The Use of X-Rays to Determine the Mitotic and Intermitotic Time of Various Mouse Tissues*

The Use of X-rays To Determine the Mitotic and Intermitotic Time of Various Mouse Tissues*

NORMANP. KNOWLTON,JR.,ANDWILLIAMR. Wi

(Los Alamos Scientific Laboratory, LoÂ«Alamos, New Mexico)

It has long been known that, as a general rule, only slight, if any, slowing of the mitotic process the sensitivity of various normal and malignant (4). The slope of the downward trend of the mitotic tissues to x-ray is proportional to the amount of index after irradiation must then be a measure of cell division which is occurring in the tissue. The the mitotic time. In other words, one would expect number of mitoses seen in a biopsy of a tumor is cells to be completing the mitotic process, but no one of the criteria for predicting the probable re cells would be entering middle prophase. Theo sponse of the tumor to irradiation (1). Since the retically, therefore, after a period equal to the amount of cell division, or more specifically the mitotic time, there would be no mitoses present mitotic index, is dependent upon the mitotic and which were later than the ones blocked at early intermitotic times (5), a study was undertaken to prophase. Actually, one would expect that a few determine which of these two factors is responsible cells would slip through the barrier at early pro- for differences in the mitotic index seen in various phase and also that a few cells which were past the tissues. barrier at the time of irradiation would be injured Many technics have been described by which the sufficiently to stop their progress through division mitotic time of various cells can be determined. (7)â€”the latter being manifested by various chro Hanging drop preparations (3, 8, 12) and tissue mosomal aberrations such as clumped metaphases, cui Iures (9, 13) have been used for direct observa chromosomal bridges of anaphase, etc. With the tion of cell division. The mitotic and intermitotic above theory in mind, a study was undertaken to times of some internal tissues have been estab determine the mitotic time of various mouse tis lished on the basis of the mitotic index and rate of sues by means of the fall in the mitotic index after growth of the tissue (2, 10). In the case of internal irradiation. organs in which there is no increase in size, this EXPERIMENTAL method is not applicable. If, however, it were pos CFy strain female mice, 8-16 weeks of age, sible to determine the mitotic time of these tissues which appeared healthy after 10 days' acclimation by some other means, then, knowing the mitotic at this laboratory, were used in these experiments. index, the intermitotic time could be calculated Control animals were subjected to exactly the (5) from the following formula: same handling procedures and confinement as the Mitotic time irradiated animals. The mice were irradiated in a Intermitotic time Mitotic index ' flat Incite cage and allowed free movement during exposure. The radiation was delivered by a 2.50-kv. peak voltage x-ray machine with no filters. The It has been shown that ionizing radiation causes inherent filtration of the x-ray tube was equiva a delay in cell division at early prophase (8, 4). At moderate dosage levels, those cells which are past lent to about 2 mm. of aluminum. The dosage de this critical stage continue through mitosis with livered to each group of mice was determined by means of a 300-r Victoreen ionisation chamber * This document is based on work performed under Con placed at the center of the cage. tract No. 7405-eng-36 for the Atomic Energy Commission. To study the effect of x-rays on the bone mar t With the technical assistance of Eletti Herring, Clare row, eight groups of seven mice were carried Morrison, Joan Thrap, and Julie Wellnitz. through the following procedure. Five animals ÃŽThe authors wish to acknowledge the assistance and advice of Gerold Tenney and his staff in the development of ir were irradiated with 400 r of x-ray given at the radiation technics and dosage measurements. rate of 100 r per minute. Animals were killed by Received for publication, September 9, 1949. crushing the cervical spine at (i, 12, 18, 24, and 30

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1950 American Association for Cancer Research. Cancer Research minutes after exposure. The control animals were calculate the normal mitotic index. The number of killed before and after the irradiated mice. A speci mitoses per 100 crypts of LieberkÃ¼hnand the aver men of hone marrow was removed from the femur age number of cells per crypt were determined for by inserting a needle into the medullary cavity and the jejunum. In the adrenal gland, the nun ber of forcing out the marrow. The specimen was mixed mitoses found in the zonae glomerulosae and fas- with a drop of fresh blood serum (human or rat) ciculatae was enumerated for 15 sections. Repre on a clean glass slide and a bone marrow smear sentative sections were then projected on white prepared. This was then air-dried, stained with paper, and the outer border of the zona glomeru- Wright's stain, and mounted in Canada balsam. losa and the inner border of the zona fasciculata The preparations were read to determine the num were outlined. The area of these two portions of ber of mitoses per 5,000 nucleated cells. Each the gland was determined by the use of a planim- mitosis was classified according to its stage of di eter. After counting the number of cells in the vision and its series, whether erythrocytic or zonae glomerulosae and fasciculatae of four of the myelocytic. Sample counts were performed on adrenal glands which had been projected, it was each bone marrow preparation to determine the possible to determine the number of cells per unit relative number of white blood cells and nucleated area of the projection. The number of mitoses per red blood cells, so that a normal mitotic index unit area and the normal mitotic index were then could be calculated for these two kinds of cells. calculated. The mitotic index of the ovarian fol licles was determined by counting the number of TABI.K1 mitoses per 50 follicles with diameters of 50 n or BONKMARROW more and then finding the average number of CHANGEIN MITOTIC ACTIVITYAFTER 400 r OF X-HAY cells per follicle. MITOSES PEH 5,000 NTCLEATED("KLLH In an effort to standardize the technic of count Time after Nucleated red ing mitotic figures, a cell was considered to be in exposure MyelocyteÂ» blood cells mitosis only between the time, in prophase, of control 11.2+1.1 8.4+1.1 (i min. 8.0 + 1.7 54 + 10 elongation of the chromosomes before the break 1Â«min. 8.4 + 1.1 4.1+0.7 down of the nuclear membrane; and the time, in 18 min. 6.8Â±1.2 8 6Â±0 8 telophase, of complete separation of the daughter Â¿4min. 3.9 + 0.9 s.a+o.ÃŸ 30 min. 2.7Â±0.9 a.o+o.s cells. This definition of mitosis was very satisfac , -V/Z (* - i tory for all tissues except the epidermis, in which Standard error \n(n-l the nuclear structure was distorted slightly by treatment with acid; therefore, the time of break To determine the mitotic time of various other down of the nuclear membrane was considered the mouse tissues, 72 mice were divided into three beginning of mitosis. groups of 24 animals each. These groups were giv en 0, 50, and 200 r of x-ray, respectively, at a rate RESULTS of 50 r per minute. Eight animals from each group There was a fairly uniform decrease in the mi were sacrificed at 10, 20, and 30 minutes after the totic activity of the myelocytes and nucleated red midpoint of exposure, or, in the case of control ani blood cells after irradiation (Table 1). mals, the midpoint of confinement. The tissues to A straight line, calculated to fit the experimen be studied were removed in a rapid manner (11) tal points for the mitotic activity in the myelo and in a definite order (6). The ears were removed cytes (Fig. 1), intersects the time ordinate at about first and placed in 0.5 per cent acetic acid; then the 35 minutes. In the case of the nucleated red blood right inguinal lymph node, a segment of jejunum, cells (Fig. 2) there is a flattening of the curve at the the right adrenal gland, and the right ovary were 24- and 30-minute points. If these points are neg removed and placed in Bouin's fixative. The epi lected, and a straight line is calculated to fit the dermis was separated from the inner surface of the 0-, 6-, 12-, and 18-minute points, this line inter ears, after 24 hours in acetic acid at a temperature sects the time ordinate at about 29 minutes. of 8Â°C.,and (Â¡-/xsectionswere prepared from the The results obtained in the other five tissues are other tissues after routine histological procedures. shown in Table 2. The responses seen in the jeju The mitotic activity of each tissue was then de num, ovary, and lymph node are almost identical termined by the methods previously described (6). (Figs. 3, 4, and 5). If the slope between the 20- and The number of mitoses per 100 standard fields, as 30-minute points for the 200-r dosage is used to outlined by a Whipple disc, and the number of determine the mitotic times of these three tissues, cells per field were determined for the epidermis they are found to be 24, 21, and 23 minutes, re and the lymph nodes, thereby making it possible to spectively. The differences between the mitotic

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1950 American Association for Cancer Research. KNOWLTONet al.â€”Determination of Period of Mito-vis 61 times arc well within the standard errors which ly for each tissue, is shown in Table 3. It is evi were determined l>y mathematically deriving the dent that the crypts of LieberkÃ¼hnofthe jejunum, formula for the line between the 20- and 30-mimite the myelocytes, the nucleated red blood cells, and points and by determining the intersections with the follicles of the ovary have the higher mitotic the normal mitotic activity and with the time ordi indicesâ€”the crypts of LieberkÃ¼hnof the jejunum, nate. The results in the epidermis and adrenal for example, have about one cell in mitosis out of gland are more difficult to interpret. If the 20- and every 100 resting cells, as compared to two out of 80-minute points on the 50-r dosage curve arc used 10,000 in the zonae glornemlosae and fasciculatae to calculate the mitotic time of the epidermis, the of the adrenal gland. The intermitotic times were calculated from the experimentally derived mitotic times and the mi- 12 CONTROL

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e 12 18 24 30 MINUTES Fio. 1.â€”Bonemarrow; myelocytic series FIG. 2.â€”Bonemarrow; erytlirocytic series

TABLE 2 CHANGEIN MITOTIC ACTIVITYAFTERIRRADIATION- CELLS PER DOSAGEINROENTGENS5020050â€¢Ã®iw5050â€¢ilK)50 E AFTEREXPOSURE CRTPT, FOLLICLE TISSUE 10 min. 20 min. SO min. ORUNITAREA Jejunum 75.3Â±4.1 69 0 + 6.8 56.1Â± 9.3 85 7 + 1.8 (Mitoses/100 crypts) 74.6Â±8.5 53.5Â±8.2 20.3Â± 2.9 cells/crypt

Ovary 6 + 5223.5 57.2 + 6 O 49.9 + 5.9 45.5Â±11 4 390 + 44 (Mitoses/50 follicles) 50.8 + 7.1 37.9 + 6.7 11.5Â± 36 cells/follicle (351 + 37) X10= Lymph node +1.415.4Â±2.817.7Â±2.128.2Â±3.4 20.0Â±1.7 16.3+ 1.8 (Mitoses/unit area) 20.8Â±1.4 16.4Â±1 3 6.3Â± 1.3 cells/unit area Epidermis 14.6 + 2.6 12.3 + 1.8 7.2+ 1.7 (205 + 2)X102 (Mitoses/unit area) 17.3 + 2.8 4.7Â±1.2 8.9Â± 1.7 cells/unit area Adrenal gland 12.7Â±4.0 7.6 + 0.9 9.6+ 3.2 (8.0 + 0.7)X10Â« (Mitoses/unit area) 200CONTROL79.4Â±3.15519.7Â±2.1 20.4 + 4 2 8.1+ 1.0 cells/unit area resultant time is 30 minutes with a relatively large totic indices (Table 3). Since the mitotic times of standard error (Table 3). The data for the epi all seven tissues are relatively constant, the inter dermis from the 200-r dosage are definitely con mitotic time is inversely proportional to the mi flicting. The large standard error at the 20-minute totic index. Those tissues with the lower mitotic point in the 200-r data makes it difficult in the case indices have the longer mitotic times. Of the tis of the adrenal gland to be accurate in establishing sues studied, the jejunum has the shortest inter the mitotic time. Judging from the shape of the mitotic time (43 + 8 hours) and the adrenal the curves obtained from the other tissues, the mitotic longest (probably more than 1000 hours). time is probably 20 or more minutes, rather than the figure of 14.4 + 4.5 minutes. A tabulation of DISCUSSION the mitotic times of the seven mouse tissues which It would appear from the experimental data were studied is presented in Table 3. that the mitotic time of various types of cells can The mitotic index, as determined experimental- be determined by delaying mitosis in early pro-

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1950 American Association for Cancer Research. Cancer Research phase by means of x-ray. In all seven types of would appear that the mitotic times for the seven cells which were studied there was a definite down different tissues are quite comparable, probably ward trend found within the first 30 minutes after varying between 20 and 35 minutes. irradiation. The slopes obtained for five of the The observation that the mitotic activity in the jejunum, ovary, and lymph node did not drop sig nificantly during the first 10 minutes after expo sure can perhaps be accounted for by the criteria used to define mitosis. The beginning of mitosis was arbitrarily defined as the time when the chro mosomes were elongated in prophase. If radiation blocked mitosis at a point about 10 minutes prior to this, there would be a number of cells still enter ing the arbitrarily defined beginning of mitosis equal to the number forming daughter cells, and the mitotic index would remain constant for this length of time. After ten minutes there would be no more cells entering the stage of chromosomal 10 20 30 40 MINUTES elongation in prophase; but there would still be FIG. 3.â€”Jejunum: Mitoses in the crypts of Lieberktlhn cells completing division, and therefore the mitotic index would begin to fall. It is apparent from the discussion above that

10 20 MINUTES FIG. 4.â€”Mitoses in the (Â¡runfianfollicles of the ovary FIG. 5.â€”Mitosesin the lymph nodes

TABLE 3 MITOTICINDEX,MITOTICTIME,ANDINTERMITOTICTIMEOFMOUSETISSUES MITOTICTIME INTEKHITOTICTIME I\ MIM'TKH TISSUE MITOTICINDEX IN ROUHS Jejunum 23.9+ 4.5 (9.3 +0.4 )10- 43+ 8 Nucleated red blood cells 29.5Â± 24 (5.0 Â±0.7 )10- 99 Â±16 Myelocytic series 35.3+ 3.0 (3.8 +0.4 )10~ 155Â±20 Ovary 21.1Â± 4.8 (2.8 +04 )1(T 123+ 33 Lymph node 23.2+ 3.3 (6.7 Â±0.8 )10 580 + 110 Epidermis 30.2+12.0 (7.5 Â±1.4 )10~ 670 + 300 Adrenal gland 14.4+ 4.5 (2.21Â±0.33)10 1090Â±430 seven tissues (the crypts of LieberkÃ¼hn,the fol the mitotic time which was measured was only the licles of the ovaries, the lymph nodes, the nucle period of time from the elongation of the chromo ated red blood cells of the bone marrow, and the eip- somes in prophase to the separation of the cyto dermis) were so comparable that the derived mi plasm of the daughter cells. In the jejunum, ovary, totic times varied only from 21 to 30 minutes. The and lymph node the delay in the fall of the mitotic myelocytes apparently have a slightly longer mi activity might be a measure of the time in pro- totic time; and the cells of the zonae glomerulosae phase that is sensitive to radiation and the elonga and fasciculatae in the adrenal gland might pos tion of the chromosomes. For these three tissues sibly have a slightly shorter one, although the data this time would appear to be 10-15 minutes. for this tissue are difficult to interpret. Thus, it Since the mitotic index is measured on the basis

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1950 American Association for Cancer Research. KNOWLTONet al.â€”Determination of Period of Mitosis of Ihe criteria for mitosis mentioned above, and the the experimentally derived mitotic time and mi mitotic time is also derived from the same arbi totic index. Differences in the mitotic indices of trary definition of mitosis, these two artificially in tissues were found to be due primarily to variations troduced errors cancel out in the determination of in the intermitotic time. the interniitotic time. Thus, the value of the aver age time between divisions is a true one and does REFERENCES not have arbitrary limitations. 1. BOYD,W. A Textbook of Pathology, pp. 274-77. Phila delphia: I-ea & Febiger, 1947. CONCLUSIONS 2. BRKES,A. M., and MARBLE,B. B. An Analysis of Mitosis A method of determining the mitotic time of in Liver Restoration. J. Exper. Med., 66:15-27, 1987. 3. CARLSON,J. G. An Analysis of X-ray Induced Single various tissues of the mouse by means of estimat Breaks in Xeuroblast Chromosomes of the Grasshopper ing the effect of x-ray on mitosis has been present (Chortophag a t iridifasciata). Proc. Nat. Acad. Se., 27:42- ed. The mitotic time was found to vary about 20- 47, 1941. ;5(iminutes in the seven tissues studied. The nor 4. HENSHAW,P. S., and COHEN,I. Further Studies on the mal mitotic index and the mitotic time of these Action of Roentgen Rays on the Gametes of Arbacia punctulata. IV. Changes in Radiosensitivity during the tissues were used to calculate the intermitotic time Cleavage Cycle. Am. J. Roentgenol., 43:917-20, 1940. from the formula: 5. HOFFMAN,J. G. Wright's Hypothesis: Its Relation to Mitotic time Volume Growth of Tissue Cells and Mitotic Index. Science, Intermitotic time = Mitotic index ' 106:343-44, 1947. 6. KNOWLTON,X.P., JR., and HEMPELMANN,I,.H. The Ef fect of X-rays on the Mitotic Activity of the Adrenal It was concluded that, since the mitotic times Gland, Jejunum, Lymph Xode, and Epidermis of the were relatively constant as contrasted to the inter Mouse. J. Cell. & Corap. Physiol., 33:73-92, 1949. mitotic times, the differences in the mitotic in 7. LEA, D. E. Actions of Radiations on Living Cells, p. 294. dices of the various tissues were due primarily to 1st ed. Xew York: Macmillan Co., 1947. 8. LEWIS, M. R., and LEWIS, W. H. Malignant Cells, of the variations in the intermitotic times. Thus, the Walker Rat Sarcoma Xo. 338. Am. J. Cancer, 16:1153-83, mitotic index is inversely proportional to the inter 1932. mitotic time, and, in general, tissue-sensitivity to 9. OLIVO,O. M., DE LoBENzi, E., and LEVI, S. G. Sulla x-ray increases, the shorter the intermitotic time. Durata dell' Intercinesi nelle Cellule Coltivate in vitro. Accad. naz. Lincei, Roma, 7:936-39, 1928. Further experiments are being planned to study 10. OLIVO,O. M., and PORTA,E. Durata delle Mitosi nelle the mitotic and intermitotic times of certain mouse Cellule Embrionali Cuore e del Fegato di Pollo. Boll. Soc. and rat tumors with the purpose of detecting any Â¡tal.biol. sper., 6:71-73, 1931. differences between normal and malignant types 11. THÃœRINGER,J.M. Studies on Cell Division in Human Epi of cells. dermis. II (a) Rate of Cell Division in Prepuce: (b) Influ ence of Various Factors on Cell Division. Anat. Ree., 40: SUMMARY 1-13, 1928. A method has been proposed to determine the 12. WILLMER,E.X. Analysis of Growth of Chick Heart Fibro- mitotic time of tissues, utilizing the effect of x-ray blasts in Hanging Drop of Fluid Medium. J. Exper. Biol., 10:323-39, 1933. on cell division. The mitotic times of seven types 13. WRIGHT,G. P. The Relative Duration of the Various of cells of the mouse were determined by this Phases of Mitosis in ('hick Fibroblasts Cultivated in rilro. method. The intermitotic time was calculated from Roy. Micr. Soc. J., 414-17. 1925.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1950 American Association for Cancer Research. The Use of X-rays to Determine the Mitotic and Intermitotic Time of Various Mouse Tissues

Norman P. Knowlton, Jr. and William R. Widner

Cancer Res 1950;10:59-63.

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