effect of vinblastine on the cell cycle and migration of ameloblasts of mouse incisors as shown by...
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Cell Tissue Kinef. (1985) 18,493-503.
Effect of vinblastine on the cell cycle and migration of ameloblasts of mouse incisors as shown by autoradiography
using 3H-thymidine
Maria Mercedes Fernandez Samperiz, Guilherme Blumen and Jose Merzel
Departamento de Morfologia, Faculdade de Odontologia de Piracicaba, Unicamp. Caixa Postal 52, 13400, Piracicaba, S.P.. Brazil
(Received 4 April 1983; Revision accepted 28 December 1984)
Abstract. The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with 3H-thymidine ([3H]TdR) and radioautography. A group of mice received 2 pglg of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1pCilg body weight of [3HlTdR, and sacrificed at time intervals from 0.75 hr to 15 days.
The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. The thymidine labelling index of the treated animals was 50% higher than the controls. The velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9- 18 pmlhr respectively) as compared with the control (3.21 cell positions/hr and 18.88 pm/hr respectively).
The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.
The effects of antimicrotubular drugs, such as colchicine, vincristine and vinblastine among others, on several cell functions, have been subject of many studies recently, some on continuous growing incisors of rodents where the cells form a renewing population and undergo a sequence of morphologic and physiologic changes, all arranged in an orderly way and in successive stages from the apical to incisal end of the tooth.
Besides a reversible arrest of mitosis in metaphase, vinblastine was shown to affect ameloblasts and odontoblasts in the stage of secretion, causing cell death and the production of abnormal dental tissues (Moe & Mikkelsen, 1977a, 1977b; Moe, 1977, 1979; Mikkelsen, 1978).
Correspondence: Dr Guilherme Blumen, Departamento de Morfologia, Faculdade de Odontologia de Piracicaba, UNICAMP, Caixa Postal 52, 13400, Piracicaba, SP., Brazil.
493
494 Maria M. F. Samperiz, G. Blumen and J . Merzel
The present work was carried out in order to study the possible effects of vinblastine on the proliferation and migration of the ameloblasts of the mouse lower incisor, by labelling the cells with 13H1TdR and radioautography.
M A T E R I A L S A N D M E T H O D S
Female mice, 5 weeks old, were divided in two groups, experimental and control, of twenty-three animals each. Animals of the experimental group received a single intraperitoneal injection of 2 pglg of body weight of vinblastine (Ely Lilly). Six hr after, these animals and those of the control group were injected, intraperitoneally, with a single dose of 1 pCi per g of body weight of 13HlTdR (specific activity 5 Ci/mmol, Amersham) and sacrificed, one animal of each group, after time intervals varying from 0.75 hr to 15 days.
Under ether anaesthesia, the mandibles were dissected, separated into hemi-mandibles, removing all the surrounding soft tissues and fixed in Bouin’s solution for 24 hr. After being decalcified with EDTA for 21 days, the pieces were dehydrated and embedded routinely in paraffin so as to get longitudinal sections of the lower incisor. Thick sections (6 pm), were transferred to gelatinated glass slides and dipped in Ilford K-5 liquid emulsion (Kopriwa & Leblond, 1962), exposed for 13 days and post-stained with haematoxylin and eosin.
The duration of the cell cycle of ameloblasts was determined by plotting the percentage of labelled metaphases, present in the proliferative zone of the odontogenic organ, against time from 0.75 to 24 hr after the injection of 13HlTdR (Quastler & Sherman, 1959). The mitotic index was calculated by counting the number of mitotic figures in the proliferative zone of all animals. Thymidine labelling index was determined by counting the number of labelled nuclei of ameloblasts 0.75 and 1 hr after the injection of f3HITdR.
The mitotic index was determined, using all animals, control and treated, by counting the number of ameloblasts and respective mitoses in each section.
Using the animals sacrificed at 1 and 96 hr after the injection of l3H1TdR, in a column of ameloblasts from the point of maximum convexity of the apical loop of the enamel organ, up to the point where the postsecretory transition starts, the number of cells, the number of labelled cells and the number of silver grains were counted.
The ameloblast migration velocity and some cell cycle parameters can be estimated by analysing the grain count dilution, employing the method described by Zajicek (1974). Within one generation the average grain count in the progenitor compartment drops to half of the initial value, and the time it takes for halving equals the mean generation time. In a system in a steady state the turnover rate constant k is:
where g stands for the progenitor grain count at time t (96 hr) and g, the progenitor grain count at the initial time (1 hr). The mean generation time (G) is:
G = 0.693Ik
The mean synthetic time ( S ) can be calculated by the equation:
where N , is the number of cells labelled at 1 hr after the injection of I3H]TdR and N p is the number of cells counted in the progenitor compartment.
Vinblastine and ameloblasts 495
The cell production rate (P), assuming that all progenitor cells divide and each dividing cell produces one new cell, is given by the equation:
P = NP - cells/hr. G
The time it takes to replace the mature ameloblasts (N , ) in a 500 pm segment (number of cells counted in the segment 501 to 1000 pm in animals sacrificed 96 hr after [)HITdR, or transit time T, is:
and the velocity ( V ) for such displacement, equals
500 I
V = - ymlhr.
RESULTS
(i) Cell cycle of ameloblasts The region of the odontogenic organ where ameloblasts divide was circumscribed to the apical end of the tooth, between the point of the greatest convexity of the apical loop and the point where odontoblasts start the secretion of pre-dentine. This region was named by Warshawsky & Smith (1974) as the region of ameloblasts facing pulp, part of the pre-secretory zone. Although mitotic figures could be seen in a more incisally-placed location (in the beginning of the region of ameloblasts facing dentine), uptake of l3H1TdR by ameloblasts was observed only in cells facing pulp.
The number of metaphase figures and the percentage of labelled mitoses are given in Table 1. As can be seen, up to 8 hr after the administration of ['HITdR, the number of metaphases among ameloblasts, either labelled or not, is considerably higher in vinblastine treated animals. In these animals, but not in the control ones, a fair number of mitotic figures located between the layer of ameloblasts and the stratum intermedium (Figs 2 and 3) were present and counted as ameloblasts.
Besides the arrest of mitoses in metaphase which, with the dose used, lasted at least 14 hr after vinblastine was injected, no signs of cell damage were seen in the proliferative compartment.
By plotting the percentage of labelled metaphases against time, one obtains the curves of Fig. 1 from which, according to Quastler & Sherman (1959), the following times can be deducted: G = generation time; S = duration of S phase of the cell cycle; G, + M +G, = duration of G , plus mitosis plus G,; and G, + 1/2M = duration of G, plus half the time of mitosis. G, + 1/2M was calculated by the difference between G and the sum of S + G, + 1/2M.
Using the same animals and by counting the number of ameloblasts and respective mitoses, the mitotic indices were calculated at 0.046 and 0.272 for control and treated animals respectively (Table 2).
According to Quastler (1963) the mitotic time (M) is given by mitotic index X generation time. Having the mitotic time, the duration of G, and G, was calculated from the data of Fig. 1 and the duration of each phase of the cell cycle is presented in Table 3.
In animals sacrificed at 0.75 and 1 hr after the injection of thymidine the labelling index
496 Table 1. Number and percentage of labelled metaphases of ameloblasts in the odontogenic organ of the lower
incisors of control and vinblastine-treated mice. at several time intervals after a single injection of I’HlTdR
Maria M . F. Samperiz, G. Blumen and J. Merzel
No. of metaphases No. of labelled metaphases Percentage of labelled metaphases Time (hr) Control Treated Control Treated Control Treated
0.75 1 .oo 1.50 2.00* 4.00 5.00 7.00 8.00
10.00 12.00 16.00 18.00 20.00 24.00
7 8
10 I 1
5 6 3 8
1 1 9
1 1 15 9 9
85 133 97 58
112 I29 27
1 1 2 18 4 3 8
1 1 10
0 1 6
10 5 6 3 6 3 1 2 7 6 2
4 13 12 6
56 109 26 74
5 1 0 3 8 5
0.0 12.5 60.0 90.9
100.0 100.0 100.0 75.0 27.3 1 1 . 1 18.2 46.7 66.7 22.2
4.7 9.8
12.4 10.3 50.0 84.5 96.3 66.1 27.8 25.0 0.0
31.5 72.7 50.0
~ ~~ ~~
Each value is the mean of counts of three different sections. * Counts only in two sections.
--- Control
- Vinblastme
T #me (ti.)
Fig. 1. Percentages of labelled metaphases of ameloblasts in the odontogenic organ of the lower incisors of control and vinhlastinc-treated mice, at several time intervals after a single injection of I3HITdR. From the curves, at the 50% level. the following times, in hours, can be determined (Quastler & Sherman, 1959):
G S G, + G,+ M G,A 1/2M G, + li2M - -
Control 17.0 7.6 9.4 1.4 8.0 Vinhlastine- 14.8 4.8 10.0 4.0 6.0
treated
Vinblastine and ameloblasts 497
Fig. 2. Longitudinal section of the odontogenic organ of the lower incisor of a mouse treated with vinblastine and killed I hr after receiving ['HITdR, showing many metaphases, specially those located between the stratum intermedium and the layer of ameloblasts (arrow). A: ameloblasts; SI: stratum intermedium; SR: stellate reticulum; ODE: outer dental epithelium; P: pulp; 0: odontoblasts. H and E x 140.
Fig. 3. Higher magnification of the area delimited in Fig. 2. Mitotic figure (arrow) was counted as an ameloblast. A: ameloblast: SR: stratum intermedium; 0: odontoblasts; P: pulp H and E x 350.
was determined as being 0 . 3 3 for the control animals and 0.49 for the vinblastine-treated ones.
(ii) Velocity of the migration of ameloblasts Since the ameloblasts are arranged in a continuous layer of cells, organized in most of its length as a simple columnar epithelium, the distance of a cell with a labelled nucleus from the
498
Table 2. Mitotic index of ameloblasts in the odontogenic organ of the lower incisor of control and vinblastine treated mice
Maria M . F. Samperiz, G. BIumen and J . Merzel
No. of ameloblasts No. of mitoses Mitotic index
Time* Control Treated Control Treated Control Treated
6.75 387 238 14 85 0.036 0.357 7.00 220 239 12 134 0.054 0,560 7.50 329 223 14 97 0.042 0.435 8.00 349 154 21 58 0.060 0.376
10.00 384 202 15 115 0.039 0.5 70 11.00 324 255 15 129 0.046 0.505 13.00 357 207 12 29 0.033 0.140 14.00 397 22 I 10 115 0.025 0.520 16.00 382 230 14 24 0.036 0.104 18.00 337 264 15 8 0.044 0.030 22.00 336 226 20 5 0.059 0.022 24.00 333 148 13 8 0.039 0.054 26.00 326 202 16 15 0.049 0.074 30.00 312 279 16 17 0.05 1 0.060
Mean 0.046 0.272
* Hours after the injection of vinblastine, in the treated animals.
Table 3. Generation time and duration of each phase of the cell cycle of ameloblasts in hr. as calculated from the values given in Fig. 1 and the mitotic time
Groups G G, S G, M
Control 17.0 7.6 7.6 1 .o 0.75 Vinblastine 14.8 4.0 4.8 2.0 4 .OO
Table 4. Cell position of the most incisally-labelled nucleus of ameloblasts in the lower incisor of control and vinblastine treated mice at several time intervals after a single injection
of ['HlTdR
Cell position Time (hr) Control Vinblastine-treated
1 24 48 72 96
120 144 168 192 240 3 60
78 146 193 269 333 382 44 1
523 943
1200
-
58 111 159 156 159 302 418 6 20 6 20 627 862
Vinblastine and ameloblasts 499
I125
'003
A Control
0 Vinblostine
Time ( h r )
Fig. 4. Regression lines of the cell position of labelled ameloblasts in the lower incisor of control and vinblastine-treated mice, as a function of time after a single injection of ['HITdR.
point of maximum convexity of the apical loop can be determined by the number of cell positions along the longitudinal axis. The cell positions, at each time interval after administration of [3HlTdR, of the most advanced ameloblast with labelled nucleus in both control and vinblastine-treated animals, are given in Table 4.
A simple linear regression, according to the model Yi = a + bXi was fitted to the data of each group, where: Yi = is the distance measured by cellular positions covered by the most advanced labelled ameloblast at the time Xi; X , = is the time after the injection of tritiated thymidine; a = is the linear coefficient of the simple linear regression; it represents the initial distance covered by the most advanced labelled cell; b = is the regression coefficient of the simple linear regression; it expresses the velocity which was assumed as being constant.
The estimated regression lines (Fig. 4) values of a and b were the following:
control: Yi = 34.4 + 3.21Xi vinblastine: Yi = 41.1 + 2.48Xi
and the correlation coefficients (R2) were respectively: 0.96 and 0.91. Since both coefficients are very high, the regression equation explains the variation of the distance as a function of time.
On the other hand, the velocity of migration given by the regression coefficient (b), for the control group (3.21 cell positions per hr), was significantly different from the one of the treated group (2.48 cell positions per hr), at the 5% level.
(iii) Ameloblasts kinetics estimated by the silver grain count Using the data given in Table 5 , where g = 123 and go = 1.596 for the control and g = 160 and go = 1.555 for the treated animals, the calculated constant k was 0.02670 and 0.02369
VI
Tabl
e 5.
Tot
al n
umbe
r of
cel
ls. g
rain
cou
nt a
nd la
belle
d ce
ll co
unt in a
n am
eloh
last
col
umn.
at e
very
I0
0p
. of
the
low
er i
ncis
or o
f m
ice
trea
ted
with
vin
blas
tine
(VB
L) a
nd c
ontr
ols
(C).
at I
and
96
hr a
fter
labe
lling
with
I 'H
ITdR
8
Cel
l cou
nt
Labe
lled
cell
coun
t G
rain
cou
nt
1 hr
96
hr
I hr
96
hr
I hr
96
hr
[lis
tanc
c (p
m)
C
VB
L C
V
BL
C
VB
L C
V
BL
C
V
BL
C
V
BL
1-10
0 10
1-2
00
20 1
-300
30
1-4
00
401-
500
Subt
otal
50 1
-600
60
1-70
0 70
1-80
0 80
1-90
0 90
1 - 10
00
1001
-1 1
00
1101
-120
0 12
01- I
300
1301
- I4
00
140 I
- 15
00
150 1
- I6
00
I60
I- I 7
00
I70
I- I8
00
1801
- I9
00
I90
1-20
00
Tot
al
25.5
28
.30
21.5
23
.0
35.5
22
.60
33.5
38
.3
49.0
25
.30
39.0
43
.3
45.0
36
.30
45.0
43
.0
45.3
25
.38
49.5
51
.6
200.
3 13
7.88
18
8.5
199.
2
43.5
59
.0
45.5
59
.3
42.0
46
.0
40.5
46
.3
32.5
46
.6
34.0
62
.3
39.5
49
.0
34.0
53
.0
33.5
49
.6
37.0
49
.3
38.0
34
.0
32.5
29
.5
29.2
3
733.
73
119.
6
11.0
7.
60
4.0
11.0
12
.60
2.5
18.0
15
.60
4.0
9.0
13.3
0 4.
5 3.
33
10.0
0*
7.5
52.8
3 59
.10
22.5
7.5
11.0
5.
5 11
.5
11.0
12
.5
15.5
13
.0
8.0
22.0
23
.0
20.5
22
.0
16.5
16
.92
238.
92
I .6
235.
5 1.
6 34
0.5
3.6
601.
5 5.
3 28
9.5
9.0
129.
23
2 I.
1 1.
596.
23
15.6
13
.6
19.0
18
.0
17.6
23
.6
24.6
17
.6
18.8
lZ
.O*
201.
5
203.
0 15
.5
318.
3 9.
0 38
5.0
15.5
37
7.0
27.5
27
1.5
56.0
1.55
4.8
123.
5
36.5
56
.5
46-5
75
.0
88.0
10
2.0
108.
5 10
2.0
106.
0 28
8.5
357.
0 35
7.5
474.
0 39
0.5
413.
07
3.12
5.07
48.3
3 13
.33
24.6
6 27
.33
46.3
3
159.
98
83.0
87
.3
131.
3 13
2.3
134.
6 19
8.3
311.
0 20
6.3
224.
8 22
8.6
1,89
7.48
Each
val
ue is
the
mea
n of
cou
nts
in th
ree
sect
ions
. Th
e su
b-to
tal c
orre
spon
ds to
the
prog
enito
r co
mpa
rtm
ent,
whi
ch w
as d
efin
ed b
y th
e m
ost
inci
sally
-lab
elle
d am
elob
last
1 hr
aft
er th
e in
ject
ion
of [
'HIT
dR.
* The
hig
hest
labe
lled
cell
was
foun
d at
a fe
w p
m le
ss th
an 5
OO
pm o
r 15
00 p
.
Vinblastine and ameloblasts 501
Table 6. Some kinetic parameters of ameloblasts devided from the silver grain count method (Zajicek. 1974) ~~~
Generation Synthetic Cell production Transit time Velocity time (G) time (S) rate (P) (T) (V)
(hr) (hr) (cell/hr) (hr) ( w / h r ) ~~
Control 25.96 6.88 7.10 26.49 18.88 Vinblastine-treated 29.25 12.50 4.12 54.45 9.18
Raw data in Table 5 . Equations are given in text.
respectively for the control and treated group. From these values, using the equations mentioned previously, the calculated kinetic parameters are given in Table 6.
As can be seen, the ameloblasts migration velocity by this method is close to the one calculated by the displacement of the most advanced labelled cell (in the sections used, the mean width of ameloblasts were 4.5 pm; so 3-21 cellslhr equals 14.45 pmlhr and 2.48 cells/hr equals 1 1.16 pm/hr), showing that the ameloblasts migration rate was slower in the treated animals.
On the other hand, the calculated times for the duration of the cell cycle and S phase are quite different from the ones determined by flux of labelled metaphase curve (Table 3). By the grain dilution method the generation time G was much longer, and even longer for the vineblastine treated animals. The synthetic time was more or less the same for the controls with both methods, but for the treated [animals it was three times longer.
D I S C U S S I O N
The generation time and the duration of each phase of the cell cycle determined for the ameloblasts of the control mice were within the limits of those calculated by Hwang, Cronkite & Tonna (1966) using a similar method.
Vinblastine-treated animals showed an arrest of mitotic cells in metaphase, as already described by Moe (1977) and Mikkelsen (1978), as a result of the drug interfering with microtubular proteins of the mitotic spindle (Sartorelli & Creasey, 1969). The duration of the cell cycle determined for vinblastine-treated animals was shorter than that of the control ones and close to the minimum values calculated by Hwang et al. (1966). The first peak of the curve of labelled metaphases (Fig. 1) however, in the treated animals, did not reach a ‘plateau’ of 100% labelling, as would be expected. This could be explained by an underestimation of the number of metaphases in the population. In these animals a fair number of mitotic figures were seen at the limit between the ameloblast layer and the stratum intermedium, of which those partly located at the level of the ameloblasts were counted as such. Probably more cells which are at the level of the stratum intermedium belong to the ameloblast population. Cells in similar situations were described by Ten Cate (1961) as ‘insinuating cells’, which would represent a recruitment of cells from the stratum intermedium to the ameloblast layer and would account for a 13% increase of the latter in human tooth germs. Chiba (1965) showed similar cells in the rat incisor, although we were not able to observe them in the control mice. If this is true, the percentage of labelled metaphase would increase, and the shape of curve of Fig. 1 would perhaps be different, leading to different time determinations.
As for the duration of each phase of the cell cycle, in the treated animals they were all quite different from that of the controls and the values given by Hwang et al. (1966). The times of G , and G , include the mitotic time which, due to the arrest of mitoses, was longer
502 Vinblastine and ameloblasts
than the controls. There is, in the literature, evidence that cycling cells in vitro are different in sensitivity to vinblastine according to the phase of the cycle they are exposed to the drug and according to Madoc-Jones & Mauro (1 968) vinblastine exerts a lethal effect in cells which are in S and late G,. This effect is dose and time-dependent. The dose used in the present experiment is the same used by Moe (1977) and Moe & Mikkelsen (1977b), who did not observe any major effect of vinblastine on ameloblasts of the proliferative compartment other than arrested mitotic figures. On the other hand, the same authors reported that ameloblasts of the secretory compartment were severely damaged and the number of cells was decimated by cellular degeneration.
According to Appleton (1983) the estimation of cell kinetic parameters from the flux of labelled mitotic curve (FLM) is unsatisfactory for disturbed systems where the steady state is not maintained. In general, the cell populations of rodent incisors are not in a steady state since there is growth involved (Smith & Warshawsky, 1977). On the other hand, vinblastine alters the distribution of the cells in mitosis because of the arrest in metaphase and, as quoted above, it causes a loss of secretory ameloblasts.
The generation and synthetic times calculated by the method of silver grain dilution (Zajicek, 1974) are quite different from the ones determined by the FLM curve. For the controls they are closer of the values determined for rat incisor (Zakicek & Bar-Lev, 1971a; 197 1 b) and for the vinblastine-treated mice they would be considerably longer. The rise in ameloblast production in vinblastine-treated animals indicated by the FLM method could be explained by the need to balance the extra loss of cells in the secretory compartment. But such increase in production is not confirmed by Zajicek’s method, which indicates a lower production, compatible with the slowing down of the ameloblast migration (discussed below).
It seems that a more accurate method for analysing the kinetics of the ameloblast progenitor compartment has to be developed, perhaps a three-dimensional approach, since there is the possibility of exchange between the several cell types found in the odontogenic organ of the rodent incisors (Smith & Warshawsky. 1975a) as the passage of cells from the stratum intermedium to the ameloblast layer as already discussed.
As for the migration of ameloblasts, the results showed a decrease in velocity in vinblastine-treated animals with both methods used. Since migration, according to Smith & Warshawsky (1975b). is a result of two components, tooth eruption and tooth growth, both could be affected by the drug. A slowing of tooth eruption due to vinblastine was shown by Chiba, Takizawa & Ohshima (1980). Tooth eruption seems to be related to the activity of fibroblasts in the periodontal ligament (Berkovitz & Thomas, 1969: Berkovitz, 1971; Beertsen. Everts & Van Den Hooff, 1974; Michaeli, Zajicek & Ginio, 1979) probably due to their mobility and or the secretion of collagen, both of which can be affected by vinblastine (Goldman, 1971; Waddell, Robson & Edwards, 1974; Ehrlich, Ross & Bornstein, 1974). Tooth growth depends on the production of new cells in the odontogenic organ, and in this regard our results are contradictory, although the determined slowing down of the velocity of ameloblast migration is compatible with a decrease of cell production as a vinblastine effect.
A C K N O W L E D G M E N T
This paper is part of the thesis presented by Maria M. Fernandez Samperiz to the FOP-UNICAMP in fulfilment of the requirements for a Master’s degree, and was carried out with the support of a grant from FAPESP (Proc. Med. 77/1479 and 78/0399). The revising of the manuscript by Dr Darcy de Oliveira Tosello is gratefully acknowledged.
Maria M . F. Samperiz, G. Blumen and J . Merzel 503 R E F E R E N C E S
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