0022_202x ;so/ 7401-0043$02.00/ 0 . TH JOURNAL OF INVESTIG ATIVE DERMATOLOG Y, 74:43-46, 1980 Vol. 74, No. I co:yright © ]980 by The Williams & Wilkins Co. Pri11t ed in U.S.A. Cell Proliferation Kinetics in the Human Hair Root

GERALD D. WEINSTEIN, M.D., AND KATHRYNE MASON MOONEY, M.D. Department of Dermatology, University of Miami School of Medicine, Miami, Florida, U.S.A.

The kinetics of cell proliferation in the matrix cells of with a 4- mm punch instrument from separ ate injection sites at the human scalp hair were determined in vivo. Autoradi­ following postinjection time intervals: 1 hr, 3 hr, 4.5 hr, 6 hr, and every ographic analysis was employed on biopsy specimens 3 hr subsequently, terminating between 21 and 27 hr postinjection. In 2 patients specimens were obtained only for the I hr analysis. Biopsy t aken at specific time .i~terv~ls after ~ntradermal injec­ tion of tritiated thymidme ("H-TdR) mto the scalps of specimens were prepared for autoradiographic analysis and histological examination as previously described [6,7]. Autoradiographic exposure v olunteer subjects. The durations of the S and Gz phases was for 1-3 weeks with most data collected from 2 week exposures. (Ts and Tg2) were 11.1 and 3.6 hr respectively, obtained Kodak NTB-2 emulsion was used. Upon staining of the developed from the composite percent labeled mitosis curve. The autoradiographic slides with hematoxylin and eosin, the matrices of the labeling index of the hair matrix cells was 28.6% ± 2.2%. hair roots were examined microscopically. Cells labeled above back­ The durations of mitosis and of the total cell cycle (Tm ground from tritium emissions were those which had incorporated the and Tgc) were calculated to be 0.8 hr and 38.8 hr respec­ injected "H-TdR. t ively. The growth fraction of hair root germinative cells The principles involved in the determination of the proliferation was estimated as 75-100% on the basis of "continuous" kinetics of human epidermal cells by autoradiographic analysis have aH-Td.R exposure over the duration of the cell cycle. been described previously [7] and are used here for hair matrix prolif­ eration kinetics. It is now well-established that the radioactive DNA It is noted that the kinetics of human hair root cells precursor ~ H - TdR is incorporated into DNA when the DNA molecule are quite similar to the kinetics of psoriatic epidermal is being synthesized. Thus it will label only those cells which are in the cells suggesting that both tissues are approaching the DNA synthesizing part of the cell cycle (S phase) at the time t hey are ma:xiroum proliferative rate of keratinogenic epithelial exposed to the isotope [8,9]. Injections of ~ H-TdR are considered to act cells. as pulse exposures of isotopic labeling. By the serial biopsy method the "H-TdR labeled S phase cells can be followed microscopically over the 27-hr period as they progress through the cell cycle, particularly the M Previous studies have suggested that the germinative cell phase, where they are seen as labeled mitoses. The goal is to determine population of the hair r~ot matrix constitutes m~e of the fast~r the duration of the cell cycle and its component phases: S (DNA proliferating cell pools m the human body. Evidence for this synthesis) , G" (premitotic rest period), and G, (interphase). was based on the high numbers of mitotic cells relative to total The duration of the S phase (Ts) was calculated by finding the percentage of mitotic cells which were labeled in the hair root matrices cells (mitotic index) seen in hair roots [1-3]. Clinical evidence at each time interval from 1 to 27 hr postinjection. An average of 101 of rapid proliferation has been interpreted indirectly from the mitoses from one or more labeled hair roots were counted for each time alopecia found as a result of the cytoxic effects of cancer interval for each patient. All hairs studied were anagen hairs as seen by chemotherapeutic drugs on the high proportion of susceptible the hair root morphology. The data was plotted as a percent labeled germinative cells. mitoses (PLM) curve. The labeled mi toses represent cells that were in Measurement of cell turnover kinetics by the use of autora­ various parts of the S phase at the time of injection having entered the diographic analysis has been carried out on many types of M phase prior to t h ~ time of the biopsy. The earliest labeled mitoses human and animal tissue but there is minimal data available seen were theoretically cells which were very near the termination of 1 on their root kinetics. Mouse and sheep hair (wool) matrix cells DNA synthesis at the time of ' H -TdR injection. In further samples have been shown to have proliferative cell cycles of 12.5 and 21 greater percentages of mitoses were labeled indicating a wave of S phase labeled cells moving through M phase. In the later specimens hr respectively [ 4,5]. In this study autoradiographic analysis most of the mitoses were again labeled, these being cells that were in was employed following local intradermal injections of tritiated the G, phase at the time of injection. thymidine (lH-TdR) into human scalp to determine the turn­ The duration of the S phase is defined here as the time interval over time of the hair root germinative population, as well as between the 37% points on the ascending and descending limbs of the the kinetics of the various phases of the cell cycle. PLM curve [10] and is the same method used in our previous studies for comparative procedures. The median G2 phase duration is defined MATERIALS AND METHODS as the number of hours from the time of injection until the labeled mitosis curve ascends to the 37% 'point. An alternate approach for Nine male patients over 40 yr of age were included in the study after determining the S and G1 phases .involves computer analysis of the obtaining informed consent. Three were normal volunteers and 6 had PLM curve. With computer analyses the S phase measurements are skin conditions not affecting the scalp. The tissue samples were taken approximately equivalent to calcul ations using the 50% points on the from nonbalding scalp, usually the occipital area. The scalp in each of PLM curve. The advantages and disadvantages of both approaches are 7 patients was injected intradermally in 9-11 separate locations, with beyond the scope of this paper and are discussed elsewhere [11,12]. In 0.1 ml (5/lC) o f ~ H - TdR (sp act= 1.9 C/mM). Serial biopsies were taken addition to data analysis of the PLM curve in this paper as described above 2 independent computerized analyses will be presented for co m­ parison. Manuscript received April 23, 1979; accepted for publication August The population of cells used in the calculations was the germinative 1, 1979. area of the hair root matrix. This was considered to be that portion of This work was supported in part by Public Health Service Grant the matrix below the critical line of Auber (i.e., the widest point of the AM-14887 from the National Institutes of Healt h and the Dermatology dermal papilla, approximately at its midpoint ) [3) (Figure 1). Van Scott, Foundation of Miami. Ekel and Auerbach found that approximately 2/ 3 of mitoses in the hair Dr. Mooney's current address is the Department of Dermatology, root occurred below the line of Auber and the remaining 1/ 3 occurred Stanford University, Palo Alto, California. between the line of Auber and the top of the dermal papilla [3]. Reprint requests to: Gerald D. Weinstein, M.D., Department or" Determinations of Ns, Nm, and Ngc were obtained by direct counting Dermatology, Med-Sci I, Hm. C-270, California College of Medicine, of matrix cells below the line of Auber in a single plane of fo cus under University of California, Irvine, Irvine, CA 92717. the light microscope at high power. Ns was found by counting the Abbreviations: number of labeled cells in uniformly labeled hair roots 1 hr after GF: growth fraction injection of "H -TdR. No correction factor [7] was used for the compu­ PLM: percent labeled mitoses tation of the labeling indices.

43 44 WEINSTE IN AND MOONEY Vol. 74, No. 1

repeat injections, adjoining scalp sites were injected with saline l.D. a~ the same time in tervals (0, 12, 24, hr) and "H-TdR at 36 hr, 1 hr prior to biopsy.

RESULTS Figure l shows a n anagen h air root 1 hr postinjection wit? 1 ' H-thymidine. This is typical of the microscopic sections of hall' roots used in t his study to d etermine the labeling index of the m atrix cells. The mean labeling index

Ns x 100 Ngc for 7 patients 1 hr postinjection was 28.6% (± 2.2% S.D.), with a ra n ge of 25.5% to 31.6% (table 1) . The duration of DNA synthesis, Ts, in the germinative cells of t he hair root was determined from the PLM curve which represented a composite (average) of the data from se~e n patients (Fig 2). The value obtained for Ts was 11.1 hr usmg the 37% level of the PLM curve. Ts obtained from the individual curves of each patient ranged from 9.8 - 12.2 hr with an almost ide ntical value of 11.0 ± 0.9 (mean ± SD) indicating a close similarity between the composite a nd individual curves (Ta ble 1 F IG 1. Autoradiograph of human hair matrix 1 hr after intradermal II). Tg ~ was 3.6 hr from the composite curve, with a range of2.9 injection of tri tiated thymidine. Numerous labeled cells are visible in to 4.7 hr from the separate curves. The mean mitotic index lower half of the hair matrix below the critical line of Auber which is drawn in (autoradiographic exposure time-2 weeks) (reduced from X Nm 274). -- X 100 Ngc

Nm was determined by counting the number of mitotic cells in the in t h e 1 hr specimen for 7 patients was 2.1% ± 1.0% SD with a same populations as S phase cells. There was no difficulty in distin­ range of 0.7 to 3.5 (Table 1) . Having obtained the labeling and guishing ce lls which were in metaphase, anaphase, or telophase. In mitotic indices, Tm a nd Tgc were calculated using t h e equiva­ order to eli minate the ambigui ty of designating cells in early prophase, lencies given in t h e methods section. Tm was 0.8 hr a nd T gc a ce ll had to meet the fo llowing criteria to be counted as a prophase 38.8 hr. By subtraction Tg, was 23.3 hr. Results of t h e computer ce ll : (l) Segmentation or disappearance of nuclear membrane, (2) a nalyses a re presented in Table II compared to data from the Sufficient condensation of chromatin so that modrately thick strands composite and individual PLM curves measured at the 37% coul d be distinguished (i. e. not t hin threads) [21,22). Thus early pro­ level. phase cells were not counted and the estimate of Nm (and therefore Tm) may be short by this factor. Ngc was the total number of cells in The growth fraction studies were carried out on 2 patients. the population from which Ns and Nm were counted. Calculation of the duration of the remaining phases of the cell cycle TABlA;; I. Labeling and mitotic indices in human hair matrix cells is based on the equation: Ns Labeling Mitotic Ns Ngc Patient Labeled Nm Ngc Cells Index (%) Index(%) Ts == T gc N == number of cells in each phase A 143 13 452 31.6 3.0 T == time in hours B 44 4 165 26.7 2.4 gc = germinative cycle c 117 3 418 28.0 0.7 Tgc = T s + T g2 + Tm + Tg1 D 87 3 34 1 25.5 0.9 F 115 10 391 29.4 2.6 T hese equivalencies merely mean that all cells in the population (i.e., H 139 8 447 31.1 1.8 Ngc) are assumed to act homogeneously and proceed through the cell I 118 15 423 27.9 3.5 cycl e at an equal rate. The hair matrix proliferative pool is also Average 28.6 ± 2.2 2.1 ± 1.0 co nsidered here to be a steady state population. Therefore, the number of cells in any one phase, eg. G2, at any time is dependent only on the

relative amount of time in the cell cycle occupied by the phase. 100 The above calculations assume a growth fraction of 100% with no cells in a "dormant" Go phase. Preliminary studies were initiated to 90 determine an approximate size of the growth fraction (GF) in human hair matrix. A major technic for GF analysis is continuous exposure of •o

tritiated thymidine to the proliferating tissue for at least the duration 70 of a cell cycl e (T c) [13]. In animal studies and some human studies (p rimarily oncologic) , ''H-TdR is continuously perfused intravenously. . hO Since this was not feasibl e for these studies, "H-TdR was injected 0 so intradermally at 12 hr in tervals for 36 hr (0 , 12, 24, and 36 hr) into the X: same site to be followed by a biopsy 1 hr after the last injection. T he ~. '•0 times selected were based on the experimental results of Ts ;;;;, 11 hr. "'. -' Almost all cells activ ely proliferating would at some time during the 36 .., )0 hr theoretically enter the S phase (since Tgc was calculated to be 39 hr) and therefore become labeled by exposure to "H-TdR. By this method the percentage of matrix cells actively cycling co uld be esti­ mated as the GF. Cells in the quiescent Go phase are not cycling and therefore would not label with intermittent "continuous" "H-TdR ex­ 12 l ~ 16 21 posure. The experiment was done using 2 male subjects and injecting llours post 3tl -thymidine i njection "H-TdR in to the same site on the occipital scalp. As a control for the F IG 2. Percent labeled mitosis curve of composite values from 7 possible stimulation or recruitment of cells into the S phase by the subjects studied. Vertical lines represent 1 SD of average values. Jan. 1980 CELL PROLIFERATION IN HUMAN HAIR ROOT 45 After 4 injections o f "H-TdR into the same s ites over 36 hr the labeling indices were 74.6% and 93.1%. (Fig 3). The control labeling indices using saline in place of the first 3 aH-TdR injections were 24.9% and. 33.6% respectively. These data s ug­ gest a large G~ of approxu_nately 75-100% human hair matrix cells. Over th1s 36-hr penod there does not seem to be a recruit m ent of cells into the DNA synthesis period as a result of the trauma of repeated injections. DISCUSSION Sever al workers have studied the labeling of the hair matrix with '1H -TdR, using either human (14] or animal hair (4,5,15- 1 7]. Shanud and Marks. found ~ labelinf? index of 35.8% in the normal human scalp hau matnx [14] shghtly higher than our average labeling index of 28.6%. The disparity in these results is likely due to differences in technique. Sharad and Marks' experiment was done in vitro, where excised skin samples were in cubated in H'1-thymidine for 2 1/~ hr before obtaining the labeling index. In the present study the labeling index was obtained from biopsies taken 1 hr postintradermal injection of H a-th y midine (in vivo). Since labeled mitoses, which will pro­ duce 2 la beled daughter cells, are already present between 1 and 3 hr postinjection (Fig 2) the 2 1/~ hr incubation duration of Sharad and Marks may account for the higher labeling index. . FIG 3. Aut?r~diograph of human h air r oot after 4 injections of The mitotic index of human scalp hair matrix cells below the tn tmted t hynudme at 12- hr mtervals. This specimen shows t hat almost critical line of Auber was determined b y Van Scott, Eke!, and all the .proliferative cells in the lower half of the matrix are labeled suggestmg. a g rowth fraction approximating 100%. (Autoradiographic Auerbach to be a bout 71 mitoses per 1,000 cells or 7.1 % (3]. exposure time-3 weeks) (reduced from x 274) . T his is more t han triple the figure we obtained of 2.1%. As discussed above, early prophase cells were excluded from the counts of mitoses because of the unreliability of accurately epithel.ium ( 2~ ]. .Interestingly t he duration of the cell cycle in identifying them and this must account partially for our lower the h a~r m at~ 1x .Is ac.tually very similar to the Tgc of 37.5 hr found 1~ p~onatic ep1dermis [7]. One can figure. We obtained our m~t?tic ~nd ex by counting the number speculate that th ese of mitoses and total cells v1s1ble m an approximate single plane 2 germmatiV e cell populations approach "maximum turnover o f focus under high power. Van Scott, Eke!, and Auerbach rate," corresponding to the minimum amounts of time spent in calculated the mitotic index by a different method, in which each P.h ase o~ the eel~ cycle _which an epithelial celJ can undergo and still retam k eratmogemc potential. t h ey counted the number of mitoses visible in entire 6 p. thick If sections, and then converted the figure to the number of mitoses th e hair root does indeed approach the maximum speed of human . cell p~o li~e ration per 1,000 matrix cells by a~suming that one matrix cell occupies it may be of potential value as a tool a volume of about 1,000 l . Aside from the prophase criteria it for the mvestJgat10n of biochemical mediators both natural and is not readily apparent why the 2 methods still give such sy~th et i c, w.hich act to alter cellular proliferation, especially of e~ Jd erm aJ t1ssues. The ease of obtaining hair root sampl different mitotic indices. es by It can b e seen that the hair matrix has the most rapid b1opsy or eve.n b y plucking, coupled with the fact that they represent a h1 ghly proliferative tissue which is normal rather proliferative cell cy cl ~ of the normal human epithelial tissues which have been studied. For example, the following cell cycles than abnormal, makes the hair root attractive for use in such have been d etermined in human tissues: >457 hr in epidermis investigations. [23], 48 hr in colonic epithelium [18,19], and 63 hr in vaginal REFERENCES l. Bullough. WS, Laurence EB: The Mitotic Activity of the Follicle, TABLE II. Duration of S and G 2 phase in human hair matrix cells ~h~ Bwlogy of Ha.t.r Growth. Edited by W Montagna, RA.A from individual and composite PLM curves and from computer b lhs, New York, Academic Press, 1958, pp 171-187 analys 2. Van S~ott EJ, Ek('!l TM: Geometric relationships between the es matnx of the hau· bulb and its dermal papilla in normal and Patient T. (hr) Tg2 (hr) alopecic scalp. J Invest Dermatol 31 :28 1, 1958 3. Va~ Scott EJ, Eke! TM, Auerbach R: Determinants of rate and A 10.6 3.2 kmet1cs of cell division in scalp hair. J Invest Dermatol 41·269- B 10.2 3.6 273, 1963 . c 11.1 2.9 4. Griem ML: Use of multiple biopsies fo r the study of the cell cycle E 12.2 4.2 of t he mouse hair follicle. Nature 2 10:2 13-21.4, 1966 G 10.9 4.5 5. Downes AM, Chapman .RE, TillAR, Wilson PA: Proliferation cycle 12.0 3.4 a ~1d fate of cell nuclei m wool follicles. Nature 212:477-478, 1966 H 6. Wernstem GD, Van Scott EJ : Autoradiographic analysis of 9.8 4.7 turnover I times of normal and p oriatic e pidermis. J. Invest Dermatol 45· Average from individual 11.0 ± .9 3.8 ± .7 257, 1965 . PLM curves 7. Weinstein GD, Frost P: Abnormal cell proliferation in psoriasis. J Invest Dermatol 50:254, 1968 Values from composite 37% level11.1 3.6 8. Baserga R: The r ~ l at io n s hip of the cell cycl e to tumor growth and PLM curve (Fig 2) at: 50% level 9.3 4.1 control of cell division. A review. Can cer Res 25:581, 1965 9. W1mler SE: Methods of studying cell proliferation with emphasis Computer analysis- on DNA labels_. Cell Proliferation. Edited by LF Lamerton, RJM G. Steel 8.9 4.9 Fry., F.A. Davis Company, Philadelphia, 1963, p 1 M. Mendelsohn 9.2 4.1 10. Qua ~t l e r H:. The analysis of cell population kinetics, Cell Prolifer­ atiOn. Edited by LF Lamerton W , RJM Fry. F.A. Davis Company, e are indebted to Dr. G. Gordon Steel of the Radiotherapy Re­ Philadelphia, 1963, p 18 search Institute of Cancer Research, Belmont, England, and Dr. Mor­ ll. Aheme WA, Camplejohn RS, Wright NA: An Introduction to Cell timer Mendelsohn of the Lawrence Livermore Laboratory, Livermore, Population Kinetics. Arnold Ltd., London, 1977, pp 31-35 E. California, for their help in the computer analysis of the PLM data. 12. Sleel G, Hanes S: The technique of l abeled mitoses: Analysis by 46 WEINSTEIN AND MOONEY Vol. 74, No. I

automatic curve-fitting. Cell Tissue Kiinetics 4:93-105, 1971 Regrowing Hair. Acta Dermatovener (Stockh.) 50:169, 1970 13. T erz J , Lawrence W, Cox B: Analysis of the cycling and noncycling 18. Lipkin M: Cell proliferation in the gastrointestinal tract of m an. cell population of human solid tumors. Cancer 40:1462-1470, 1977 Fed Proc 24: 10, 1965 14. Sharad P, Marks R: Hair Follicle Kinetics in Psoriasis. Br J 19. Lipkin M, Deschner E: Early proliferation changes in intestinal Dermatol 94:7, 1976 cells. Cancer Res 36:2665, 1976 15. Potten CS: Tritiated thymidine incorporation into hair follicle 20. Averette H, Weinstein G, Frost P: Auto radiographic analysis of cell matrix and epidermal basal cells after stimulation. J Invest proliferation kinetics in human genital tissues. 1. Normal cervix Dermatol 56:311, 1971 and vagina. J Obstet Gynec 108:8-17, 1970 16. Mathur M, Neo MG: Kinetics of proliferation and differentiation 21. Mazia D: How Cells Divide. Sci Am 205: 100, 1961 in the hair follicle a nd epidermis in neonatally undernourished 22. Ambrose E , Easty D: Stages of mitosis, Cell Biology. Thomas rats. Am J Pathol 82:9, 1976 Nelson & Sons Ltd., 1970, pp. 290-299 17. Allegra F, Giacometti L, Uno H, Adachi K: Studies of common 23. Weinstein G, Frost P: Methotrexate for psoriasis. Arch. Dermatol baldness in the stumptailed m acague. III. DNA Synthesis in 103:33-38, 1971

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