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Modification of photocarcinogenesis by two immunosuppressive agents
Cancer Letters, 1 (1976) 243--247
243
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
IMMUNOM O D IOF PHOTOCARCINOGENESIS F I C A T IBY TWO O N SUPPRESSIVE AGENTS*
ROBERT B. NATHANSON**, P. DONALD FORBES and FREDERICK URBACH
The Skin and Cancer Hospital, Photobiology Program, Temple University Health Sciences Center, 3322 N. Broad Street, Philadelphia, Pa. 19140 (U.S.A.) (Received 22 January 1976)
SUMMARY
The carcinogenic effect of ultraviolet radiation (UVR) on the skin of hairless (hr) mice was modified by two immunosuppressive agents, rabbit antimouse lymphocytic serum (ALS), and 6-mercaptopurine (6MP). Daily exposure of mice to UVR resulted in multiple tumor production. Carcinogenesis was measured in terms of affected mice (prevalence) and numbers of tumors produced. By both criteria, photocarcinogenesis was enhanced by ALS but inhibited by 6MP.
INTRODUCTION
Several lines of evidence indicate that immunology and carcinogenesis are potentially interactive processes [1 ]. Increased cancer risk in humans under prolonged immunosuppressive treatment following organ transplantation has been reported [2,9,11]. Some laboratory data suggest that chemical carcinogenesis in animals can be enhanced by treatment with immunosuppressive drugs, although the mechanism of enhancement is not clear; there are in fact, data indicating lack of tumor enhancement even in the presence of demonstrated immunosuppression or immune deficiency [6,10]. Ultraviolet radiation (UVR) is a naturally-occurring carcinogen in the human environment, as well as a carcinogen that can be readily manipulated in the laboratory. We have undertaken a series of studies to determine whether UVR photocarcinogenesis either affects, or is affected by, the host's immune capabilities. This report presents results o f a preliminary experiment which was designed to determine whether any component of two immunosuppressive preparations could influence UVR-induced tumor production in the skin of mice [8]. * This work was supported by USPHS Grants ES 00629 and CA 11536. 3, Present Address: 120 Lawler Road, West Hartford, Conn. 06117.
244 MATERIALS AND METHODS
Thirty-six 8-week-old male hairless (hr) mice [5,7] of the outbred stock Skh: hairless-1 were separated into 3 groups. Each group was maintained in a stainless steel cage designed to permit uniform irradiation of 12 mice, and each animal was housed in a separate cubicle [4] with free access to commercial mouse chow and tap water. The U V R source was a bank of four FS40T12 Westinghouse fluorescent sun lamps emitting U V R principally at 280--320 nm. A WL-767 p h o t o t u b e was used to measure the relative U V R o u t p u t in terms of erythema effective energy (EEE; see reference [ 5] ). The mice were irradiated five days per week, Monday through Friday, for 30 weeks. The daily dose of 470 jm- 2 (EEE) produced no visible acute changes in the skin. The exposure period averaged 7.5 min per day. The acumulated dose after 30 weeks was 70 × 10 ~ jm- 2 (EEE). Intensities and doses are calculated and reported for skin surface level; the actual radiation received b y each animal was influenced by the animal's position and motion during exposure. Mice in one group received 0.1 ml rabbit anti-mouse l y m p h o c y t e serum (ALS; Microbiological Associates, catalog number 57-111) subcutaneously, twice weekly; the second group received 6-mercaptopurine (6MP; Purinethol, Burroughs Wellcome Co.) intraperitoneally five times per week at the rate of 12 mg/kg b o d y weight. The third group received isotonic saline solution intraperitoneally, 5 times weekly. Each injection was a volume of 0.1 ml. All surviving mice were injected for 20 weeks, beginning 2 weeks before the first U V R exposure. Animals were examined weekly for skin changes, and the t u m o r count was recorded for each mouse. Moribund and dead mice were autopsied; tumors and other tissues were prepared for routine histologic examination. RESULTS AND DISCUSSION
Irradiated animals began to show cutaneous s y m p t o m s of U V R treatment during the fifth to eighth weeks of exposure. The skin appeared smoother, somewhat thickened and more keratotic than unirradiated hairless mice [7]. A mild, patchy transient erythema developed during each week's exposure, usually resolving over the weekend. Impacted pilosebaceous cysts, white and hyaline in appearance, occasionally developed. Each t u m o r began as a slightly reddened, cornified papule, first recognized when a b o u t 0.5 mm in diameter. Tumors sometimes grew to several centimeters in diameter. Adjacent tumors frequently merged, but otherwise the t u m o r loss rate was very low. Histologically, 90% of the tumors were squamous cell carcinomas; the remainder were sarcomas or hemangiomas or tumors of mixed cell type. There were no pilosebaceous adenomas. Most autopsied mice had enlarged lymph nodes; one node contained a focus of squamous cell carcinoma.
245 E n u m e r a t i o n d a t a are p r e s e n t e d graphically in Fig. 1 in t e r m s o f animal survival, o f p r e v a l e n c e ( a f f e c t e d m i c e / m i c e surviving at each o b s e r v a t i o n period), and o f t u m o r yield ( t u m o r s p r e s e n t / m i c e surviving at each o b s e r v a t i o n period). No m o r t a l i t y o c c u r r e d until n e a r l y t w o m o n t h s a f t e r t h e fir3t t u m o r a p p e a r e d and b y this t i m e clear d i f f e r e n c e s in i n c i d e n c e and t u m o r yield had already b e e n established. A t 13 weeks, t h e ALS g r o u p had a significantly greater p r e v a l e n c e t h a n t h e saline or 6MP groups ( 1 2 / 1 2 vs. 6 / 1 2 vs. 3/12; p = < 0.005). T h e W i l c o x o n r a n k sum test [12] was used t o evaluate t u m o r yield. A t 13 weeks, t h e m e a n n u m b e r s o f t u m o r s per survivor were: ALS, 6.5; saline, 1.08; 6MP, 0.25. T h e A L S g r o u p h a d a significantly greater t u m o r yield t h a n did t h e o t h e r groups (p = < 0.01).
1.0.
~
c o n ~MP
,,Z, .7_ ~,6w.5 ~4_ 3_ 2
40
ALS
3°i u4 2~
con
g
/
10
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 W E E K S OF U V T R E A T M E N T
Fig,1. Results of the experiment. Upper panel: Survival data, showing number of mice alive during each week of the experiment. Con = isotonic saline, 6MP = 6 mercaptopurine, ALS = antilymphocyte serum. All mice exposed to UVR daily for 30 weeks. Center panel: Tumor prevalence, expressed as (mice with one or more tumors)/(survivors), at each observation period. Lower panel: Tumor yield, expressed as tumors/survivors, at each observation period.
246
By 15 weeks, the prevalence figures were: ALS vs. saline (12/12 vs. 9/12) p = < 0.05; ALS vs. 6MP (12/12 vs. 2/12), p = < 0.005; saline vs. 6MP (9/12 vs. 2/12), p = < 0.005. The mean numbers of tumors per survivor were: ALS, 9.25; saline, 2.0; 6MP, 0.33. These numbers were all significantly different from each other (p = < 0.01). At 20 weeks, only one death had occurred, and all b u t one survivor o u t of 35 had at least one tumor. The mean numbers of tumors per survivor were: ALS, 16.9; saline, 7.5; 6MP, 2.9. These numbers were all significantly different from each other (p = < 0.01). : Survival of drug-treated mice became significantly less than controls at 22 weeks (ALS) and 26 weeks (6MP) respectively (p = < 0.05). From 16 through 30 weeks, survival in the ALS group was less than in the 6MP group, but the difference was not statistically significant (p = > 0.1). Effects of b o t h immunosuppressive drugs on carcinogenesis were significant b u t the mode of action is not clear. The anti-metabolic and antitumor activity of 6MP [3] may be related to the suppression of photocarcinogenesis reported here. A more recent study indicates that the enhancement of photo-carcinogenesis by ALS was not attributable to the presence of rabbit serum itself (to be published). Since both ALS and 6MP were administered before and during the U V R treatment period, the compounds were present during t u m o r initiation and development. Thus, either c o m p o u n d could have exerted its influence on any stage of the carcinogenic process. The extent to which various components of the immune and other systems are responsible for modifying t u m o r production cannot be determined from this experiment. The results, in fact, would suggest that no simple relationship can be drawn between drugs considered to be immunosuppressive, and t h e progress of photocarcinogenesis. The effects of these c o m p o u n d s under other treatment schedules, as well as the mechanism of action remain to be clarified. REFERENCES 1 Conference on Immunology of Carcinogenesis (1972) Natl. Cancer Inst. Monograph 35, DHEW Publication (NI_H) 72--334 NCI, Bethesda, Maryland. 2 Doak, P.B., Montgomerie, T.Z., North, J.D. and Smith, A. (1968) Reticulum cell sarcoma after renal homotransplantation and azathioprine and prednisone therapy. Br. Med. J., 4, 746--748. 3 Elion, G.B. and Hitchings, G.H. (1965) Metabolic basis for the actions of analogs of purines and pyrimidines. Adv. Chemother., 2, 91--177. 4 Forbes, P.D. and Urbach, F. (1969) Vascular and neoplastic changes in mice following ultraviolet radiation. In: The Biological Effects of Ultraviolet Radiation, pp. 279--289. Editor: F. Urbach. Pergamon Press, London. 5 Forbes, P.D. and Urbach, F. (1975) Experimental modification of photocarcinogenesis III. Simulation of exposure to sunlight and fluorescent whitening agents. F o o d Cosmet. Toxicol., 3, 234--345. 6 Kripke, M.L. and Borsos, T. (1974) Immunosuppression and carcinogenesis. Israel J. Med. Sci., 10, 888--903.
247
7 Mann, S.J. (1971) Hair loss and cyst formation in hairless and rhino mutant mice. Anat. Rec., 170,485--500. 8 Nathanson, R.B., Forbes, P.D. and Urbach, F. (1973) UV photocarcinogenesis: Modification by anti-lymphocytic serum or 6-marcaptopurine. Proc. Am. Assoc. Cancer Res., Abstract 182, 14, 46. 9 Penn I. (1970) Malignant tumors in organ transplant recipients. Recent Results Cancer Res., 35:1. Springer-Verlag, New York. 10 Stutman, O. (1974) Tumor development after 3-methylcholanthrene in immunologically deficient athymic-nude mice. Science, 183,534. 11 Walder, B.K., Robertson, N.R. and Jermy, D. (1971) Skin cancer and immunosuppression. Lancet, 2, 1282--1283. 12 Wilcoxon, F. and Wilcox, R. (1964) In: Some Rapid Approximate Statistical Procedures. Lederle Laboratories, Pearl River, New Jersey.
243
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
IMMUNOM O D IOF PHOTOCARCINOGENESIS F I C A T IBY TWO O N SUPPRESSIVE AGENTS*
ROBERT B. NATHANSON**, P. DONALD FORBES and FREDERICK URBACH
The Skin and Cancer Hospital, Photobiology Program, Temple University Health Sciences Center, 3322 N. Broad Street, Philadelphia, Pa. 19140 (U.S.A.) (Received 22 January 1976)
SUMMARY
The carcinogenic effect of ultraviolet radiation (UVR) on the skin of hairless (hr) mice was modified by two immunosuppressive agents, rabbit antimouse lymphocytic serum (ALS), and 6-mercaptopurine (6MP). Daily exposure of mice to UVR resulted in multiple tumor production. Carcinogenesis was measured in terms of affected mice (prevalence) and numbers of tumors produced. By both criteria, photocarcinogenesis was enhanced by ALS but inhibited by 6MP.
INTRODUCTION
Several lines of evidence indicate that immunology and carcinogenesis are potentially interactive processes [1 ]. Increased cancer risk in humans under prolonged immunosuppressive treatment following organ transplantation has been reported [2,9,11]. Some laboratory data suggest that chemical carcinogenesis in animals can be enhanced by treatment with immunosuppressive drugs, although the mechanism of enhancement is not clear; there are in fact, data indicating lack of tumor enhancement even in the presence of demonstrated immunosuppression or immune deficiency [6,10]. Ultraviolet radiation (UVR) is a naturally-occurring carcinogen in the human environment, as well as a carcinogen that can be readily manipulated in the laboratory. We have undertaken a series of studies to determine whether UVR photocarcinogenesis either affects, or is affected by, the host's immune capabilities. This report presents results o f a preliminary experiment which was designed to determine whether any component of two immunosuppressive preparations could influence UVR-induced tumor production in the skin of mice [8]. * This work was supported by USPHS Grants ES 00629 and CA 11536. 3, Present Address: 120 Lawler Road, West Hartford, Conn. 06117.
244 MATERIALS AND METHODS
Thirty-six 8-week-old male hairless (hr) mice [5,7] of the outbred stock Skh: hairless-1 were separated into 3 groups. Each group was maintained in a stainless steel cage designed to permit uniform irradiation of 12 mice, and each animal was housed in a separate cubicle [4] with free access to commercial mouse chow and tap water. The U V R source was a bank of four FS40T12 Westinghouse fluorescent sun lamps emitting U V R principally at 280--320 nm. A WL-767 p h o t o t u b e was used to measure the relative U V R o u t p u t in terms of erythema effective energy (EEE; see reference [ 5] ). The mice were irradiated five days per week, Monday through Friday, for 30 weeks. The daily dose of 470 jm- 2 (EEE) produced no visible acute changes in the skin. The exposure period averaged 7.5 min per day. The acumulated dose after 30 weeks was 70 × 10 ~ jm- 2 (EEE). Intensities and doses are calculated and reported for skin surface level; the actual radiation received b y each animal was influenced by the animal's position and motion during exposure. Mice in one group received 0.1 ml rabbit anti-mouse l y m p h o c y t e serum (ALS; Microbiological Associates, catalog number 57-111) subcutaneously, twice weekly; the second group received 6-mercaptopurine (6MP; Purinethol, Burroughs Wellcome Co.) intraperitoneally five times per week at the rate of 12 mg/kg b o d y weight. The third group received isotonic saline solution intraperitoneally, 5 times weekly. Each injection was a volume of 0.1 ml. All surviving mice were injected for 20 weeks, beginning 2 weeks before the first U V R exposure. Animals were examined weekly for skin changes, and the t u m o r count was recorded for each mouse. Moribund and dead mice were autopsied; tumors and other tissues were prepared for routine histologic examination. RESULTS AND DISCUSSION
Irradiated animals began to show cutaneous s y m p t o m s of U V R treatment during the fifth to eighth weeks of exposure. The skin appeared smoother, somewhat thickened and more keratotic than unirradiated hairless mice [7]. A mild, patchy transient erythema developed during each week's exposure, usually resolving over the weekend. Impacted pilosebaceous cysts, white and hyaline in appearance, occasionally developed. Each t u m o r began as a slightly reddened, cornified papule, first recognized when a b o u t 0.5 mm in diameter. Tumors sometimes grew to several centimeters in diameter. Adjacent tumors frequently merged, but otherwise the t u m o r loss rate was very low. Histologically, 90% of the tumors were squamous cell carcinomas; the remainder were sarcomas or hemangiomas or tumors of mixed cell type. There were no pilosebaceous adenomas. Most autopsied mice had enlarged lymph nodes; one node contained a focus of squamous cell carcinoma.
245 E n u m e r a t i o n d a t a are p r e s e n t e d graphically in Fig. 1 in t e r m s o f animal survival, o f p r e v a l e n c e ( a f f e c t e d m i c e / m i c e surviving at each o b s e r v a t i o n period), and o f t u m o r yield ( t u m o r s p r e s e n t / m i c e surviving at each o b s e r v a t i o n period). No m o r t a l i t y o c c u r r e d until n e a r l y t w o m o n t h s a f t e r t h e fir3t t u m o r a p p e a r e d and b y this t i m e clear d i f f e r e n c e s in i n c i d e n c e and t u m o r yield had already b e e n established. A t 13 weeks, t h e ALS g r o u p had a significantly greater p r e v a l e n c e t h a n t h e saline or 6MP groups ( 1 2 / 1 2 vs. 6 / 1 2 vs. 3/12; p = < 0.005). T h e W i l c o x o n r a n k sum test [12] was used t o evaluate t u m o r yield. A t 13 weeks, t h e m e a n n u m b e r s o f t u m o r s per survivor were: ALS, 6.5; saline, 1.08; 6MP, 0.25. T h e A L S g r o u p h a d a significantly greater t u m o r yield t h a n did t h e o t h e r groups (p = < 0.01).
1.0.
~
c o n ~MP
,,Z, .7_ ~,6w.5 ~4_ 3_ 2
40
ALS
3°i u4 2~
con
g
/
10
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 W E E K S OF U V T R E A T M E N T
Fig,1. Results of the experiment. Upper panel: Survival data, showing number of mice alive during each week of the experiment. Con = isotonic saline, 6MP = 6 mercaptopurine, ALS = antilymphocyte serum. All mice exposed to UVR daily for 30 weeks. Center panel: Tumor prevalence, expressed as (mice with one or more tumors)/(survivors), at each observation period. Lower panel: Tumor yield, expressed as tumors/survivors, at each observation period.
246
By 15 weeks, the prevalence figures were: ALS vs. saline (12/12 vs. 9/12) p = < 0.05; ALS vs. 6MP (12/12 vs. 2/12), p = < 0.005; saline vs. 6MP (9/12 vs. 2/12), p = < 0.005. The mean numbers of tumors per survivor were: ALS, 9.25; saline, 2.0; 6MP, 0.33. These numbers were all significantly different from each other (p = < 0.01). At 20 weeks, only one death had occurred, and all b u t one survivor o u t of 35 had at least one tumor. The mean numbers of tumors per survivor were: ALS, 16.9; saline, 7.5; 6MP, 2.9. These numbers were all significantly different from each other (p = < 0.01). : Survival of drug-treated mice became significantly less than controls at 22 weeks (ALS) and 26 weeks (6MP) respectively (p = < 0.05). From 16 through 30 weeks, survival in the ALS group was less than in the 6MP group, but the difference was not statistically significant (p = > 0.1). Effects of b o t h immunosuppressive drugs on carcinogenesis were significant b u t the mode of action is not clear. The anti-metabolic and antitumor activity of 6MP [3] may be related to the suppression of photocarcinogenesis reported here. A more recent study indicates that the enhancement of photo-carcinogenesis by ALS was not attributable to the presence of rabbit serum itself (to be published). Since both ALS and 6MP were administered before and during the U V R treatment period, the compounds were present during t u m o r initiation and development. Thus, either c o m p o u n d could have exerted its influence on any stage of the carcinogenic process. The extent to which various components of the immune and other systems are responsible for modifying t u m o r production cannot be determined from this experiment. The results, in fact, would suggest that no simple relationship can be drawn between drugs considered to be immunosuppressive, and t h e progress of photocarcinogenesis. The effects of these c o m p o u n d s under other treatment schedules, as well as the mechanism of action remain to be clarified. REFERENCES 1 Conference on Immunology of Carcinogenesis (1972) Natl. Cancer Inst. Monograph 35, DHEW Publication (NI_H) 72--334 NCI, Bethesda, Maryland. 2 Doak, P.B., Montgomerie, T.Z., North, J.D. and Smith, A. (1968) Reticulum cell sarcoma after renal homotransplantation and azathioprine and prednisone therapy. Br. Med. J., 4, 746--748. 3 Elion, G.B. and Hitchings, G.H. (1965) Metabolic basis for the actions of analogs of purines and pyrimidines. Adv. Chemother., 2, 91--177. 4 Forbes, P.D. and Urbach, F. (1969) Vascular and neoplastic changes in mice following ultraviolet radiation. In: The Biological Effects of Ultraviolet Radiation, pp. 279--289. Editor: F. Urbach. Pergamon Press, London. 5 Forbes, P.D. and Urbach, F. (1975) Experimental modification of photocarcinogenesis III. Simulation of exposure to sunlight and fluorescent whitening agents. F o o d Cosmet. Toxicol., 3, 234--345. 6 Kripke, M.L. and Borsos, T. (1974) Immunosuppression and carcinogenesis. Israel J. Med. Sci., 10, 888--903.
247
7 Mann, S.J. (1971) Hair loss and cyst formation in hairless and rhino mutant mice. Anat. Rec., 170,485--500. 8 Nathanson, R.B., Forbes, P.D. and Urbach, F. (1973) UV photocarcinogenesis: Modification by anti-lymphocytic serum or 6-marcaptopurine. Proc. Am. Assoc. Cancer Res., Abstract 182, 14, 46. 9 Penn I. (1970) Malignant tumors in organ transplant recipients. Recent Results Cancer Res., 35:1. Springer-Verlag, New York. 10 Stutman, O. (1974) Tumor development after 3-methylcholanthrene in immunologically deficient athymic-nude mice. Science, 183,534. 11 Walder, B.K., Robertson, N.R. and Jermy, D. (1971) Skin cancer and immunosuppression. Lancet, 2, 1282--1283. 12 Wilcoxon, F. and Wilcox, R. (1964) In: Some Rapid Approximate Statistical Procedures. Lederle Laboratories, Pearl River, New Jersey.