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Resistance to moloney murine sarcoma virus-induced tumorigenesis in NK-deficient beige mice
CELLULAR
IMMUNOLOGY
74, 120-125 (1982)
Resistance to Moloney Murine Sarcoma Virus-Induced Tumorigenesis in NK-Deficient beige Mice’ ANNE T. TRUESDALE,~ DAVID A. JOHNSON,~ HENDRICK G. BEDIGIAN, HENRY C. OUTZEN, AND GEORGE A. CARLSO~ The Jackson Laboratory, Bar Harbor, Maine 04609 Received July 13, 1982; accepted September I, 1982 C57BL/6J-bg’ bg/bg mice are reported to be lesssusceptible to tumor induction by threshold dosesof Moloney murine sarcoma virus than their +/bg littermates, and there are no significant differences between bg/bg and +/bg mice in which tumors were induced with respect to tumor latency, size, and regression rate. The difference in tumor frequency cannot be accounted for by M-MSV boosting of activity in bg/bg mice or by depression of activity in +/bg animals.
INTRODUCTION Mice homozygous for the mutation beigehave greatly reduced, though not a total loss of, natural killer (NK) cell activity ( 1). NK cells are a nonadaptive and spontaneously cytolytic lymphocyte subpopulation whose activity has been shown to be directed against a variety of cell types in vitro and in vivo. These cells have been postulated to serve as a surveillance mechanism against neoplasia and to prevent the metastatic spread of tumors (2). Appealing evidence in support of this was the finding that nude mice, which are surprisingly tumor resistant (3,4), possessedhigh NK-cell activity (5). Because the beige mutation was reported to be relatively selective for NK activity (6), the beige mouse has been frequently used to provide direct evidence for the significance of NK cells in antitumor surveillance. For example Karre et al. (7) found that bgJ/bgJ mice were more susceptible than +/bgJ animals to two transplantable lymphomas, EL4 and P52- 127-166, while Talmadge et al. (8) reported similar findings with the B16 melanoma line. However, it is difficult to draw a general conclusion regarding the role of NK cells in antitumor surveillance when transplantable cell lines known to be susceptible to NK-mediated lysis in vitro are used. It is more appropriate to examine the susceptibility of beige mice to induction of autochthonous tumors before reaching a decision on the effectiveness of the NK system in surveillance. In this communication we report that bg/bg mice actually were less susceptible to tumor induction by threshold doses of Moloney murine sarcoma virus (M-MSV) than their +/bg littermates, and that there ’ This investigation was supported by Grants CA 28231, CA 24901, CA 25944, CA 31102, and CP 33255 from the National Cancer Institute, USPHS. 2 Present address: Reed College, Portland, Oreg. 97202. 3 Present address: Scripps Clinic and Research Foundation, La Jolla, Calif. 92037. 4 To whom correspondence should be addressed. 120 0008-8749/82/ 170120-06$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
VIRAL
ONCOGENESIS
IN BEIGE MICE
121
were no significant differences between bg/bg and +/bg mice in which tumors were induced with respect to tumor latency, size, and regression rate. The difference in tumor frequency could not be accounted for by either M-MSV boosting NK activity in bg/bg mice or depressing activity in +/bg animals. MATERIALS
AND METHODS
Mice. C57BL/6J-bgJ/bgJ and +/bgJ mice were either purchased from Animal Resources at the Jackson Laboratory or obtained from our own colony. The mice used were between 6 and 9 weeks of age. Moloney murine sarcoma virus. M-MSV was obtained from Research Resources, Biological Carcinogenesis Program of the U.S. National Cancer Institute (lots MSVB-162 and MSV-B-165). A virus stock was also prepared from tumor homogenates of DBA/2J female mice injected with NC1 stock virus. This preparation was titered for focus formation on ARK-2B cells and found to contain approximately 3 X IO3 focus-forming units (FFU) per milliliter. Tumor induction and measurement. Virus preparations were diluted in phosphatebuffered saline and 0.05 or 0.1 ml injected into the gastrocnemius muscle. Leg measurements in two dimensions were taken daily with calipers and an approximation of the ellipsoid volume was calculated. Probability values were calculated using the Student t test, the x2 test, and the sign test for paired animals where appropriate (9). Natural killer cell assay. YAC-1 cells (obtained through the courtesy of G. Cudkowicz, State University of New York at Buffalo) were labeled with 51Cras sodium chromate (New England Nuclear) for use as targets in a standard 4-hr assayof splenic NK activity using 96-well microtiter plates and RPM1 1640 supplemented with 10% fetal bovine serum. The percentage specific release was calculated as follows: percent release = (sample release - spontaneous release) x 1oo ’ (total - spontaneous release) “Total” represents the amount of label actually in the target cells and “spontaneous release” is the amount of radioactivity released in the absence of spleen cells. RESULTS Tumor induction by threshold doses of M-MS’ in bg/bg and +/bg Mice.. A total of 25 bg/bg and +/bg male C57BL/6J-bgJ mice were littermate paired and groups of six or seven given 0.1 ml of two-fold serial dilutions from undiluted to 1:8 of our virus preparation (300-37.5 FFU). This range of dilutions was chosen after preliminary experiments titrating the virus in C57BL/6J mice. Each pair was numbered and assigned randomly to a cage. Thus each cage contained both bg/bg and +/bg mice and most often recipients at at least two different dilutions of virus, thereby reducing the possibility of “cage effects” as a factor in our results. Leg measurements were taken daily for 22 days. The percentagesof bg/bg and +/bg mice with measurable tumors at each dilution are shown in Fig. 1. At a later date, five bg/bg and five +/ bg female C57BL/6J-bg’ mice were given 0.1 ml of the undiluted stock, and these results are also shown in Fig. 1. At all dilutions and in both sexes,tumor frequency was greater in +/bg than in bg/bg mice, with approximately 64 FFU required for 50% tumor incidence in +/bg while 110 FFU were required in bg/bg animals. Using
122
TRUESDALE
ET AL.
2
0 Log.2
MMSV
3
Dilution
FIG. 1. Tumor induction in C57BL/6J-bg/bg and +/bg mice by threshold inocula of M-MSV. The circles represent percentage tumors in groups of six male mice (seven per group at a virus log2 dilution = 1) innoculated with dilutions of virus. The squares represent five female mice each. Open symbols represent +/bg mice and closed symbols, bg/bg mice. See text for details.
the x2 test, cumulative tumor incidence in bg/bg mice was significantly lower than in +/bg animals (p = 0.039). Since normal and mutant littermates were paired, significance was also analyzed using the sign test (9). Of the 14 pairs in which one mouse developed a tumor while the other did not, 12 pairs had +/bg as the tumor bearer and only two pairs were the reverse (P = 0.0 12). Using the sign test on paired mice which both developed tumors did not. reveal any significant differences in tumor latency, size, or regression times between bg/bg and +/bg mice. Tumor growth in bg/bg and +/bg mice, In order to confirm that the growth and regression of M-MSV-induced tumors did not differ significantly between bg/bg and +/bg mice, larger inocula of virus were used. Two experiments were carried out, the results of which are shown in Fig. 2. In the first experiment, C57BL/6J female bg/bg and +/bg mice were injected with 2 X lo5 FFU M-MSV. In both groups, six of seven injected mice developed tumors which appeared clinically between 2 to 4 days postinjection, peaked at 4 to 5 days, and then regressed.At no point were the differences in tumor size between the two groups of mice statistically significant, although tumor growth appeared to be slightly lower in the NK-deficient bg/bg animals. In the second experiment, groups of 14 male +/+ and bg/bg mice were injected with 2 X lo5 FFU M-MSV. Tumors appeared in all animals of both groups starting by 4 days and peaking at 6 days postinjection. The tumors in both groups were slightly larger than in the first experiment, but there was no significant difference in tumor growth between the two groups of mice. It is of interest to note, however, that in this experiment using male mice, the tumors were slightly larger in the bg/ bg mice, while the opposite effect was seen in the first experiment with female mice. NK activity ofM-MSV-infected mice. In preliminary experiments (data not shown) C57BL/6J mice were inoculated with twofold dilutions of M-MSV ranging from 5 X lo4 to 3 1 FFU and NK activity determined 1, 4, 8 and 11 days postinjection. In contrast to the results of Gerson et al. (10) and Becker and Klein (1 I), we found no significant difference in NK activity between M-MSV injected and control mice. Figure 3 illustrates the results of one experiment using C57BL/6J-bgJ mice. Four
VIRAL ONCOGENESIS IN BEIGE MICE
09
bgJ/+
-
bgJ/bgJ
---
+/+
0
2
4
6
6
IO I2
DAYS PDSTMSV
14 16
123
-
IS 20
CHALLENGE
FIG. 2. Tumor growth in bg/bg and phenotypically normal C57BL/6J mice. Mice were injected im with 2 X IO5 FFU of M-MSV. No signficant change in leg muscle size occurred in uninjected controls (not shown). Standard errors are indicated by the bars. In the first experiment (upper panel) groups of seven female mice were used. In the second experiment (lower panel) groups of 14 male mice were used.
+/bg and four bg/bg mice were injected with 150 FFU of M-MSV and splenic NK activity against YAC-1 was tested 3 days later. Although NK levels in the virusinjected mice were slightly higher than those in the controls, the differences were not statistically significant. From these results it was evident that neither a boost in bg/bg NK activity nor a depression of +/bg levels could account for the difference in tumor incidence. DISCUSSION The surprising findings that NK-deficient beigemice were lesssusceptible to tumor induction by threshold inocula of M-MSV and that the growth patterns of the tumors appearing did not differ significantly between bg/bg and +/bg mice superficially could be taken as evidence that NK cells do not play a significant role in surveillance against transformed cells. However, it is important to emphasize that the natural resistance system is complex, and that the beigemutation does not cause malfunction of all subpopulations of NK cells ( 12, 13, and Carlson et al., submitted). Nonetheless, it is clear that the NK defect of beige mice did not result in increased susceptibility to M-MSV tumorigenesis. There are several possible explanations for the lower susceptibility of beige mice to threshold inocula of M-MSV. Tumors induced by this virus are primarily composed of infiltrating host cells, and not by transformed cells alone (14). In addition to NK cells, macrophage ( 15), granulocyte ( 16), and cytotoxic T-cell ( 17) functions are affected by the beige mutation, and hence the lower tumor incidence in the beige animals may reflect a reduced inflammatory response rather than a lower susceptibility to transformation. Of possible relevance is our recent finding that the
124
TRUESDALE
ET AL.
-_ 26
-
22
-
6-
EFFECTOR
: TARaET
FIG. 3. NK activity in M-MSV-injected bg/bg and +/bg mice. Four bg/bg and four +/bg mice were injected im with 150 FFU of M-MSV and splenic activity against YAC-I tested 3 days later (0 = +/bg q = bg/bg). Two mice of each genotype served as uninjected controls (0 = +/bg n = bg/bg). Average specific lysis for each group is shown. There was no statistically significant difference between virusinoculated and uninjected mice.
immune rejection response against allogeneic tumor cells occurs later in bg/bg than in +/bg animals (manuscript in preparation). In this regard, immunostimulation of tumor growth (18) or induction of suppressor cells due to impaired immunoregulation in NK-deficient mice (19) warrant consideration as factors which could account for the difference in tumor frequency between bg/bg and +/bg mice. On the other hand, the finding that tumor latency, growth, and regression rate did not differ significantly between mutant and phenotypically normal animals might suggest that differential susceptibility to transformation by M-MSV is responsible. For example, the beige mutation affects lysosome morphology, possibly via a defect in lipid metabolism (20) which might alter the susceptibility of the target tissue to infection or transformation by the virus. No evidence is available on this point, however. In conclusion, relatively few data are available on the susceptibility of beige mice to autochthonous tumor induction. In contrast to the results presented here, Argov et al. (2 1) reported that bg/bg mice developed tumors slightly earlier than +/bg mice after feeding with DMBA. The lack of a dramatic difference in tumor incidence was attributed to suppression of NK activity in the phenotypically normal animals by the carcinogen. However, caution should be exercised in attributing differences in tumor incidence between bg/bg and +/bg mice solely to NK cells due to the pleiotropic effects of this mutation.
VIRAL ONCQGENESIS IN BEIGE MICE
125
REFERENCES 1. Roder, J., and Duwe, A., Nature (London) 278, 451, 1979. 2. Kiessling, R., and Wigzell, H., Immunol. Rev. 44, 165, 1979. 3. Outzen, H. C., Custer, R. P., Eaton, G. J., and Prehn, R. T., J. Reticuloendothelial. Sot. 17, 1, 1975. 4. Rygaard, J., and Povlsen, C. O., Transplant. Rev. 28, 43, 1976. 5. Herberman, R. B., and Holden, H. T., Adv. Cancer Res. 27, 305, 1978. 6. Roder, J. C., Lohmann-Matthes, M., Domzig, W., and Wigzell, H., J. Immunol. 123, 2174, 1979. 7. Karre, K., Klein, G. O., Kiessling, R., Klein, G., and Roder, J. C., Nature (London) 284, 624, 1980. 8. Talmadge, J. E., Meyers, K. M., Prieur, D. J., and Starkey, J. R., Nature (London) 284, 622, 1980. 9. Siegel, S., “Nonparametric Statistics for the Behavioral Sciences.” McGraw-Hill, New York, 1956. 10. Gerson, J. M., Varesio, L., and Herberman, R. B., Int. J. Cancer 27, 243, 1981. I I. Becker, S., and Klein, E., J. Immunol. 6, 892, 1976. 12. Lust, J. A., Kumar, V., Burton, R. C., Bartlett, S. P., and Bennett, M., J. Exp. Med. 154, 306. 1981. 13. Minato, N., Reid, L., and Bloom, B. R., J. Exp. Med. 154, 750, 1981. 14. Stanton, M. F., Law, L. W., and Ting, R. C., J. Nat. Cancer Inst. 40, 1113, 1968. 15. Morahan, P. S., Law, L. W., and Ting, R. C., In “Genetic Control of Natural Resistance to Infection and Malignancy” (E. Skamene, P. A. L. Kongshavn, and M. Landy, Eds.), p. 575. Academic Press, New York, 1980. 16. Gallin, J. F., Bujak, J. S., Patten, E., and Wolff, S. M., Blood 43, 201, 1974. 17. Saxena, R. K., Saxena, Q. B., and Adler, W. H., Nature (London) 295, 240, 1982. 18. Prehn, R. T., J. Reticuloendothel. Sac. 10, 1, 1971. 19. Plata, F., MacDonald, R., and Shain, B., J. Immunol. 123, 852, 1979. 20. Brandt, E. J., Swank, R. T., and Novak, E. K., In, “Immunologic Defects in Laboratory Animals” (M. E. Gershwin, and B. Merchant, Eds.), Vol. I, p. 99. Plenum, New York, 1981. 21. Argov, S., Cochran, A. J., Karre, K., Klein, G. O., and Klein, G., Int. J. Cancer 28, 739, 1981.
IMMUNOLOGY
74, 120-125 (1982)
Resistance to Moloney Murine Sarcoma Virus-Induced Tumorigenesis in NK-Deficient beige Mice’ ANNE T. TRUESDALE,~ DAVID A. JOHNSON,~ HENDRICK G. BEDIGIAN, HENRY C. OUTZEN, AND GEORGE A. CARLSO~ The Jackson Laboratory, Bar Harbor, Maine 04609 Received July 13, 1982; accepted September I, 1982 C57BL/6J-bg’ bg/bg mice are reported to be lesssusceptible to tumor induction by threshold dosesof Moloney murine sarcoma virus than their +/bg littermates, and there are no significant differences between bg/bg and +/bg mice in which tumors were induced with respect to tumor latency, size, and regression rate. The difference in tumor frequency cannot be accounted for by M-MSV boosting of activity in bg/bg mice or by depression of activity in +/bg animals.
INTRODUCTION Mice homozygous for the mutation beigehave greatly reduced, though not a total loss of, natural killer (NK) cell activity ( 1). NK cells are a nonadaptive and spontaneously cytolytic lymphocyte subpopulation whose activity has been shown to be directed against a variety of cell types in vitro and in vivo. These cells have been postulated to serve as a surveillance mechanism against neoplasia and to prevent the metastatic spread of tumors (2). Appealing evidence in support of this was the finding that nude mice, which are surprisingly tumor resistant (3,4), possessedhigh NK-cell activity (5). Because the beige mutation was reported to be relatively selective for NK activity (6), the beige mouse has been frequently used to provide direct evidence for the significance of NK cells in antitumor surveillance. For example Karre et al. (7) found that bgJ/bgJ mice were more susceptible than +/bgJ animals to two transplantable lymphomas, EL4 and P52- 127-166, while Talmadge et al. (8) reported similar findings with the B16 melanoma line. However, it is difficult to draw a general conclusion regarding the role of NK cells in antitumor surveillance when transplantable cell lines known to be susceptible to NK-mediated lysis in vitro are used. It is more appropriate to examine the susceptibility of beige mice to induction of autochthonous tumors before reaching a decision on the effectiveness of the NK system in surveillance. In this communication we report that bg/bg mice actually were less susceptible to tumor induction by threshold doses of Moloney murine sarcoma virus (M-MSV) than their +/bg littermates, and that there ’ This investigation was supported by Grants CA 28231, CA 24901, CA 25944, CA 31102, and CP 33255 from the National Cancer Institute, USPHS. 2 Present address: Reed College, Portland, Oreg. 97202. 3 Present address: Scripps Clinic and Research Foundation, La Jolla, Calif. 92037. 4 To whom correspondence should be addressed. 120 0008-8749/82/ 170120-06$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
VIRAL
ONCOGENESIS
IN BEIGE MICE
121
were no significant differences between bg/bg and +/bg mice in which tumors were induced with respect to tumor latency, size, and regression rate. The difference in tumor frequency could not be accounted for by either M-MSV boosting NK activity in bg/bg mice or depressing activity in +/bg animals. MATERIALS
AND METHODS
Mice. C57BL/6J-bgJ/bgJ and +/bgJ mice were either purchased from Animal Resources at the Jackson Laboratory or obtained from our own colony. The mice used were between 6 and 9 weeks of age. Moloney murine sarcoma virus. M-MSV was obtained from Research Resources, Biological Carcinogenesis Program of the U.S. National Cancer Institute (lots MSVB-162 and MSV-B-165). A virus stock was also prepared from tumor homogenates of DBA/2J female mice injected with NC1 stock virus. This preparation was titered for focus formation on ARK-2B cells and found to contain approximately 3 X IO3 focus-forming units (FFU) per milliliter. Tumor induction and measurement. Virus preparations were diluted in phosphatebuffered saline and 0.05 or 0.1 ml injected into the gastrocnemius muscle. Leg measurements in two dimensions were taken daily with calipers and an approximation of the ellipsoid volume was calculated. Probability values were calculated using the Student t test, the x2 test, and the sign test for paired animals where appropriate (9). Natural killer cell assay. YAC-1 cells (obtained through the courtesy of G. Cudkowicz, State University of New York at Buffalo) were labeled with 51Cras sodium chromate (New England Nuclear) for use as targets in a standard 4-hr assayof splenic NK activity using 96-well microtiter plates and RPM1 1640 supplemented with 10% fetal bovine serum. The percentage specific release was calculated as follows: percent release = (sample release - spontaneous release) x 1oo ’ (total - spontaneous release) “Total” represents the amount of label actually in the target cells and “spontaneous release” is the amount of radioactivity released in the absence of spleen cells. RESULTS Tumor induction by threshold doses of M-MS’ in bg/bg and +/bg Mice.. A total of 25 bg/bg and +/bg male C57BL/6J-bgJ mice were littermate paired and groups of six or seven given 0.1 ml of two-fold serial dilutions from undiluted to 1:8 of our virus preparation (300-37.5 FFU). This range of dilutions was chosen after preliminary experiments titrating the virus in C57BL/6J mice. Each pair was numbered and assigned randomly to a cage. Thus each cage contained both bg/bg and +/bg mice and most often recipients at at least two different dilutions of virus, thereby reducing the possibility of “cage effects” as a factor in our results. Leg measurements were taken daily for 22 days. The percentagesof bg/bg and +/bg mice with measurable tumors at each dilution are shown in Fig. 1. At a later date, five bg/bg and five +/ bg female C57BL/6J-bg’ mice were given 0.1 ml of the undiluted stock, and these results are also shown in Fig. 1. At all dilutions and in both sexes,tumor frequency was greater in +/bg than in bg/bg mice, with approximately 64 FFU required for 50% tumor incidence in +/bg while 110 FFU were required in bg/bg animals. Using
122
TRUESDALE
ET AL.
2
0 Log.2
MMSV
3
Dilution
FIG. 1. Tumor induction in C57BL/6J-bg/bg and +/bg mice by threshold inocula of M-MSV. The circles represent percentage tumors in groups of six male mice (seven per group at a virus log2 dilution = 1) innoculated with dilutions of virus. The squares represent five female mice each. Open symbols represent +/bg mice and closed symbols, bg/bg mice. See text for details.
the x2 test, cumulative tumor incidence in bg/bg mice was significantly lower than in +/bg animals (p = 0.039). Since normal and mutant littermates were paired, significance was also analyzed using the sign test (9). Of the 14 pairs in which one mouse developed a tumor while the other did not, 12 pairs had +/bg as the tumor bearer and only two pairs were the reverse (P = 0.0 12). Using the sign test on paired mice which both developed tumors did not. reveal any significant differences in tumor latency, size, or regression times between bg/bg and +/bg mice. Tumor growth in bg/bg and +/bg mice, In order to confirm that the growth and regression of M-MSV-induced tumors did not differ significantly between bg/bg and +/bg mice, larger inocula of virus were used. Two experiments were carried out, the results of which are shown in Fig. 2. In the first experiment, C57BL/6J female bg/bg and +/bg mice were injected with 2 X lo5 FFU M-MSV. In both groups, six of seven injected mice developed tumors which appeared clinically between 2 to 4 days postinjection, peaked at 4 to 5 days, and then regressed.At no point were the differences in tumor size between the two groups of mice statistically significant, although tumor growth appeared to be slightly lower in the NK-deficient bg/bg animals. In the second experiment, groups of 14 male +/+ and bg/bg mice were injected with 2 X lo5 FFU M-MSV. Tumors appeared in all animals of both groups starting by 4 days and peaking at 6 days postinjection. The tumors in both groups were slightly larger than in the first experiment, but there was no significant difference in tumor growth between the two groups of mice. It is of interest to note, however, that in this experiment using male mice, the tumors were slightly larger in the bg/ bg mice, while the opposite effect was seen in the first experiment with female mice. NK activity ofM-MSV-infected mice. In preliminary experiments (data not shown) C57BL/6J mice were inoculated with twofold dilutions of M-MSV ranging from 5 X lo4 to 3 1 FFU and NK activity determined 1, 4, 8 and 11 days postinjection. In contrast to the results of Gerson et al. (10) and Becker and Klein (1 I), we found no significant difference in NK activity between M-MSV injected and control mice. Figure 3 illustrates the results of one experiment using C57BL/6J-bgJ mice. Four
VIRAL ONCOGENESIS IN BEIGE MICE
09
bgJ/+
-
bgJ/bgJ
---
+/+
0
2
4
6
6
IO I2
DAYS PDSTMSV
14 16
123
-
IS 20
CHALLENGE
FIG. 2. Tumor growth in bg/bg and phenotypically normal C57BL/6J mice. Mice were injected im with 2 X IO5 FFU of M-MSV. No signficant change in leg muscle size occurred in uninjected controls (not shown). Standard errors are indicated by the bars. In the first experiment (upper panel) groups of seven female mice were used. In the second experiment (lower panel) groups of 14 male mice were used.
+/bg and four bg/bg mice were injected with 150 FFU of M-MSV and splenic NK activity against YAC-1 was tested 3 days later. Although NK levels in the virusinjected mice were slightly higher than those in the controls, the differences were not statistically significant. From these results it was evident that neither a boost in bg/bg NK activity nor a depression of +/bg levels could account for the difference in tumor incidence. DISCUSSION The surprising findings that NK-deficient beigemice were lesssusceptible to tumor induction by threshold inocula of M-MSV and that the growth patterns of the tumors appearing did not differ significantly between bg/bg and +/bg mice superficially could be taken as evidence that NK cells do not play a significant role in surveillance against transformed cells. However, it is important to emphasize that the natural resistance system is complex, and that the beigemutation does not cause malfunction of all subpopulations of NK cells ( 12, 13, and Carlson et al., submitted). Nonetheless, it is clear that the NK defect of beige mice did not result in increased susceptibility to M-MSV tumorigenesis. There are several possible explanations for the lower susceptibility of beige mice to threshold inocula of M-MSV. Tumors induced by this virus are primarily composed of infiltrating host cells, and not by transformed cells alone (14). In addition to NK cells, macrophage ( 15), granulocyte ( 16), and cytotoxic T-cell ( 17) functions are affected by the beige mutation, and hence the lower tumor incidence in the beige animals may reflect a reduced inflammatory response rather than a lower susceptibility to transformation. Of possible relevance is our recent finding that the
124
TRUESDALE
ET AL.
-_ 26
-
22
-
6-
EFFECTOR
: TARaET
FIG. 3. NK activity in M-MSV-injected bg/bg and +/bg mice. Four bg/bg and four +/bg mice were injected im with 150 FFU of M-MSV and splenic activity against YAC-I tested 3 days later (0 = +/bg q = bg/bg). Two mice of each genotype served as uninjected controls (0 = +/bg n = bg/bg). Average specific lysis for each group is shown. There was no statistically significant difference between virusinoculated and uninjected mice.
immune rejection response against allogeneic tumor cells occurs later in bg/bg than in +/bg animals (manuscript in preparation). In this regard, immunostimulation of tumor growth (18) or induction of suppressor cells due to impaired immunoregulation in NK-deficient mice (19) warrant consideration as factors which could account for the difference in tumor frequency between bg/bg and +/bg mice. On the other hand, the finding that tumor latency, growth, and regression rate did not differ significantly between mutant and phenotypically normal animals might suggest that differential susceptibility to transformation by M-MSV is responsible. For example, the beige mutation affects lysosome morphology, possibly via a defect in lipid metabolism (20) which might alter the susceptibility of the target tissue to infection or transformation by the virus. No evidence is available on this point, however. In conclusion, relatively few data are available on the susceptibility of beige mice to autochthonous tumor induction. In contrast to the results presented here, Argov et al. (2 1) reported that bg/bg mice developed tumors slightly earlier than +/bg mice after feeding with DMBA. The lack of a dramatic difference in tumor incidence was attributed to suppression of NK activity in the phenotypically normal animals by the carcinogen. However, caution should be exercised in attributing differences in tumor incidence between bg/bg and +/bg mice solely to NK cells due to the pleiotropic effects of this mutation.
VIRAL ONCQGENESIS IN BEIGE MICE
125
REFERENCES 1. Roder, J., and Duwe, A., Nature (London) 278, 451, 1979. 2. Kiessling, R., and Wigzell, H., Immunol. Rev. 44, 165, 1979. 3. Outzen, H. C., Custer, R. P., Eaton, G. J., and Prehn, R. T., J. Reticuloendothelial. Sot. 17, 1, 1975. 4. Rygaard, J., and Povlsen, C. O., Transplant. Rev. 28, 43, 1976. 5. Herberman, R. B., and Holden, H. T., Adv. Cancer Res. 27, 305, 1978. 6. Roder, J. C., Lohmann-Matthes, M., Domzig, W., and Wigzell, H., J. Immunol. 123, 2174, 1979. 7. Karre, K., Klein, G. O., Kiessling, R., Klein, G., and Roder, J. C., Nature (London) 284, 624, 1980. 8. Talmadge, J. E., Meyers, K. M., Prieur, D. J., and Starkey, J. R., Nature (London) 284, 622, 1980. 9. Siegel, S., “Nonparametric Statistics for the Behavioral Sciences.” McGraw-Hill, New York, 1956. 10. Gerson, J. M., Varesio, L., and Herberman, R. B., Int. J. Cancer 27, 243, 1981. I I. Becker, S., and Klein, E., J. Immunol. 6, 892, 1976. 12. Lust, J. A., Kumar, V., Burton, R. C., Bartlett, S. P., and Bennett, M., J. Exp. Med. 154, 306. 1981. 13. Minato, N., Reid, L., and Bloom, B. R., J. Exp. Med. 154, 750, 1981. 14. Stanton, M. F., Law, L. W., and Ting, R. C., J. Nat. Cancer Inst. 40, 1113, 1968. 15. Morahan, P. S., Law, L. W., and Ting, R. C., In “Genetic Control of Natural Resistance to Infection and Malignancy” (E. Skamene, P. A. L. Kongshavn, and M. Landy, Eds.), p. 575. Academic Press, New York, 1980. 16. Gallin, J. F., Bujak, J. S., Patten, E., and Wolff, S. M., Blood 43, 201, 1974. 17. Saxena, R. K., Saxena, Q. B., and Adler, W. H., Nature (London) 295, 240, 1982. 18. Prehn, R. T., J. Reticuloendothel. Sac. 10, 1, 1971. 19. Plata, F., MacDonald, R., and Shain, B., J. Immunol. 123, 852, 1979. 20. Brandt, E. J., Swank, R. T., and Novak, E. K., In, “Immunologic Defects in Laboratory Animals” (M. E. Gershwin, and B. Merchant, Eds.), Vol. I, p. 99. Plenum, New York, 1981. 21. Argov, S., Cochran, A. J., Karre, K., Klein, G. O., and Klein, G., Int. J. Cancer 28, 739, 1981.