close

Вход

Забыли?

вход по аккаунту

?

Localization of maximum cell division in epidermis.

код для вставкиСкачать
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.
Документ
Категория
Без категории
Просмотров
1
Размер файла
443 Кб
Теги
division, epidermis, maximum, localization, cells
1/--страниц
Пожаловаться на содержимое документа