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Resistance against isotransplantation of mouse tumors induced by Rous sarcoma virus
H. 0. Sjiigren
and N. Jonsson
and lower oviducal segments might be correlated with the deposition around the egg of molecular configurations which promote the adhesion and penetration of spermatozoa. However, the increase in fertilizability of eggs from the ovisac as compared with oviducal eggs could not be explained on this basis. Possibly, physiological changes occur in eggs in this region of the female reproductive tract leading to greater fertilizability, or there are changes at the egg surface or in the jelly layers which enhance the susceptibility of the egg to the male gamete. Experiments are in progress to test these possibilities. As to the faster rate of cleavage of oviducal eggs, as compared with those from the ovisac, there seems to be no obvious a priori explanation. Two possibilities suggest themselves for further testing: (1) the absence in oviducal eggs of an inhibitory factor causing a slower cleavage rate, which may be present in eggs from the ovisac; (2) the presence in the oviducal environment of a factor causing accelerated cleavage, the absence of which in the ovisac restores the normal rate of division. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
APLINGTON, H. W., JR., Ohio J. Sci. 57, 91 (1957). BARCH, S. H. and SHAVER, J. R., Am. Zool. 3, 157 (1963). BATAILLON, E. and TCHOU-SU, Arch. Biol. 40, 439 (1930). DALCQ, A., PASTEELS, J. and BRACHET, J., Mem. Mus. Roy. Hisf. Nat. IIe st!r., 882 (1936). LINDQUIST, E. F., in Design and analysis of experiments in psychology and education, pp. 266-273. Houghton Mifflin Co., Boston, 1953. RUGH, R., J. Exptl Zool. 71, 163 (1935). SHAVER, J. R. and BARCH, S. H., Acta Embryol. Morphol. Exptl 3, 180 (1960). SHAVER, J. R., BARCH, S. H. and SHIVERS, C. A., J. Exptl Zool. 151, 95 (1962). SHIVERS, C. A., Am. Zool. 2, 448 (1962). SXEDECOR, G. W., in Statistical Methods, pp. 237-238. 5th ed. The Iowa State CollegePress,
1956.
RESISTANCE
AGAINST
ISOTRANSPLANTATION
INDUCED
BY
ROUS
H. 0. SJOGREN Institute
for
Tumor Biology, Karolinska Institute of Pathology, Received
OF MOUSE
SARCOMA
TUMORS
VIRUS1
and N. JONSSON Institutet University
November
Medical of Lund,
School, Sweden
Stockholm
and
11, 1963
IT has been demonstrated that specific transplantation resistance can be induced against isografts of certain tumors of known viral origin. Animals inoculated with polyoma, SV40, Gross, Graffi or Moloney virus showed various degrees of re1 This work has been supported by grants C-4747 and CA 06415-01 from Institute, United States Public Health Service, by grants from the Damon Fund (DRG-598), and the Swedish Cancer Society. Part
of the results
of this
study
was reported
Virus Group in London (Sept. 1963). Experimental
Cell Research
32
at the first
meeting
of The
the National Cancer Runyon Memorial European
Tumour
Resistance to isografting
of Rous sarcomas
619
sistance against the subsequent grafting of genetically compatible neoplastic cells induced by the same virus while no clear-cut cases of cross resistance have been found between tumors induced by different viruses [4, 6, 7, 8, IO, II, 12, 151. In the case of polyoma tumors and Shope papillomas a similar resistance could be induced by pretreatment with tumor cells not releasing infectious virus [5, 141 indicating that TABLE
Type
I. Pooled
results of 14 tests with four
of recipients
Untreated controls Untreated controls Rous virus infected Rous virus infected Polyoma virus infected Polyoma virus infected Homografted with Rous tumors Homografted with Rous tumors Homografted with non-Rous tumors Homografted with non-Rous tumors
Wholebody X-bradiationa -
Takesb 7 0.2 x D,
400 r 400 r
400 r
after
subcutaneous cell numberC D,
mouse tumors. inoculaton indicated
iOxD,
19/27
25125
lSj19
S/20
414 lS/ZS
314 12/15
19/21
8/8 18/21
400 1‘ 400 r -
Rous virus-induced
of the
100 x D,
200 x D,
O/4 O/3
6/7 517
717
Sk3 3/3
414 O/24 3/12
O/l8 2/11
16/19
S/l0
12/12
717
r Irradiation performed 24 hr prior to test challenge. The figures denote the number of animals with progressively growing tumors over the total number tested. ’ The cell number is indicated as multiples of the minimal cell dose necessary for growth in 100 per cent of the untreated controls ( = Dm). The D,‘s for the four tumors were 102, 103, 5 x 103, and 5 x lo3 cells respectively.
different tumors induced by the samevirus possesscommon, tumor-specific antigen(s) whosepresencedoesnot depend on the synthesis of mature virus particles and which do not necessarily represent any viral antigen. The present investigation extends these findings to tumors induced by the inoculation of Rous virus into newborn mice. Since the number of different species shown to be susceptibleto the oncogenic effect of the virus strain used is larger than for any other known virus, tumors induced with this virus would be especially suitable for an investigation of the possibleexistence of common antigens in tumors induced by the samevirus in different species. Virus.-The Schmidt-Ruppin strain of Rous virus was used [13]. This strain induces tumors in birds and mammals as well. Its action spectrum includes fowls, ducks, mice, rats, guinea pigs, hamsters and rabbits [l, 2, 91. A homogenate of the Rous chicken sarcomatissue was used asth \rirus sourcein the present investigation, prepared in 0.1 M potassium citrate. The material was stored at -79°C. Tumors.-Tumors were induced by inoculating a fresh suspensionof living Rous Experimental
Cell Research
32
H. 0. Sjiigren
and N. Jonsson
(Schmidt-Ruppin strain) chicken sarcoma cells subcutaneously into newborn mice of the strains A/%, A.BY, A.CA and C57BL/KL. Established tumors were passaged serially in genetically compatible mice. Four different tumors (one in each of the mouse strains) from the third to the twelfth passages were used in the experiments. Mechanically prepared crude cell suspensions were used for passage and for homografting (see below). Tumor suspensions used for isologous challenge inoculations were prepared by trypsinization. The approximate number of viable cells was estimated by trypan blue staining according to Boyse et al. [3], and a known inoculum dose was deposited in the right flank. In preliminary experiments the minimum cell dose required for the take and progressive growth of tumors in 100 per cent of untreated isologous controls ( =D,) was determined for each neoplasm. Tumors of non-Rous origin were used for homograft pretreatments. These tumors included two spontaneous mammary carcinomas, one methylcholantrene-induced sarcoma and three polyoma-induced tumors, two of which were induced in vivo and one in vitro, The following groups of adult recipients were challengedwith genetically compatible tumor cells: (1) untreated mice; (2) mice inoculated 2-6 times intraperitoneally with 0.1 ml of the Schmidt-Ruppin virus pool, the last dose given 5-10 days prior to the test challenge; (3) mice inoculated with polyoma virus intraperitoneally (tissue culture medium of polyoma-infected mouse-embryo cells); (4) mice pretreated 2-6 times with subcutaneous homografts of Rous sarcoma induced in foreign mouse strains, the last inoculation given 5-10 days prior to the test challenge; (5) mice pretreated with homografts of other mouse tumors of various origin, including neoplasmsinduced by the polyoma virus, by methylcholantrene or arising spontaneously. Some of the experiments also included mice that received whole-body X-irradiation (400 r) 24 hr prior to the test challenge. Results.-The results of 14 tests with four different Rous virus-induced tumors are summarized in Table I. Mice that had received Rous tumor homografts prior to the test challenge showed a clear-cut transplantation resistance, while homografting with non-Rous tumors induced no resistance. The resistance induced by Rous tumor homografts was not abolished by 400 r X-rays 24 hr prior to the test challenge. This finding, together with the absenceof resistance after pretreatment with nonRous tumor homografts rules out the possibility that an unspecific stimulation of the primary immune reaction against antigenic tumor cells was responsible for the resistance detected. The Rous virus in the form of chicken Rous tumor homogenate induced a weak resistance, while polyoma virus induced no resistance. The Rous virus-induced resistance, however, was either not specific or was very weak since it was not detectable in mice that received 400 r X-irradiation 24 hr prior to the test challenge. The specific resistance against isologous Rous tumors demonstrated in mice homografted with other Rous tumors indicates that these tumors possesscommon antigen(s). These might be identical with virus antigen(s) or they may represent cellular antigens peculiar to Rous tumors. It is not clear at present whether the Rous virus-induced mouse tumors release virus or not. The absenceor very weak restistance in mice pretreated with Rous viruscontaining material tends to indicate, however, that the transplantation resistance is not dependent on an antiviral immune responseof the recipients. Experimental
Cell Research
32
Resisfance fo isograffing
of Rous sarcomas
621
REFERENCES 1. AHLSTR~M,
C.
Cancrum 2.
AHLSTRGM,
G.,
BERGMAN,
S.,
FORSBY,
N.
and
JONSSON,
N.,
Acta
Unio
Intern.
Contra
19, 294 (1963). C. G.,
JONSSON,
N.
and
FORSBY,
N.,
Acta
Pathol.
Jlicrobiol.
Scand.
Suppt.
154,
127 (1962). 3. 4. 5. 6.
7. 8.
9. 10. 11.
12. 13. 14. 15.
BOYSE, E. A., OLD, L. J. and THOMM, G., Transplant. Bull. 29, 64 (1962). DEFESDI, V., Proc. Sot. Exptt Biot. Med. 113, 12 (1963). EVANS, C. A., WEISER, R. S. and ITO, Y., Cold Spring Harbor Symp. Quant. Biol. 27, (1962). HABEL, K., Proc. Sot. Exptl Biol. Xed. 106, 722 (1961). HABEL, K. and EDDY, B. E., ibid. 113, 1 (1963). KLEIN, G., SJGGREN, H. 0. and KLEIN, E., Cancer Res. 22, 955 (1962). KLEMENT, V. and SVOBODA, J., Folia biot. 9, 181 (1963). KOCH, M. A. and SABIN, A. B., Proc. Sot. Exptl Biol. Xed. 113, 4 (1963). PASTERNAK, G. and GRAFFI, A., Brit. J. Cancer. In press. SACHS, L., J. Natl. Cancer Inst. 29, 759 (1962). SCHMIDT-RUPPIN, H., Krebsforsch. Krebsbekiimpfung 3, 26, Strahlentherapie Sonderband (1959). SJGGREN, H. O., Virology 15, 214 (1961). SJGGRES, H. O., HELLSTRGM, I. and KLEIS, G., Cancer Res. 21, 329 (1961).
Experimental
Cell
Research
453
41
32
and N. Jonsson
and lower oviducal segments might be correlated with the deposition around the egg of molecular configurations which promote the adhesion and penetration of spermatozoa. However, the increase in fertilizability of eggs from the ovisac as compared with oviducal eggs could not be explained on this basis. Possibly, physiological changes occur in eggs in this region of the female reproductive tract leading to greater fertilizability, or there are changes at the egg surface or in the jelly layers which enhance the susceptibility of the egg to the male gamete. Experiments are in progress to test these possibilities. As to the faster rate of cleavage of oviducal eggs, as compared with those from the ovisac, there seems to be no obvious a priori explanation. Two possibilities suggest themselves for further testing: (1) the absence in oviducal eggs of an inhibitory factor causing a slower cleavage rate, which may be present in eggs from the ovisac; (2) the presence in the oviducal environment of a factor causing accelerated cleavage, the absence of which in the ovisac restores the normal rate of division. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
APLINGTON, H. W., JR., Ohio J. Sci. 57, 91 (1957). BARCH, S. H. and SHAVER, J. R., Am. Zool. 3, 157 (1963). BATAILLON, E. and TCHOU-SU, Arch. Biol. 40, 439 (1930). DALCQ, A., PASTEELS, J. and BRACHET, J., Mem. Mus. Roy. Hisf. Nat. IIe st!r., 882 (1936). LINDQUIST, E. F., in Design and analysis of experiments in psychology and education, pp. 266-273. Houghton Mifflin Co., Boston, 1953. RUGH, R., J. Exptl Zool. 71, 163 (1935). SHAVER, J. R. and BARCH, S. H., Acta Embryol. Morphol. Exptl 3, 180 (1960). SHAVER, J. R., BARCH, S. H. and SHIVERS, C. A., J. Exptl Zool. 151, 95 (1962). SHIVERS, C. A., Am. Zool. 2, 448 (1962). SXEDECOR, G. W., in Statistical Methods, pp. 237-238. 5th ed. The Iowa State CollegePress,
1956.
RESISTANCE
AGAINST
ISOTRANSPLANTATION
INDUCED
BY
ROUS
H. 0. SJOGREN Institute
for
Tumor Biology, Karolinska Institute of Pathology, Received
OF MOUSE
SARCOMA
TUMORS
VIRUS1
and N. JONSSON Institutet University
November
Medical of Lund,
School, Sweden
Stockholm
and
11, 1963
IT has been demonstrated that specific transplantation resistance can be induced against isografts of certain tumors of known viral origin. Animals inoculated with polyoma, SV40, Gross, Graffi or Moloney virus showed various degrees of re1 This work has been supported by grants C-4747 and CA 06415-01 from Institute, United States Public Health Service, by grants from the Damon Fund (DRG-598), and the Swedish Cancer Society. Part
of the results
of this
study
was reported
Virus Group in London (Sept. 1963). Experimental
Cell Research
32
at the first
meeting
of The
the National Cancer Runyon Memorial European
Tumour
Resistance to isografting
of Rous sarcomas
619
sistance against the subsequent grafting of genetically compatible neoplastic cells induced by the same virus while no clear-cut cases of cross resistance have been found between tumors induced by different viruses [4, 6, 7, 8, IO, II, 12, 151. In the case of polyoma tumors and Shope papillomas a similar resistance could be induced by pretreatment with tumor cells not releasing infectious virus [5, 141 indicating that TABLE
Type
I. Pooled
results of 14 tests with four
of recipients
Untreated controls Untreated controls Rous virus infected Rous virus infected Polyoma virus infected Polyoma virus infected Homografted with Rous tumors Homografted with Rous tumors Homografted with non-Rous tumors Homografted with non-Rous tumors
Wholebody X-bradiationa -
Takesb 7 0.2 x D,
400 r 400 r
400 r
after
subcutaneous cell numberC D,
mouse tumors. inoculaton indicated
iOxD,
19/27
25125
lSj19
S/20
414 lS/ZS
314 12/15
19/21
8/8 18/21
400 1‘ 400 r -
Rous virus-induced
of the
100 x D,
200 x D,
O/4 O/3
6/7 517
717
Sk3 3/3
414 O/24 3/12
O/l8 2/11
16/19
S/l0
12/12
717
r Irradiation performed 24 hr prior to test challenge. The figures denote the number of animals with progressively growing tumors over the total number tested. ’ The cell number is indicated as multiples of the minimal cell dose necessary for growth in 100 per cent of the untreated controls ( = Dm). The D,‘s for the four tumors were 102, 103, 5 x 103, and 5 x lo3 cells respectively.
different tumors induced by the samevirus possesscommon, tumor-specific antigen(s) whosepresencedoesnot depend on the synthesis of mature virus particles and which do not necessarily represent any viral antigen. The present investigation extends these findings to tumors induced by the inoculation of Rous virus into newborn mice. Since the number of different species shown to be susceptibleto the oncogenic effect of the virus strain used is larger than for any other known virus, tumors induced with this virus would be especially suitable for an investigation of the possibleexistence of common antigens in tumors induced by the samevirus in different species. Virus.-The Schmidt-Ruppin strain of Rous virus was used [13]. This strain induces tumors in birds and mammals as well. Its action spectrum includes fowls, ducks, mice, rats, guinea pigs, hamsters and rabbits [l, 2, 91. A homogenate of the Rous chicken sarcomatissue was used asth \rirus sourcein the present investigation, prepared in 0.1 M potassium citrate. The material was stored at -79°C. Tumors.-Tumors were induced by inoculating a fresh suspensionof living Rous Experimental
Cell Research
32
H. 0. Sjiigren
and N. Jonsson
(Schmidt-Ruppin strain) chicken sarcoma cells subcutaneously into newborn mice of the strains A/%, A.BY, A.CA and C57BL/KL. Established tumors were passaged serially in genetically compatible mice. Four different tumors (one in each of the mouse strains) from the third to the twelfth passages were used in the experiments. Mechanically prepared crude cell suspensions were used for passage and for homografting (see below). Tumor suspensions used for isologous challenge inoculations were prepared by trypsinization. The approximate number of viable cells was estimated by trypan blue staining according to Boyse et al. [3], and a known inoculum dose was deposited in the right flank. In preliminary experiments the minimum cell dose required for the take and progressive growth of tumors in 100 per cent of untreated isologous controls ( =D,) was determined for each neoplasm. Tumors of non-Rous origin were used for homograft pretreatments. These tumors included two spontaneous mammary carcinomas, one methylcholantrene-induced sarcoma and three polyoma-induced tumors, two of which were induced in vivo and one in vitro, The following groups of adult recipients were challengedwith genetically compatible tumor cells: (1) untreated mice; (2) mice inoculated 2-6 times intraperitoneally with 0.1 ml of the Schmidt-Ruppin virus pool, the last dose given 5-10 days prior to the test challenge; (3) mice inoculated with polyoma virus intraperitoneally (tissue culture medium of polyoma-infected mouse-embryo cells); (4) mice pretreated 2-6 times with subcutaneous homografts of Rous sarcoma induced in foreign mouse strains, the last inoculation given 5-10 days prior to the test challenge; (5) mice pretreated with homografts of other mouse tumors of various origin, including neoplasmsinduced by the polyoma virus, by methylcholantrene or arising spontaneously. Some of the experiments also included mice that received whole-body X-irradiation (400 r) 24 hr prior to the test challenge. Results.-The results of 14 tests with four different Rous virus-induced tumors are summarized in Table I. Mice that had received Rous tumor homografts prior to the test challenge showed a clear-cut transplantation resistance, while homografting with non-Rous tumors induced no resistance. The resistance induced by Rous tumor homografts was not abolished by 400 r X-rays 24 hr prior to the test challenge. This finding, together with the absenceof resistance after pretreatment with nonRous tumor homografts rules out the possibility that an unspecific stimulation of the primary immune reaction against antigenic tumor cells was responsible for the resistance detected. The Rous virus in the form of chicken Rous tumor homogenate induced a weak resistance, while polyoma virus induced no resistance. The Rous virus-induced resistance, however, was either not specific or was very weak since it was not detectable in mice that received 400 r X-irradiation 24 hr prior to the test challenge. The specific resistance against isologous Rous tumors demonstrated in mice homografted with other Rous tumors indicates that these tumors possesscommon antigen(s). These might be identical with virus antigen(s) or they may represent cellular antigens peculiar to Rous tumors. It is not clear at present whether the Rous virus-induced mouse tumors release virus or not. The absenceor very weak restistance in mice pretreated with Rous viruscontaining material tends to indicate, however, that the transplantation resistance is not dependent on an antiviral immune responseof the recipients. Experimental
Cell Research
32
Resisfance fo isograffing
of Rous sarcomas
621
REFERENCES 1. AHLSTR~M,
C.
Cancrum 2.
AHLSTRGM,
G.,
BERGMAN,
S.,
FORSBY,
N.
and
JONSSON,
N.,
Acta
Unio
Intern.
Contra
19, 294 (1963). C. G.,
JONSSON,
N.
and
FORSBY,
N.,
Acta
Pathol.
Jlicrobiol.
Scand.
Suppt.
154,
127 (1962). 3. 4. 5. 6.
7. 8.
9. 10. 11.
12. 13. 14. 15.
BOYSE, E. A., OLD, L. J. and THOMM, G., Transplant. Bull. 29, 64 (1962). DEFESDI, V., Proc. Sot. Exptt Biot. Med. 113, 12 (1963). EVANS, C. A., WEISER, R. S. and ITO, Y., Cold Spring Harbor Symp. Quant. Biol. 27, (1962). HABEL, K., Proc. Sot. Exptl Biol. Xed. 106, 722 (1961). HABEL, K. and EDDY, B. E., ibid. 113, 1 (1963). KLEIN, G., SJGGREN, H. 0. and KLEIN, E., Cancer Res. 22, 955 (1962). KLEMENT, V. and SVOBODA, J., Folia biot. 9, 181 (1963). KOCH, M. A. and SABIN, A. B., Proc. Sot. Exptl Biol. Xed. 113, 4 (1963). PASTERNAK, G. and GRAFFI, A., Brit. J. Cancer. In press. SACHS, L., J. Natl. Cancer Inst. 29, 759 (1962). SCHMIDT-RUPPIN, H., Krebsforsch. Krebsbekiimpfung 3, 26, Strahlentherapie Sonderband (1959). SJGGREN, H. O., Virology 15, 214 (1961). SJGGRES, H. O., HELLSTRGM, I. and KLEIS, G., Cancer Res. 21, 329 (1961).
Experimental
Cell
Research
453
41
32