Kinetics of Human Epidermal Cell Proliferation: Diurnal Variation*

Vol. 50, No. 6

THE JOURNAL 05' INVESTIGATIVE DERMATOLOGY

Copyright

Printed in U.S.A.

1968 by The Williams & Wilkins Co.

KINETICS OF HUMAN EPIDERMAL CELL PROLIFERATION: DIURNAL VARIATION* GUINTER KAHN, M.D.,t GERALD D. WEINSTEIN, M.D. AND PHILLIP FROST, M.D.

being over 50 years old. The patients had been hospitalized for physical rehabilitation because

In studies of animal epithelial tissues in Which mitoses Were counted at different times of day, the time of maximal and minimal activity has varied greatly depending upon the species and

of orthopedic problems. Only one patient (JF) had a systemic illness, mild diabetes, under eoatrol. The biopsy specimens were processed for autoradiog-

experimental conditions. Most investigators raphy with 6 micron histologic sections and ex-

have concluded that mitotie activity is greatest posed to photographic emulsion for 3—4 weeks. When an animal is at relative rest. These stud- Autoradiographs were analyzed by projecting their images magnified 175 times onto paper and ies are summarized in Table I.

tracing the perimeter of the epidermis. The length of the basal cell layer was measured and from this the number of basal cells in a section was deter-

In previous studies of diurnal variation of proliferative activity in human tissues, mitoses were counted. Autoradiographie methods,

mined as follows:

Which utilize tritiated thymidine as a marker for cells synthesizing DNA in preparation for mitosis, may also be employed to measure the proliferative activity of a cell population (1, 2). With this technic cellular proliferation can be analyzed more easily and with greater accuracy since there are more cells in DNA synthesis than in mitosis at a given moment and since labeled cells are easier to identify than mitoses. The present study was performed to determine whether diurnal variation of epidermal cell proliferation is present in human epidermis by tritiated thymidine labeling experiments. It would be important to be aware of such diurnal variations in view of the many

number of basal cells in a section

studies on cellular kinetics now being performed using tritiated thymidine teehnies.

= basal cell layer length width of average basal cell Areas of epidermal thickening associated with hair follicles and sweat gland ducts were not included in the measurements. Using smaller areas of epidermis under higher

magnification the width of an average basal cell was calculated as follows:

average basal cell width

=

One-tenth ml (5 microcuries) of thymidine-1H 1.9 C/mM) was injected intradermally into each of 4 areas of normal lumbar skin. The thymidioe-1H was injected at 0600, 1200, 1800, and 2400 hours into 7 men and 2 women. Injection sites

cell layer length number of basal cells

The labeling index, i.e. the percentage of basal cells labeled by thymidine-5H thirty minutes after injection, was thea determined: labeling index number of labeled cells X 100% total number of cells in the basal layer

MATERIAL5 AND METHODS

(sp act =

basal

The variability and reproducibility of the experimental technic were tested by performing duplicate or triplicate injections and biopsies at

were marked and 30 minutes later 4 mm punch the same time in five patients. biopsy specimens were taken. The average age of RE 5TJLTS the patients was 61, seven of the nine subjects

This work was supported by research grants

from the National Institutes of Health (CA10292) and the Hartford Foundation. Accepted for publication December 23, 1967.

The labeling index for each patient at different hours is presented in Table II. An average of 146 labeled cells were counted at each time in each patient. When the data from each

* From the Department of Dermatology, University of Miami School of Medicine, Miami, patient were plotted on a graph, no pattern of diurnal variation among the different subFlorida, 33136. t Present address: Division of Dermatology, jects was apparent. However, there was eviUniv. of Colorado Medical Center, Denver, dence for a slight increase in the rate of cell Colorado. 459

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TABLE I Diurnal variation of mitotic activity System

Tissue

Age

Kittens (6)

Newborn

Mice

Not stated Lymphatic

White mice (6)

28 Days

Lymphatic

Rat)o9f Ref

Time oi1maximum

Time oçminimum

2230—0230

1030

2.1:1

3

1100

0300

2.5:1

4

1200

2000—2400

3.0:1

15

2.0:1

16

Epidermis, lumbar area

White mice (48)

1—7 Days

Abdomen

2000

1200

Human (males) (13)

8 Days

Epidermis, prepuce

2100

1000

Human (males) (57)

6—11 Days

Epidermis, prepuce

2200

0500—1000

3.6:1

10

Human (males) (14)

Infant

Epidermis, prepuce

2100

1000

2.0:1

11

Epidermis, abdomen

0800

2000

3.5:1

17

3.2:1

6

Albino rats (males) 28 Days

9

(98)

Male mice

2—4 mo.

Epidermis, ear, back; 0600 epithelia of esoph- and 1400 agus, duodenum, epididymis

Epidermis, shoulder

Human (males) (12)

20 years

Hairless mice (48)

Not stated Epidermis, back

1000

and (two max- 2000 ima)

(two mmima)

3.2:1

2400—0300

1000—1300

6.0:1

18

2400

1400

4.7:1

5

Numbers in parentheses indicate number of subjects in each study.

proliferation at midnight when the data were

(3, 4) and has since been observed in many

analyzed in the following manner: The mean of the four labeling indices for each subject was

other animals. As seen in Table I there has been no consistency in the time when maximum and

calculated and the percent variation at each minimum mitotic activity occurs. Important different hour from the individual's mean was determinants of proliferative activity to be determined (Fig. 1). These data were examined statistically by Wilcoxon's Signed-Ranks Test, which revealed that at 2400 hours (midnight) there was a small but significant increase in la-

considered include age, sex, physical condition, environment, metabolic rate, intracellular car-

bohydrate levels, and the proximity of other animals (5—8). Physical activity of the animal

beling indices above the individual's means is reported to result in lower mitotic counts (p < 0.05). No significant differences were (6). The diurnal variation of physical activity found at the other hours. in most species must, therefore, also be conIn the experiments designed to test the re- sidered. Cooper and Broders et al. have shown liability of each value, it was found that the that newborn infants (whose activity and enaverage variance in each patient was less than vironment are presumably uniform over 24 hours) have a definite cireadian rhythm of epi15% (Tab. III). dermal cell mitoses (9—11). Scheving conDISCUsSION

firmed this finding in the shoulder skin of adult

Diurnal variation of epidermal mitotic ac- males (18). These studies therefore indicate tivity was originally noted in kittens and mice that there is at least a two fold increase in the

461

DIURNAL VARIATIONS

TABLE II Labeling index at different hours (% basal cells labeled) Tritiated thymidine injected intradermally at the times stated. Biopsies for autoradiographic proces-.

sing taken 30 minutes after each injection (see text). The labeling index at different times of day is shown. No consistent pattern of diurnal variation in labeling indices is apparent. Note: The data as presented are not corrected for the thickness of the histologic sections used for autoradiography. Multiplying all the labeling indices by 1.4, the correction factor under these conditions, will give the absolute values for the number of cells labeled (21, 22). 0600

(Midnight) 2400

Patient

S of labeled cells counted per specimen

2 3

4 5 6 7 8 9

% cells labeled

S of labeled cells counted

(Noon) 1200

% of cells labeled

126 123 129 121 136 88 274

3.8 2.7 3.8 2.8 3.6

62 185 170 110

1.8 3.1

3.5

100 182

144

3.6 5.8

2.3 4.0 2.3

4.8

230

97

% of cells labeled

5' of labeled cells counted

5.1 2.2 3.2 2.7 3.5 2.3 5.4 2.7 3.9

243 104 156 169 129 64 209 162 188

3.0

3.8

Average

3.6 3.8

S of labeled cells counted

1800

% of cells labeled

3.4 2.8 2.4 2.4 3.4 2.4 3.6 3.8 6.4

144 123 93 120 97 41 184 194 256

3.5

3.4

Std. Dev.

TABLE III

aa;

I

+50 0 +40 +30 E +20 0 IS Li a

'C

a; C

a; C a; .0 0 -J

+ 10

0 —

0

I S

-I-

I

I

I

S

•

—20

0 —30 C 0 —40 a

The labeling index and number of labeled cells

counted per section in duplicate and triplicate

$

.

Labeling index at 1800 hours (% basal cells labeled)

I

•

-I•

specimens at the same time of day. Biopsy Si Patient

per

S

I• B C

I I

2400 (MIDNIGHT)

I

0600

t

(200

I

800

cells labeled

D E

56 55 149 119 89

1.9 1.4 5.3 4.7 2.4

Biposy S3

Sof

Sot

% of

section

A

a -50

Sot

labeled cells counted

Biopsy 52

labeled labeled % of cells % of cells cc counted labeled counted labeled per per section section

76 71 223 108 66

2.3 1.6 5.3 3.5 2.6

86 76 86

— —

2.5 1.6 4.1 — —

TIME of BIOPSY

FIG. 1. Each point represents the variation at that time from the mean of an individual's labeling indices. The bar is the average of the values at each time. This average is slightly greater at midnight than at other times.

Autoradiographic methods with tritiated thymidine have been utilized in animals to investigate cell proliferation kinetics. In one study of mouse esophagus, stomach, tongue, and abdom-

inal skin, a maximum number of cells in-

mitotic count at night (Table I). This increase corporated thymidine-'H at about 1730 hours. can be due to a greater rate of cell proliferation However, kidney tubule and jejunum showed no diurnal variation of labeling indices (12). A. or to a longer duration of the mitotic event.

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

recent study on rat corneol epithelium indi-

H. J. M., F. A. Davis Company, Philadelphia,

cated that diurnal variation of labeling indices

2. Weinstein, C. D. and Van Scott, E. J.: Turnover time of human normal and psoriatic epidermis by autoradiographic analysis. J. Invest. Derm., 45: 257, 1965. 3. Fortuyn von Leyden, C. E. D.: Observations on periodic nuclear division in the cat. Proc.

is present (13) ; in another study on rat lens cpithclium, no variation was found (14).

The number of cells synthesizing DNA can be considered a measure of cell proliferation assuming the duraton of the DNA synthesis period remains constant throughout a 24 hour period. If the DNA synthesis period itself has diurnal variations, it would affect the rate of cell proliferation as measured by a labeling index. Indeed, in recent reports of studies performed on mouse and salamander epidermis it is suggested that the length of the DNA syn-

thesis period may vary over a 24 hour period (19, 20). However, no information is available yet on such a variation of DNA synthesis in human tissues. A similar situation also obtains in studies utilizing mitotic counts without information concerning mitotic duration. Even-

sen has, in fact, presented evidence that diurnal variations of mitotic counts arc mainly due to variation of mitotic duration and not mitotic rate (5).

This is the first reported study of diurnal

1963.

Akad. Wet. Amsterdam, 19:

38, 1917.

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6. Bullough, W. S.: Mitotic activity in the adult

male mouse. Proc. of Roy. Soc. London, Bi35: 212, 1948.

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totic activity. Biol. Rev., 27: 133, 1952. 8. Holberg, F.: Temporal coordination of physiological function. Cold Spring Harbor Symp. Quant. Biol., 25: 289, 1960.

9. Cooper, Z. K. and Schiff, A.: Mitotic rhythm in human epidermis. Proc. Soc. Exp. Biol. and Med., 39: 323, 1938.

10. Cooper, Z. K.: Mitotic rhythm in human epidermis. J. Invest. Derm., 2: 289, 1939. 11. Broclers, A. C.: Rhythmicity of mitosis in the epidermis of human beings. Proc. Staff. Meet. Mayo Clinic, 14: 423, 1939.

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Human cpidermal cell proliferation was stud-

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