treated by radium. Fig. 1 shows the data obtained. It can be seen that the frequency of cells with chromosome injuries follows closely the estimated radiation dose, it decreases with the distance from the radium source, while the number of cells with pyknotic nuclei (indirect effect) increases with the distance. The fact that 80,000-150,000 rads are required to inhibit the growth of Experimental Cell Research, Suppl. 9
P. C. Keller tumor explant in culture [Z], while in vivo the growth of tumors can be inhibited by doses hundred times less, demonstrate the importance of interaction between tumor cells and the host environment. REFERENCES 1. EVANS, H. J. and SPARROW, A. H., Brookhauen Symposia in Biology, No. 14 (1961). 2. GOLDFEDER, A., Arm. N. Y. Acad. Sci. 95, 13 (1961). 3. KOLLER, P. C., Biological Basis of Radiotherapy in Cancer. P. RAVEN (ed.) Butterworths,
1959. 4. RbvBsz, L. and NORMAN, U., J. natl. Cancer Inst., 25, 5 (1960). 5. RICHARDS, B. M. and ATKIN, N. B., &it. J. Cancer 13, 788 (1959). 6. UPTON, A. C., Eqtl. Cell Res. Suppl. 9, 538 (1963).
Experimental
Cell Research, Suppl. 9