References
(10) ARMAND JP, CVITKOVIC E: Suramin: A new
therapeutic concept. Eur J Cancer 26:417—419, 1990 (//) ROCHEFORT H: Biological and clinical significance of cathepsin D in breast cancer. In Seminars in Cancer Biology (Gottesman MM, ed), vol 1. London: Saunders Scientific Publications, 1990, pp 153-160 (12) FREISS G, VIGNON F, PAU B, ET AL: A two-site
immunoenzymometric assay of 52-kDa procathepsin D, and its use in human breast diseases. Clin Chem 35:234-237, 1989 (13) BRIOZZO P, MORISSET M, CAPONY F, ET AL: In
vitro degradation of extracellular matrix with Mr 52,000 cathepsin D secreted by breast cancer cells. Cancer Res 48:3688-3692, 1988
205) in animal tissues. Nature 170:567-569, 1952 (2) AKANJI MA: Rat kidney lysosomal membrane damage induced by suramin in vitro and in vivo. Pharmacol Toxicol 62:318-321, 1988 (3) CONSTANTOPOULOS G, REES S, CRAGG BG, ET
Autocrine growth stimulation of the MCF 7 breast cancer cells by the estrogen-regulated 52 K protein. Endocrinology 118:1537-1545, 1986 (15) CAPONY F, MORISSET M, BARRETT AJ, ET AL:
Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells. J Cell Biol 104:253-262, 1987 (16) LIPPMAN ME, DICKSON RB: Mechanisms of
growth control in normal and malignant breast epithelium. Recent Prog Horm Res 45:383435, 1989 (17) FREISS G, PREBOIS C, ROCHEFORT H, ET AL:
Anti-steroidal and anti-growth factor activities of anti-estrogens. J Steroid Biochem Mol Biol 37:777-781,1990 (18) CAVAILLES V, AUGEREAU P, GARCIA M, ET AL: Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breast cancer cells. Nucleic Acids Res 16:1903-1919,1988 Suramin EGF mediated reversible inhibition of hormone dependent growth in the MCF7 cell line. Proc Am Assoc Cancer Res, abstr 339, 1990 (20) HENDERSON IC: Endocrine therapy of breast cancer. In Breast Diseases (Harris IR, Hellman S, Henderson IC, et al, eds). Philadelphia: Lippincott, 1987, pp 398-479
(21) BERNS EM, SCHUURMANS AL, BOLT J, ET AL: AL: Experimental animal model for mucopolyAntiproliferative effects of suramin on saccharidosis: Suramin-induced glycosaminoandrogen responsive tumour cells. Eur J Canglycan and sphingolipid accumulation in the cer 26:470-474, 1990 rat. Proc Natl Acad Sci USA 77:3700-3704, 1980 (22) VIGNON F, DEROCQ D, CHAMBON M, ET AL: Estrogen-induced proteins secreted by MCF7 (4) HORNE MK III, STEIN CA, LA ROCCA RV, ET human breast cancer cells stimulate their AL: Circulating glycosaminoglycan anticoproliferation. C R Seances Acad Sci 296:151agulants associated with suramin treatment. 156,1983 Blood 71:273-279, 1988 (5) LA ROCCA
RV,
STEIN
CA,
MYERS
CE:
platelet-derived growth factor receptor in sistransformed cells and reversal by suramin. J Biol Chem 263:12608-12618,1988
(6) SCHNEIDER WJ, BEISIEGEL U, GOLDSTEIN JL,
(24) FLEMING TP, MATSUI T, MOLLOY CJ, ET AL:
(8) COFFEY RJ JR, LEOF EB, SHIPLEY GD, ET AL:
Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells. J Cell Physio! 132:143-148, 1987 (9) BETSHOLTZ C, JOHNSSON A, HELDIN CH, ET AL:
Efficient reversion of simian sarcoma virustransformation and inhibition of growth factorinduced mitogenesis by suramin. Proc Natl Acad Sci USA 83:6440-6444, 1986
42
(23) HUANG SS, HUANG JS: Rapid turnover of the
Suramin: Prototype of a new generation of antitumor compounds. Cancer Cells 2:106115,1990 ET AL: Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight. J Biol Chem 257:2664-2673,1982 (7) HOSANG M: Suramin binds to platelet-derived growth factor and inhibits its biological activity. J Cell Biochem 29:265-273, 1985
Sanjeev Kumar, G. P. Talwar, Debajit K. Biswas*
(14) VIGNON F, CAPONY F, CHAMBON M, ET AL:
(19) BERTHOIS Y, MARTIN PM, DONG XF, ET AL:
(/) JANSCO N, JANSCO-GABOR A: Suramin (Bayer
Necrosis and Inhibition of Growth of Human Lung Tumor by Anti-oc-Human Chorionic Gonadotropin Antibody
Autocrine mechanism for v-sis transformation requires cell surface localization of internally activated growth factor receptors. Proc Natl Acad Sci USA 86:8063-8067,1989 (25) KEATING
MT,
WILLIAMS
LT:
Autocrine
stimulation of intracellular PDGF receptors in v-sis-transformed cells. Science 239:914-916, 1988 (26) GARCIA M, DEROCQ D, PUJOL P, ET AL: Over-
expression of transfected cathepsin D in transformed cells increases their malignant phenotype and metastatic potency. Oncogene 5:1809-1814,1990
Human lung tumor cells (ChaGo) established in culture from a bronchogenie squamous cell carcinoma synthesize and secrete large amounts of human chorionic gonadotropin (HCG), predominantly the a subunit of the glycoprotein hormone. ChaGo cells lose their transformation phenotypes following treatment with anti-a-HCG antibody or following inhibition of intracellular synthesis of a-HCG by the anti-sense RNA technique. We report that tumors induced by ChaGo cells in female athymic mice undergo necrotic degeneration following local or intraperitoneal administration of a-HCGspecific-antibody. The a-HCG antibody did not affect the growth of tumors induced by a-HCG nonproducing human tumor cells. Histopathological examinations of the anti-a-HCG antibody-treated tumor tissues showed active necrosis. When the antibody treatment was discontinued, the tumorigenesis process resumed. When anti-a- HCG antibody was administered simultaneously with ChaGo cells, a concentration-dependent inhibition of tumor growth in athymic mice was completely prevented at higher concentrations of the specific antibody. [J Natl Cancer Inst 84:42-47,1992]
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
marker of poor prognosis for breast cancer patients with both node-negative and node-positive tumors ( / / ) . We have proposed that the increased secretion of this lysosomal enzyme by tumors might favor breast cancer cell growth (14) and metastasis (26). The action of suramin on lysosomes in vivo was associated with stimulation or depletion of enzyme activities (2J). Cathepsin D is up-regulated by E2 and growth factors in ER+ cells, but it is also constitutively produced at various rates by ER- cells. The effect of suramin on the accumulation and secretion of this factor reinforces the clinical potential of this drug, which can interfere with the metastatic process of both types of tumors. This result also focuses on another new mechanism for the antimetastatic activity of this agent. In conclusion, because of its effects on growth and cathepsin D secretion, suramin might prove to be a useful therapeutic agent for breast cancer patients, especially for patients with ER- tumors which are insensitive to antihormonal strategies.
Epidemiological surveys indicate that human lung cancers are often associated with ectopic synthesis of hormones, predominantly human chorionic gonadotropin (HCG) (7-7). Increased circulating levels of HCG and its subunits are often used as biochemical markers for malignancy, and decreased levels of the hormone HCG are often used as markers for successful surgery in human lung tumors. Under physiological conditions, HCG is Journal of the National Cancer Institute
Received August 1, 1991; accepted September 30, 1991. . Supported in part by grants from the Department of Biotechnology, Government of India, the IDRC of Canada, the Rockefeller Foundation, and by the South-to-South Cooperation Program in Reproductive Health and the Milton Fund of the Harvard Medical School. S. Kumar was a World Health Organization visiting fellow at the Harvard School of Dental Medicine. S. Kumar, G. P. Talwar, National Institute of Immunology, New Delhi, India. D. K. Biswas, Laboratory of Molecular Biology, Harvard School of Dental Medicine, Boston, Mass. We thank Dr. Gerald Shklar for his consistent help and expert opinion on experiments involving histopathology and Mrs. Diane Trickier for processing the tissues for histopathological examination. *Correspondence to: Debajit K. Biswas, Ph.D., D.Sc, Laboratory of Molecular Biology, Harvard School of Dental Medicine, 188 Longwood Ave., Boston, MA 02115.
Vol. 84, No. 1, January 1, 1992
passive immunization with anti-a-HCG antibody. Vaccines inducing the formation of anti-HCG antibodies have now been developed (9) and are in phase I and phase II (70) clinical trials in which antifertility activity of the vaccine is being examined. If a role for a-HCG in tumorigenesis is established, a similar therapeutic approach for treatment of human lung cancer may be feasible in the near future.
Materials and Methods Tissue Culture ChaGo cells were grown in Ham's F10 medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal bovine serum at 37 °C in 95% air and 5% CO2. The details of the soft-agar technique were described previously (8). Anti-a-HCG Antibody The a subunit of the human chorionic gonadotropin (a-HCG), at 100 ng/mL in saline, was mixed with equal amounts of complete Freund adjuvant, and 2 mL of this mixture was administered intradermally to goats. The purity of the a-HCG samples used for raising the antibody was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single protein band was observed with electrophoretic mobility comparable to that of the authentic a-HCG obtained from the Hormone Distribution Agency (National Institutes of Health, Bethesda, Md.). Booster injections, with the same amount of antigen in incomplete Freund adjuvant, were given three times every 30 days after the primary injection. Animals were then rested for 6 months and given booster injections again with the same amount of antigen in incomplete FreUnd adjuvant. These goats were bled after 12 days, and the antigen-binding capacity of the serum was titrated by radioimmunoassay. The specific batch of polyvalent antibodies used in this investigation has the antigen-binding capacity of 1892 (Xg/mL. The serum was diluted with phosphatebuffered saline to obtain the desired concentrations for administration to athymic mice. Control serum was obtained from the same goat prior to immunization. This normal goat serum was also diluted to the same degree and administered to the control animals.
Previously, we reported that the growth of ChaGo cells in tissue culture and in soft agar was inhibited in the presence of anti-a-HCG antibody (8). These properties of the new batch of antibody used in this investigation were re-examined and confirmed. Tumor Induction in Athymic Mice Female athymic mice (nu/nu mice, 3-4 weeks old) were obtained from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, Md.). These animals were housed in sterile cages, maintained on sterile food and water, and separated from other animals. Tumors were induced by the implantation of 1-2 x 106 ChaGo cells under the dorsal skin of the mice. The animals were examined routinely for palpable tumor growth. Tumor growth was monitored by routine photography. Tumors were induced in 95% of the animals in a predicted time sequence by one implantation of the indicated number of ChaGo cells.
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
synthesized during the early stages of pregnancy by trophoblastic cells. The ectopic synthesis of the hormone by lung cells thus is an inappropriate expression of these genes. The role of the commonly observed overproduction of HCG by human lung tumor cells in the transformation process has not been elucidated. We have examined the role of ectopic synthesis of the a subunit of HCG in the transformation process in a cultured cell line (ChaGo). This cell line was derived from a human bronchogenic squamous cell carcinoma (7,8). ChaGo cells synthesize and secrete both the a and the fS subunits of the glycoprotein hormone (7). The clonal strain of ChaGo cells used in our investigation produces predominantly large amounts of a-HCG (7). We have previously reported that ChaGo cells lost their characteristic transformation phenotypes, including the tumorigenic potential in athymic mice, following inhibition of intracellular synthesis of a-HCG by the anti-sense RNA technique (5). In contrast, the stimulation of a-HCG synthesis by cyclic adenosine monophosphate in these cells stimulated cell proliferation and cell progression into the S phase (8). These results prompted us to postulate that a-HCG acts as an autocrine growth factor and plays an important role in the transformation process by maintaining the cells in a continuously proliferating state. In this investigation, we verify this postulate by altering the level of a-HCG during the ChaGo cell-induced in vivo tumorigenesis process in athymic mice by
Histology Tumors at different stages of development were surgically removed from treated and control animals and a small fragment of each tumor was fixed in 10% formalin. Paraffin sections of these tumor fragments were stained with hematoxylin and eosin, after which they were examined and photographed under a phasecontrast microscope at the magnifications indicated in the legends to the figures.
Results Induction of Necrosis of ChaGo CellInduced Tumor in Athymic Mice by Anti-a-HCG We examined the influence of local administration of the anti-a-HCG antibody on the growth of the ChaGo cell-induced (1 x 106) tumors. Two weeks after ChaGo cells were implanted, the tumor attained the sizes shown in panels A and E of Fig. 1. At that time, 500 ng of anti-a-HCG antibody was administered on the same site twice a week under the tumor (indicated by arrow in Fig. 1, E). Control animals routinely received equivalent amounts of normal goat serum. The upper panels of Fig. 1 show the development of ChaGo cell-induced tumors photographed at REPORTS
43
Fig. 1. Panels A-D: control. Panels E-H: a-HCG antibody. Necrosis of ChaGo cell-induced tumors by antioc-HCG antibody. The protocol for induction of tumors in athymic mice is detailed in the "Materials and Methods" section. One transplantation of ChaGo cells induced tumors of the sizes shown in panels A and E in 2 weeks. At that time, 0.5 mL of the a-HCG antibody sample (500 ng antigen-binding capacity) was injected intradermally under the tumor (indicated by arrow in panel E) into five of the tumor-bearing animals, and 0.5 mL of diluted normal goat serum was also administered similarly to another two animals. The aHCG antibody treatment protocol was repeated twice a week. Growth of tumors was then monitored and photographed 2 weeks (panels B and F), 3 weeks (panels C and G), and 5 weeks (panels D and H) after the first administration of the a-HCG antibody-containing serum or the normal goat serum. This experiment was repeated three times with the same number of animals in the control group (total of six) and in the treatment group (total of 15). All animals showed a similar pattern of tumor growth in the control groups and the same degree of necrosis in the treated groups.
regular intervals in athymic mice in the presence of normal goat serum. Such tumors attained the size shown in Fig. 1, panel D, at the end of the 7th week after cell implantation. The results presented in Fig. 1 are typical of three such experiments conducted with a total of six animals in the control group and 15 animals in the treated group. All six controls developed tumors in the same time sequences as shown in the upper panels of Fig. 1. Similarly, all 15 animals in the a-HCG antibody-treated group demonstrated a typically similar pattern of tumor growth and simultaneous necrosis (Fig. 1, panels E, F, G, H). The photograph (Fig. 1, panel H) taken at the end of the 7th week after cell implanta-
44
tion and following 5 weeks of continuous treatment with anti-a-HCG antibody shows destruction of a significant amount of tumor tissue. When the anti-a-HCG antibody treatment was stopped, the tumorigenesis process resumed in all three experimental animals (Fig. 2). In the absence of the specific antibody, the residual cells proliferated, and the tumor mass progressively regenerated from the residual tumor cells within 4 weeks after the antibody treatment was discontinued (Fig. 2, panels B-D). These results demonstrate that tumor necrosis was specifically induced by the a-HCG antibody in the administered serum. The tumor cells were deprived of the autocrine growth factor
Inhibition of Growth of ChaGo CellInduced Tumor by Simultaneous Administration of Anti-a-HCG Antibody The results above show necrosis of tumor cells and regression of tumors by a-HCG antibody treatment. The results presented in this section demonstrate a concentration-dependent inhibition of tumor growth and tumor induction in athymic mice in the presence of a-HCG antibody. The same number of ChaGo cells (1 x 106) suspended in 0.5 mL of phosphate-buffered saline containing 50500 ng of a-HCG antibody were transplanted under the dorsal skin of athymic mice (three animals in each treatment group). The control animals (three animals) also received the same number of cells with equivalent amounts of normal goat serum. The antibody treatment was continued at the respective concentrations, administered twice a week locally at the site of tumor cell transplantation. The results (Fig. 4) show the pattern of tumor growth in the absence and in the presence of indicated amounts of antibody as recorded by photography at dif-
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
a-HCG, presumably due to the formation of the complex with antibody. Depriving the cells of a-HCG not only inhibited tumor growth but also induced extensive tumor necrosis. The process of active necrosis in the presence of anti-a-HCG antibody was more evident from the histopathological examination of the treated tumor tissue. The results (Fig. 3, panel A) demonstrate that the untreated tumor cells were proliferating with a high mitotic index. However, the histopathology of the treated tumor showed increased cellular necrosis with longer treatment with the aHCG antibody. After 5 weeks of a-HCG antibody treatment, patches of tumor cells were surrounded by massive necrotic tissue (Fig. 3, panel B). Pinocytosis and extensive tumor cell damage were observed (Fig. 3, panel C). These results demonstrate regression of ChaGo cell-induced tumors by the local administration of anti-a-HCG antibody, presumably because the cells were deprived of the essential growth factor produced by the cells themselves.
B
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
500 ng, there was no palpable tumor growth, even 10 weeks after transplantation of the tumor cells in the presence of continued antibody treatment. Histopathological examination of tumor tissues treated with 50 and 100 ng revealed partial necrosis. These results demonstrate that tumor growth can be inhibited and completely prevented by depriving the cells of a-HCG by continued exogenous administration of the antibody. A similar pattern of tumor growth inhibition was observed following intraperitoneal administration of a-HCG antibody simultaneously with the transplantation of ChaGo cells at the dorsal surface of the animals. Such continued passive immunization (twice a week) with 500 and 1000 ng of anti-a-HCG antibody prevented tumor growth completely (data not shown). We further verified the specificity of the anti-a-HCG antibody effect on the ChaGo cell-induced tumor by examining the effect of anti-a-HCG on the growth of tumors induced by two HCG-nonproducing human tumor cells in culture. Human cell lines A431 (epidermoid carcinoma) and T24 (bladder carcinoma) induced tumors in athymic mice, but the growth of both of these two types of tumors was not affected by a-HCG antibody treatment.
Discussion
Fig. 2. Effect of withdrawal of a-HCG antibody treatment on tumor growth. Treatment was stopped after 5 weeks in three animals, and the effect of this treatment withdrawal on tumor growth was monitored at regular intervals by photography. Photographs of the same animal in the treated group as shown in panel H of Fig. I are shown in panels A, B, C, and D of Fig. 2 at 1, 2, 3, and 4 weeks, respectively, after anti-a-HCG antibody administration was stopped.
ferent intervals (2 weeks after tumor cell transplantation, A series of panels; 4 weeks, B series; 6 weeks, C series; 8 weeks, D series; and 10 weeks, E series). Tumors grew in the control animals in an unrestricted manner and attained the indicated size within 8 weeks. Histopathological examination of sections of the ChaGo cell-induced tumors showed cell Vol. 84, No. 1, January I, 1992
proliferation with a high mitotic index. No visible necrosis of the tumor cells was detected. Although the continued treatment with 50 and 100 ng of a-HCG antibody did not substantially inhibit tumor growth, necrosis of tumor tissue was evident. Treatment with 200 ng of anti-aHCG antibody, on the other hand, significantly inhibited tumor growth. At
Our results further support a role for aHCG in the in vivo genesis of a hormoneproducing human lung tumor. They also demonstrate a reversal of tumor phenotypes by the specific antibody. The fact that tumor growth inhibition and prevention were observed only with the a-HCG antibody-containing serum, and not with the serum obtained from the same goat prior to immunization, strongly suggests that this effect is due to the specific antibody and not to some other agents in the serum. The two sera are isotypically matched, except that the one obtained after immunization contained the specific antibody. The results of the experiments on treatment withdrawal further substantiate the specificity of the effect of administered a-HCG antibody. Removal of the a-HCG antibody reversed the tumor growth-inhibitory effect, even in the presence of normal goat serum. In the absence of the REPORTS
45
a-HCG antibody, the tumor grew back, presumably from the residual active cells. The fact that the a-HCG antibody treatment was effective specifically in the aHCG-producing cells was demonstrated by the ineffectiveness of similar treatment on the growth of tumors induced by HCG-nonproducing human cells. We postulate from these results that the growth and manifestation of transformation phenotypes of this human lung tumor are dependent on the level of a-HCG synthesis. These results suggest an autocrine growth factor-like role for this hormone in cell proliferation and in the tumorigenesis process. The carcinogenesis process is believed to be mediated via the loss of regulation of gene expression, which leads to the inappropriate expression, overexpression, or suppression of genes inducing cellular dedifferentiation. The common phenomenon of ectopic hormone production associated with several types of human tumors suggests the involvement of the hormone molecules in cellular dedifferentiation and neoplastic transformation.
41
The most convincing evidence that hormones play a role in the transformation process originated from the work by Lippman and his colleagues (//) on the role of estradiol in the hormone-dependent human breast cancer cells. These investigators reported that estradiol treatment of hormone-dependent breast cancer MCF-7 cells in culture increased the Sphase fraction and tamoxifen inhibited the growth of the MCF-7 cells by arresting the cells in the Gj phase of the cycle
{U.12). We have reported previously that deprivation of the hormone in the aHCG-producing human lung tumor cells induced concomitant loss of all the transformation phenotypes by antibody treatment or by a decrease in the intracellular a-HCG level by the anti-sense RNA technique {8). Mechanistically, the role of aHCG on ChaGo cell proliferation is somewhat analogous to estradiol-induced stimulation of growth of MCF-7 cells. The reduced level of a-HCG decreased the S-phase fraction of cells, and stimulation of a-HCG synthesis by cyclic
References (/)
ODELL WD,
WOLFSON AR:
Hormonal
syn-
dromes associated with cancer. Ann Rev Med 29:379^06,1985 (2) DARNELL RB: Independent regulation by sodium butyrate of gonadotropin alpha gene expression and cell cycle progression in HeLa cells. Mol Cell Biol 4:829-839, 1984
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
Fig. 3. Histology of control and a-HCG antibody-treated tumors. Panel A shows the histological features of the tissue sections of ChaGo cell-induced tumor from animals 7 weeks after implantation of cells. White arrowheads show a few of the several mitotic cells (original magnification x400). Panels B and C show progression of the necrosis of tumor tissue after 5 weeks of a-HCG antibody treatment (original magnifications, x80 and X400, respectively). Area with tumor cells is designated by T with arrows, and necrotic area is designated by N with arrows.
adenosine monophosphate treatment increased by the same amount {8). The results suggest a role for these hormones in the in vivo genesis of the tumor, presumably mediated by their influence on the cell's growth cycle. We further verified this postulate by examining the tumorigenesis process in vivo in an environment in which the level of the specific hormone was reduced. Under this condition, the tumor cells stopped growing, and necrotic degeneration of tumor cells was observed. The lung tumor cells stopped their continuous proliferation and lost their characteristic tumor phenotypes, suggesting that tumorigenesis is dependent on hormone production and that the hormone plays the role of an autocrine growth factor. The major difference between the in vitro and the in vivo experiments was that in the in vitro experiments a reduced level of hormone synthesis seemed to arrest the cells in the Gi phase, whereas in the in vivo experiments deprivation of the hormone to the cells not only inhibited cell proliferation but also induced cell death. The in vivo tumor growth may be explained on the basis of the hormone's role in maintaining the cells continuously in the proliferation state by inducing the cells to progress into the S phase. Reduction of the hormone levels by the a-HCG antibody blocks the progression of the cells into the S phase and inhibits tumor growth. We have demonstrated a role for ectopic a-HCG synthesis in the transformation of human lung tumor cells both in culture by a molecular approach (8) and in intact animals by passive immunization. The specific antibody blocks the proliferation of the tumor cells and causes the tumors to undergo active necrosis and regression under in vivo conditions. These results obtained from both in vitro and in vivo experiments thus provide a basis for a therapeutic approach for the treatment of hormone-producing human lung cancers.
of anti-human chorionic gonadotropin vaccine. Contraception41:301-316,1990 (11)
BRUNNER N, BRONZERT D, VINDELOV LL, ET
AL: Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice. Cancer Res 49:1515-1520, 1989 (12)
SUTHERLAND RL, GREEN MD, HALL RE, ET AL:
Tamoxifen induces accumulation of MCF-7 human mammary carcinoma cells in the Go-Gi phase of the cell cycle. Eur J Cancer Clin Oncol 19:615-621, 1983
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
Effect of Vitamin A, C, and E Supplementation on Rectal Cell Proliferation in Patients With Colorectal Adenomas Gian Maria Paganelli* Guido Biasco, Giovanni Brandi, Renato Santucci, Giuseppe Gizzi, Valeria Villani, Massimo Cianci, Mario Miglioli, Luigi Barbara
Fig. 4. Inhibition of tumor induction by anti-a-HCG antibody. ChaGo cells, 1 x 10 in 0.5 mL of phosphatebuffered saline containing 50-500 ng of a-HCG antibody, were transplanted under the dorsal skin of athymic mice (three animals in each group). The control group was given transplants of the same amount of cells and an equivalent amount of normal goat serum (designated as 0 ng of a-HCG antibody [a-HCG-ab]). Series of panels under A, B, C, D, and E show tumor sizes photographed 2, 4, 6, 8, and 10 weeks, respectively, after transplantation of cells with indicated concentrations of antibody. Pictures in all panels were taken of the same animal in each group.
FISHERMAN PH, BRADLEY RM, REBOIS RV, ET
(7) TASHJIAN AH JR, WEINTRAUB BD, BAROWSKY
AL: The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells. J Biol Chem 259:7983-7989,1984 (4) GHOSH MK, COX RP: Production of human chorionic gonadotropin in HeLa cells. Nature 259:416-417,1976
NJ, ET AL: Subunits of human chorionic gonadotropin: Unbalanced synthesis and secretion by clonal strains derived from bronchogenic carcinoma. Proc Natl Acad Sci USA 70:14191422,1973
(3)
(5)
MINNA JD, HIGGINS GA, GLATSTEIN EJ:
Vol. 84, No. 1, January 1, 1992
(9)
PASION SG,
WONG DT,
ET AL:
TALWAR GP, SJARMA NC, DUBEY SK, ET AL:
Isoimmunization against human chorionic gonadotropin with conjugates of processed beta-subunit of the hormone and tetanus toxoid. Proc Natl Acad Sci USA 73:218-222, 1976
Can-
cer of the lung. In Principles and Practice of Oncology (DeVita VT Jr, Hellman S, Rosenberg SA, eds). Philadelphia: Lippincott, 1982, pp 396-402
RIVERA RT,
Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin. J Cell Biol 108:2423-2434, 1989
PETERS BP, KRZESICKI RF, HARTLE RJ, ET AL:
A kinetic comparison of the processing and secretion of the alpha and beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells. J Biol Chem 259:15123-15130,1984 (6)
(8)
(10)
TALWAR GP, HINGORANI V, KUMAR S, ET AL:
Phase I clinical trials with three formulations
Studies suggest that cell proliferation abnormalities of the colorectal mucosa are associated with risk of neoplasia, and most cancers of the large bowel are thought to arise from adenomas. The results of other studies suggest that vitamins A, C, and E have chemopreventive efficacy against colon cancer in animal models. This study evaluates the effect of dietary vitamin supplementation on cell kinetics in uninvolved rectal mucosa in patients with colorectal adenomas. Twenty patients with colorectal adenomas were given vitamins A, C, and E for 6 months after complete polypectomy, and 21 patients with adenomas received placebo. In each patient, six biopsy specimens were taken from normal-appearing rectal mucosa before treatment and after 3 and 6 months of treatment and were incubated with tritiated thymidine ([3H]thymidine), and the [3H]thymidine-labeled cells were counted by use of autoradiography. Two parameters of cell proliferation were evaluated: 1) the ratio of the number of labeled cells to the total number of cells (thymidine labeling index) and 2) the ratio of the number of labeled cells in the upper REPORTS
47
(10) ARMAND JP, CVITKOVIC E: Suramin: A new
therapeutic concept. Eur J Cancer 26:417—419, 1990 (//) ROCHEFORT H: Biological and clinical significance of cathepsin D in breast cancer. In Seminars in Cancer Biology (Gottesman MM, ed), vol 1. London: Saunders Scientific Publications, 1990, pp 153-160 (12) FREISS G, VIGNON F, PAU B, ET AL: A two-site
immunoenzymometric assay of 52-kDa procathepsin D, and its use in human breast diseases. Clin Chem 35:234-237, 1989 (13) BRIOZZO P, MORISSET M, CAPONY F, ET AL: In
vitro degradation of extracellular matrix with Mr 52,000 cathepsin D secreted by breast cancer cells. Cancer Res 48:3688-3692, 1988
205) in animal tissues. Nature 170:567-569, 1952 (2) AKANJI MA: Rat kidney lysosomal membrane damage induced by suramin in vitro and in vivo. Pharmacol Toxicol 62:318-321, 1988 (3) CONSTANTOPOULOS G, REES S, CRAGG BG, ET
Autocrine growth stimulation of the MCF 7 breast cancer cells by the estrogen-regulated 52 K protein. Endocrinology 118:1537-1545, 1986 (15) CAPONY F, MORISSET M, BARRETT AJ, ET AL:
Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells. J Cell Biol 104:253-262, 1987 (16) LIPPMAN ME, DICKSON RB: Mechanisms of
growth control in normal and malignant breast epithelium. Recent Prog Horm Res 45:383435, 1989 (17) FREISS G, PREBOIS C, ROCHEFORT H, ET AL:
Anti-steroidal and anti-growth factor activities of anti-estrogens. J Steroid Biochem Mol Biol 37:777-781,1990 (18) CAVAILLES V, AUGEREAU P, GARCIA M, ET AL: Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breast cancer cells. Nucleic Acids Res 16:1903-1919,1988 Suramin EGF mediated reversible inhibition of hormone dependent growth in the MCF7 cell line. Proc Am Assoc Cancer Res, abstr 339, 1990 (20) HENDERSON IC: Endocrine therapy of breast cancer. In Breast Diseases (Harris IR, Hellman S, Henderson IC, et al, eds). Philadelphia: Lippincott, 1987, pp 398-479
(21) BERNS EM, SCHUURMANS AL, BOLT J, ET AL: AL: Experimental animal model for mucopolyAntiproliferative effects of suramin on saccharidosis: Suramin-induced glycosaminoandrogen responsive tumour cells. Eur J Canglycan and sphingolipid accumulation in the cer 26:470-474, 1990 rat. Proc Natl Acad Sci USA 77:3700-3704, 1980 (22) VIGNON F, DEROCQ D, CHAMBON M, ET AL: Estrogen-induced proteins secreted by MCF7 (4) HORNE MK III, STEIN CA, LA ROCCA RV, ET human breast cancer cells stimulate their AL: Circulating glycosaminoglycan anticoproliferation. C R Seances Acad Sci 296:151agulants associated with suramin treatment. 156,1983 Blood 71:273-279, 1988 (5) LA ROCCA
RV,
STEIN
CA,
MYERS
CE:
platelet-derived growth factor receptor in sistransformed cells and reversal by suramin. J Biol Chem 263:12608-12618,1988
(6) SCHNEIDER WJ, BEISIEGEL U, GOLDSTEIN JL,
(24) FLEMING TP, MATSUI T, MOLLOY CJ, ET AL:
(8) COFFEY RJ JR, LEOF EB, SHIPLEY GD, ET AL:
Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells. J Cell Physio! 132:143-148, 1987 (9) BETSHOLTZ C, JOHNSSON A, HELDIN CH, ET AL:
Efficient reversion of simian sarcoma virustransformation and inhibition of growth factorinduced mitogenesis by suramin. Proc Natl Acad Sci USA 83:6440-6444, 1986
42
(23) HUANG SS, HUANG JS: Rapid turnover of the
Suramin: Prototype of a new generation of antitumor compounds. Cancer Cells 2:106115,1990 ET AL: Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight. J Biol Chem 257:2664-2673,1982 (7) HOSANG M: Suramin binds to platelet-derived growth factor and inhibits its biological activity. J Cell Biochem 29:265-273, 1985
Sanjeev Kumar, G. P. Talwar, Debajit K. Biswas*
(14) VIGNON F, CAPONY F, CHAMBON M, ET AL:
(19) BERTHOIS Y, MARTIN PM, DONG XF, ET AL:
(/) JANSCO N, JANSCO-GABOR A: Suramin (Bayer
Necrosis and Inhibition of Growth of Human Lung Tumor by Anti-oc-Human Chorionic Gonadotropin Antibody
Autocrine mechanism for v-sis transformation requires cell surface localization of internally activated growth factor receptors. Proc Natl Acad Sci USA 86:8063-8067,1989 (25) KEATING
MT,
WILLIAMS
LT:
Autocrine
stimulation of intracellular PDGF receptors in v-sis-transformed cells. Science 239:914-916, 1988 (26) GARCIA M, DEROCQ D, PUJOL P, ET AL: Over-
expression of transfected cathepsin D in transformed cells increases their malignant phenotype and metastatic potency. Oncogene 5:1809-1814,1990
Human lung tumor cells (ChaGo) established in culture from a bronchogenie squamous cell carcinoma synthesize and secrete large amounts of human chorionic gonadotropin (HCG), predominantly the a subunit of the glycoprotein hormone. ChaGo cells lose their transformation phenotypes following treatment with anti-a-HCG antibody or following inhibition of intracellular synthesis of a-HCG by the anti-sense RNA technique. We report that tumors induced by ChaGo cells in female athymic mice undergo necrotic degeneration following local or intraperitoneal administration of a-HCGspecific-antibody. The a-HCG antibody did not affect the growth of tumors induced by a-HCG nonproducing human tumor cells. Histopathological examinations of the anti-a-HCG antibody-treated tumor tissues showed active necrosis. When the antibody treatment was discontinued, the tumorigenesis process resumed. When anti-a- HCG antibody was administered simultaneously with ChaGo cells, a concentration-dependent inhibition of tumor growth in athymic mice was completely prevented at higher concentrations of the specific antibody. [J Natl Cancer Inst 84:42-47,1992]
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
marker of poor prognosis for breast cancer patients with both node-negative and node-positive tumors ( / / ) . We have proposed that the increased secretion of this lysosomal enzyme by tumors might favor breast cancer cell growth (14) and metastasis (26). The action of suramin on lysosomes in vivo was associated with stimulation or depletion of enzyme activities (2J). Cathepsin D is up-regulated by E2 and growth factors in ER+ cells, but it is also constitutively produced at various rates by ER- cells. The effect of suramin on the accumulation and secretion of this factor reinforces the clinical potential of this drug, which can interfere with the metastatic process of both types of tumors. This result also focuses on another new mechanism for the antimetastatic activity of this agent. In conclusion, because of its effects on growth and cathepsin D secretion, suramin might prove to be a useful therapeutic agent for breast cancer patients, especially for patients with ER- tumors which are insensitive to antihormonal strategies.
Epidemiological surveys indicate that human lung cancers are often associated with ectopic synthesis of hormones, predominantly human chorionic gonadotropin (HCG) (7-7). Increased circulating levels of HCG and its subunits are often used as biochemical markers for malignancy, and decreased levels of the hormone HCG are often used as markers for successful surgery in human lung tumors. Under physiological conditions, HCG is Journal of the National Cancer Institute
Received August 1, 1991; accepted September 30, 1991. . Supported in part by grants from the Department of Biotechnology, Government of India, the IDRC of Canada, the Rockefeller Foundation, and by the South-to-South Cooperation Program in Reproductive Health and the Milton Fund of the Harvard Medical School. S. Kumar was a World Health Organization visiting fellow at the Harvard School of Dental Medicine. S. Kumar, G. P. Talwar, National Institute of Immunology, New Delhi, India. D. K. Biswas, Laboratory of Molecular Biology, Harvard School of Dental Medicine, Boston, Mass. We thank Dr. Gerald Shklar for his consistent help and expert opinion on experiments involving histopathology and Mrs. Diane Trickier for processing the tissues for histopathological examination. *Correspondence to: Debajit K. Biswas, Ph.D., D.Sc, Laboratory of Molecular Biology, Harvard School of Dental Medicine, 188 Longwood Ave., Boston, MA 02115.
Vol. 84, No. 1, January 1, 1992
passive immunization with anti-a-HCG antibody. Vaccines inducing the formation of anti-HCG antibodies have now been developed (9) and are in phase I and phase II (70) clinical trials in which antifertility activity of the vaccine is being examined. If a role for a-HCG in tumorigenesis is established, a similar therapeutic approach for treatment of human lung cancer may be feasible in the near future.
Materials and Methods Tissue Culture ChaGo cells were grown in Ham's F10 medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal bovine serum at 37 °C in 95% air and 5% CO2. The details of the soft-agar technique were described previously (8). Anti-a-HCG Antibody The a subunit of the human chorionic gonadotropin (a-HCG), at 100 ng/mL in saline, was mixed with equal amounts of complete Freund adjuvant, and 2 mL of this mixture was administered intradermally to goats. The purity of the a-HCG samples used for raising the antibody was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single protein band was observed with electrophoretic mobility comparable to that of the authentic a-HCG obtained from the Hormone Distribution Agency (National Institutes of Health, Bethesda, Md.). Booster injections, with the same amount of antigen in incomplete Freund adjuvant, were given three times every 30 days after the primary injection. Animals were then rested for 6 months and given booster injections again with the same amount of antigen in incomplete FreUnd adjuvant. These goats were bled after 12 days, and the antigen-binding capacity of the serum was titrated by radioimmunoassay. The specific batch of polyvalent antibodies used in this investigation has the antigen-binding capacity of 1892 (Xg/mL. The serum was diluted with phosphatebuffered saline to obtain the desired concentrations for administration to athymic mice. Control serum was obtained from the same goat prior to immunization. This normal goat serum was also diluted to the same degree and administered to the control animals.
Previously, we reported that the growth of ChaGo cells in tissue culture and in soft agar was inhibited in the presence of anti-a-HCG antibody (8). These properties of the new batch of antibody used in this investigation were re-examined and confirmed. Tumor Induction in Athymic Mice Female athymic mice (nu/nu mice, 3-4 weeks old) were obtained from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, Md.). These animals were housed in sterile cages, maintained on sterile food and water, and separated from other animals. Tumors were induced by the implantation of 1-2 x 106 ChaGo cells under the dorsal skin of the mice. The animals were examined routinely for palpable tumor growth. Tumor growth was monitored by routine photography. Tumors were induced in 95% of the animals in a predicted time sequence by one implantation of the indicated number of ChaGo cells.
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
synthesized during the early stages of pregnancy by trophoblastic cells. The ectopic synthesis of the hormone by lung cells thus is an inappropriate expression of these genes. The role of the commonly observed overproduction of HCG by human lung tumor cells in the transformation process has not been elucidated. We have examined the role of ectopic synthesis of the a subunit of HCG in the transformation process in a cultured cell line (ChaGo). This cell line was derived from a human bronchogenic squamous cell carcinoma (7,8). ChaGo cells synthesize and secrete both the a and the fS subunits of the glycoprotein hormone (7). The clonal strain of ChaGo cells used in our investigation produces predominantly large amounts of a-HCG (7). We have previously reported that ChaGo cells lost their characteristic transformation phenotypes, including the tumorigenic potential in athymic mice, following inhibition of intracellular synthesis of a-HCG by the anti-sense RNA technique (5). In contrast, the stimulation of a-HCG synthesis by cyclic adenosine monophosphate in these cells stimulated cell proliferation and cell progression into the S phase (8). These results prompted us to postulate that a-HCG acts as an autocrine growth factor and plays an important role in the transformation process by maintaining the cells in a continuously proliferating state. In this investigation, we verify this postulate by altering the level of a-HCG during the ChaGo cell-induced in vivo tumorigenesis process in athymic mice by
Histology Tumors at different stages of development were surgically removed from treated and control animals and a small fragment of each tumor was fixed in 10% formalin. Paraffin sections of these tumor fragments were stained with hematoxylin and eosin, after which they were examined and photographed under a phasecontrast microscope at the magnifications indicated in the legends to the figures.
Results Induction of Necrosis of ChaGo CellInduced Tumor in Athymic Mice by Anti-a-HCG We examined the influence of local administration of the anti-a-HCG antibody on the growth of the ChaGo cell-induced (1 x 106) tumors. Two weeks after ChaGo cells were implanted, the tumor attained the sizes shown in panels A and E of Fig. 1. At that time, 500 ng of anti-a-HCG antibody was administered on the same site twice a week under the tumor (indicated by arrow in Fig. 1, E). Control animals routinely received equivalent amounts of normal goat serum. The upper panels of Fig. 1 show the development of ChaGo cell-induced tumors photographed at REPORTS
43
Fig. 1. Panels A-D: control. Panels E-H: a-HCG antibody. Necrosis of ChaGo cell-induced tumors by antioc-HCG antibody. The protocol for induction of tumors in athymic mice is detailed in the "Materials and Methods" section. One transplantation of ChaGo cells induced tumors of the sizes shown in panels A and E in 2 weeks. At that time, 0.5 mL of the a-HCG antibody sample (500 ng antigen-binding capacity) was injected intradermally under the tumor (indicated by arrow in panel E) into five of the tumor-bearing animals, and 0.5 mL of diluted normal goat serum was also administered similarly to another two animals. The aHCG antibody treatment protocol was repeated twice a week. Growth of tumors was then monitored and photographed 2 weeks (panels B and F), 3 weeks (panels C and G), and 5 weeks (panels D and H) after the first administration of the a-HCG antibody-containing serum or the normal goat serum. This experiment was repeated three times with the same number of animals in the control group (total of six) and in the treatment group (total of 15). All animals showed a similar pattern of tumor growth in the control groups and the same degree of necrosis in the treated groups.
regular intervals in athymic mice in the presence of normal goat serum. Such tumors attained the size shown in Fig. 1, panel D, at the end of the 7th week after cell implantation. The results presented in Fig. 1 are typical of three such experiments conducted with a total of six animals in the control group and 15 animals in the treated group. All six controls developed tumors in the same time sequences as shown in the upper panels of Fig. 1. Similarly, all 15 animals in the a-HCG antibody-treated group demonstrated a typically similar pattern of tumor growth and simultaneous necrosis (Fig. 1, panels E, F, G, H). The photograph (Fig. 1, panel H) taken at the end of the 7th week after cell implanta-
44
tion and following 5 weeks of continuous treatment with anti-a-HCG antibody shows destruction of a significant amount of tumor tissue. When the anti-a-HCG antibody treatment was stopped, the tumorigenesis process resumed in all three experimental animals (Fig. 2). In the absence of the specific antibody, the residual cells proliferated, and the tumor mass progressively regenerated from the residual tumor cells within 4 weeks after the antibody treatment was discontinued (Fig. 2, panels B-D). These results demonstrate that tumor necrosis was specifically induced by the a-HCG antibody in the administered serum. The tumor cells were deprived of the autocrine growth factor
Inhibition of Growth of ChaGo CellInduced Tumor by Simultaneous Administration of Anti-a-HCG Antibody The results above show necrosis of tumor cells and regression of tumors by a-HCG antibody treatment. The results presented in this section demonstrate a concentration-dependent inhibition of tumor growth and tumor induction in athymic mice in the presence of a-HCG antibody. The same number of ChaGo cells (1 x 106) suspended in 0.5 mL of phosphate-buffered saline containing 50500 ng of a-HCG antibody were transplanted under the dorsal skin of athymic mice (three animals in each treatment group). The control animals (three animals) also received the same number of cells with equivalent amounts of normal goat serum. The antibody treatment was continued at the respective concentrations, administered twice a week locally at the site of tumor cell transplantation. The results (Fig. 4) show the pattern of tumor growth in the absence and in the presence of indicated amounts of antibody as recorded by photography at dif-
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
a-HCG, presumably due to the formation of the complex with antibody. Depriving the cells of a-HCG not only inhibited tumor growth but also induced extensive tumor necrosis. The process of active necrosis in the presence of anti-a-HCG antibody was more evident from the histopathological examination of the treated tumor tissue. The results (Fig. 3, panel A) demonstrate that the untreated tumor cells were proliferating with a high mitotic index. However, the histopathology of the treated tumor showed increased cellular necrosis with longer treatment with the aHCG antibody. After 5 weeks of a-HCG antibody treatment, patches of tumor cells were surrounded by massive necrotic tissue (Fig. 3, panel B). Pinocytosis and extensive tumor cell damage were observed (Fig. 3, panel C). These results demonstrate regression of ChaGo cell-induced tumors by the local administration of anti-a-HCG antibody, presumably because the cells were deprived of the essential growth factor produced by the cells themselves.
B
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
500 ng, there was no palpable tumor growth, even 10 weeks after transplantation of the tumor cells in the presence of continued antibody treatment. Histopathological examination of tumor tissues treated with 50 and 100 ng revealed partial necrosis. These results demonstrate that tumor growth can be inhibited and completely prevented by depriving the cells of a-HCG by continued exogenous administration of the antibody. A similar pattern of tumor growth inhibition was observed following intraperitoneal administration of a-HCG antibody simultaneously with the transplantation of ChaGo cells at the dorsal surface of the animals. Such continued passive immunization (twice a week) with 500 and 1000 ng of anti-a-HCG antibody prevented tumor growth completely (data not shown). We further verified the specificity of the anti-a-HCG antibody effect on the ChaGo cell-induced tumor by examining the effect of anti-a-HCG on the growth of tumors induced by two HCG-nonproducing human tumor cells in culture. Human cell lines A431 (epidermoid carcinoma) and T24 (bladder carcinoma) induced tumors in athymic mice, but the growth of both of these two types of tumors was not affected by a-HCG antibody treatment.
Discussion
Fig. 2. Effect of withdrawal of a-HCG antibody treatment on tumor growth. Treatment was stopped after 5 weeks in three animals, and the effect of this treatment withdrawal on tumor growth was monitored at regular intervals by photography. Photographs of the same animal in the treated group as shown in panel H of Fig. I are shown in panels A, B, C, and D of Fig. 2 at 1, 2, 3, and 4 weeks, respectively, after anti-a-HCG antibody administration was stopped.
ferent intervals (2 weeks after tumor cell transplantation, A series of panels; 4 weeks, B series; 6 weeks, C series; 8 weeks, D series; and 10 weeks, E series). Tumors grew in the control animals in an unrestricted manner and attained the indicated size within 8 weeks. Histopathological examination of sections of the ChaGo cell-induced tumors showed cell Vol. 84, No. 1, January I, 1992
proliferation with a high mitotic index. No visible necrosis of the tumor cells was detected. Although the continued treatment with 50 and 100 ng of a-HCG antibody did not substantially inhibit tumor growth, necrosis of tumor tissue was evident. Treatment with 200 ng of anti-aHCG antibody, on the other hand, significantly inhibited tumor growth. At
Our results further support a role for aHCG in the in vivo genesis of a hormoneproducing human lung tumor. They also demonstrate a reversal of tumor phenotypes by the specific antibody. The fact that tumor growth inhibition and prevention were observed only with the a-HCG antibody-containing serum, and not with the serum obtained from the same goat prior to immunization, strongly suggests that this effect is due to the specific antibody and not to some other agents in the serum. The two sera are isotypically matched, except that the one obtained after immunization contained the specific antibody. The results of the experiments on treatment withdrawal further substantiate the specificity of the effect of administered a-HCG antibody. Removal of the a-HCG antibody reversed the tumor growth-inhibitory effect, even in the presence of normal goat serum. In the absence of the REPORTS
45
a-HCG antibody, the tumor grew back, presumably from the residual active cells. The fact that the a-HCG antibody treatment was effective specifically in the aHCG-producing cells was demonstrated by the ineffectiveness of similar treatment on the growth of tumors induced by HCG-nonproducing human cells. We postulate from these results that the growth and manifestation of transformation phenotypes of this human lung tumor are dependent on the level of a-HCG synthesis. These results suggest an autocrine growth factor-like role for this hormone in cell proliferation and in the tumorigenesis process. The carcinogenesis process is believed to be mediated via the loss of regulation of gene expression, which leads to the inappropriate expression, overexpression, or suppression of genes inducing cellular dedifferentiation. The common phenomenon of ectopic hormone production associated with several types of human tumors suggests the involvement of the hormone molecules in cellular dedifferentiation and neoplastic transformation.
41
The most convincing evidence that hormones play a role in the transformation process originated from the work by Lippman and his colleagues (//) on the role of estradiol in the hormone-dependent human breast cancer cells. These investigators reported that estradiol treatment of hormone-dependent breast cancer MCF-7 cells in culture increased the Sphase fraction and tamoxifen inhibited the growth of the MCF-7 cells by arresting the cells in the Gj phase of the cycle
{U.12). We have reported previously that deprivation of the hormone in the aHCG-producing human lung tumor cells induced concomitant loss of all the transformation phenotypes by antibody treatment or by a decrease in the intracellular a-HCG level by the anti-sense RNA technique {8). Mechanistically, the role of aHCG on ChaGo cell proliferation is somewhat analogous to estradiol-induced stimulation of growth of MCF-7 cells. The reduced level of a-HCG decreased the S-phase fraction of cells, and stimulation of a-HCG synthesis by cyclic
References (/)
ODELL WD,
WOLFSON AR:
Hormonal
syn-
dromes associated with cancer. Ann Rev Med 29:379^06,1985 (2) DARNELL RB: Independent regulation by sodium butyrate of gonadotropin alpha gene expression and cell cycle progression in HeLa cells. Mol Cell Biol 4:829-839, 1984
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
Fig. 3. Histology of control and a-HCG antibody-treated tumors. Panel A shows the histological features of the tissue sections of ChaGo cell-induced tumor from animals 7 weeks after implantation of cells. White arrowheads show a few of the several mitotic cells (original magnification x400). Panels B and C show progression of the necrosis of tumor tissue after 5 weeks of a-HCG antibody treatment (original magnifications, x80 and X400, respectively). Area with tumor cells is designated by T with arrows, and necrotic area is designated by N with arrows.
adenosine monophosphate treatment increased by the same amount {8). The results suggest a role for these hormones in the in vivo genesis of the tumor, presumably mediated by their influence on the cell's growth cycle. We further verified this postulate by examining the tumorigenesis process in vivo in an environment in which the level of the specific hormone was reduced. Under this condition, the tumor cells stopped growing, and necrotic degeneration of tumor cells was observed. The lung tumor cells stopped their continuous proliferation and lost their characteristic tumor phenotypes, suggesting that tumorigenesis is dependent on hormone production and that the hormone plays the role of an autocrine growth factor. The major difference between the in vitro and the in vivo experiments was that in the in vitro experiments a reduced level of hormone synthesis seemed to arrest the cells in the Gi phase, whereas in the in vivo experiments deprivation of the hormone to the cells not only inhibited cell proliferation but also induced cell death. The in vivo tumor growth may be explained on the basis of the hormone's role in maintaining the cells continuously in the proliferation state by inducing the cells to progress into the S phase. Reduction of the hormone levels by the a-HCG antibody blocks the progression of the cells into the S phase and inhibits tumor growth. We have demonstrated a role for ectopic a-HCG synthesis in the transformation of human lung tumor cells both in culture by a molecular approach (8) and in intact animals by passive immunization. The specific antibody blocks the proliferation of the tumor cells and causes the tumors to undergo active necrosis and regression under in vivo conditions. These results obtained from both in vitro and in vivo experiments thus provide a basis for a therapeutic approach for the treatment of hormone-producing human lung cancers.
of anti-human chorionic gonadotropin vaccine. Contraception41:301-316,1990 (11)
BRUNNER N, BRONZERT D, VINDELOV LL, ET
AL: Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice. Cancer Res 49:1515-1520, 1989 (12)
SUTHERLAND RL, GREEN MD, HALL RE, ET AL:
Tamoxifen induces accumulation of MCF-7 human mammary carcinoma cells in the Go-Gi phase of the cell cycle. Eur J Cancer Clin Oncol 19:615-621, 1983
Downloaded from http://jnci.oxfordjournals.org/ at Columbia University Libraries on February 2, 2015
Effect of Vitamin A, C, and E Supplementation on Rectal Cell Proliferation in Patients With Colorectal Adenomas Gian Maria Paganelli* Guido Biasco, Giovanni Brandi, Renato Santucci, Giuseppe Gizzi, Valeria Villani, Massimo Cianci, Mario Miglioli, Luigi Barbara
Fig. 4. Inhibition of tumor induction by anti-a-HCG antibody. ChaGo cells, 1 x 10 in 0.5 mL of phosphatebuffered saline containing 50-500 ng of a-HCG antibody, were transplanted under the dorsal skin of athymic mice (three animals in each group). The control group was given transplants of the same amount of cells and an equivalent amount of normal goat serum (designated as 0 ng of a-HCG antibody [a-HCG-ab]). Series of panels under A, B, C, D, and E show tumor sizes photographed 2, 4, 6, 8, and 10 weeks, respectively, after transplantation of cells with indicated concentrations of antibody. Pictures in all panels were taken of the same animal in each group.
FISHERMAN PH, BRADLEY RM, REBOIS RV, ET
(7) TASHJIAN AH JR, WEINTRAUB BD, BAROWSKY
AL: The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells. J Biol Chem 259:7983-7989,1984 (4) GHOSH MK, COX RP: Production of human chorionic gonadotropin in HeLa cells. Nature 259:416-417,1976
NJ, ET AL: Subunits of human chorionic gonadotropin: Unbalanced synthesis and secretion by clonal strains derived from bronchogenic carcinoma. Proc Natl Acad Sci USA 70:14191422,1973
(3)
(5)
MINNA JD, HIGGINS GA, GLATSTEIN EJ:
Vol. 84, No. 1, January 1, 1992
(9)
PASION SG,
WONG DT,
ET AL:
TALWAR GP, SJARMA NC, DUBEY SK, ET AL:
Isoimmunization against human chorionic gonadotropin with conjugates of processed beta-subunit of the hormone and tetanus toxoid. Proc Natl Acad Sci USA 73:218-222, 1976
Can-
cer of the lung. In Principles and Practice of Oncology (DeVita VT Jr, Hellman S, Rosenberg SA, eds). Philadelphia: Lippincott, 1982, pp 396-402
RIVERA RT,
Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin. J Cell Biol 108:2423-2434, 1989
PETERS BP, KRZESICKI RF, HARTLE RJ, ET AL:
A kinetic comparison of the processing and secretion of the alpha and beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells. J Biol Chem 259:15123-15130,1984 (6)
(8)
(10)
TALWAR GP, HINGORANI V, KUMAR S, ET AL:
Phase I clinical trials with three formulations
Studies suggest that cell proliferation abnormalities of the colorectal mucosa are associated with risk of neoplasia, and most cancers of the large bowel are thought to arise from adenomas. The results of other studies suggest that vitamins A, C, and E have chemopreventive efficacy against colon cancer in animal models. This study evaluates the effect of dietary vitamin supplementation on cell kinetics in uninvolved rectal mucosa in patients with colorectal adenomas. Twenty patients with colorectal adenomas were given vitamins A, C, and E for 6 months after complete polypectomy, and 21 patients with adenomas received placebo. In each patient, six biopsy specimens were taken from normal-appearing rectal mucosa before treatment and after 3 and 6 months of treatment and were incubated with tritiated thymidine ([3H]thymidine), and the [3H]thymidine-labeled cells were counted by use of autoradiography. Two parameters of cell proliferation were evaluated: 1) the ratio of the number of labeled cells to the total number of cells (thymidine labeling index) and 2) the ratio of the number of labeled cells in the upper REPORTS
47
