W. A. THOMAS, R. A. FLORENTIN, S. C. NAM, J. M. REINER AND K. T. LEE 
869 
terol diet can not be determined without more 
i extended experiments designed to test this 
point. 
The possibilities can be narrowed down, how- 
ever. In a homogeneous population (no Go com- 
ponent) with uniform age distribution, the la- 
beling index (ratio of cells in S to total cells in 
the population at any moment) is equal to the 
ratio of S time to generation time.^^ The label- 
ing index of our tissues at the beginning of the 
experiment was about 0.7 percent. Taking the S 
time as 8 hours, we calculate a generation time 
of 48 days. 
This value is maximal (if S is really 8 
hours). The assumption of an exponential age 
distribution would result in multiplying the 
value of loge 2 = 0.693, giving 33 days.^^ If the 
population were not homogeneous, and con- 
tained a Go component, the denominator of the 
labeling index would be a fraction of the total 
population — ^that part of it in the cycle at a 
given moment. With growth fraction 2/3, the 
generation time would then be 32 days. An in- 
creasing proportion of Go phase (decreasing 
growth fraction) would bring the generation 
time into a range (less than 28 days) from 
which we can exclude it experimentally. 
Granted the validity of our observations and 
an S of 8 hours, we can bracket the generation 
time between 28 and 48 days. If our prelimi- 
nary estimate of 8 hours for S proves to be low, 
the calculated generation time would of course 
be longer. As to the generation time of the ex- 
cess post-pulse labeled cells that have been re- 
cruited by the cholesterol diet, we can say noth- 
ing, and must await the results of observations 
of longer periods. The fact that mitoses were 
numerous among these cells at 2 and 7 days 
(that is, during the period of post-pulse label- 
ing) might plausibly be expected, and the fact 
that they were negligible at 15 days and 30 days 
suggests that these cells, like the original 
pulse-labeled group, have generation times 
greater than 28 days. 
Because of the limited time of observation, 
we can do no more than guess as to whether the 
newly recruited cells were derived from Go or 
resulted from acceleration of cells slowly mean- 
dering through Gi. If longer periods of obser- 
vation indicate that the original pulse-labeled 
cells are no faster than they were initially, the 
decision will be clear. Moreover, knowledge of 
the generation time of this population would 
permit us, by calculations similar to those just 
discussed, to estimate the magnitude of Go 
(which evidently might be negligible). As to a 
non-dividing population, our experiments give 
no clue as to whether it even exists in aortic 
SMC, let alone what its properties might be. In 
Table IX a summary is given of some of the 
data we now know or can surmise regarding 
distribution of arterial SMC in relation to the 
cell cycle. Clearly the cells in Gi or Go are the 
ones on which we now need to focus our atten- 
tion. 
It is interesting to note that the effect of most 
other known stimulators of DNA synthesis is 
on Gi or Go cells. These include the effect of 
partial hepatectomy on hepatic cells^^ and of is- 
oproterenol on the salivary glands.^^ The bio- 
chemical mechanisms that account for the stimu- 
lation are not known for any type of stimulus. 
With most, either a rise in RNA synthesis has 
been the first change observed or the effect has 
been abolished by small doses of actinomycin 
This has led many investigators to postu- 
late that the stimulatory effect was on tran- 
scription. The postulated effect could be pro- 
duced either by removal of an inhibitor that is 
normally present or through some direct inter- 
action. RNA synthesis and the effect of actino- 
mycin D have not yet been investigated in cho- 
lesterol-fed swine. 
Our attention is now turning to investigation 
of mechanisms that control whether or not an 
arterial SMC is going to synthesize DNA and 
divide. We have evidence from in vitro studies 
suggesting that there is something in the serum 
of hypercholesterolemic swine that is involved 
in control mechanisms,^^ A greater proportion 
of cultured arterial SMC are synthesizing DNA 
Table IX. — Information now surmised on arterial SMC 
in relation to cell cycle 
Outset 
Now 
Cells in: S 
~0.79r 
~0.7% 
G» 
<l-987r 
<1% 
M - - - 
-- <1% 
<1% 
G, + G„ 
<l-989'r 
BO-98% 
Dividing population 
2-98% 
50-100% 
Non-dividing population 
0-98% 
0-50% 
Generation time 
7 
>28 days 
