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The effect of pH on transformation of BHK21 cells by polyoma virus
VIROLOGY
28,
188-201 (1966)
The Effect
of pH on Transformation Polyoma II. Kinetic
ALEXANDER Medical
Research Council
L. KISCH1,2
Experimental
of BHK21 Cells
by
Virus
Considerations AND
JOHK
H. SUBAK-SHARPE
Virus Research L’nit, Institute Glasgow, Scotland
of Virology,
University
of Glasgow,
Accepted September 8, 1965 Changes of the sequence or of the relative duration of combined high and low pH incubation affect the frequency with which transformed colonies arise in cultures of polyoma virus-infected BHK21 cells. In general, high pH incubation increases the number of transformed colonies but reduces the plating efficiency of nontransformed infected cells while low pH incubation has opposite effects. However the effect of pH on t,ransformationisindependent of its effect on the plating efficiency of nontransformed cells. Low pH incubation also increases the rat,io of “mixed” to “pure” transformed colonies. Two-day “pulses” of low pH reduce the number of transformed colonies, but this “pulse” effect diminishes progressively with time, apparently because of a progrescolonies. The effect sively diminishing inhibition of formation of “pure ” transformed of pH on plating efficiency is greatest on the first day post infection and then rapidly declines at a different rat,e than the effect on transformation. transformed colonies arise continuously aft,er The data confirm that “mixed” transformed colonies also continue to arise for infection and suggest that “pure” 3-4 days after infection. They further indicate that the development of “mixed” and “pure” transformed colonies is affected difl’erently by high and low pH. The concept of “nascent” transformed cells as opposed to established transformed cells is discussed INTRODUCTION
It has been reported (Kisch and Ii‘raser, 1964) that the pH at which BHK21 cells infected with polyoma virus (PV)3 are incubated affects the frequency with which 1 Aided by a grant for a Postdoctoral Research Scholarship from the American Cancer Society. 2 Present address: Department, of Medicine, School of Medicine, University of New Mexico, Albuquerque, New Mexico. 3 Abbreviations used in text: PV = polyoma colonies; y0 t/cell = virus; t = all transformed number of transformed colonies per 100 infected cells plated; 7c t/co1 = number of transformed colonies per 100 colonies scored; M = mixed kansformed colonies; P = pure transformed colonies; pC0~ = partial pressure of carbon dioxide. 188
transformed colonies arise. The experiments described here were intended to establish whether this enhancing effect of high pH on transformation results from a net increase in the number of transformed colonies, or merely from different’ial select’ion at the cellular level. To investigate the kinetics with which transformed colonies arise, the pH of PV infected cell cultures was altered during the incubation period at varying time intervals following infection. Stoker (1963) observed in microdrop cultures that transformation may be delayed for at least 5 cell divisions in colonies derived from single PV-infected cells. It’ therefore seemed relevant t,o determine whether trans-
pH EFFECT
ON BHK21 CELL THANSFOILMATION.
format ion in pure as well as in mixed transformed colonies is subject to delay, or whether it occurs only within a critical short time after infection. Finally, it seemed of int’erest to determine whether any relationship between the effect of pH on the plating efficiency of infected cells (Kisch and Fraser, 1964) and its effect on trltnsformation could be demonstrated. MATERIALS
AND METHODS
T7irus, cells, medium, and transfomation assay. The origin of the stock polyoma virus and of t,he BHIi21/C13 cells employed in all assays, the constit’ution of the media used, ard d&ails of the method of pH measurement a,s well as of the transformation assay techniques employed have been described previously (Kisch and Fraser, 1964). In the present series of experiments all transformed colonies (t) have been scored by a single observer as either “pure transformed” (P) or as i‘mixed transformed” (XI). Characteristically 1\1 colonies cont’ained a sector of cells showing random orientat’ion, the remainder of the colony exhibiting bhe regular parallel orientation of cells typical of normal colonies (St’oker, 1963). Design of experiments. Two experimental designs were employed. In the first or “switch experiment”, cultures of PV infected cells were kept at, high pH (e.g., Expt. 2, 7.6-7.8) following infection, while a duplicate set, of infected cultures was incubated at low pH (6.9-7.1). After varying periods of incubation, the plates were switched reciprocally from high to low pH or vice versa. T\‘o culture was switched more than OIICC. After a total of 7 days’ incubation t,he cultures were scored for the presence of M, P, and nontransformed colonies. The pH switch was accomplished by changing the pCOz of the incubator jar. In the second type of experiment) (“pulse experiment”), replicate cultures were exposed t)o a two-day pulse of low pH, either immediately or after an initial incubat’ion period at, high pH, and t’hen returned t,o high pH incubation. There are two ways in which the number of transformed colonies is usually recorded (Stoker and Abel, 1962). The first, the % t/
I
189
cell, relat’es the number of transformed colonies scored to the number of infected cells initially plated. The second, the % t/col, relat’es the number of transformed colonies scored to the total number of transformed and normal colonies observed. The % t/cell, unlike the % l/co& is independent of the plating efficiency of “normal” cells provided there is no interaction between normal and transformed colonies which varies over the range of colony numbers in the experiments. Assuming no such interaction, the 70 t/cell is the plat’ing efficiency of “nascent”’ transformed cells, and comparisons of % t/cell values reflect solely the kinetics of transformed colony formation. A “nascent” transformed cell should be distinguished from an established transformed cell. As used here it, describes a newly arisen, phenotypically t’ransformed cell whose immediate parent cell was bot’h phenotypically untransformed and descended from untransformed lineage. As the physiological homeostatic equilibrium at this point in descent probably undergoes drastic changes, one cannot a prio~i assume t’hat a “nascent” transformed cell behaves either like a normal or like an established transformed cell. As we have no information regarding possible treatment-dependent variation in in the relative plating efficiency of normal and nascent transformed cells, the data have been expressed in terms of 70 t/cell vslues. RESULTS 1. Effect o.f Initial
pH on Transformation
(5% t/cell) The effect which initial pH exerted on transformation is shown by the data in Table 1, column 8, and also by the analysis of variance of the results of experiments la and lb given in Table 2(B). For convenience the data obtained from plating the higher cell inocula only are plotted in Fig. 1 although the remaining data also complet’ely support the following conclusions: In contrast to the rapidly diminishing effect of pH on plating efficiency (see Section 3) maintenance of an initial high pH progressively increased t’he number of transformed colo-
190
KISCH
AND
SUBAK-SHARPE
TABLE 1 TRANSFORMED COLONYTOTAL,% ~/CELL,% PLATINGEFFICIENCYAND~
2
3
Expt. no.b
Final pH
4 5 Transformed colonies “pure )I “mixed”
6
7
8
9
Replicates
“/n Plating efficiency
Ojof/cell
M/PC
High
Low
7
-
la lb 2
7.6 7.6 7.6
30; fid 33;12 56; 6
77;lO 47 $4 71;ll
6;2 6;2 8;2
41.9;44.8 43.1;54.0 21.8;24.2
4.45;4.00 3.33;6.50 3.18;3.40
2.42 1.36 1.32
5
-
la lb 2, 3
6.8 6.8 7.0
41; 7 27; 8 65;ll
44;ll 52; 6 41; 9
5;2 6;2 8;2
43.3;47.8 34.6;35.5 21.6;26.0
4.25;4.50 3.29;3.50 2.65;4.00
1.15 1.66 0.66
4
-
2
7.1
36; 7
43; 8
8;2
21.7;29.0
1.98;3.00
1.19
i 3
-
:;I; 2
6.8 6.8 7.1
8; 4 8; 7 39; 5
17; 7 20; 8 46; 9
2;2 4;2 8;2
45.3;50.8 40.5;47.0 20.6;22.8
3.13;2.75 1.75;3.75 2.13;2.80
2.00 1.87 1.25
2
-
2
6.9
24; 7
41; 3
8;2
21.3;21.2
1.63;2.00
1.42
-
la lb 2
6.7 6.7 7.0
3; 1 2;; x
4; 0 5; 1 28; 8
2;l 3;l 8;2
46.0;48.5 42.8;49.5 25.1;25.6
0.88;0.50 0.83;1.00 1.40;2.80
1.00 1.00 1.04
k 2
6.6 6.6 7.0
4; 2 6; 1 11; 0
25; 4 16; 5 26; 1
6;2 6;2 8;2
55.4;50.8 51.5;52.5 30.6;36.4
1.21;1.50 0.92;1.50 0.93;0.20
4.83 3.00 2.45
i:
la lb 2
7.5 7.5 7.7
11; 2 8; 3 32; 8
31; 4 18; 8 34; 7
6;2 5;2 a;2
48.5;53.5 49.6;52.0 30.4;31.6
1.75;1.50 1.30;2.75 1.65;3.06
2.F9 2.36 1.03
4
2
7.7
28; 7
33; 3
8;2
32.2;39.4
1.53;2.00
1.03
-
3 i
la lb 2
7.3 7.3 7.7
10; 1 16; 2 22; 3
29; 9 35; 5 53; 6
4;l 5;l 8;2
50.8;48.0 43.8;47.0 30.9;31.2
2.44;5.00 2.55;3.50 1.88;1.80
3.45 2.22 2.36
-
2
2, 3e
7.8
43; 2
40; 5
8;2
28.1;28.8
2.08;1.40
1.00
-
1 1 1
:;1 2
7.3 7.3 7.7
14; 6 30; 5 40; 3
40; 7 46; 9 62; 8
6;2 6;2 8;2
51.0;52.8 50.8;56.8 27.0;34.4
2.25;3.25 3.17;3.50 2.55;2.20
2.35 1.57 1 .63
i: 3
:*7” 717
45; 4 44; 3 49;lO
45; 9 53; 6 37 ;lO
8;2 8;2 8;2
25.6;29.2 24.8;28.8 23.1;29.6
2.25;2.60 2.43;1.80 2.15;4.00
1.10 1.26 0.80
7’ 5
1 : -
; 7
-
9 4
5
-
Q In “switch” experiment,s (la, lb, 2) cultures of PV-infected BHK21 cells were incubated for the specified number of days at initial pH and then reciprocally switched from high to low pH or vice versa. In “pulse” experiment (3),, replicate cultures were exposed to a-day pulse of low pH, either immediately or after an initial incubation period at high pH and then returned to high pH incubation. Duration of all experiments was 7 days. Data refer to the totals obtained by summing t,he replicate plate observations at each ooint. b Polyoma &us was stock PlOl. Exposed multiplicity of infection per cell for experiments la, 2, and 3 was 5300 PFU and for lb 2650 PFU. all “mixed” transformed colonies cM/P = all “pure” transformed colonies d Values listed in columns 2-6 to the left of a semicolon refer to groups of plates seeded with 400 (Expts. la and lb) or 500 (Expts. 2 and 3) infected cells per plate, while values listed to the right of a semicolon refer to groups in which each plate was seeded with 200 (Expts. la and lb) or 250 (Expts. 2 and 3) infected cells each. 6 Point which features jointly in experiments 2 and 3.
pH EFFECT ON BHK21CELL TRANSFORMATION.I TABLE
2
ANALYSIS OF VARIANCE OF EXPERIMENTS Item Part
A: Effect of initial
Degrees of freedom pH on plating
la AND 1bU Sum of squares
Probability
72.262 44.3% 7.942 7.776 7.320 13.088 4.8G7
66.910 9.650 1.669 4.511 51.080
43.6G4 4.474 2.386 0.55G
eficiency 15 1 1 3 1 10 3
Total Effect of pHh Equality of VD Effect of day Initial periodd Interactions Interaction day X pH Part B: Effect of initial pH on yo t/cell Total Effect, of pHh Equality of V1lc Effect of day Int,eract ionse Interactions Hay Day pH Day
191
x pHe x VI> X VI) x pH x VI)
15 1 1 3 10 broken down 3 3 1 3
n In Part A t,he differences in plating efficiency of the 16 experimental points of experiment la and items are responsible for the observed differences. Part, B lb are analyzed to discover what “effect” shows similar analysis with respect to differences in the 70 t/cell. * Tests whether the INITZAAL pH (either high or low) influences the results; highly significant, both with plating efficiency and 70 t/cell. c VD is virus dilution. This significantly affects the plating efficiency. d Shows that the entire effect is exerted during the initial period. e Shows that the only important interactions that influence the 70 t/cell are those between pH and day of pH switch.
nies, while maintenance at an initial low pH progressively reduced their number. In either case the distribution of successive points suggests an approximately linear relationship between duration of the initial pH and t,he observed 70 t/cell. The calculated first-order regressions (Mather, 1949) for the six series plotted in Fig. 1 are presented in Table 3A. The slopes of 5 of t’he calculated regressions deviated significantly from the “null” hypothesis of zero slope, while the sixth just failed to do so. Since the three high pH regressions as well as the three low pH regressions were statistically c*onsistent, appropriate “mean” regression lines were calculated. Here experiment’ 2 resuhs received twice the weight of t’he results from experiment la or lb as each point arose from 4000 cells plated, while t,he results of experiment la or lb arose on average from about 2000 cells plated.
The calculated “mean” regressions suggest that high pH increased the % t/cell on average by 0.39 per day; and conversely that low pH decreased the % t/cell by 0.33 per day. But this calculation is based on the assumption that the effect of pH per day was constant over the duration of the experiment. The data obtained from the pulse experiment permit evaluation of the validity of this assumption and of the effectiveness (in reducing the % t/cell) of standard a-day pulses of low pH applied at various times aft,er plating. Here each culture was incubated for a total of 5 days at high pH and 2 days at low pH, the only variable being the timing of the low pH pulse. The relevant data from Table 1 are plotted in Fig. 2 (P + RI) together with the calculated first-order regression line, which is significant’ (RcP+M), Table 3B). This establishes
192
KISCH DAYS
AND
MAINTAINED
AT
SUBAK-SHABPE
INITIAL
pH
UNTIL
CHANGEOVER
HIGH
p”;
I*
0
LCIW
pH;
DILUTED
1.3
EXPT.
la
4
*I
‘I
‘I
-I
I-
1’6
I’
lb
4
I’
,.
.*
‘*
**
1.6
..
Ib
,
.I
,.
..
..
3,
I3
,’
2
u
-
.I
,,
II
.I
13
.’
2
.
INITIAL
VIRUS
DlLUTED
13EXPT
,NITIAL
VIRUS
Fig. 1. Effect of duration of initial pH on the “/b t/cell. In experiments la and 2 polyoma virus stock 101 was diluted 1:3 and in experiment, lb, 1:6. The experiments were carried out as described for Fig, 3, except that experiments la and lb employed only 6 replicate petri dishes per group, each seeded with 400 infect,ed cells. The combined “mixed” and “pure,” transformed colony totals per 100 cells plat,ed are plot.ted against the period during which the initial pH was maintained until pH switch. In some groups plates were lost, as showninTable 1. Initial highpH: l , virusdiluted 1:3, Expt. la; A, 1:6, Expt. lb; v, 1:3, Expt. 2. Initial low pH: 0, virus diluted 1:3, Expt. la; A, 1:6, Expt. lb; V, 1:3, Expt. 2.
that the effect of a a-day low pH pulse progressively declined with time. The simple first-order regressions fitted to the switch data are therefore not an adequate model, and a more complex relationship must hold. Previous considerations suggest. a model combining (1) the linear enhancing effect of incubation time at high pH on the number of transformed colonies; and (2) the linear decrease in effectiveness of high pH with the day post infection which high pH was experienced. Algebraically expressed this means
where T = total number
of kansformed
colonies; a, b, and c are constants; c = sum of; q = 1 for each day at high pH and 0 for each day at low pH; and t = 1 on day 1, 2 on day 2, and so on. This equation was fitted to the “high cell number plat’ed” data of (switch) experiment 2 and (pulse) experiment 3 (Table 1, column 8). Table 4 shows the excellent fit, of this relatively simple unified model wit,h all the data. Therefore the % t/cell obtained 7 days post infection can be considered as a simple function of the total time spent at a high pH and of the precise days post infection at which this pH was experienced. We are indebted to Mr. M. W. Birch of the Mathematics Department’ for both the
colonies;
0.05-0.01 0.05-0.01 >0.2
a Discussed at length in the text. * R~+M, Kegressioll of all transformed only.
RM
RP
PLP.td
of “pure”
transformed
Y = (2.36+0.04)+(0.142f0.025) Y = (1.23f0.05)+(0.139f0.029) Y = (1.13+0.0G)+(0.003f0.037)
to the pulse experiment
RF, regression
of 2-day pulse of low pH, jtted
colonies
only;
(r-3.2) (z-3.2) (x-3.2)
RM, regression
(z-3.2)
Y = (2.153f0.154)+(-0.331f0.063)
Mean Low pH Part. B. On midpoint
(z-3.2) (x-3.2) (r-3.1)
(z-3.2)
(x-3.2) (r-3.2) (2-3.1)
1’ = (2.421&0.342)+(-0.3G7ztO.133) Y = (2.25&0.104)+(-0.375fO.042) Y = (1.968+0.085)+(-0.29OzkO.038)
0.1-0.05 0.01-0.001
pH, jitted to the data from the switch experiments 0.05-0.01 Y = (2.783+0.275)+(0.554&0.108) 0.01~.001 Y = (2.108~0.158)+(0.383zt0.063)
la Low pH lb Low pH 2 Low pH
at initial
Regression line E’ = (,?j f s&J -+ (b f Sb) (x -P)
t/&ID
Y = (2.21&0.140)+(0.389~z0.057)
On days maintained pII pH pH
Probability of null hypothesis
3
Mea11 High pH
Part A: la High lb High 2 High
Item
TABLE LINEAR REGRESSIONS OF “/c
of “mixed”
0.95
0.14.05
ha - bib
transformed
0.054.02
6,-b,
O.G-o.5
0.3-0.2
ha, - bi:
colonies
0.2411
0.4-0.3
hi, - bz
Probability of regressions being the same
194
KISCH
AND
SUBAK-SHARPE
3.0
2.0
2 zl 0 4 D a
1.0
0 0
I
I
I
I
I
I
I
2
3
4
5
6
MIDPOINT
OF
TWO
DAY
PULSE
OF
LOW
I
pH.
Fig. 2. Effect of time of application of a 2-day pulse of low pH on the c/c t/cell. Conditions were asfor Fig. 4. Plotted are (1) all transformed colonies (P + M): 0; (2) “pure” transformed colonies (P) A; and (3) mixed transformed colonies (M), 0. The P + M totals observed after 7 days at high pH and after 7 days at low pH are shown for reference. Also plotted are the calculated regression lines (Table transformed colonies (Rp), and “mixed” transformed 3B) for all transformed colonies (&=+M), “pure” colonies (RM).
likelihood estimation of the constants and the test for goodness of fit’.
maximum
2. Differential E$ect of Low pH on Development of P and Al T~ansjormed Colonies Differential effects of maintained pH on P and M colonies can be detected from comparisons of M/P ratios. Where P decreases relatively more or increases relatively less t’han M, this ratio will increase. Table 5 presents the M/P ratios observed after incubation for 7 days when the duration of the initial incubation period at high or low pH was varied (columns 3 and 4). Appropriate
comparison of these values in point for point comparisons (column 5), or with the M/P ratios obtained after the standard 7-day period of incubation at low pH (columns 6 and 7) are also included. Column 5 shows that at almost every point relatively more M (or fewer P) colonies were produced when the initial maintained pH was low. Columns 6 and 7 show that relatively more M (or fewer P) colonies arose after 7 days of maintained low pH than with any other treatment. Analysis of the data available does not reveal a significant progressive change in M/P as the duration of initial low pH incu-
pH EFFECT TABLE FITTING
4
Expectation
LOW
7 5 4 3 2 1
Pnlse
CELL
OF THE EQUATION T = a+b xq+cxtq TO THE DATA OF SWITCH EXPICRIMENT 2 AND THE PULSE EXPERIMENT 3,*
Days at initial pH .High -
ON BHK21
7 5 4 3 2 1 2 3 4
a + a + a + a + a + a+ a a + a + a + a + a + a + a + a +
7b + 5b + 4b + 3b + 2b + b+
28~ 15~ 10~ 6Gc 3c c
2b 3b 4b 5b 6b 5b 5b 5b
13~ 1%~ 22c 25~ 27~ 23~ 21~ 19c
+ + + + + + + +
Transformed colonies (P + M) Estimated
Observed
116.4 99.8 89.9 78.9 66 8 53.6 39.4 56.0 G5.9 7G.9 89.0 102.2 91.2 93.3 95.5
127 106 79 85 65 56 37 66 61 75 83 102 90 97 86
n Goodness Maximnm
of fit x& = 7.26; P = 0.9-0.8 likelihood estimates: a = 39.42 b = 15.31 c = -1.08 Standard error covariance matrix +18.30 -5.06 +0.23 -5.06 +F.63 -1.24 -1.24 +0.29 [ +0.23 * Symbols are explained in the text.
1
bation or high pH incubation is increased. Hdwever, this test was rather insensitive as it depended frequently on ratios derived from small numbers of observed colonies and further data would be of int)erest. Having shown that the effectiveness of a low pH pulse (in reducing t,he % t/cell) progressively diminished with time post infection, it was examined whether P and M colonies were equally affected. The relevant analysis given in Table 3B and Fig. 2 shows that the actual time post infection at which cells were exposed to a low pH pulse wan irrelevant to the number of Xl colonies observed, but only affected the number of P colonies. The regression of P colonies with time of pulse significantly deviates from zero and from that of M colonies, while this latter regression does not deviate significantly from zero (Table 3B). The progres-
TRANSFORMATION.
I
195
sive loss of effectiveness of low pH on % t/cell was thus due to t’he progressively increasing escape from suppression of pure transformed colonies, not to suppression or delay of appearance, or increased difficulty in scoring, of mixed transformed colonies. Thus, there are two independent) sources of evidence t,hat low pH differentially affected P and 111 colonies, resulting in an increased M/P ratio. In the pulse experiment this was due to progressive increase in t,he number of P colonies. In the switch experiments AI/P ratios mere generally higher at low initial pH, and in all three experiments of this type, were highest aft’er 7 days of low pH incubation. The act(ua1 colony numbers in Table 1 (columns 4 and 5) show that t.his effect was not due to higher plating efficiency of normal but potentially mixed colonies at low pH. Additional data are needed to extjend this investigation. 3. E$ect of pH on. Plating Ejiciency The effect which the duration of the initial pH exerted on the efficiency of plating is illustrated by data from experiment 2 plotted in Fig. 3. In all switch experiments low initial pH invariably led to a 20-50 % greater plating efficiency than high pH. Moreover, the influence of initial pH on the plat,ing efliciency was obviously greatest on the first day, after which it rapidly declined. The influence of the t,ime of application of pH on plating efficiency was observed in the pulse experiment. The data are plotted in Fig. 4. These data were analyzed by taking the total increase in plating efficiency at 7 days (of “low pH treated” over “high pH treated” cells) as 100 %. Then 2-day low pH pulses 011 days O-2, 1-3, 24, 3-5, and 5-7 resulted in respect,ive increases of 66.3, 41.6, 33.7, 16.9, and 0.0%. This yields the following estimates of relative effectiveness for each day: 1 = 41.5 %; 2 = 24.8 %; 3 = 16.8%; 4 = 16.9 %; 5, 6, 7 = 0.0 %. The relative effectiveness of day one can be calculated independently from the data of the simultaneously performed switch experiment (experiment 2) as 67.4 % and 52.8 %. Thus low pH uniformly resulted in greater plating efficiency and was most effective on t)he first day with fairly rapid
196
KISCH
AND
SUBAK-SHARPE
TABLE DEPENDENCE
OF THE RATIO “MIXED”
Column: 1 Day of maintained initial pH
* L/H
2 Expt. no.
5
THANSFORMED: “ PURE”
3 Initial high PH (HI
5
6
L/H”
L7/H
7 U/L
4.83 3.00 2.45
2.0 2.2 1.9
2.69 2.30 1.06
2.3 1.4 1.6
4.2 1.8 3.7
1.8 1.3 2.3
1.19
1.03
0.9
2.1
2.4
la lb 2
2.00 1.87 1.25
3.45 2.22 2.36
1.7 1.2 1.9
2.4 1.6 1.9
1.4 1.4 1.1
2
2
1.42
1.00
0.7
1.7
2.4
1 1 1
la lb 2
1.00 1.00 1.04
2.35 1.57 1.63
2.3 1.8 1 .6
4.8 3.0 2.4
2.1 1.9 1.5
7 7 7
la lb 2
5 5 5
la lb 2
4
2
3 3 3
2.42h 1.36 1.32
4 Initial low PH (L)
TRANSFORMED COLONIES UPON pH
probability
of 12:2 result if no difference P Yat,es correction p; x: = 7.14 0.02-0.01
decline reaching zero by the fifth day. This is also confirmed by the analysis of variance of the data of swit,ch experiments la and lb (Table 2A). As was shown earlier, high pH incubation progressively increased the % t/cell but was initially detrimental to plating efficiency. Data from experiments la and lb plotted in Fig. 5 indicate that no general correlation existed between % t/cell and plat,ing efficiency, irrespective of cell numbers seeded per culture. However the number of cells plated affected both the plating efficiency and % t/cell. (Table 1, columns 7 and 8). In 28 of 31 comparisons (14/16 in experiments la and lb and 14/15 in experiments 2 and 3) plates seeded with the lower cell number had higher plating efficiency-on average 3.4 %. While the number of cells plated did not significantly modify M/P rat’ios under the pH conditions tested, it did
affect % t/cell values, for 22 of 31 comparisons showed a higher % t/cell when plates were seeded with the lower cell numbers (12/16 in experiments la and lb and lo/15 in experiments 2 and 3). On average this advantage was 0.72 in experiments la and lb and 0.44 in experiments 2 and 3. These findings differ from those of Stoker and Abel (1962), who found that under their experimental conditions the % t/cell was unaffected by the total number of cells per plate. It is unlikely that these findings stem from easier recognition and enumeration of normal as well as P and ?(/I transformed colonies when the colony density per culture is relatively low. Inherent in such an hypothesis is the expectation t’hat t/co1 values should remain unchanged, whereas in fact t/co1 values from plates seeded with the lower cell number exceeded t/co1 values from plates seeded with high cell numbers in
pH EFFECT
I
1 0
ON BHK21
f 1
CELL
I 2 DAYS
OF
TRANSFOKMATION.
I 3 INITIAL
I b pH
UNTIL
I 5
197
I
I 6
I 7
CHANGEOVER
Fig. 3. Effect of duration of initial pH on plating efficiency. Each of 96 petri dishes with feeder layers was inoculated with 500 polyoma virus-infected cells, then t,he dishes were distributed into 12 groups of 8 replicates each, and incubated at either high (7.7) or low (7.0) pH. After different periods of time for different groups, all replicates in a group were changed from high to low pH or vice versa by adjustment of the pC0, of the incubator jar. After a total of 7 days’ incubation, all cultures were scored for “mixed” transformed, and nontransformed colonies. Every point represents a group mean transformed, “pure” derived from 4000 plated cells. Data shown are derived from experiment 2. Similar results were obtained in switch experiments la and lb except, that plating efficiencies were higher. Initial low pH A: initial high pH, A.
20/31 cases with an average advantage of 0.9 %. Thus the cell number plated modified both the plating efficiency and the % t/cell, but did not affect I’ and 31 colonies differentially. 4. Zffect of Virus Dilution The effect of a twofold virus dilution on plating efficiency could be examined in experiments la and lb and was highly significant (Table 2A). Plating efficiency was consistently greater at both low and high pH when cells had been exposed to the more concentrated virus suspension. Hence either nontransforming virus particles, or unidentified components in the original polyoma virus suspension increased the plating efficiency of cells destined to produce normal colonies. As no significant clumping of cells by virus was observed in any of these experiments, this cannot be invoked in the explanation. Over the range of virus concentration used, significant effect’s on 70 t/cell were not observed (Table 2B). This is in agreement with t,he findings of Stoker and Abel (1962).
5. Consistency of Data, Absence of Bias in Scoring Table 1 presents the data obtained in “switch” (la, lb, 2) and “pulse” (3) experiments. To ensure the validity of t’he conclusions drawn regarding the influence of the various factors under consideration, the consistency and lack of bias of the actual replicate observations were tested. Internal consistency of replicate observations with respect t,o the proportion of transformed to normal colonies (% t/col), was tested for and found in 2 X f contingency x2 tests (Mather, 1949) carried out on the fifteen separate overall % t/co1 values of switch experiment 2 and the pulse experiment 3 (Table 6). Similar analysis of experiments la and lb was not possible as t’otal colonies had only been counted on two or three replicate plates in each group. Individual plate expectations were too small to permit analogous analysis regarding t,ype (P and M) of transformed colony. Bartlett’s (1937) test for homogeneity of
KISCH
AND
SUBAK-SHARPE
7 DAY LOW PE
‘DAY HIGH PE
^
IJ
I
I
I
1
I
I
1
z
3
4
5
6
MIDPOINT
OF
TWO
DAY
PULSE
OF
LOW
pH.
FIG. 4. Effect of time of application of a 2-day “pulse” of low pH on plating efficiency. In pulse experiment 3, 40 petri dishes with feeder layers were inoculated, each with 500 polyoma virus-infected cells, and placed into 5 groups of 8 replicates each. One group was incubated at low pH for 2 days, then changed to high pH and incubat,ed for a further 5 days. The four other groups were initially incubated at high pH, then at different times given a a-day pulse of low pH, followed by further incubation at high pH. All other conditions were as for Fig. 3.
variances was utilized to demonstrate that all the % t/cell and plating efficiency variances respectively were homogeneous (Table 7). These tests, t’aken together, rule out’ any possibility that extra P colonies might have originated at high pH through transformed (but not nontransformed) cells floating off from established colonies and initiating new colonies with high efficiency. Firstly t’his should have frequently resulted in localized areas containing ‘kwarms” of transformed colonies. Such “swarms” were not observed at the time of the scoring. Secondly, this
should generate high replicate plate variation, and hence significant % t/co1 differences within samples, whereas Table 6 shows t’hat this was not the case. Thirdly, this would imply heterogeneity of variances with high pH variances greater than low pH variances. This too was not found (Table 7). In scoring transformed colonies as P or &I, possible observer bias must be taken into account. If present, observer bias should reduce variation between replicate plates and thus generally decrease the significance levels, pushing the probability distribution of the
pH EFFECT
ON BHK21
CELL
TI’LANSFOIIMATION.
199
I
“blind” by a single observer, and at the time of scoring possible systematic differences between P and 11 colonies were not anticipated. For these reasons observer bias may be dismissed from consideration.
contingency x2 to the left. But Table 6 gives no indication of such influence. Other tests which investigated the behaviour of the M/P ratio also failed to indicate bias. Furt,hermore all cultures had been scored
0
5 0
0 0
0 0 0
0 0
3.75
0
0
0 : :
0 al
0
2.5
.A . s-
0
0
0
0 0
0
0 0
1.25
0
0 0
0
0 0
0
0
0 0
I
1
35
40
1 45 %
PLATING
I
I
50
55
EFFICIENCY
FIG. 5. Lack of relationship between plating efficiency and the yO f/cell. The data are from experiments la and lb. All results from plating 200 cells per plate (0) and 400 cells per plate (a) have been plotted separately. Plating efficiency and y0 t/cell are not correlated.
PWBABILITY
TABLE 6 UISTRIBLJTION OF CONTINGENCY x2 TESTS ON TO t/co1 FOK THE 15 GROUPS OF REPLICATES IN EXPERIMENTS 2 ASU 3”
Probabilit) Groups
0.99-0.95~.9-0.8~.7-0.5~).3~.2-0.1~.05~.01 1 222322
(1Every group consists of 8 replica plates. The proportions on individual replica plates is tested for internal consistency
1
of transformed to normal group by group.
colonies
scored
TABLE 7 BAR’I’LETT’S TEST FOR HOMOGENEITY OF s, t/cell and PLATING EFFICIENCY VARIANCES~ l%pt. no. 2 3
%t/cell XL Xi
17.040 8.324
Plating efficiency P = 0.2-0.1 P = 0.1-0.05
6.628 4.425
P = 0.94.8 P = 0.5-0.3
a The eight replica plates of every group vary, firstly in the y0 t/cell and secondly in the plating efficiency of individual replica plates. This variation is measured by the variance. The four tests show that within both experiments all the groups’ variances may be considered homogeneous.
200
KISCH
AND SUBAK-SHARPE
DISCUSSION
A prime objective of the experiments was to establish whether the enhancing effect of high pH upon transformation (Kisch and Fraser, 1964) was due to a real increase in the number of transformed colonies, or whether differential selection resulted in an apparent increase. The present analysis, which is independent of t’he plating efficiency of nontransformed cells and therefore of differential selection, establishes COIUAUsively that high pH results in a real and substantial increase in the number of transformed colonies observable after 7 days of incubat’ion. When the pH difference was maintained over the whole incubation period, the percentage increase in %’ t/cell over values obtained at’ low pH reached 200-300 %. How pH operates is not clear. High pH may selectively favour “nascently” transformed cells, allowing them to form colonies. pH does not appear to select differentially the established transformed cells grown in the presence of nontransformed cells (Kisch, unpublished). Alternatively high pH may cause changes in cell physiology which increase t,he probability of the transforming event itself. Preincubation experiments in which cells previously grown at high or low pH are exposed to PV and subsequently incubated at the homologous or heterologous pH could provide interesting supplementary information. In our experiments the final % t/cell observed could always be ascribed to two separable linear functions of high pH. The first and more important of these depends only on the total duration of exposure to high pH: the second depends on the actual time after infection when exposure to high pH took place, since the effectiveness of high pH in increasing the % t/cell was found to diminish progressively with time. The results indicate that the total time of exposure affects the number of both P and M colonies which develop, t’hough to different extent as shown by the generally lower M/P ratio found at high pH. This change in M/P is not due to suppression, or delay of appearance or increased difficulty of scoring of M colonies. The pulse experiment indi-
cates that the effect of pH on development of P colonies is a progressively diminishing one, whereas t’he effect on 11 colonies is not,. This finding requires confirmation as it derives only from this one experiment. Delayed transformat’ion giving rise to M colonies has previously been described (Stoker, 1963). On t’he basis of all t’he data presented here it is concluded that P colonies also continue to arise for at least 3-4 days after infection. This implies t’hat many “nascent” t)ransformed cells delay their first division cycle and remain for some time, viable but dormant, on the feeder layer, where low pH prevents them from forming colonies. Alternatively, high pH may induce physiological changes in viable but dormant, infected cells and so increase t,he probability of their undergoing the transforming event prior to initiation of cell division. Low pH does not delay transformation, for this would result in an increase in the number of mixed colonies. Two further hypotheses, neither of which seems attractive, should be ment’ioned: (1) Potentially transformed cells may divide forming potentially transformed clones, all of whose cells could still be affected synchronously by pH. Mechanistically this seems highly improbable. (2) The ultimate fate of the early arisen ,1L colonies may be pH dependent; e.g., low pH may favour overgrowth by the nontransformed component, and the converse occur at high pH (Stoker, 1963). However, t*his would imply that at, an intermediate pH the proportion of M colonies should be maximal. Data supporting this hypothesis have not been obtained. The very pronounced and irreversible effect of initial pH on plating efficiency is of interest as it ran counter to the effect of pH on transformation. The effectiveness of low pH in raising plating efficiency was greatest on t’he first day after infection and then rapidly declined. This suggests that the plating efficiency of nontransformed cells was only modifiable during the very earliest stages of colony formation and that pH st lect’s cells manifesting physiological, not genet’ical, differences. Low pH may increase the potentiality of cells to attach to glass and undergo t,heir first division.
pH EFFECT
ON BHK21
CELL
ACKNOWLEDGMENT We are indebted to Professor M. C. P. Stoker for his kind interest and many helpful suggestions iii the preparation of the manuscript. One of us (.4. L. K.), would also like to express his thanks to the members of the Inst,itut,e of Virology for their friendly hospitality and to Mrs. M. Macnamara for excellent, technical assistance. REFERENCES M. S. (1937). Properties of sufficiency and statistical tests. Proc. Roy. Sot. A160, 268. KISCH, A. L., and FRASER, K. B. (1964). The effect of pH on transformation of BHK21 cells by B.~RTLETT,
TRANSFORMATION.
I
201
polvoma virus. I. Relationship between transformation rate and synthesis -of viral antigen. Virology 24, 186-192. MXPHERSON, I., and MONTBGNIER, L. (1964). Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291-294. M.~THER, K. (1949). “Statistical Analysis in Biologv.” Methuen. London. STOKER,” M. (1963). belayed transformation by polyoma virus. Virology 20, 366-371. P. (1962). Conditions STOKER, M., and ABEL, affecting transformation by polyoma virus. Cold Spring Harbor Symp. Quant. Biol. 27, 375386.
28,
188-201 (1966)
The Effect
of pH on Transformation Polyoma II. Kinetic
ALEXANDER Medical
Research Council
L. KISCH1,2
Experimental
of BHK21 Cells
by
Virus
Considerations AND
JOHK
H. SUBAK-SHARPE
Virus Research L’nit, Institute Glasgow, Scotland
of Virology,
University
of Glasgow,
Accepted September 8, 1965 Changes of the sequence or of the relative duration of combined high and low pH incubation affect the frequency with which transformed colonies arise in cultures of polyoma virus-infected BHK21 cells. In general, high pH incubation increases the number of transformed colonies but reduces the plating efficiency of nontransformed infected cells while low pH incubation has opposite effects. However the effect of pH on t,ransformationisindependent of its effect on the plating efficiency of nontransformed cells. Low pH incubation also increases the rat,io of “mixed” to “pure” transformed colonies. Two-day “pulses” of low pH reduce the number of transformed colonies, but this “pulse” effect diminishes progressively with time, apparently because of a progrescolonies. The effect sively diminishing inhibition of formation of “pure ” transformed of pH on plating efficiency is greatest on the first day post infection and then rapidly declines at a different rat,e than the effect on transformation. transformed colonies arise continuously aft,er The data confirm that “mixed” transformed colonies also continue to arise for infection and suggest that “pure” 3-4 days after infection. They further indicate that the development of “mixed” and “pure” transformed colonies is affected difl’erently by high and low pH. The concept of “nascent” transformed cells as opposed to established transformed cells is discussed INTRODUCTION
It has been reported (Kisch and Ii‘raser, 1964) that the pH at which BHK21 cells infected with polyoma virus (PV)3 are incubated affects the frequency with which 1 Aided by a grant for a Postdoctoral Research Scholarship from the American Cancer Society. 2 Present address: Department, of Medicine, School of Medicine, University of New Mexico, Albuquerque, New Mexico. 3 Abbreviations used in text: PV = polyoma colonies; y0 t/cell = virus; t = all transformed number of transformed colonies per 100 infected cells plated; 7c t/co1 = number of transformed colonies per 100 colonies scored; M = mixed kansformed colonies; P = pure transformed colonies; pC0~ = partial pressure of carbon dioxide. 188
transformed colonies arise. The experiments described here were intended to establish whether this enhancing effect of high pH on transformation results from a net increase in the number of transformed colonies, or merely from different’ial select’ion at the cellular level. To investigate the kinetics with which transformed colonies arise, the pH of PV infected cell cultures was altered during the incubation period at varying time intervals following infection. Stoker (1963) observed in microdrop cultures that transformation may be delayed for at least 5 cell divisions in colonies derived from single PV-infected cells. It’ therefore seemed relevant t,o determine whether trans-
pH EFFECT
ON BHK21 CELL THANSFOILMATION.
format ion in pure as well as in mixed transformed colonies is subject to delay, or whether it occurs only within a critical short time after infection. Finally, it seemed of int’erest to determine whether any relationship between the effect of pH on the plating efficiency of infected cells (Kisch and Fraser, 1964) and its effect on trltnsformation could be demonstrated. MATERIALS
AND METHODS
T7irus, cells, medium, and transfomation assay. The origin of the stock polyoma virus and of t,he BHIi21/C13 cells employed in all assays, the constit’ution of the media used, ard d&ails of the method of pH measurement a,s well as of the transformation assay techniques employed have been described previously (Kisch and Fraser, 1964). In the present series of experiments all transformed colonies (t) have been scored by a single observer as either “pure transformed” (P) or as i‘mixed transformed” (XI). Characteristically 1\1 colonies cont’ained a sector of cells showing random orientat’ion, the remainder of the colony exhibiting bhe regular parallel orientation of cells typical of normal colonies (St’oker, 1963). Design of experiments. Two experimental designs were employed. In the first or “switch experiment”, cultures of PV infected cells were kept at, high pH (e.g., Expt. 2, 7.6-7.8) following infection, while a duplicate set, of infected cultures was incubated at low pH (6.9-7.1). After varying periods of incubation, the plates were switched reciprocally from high to low pH or vice versa. T\‘o culture was switched more than OIICC. After a total of 7 days’ incubation t,he cultures were scored for the presence of M, P, and nontransformed colonies. The pH switch was accomplished by changing the pCOz of the incubator jar. In the second type of experiment) (“pulse experiment”), replicate cultures were exposed t)o a two-day pulse of low pH, either immediately or after an initial incubat’ion period at, high pH, and t’hen returned t,o high pH incubation. There are two ways in which the number of transformed colonies is usually recorded (Stoker and Abel, 1962). The first, the % t/
I
189
cell, relat’es the number of transformed colonies scored to the number of infected cells initially plated. The second, the % t/col, relat’es the number of transformed colonies scored to the total number of transformed and normal colonies observed. The % t/cell, unlike the % l/co& is independent of the plating efficiency of “normal” cells provided there is no interaction between normal and transformed colonies which varies over the range of colony numbers in the experiments. Assuming no such interaction, the 70 t/cell is the plat’ing efficiency of “nascent”’ transformed cells, and comparisons of % t/cell values reflect solely the kinetics of transformed colony formation. A “nascent” transformed cell should be distinguished from an established transformed cell. As used here it, describes a newly arisen, phenotypically t’ransformed cell whose immediate parent cell was bot’h phenotypically untransformed and descended from untransformed lineage. As the physiological homeostatic equilibrium at this point in descent probably undergoes drastic changes, one cannot a prio~i assume t’hat a “nascent” transformed cell behaves either like a normal or like an established transformed cell. As we have no information regarding possible treatment-dependent variation in in the relative plating efficiency of normal and nascent transformed cells, the data have been expressed in terms of 70 t/cell vslues. RESULTS 1. Effect o.f Initial
pH on Transformation
(5% t/cell) The effect which initial pH exerted on transformation is shown by the data in Table 1, column 8, and also by the analysis of variance of the results of experiments la and lb given in Table 2(B). For convenience the data obtained from plating the higher cell inocula only are plotted in Fig. 1 although the remaining data also complet’ely support the following conclusions: In contrast to the rapidly diminishing effect of pH on plating efficiency (see Section 3) maintenance of an initial high pH progressively increased t’he number of transformed colo-
190
KISCH
AND
SUBAK-SHARPE
TABLE 1 TRANSFORMED COLONYTOTAL,% ~/CELL,% PLATINGEFFICIENCYAND~
2
3
Expt. no.b
Final pH
4 5 Transformed colonies “pure )I “mixed”
6
7
8
9
Replicates
“/n Plating efficiency
Ojof/cell
M/PC
High
Low
7
-
la lb 2
7.6 7.6 7.6
30; fid 33;12 56; 6
77;lO 47 $4 71;ll
6;2 6;2 8;2
41.9;44.8 43.1;54.0 21.8;24.2
4.45;4.00 3.33;6.50 3.18;3.40
2.42 1.36 1.32
5
-
la lb 2, 3
6.8 6.8 7.0
41; 7 27; 8 65;ll
44;ll 52; 6 41; 9
5;2 6;2 8;2
43.3;47.8 34.6;35.5 21.6;26.0
4.25;4.50 3.29;3.50 2.65;4.00
1.15 1.66 0.66
4
-
2
7.1
36; 7
43; 8
8;2
21.7;29.0
1.98;3.00
1.19
i 3
-
:;I; 2
6.8 6.8 7.1
8; 4 8; 7 39; 5
17; 7 20; 8 46; 9
2;2 4;2 8;2
45.3;50.8 40.5;47.0 20.6;22.8
3.13;2.75 1.75;3.75 2.13;2.80
2.00 1.87 1.25
2
-
2
6.9
24; 7
41; 3
8;2
21.3;21.2
1.63;2.00
1.42
-
la lb 2
6.7 6.7 7.0
3; 1 2;; x
4; 0 5; 1 28; 8
2;l 3;l 8;2
46.0;48.5 42.8;49.5 25.1;25.6
0.88;0.50 0.83;1.00 1.40;2.80
1.00 1.00 1.04
k 2
6.6 6.6 7.0
4; 2 6; 1 11; 0
25; 4 16; 5 26; 1
6;2 6;2 8;2
55.4;50.8 51.5;52.5 30.6;36.4
1.21;1.50 0.92;1.50 0.93;0.20
4.83 3.00 2.45
i:
la lb 2
7.5 7.5 7.7
11; 2 8; 3 32; 8
31; 4 18; 8 34; 7
6;2 5;2 a;2
48.5;53.5 49.6;52.0 30.4;31.6
1.75;1.50 1.30;2.75 1.65;3.06
2.F9 2.36 1.03
4
2
7.7
28; 7
33; 3
8;2
32.2;39.4
1.53;2.00
1.03
-
3 i
la lb 2
7.3 7.3 7.7
10; 1 16; 2 22; 3
29; 9 35; 5 53; 6
4;l 5;l 8;2
50.8;48.0 43.8;47.0 30.9;31.2
2.44;5.00 2.55;3.50 1.88;1.80
3.45 2.22 2.36
-
2
2, 3e
7.8
43; 2
40; 5
8;2
28.1;28.8
2.08;1.40
1.00
-
1 1 1
:;1 2
7.3 7.3 7.7
14; 6 30; 5 40; 3
40; 7 46; 9 62; 8
6;2 6;2 8;2
51.0;52.8 50.8;56.8 27.0;34.4
2.25;3.25 3.17;3.50 2.55;2.20
2.35 1.57 1 .63
i: 3
:*7” 717
45; 4 44; 3 49;lO
45; 9 53; 6 37 ;lO
8;2 8;2 8;2
25.6;29.2 24.8;28.8 23.1;29.6
2.25;2.60 2.43;1.80 2.15;4.00
1.10 1.26 0.80
7’ 5
1 : -
; 7
-
9 4
5
-
Q In “switch” experiment,s (la, lb, 2) cultures of PV-infected BHK21 cells were incubated for the specified number of days at initial pH and then reciprocally switched from high to low pH or vice versa. In “pulse” experiment (3),, replicate cultures were exposed to a-day pulse of low pH, either immediately or after an initial incubation period at high pH and then returned to high pH incubation. Duration of all experiments was 7 days. Data refer to the totals obtained by summing t,he replicate plate observations at each ooint. b Polyoma &us was stock PlOl. Exposed multiplicity of infection per cell for experiments la, 2, and 3 was 5300 PFU and for lb 2650 PFU. all “mixed” transformed colonies cM/P = all “pure” transformed colonies d Values listed in columns 2-6 to the left of a semicolon refer to groups of plates seeded with 400 (Expts. la and lb) or 500 (Expts. 2 and 3) infected cells per plate, while values listed to the right of a semicolon refer to groups in which each plate was seeded with 200 (Expts. la and lb) or 250 (Expts. 2 and 3) infected cells each. 6 Point which features jointly in experiments 2 and 3.
pH EFFECT ON BHK21CELL TRANSFORMATION.I TABLE
2
ANALYSIS OF VARIANCE OF EXPERIMENTS Item Part
A: Effect of initial
Degrees of freedom pH on plating
la AND 1bU Sum of squares
Probability
72.262 44.3% 7.942 7.776 7.320 13.088 4.8G7
66.910 9.650 1.669 4.511 51.080
43.6G4 4.474 2.386 0.55G
eficiency 15 1 1 3 1 10 3
Total Effect of pHh Equality of VD Effect of day Initial periodd Interactions Interaction day X pH Part B: Effect of initial pH on yo t/cell Total Effect, of pHh Equality of V1lc Effect of day Int,eract ionse Interactions Hay Day pH Day
191
x pHe x VI> X VI) x pH x VI)
15 1 1 3 10 broken down 3 3 1 3
n In Part A t,he differences in plating efficiency of the 16 experimental points of experiment la and items are responsible for the observed differences. Part, B lb are analyzed to discover what “effect” shows similar analysis with respect to differences in the 70 t/cell. * Tests whether the INITZAAL pH (either high or low) influences the results; highly significant, both with plating efficiency and 70 t/cell. c VD is virus dilution. This significantly affects the plating efficiency. d Shows that the entire effect is exerted during the initial period. e Shows that the only important interactions that influence the 70 t/cell are those between pH and day of pH switch.
nies, while maintenance at an initial low pH progressively reduced their number. In either case the distribution of successive points suggests an approximately linear relationship between duration of the initial pH and t,he observed 70 t/cell. The calculated first-order regressions (Mather, 1949) for the six series plotted in Fig. 1 are presented in Table 3A. The slopes of 5 of t’he calculated regressions deviated significantly from the “null” hypothesis of zero slope, while the sixth just failed to do so. Since the three high pH regressions as well as the three low pH regressions were statistically c*onsistent, appropriate “mean” regression lines were calculated. Here experiment’ 2 resuhs received twice the weight of t’he results from experiment la or lb as each point arose from 4000 cells plated, while t,he results of experiment la or lb arose on average from about 2000 cells plated.
The calculated “mean” regressions suggest that high pH increased the % t/cell on average by 0.39 per day; and conversely that low pH decreased the % t/cell by 0.33 per day. But this calculation is based on the assumption that the effect of pH per day was constant over the duration of the experiment. The data obtained from the pulse experiment permit evaluation of the validity of this assumption and of the effectiveness (in reducing the % t/cell) of standard a-day pulses of low pH applied at various times aft,er plating. Here each culture was incubated for a total of 5 days at high pH and 2 days at low pH, the only variable being the timing of the low pH pulse. The relevant data from Table 1 are plotted in Fig. 2 (P + RI) together with the calculated first-order regression line, which is significant’ (RcP+M), Table 3B). This establishes
192
KISCH DAYS
AND
MAINTAINED
AT
SUBAK-SHABPE
INITIAL
pH
UNTIL
CHANGEOVER
HIGH
p”;
I*
0
LCIW
pH;
DILUTED
1.3
EXPT.
la
4
*I
‘I
‘I
-I
I-
1’6
I’
lb
4
I’
,.
.*
‘*
**
1.6
..
Ib
,
.I
,.
..
..
3,
I3
,’
2
u
-
.I
,,
II
.I
13
.’
2
.
INITIAL
VIRUS
DlLUTED
13EXPT
,NITIAL
VIRUS
Fig. 1. Effect of duration of initial pH on the “/b t/cell. In experiments la and 2 polyoma virus stock 101 was diluted 1:3 and in experiment, lb, 1:6. The experiments were carried out as described for Fig, 3, except that experiments la and lb employed only 6 replicate petri dishes per group, each seeded with 400 infect,ed cells. The combined “mixed” and “pure,” transformed colony totals per 100 cells plat,ed are plot.ted against the period during which the initial pH was maintained until pH switch. In some groups plates were lost, as showninTable 1. Initial highpH: l , virusdiluted 1:3, Expt. la; A, 1:6, Expt. lb; v, 1:3, Expt. 2. Initial low pH: 0, virus diluted 1:3, Expt. la; A, 1:6, Expt. lb; V, 1:3, Expt. 2.
that the effect of a a-day low pH pulse progressively declined with time. The simple first-order regressions fitted to the switch data are therefore not an adequate model, and a more complex relationship must hold. Previous considerations suggest. a model combining (1) the linear enhancing effect of incubation time at high pH on the number of transformed colonies; and (2) the linear decrease in effectiveness of high pH with the day post infection which high pH was experienced. Algebraically expressed this means
where T = total number
of kansformed
colonies; a, b, and c are constants; c = sum of; q = 1 for each day at high pH and 0 for each day at low pH; and t = 1 on day 1, 2 on day 2, and so on. This equation was fitted to the “high cell number plat’ed” data of (switch) experiment 2 and (pulse) experiment 3 (Table 1, column 8). Table 4 shows the excellent fit, of this relatively simple unified model wit,h all the data. Therefore the % t/cell obtained 7 days post infection can be considered as a simple function of the total time spent at a high pH and of the precise days post infection at which this pH was experienced. We are indebted to Mr. M. W. Birch of the Mathematics Department’ for both the
colonies;
0.05-0.01 0.05-0.01 >0.2
a Discussed at length in the text. * R~+M, Kegressioll of all transformed only.
RM
RP
PLP.td
of “pure”
transformed
Y = (2.36+0.04)+(0.142f0.025) Y = (1.23f0.05)+(0.139f0.029) Y = (1.13+0.0G)+(0.003f0.037)
to the pulse experiment
RF, regression
of 2-day pulse of low pH, jtted
colonies
only;
(r-3.2) (z-3.2) (x-3.2)
RM, regression
(z-3.2)
Y = (2.153f0.154)+(-0.331f0.063)
Mean Low pH Part. B. On midpoint
(z-3.2) (x-3.2) (r-3.1)
(z-3.2)
(x-3.2) (r-3.2) (2-3.1)
1’ = (2.421&0.342)+(-0.3G7ztO.133) Y = (2.25&0.104)+(-0.375fO.042) Y = (1.968+0.085)+(-0.29OzkO.038)
0.1-0.05 0.01-0.001
pH, jitted to the data from the switch experiments 0.05-0.01 Y = (2.783+0.275)+(0.554&0.108) 0.01~.001 Y = (2.108~0.158)+(0.383zt0.063)
la Low pH lb Low pH 2 Low pH
at initial
Regression line E’ = (,?j f s&J -+ (b f Sb) (x -P)
t/&ID
Y = (2.21&0.140)+(0.389~z0.057)
On days maintained pII pH pH
Probability of null hypothesis
3
Mea11 High pH
Part A: la High lb High 2 High
Item
TABLE LINEAR REGRESSIONS OF “/c
of “mixed”
0.95
0.14.05
ha - bib
transformed
0.054.02
6,-b,
O.G-o.5
0.3-0.2
ha, - bi:
colonies
0.2411
0.4-0.3
hi, - bz
Probability of regressions being the same
194
KISCH
AND
SUBAK-SHARPE
3.0
2.0
2 zl 0 4 D a
1.0
0 0
I
I
I
I
I
I
I
2
3
4
5
6
MIDPOINT
OF
TWO
DAY
PULSE
OF
LOW
I
pH.
Fig. 2. Effect of time of application of a 2-day pulse of low pH on the c/c t/cell. Conditions were asfor Fig. 4. Plotted are (1) all transformed colonies (P + M): 0; (2) “pure” transformed colonies (P) A; and (3) mixed transformed colonies (M), 0. The P + M totals observed after 7 days at high pH and after 7 days at low pH are shown for reference. Also plotted are the calculated regression lines (Table transformed colonies (Rp), and “mixed” transformed 3B) for all transformed colonies (&=+M), “pure” colonies (RM).
likelihood estimation of the constants and the test for goodness of fit’.
maximum
2. Differential E$ect of Low pH on Development of P and Al T~ansjormed Colonies Differential effects of maintained pH on P and M colonies can be detected from comparisons of M/P ratios. Where P decreases relatively more or increases relatively less t’han M, this ratio will increase. Table 5 presents the M/P ratios observed after incubation for 7 days when the duration of the initial incubation period at high or low pH was varied (columns 3 and 4). Appropriate
comparison of these values in point for point comparisons (column 5), or with the M/P ratios obtained after the standard 7-day period of incubation at low pH (columns 6 and 7) are also included. Column 5 shows that at almost every point relatively more M (or fewer P) colonies were produced when the initial maintained pH was low. Columns 6 and 7 show that relatively more M (or fewer P) colonies arose after 7 days of maintained low pH than with any other treatment. Analysis of the data available does not reveal a significant progressive change in M/P as the duration of initial low pH incu-
pH EFFECT TABLE FITTING
4
Expectation
LOW
7 5 4 3 2 1
Pnlse
CELL
OF THE EQUATION T = a+b xq+cxtq TO THE DATA OF SWITCH EXPICRIMENT 2 AND THE PULSE EXPERIMENT 3,*
Days at initial pH .High -
ON BHK21
7 5 4 3 2 1 2 3 4
a + a + a + a + a + a+ a a + a + a + a + a + a + a + a +
7b + 5b + 4b + 3b + 2b + b+
28~ 15~ 10~ 6Gc 3c c
2b 3b 4b 5b 6b 5b 5b 5b
13~ 1%~ 22c 25~ 27~ 23~ 21~ 19c
+ + + + + + + +
Transformed colonies (P + M) Estimated
Observed
116.4 99.8 89.9 78.9 66 8 53.6 39.4 56.0 G5.9 7G.9 89.0 102.2 91.2 93.3 95.5
127 106 79 85 65 56 37 66 61 75 83 102 90 97 86
n Goodness Maximnm
of fit x& = 7.26; P = 0.9-0.8 likelihood estimates: a = 39.42 b = 15.31 c = -1.08 Standard error covariance matrix +18.30 -5.06 +0.23 -5.06 +F.63 -1.24 -1.24 +0.29 [ +0.23 * Symbols are explained in the text.
1
bation or high pH incubation is increased. Hdwever, this test was rather insensitive as it depended frequently on ratios derived from small numbers of observed colonies and further data would be of int)erest. Having shown that the effectiveness of a low pH pulse (in reducing t,he % t/cell) progressively diminished with time post infection, it was examined whether P and M colonies were equally affected. The relevant analysis given in Table 3B and Fig. 2 shows that the actual time post infection at which cells were exposed to a low pH pulse wan irrelevant to the number of Xl colonies observed, but only affected the number of P colonies. The regression of P colonies with time of pulse significantly deviates from zero and from that of M colonies, while this latter regression does not deviate significantly from zero (Table 3B). The progres-
TRANSFORMATION.
I
195
sive loss of effectiveness of low pH on % t/cell was thus due to t’he progressively increasing escape from suppression of pure transformed colonies, not to suppression or delay of appearance, or increased difficulty in scoring, of mixed transformed colonies. Thus, there are two independent) sources of evidence t,hat low pH differentially affected P and 111 colonies, resulting in an increased M/P ratio. In the pulse experiment this was due to progressive increase in t,he number of P colonies. In the switch experiments AI/P ratios mere generally higher at low initial pH, and in all three experiments of this type, were highest aft’er 7 days of low pH incubation. The act(ua1 colony numbers in Table 1 (columns 4 and 5) show that t.his effect was not due to higher plating efficiency of normal but potentially mixed colonies at low pH. Additional data are needed to extjend this investigation. 3. E$ect of pH on. Plating Ejiciency The effect which the duration of the initial pH exerted on the efficiency of plating is illustrated by data from experiment 2 plotted in Fig. 3. In all switch experiments low initial pH invariably led to a 20-50 % greater plating efficiency than high pH. Moreover, the influence of initial pH on the plat,ing efliciency was obviously greatest on the first day, after which it rapidly declined. The influence of the t,ime of application of pH on plating efficiency was observed in the pulse experiment. The data are plotted in Fig. 4. These data were analyzed by taking the total increase in plating efficiency at 7 days (of “low pH treated” over “high pH treated” cells) as 100 %. Then 2-day low pH pulses 011 days O-2, 1-3, 24, 3-5, and 5-7 resulted in respect,ive increases of 66.3, 41.6, 33.7, 16.9, and 0.0%. This yields the following estimates of relative effectiveness for each day: 1 = 41.5 %; 2 = 24.8 %; 3 = 16.8%; 4 = 16.9 %; 5, 6, 7 = 0.0 %. The relative effectiveness of day one can be calculated independently from the data of the simultaneously performed switch experiment (experiment 2) as 67.4 % and 52.8 %. Thus low pH uniformly resulted in greater plating efficiency and was most effective on t)he first day with fairly rapid
196
KISCH
AND
SUBAK-SHARPE
TABLE DEPENDENCE
OF THE RATIO “MIXED”
Column: 1 Day of maintained initial pH
* L/H
2 Expt. no.
5
THANSFORMED: “ PURE”
3 Initial high PH (HI
5
6
L/H”
L7/H
7 U/L
4.83 3.00 2.45
2.0 2.2 1.9
2.69 2.30 1.06
2.3 1.4 1.6
4.2 1.8 3.7
1.8 1.3 2.3
1.19
1.03
0.9
2.1
2.4
la lb 2
2.00 1.87 1.25
3.45 2.22 2.36
1.7 1.2 1.9
2.4 1.6 1.9
1.4 1.4 1.1
2
2
1.42
1.00
0.7
1.7
2.4
1 1 1
la lb 2
1.00 1.00 1.04
2.35 1.57 1.63
2.3 1.8 1 .6
4.8 3.0 2.4
2.1 1.9 1.5
7 7 7
la lb 2
5 5 5
la lb 2
4
2
3 3 3
2.42h 1.36 1.32
4 Initial low PH (L)
TRANSFORMED COLONIES UPON pH
probability
of 12:2 result if no difference P Yat,es correction p; x: = 7.14 0.02-0.01
decline reaching zero by the fifth day. This is also confirmed by the analysis of variance of the data of swit,ch experiments la and lb (Table 2A). As was shown earlier, high pH incubation progressively increased the % t/cell but was initially detrimental to plating efficiency. Data from experiments la and lb plotted in Fig. 5 indicate that no general correlation existed between % t/cell and plat,ing efficiency, irrespective of cell numbers seeded per culture. However the number of cells plated affected both the plating efficiency and % t/cell. (Table 1, columns 7 and 8). In 28 of 31 comparisons (14/16 in experiments la and lb and 14/15 in experiments 2 and 3) plates seeded with the lower cell number had higher plating efficiency-on average 3.4 %. While the number of cells plated did not significantly modify M/P rat’ios under the pH conditions tested, it did
affect % t/cell values, for 22 of 31 comparisons showed a higher % t/cell when plates were seeded with the lower cell numbers (12/16 in experiments la and lb and lo/15 in experiments 2 and 3). On average this advantage was 0.72 in experiments la and lb and 0.44 in experiments 2 and 3. These findings differ from those of Stoker and Abel (1962), who found that under their experimental conditions the % t/cell was unaffected by the total number of cells per plate. It is unlikely that these findings stem from easier recognition and enumeration of normal as well as P and ?(/I transformed colonies when the colony density per culture is relatively low. Inherent in such an hypothesis is the expectation t’hat t/co1 values should remain unchanged, whereas in fact t/co1 values from plates seeded with the lower cell number exceeded t/co1 values from plates seeded with high cell numbers in
pH EFFECT
I
1 0
ON BHK21
f 1
CELL
I 2 DAYS
OF
TRANSFOKMATION.
I 3 INITIAL
I b pH
UNTIL
I 5
197
I
I 6
I 7
CHANGEOVER
Fig. 3. Effect of duration of initial pH on plating efficiency. Each of 96 petri dishes with feeder layers was inoculated with 500 polyoma virus-infected cells, then t,he dishes were distributed into 12 groups of 8 replicates each, and incubated at either high (7.7) or low (7.0) pH. After different periods of time for different groups, all replicates in a group were changed from high to low pH or vice versa by adjustment of the pC0, of the incubator jar. After a total of 7 days’ incubation, all cultures were scored for “mixed” transformed, and nontransformed colonies. Every point represents a group mean transformed, “pure” derived from 4000 plated cells. Data shown are derived from experiment 2. Similar results were obtained in switch experiments la and lb except, that plating efficiencies were higher. Initial low pH A: initial high pH, A.
20/31 cases with an average advantage of 0.9 %. Thus the cell number plated modified both the plating efficiency and the % t/cell, but did not affect I’ and 31 colonies differentially. 4. Zffect of Virus Dilution The effect of a twofold virus dilution on plating efficiency could be examined in experiments la and lb and was highly significant (Table 2A). Plating efficiency was consistently greater at both low and high pH when cells had been exposed to the more concentrated virus suspension. Hence either nontransforming virus particles, or unidentified components in the original polyoma virus suspension increased the plating efficiency of cells destined to produce normal colonies. As no significant clumping of cells by virus was observed in any of these experiments, this cannot be invoked in the explanation. Over the range of virus concentration used, significant effect’s on 70 t/cell were not observed (Table 2B). This is in agreement with t,he findings of Stoker and Abel (1962).
5. Consistency of Data, Absence of Bias in Scoring Table 1 presents the data obtained in “switch” (la, lb, 2) and “pulse” (3) experiments. To ensure the validity of t’he conclusions drawn regarding the influence of the various factors under consideration, the consistency and lack of bias of the actual replicate observations were tested. Internal consistency of replicate observations with respect t,o the proportion of transformed to normal colonies (% t/col), was tested for and found in 2 X f contingency x2 tests (Mather, 1949) carried out on the fifteen separate overall % t/co1 values of switch experiment 2 and the pulse experiment 3 (Table 6). Similar analysis of experiments la and lb was not possible as t’otal colonies had only been counted on two or three replicate plates in each group. Individual plate expectations were too small to permit analogous analysis regarding t,ype (P and M) of transformed colony. Bartlett’s (1937) test for homogeneity of
KISCH
AND
SUBAK-SHARPE
7 DAY LOW PE
‘DAY HIGH PE
^
IJ
I
I
I
1
I
I
1
z
3
4
5
6
MIDPOINT
OF
TWO
DAY
PULSE
OF
LOW
pH.
FIG. 4. Effect of time of application of a 2-day “pulse” of low pH on plating efficiency. In pulse experiment 3, 40 petri dishes with feeder layers were inoculated, each with 500 polyoma virus-infected cells, and placed into 5 groups of 8 replicates each. One group was incubated at low pH for 2 days, then changed to high pH and incubat,ed for a further 5 days. The four other groups were initially incubated at high pH, then at different times given a a-day pulse of low pH, followed by further incubation at high pH. All other conditions were as for Fig. 3.
variances was utilized to demonstrate that all the % t/cell and plating efficiency variances respectively were homogeneous (Table 7). These tests, t’aken together, rule out’ any possibility that extra P colonies might have originated at high pH through transformed (but not nontransformed) cells floating off from established colonies and initiating new colonies with high efficiency. Firstly t’his should have frequently resulted in localized areas containing ‘kwarms” of transformed colonies. Such “swarms” were not observed at the time of the scoring. Secondly, this
should generate high replicate plate variation, and hence significant % t/co1 differences within samples, whereas Table 6 shows t’hat this was not the case. Thirdly, this would imply heterogeneity of variances with high pH variances greater than low pH variances. This too was not found (Table 7). In scoring transformed colonies as P or &I, possible observer bias must be taken into account. If present, observer bias should reduce variation between replicate plates and thus generally decrease the significance levels, pushing the probability distribution of the
pH EFFECT
ON BHK21
CELL
TI’LANSFOIIMATION.
199
I
“blind” by a single observer, and at the time of scoring possible systematic differences between P and 11 colonies were not anticipated. For these reasons observer bias may be dismissed from consideration.
contingency x2 to the left. But Table 6 gives no indication of such influence. Other tests which investigated the behaviour of the M/P ratio also failed to indicate bias. Furt,hermore all cultures had been scored
0
5 0
0 0
0 0 0
0 0
3.75
0
0
0 : :
0 al
0
2.5
.A . s-
0
0
0
0 0
0
0 0
1.25
0
0 0
0
0 0
0
0
0 0
I
1
35
40
1 45 %
PLATING
I
I
50
55
EFFICIENCY
FIG. 5. Lack of relationship between plating efficiency and the yO f/cell. The data are from experiments la and lb. All results from plating 200 cells per plate (0) and 400 cells per plate (a) have been plotted separately. Plating efficiency and y0 t/cell are not correlated.
PWBABILITY
TABLE 6 UISTRIBLJTION OF CONTINGENCY x2 TESTS ON TO t/co1 FOK THE 15 GROUPS OF REPLICATES IN EXPERIMENTS 2 ASU 3”
Probabilit) Groups
0.99-0.95~.9-0.8~.7-0.5~).3~.2-0.1~.05~.01 1 222322
(1Every group consists of 8 replica plates. The proportions on individual replica plates is tested for internal consistency
1
of transformed to normal group by group.
colonies
scored
TABLE 7 BAR’I’LETT’S TEST FOR HOMOGENEITY OF s, t/cell and PLATING EFFICIENCY VARIANCES~ l%pt. no. 2 3
%t/cell XL Xi
17.040 8.324
Plating efficiency P = 0.2-0.1 P = 0.1-0.05
6.628 4.425
P = 0.94.8 P = 0.5-0.3
a The eight replica plates of every group vary, firstly in the y0 t/cell and secondly in the plating efficiency of individual replica plates. This variation is measured by the variance. The four tests show that within both experiments all the groups’ variances may be considered homogeneous.
200
KISCH
AND SUBAK-SHARPE
DISCUSSION
A prime objective of the experiments was to establish whether the enhancing effect of high pH upon transformation (Kisch and Fraser, 1964) was due to a real increase in the number of transformed colonies, or whether differential selection resulted in an apparent increase. The present analysis, which is independent of t’he plating efficiency of nontransformed cells and therefore of differential selection, establishes COIUAUsively that high pH results in a real and substantial increase in the number of transformed colonies observable after 7 days of incubat’ion. When the pH difference was maintained over the whole incubation period, the percentage increase in %’ t/cell over values obtained at’ low pH reached 200-300 %. How pH operates is not clear. High pH may selectively favour “nascently” transformed cells, allowing them to form colonies. pH does not appear to select differentially the established transformed cells grown in the presence of nontransformed cells (Kisch, unpublished). Alternatively high pH may cause changes in cell physiology which increase t,he probability of the transforming event itself. Preincubation experiments in which cells previously grown at high or low pH are exposed to PV and subsequently incubated at the homologous or heterologous pH could provide interesting supplementary information. In our experiments the final % t/cell observed could always be ascribed to two separable linear functions of high pH. The first and more important of these depends only on the total duration of exposure to high pH: the second depends on the actual time after infection when exposure to high pH took place, since the effectiveness of high pH in increasing the % t/cell was found to diminish progressively with time. The results indicate that the total time of exposure affects the number of both P and M colonies which develop, t’hough to different extent as shown by the generally lower M/P ratio found at high pH. This change in M/P is not due to suppression, or delay of appearance or increased difficulty of scoring of M colonies. The pulse experiment indi-
cates that the effect of pH on development of P colonies is a progressively diminishing one, whereas t’he effect on 11 colonies is not,. This finding requires confirmation as it derives only from this one experiment. Delayed transformat’ion giving rise to M colonies has previously been described (Stoker, 1963). On t’he basis of all t’he data presented here it is concluded that P colonies also continue to arise for at least 3-4 days after infection. This implies t’hat many “nascent” t)ransformed cells delay their first division cycle and remain for some time, viable but dormant, on the feeder layer, where low pH prevents them from forming colonies. Alternatively, high pH may induce physiological changes in viable but dormant, infected cells and so increase t,he probability of their undergoing the transforming event prior to initiation of cell division. Low pH does not delay transformation, for this would result in an increase in the number of mixed colonies. Two further hypotheses, neither of which seems attractive, should be ment’ioned: (1) Potentially transformed cells may divide forming potentially transformed clones, all of whose cells could still be affected synchronously by pH. Mechanistically this seems highly improbable. (2) The ultimate fate of the early arisen ,1L colonies may be pH dependent; e.g., low pH may favour overgrowth by the nontransformed component, and the converse occur at high pH (Stoker, 1963). However, t*his would imply that at, an intermediate pH the proportion of M colonies should be maximal. Data supporting this hypothesis have not been obtained. The very pronounced and irreversible effect of initial pH on plating efficiency is of interest as it ran counter to the effect of pH on transformation. The effectiveness of low pH in raising plating efficiency was greatest on t’he first day after infection and then rapidly declined. This suggests that the plating efficiency of nontransformed cells was only modifiable during the very earliest stages of colony formation and that pH st lect’s cells manifesting physiological, not genet’ical, differences. Low pH may increase the potentiality of cells to attach to glass and undergo t,heir first division.
pH EFFECT
ON BHK21
CELL
ACKNOWLEDGMENT We are indebted to Professor M. C. P. Stoker for his kind interest and many helpful suggestions iii the preparation of the manuscript. One of us (.4. L. K.), would also like to express his thanks to the members of the Inst,itut,e of Virology for their friendly hospitality and to Mrs. M. Macnamara for excellent, technical assistance. REFERENCES M. S. (1937). Properties of sufficiency and statistical tests. Proc. Roy. Sot. A160, 268. KISCH, A. L., and FRASER, K. B. (1964). The effect of pH on transformation of BHK21 cells by B.~RTLETT,
TRANSFORMATION.
I
201
polvoma virus. I. Relationship between transformation rate and synthesis -of viral antigen. Virology 24, 186-192. MXPHERSON, I., and MONTBGNIER, L. (1964). Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291-294. M.~THER, K. (1949). “Statistical Analysis in Biologv.” Methuen. London. STOKER,” M. (1963). belayed transformation by polyoma virus. Virology 20, 366-371. P. (1962). Conditions STOKER, M., and ABEL, affecting transformation by polyoma virus. Cold Spring Harbor Symp. Quant. Biol. 27, 375386.