LOCALIZATION O F MAXIMUM CELL DIVISION I N EPIDERMIS E. V. COWDRY AND HENRY C. THOMPSONJ JR. Anatorniwl Laboratory, Washington University and Barnard Free Skin and cancer Hospital, S t . Louis, &fissouri There is difference of opinion as to the layer in the epidermis in which cell division is maximal. Pinkus ( '27) believes that it is the basal layer and goes so far as to state that mitosis in this layer is the only normal mode of epidermal replacement. Thuringer ( '24 and 'as), on the other hand, has found more mitoses in the spinous than in the basal layer. He has, moreover, presented evidence ('39) that on stimulation these two strata respond by increase in mitosis independently. This may afford some clue to the origin and sharply contrasting properties of basal and spinous (squamous) cell carcinoma. For our own studies in Barnard Hospital the whole problem of cellular relations in the epidermis needs clarification. Among the changes in epidermis under the influence of rnethylcholanthrene that are being investigated, alterations in mitotic frequency are important. These have been studied in whole mounts of epidermis, viewed vertically, by Cooper and Reller ( '42) and by Reller and Cooper ( '44). The whole mount method has great advantages over the examination of sections in the making of total counts involving all the layers. But sections are essential for differential counts of mitoses in the several layers. The trouble has been that mitoses are so rare that great numbers of nuclei have to be examined before a sufficient number of mitoses are located to give an accurate measure of their frequencies. For instance in the epidermis of the foot pads of cats, Thuringer ('39) found only 500 mitoses in 210,765 cells, a ratio of 1: 421 ; while Cooper and Franklin ( '40) found in epidermis of the ears of mice a maximum of fifty-nine mitoses per 15,000 nuclei, a ratio of 1:250. The object of this study was to test the applicability of a method of concentrating the mitoses, which has proved useful in other tissue, and possibly with its help to obtain data on normal epidermis which would serve as a basis for later studies on epidermal carcinogenesis. 'Aided by grants from an Anonymous donor, and from the U. S. Public Health Service, the latter on recommendation of the National Advisory Cancer Council. 403 404 E. V. COWDRY AND HENRY C. THOMPSON, JR.. MATERIAL AND METHODS Mice of new Buffalo strain were chosen as material because of their use by others in this hospital concentrating their attention on epidermal carcinogens. It was not feasible to work with normal epidermis of the ears or back because of its extreme thinness. Preliminary observations showed that the regionally hyperplastic epidermis of the hind foot pads was satisfactory. Though rather more cornified, it is of approximately the same thickness and exhibits about the same layering as the epidermis of the back made hyperplastic with methylcholanthrene in which malignant foci are likely to appear. It was decided to limit the observations to young actively growing mice because in them one would expect the layer of maximum cell division to be more sharply marked than in adults. The method of concentrating mitoses was the same as that employed with conspicuous success by Allen, Smith and Gardner ( '37) in localizing the growth effect of theelin in the female genital tract. It depends on the action of colchicine in allowing mitosis to begin and in holding up completion of the process so that they accumulate. I n a preliminary experiment the optimum dosage was determined. F o r the main experiment a litter of six 10-day-old mice was selected. At 8 A.M., 12 noon, 4 P.M., 8 P.M. and midnight one mouse was injected subcutaneously with 0.25 cc. of 1:10,OOO aq. colchicine. At 4 A.M. the next morning all were sacrificed together with one untreated mouse to serve as control. The hind footpads were excised and fixed in Bouin's fluid. After a few minutes they were trimmed with a sharp razor blade, imbedded in paraffin in the usual way, sectioned at 8j.1 and stained with hematoxylin and eosin. OBSERVATIONS All the counts were made by one of us (H. C. T. Jr.) with the aid of a mechanical stage. The nuclei not in mitosis were enumerated by an ordinary mechanical counter, while the mitoses were recorded by hand on a sheet of paper (table 1). Thus, in the normal untreated control epidermis 1826 nuclei of basal cells (actually in touch proximally with the basement membrane) were counted. Among these, forty-six were in mitosis so that the mitotic ratio was 1:39.7. I n the remaining epidermis, consisting of cells distal to the basal ones, it was not possible sharply to delimit the distal margin of the spinous layer. Consequently all the non-basal nuclei were included. 405 EPIDER,MAL CELL DIVISION TABLE 1 Mitotio ratio i n basal layer and rest of epidermis. RHIST OF EPIDERMIS BABAL IIAYEB Nuclei in mitosis TotaX nuclei Ratio Nuclei in mitosis Other nuclei Ratio Control 46 1826 1 : 39.7 121 4400 1 : 37.1 4 hrs. (midnight t o 4 A.M.) 12 524 1 : 43.6 40 1300 1 :32.5 8 P.M.4 A.M.) 43 952 1 : 22.1 158 2952 1 : 18.6 12 hrs. ( 4 P.M.4 A.M.) 6 792 1 : 132 54 1449 1 : 26.7 16 hrs. (Noon4 A.M.) 6 584 1: 97.3 54 1276 1: a3.6 20 hrs. (8 A.M.4 A.M.) 6 625 1 : 104 42 1622 1: 38.6 119 5303 1 : 44.5 469 12999 1 : 27.7 8 hrs. Totals These amounted in the normal epidermis to 4400 among which 121 were in mitosis so that the ratio was 1: 37.1. Ratios for the specimens a t various times after the injection of colchicine follow in the table data on the normal epidermis. It is clear that throughout the series the ratios are higher for the suprabasal nuclei than for the basal ones. The highest ratios were found in both 8 hours after colchicine injection at which time its action is generally considered to be maximal. When the mitoses were counted in the suprabasal layers a record was kept of whether they were located in the inner, middle or outer third of this portion of the epidermal sheet. For the sake of clearness these data are presented in a second table (table 2). They show that, in all the treated epidermises, more mitoses. were always observed in the middle than in either the proximal or the distal thirds of this suprabasal sheet. The cellular population of this middle third obviously differs from that of the other two. The cells are larger and possess more pronounced 406 E. V. COWDRY AND HENRY C. THOMPSON, J R . spines than in the proximal third. They are also larger and do not show the granulation and cornification evident in many cells of the distal third. The fact that in the control epidermis more mitoses were found in the proximal third than in the middle third is important because it shows that level of maximum mitosis is not fixed. TABLE 2 Incidence of mitoses in parts of suprabasal epidermis. TIME APTEB COLCEICINE INJEWION Control 4 hrs. 8 hrs. 12 hrs. 16 hrs. 20 hrs. PROXIMAL MIDDLl DISTAL 36 1h 'h TOTAL 77 8 38 12 14 12 33 24 103 33 34 27 11 8 17 9 6 3 4400 1300 2952 1449 1276 1622 DISCUSSION Cautious interpretation of these results is indicated, because there are several factors that may possibly have influenced them. Cooper and Franklin ('40) have discovered that a diurnal rhythm exists in frequency of mitosis in epidermis of the ears of mice with gradual increase to a maximum at 10 A.M. and decrease to a minimum at 10 P.M. Consequently it is likely that the frequency of cells entering into mitosis in the epidermis of footpads will not have been uniform throughout the period of our experiment extending from 8 A.M. to 4 A.M. It is doubtful, however, whether this alone would materially influence the location of maximum cell division. Only further work will show whether during the rhythm there is a change in level of maximum mitosis. This is not suggested, but is only held as a possibility. Even so the magnitude of the shift might not be sufficient temporarily to govern the level in our specimens. I n another paper of this series by Paletta and Cowdry ( '42) mention has been made of the possibility, noted occasionally by investigators working with other tissue, that colchicine may to some extent either increase or decrease the number of nuclei entering into mitosis. Unless this influence, one way o r the other, acted differently on the several layers it might be expected t o bring about an increase or a decrease in the maximum frequency of mitosis, wherever that is located, without causing any shift in location. Until evidence is presented of the exist- EPIDERMAL CELL DIVISION 407 ence of a mitosis stimulating or inhibiting influence of colchicine acting differentially on the layers of mouse epidermis this possibly disturbing factor, though kept in mind, need not be further discussed. It is also conceivable that the summation effect of the drug on mitoses will be greater in the basal layer of cells, which receives it first and most directly from the tissue fluid beneath, than in the more distant suprabasal cells. If this is so, and the cells were otherwise equal in mitotic activity, one would look for a higher incidence of arrested mitoses in the basal than in the suprabasal cells. This gives added significance to the fact that the highest incidence in the colchicine treated epidermises was not only in the suprabasal epidermis but in its middle third; for, on this hypothesis, a normally higher incidence of mitosis in this third might be somewhat reduced relatively by greater summation of mitoses in the basal cells. There is a second consideration which strengthens the conclusion that, in the treated specimens examined, the highest incidence of mitosis was actually in the middle epidermis. I n dividing this into three parts, based on thickness of tissue distal to the basal layer, fewer cells were included in the middle third than in either of the other two ; because in it the cells were larger than in the proximal and distal thirds. If the frequency of mitosis were the same one would anticipate the finding of fewer mitoses in the middle third than in the proximal third. For a small number of cells to have exhibited more mitoses than a larger number means that the frequency was relatively even higher in the middle than the figures indicate. Observations by Thuringer (’39) are of interest in connection with ours, because he used the same kind of epidermis (palmar and plantar pads) though not of mice but of cats. He observed the distribution of 500 mitoses, expressed in percentages, to be : basal layer, 44.5 ; proximal third spinosum, 54.5; middle third spinosum, 1; and outer third spinosum, 0. From this he concluded that “the lower one third of the stratum spinosum was more active than the stratum basale” which is obviously justified from his data. His published figures do not, however, permit calculation of relative mitotic frequencies in these layers ; because, while the number of mitoses in each are given, the number of cells is not. If the number of cells examined were equal in both, then it could be said that the mitotic frequency was slightly higher in the proximal third of the spinosum than in the basal layer but if it were significantly greater in the former this would not be the case. On the whole it seems that our observations place the stratum of maximum frequency of mitosis in the tissues we studied more distally 408 E. V. COWDRY AND HENRY C. THOMPSON, J R . than is likely to have been the case in Thuringer’s “resting” material; for, it must be remembered that the suprabasal epidermis, which we artificially divided into three parts, included some tissue distal to the true spinous layer so that our proximal and middle thirds of the suprabasal epidermis included more than two thirds of the spinous layer. On electrical stimulation Thuringer discovered an impressive shift in the location of the greatest number of mitoses. Those in the lower third of the spinosum exceeded those in the stratum basale by as much as 653 to 100. I n view of this finding, and of our observation of maximum mitoses in the proximal third of the suprabasal epidermis of the untreated specimen whereas the maximum was always in the middle third of the treated specimens (table 2), it would be unwise to assume that the level of maximum incidence of mitoses in epidermis is fixed. On the contrary this level can probably rise and fall in different physiological and pathological-conditions depending on many factors including increase and decrease in the amount and character of fluids seeping u p into the epidermis from the tissue fluid beneath. The discovery in our material and in Thuringer’s that the maximum frequency of mitosis was centered somewhere in the spinous layer is not to be interpreted as indiaating that cells are mainly produced by multiplication of preexisting ones in this layer and are displaced in both direct ions. All the evidence points to removal of worn out cells from the distal surface of the epidermis. Signs of death of basal cells are few and far between. Consequently there is as yet no valid reason to recast the old idea that the epidermis is constructed of layers of cells shifting in a distal direction and taking origin ultimately from basal cells in contact with the basement membrane. We can picture these basal cells as vegetative interrnitotics in the sense defined by Cowdry ( ’42). Their individual lives are intermitotic, since they extend from one mitosis to another. They can be regarded as vegetative ; since, on multiplication, some of the daughter cells remain as basal cells close to the basement membrane, vegetative and no more differentiated than their forebears. Other daughter cells are displaced distally and start out on the path of differentiation. These, by contrast, are differentiating intermitotics, whose lives extend likewise from one mitosis to another. Each one begins its individual life a little further differentiated than its predecessor so that differentiation is in a series of ascending cellular steps until cells are produced, which are handicapped by increasing distance from the life giving tissue fluids, and which are postmitotics in the sense that their individual lives end in death, not in another mitosis. EPIDER,MAL CELL DIVISION 409 The finding of maximum mitotic frequency in the spinous cells of the middle third of the treated suprabasal epidermis in our specimens simply means that these particular differentiating intermitotics are multiplying more rapidly than the other cells just as primary spermatocytes of the testicle can and often do divide more frequently than the spermatogonia, the order of replacement being the same in the testicle as in the epidermis. SUMMARY I n the epidermis of the hind foot pads of an untreated 10-day-old new Buffalo mouse the ratio of mitoses to nondividing nuclei was 1:39.7 in the basal layer and 1:37.1 in all the suprabasal layers taken together. I n the litter mates, each of which received 0.25 cc. 1: 10,000 aq. colchicine subcutaneously, the ratios increased to a maximum of 1:22.1 and 1:18.6, both 8 hours after the injection. I n the untreated animal the site of maximum mitotic frequency was in the spinous cells of the proximal third of the suprabasal epidermis, while in all of the treated ones it was located in those of the middle third. The possibility is suggested that the level of maximum mitoses in the epidermis is not fixed but subject to change in different physiological and pathological conditions. LITERATURE CITED M. SMITH AND WILLIAMU. GARDNER 1937 Accentuation of the growth ALLEN,EWAR,GEORGE effect of theelin on genital tissues of the ovarieetomized mouse by arrest of mitosis with colchicine. Am. J. Anat., vol. 61, pp. 321-329. COOPER,Z. K. 1939 Mitotic rhythm in human epidermis; introduction and review of literature. J. Inv. Dermat., vol. 2, pp. 289-300. COOPER,Z. K., AND H. C. FRANKLIN1940 Mitotic rhythm in the epidermis of the mouse. Anat. Rec., vol. 78, pp. 1-8. COOPER,Z. K., AND H. C. RELLER 1942 Mitotic frequency in methylcholanthrene epidermal carcinogenesis in mice. J. Nat. Cancer Inst., vol. 2, pp. 335-344. COOP^, Z. K., A N D A. SCHIFF 1938 Mitotic rhythm in human epidermis. Proc. Soc. Exp. Biol. and Med., vol. 39, pp. 323-324. COWDRY,E. V. 1942 Chapter on Ageing of Tissue Cells i n Cowdry’s Problem of Ageing. Baltimore: Williams & Wilkius, 936 pp. PALETTA, F. X., AND E. V. COWDRY 1942 Influence of colchicine during methylcholarithrene epidermai carcinogenesis i n mice. Am. J. Path., vol. 18, pp. 291-311. PINKUS, H. 1927 Anatomie der E a u t , in Handbuch der H a u t und Geschlechtskrankheiten, vol. 1 ( l ) , p. 89. EELLER, H. C., AND Z. K. COOPER 1943 Mitotic incidence in the first 48 hours of methylcholanthrene epidermal carcinogenesis. Cancer Res., (in press). THUBINGER,J. M. 1924 Regeneration of stratified squamous epithelium. Anat. Rec., vol. 28, pp. 31-38. 1928 Studies on cell division in the human epidermis. Anat. Rec., vol. 40, pp. 1-13. 1939 The mitotic index of the palmar and plantar epidermis in response to stimulation. J. Inv. Dermat., vol. 2, pp. 213-326.