- Email: [email protected]
Further studies on the induced resistance against isotransplantation of polyoma tumors
214
DISCUSSION
AND
PRELIMINARY
until the second agar overlay did not alter plaque formation or survival of cells. Microof plaques revealed scopic examination many intact stained cells amidst cellular debris. The number of plaques was proportional to the dilution inoculated and the number of plaque-forming units (PFU) was 8.9 X lo7 per milliliter (average of two titrations of E strain). Simultaneous titration of E strain in eggs gave 1.1 X lo8 egg infectious units (EIU) per milliliter indicating an EIU :PFU ratio of essentially one. Allantoic fluid from the first egg passage.of material picked from an E strain plaque on the fourteenth day contained hemagglutinin which was indistinguishable from E strain by hemagglutination inhibition test. Material picked from E plaques on the sixteenth day contained 3 to 6 x lo3 PFU per plaque. The U strain was tested for plaque formation in HeLa cells and a titer of 7.7 x lo7 PFU per milliliter was obtained. Plaques were similar to those seen in chick embryo cult’ures but were first visible on the seventh day and increased in number and size until the eleventh day, after which no changes were noted. The HeLa cell cultures degenerat’ed soon after the fifteenth day. REFERENCES 1. WELLER, T. H., and ENDERS, J. F., Proc. Sot. Exptl. Biol. Med. 69, 124-128 (1948). 2. HENLE, G., and DEINHARDT, F., Proc. Sot. Exptl. Biol. Med. 89, 556-560 (1955). 3. SHELOKOV, A., VOGEL, J. E., and CHI, L., Proc. Sot. Exptl. Biol. Med. 97, 802-809 (1958). 4. BRANDT, C. D., Virology 14, l-10 (1961). 5. GRANOFF, A., Virology 9,636-648 (1959). 6. MANDEL, B., Virology 6,424-447 (1958). 7. MANDEL, B., Virology 14, 316-328 (1961). THOMAS E. FROTHINGHAM~ ALLAN GRANOFF Divisions of Epidemiology and Infectious Diseases The Public Health Research Institute of the City of New York, Inc. New York 9, New York Received August 8, 1961 ’ Present address: Department of Tropical Health, Harvard University School of Health, Boston 15, Massachusetts.
Public Public
Further
REPORTS Studies against
on the
Induced
lsotransplantation Polyoma
Resistance of
Tumors’
In previous studies it was found (1, 2) that pretreatment of adult or newborn mice with polyoma virus induces a state of relative resistance against the isotransplantation of established tumors, originally induced by polyoma virus. No such resistance could be demonstrated against tumors induced by other means. The question arose whether the resistance was mediated by the antiviral antibodies or was induced by some cellular antigen peculiar to polyoma-infected cells. As one approach to this problem, mice were homografted with polyoma tumors. Subsequent to the regression of the homografts, the mice received a polyoma tumor isograft. Since transplanted polyoma tumor cells are known often to release no or but small amounts of virus (3) and to induce no or only low titers of antiviral antibodies (3, 4), it was of particular interest to examine the resistance status of such mice. If resistance were due to a cellular antigen or antigens, shared by polyoma-induced tumors, the animals could be expected to show the same behavior as previously found after virus pretreatment but in the absence of high antiviral antibody titers. Mice of the inbred strain A/Sri and its three coisogenic resistant IR sublines ASW, A.CA, and A.BY (5) were used, together with different F, hybrid combinations. All three sublines have an A strain background but carry different alleles at the histocompatibility-2 (H-2) locus. The homozygosis status of the mouse strains used has been recently checked by skin and tumor transplantation (6). The A strain turned out to be fully skin compat,ible, whereas the IR sublines showed considerable residual heterozygosis manifested by the rejection of intrastrain skin grafts in contrast to tumor grafts which grew as well as within the A strain or other standard inbred strains. The mice were bred in a building where no poly1 This work has been supported by grant C-4747 from the National Cancer Institute, United States Public Health Service, and by grants from the Swedish Cancer Society.
DZSCUSSION AXD PRELIMINARY
oma-infected animals have ever been introduced. Sera of randomly selected animals from this colony were routinely tested, in the course of these experiments, for antiviral antibodies by the hemagglutination inhibition (HI) test (7). Titers in the range of 1:30-l : 240 were found. This indicates that no polyoma contamination has occurred. All polyoma experiments were carried out in another building about a mile and a half away. Tumors were induced by polyoma virus inoculated into newborn mice (8). The transplantation behavior of all tumors except one (SECH) has been described previously (1, a), Tumor cells were brought into suspension by forcing the tissue through a @)-mesh stainless steel screen into Ringer’s solut’ion on a 1: 5 volume basis. The macroscopic tissue fragments mere allowed to sediment for about 5 minutes at O”, then the approximate number of viable cells was cstimated in the supernatant according to Schrek’s method (9). The lowest cell dose required to give rise to progressively growing tumors in 100% of adult, untreated isologous mice (I), (the “minimal cell dose”) n-as inoculated subcutaneously into the right flank of isologous hosts. The following five different groups of adult recipients were used. 1. Two to 5 months old, untreated, isologous mice (groups IJ in Table 1). 2. Three to 6 months old, isologous mice pretreat’ed with polyoma virus at adult age (groups VA in Table 1). The virus was partly purified by mixing guinea pig erythrocytes at 0” with medium from polyoma-infected cultures of mouse embryo cells. After 2-4 hours the red cells were washed and the virus was elmed at 37” (1). Doses corresponding to HA titers in the range 1:32I : 128, were injected subcutaneousIy; three or four inoculations were given at intervals of 1-2 weeks; this was the same scheme as that used in the previous studies (1). 3. Two to 4 months old, isologous mice inoculated with virus when newborn (groups VN in Table 1). These animals were used before they had developed any palpable primary tumor of their own. 4. Four to 6 months old, isologous mice
REPORTS
215
pretreated with polyoma tumor homografts (groups HP in Table 1). Suspensions of living polyoma tumor cells induced in genetically foreign mouse strains, differing at the H-2 locus, were inoculated to mice 2-3 months old. Three inoculations were given. The homografted tumors regressed uniformly after a period of temporary growth. Each inoculation followed immediately after the disappearance of the previous, regressing tumor. The intervals between inoculations varied between 3 and 6 weeks, depending on the speed of regression, which was in turn dependent on the tumor-host combination. 5. Four t,o 6 months old, isologous mice pretreated with homografts of other tumors, induced by other means than by polyoma virus and derived from the virus-free colony (groups HNP in Table lj. Mice 2-3 months old were inoculated three times with spont,aneous mammary carcinomas originating in a foreign mouse strain. The schedule of pretreatment was the same as in group 4. Following the inoculation of the polyoma t’unior isografts, all groups of mice were inspected and palpated twice a meek and the appearance of tumors was registered. Ail tumors were measured by caliper. Three different diameters were measured, and the geometrical mean was calculated. RESUJTS
The results are summarized in Table 1. As in previous experiments (1) the isologous mice pretreated with purified virus (groups VA in Table 1) and mice inoculated with virus when newborn (groups VN in Table 1) were wholly or partially resistant against the transplantation of small numbers of isologous polyoms tumor cells. R.esistance was manifest.ed with aIi tumors tested and was expressed in a reduced frequency of takes, in prolonged latency periods, or in delayed growth of the tumor. Isologous recipients pretreated with polyoma tumor homografts (groups HP in Table 1) exhibited the same type of resistance m&h all six tumors tested. In contrast, pretreatment with homografts of nonpolyoma tumors induced no detectable resistance (groups HNP in Table 1).
Expt.
’ osteogenic S&1‘coma
Epithelial thy1ncma
SEWA
SEWE
Subcotanecm fibromrcoma
A.SW
A.SW
A CA
A.CA
Mixed
SECBT
SECH
A/h
Hair follicle trmm
SESR
type thyr!lCrnB
A/%
origin
s9
Undifferentiated subou taleC”8 ear00ma
TYPO
TUtl0r
SESO
Designation*
I TI-
-
U VA VN HPh
-
6-8
-
::: -O/lo(-) -O/10(-) -O/10(-) - O/4(-)
-
85 99 50
:::
-
o/lo(-) O/10(-) O/O(-) O/4(-)
O/5(-) O/5 (6) O/4(-) O/4(-)
2/4 (6.7) 4/4(11.3) 3/3(16.9)
-
2/4 (5.8) 4/4 (9.7) 3/3(16.0)
4/4(19.1) 4/4(16.6)
3/3 (6.3) O/4(-) 4/4 (7.7)
o/3(-) O/3(-) O/3(-) O/3(-)
o/4(-) O/3(-) O/5(-)
9-12
-44/4(16.3) -44/4(15.6)
/4 2/3/(l.7) /40/4(-) /4 2/4 (1.3)
--o/3(-) -O/3(-) --o/3(-) -O/3(-)
/4 o/4(-) /30/3(-) /50/5(-)
-5
104 89 56
U VA VN HP
18
62 74 72
67 91 70 97
-
18 28
U VA VN HPh HNPh
U VA VN HPh
U VA HPh
12
4
TABLE
1
O/lo(-) O/10(-) O/10(-) O/4 (-)
2/5 (1.0) O/5 (-) O/4(-) O/4(-)
2/4 (6.7) 3/4(l2.4) 3/3(22.7)
4/4(23.1) 4/4(20.4)
3/3 (9.8) O/4(-) 4/4 (9.0)
3/3 (5.5) O/3(-) l/3 (0.8) O/3(-)
3/4 (5.0) O/3(-) O/5(-)
13-16
17-20
(5.5) (0.6) (9.2)
(11.2) (2.0) (3.0) (2.3)
(9.4) (9.4) (26.1)
O/10(-) O/10(-) O/10(-) O/4(- 1
4/5 (4.5) O/5 (6) O/4(-) O/4(-)
2/4 3/4 3/3
3/3e(27.9) 4/4 (19.7)
3/3 l/4 4/4
3/3 l/3 1/3 l/3
21-24
after
(11.0) (11.0) -
-
(6.3) (0.6) (9.4)
(12.7) (3.7) (5.8) (3.3)
O/10(-) l/10 (0.3) O/10(-) l/4 (1.5)
4/4e(10.4) O/5 (-) O/4(-) O/4 C-1
Z/4 3/4
3/3 l/4 4/4
3/3 2/3 3/3 l/3
-
O/10(-) l/10(0.3) O/10(-) l/4 (1.5)
4/4(11.8) O/5 c-1 O/4(-) O/4(-)
2/3c(6.5) 3/4(11.0) -
1
Z/3 (4.6) l/4 (1.8) 4/4(10.2)
3/3(17.8) 2/3 (5.3) 3/3(10.5) l/3 (4.6)
4/4(15.0) O/3(-) l/5 (2.4)
(8.8) (14.1)
(4.7) (1.7) (10.8)
(20.1) (5.3) (12.4) (6.1)
2/10 (0.8) l/10 (0.3) O/10(-) l/4 (1.5)
3/3<(15.3) O/5 (6-j O/4(-) O/4(-)
Z/3 3/4
Z/3 l/4 4/4
3/3 2/3 3/3 l/3
3/3Y18.1) O/3(-) 2/5 (4.3)
___~
29-32
cell inoculationa
25-28
tumor
!/2e(22.2) l/3 (6.7) 1/3 (16.7) l/3 (8.5)
l/3(-) !/5 (6.3)
37-40
)li
-
-
(6.2) (0.8) (1.2) (3.7)
)S !/Ze(17.6) (l/5(-) ( v4 (-1 1/4 (1.3)
l/3 (13.3) )i !/3e(17.4)
)/lo 7/10(3.1 Ii ./lo l/10(0.4 l/10(1.0 )1 ./lo l/4 (1.3 ): !/4
3/3(17.7 O/5 (-) O/4 C-1 O/4 C-1
3/3(12.3 3/4(14.7
-
!/3 (5.5) 2/3 (5.5 (2.1) l/4 (1.9 )I /4 4/4(11.4 )4 L/4 (12.9)
3/3(21.8 3/3 (6.3 3/3(14.0 1/3 (7.3
3/3(22.0 O/3(-) Z/5 (5.6
33-36
-
WITH HOMOGRAFTS OFTUMORS MEANS
OTHER
4/4 (14.0) O/3(-) l/5 (1.2)
of days
4/4 (8.9) O/3(-) O/5(-)
Number
POLYOMA TUMORS TO ISOLOGOUS HOSTS PRETREATED STRAINS BY POLYOMA VIRUS OR BY FOREIGN
147 8
-
OF
U HPh HNPh
10
7
14
TRANSPLANTATION
(20.6
9/10 l/l0 l/10 2/4
(9.3 (1.0 (1.3 (4.1
O/5(-) O/4 C-1 l/4 (2.4
2/2
-
-
-
-
3/3 (11.1 2/2”(17.5 l/3 (9.7
O/3(-) 2/5 (7.9
41-44
INDUCED
9/10 2/10 l/10 3/4Q
5/5 O/5 O/4 l/4
4/4 4/4 4/4 3/4 3/3
1/4 414
3/3Q
313 3/3 3/3 l/3
4/4 O/3 215
IN
I
0
13 14 28
22 22 22
15 15 15 15 15
14 14 14
20 20 20 20
-
HI titers serum
l/2
,
O/4
8/8
4/4
-
3/3 4/4 O/3
-
4/4 l/3< i/3i
O/l
O/3 1/3i
o/2
O/3
O/l
2/z 212
of
d Sera taken from the recipients in the course of each experiment were teated for antiviral antibodies by the hemagglutination inhibition (HI) test. Mice that have not been in contact with polyoma. virus bad titers in the range 1:3&1:240. Titers of I:480 or higher are taken aa positive.
of tumors.
a The figures denote the number of mice with palpable tumors over the total number inooulated. Figures in parenthesis indicate average tumor diameter in millimeters. b The tumors have been designated according to the following principles: the first two letters S and E refer to the induction of the tumor by the SE polyoma virus. The next letter indicates the strain of origin, S, Y, C, and W standing for A/Sri, A.BY, A.CA, and A.SW, respectively, while NS symbolizes the A X A.SW F1 hybrid. The last letter is the seritrl designtttion of the individual tumors, applied in alphabetic order according to their chronological appesrance. The Tin SECBT denotes the thymoma in an A.CA mouse which developed two primary tumors. C U = untreated isologous mice; VA = isologous mice pretreated with virus at adult age; VN = isologous mice pretreated with virus 88 newborn; HP = isologous mice pretreated with polyoma tumor homografts; HNP = isologous mice pretreated with homografts of other types
positive positive
’ The
HI
titer
HI titer
= 1:480.
= 1: 1920.
HNP, experiment 3: S13S (Sth, lOth, and 11th transfer generations of a spontaneous carcinoma of strain A/Sri origin). HNP, experiment 4: 5145 (the primary tumor and its two first transfer generations.
j The
Group rnmnmary Group
Group HP, experimenbs 3 and 4: SENSV (4th transfer generation of an osteogenic %~rcoma of A X A.SW origin), SEYF (10th tritnsfergeoeration of a subcutaneous fibrosarcomaof A.BY origin) and 7th transfer generation of SESO. Group HP, experiments 5 and 6: SEYB (7th transfer generation), SEYLM (2nd transfer generation), and SESO (7th transfer generation).
e One animal dead with tumor. 1 One animal dead without tumor. * One animal developed a tumor more than 44 days after the inocnlstion. h For the homograft pretreatment the following tumors were used: group HP, experiments 1 and 2: SEYB (7th transfer generation of an osteogenic sarcomaof A.BY origin), lltbgeneration of SEWA and SEYM (3rd transfer generation of an osteogenic sarcoma of 9.BY origin).
;3
g
g
H
5
ii! 2
$
z
d
$
g
z
3
218
DISCUSSION
AND
PRELIMINARY
The sera of the recipients were tested for hemagglutination-inhibiting (HI) antiviral antibodies. In untreated control animals the titer was equal to or less than 1: 240, and in recipients pretreated with homologous nonpolyoma tumor cells the titer was not higher than 1: 240, except in two animals (experiments 3 and 4)) in which a titer of 1: 480 was found. In mice pretreated with virus as adults or when newborn, the titer ranged between 1:960 and 1: 15,360. A single exception was found in one animal pretreated as adult (experiment 4). Here the titer was 1:120. When mice receive inoculations of serially transplanted polyoma tumor cells from a foreign strain it is the rule that most animals develop no antiviral HI antibodies. In a few cases, however, positive titers up to 1: 7680 have been found in the sera of mice that had received one or more inoculations of cells from the following tumors: SEYB, SEYM, and SEYF (4). Three of the tumors used in this investigation, SESO, SEYF, and SEWA, have been tested in vitro and found t,o release virus in some of the tested transfer generations whereas in other passages no release was found (10). This finding is in agreement with that of Habel and Silverberg (S), working with other mouse tumors. For the present experiments 6 groups of mice were selected in which no or only one animal had titers higher than 1:240. In two cases (experiments 1 and 4) one animal in each group had a titer of 1:1920, while the other mice in these two groups had a titer of 1: 240.
REPORTS
oma tumor homografts induces the same type of resistance although most animals had no humoral antiviral antibodies detect’able by the HI method. This weakens the possibility that resistance is due to humoral antiviral antibodies. An immunization against cellular antigens becomes more probable. The antigens that can be considered fall into three major categories: ordinary isoantigens due to the heterozygosis of some of the st,rains used; tissue-specific antigens; and specific ant,igens common to polyoma-induced tumors. The heterozygosis of the mouse strains can be ruled out as an explanation since the resist,ance was clearly demonstrable with two A strain tumors and this strain was fully homozygous according to intrastrain skin grafting tests (6). Tissuespecific antigenicity might be considered since all polyoma tumor homografts used for pretreatment mere sarcomas while the spontaneous tumors were mammary carcinomas. However, in experiment 2, resistance could be demonstrated with an epithelial polyoma tumor and the assumption of tissue specific antigens became improbable. The possible exist,ence of a cellular antigen specific to polyoma tumor cells would fit well with the present results. Provided that this antigenicity were shared by polyoma-infected normal cells, the resistance of virus-treated animals could be due to the same mechanism. Further experiments are in progress to elucidate the situation. As a possibly analogous situation, some phenomena of lysogenic conversion may serve as models (11). ACKNOWLEDGMENTS
DISCUSSION
Inoculation of polyoma virus into adult or newborn mice is known to induce humoral antiviral HI ant,ibodies. Mice inoculated with virus as newborn develop various primary tumors concurrently with a high ant.iviral antibody titer in serum. It was found in previous work (1, 2) that virus treatment of newborn as well as adult mice also induces a state of relative resistance against isotransplantation of established polyoma tumor cells, but not against tumors induced by other means. The present results show that pretreatment of mice with poly-
The author wishes to thank Professor G. Klein for his advice and support in the course of these experiments, and Professor Nils Ringertz for providing laboratory facilities for these experiments at Sabbatsberg’sHospital. The skillful assistance of my wife, Mrs. Barbro SjGgren,is gratefully acknowledged. REFERENCES 1. SJ~GREN, H. O., HELLSTR~M, I., and KLEIN, G., Exptl. Cell Research 23, 204-208 (1961). 2. SJ~GREN, H. O., HELLSTR~M, I., and KLEIN, G., Cancer Research 21, 329-337 (1961). 3. HABEL, K., and SILVERBERG,R. J., Virology 12, 463-476 (1960).
DISCUSSION
AND
PRELIMINARY
4. SJ~FREN, H. O., unpublished. 5. S~YEI,L, G. D., YranspZn&ntion Bull. 2, 6-8 (1955). 6. IJYDER, O., and KLEIX, E., /. Nntl. Cancer Inst. 24, 707-720 ( 1960). 7. EDDY. B. E., Rowz, W. P., HARTLEY, J. I?-., STEWART. S. E., and HUEBMX, R. J., virology 6, 290-291 (1958). 8. SJ~CREN, H. O., and RISGERTZ, N.. To be published. 9. SCHREK. R., Am. J. Cancer 28, 389-392 (1936). 10. HELLSTRRM, I., unpublished. 3, 261il. LLYRI.4. S. 8;., E’t.oc. cm. Crtr~co Cmf. 270 ( 1959). Hairs OLOF SJ~GRES Institute for Tumor Biology Karolin,skn Institutet Medical School Stockholm, Sweden
A General
Survival
with
Damaged
Law for Virus Genetic
and Cells
Material’
A formula for survival of irradiat,ed phage in multiply infected bacteria (the phenomenon called multiplicity reactivation, shortly designakd MR) is derived. The formula, based on t’he “chromosomic recombination theory” (I), is more precise than earlier formulas and can be applied at any dose and with any size of damage. By this formula it, is possible to investigate cases in which there is reason to suspect large-sized damages. The same formula applies also to t,he survival of diploid and polyploid cells in t’he special case in which only recessive damages are caused by the inactivating agent used. A theory that has been used to calculate the survival probability of viruses in multiply infected cells (multicomplexes) and which, for the special case of recessive damages only, may apply also to radiation resistance of cells, is the following: The cell or t,he multicomplex will survive if, and only if, there is no segment of genetic material which is damaged in every one of the homologous sets of chromosomes of a diploid or polyploid cell, or in every one of the in1 This investigation was supported by research grant RG-6980 from the Division of General Medical Sciences of the National Institutes of Health, United States Public Health Service.
219
REPORTS
fecting viruses. An earlier approximate formula for this survival probability (1) is not applicable for large damages nor for very high doses of radiaGon or other inactivating agents. The purpose of this paper is to present’ a formula which is valid at, any dose and wit,h any size of damage. SURVIVAL
LAW
Let m be the degree of polyploidy (m = 2 for diploid cells, m = 3 for triploid cells, etc.) or the mukiplicity of infection. Let W, be the survival probability, h the number of lethal hit,s (dose), and L the mean lengt,h [measured in target units, see Barricelli (W)] of the damage caused in the genetic material by a let’hal hit. If W,(A) is the probabilit’y that, no single point> of a given genet,ic segment of target length A is damaged in every one of t’he m genomes, the probability W,(A + dA) will be given by:
hdA
1 (1)
where 0, is the probability that all the m genomes are damaged at a specific point, for instance, at the end of the above segment of target length A (defined as point A), and O,-1 is the probability t,hat’ m - 1 out of the m genomes are damaged and one of them is undamaged at the point A. 0, = (1 - e-Lh)m
and
(),-1 = m(l - e-Lh)m-le-Lh
(4
Thus 0,-1/l - 0, will be the probability of m - 1 damaged and one undamaged genomes at a point where it is known t’hat not all the m genomes are damaged. W,( /\) (O&l - 0,) will therefore be the probabilit’y t’hat no point of the segment A is damaged in all m genomes and that m - 1 genomes are damaged and one is undamaged at the end of this segment (point A). If this probability is multiplied by hdA (which, at the dose h, is the probability that a damage shall start between /j and A + d/j in the only genome which is undamaged at the point A), one obviously obt’ains the quan-
DISCUSSION
AND
PRELIMINARY
until the second agar overlay did not alter plaque formation or survival of cells. Microof plaques revealed scopic examination many intact stained cells amidst cellular debris. The number of plaques was proportional to the dilution inoculated and the number of plaque-forming units (PFU) was 8.9 X lo7 per milliliter (average of two titrations of E strain). Simultaneous titration of E strain in eggs gave 1.1 X lo8 egg infectious units (EIU) per milliliter indicating an EIU :PFU ratio of essentially one. Allantoic fluid from the first egg passage.of material picked from an E strain plaque on the fourteenth day contained hemagglutinin which was indistinguishable from E strain by hemagglutination inhibition test. Material picked from E plaques on the sixteenth day contained 3 to 6 x lo3 PFU per plaque. The U strain was tested for plaque formation in HeLa cells and a titer of 7.7 x lo7 PFU per milliliter was obtained. Plaques were similar to those seen in chick embryo cult’ures but were first visible on the seventh day and increased in number and size until the eleventh day, after which no changes were noted. The HeLa cell cultures degenerat’ed soon after the fifteenth day. REFERENCES 1. WELLER, T. H., and ENDERS, J. F., Proc. Sot. Exptl. Biol. Med. 69, 124-128 (1948). 2. HENLE, G., and DEINHARDT, F., Proc. Sot. Exptl. Biol. Med. 89, 556-560 (1955). 3. SHELOKOV, A., VOGEL, J. E., and CHI, L., Proc. Sot. Exptl. Biol. Med. 97, 802-809 (1958). 4. BRANDT, C. D., Virology 14, l-10 (1961). 5. GRANOFF, A., Virology 9,636-648 (1959). 6. MANDEL, B., Virology 6,424-447 (1958). 7. MANDEL, B., Virology 14, 316-328 (1961). THOMAS E. FROTHINGHAM~ ALLAN GRANOFF Divisions of Epidemiology and Infectious Diseases The Public Health Research Institute of the City of New York, Inc. New York 9, New York Received August 8, 1961 ’ Present address: Department of Tropical Health, Harvard University School of Health, Boston 15, Massachusetts.
Public Public
Further
REPORTS Studies against
on the
Induced
lsotransplantation Polyoma
Resistance of
Tumors’
In previous studies it was found (1, 2) that pretreatment of adult or newborn mice with polyoma virus induces a state of relative resistance against the isotransplantation of established tumors, originally induced by polyoma virus. No such resistance could be demonstrated against tumors induced by other means. The question arose whether the resistance was mediated by the antiviral antibodies or was induced by some cellular antigen peculiar to polyoma-infected cells. As one approach to this problem, mice were homografted with polyoma tumors. Subsequent to the regression of the homografts, the mice received a polyoma tumor isograft. Since transplanted polyoma tumor cells are known often to release no or but small amounts of virus (3) and to induce no or only low titers of antiviral antibodies (3, 4), it was of particular interest to examine the resistance status of such mice. If resistance were due to a cellular antigen or antigens, shared by polyoma-induced tumors, the animals could be expected to show the same behavior as previously found after virus pretreatment but in the absence of high antiviral antibody titers. Mice of the inbred strain A/Sri and its three coisogenic resistant IR sublines ASW, A.CA, and A.BY (5) were used, together with different F, hybrid combinations. All three sublines have an A strain background but carry different alleles at the histocompatibility-2 (H-2) locus. The homozygosis status of the mouse strains used has been recently checked by skin and tumor transplantation (6). The A strain turned out to be fully skin compat,ible, whereas the IR sublines showed considerable residual heterozygosis manifested by the rejection of intrastrain skin grafts in contrast to tumor grafts which grew as well as within the A strain or other standard inbred strains. The mice were bred in a building where no poly1 This work has been supported by grant C-4747 from the National Cancer Institute, United States Public Health Service, and by grants from the Swedish Cancer Society.
DZSCUSSION AXD PRELIMINARY
oma-infected animals have ever been introduced. Sera of randomly selected animals from this colony were routinely tested, in the course of these experiments, for antiviral antibodies by the hemagglutination inhibition (HI) test (7). Titers in the range of 1:30-l : 240 were found. This indicates that no polyoma contamination has occurred. All polyoma experiments were carried out in another building about a mile and a half away. Tumors were induced by polyoma virus inoculated into newborn mice (8). The transplantation behavior of all tumors except one (SECH) has been described previously (1, a), Tumor cells were brought into suspension by forcing the tissue through a @)-mesh stainless steel screen into Ringer’s solut’ion on a 1: 5 volume basis. The macroscopic tissue fragments mere allowed to sediment for about 5 minutes at O”, then the approximate number of viable cells was cstimated in the supernatant according to Schrek’s method (9). The lowest cell dose required to give rise to progressively growing tumors in 100% of adult, untreated isologous mice (I), (the “minimal cell dose”) n-as inoculated subcutaneously into the right flank of isologous hosts. The following five different groups of adult recipients were used. 1. Two to 5 months old, untreated, isologous mice (groups IJ in Table 1). 2. Three to 6 months old, isologous mice pretreat’ed with polyoma virus at adult age (groups VA in Table 1). The virus was partly purified by mixing guinea pig erythrocytes at 0” with medium from polyoma-infected cultures of mouse embryo cells. After 2-4 hours the red cells were washed and the virus was elmed at 37” (1). Doses corresponding to HA titers in the range 1:32I : 128, were injected subcutaneousIy; three or four inoculations were given at intervals of 1-2 weeks; this was the same scheme as that used in the previous studies (1). 3. Two to 4 months old, isologous mice inoculated with virus when newborn (groups VN in Table 1). These animals were used before they had developed any palpable primary tumor of their own. 4. Four to 6 months old, isologous mice
REPORTS
215
pretreated with polyoma tumor homografts (groups HP in Table 1). Suspensions of living polyoma tumor cells induced in genetically foreign mouse strains, differing at the H-2 locus, were inoculated to mice 2-3 months old. Three inoculations were given. The homografted tumors regressed uniformly after a period of temporary growth. Each inoculation followed immediately after the disappearance of the previous, regressing tumor. The intervals between inoculations varied between 3 and 6 weeks, depending on the speed of regression, which was in turn dependent on the tumor-host combination. 5. Four t,o 6 months old, isologous mice pretreated with homografts of other tumors, induced by other means than by polyoma virus and derived from the virus-free colony (groups HNP in Table lj. Mice 2-3 months old were inoculated three times with spont,aneous mammary carcinomas originating in a foreign mouse strain. The schedule of pretreatment was the same as in group 4. Following the inoculation of the polyoma t’unior isografts, all groups of mice were inspected and palpated twice a meek and the appearance of tumors was registered. Ail tumors were measured by caliper. Three different diameters were measured, and the geometrical mean was calculated. RESUJTS
The results are summarized in Table 1. As in previous experiments (1) the isologous mice pretreated with purified virus (groups VA in Table 1) and mice inoculated with virus when newborn (groups VN in Table 1) were wholly or partially resistant against the transplantation of small numbers of isologous polyoms tumor cells. R.esistance was manifest.ed with aIi tumors tested and was expressed in a reduced frequency of takes, in prolonged latency periods, or in delayed growth of the tumor. Isologous recipients pretreated with polyoma tumor homografts (groups HP in Table 1) exhibited the same type of resistance m&h all six tumors tested. In contrast, pretreatment with homografts of nonpolyoma tumors induced no detectable resistance (groups HNP in Table 1).
Expt.
’ osteogenic S&1‘coma
Epithelial thy1ncma
SEWA
SEWE
Subcotanecm fibromrcoma
A.SW
A.SW
A CA
A.CA
Mixed
SECBT
SECH
A/h
Hair follicle trmm
SESR
type thyr!lCrnB
A/%
origin
s9
Undifferentiated subou taleC”8 ear00ma
TYPO
TUtl0r
SESO
Designation*
I TI-
-
U VA VN HPh
-
6-8
-
::: -O/lo(-) -O/10(-) -O/10(-) - O/4(-)
-
85 99 50
:::
-
o/lo(-) O/10(-) O/O(-) O/4(-)
O/5(-) O/5 (6) O/4(-) O/4(-)
2/4 (6.7) 4/4(11.3) 3/3(16.9)
-
2/4 (5.8) 4/4 (9.7) 3/3(16.0)
4/4(19.1) 4/4(16.6)
3/3 (6.3) O/4(-) 4/4 (7.7)
o/3(-) O/3(-) O/3(-) O/3(-)
o/4(-) O/3(-) O/5(-)
9-12
-44/4(16.3) -44/4(15.6)
/4 2/3/(l.7) /40/4(-) /4 2/4 (1.3)
--o/3(-) -O/3(-) --o/3(-) -O/3(-)
/4 o/4(-) /30/3(-) /50/5(-)
-5
104 89 56
U VA VN HP
18
62 74 72
67 91 70 97
-
18 28
U VA VN HPh HNPh
U VA VN HPh
U VA HPh
12
4
TABLE
1
O/lo(-) O/10(-) O/10(-) O/4 (-)
2/5 (1.0) O/5 (-) O/4(-) O/4(-)
2/4 (6.7) 3/4(l2.4) 3/3(22.7)
4/4(23.1) 4/4(20.4)
3/3 (9.8) O/4(-) 4/4 (9.0)
3/3 (5.5) O/3(-) l/3 (0.8) O/3(-)
3/4 (5.0) O/3(-) O/5(-)
13-16
17-20
(5.5) (0.6) (9.2)
(11.2) (2.0) (3.0) (2.3)
(9.4) (9.4) (26.1)
O/10(-) O/10(-) O/10(-) O/4(- 1
4/5 (4.5) O/5 (6) O/4(-) O/4(-)
2/4 3/4 3/3
3/3e(27.9) 4/4 (19.7)
3/3 l/4 4/4
3/3 l/3 1/3 l/3
21-24
after
(11.0) (11.0) -
-
(6.3) (0.6) (9.4)
(12.7) (3.7) (5.8) (3.3)
O/10(-) l/10 (0.3) O/10(-) l/4 (1.5)
4/4e(10.4) O/5 (-) O/4(-) O/4 C-1
Z/4 3/4
3/3 l/4 4/4
3/3 2/3 3/3 l/3
-
O/10(-) l/10(0.3) O/10(-) l/4 (1.5)
4/4(11.8) O/5 c-1 O/4(-) O/4(-)
2/3c(6.5) 3/4(11.0) -
1
Z/3 (4.6) l/4 (1.8) 4/4(10.2)
3/3(17.8) 2/3 (5.3) 3/3(10.5) l/3 (4.6)
4/4(15.0) O/3(-) l/5 (2.4)
(8.8) (14.1)
(4.7) (1.7) (10.8)
(20.1) (5.3) (12.4) (6.1)
2/10 (0.8) l/10 (0.3) O/10(-) l/4 (1.5)
3/3<(15.3) O/5 (6-j O/4(-) O/4(-)
Z/3 3/4
Z/3 l/4 4/4
3/3 2/3 3/3 l/3
3/3Y18.1) O/3(-) 2/5 (4.3)
___~
29-32
cell inoculationa
25-28
tumor
!/2e(22.2) l/3 (6.7) 1/3 (16.7) l/3 (8.5)
l/3(-) !/5 (6.3)
37-40
)li
-
-
(6.2) (0.8) (1.2) (3.7)
)S !/Ze(17.6) (l/5(-) ( v4 (-1 1/4 (1.3)
l/3 (13.3) )i !/3e(17.4)
)/lo 7/10(3.1 Ii ./lo l/10(0.4 l/10(1.0 )1 ./lo l/4 (1.3 ): !/4
3/3(17.7 O/5 (-) O/4 C-1 O/4 C-1
3/3(12.3 3/4(14.7
-
!/3 (5.5) 2/3 (5.5 (2.1) l/4 (1.9 )I /4 4/4(11.4 )4 L/4 (12.9)
3/3(21.8 3/3 (6.3 3/3(14.0 1/3 (7.3
3/3(22.0 O/3(-) Z/5 (5.6
33-36
-
WITH HOMOGRAFTS OFTUMORS MEANS
OTHER
4/4 (14.0) O/3(-) l/5 (1.2)
of days
4/4 (8.9) O/3(-) O/5(-)
Number
POLYOMA TUMORS TO ISOLOGOUS HOSTS PRETREATED STRAINS BY POLYOMA VIRUS OR BY FOREIGN
147 8
-
OF
U HPh HNPh
10
7
14
TRANSPLANTATION
(20.6
9/10 l/l0 l/10 2/4
(9.3 (1.0 (1.3 (4.1
O/5(-) O/4 C-1 l/4 (2.4
2/2
-
-
-
-
3/3 (11.1 2/2”(17.5 l/3 (9.7
O/3(-) 2/5 (7.9
41-44
INDUCED
9/10 2/10 l/10 3/4Q
5/5 O/5 O/4 l/4
4/4 4/4 4/4 3/4 3/3
1/4 414
3/3Q
313 3/3 3/3 l/3
4/4 O/3 215
IN
I
0
13 14 28
22 22 22
15 15 15 15 15
14 14 14
20 20 20 20
-
HI titers serum
l/2
,
O/4
8/8
4/4
-
3/3 4/4 O/3
-
4/4 l/3< i/3i
O/l
O/3 1/3i
o/2
O/3
O/l
2/z 212
of
d Sera taken from the recipients in the course of each experiment were teated for antiviral antibodies by the hemagglutination inhibition (HI) test. Mice that have not been in contact with polyoma. virus bad titers in the range 1:3&1:240. Titers of I:480 or higher are taken aa positive.
of tumors.
a The figures denote the number of mice with palpable tumors over the total number inooulated. Figures in parenthesis indicate average tumor diameter in millimeters. b The tumors have been designated according to the following principles: the first two letters S and E refer to the induction of the tumor by the SE polyoma virus. The next letter indicates the strain of origin, S, Y, C, and W standing for A/Sri, A.BY, A.CA, and A.SW, respectively, while NS symbolizes the A X A.SW F1 hybrid. The last letter is the seritrl designtttion of the individual tumors, applied in alphabetic order according to their chronological appesrance. The Tin SECBT denotes the thymoma in an A.CA mouse which developed two primary tumors. C U = untreated isologous mice; VA = isologous mice pretreated with virus at adult age; VN = isologous mice pretreated with virus 88 newborn; HP = isologous mice pretreated with polyoma tumor homografts; HNP = isologous mice pretreated with homografts of other types
positive positive
’ The
HI
titer
HI titer
= 1:480.
= 1: 1920.
HNP, experiment 3: S13S (Sth, lOth, and 11th transfer generations of a spontaneous carcinoma of strain A/Sri origin). HNP, experiment 4: 5145 (the primary tumor and its two first transfer generations.
j The
Group rnmnmary Group
Group HP, experimenbs 3 and 4: SENSV (4th transfer generation of an osteogenic %~rcoma of A X A.SW origin), SEYF (10th tritnsfergeoeration of a subcutaneous fibrosarcomaof A.BY origin) and 7th transfer generation of SESO. Group HP, experiments 5 and 6: SEYB (7th transfer generation), SEYLM (2nd transfer generation), and SESO (7th transfer generation).
e One animal dead with tumor. 1 One animal dead without tumor. * One animal developed a tumor more than 44 days after the inocnlstion. h For the homograft pretreatment the following tumors were used: group HP, experiments 1 and 2: SEYB (7th transfer generation of an osteogenic sarcomaof A.BY origin), lltbgeneration of SEWA and SEYM (3rd transfer generation of an osteogenic sarcoma of 9.BY origin).
;3
g
g
H
5
ii! 2
$
z
d
$
g
z
3
218
DISCUSSION
AND
PRELIMINARY
The sera of the recipients were tested for hemagglutination-inhibiting (HI) antiviral antibodies. In untreated control animals the titer was equal to or less than 1: 240, and in recipients pretreated with homologous nonpolyoma tumor cells the titer was not higher than 1: 240, except in two animals (experiments 3 and 4)) in which a titer of 1: 480 was found. In mice pretreated with virus as adults or when newborn, the titer ranged between 1:960 and 1: 15,360. A single exception was found in one animal pretreated as adult (experiment 4). Here the titer was 1:120. When mice receive inoculations of serially transplanted polyoma tumor cells from a foreign strain it is the rule that most animals develop no antiviral HI antibodies. In a few cases, however, positive titers up to 1: 7680 have been found in the sera of mice that had received one or more inoculations of cells from the following tumors: SEYB, SEYM, and SEYF (4). Three of the tumors used in this investigation, SESO, SEYF, and SEWA, have been tested in vitro and found t,o release virus in some of the tested transfer generations whereas in other passages no release was found (10). This finding is in agreement with that of Habel and Silverberg (S), working with other mouse tumors. For the present experiments 6 groups of mice were selected in which no or only one animal had titers higher than 1:240. In two cases (experiments 1 and 4) one animal in each group had a titer of 1:1920, while the other mice in these two groups had a titer of 1: 240.
REPORTS
oma tumor homografts induces the same type of resistance although most animals had no humoral antiviral antibodies detect’able by the HI method. This weakens the possibility that resistance is due to humoral antiviral antibodies. An immunization against cellular antigens becomes more probable. The antigens that can be considered fall into three major categories: ordinary isoantigens due to the heterozygosis of some of the st,rains used; tissue-specific antigens; and specific ant,igens common to polyoma-induced tumors. The heterozygosis of the mouse strains can be ruled out as an explanation since the resist,ance was clearly demonstrable with two A strain tumors and this strain was fully homozygous according to intrastrain skin grafting tests (6). Tissuespecific antigenicity might be considered since all polyoma tumor homografts used for pretreatment mere sarcomas while the spontaneous tumors were mammary carcinomas. However, in experiment 2, resistance could be demonstrated with an epithelial polyoma tumor and the assumption of tissue specific antigens became improbable. The possible exist,ence of a cellular antigen specific to polyoma tumor cells would fit well with the present results. Provided that this antigenicity were shared by polyoma-infected normal cells, the resistance of virus-treated animals could be due to the same mechanism. Further experiments are in progress to elucidate the situation. As a possibly analogous situation, some phenomena of lysogenic conversion may serve as models (11). ACKNOWLEDGMENTS
DISCUSSION
Inoculation of polyoma virus into adult or newborn mice is known to induce humoral antiviral HI ant,ibodies. Mice inoculated with virus as newborn develop various primary tumors concurrently with a high ant.iviral antibody titer in serum. It was found in previous work (1, 2) that virus treatment of newborn as well as adult mice also induces a state of relative resistance against isotransplantation of established polyoma tumor cells, but not against tumors induced by other means. The present results show that pretreatment of mice with poly-
The author wishes to thank Professor G. Klein for his advice and support in the course of these experiments, and Professor Nils Ringertz for providing laboratory facilities for these experiments at Sabbatsberg’sHospital. The skillful assistance of my wife, Mrs. Barbro SjGgren,is gratefully acknowledged. REFERENCES 1. SJ~GREN, H. O., HELLSTR~M, I., and KLEIN, G., Exptl. Cell Research 23, 204-208 (1961). 2. SJ~GREN, H. O., HELLSTR~M, I., and KLEIN, G., Cancer Research 21, 329-337 (1961). 3. HABEL, K., and SILVERBERG,R. J., Virology 12, 463-476 (1960).
DISCUSSION
AND
PRELIMINARY
4. SJ~FREN, H. O., unpublished. 5. S~YEI,L, G. D., YranspZn&ntion Bull. 2, 6-8 (1955). 6. IJYDER, O., and KLEIX, E., /. Nntl. Cancer Inst. 24, 707-720 ( 1960). 7. EDDY. B. E., Rowz, W. P., HARTLEY, J. I?-., STEWART. S. E., and HUEBMX, R. J., virology 6, 290-291 (1958). 8. SJ~CREN, H. O., and RISGERTZ, N.. To be published. 9. SCHREK. R., Am. J. Cancer 28, 389-392 (1936). 10. HELLSTRRM, I., unpublished. 3, 261il. LLYRI.4. S. 8;., E’t.oc. cm. Crtr~co Cmf. 270 ( 1959). Hairs OLOF SJ~GRES Institute for Tumor Biology Karolin,skn Institutet Medical School Stockholm, Sweden
A General
Survival
with
Damaged
Law for Virus Genetic
and Cells
Material’
A formula for survival of irradiat,ed phage in multiply infected bacteria (the phenomenon called multiplicity reactivation, shortly designakd MR) is derived. The formula, based on t’he “chromosomic recombination theory” (I), is more precise than earlier formulas and can be applied at any dose and with any size of damage. By this formula it, is possible to investigate cases in which there is reason to suspect large-sized damages. The same formula applies also to t,he survival of diploid and polyploid cells in t’he special case in which only recessive damages are caused by the inactivating agent used. A theory that has been used to calculate the survival probability of viruses in multiply infected cells (multicomplexes) and which, for the special case of recessive damages only, may apply also to radiation resistance of cells, is the following: The cell or t,he multicomplex will survive if, and only if, there is no segment of genetic material which is damaged in every one of the homologous sets of chromosomes of a diploid or polyploid cell, or in every one of the in1 This investigation was supported by research grant RG-6980 from the Division of General Medical Sciences of the National Institutes of Health, United States Public Health Service.
219
REPORTS
fecting viruses. An earlier approximate formula for this survival probability (1) is not applicable for large damages nor for very high doses of radiaGon or other inactivating agents. The purpose of this paper is to present’ a formula which is valid at, any dose and wit,h any size of damage. SURVIVAL
LAW
Let m be the degree of polyploidy (m = 2 for diploid cells, m = 3 for triploid cells, etc.) or the mukiplicity of infection. Let W, be the survival probability, h the number of lethal hit,s (dose), and L the mean lengt,h [measured in target units, see Barricelli (W)] of the damage caused in the genetic material by a let’hal hit. If W,(A) is the probabilit’y that, no single point> of a given genet,ic segment of target length A is damaged in every one of t’he m genomes, the probability W,(A + dA) will be given by:
hdA
1 (1)
where 0, is the probability that all the m genomes are damaged at a specific point, for instance, at the end of the above segment of target length A (defined as point A), and O,-1 is the probability t,hat’ m - 1 out of the m genomes are damaged and one of them is undamaged at the point A. 0, = (1 - e-Lh)m
and
(),-1 = m(l - e-Lh)m-le-Lh
(4
Thus 0,-1/l - 0, will be the probability of m - 1 damaged and one undamaged genomes at a point where it is known t’hat not all the m genomes are damaged. W,( /\) (O&l - 0,) will therefore be the probabilit’y t’hat no point of the segment A is damaged in all m genomes and that m - 1 genomes are damaged and one is undamaged at the end of this segment (point A). If this probability is multiplied by hdA (which, at the dose h, is the probability that a damage shall start between /j and A + d/j in the only genome which is undamaged at the point A), one obviously obt’ains the quan-