Changes of Hair Follicular Cells after a Single Painting of Methylcholanthrene in Mice*

Transcription

1 Changes of Hair Follicular Cells after a Single Painting of Methylcholanthrene in Mice* HSU-MU LlANcf AND E. V. CoWDRY (Wernse Cancer Research Laboratory and Department of Anatomy, Washington University, St. Louis, Jfo.) Early changes in the epidermis in methylcholanthrene-induced carcinogenesis have been extensively studied both microscopically and chemically (11-14). Less is known about altera tions in hair follicles detectable microscopically, and the results of chemical analysis of hair fol licles are conspicuous by their absence. Yet the follicles are epidermal derivatives capable of giving rise to new epidermis to replace areas of epidermis that have been removed surgically or chemically, as has been best demonstrated by Bishop (4) in his own skin. Their component cells are anatomi cally continuous with epidermal cells, which they resemble in many respects. That they may con stitute the source of some of the squamous-cell cancers produced by cutaneous applications of methylcholanthrene dissolved in benzene cannot be ignored. We have in mind some, not all, of the said cancers, for evidence of the epidermal origin of some of them is convincing. Though the epi dermis is more directly and immediately exposed to this carcinogen, the exposure of follicular cells lining the pits from which the hairs project may be equally effective, owing to the tendency of a fluid applied to the surface to lodge in such pits and to be brushed off from the free epidermal surface. For these reasons, it is deemed important, as part of a Barnard Hospital-Washington Uni versity project, which has been carried on for 16 years (13), systematically to investigate the fol licular changes, despite the fact that it will not be feasible to supplement microscopic findings by chemical analysis, because follicular material can not be collected, like epidermis, en masse in a condition suitable for chemical analysis. The series of studies on hair follicles, of which this is the second, was initiated in the belief that the investigation could be made more fruitful than * Aided by grants from the Charles F. Kettering Founda tion and from the American Cancer Society. t Fellow of the American Bureau for Medical Aid to China. Address: Biomorphic Department, National Defense Medical Center, Taipei, Taiwan, China. Received for publication October 31, previous attempts by other workers. In the first contribution (18), advantage was taken of a technic whereby whole sheets of epidermis with at tached hair follicles can be examined in place of sections, which latter yield only a very restricted view of events. The observations were limited to the period from 7-30 days after the initial applica tion of the carcinogen and included: 1. The identification of two types of hair follicles. 2. The demonstration of minute local modifica tions in both epidermis and follicles. 3. The recognition of three types of change in the follicles : a) Degeneration and disappearance, 340 &) The enlargement of individual follicles, and c) The fusion together of neighboring follicles. 4. The characterization of focal areas of ulcã ra tion, involving hair follicles. The work here represented relates to the micro scopic study of sections and thus supplements the first contribution. It is also a continuation of studies of hair follicles in this project by Cramer and Stowell (15) and by Suntzeff, Cowdry, and Carruthers (24) strengthened by a vivid apprecia tion of the cyclic activity of hair follicles providing a succession of constructive and destructive phases as long as the follicles endure, described years ago for mice by Dry (16) and confirmed by others (2, 6, 8, 26). Dry introduced the nomen clature that will be described later. The illuminat ing facts are (a) that atrophy regularly follows hyperplasia, which means that cells in both phases are subjected to the carcinogen and (6) that neighboring follicles are not necessarily syn chronized but may be observed in different phases of cyclical activity. MATERIALS AND METHODS Sixty female Swiss mice, 5 weeks old, were used. Interscapular hair was shaved, and 0.6 per cent methylcholanthrene in pure benzene was applied with a No. 4 camel's hair brush to the shaved area of each animal on the same day. Only one application was made. Animals were sacrificed from 24 hours to 15 days after painting. Three mice, chosen at random, were killed on each day. A rectangular piece of skin from the treated area, approximately 5X7 mm. in size, with its length

2 LIANGANDCOWDRYâ MethylcholanthreneChanges in Hair Follicular Cells 341 parallel to the long axis of the animal, was excised. This was properly oriented, placed on a piece of filter paper, and fixed in Bouin's fluid. Serial paraffin sections of the skins, 5 Â t in thick ness, were stained with hematoxylin and eosin. Axial sections were chosen in order to cut the hair follicles longitudinally so that changes taking place in the follicles would be more completely revealed. Fifteen similar animals served as untreated controls. These were sacrificed on the same day that the others were painted. Examination of the specimens brought to light stages in hair follicular cycle presumably like those present in the experimental mice just before the single application of carcinogen. This knowledge of what preceded the modifica tions induced by methylcholanthrene supplied an essential basis for the observations. OBSERVATIONS NORMALCONTROLS(Fios. 1-4) Marked individual differences in follicular ac tivity were observed in these fifteen normal con trols despite the fact that the same area of skin was examined in animals of the same closely in bred strain, age, and sex. According to Dry (16) the activity of hair fol licles in mice is divisible into three phases. 1. The anagen phase lasts about 17 days from the initiation of growth to the cessation of matrix cell proliferation in the bulb, which is at its maxi mum diameter. 2. The catagen phase persists about 2 days. The inner root sheath disappears, and the club sheath forms. The hair germ of the next generation appears as a layer of cells below the club. The root decreases in size. The hair club and its sheath rise to a level below the sebaceous glands. 3. The telogen phase lasts during the dormancy of the hair germ or remaining part of the follicle. When this starts to grow it is invaded by the papilla, and a new anagen phase begins. The sebaceous glands with their ducts and the part of the follicles distal to the openings of these ducts, formed in early life, for the most part normally persist throughout life, while the root sheathes and the hair shafts are renewed with each cycle. The length of the cycle is obviously condi tioned by many factors. In human skin it is customary to speak of two layers of dermis: (a) A superficial layer, which is outermost and which is sometimes also called the "papillary layer"; because, where thickened ridges of epidermis exist and these are seen in sections cut transversely, the superficial layer seems to project distally into the epidermis like papillae. In the majority of cases, however, these are not papillae but dermal ridges projecting be tween epidermal ridges or thickenings. Therefore, the designation "papillary layer" is usually a misnomer. (6) A deep layer, which is innermost and which may with some slight justification be referred to as a reticular layer, by contrast with the superficial layer is much more fibrous. The fibers are mostly of the collagenic variety and form, with some elastic fibers, a closely woven sheet which is almost netlike or reticular. In mice the epidermis is much smoother than in human beings. Since a "papillary layer" is seldom recognizable, and the distinction between super ficial and deep layers is ordinarily not clear, reference is made in this paper simply to dermis without qualification. The term subcutis, em ployed by Pullinger (20) in his excellent paper describing early responses to methylcholanthrene to designate the layer of fatty tissue often inter posed between the fibrous inner layer of dermis and underlying muscle, is here used in the same sense. HAIR FOLLICULAHCYCLEANDCYCLICCHANGES IN THESKIN The telogen phase of the follicular cycle of normal untreated mice is shown in Figure 1; in this example two generations of hairs have been completed. This is expressed by the presence of two club hairs in a composite follicle. The hair of the first generation is uppermost. Below the sheath of the second generation is the hair germ of the third generation. Beneath that is the darkly stained basal saccule. Such skins usually consist of thick epidermis and thin subcutis. Their hairs are confined only to the dermis, never extend beyond it. The follicle shown in Figure 2 is in the early anagen phase. The hair germ of the succeeding generation has started to grow. Follicles of the mid-anagen phase, characterized by the big bulbs and narrow papillary cavities, are illustrated in Figure 3. Note the large shafts and bulbs of the growing hair lying deep in the much thickened subcutis. The sebaceous gland and the hair club of the preceding generation are seen in the dermis at one side of the new hair sheath. The epidermis of such skin usually becomes thinner, because of distention exercised by the growing hairs and increase of subcutaneous tissue. Typical catagen phases were not found in the controls, probably because of their short duration. They last for approximately 2 days (16). Figure 4 shows a late catagen phase similar to that in Figure 1, except that a part of the club hair extends a little beyond the dermis, and the sub cutis is thicker than that in the telogen phase. Accordingly, the amount of subcutaneous tissue and, to some extent, the thickness of epidermis and dermis vary with the phases of the hair cycle. The increase of subcutaneous tissue reaches a

3 342 Cancer Research maximum in mid and late anagen phases (Fig. 3). Thereafter it is reduced in thickness, together with the reduction in size of the hair roots during the catagen phase. In the telogen phase, only a thin layer of subcutis remains (Fig. 1). The measurements in millimeters (mean from different portions without compensating for the shrinkage due to fixation and dehydration) from two photomicrographs (Figs. 1 and 3) enlarged to the same magnification of two control animals of the telogen and the mid-anagen phases are as follows: Telogen Mid-anagen Thickness of the whole skin Thickness of epidermis Thickness of dermis Thickness of subcutis Length of hair follicle This shows that the total thickness of the skin increases more than twice during mid-anagen phase over that of telogen phase. This thickness is due to increase in the subcutis, which is 4 times as thick as during telogen. Thus is provided space for the accommodation of the actively growing hair follicles which increase more than threefold in length during the mid-anagen phase. While the dermis and epidermis become slightly thinner than those in telogen phase, similar changes in the skin in relation to the hair growth cycle have been ob served by Chase and his collaborators in C57BL male mice (7), and by Andreasen in inbred strain St/Eh mice (1). Stages of the hair cycle in a particular region of an animal are so marked in our series that they can even be roughly estimated by inspection and palpation of the skin after shaving. The skin is thin and pink in color during the telogen phase. It becomes thick and milky during the anagen phase. However, in the determination of phases of the cycle one must not rely solely on gross inspec tion without histological check-up. CARCINOGENICSERIES Consider viewed at first the alterations a low magnification in hair follicles of 150 (Figs. 5-7) and, second, the cellular changes in them seen at a higher magnification of 325 (Figs. 8-15, except 10). The same individual variations of hair cycles were also found in painted animals as in untreated animals, progressively modified, of course, by the action of the carcinogen. Hair follicles are such dynamic organs, con tinuously changing their structural features during the different phases of their cycle, that localization of cytological responses is difficultâ especially when natural (cyclic) and carcinogen-induced atrophy and hyperplasia must be disentangled. Because of the striking differences among individ ual animals in the reaction of the skin to the carcinogen, it is to be noted that the changes men tioned and their time of appearance hold only for the particular specimen being described. However, some interesting common features were observed in the responses of the animals : a) Hair follicular cycle and response.â In the painted series, even though the hair follicles are modified, one can still tell the stage of the hair follicular cycle at the time the animal was sacri ficed by the thickness of subcutis and the length and the condition of the (modified) hair follicles. With this information, and our knowledge of the average duration of each phase of the normal hair follicular cycle and the period of time that has elapsed since painting, we can estimate the phase of the hair follicles at the time of painting. In specimens 5-15 days after painting, nearly all the altered follicles appeared in the telogen phase, and in no instance were these changed fol licles accompanied by sebaceous glands; in all but one specimen (Fig. 10), in which late anagen phases predominated, the follicular cells seemed more or less normal, but the surface epidermis usually showed a more advanced degree of hyper plasia and marked hyperkeratosis forming a thick crust over the painted area. This indicates that hair follicles in the period of telogen and late catagen phases are more susceptible to the carcin ogen than in the other phases, and in the period of mid-anagen are more resistant. 6) Cystic transformation of hair follicles.â Figure 5 is a specimen, 5 days after painting, which showed early modifications in the hair follicles. They either appeared as short clublike solid clus ters of cells attached to the epidermis or as elon gated epithelial projections, deprived of hair and sebaceous glands, stretching into the dermis-like papillae. In some specimens they appeared as isolated masses of cells lying in the dermis (Fig. 7). But the arrector phili muscles were often still in tact. These follicles were many times increased in diameter, compared to that of follicles on the normal corresponding phase of the follicular cycle (Fig. 1). Their cells, like those of the epidermis, were also hypertrophied and contained large nu clei with distinct nucleoli. Basal saccules (darkly stained cells at the bases of the follicles) were recognizable, indicating that the follicles were either very young or very old. At the right upper corner of this figure (5) two hairs can be seen in the process of separating, together with the keratin crust, from the differentiated hyperplastic epi dermis. These were hairs of the preceding genera tion, cast off owing to the action of the carcinogen. Apparently, some made-over follicles underwent rã sorption, or possibly reorganization. Such follicles,

4 LIANGANDCOWDRYâ MethykholanthreneChanges in Hair Follicular Cells 343 if severely altered, were unable to produce hair. Seven days after painting, groups of degener ated keratinized cells appeared in some of the changed follicles, as shown in Figure 6. These were early signs of the epidermoid cystic or epithelial pearl formations which are illustrated in Figure 7 of a 15-day specimen. These cysts were either con nected by elongated epithelial stumps to the epi dermis or completely enclosed by hyperplastic epithelial walls lying in the dermis. Again, no se baceous glands and hairs were observed associated with such follicles. But, at the left side of the same figure, a normal-looking follicle is seen with darkly stained hair shaft, sebaceous gland, and arrector phili muscle. By comparison with the other fol licles this demonstrates the lack of uniformity in response of hair follicles equally exposed to the same carcinogen within the same area of skin of the same individual animal. In other words, some follicles remained microscopically normal, while others were morphologically and physiologically disturbed ; instead of producing hairs, they formed epidermoid cysts. The fate of such epidermoid cysts has been followed in later stages of carcinogenesis and will be described and discussed in a separate paper. c) Cellular changes in hair follicles.â Various abnormalities of epidermal cells, described pre viously by different authors, were observed in the hyperplastic epidermis, as well as in the altered hair follicles. Figure 8 represents a 7-day specimen. It shows degenerated epidermal cells with pyknotic nuclei and cloudy cytoplasm. The same type of de generation may occur in any portion of a hair fol licle. It may extend from the epidermis into the upper part of the follicle, as shown in Figure 9. It may also appear in the middle third of the follicle, as illustrated in Figure 10 at a lower magnification. In some instances, entire follicles were involved, resulting in complete destruction and permanent disappearance of many follicles (15, 18). When degeneration involved only a portion of a follicle, the continuity between epidermal and follicular cells was broken, leaving isolated masses of fol licular cells lying in the dermis. Some of these masses become transformed into epidermoid cysts (Fig. 7). Cells with lobed or paired nuclei were most fre quently seen 10 days after painting, in the spinous or in the granular layers of hyperplastic epidermis (see Figs. 5 and 11). In the altered follicles, binucleated cells were found occupying the central layer which corresponds to the spjnous or granular layer of the epidermis (Fig. 12). In one specimen a giant binucleated cell was observed in a severely altered follicle (Fig. 13). Cells with lobed nuclei, not mentioned by other workers, were not found in the altered follicles but were present in the hyperplastic epidermis. Their nuclei were mostly bilobed, the two halves being separated by a narrow indentation on one side. A bridge of nucleoplasm was usually observed con necting the two halves of the nucleus. Occasional ly, one lobe was much larger than the other (Figs. Hand 12). Giant cells varied in shape and size. They were about 2-6 times larger than ordinary cells. One is seen with a large oval nucleus at the right side of Figure 11, beneath a binucleated cell. Its elon gated cytoplasm occupies the whole basal cell layer and extends into the upper portion of an altered follicle. Similar giant cells, either isolated or aggregated in groups, were often found located in the central portions of the altered follicles. Their cytoplasm usually stained deeply with hematoxylin and eosin. They seemed to have undergone some degeneration (Fig. 13), but ac tively dividing giant cells were also detected (Fig. 14). Solitary cells with eccentric nuclei and with (keratinoid) acidophilic clumps of material in their cytoplasms were found both in the spinous layer of the hyperplastic epidermis (Fig. 15) and the corresponding layer of altered hair follicles (Fig. 14). They seemed to constitute the nuclei, or central parts, of epidermal pearls and of kera tinized cysts of follicular origin. DISCUSSION In the first 15 days after a single application of carcinogen important changes are to be expected in hair follicles if their response approaches in promptness that of epidermis as seems likely. Among early chemical changes in epidermis the following may be cited as occurring within 10 days after a single application: Decreases in lipid-protein nitrogen ratio from 5.25 to 2.53 (25), in pg calcium/100 mg epidermis from 43.5 to 22.2 (23), and in fig iron/100 mg epidermis from 6.4 to 3.2 (5). However, technics have not yet been devised by which hair follicles responding to the carcinogen can be collected en masse for singular chemical determinations. Since the main purpose of this study is to evalu ate the early microscopic modifications in hair follicles as possibly setting the stage for the malignant transformation that takes place in the later stages of their response to methylcholanthrene, it is important to compare them to those in epidermis. Initial rhythm.â In mouse epidermis Cooper and Franklin (9) observed a rhythmic change in the frequency of mitoses. By night the frequency

5 344 Cancer Research was about twice as great as by day. Whether a similar rhythm exists in the hair follicles is not known but should be discovered. In the epidermis the cells shift to the surface in an orderly way and are lost by desquamation. In the epithelium of the hair follicles there are also cyclic changes of longer duration involving also death and loss of some cells. The follicles act as individuals, neighboring ones not always being in the same phase of the cycle. Because of this conspicuous regional dif ference in phase and in properties of follicles, the response of the follicles to the carcinogen is likely to show more regional differences than that of epidermis. The epidermis is more uniform in mitotic rhythm, although the possibility of dif ferences must be entertained, perhaps conditioned by changes in sebaceous gland secretion, me chanical trauma, and other factors. That all the sebaceous glands are not simultaneously and equally modified by the carcinogen has been demonstrated (17). Exposure to carcinogen.â The initial action of the carcinogen on deep-lying, keratin-protected, living epidermal cells may be even less than that on the cells of hair follicles because the latter are situated in pits in which it may lodge. The carcino gen accumulates in large quantities in the se baceous glandular appendages of the follicles in 3 minutes' time (22) before any of it can be detected in the deeper layer of epidermis made up of living and reactive cells. Lipase activity of the sebaceous glands begins to fail 1 day after a single application and was not detected by Kung (17) after 3 days. Destruction of cells.â This is a conspicuous feature of the epidermis and hair follicles of the center of the painted area 3 days after a single application as reported by Cramer and Stowell (15). The fact that Cooper and Relier (10) and Relier and Cooper (21) did not observe a similar destructive action of the carcinogen on epidermis of the backs of the ears is, however, to be noted. a) The degeneration and destruction of hair follicles herein reported recalls the early destruc tion of epidermis as described by Cramer and Stowell (15), except that it was restricted to cer tain follicles and even to limited parts of the fol licles, as shown in Figure 10, in which it is depicted at the middle portion of a follicle. This leaves the active bulb of the follicle isolated in the dermis where it is transformed into cystic formations (Fig. 7), whereas the epidermal destruction in volved quite uniformly the large area of the painted region. 6) Some follicles became enlarged and hyperplastic, calling to mind the well known thickening and hyperplasia of epidermis. Since the making of accurate mitotic counts in whole mounts of many follicles was not feasible, curves showing increases in frequency of mitosis, like those prepared by Cooper and Relier (10) for epidermis, could not be prepared. Probably, however, the carcinogen acted as a mitotic stimulant in some follicles as it does in epidermis. c) Modifications suggestive of unusual or re tarded cellular division were noted in the follicles, especially in cells with binucleated cells with the two nuclei of equal or unequal size, as has also been reported in epidermis. However, chromosome ab normalities, such as those described by Biesele and Cowdry (3), were not seen, perhaps because ade quate search was not made for them by the squash technic. d) Cellular enlargement was noted in some fol licles as it has been reported likewise but more uniformly in epidermis by Page (19). In the fol licles also a few gigantic cells were observed again, as in the epidermis (14), but it is doubtful whether they were quite as large. An interesting observation is that, in the early stages of the response to methylcholanthrene, nearly all the microscopically altered follicles were in the telogen phase of their cycle and had appar ently lost their previously attached sebaceous glands, whereas those not so modified were in Fio. 1.â Normal skin, showing two generations of club hairs in a composite follicle at the telogen phase; the club hairs have risen to their final position limited to the dermis; the quiescent hair-germ is located below the club sheath; beneath that is the darkly stained basal saccule. Note that the skin is thin and the subcutaneous fat is scanty. X150. FIG. 2.â Normal skin, showing a hair follicle in the early anagen phase. Note the growing hair-germ and its new papilla. The subcutis is thicker. X150. Fio. 8.â Normal skin, showing hair follicles in the midanagen phase. Note the narrow papillary cavities and the size of the growing hair bulbs situated deeply in the much thickened subcutis. XI50. Fio. 4.â Normal skin, showing a hair follicle in the late catagen phase. The club hair has not ascended to its final position and the subcutis is still thick. X150. Fio. 5.â Fivedays after painting, showing early changes of the hair follicles. Note the conically shaped enlarged follicle with darkly stained basal saccule at its tip. At the center of the hyperplastic epidermis a binucleated cell is to be seen near the basal layer. X150. Fio. 6.â Seven days after painting, showing a group of degenerated cells in the center of one of the altered hair fol licles. X150. Fio. 7.â Fifteen days after painting, showing the trans formation of altered hair follicles into cystic formations; at the left a normal looking follicle with intact sebaceous gland. X150.

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7 FIG. 8.â Seven days after painting, showing degenerated epidermal cells with pyknotie nuclei and cloudy cytoplasm. X325. FIG. 9.â Another field of Figure 8 showing the same type of degeneration extending to the upper portion of a follicle. X325. FIG. 10.â Eightdays after painting, showing the degenera tion of follicular cells involving the middle third of a follicle in the late imagen phase. X150. FIG. 11.â Ten days after painting, showing hyperplastic epidermis; at the right, note a hinucleated cell and an elongated giant cell; at the left, two cells with bilobed nuclei. X325. FIG. 12.â Tendays after painting, showing a cell with un equally sized bilobed nucleus in the upper center of the epi dermis and a » nucleated cell in the altered follicle. X325. FIG. 13.â Thirteen days after painting, showing a giant cell and a giant (»nucleatedcell in an altered hair follicle. Note the basal saccule at its tip. X325. FIG. 14.â Ten days after painting, showing two altered folliclesâ onewith a dividing giant cell, the other with a giant cell containing an eccentric nucleus and a keratinoid cytoplasmic mass. Compare with Figures 5, 6, 7, 12, and 13 and note the similarities in their follicular phases. X325. FIG. 15.â Ten days after painting, showing hyperplastic epidermis; at the lower right, note the giant cell with eccentric nucleus and keratinoid cytoplasmic clump. Compare with that in the follicle altered (Fig. 14). X325.

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9 LIANG AND COWDRYâ Methylcholanthrene Changes in Hair Follicular Cells 345 other phases of their cycle and possessed sebaceous glands. It is not possible to say whether this great er vulnerability of the altered follicles was owing (a) to the fact that they were in a stage prepared to grow rapidly or (o) to some compromise which they suffered associated with the loss of sebaceous glands or in their secretion. Kung (17) has found that even before disappearance of sebaceous glands some of these glands lose their demonstra ble lipase activity as early as 1 day after a single application of methylcholanthrene. SUMMARY Forty-five 5-week-old female Swiss mice were subjected to a single interscapular painting of 0.6 per cent methylcholanthrene in benzene. Micro scopic changes of hair follicles in the painted area were followed to 15 days after painting. Normal skins from the same region of fifteen animals were used as controls. Cyclic changes were observed in the thickness of the subcutis, indicating substantial alterations in blood supply and fat and striking individual variations of the hair follicular cycles in the same region (interscapular) of the skin in animals of even the same strain, age, and sex. The response of hair follicles to the carcinogen is a highly localized phenomenon. Altered hair follicles, as well as normal follicles, were both present in the painted area. The changed follicles were first transformed into clublike solid clusters of cells connected to or isolated from the epidermis and then into cystic formations. There is a close association in the response of hair follicles and sebaceous glands. In no instance were these al tered follicles observed to have attached sebaceous glands. Hair follicles in the period of late catagen or telogen phase (waiting to grow) seem to be more susceptible to the carcinogen than follicles in other phases. Follicles in the mid-anagen phase (vigorously growing period) are more resistant. Cytological evidence indicates that the stage is set in both epidermis and follicles in the first 15 days after a single application in much the same way in both tissues. Degenerated, binucleated, and giant cells, cells with accentric nuclei and keratinated cytoplasmic clumps, and increases of cellular, nuclear, and nucleolar sizes were ob served. REFERENCES 1. ANDBEASEN, E. Cyclic Changes in the Skin of the Mouse. Acta Path, et Microbiol. Scandinav., 32:157-64, ANDREASEN,E., and ENGELBRETH-HOLM, J. On the Signi ficance of the Mouse Hair Cycle in Experimental Carcinogenesis. Acta Path, et Microbiol. Scandinav., 32:165-69, BIESELE,J. J., and COWDHY, E. V. Chromosomal Changes in Epidermal , Carcinogenesis. J. Nat. Cancer List., 4: 4. BISHOP,G. M. Regeneration after Experimental Removal of Skin in Man. Am. J. Anat., 76:153-81, CARRUTHERS, C., and SUNTZEFF,V. Influence of Limited Application of Methylcholanthrene upon Epidermal Iron and Ascorbic Acid. J. Nat. Cancer List., 3:217-20, CHASE, H. B., and MONTAGNA,W. Relation of Hair Proliferation to Damage Induced in the Mouse Proc. Soc. Exper. Biol. & Med., 76:35-37, Skin. 7. CHASE, H. B.; MONTAGNA,W.; and MALONE,J. D. Changes in the Skin in Relation to the Hair Cycle. Anat. Ree., 116:75-82, Growth 8. CHASE,H. B.; RADCH,H.; and SMITH,V. Critical Stages of Hair Development Physio. Zool., 24:1-8, and Pigmentation in the Mouse. 9. COOPER,Z. K., and FRANKLIN,H. Mitotic Rhythm in the Epidermis of the Mouse. Anat. Ree., 78:1-8, COOPER,Z. K., and RELLEH,H. C. Mitotic Rhythm in Methylcholanthrene Epidermal Carcinogenesis in Mice. J. Nat. Cancer List., 2:335-44, COWDRY,E. V. Epidermal 135:408-11, Carcinogenesis. J.A.M.A., 12.. Properties of Cancer Cells. Arch. Path., 30: , Epidermal Carcinogenesis. In J. P. Greenstein, and A. 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Mitotic Incidence in the First 48 Hours of Methylcholanthrene Epidermal Carcino genesis. Cancer Research, 4:236-39, SIMPSON,W. L., and CRAMER,W. Fluorescence Studies of Carcinogens in Skin. Cancer Research, 6:449-65, SUNTZEFF,V., and CARRUTHERS, C. The Effect of Methyl cholanthrene upon Epidermal Sodium and Calcium. Cancer Research, 3:431-33, SUNTZEFF,V.; CARHUTHEHS, C.; and COWDRY,E. V. The Role of Sebaceous Glands and Hair Follicles in Epidermal Carcinogenesis in Mice. Cancer Research, 7:439-43, WICKS, L. F., and SUNTZEFF,V. Reduction of Total Lipid-Protein Nitrogen Ratio of Mouse Epidermis by a Single Application of Methylcholanthrene. J. Nat. Cancer Inst., 3:221-26, WOLBACH,S. B. The Hair Cycle of the Mouse and Its Importance in the Study of Sequences of Experimental Carcinogenesis. Ann. N.Y. Acad. Sc., 63:517-36, 1951.

10 Changes of Hair Follicular Cells after a Single Painting of Methylcholanthrene in Mice Hsu-mu Liang and E. V. Cowdry Cancer Res 1954;14: Updated version Access the most recent version of this article at: alerts Sign up to receive free -alerts related to this article or journal. Reprints and Subscriptions Permissions To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, use this link Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.