ANATOMY AND PATHOLOGY 
864 
ably unable to return under any circumstances. 
Others can probably re-enter if stimulated ap- 
propriately. 
Therefore, a study was designed which had 
as its basic aim interpretation, in terms of the 
cell cycle, of the observations already described 
regarding the effect of mild cholesterol diets on 
DNA synthesis and division of arterial SMC. At 
the time these studies were begun very little 
was known about the nature and size of the di- 
viding population of arterial SMC or their cell 
cycle (Table IV). For example, the dividing 
population might have been a small percentage 
of the total SMC that divided every few days 
(short generation time), retaining some prog- 
eny and releasing others into the non-dividing 
population. On the other hand, the majority of 
the arterial SMC, even though functioning in 
contraction, could have been in the dividing 
population, with DNA synthesis and division 
taking place only after long intervals in Gi 
(long generation times) or Go." Another pos- 
sibility that could also have resulted in long 
generation times was t?iat a large proportion of 
the dividing population at any one time might 
have been in Go, with divisions taking place 
only after a long sojourn.'^ 
Obviously we needed to know "'^•hich of these 
possibilities is correct before attemi ..g to de- 
fine the biochemical nature of the effect of cho- 
lesterol diets on arterial SMC reproduction ki- 
netics. In particular we needed to know what 
group of SMC to investigate m future studies, 
i.e., (1) a small population dividing frequently, 
or (2) a large population in Gi or Go, or (3) a 
large population in Go. 
In one experiment 20 swine were given ^H- 
thymidine intravenously at day 0. Ten were 
placed on the stock diet and 10 on the choles- 
terol diet. A pair consisting of one from each 
group was sacrificed at 2 hours and the remain- 
ing pairs sacrificed at 5-hour intervals up to 47 
Table IV. — Informatioji available at outset of study on 
arterial SMC in relation to cell cycle 
Cells in: S ~0.7% 
Gj _ <l-98% 
M : <1% 
G, + G„ _ <l-98% 
Dividing Population 2-98% 
Non-dividing population 0-98% 
Generation time . ? 
hours. All except the 2-hour pair (which were 
used to construct a generation table) were 
given colchicine 5 hours prior to sacrifice, thus 
providing sequential 5-hour harvests of labeled 
and unlabeled mitoses. Cross-sections of the 
aortic trifurcation were processed for autora- 
diography and grain counts per labeled cell re- 
corded. Labeled and unlabeled mitoses were also 
counted. Only cells with 4 or more grains were 
considered to be labeled. The results of this ex- 
periment are summarized in Figure 2. 
The time curve of the percentage of labeled 
mitoses furnishes a method for estimating the 
duration of the different phases of the cell 
cycle. The results in Figure 2 show that G2 is 
at least 7 hours long, since no labeled mitoses 
were seen in the swine sacrificed at 7 hours 
after receiving ^H-thymidine. We cannot esti- 
mate the duration of S in the usual manner be- 
cause of the 5-hour time spread produced by 
colchicine. We can say that it is less than 15 
hours, probably 8-10 hours (from another ex- 
periment currently in progress) . The duration 
of S + Go + M is less than 37 hours because 
the curves (Figure 2) suggest that division of 
almost all of the labeled cells was complete by 
37 hours. We checked this point by absorption 
microspectrophotometry of Feulgen-stained sec- 
TIME IN HOURS 
Figure 2. — Graphic presentation of data showing per- 
centage of mitoses that had > 4 grains in each time 
period. It is apparent that a wave of mitoses is com- 
pleted by 37 hours, suggesting that the partial cycle 
time from S through M inclusive is no more than 37 
hours. The graph also shows what appears to be the 
beginning of a second wave of mitoses in the choles- 
terol swine. Evidence is presented in the text and 
Figure 4 supporting the idea that the second wave 
represents the beginning of post-pulse labeling and 
not a second division of the labeled cells. 
