Response of T and B lymphocytes in the spleen, lymph nodes and mammary tumors in rats treated with human soluble tumor-associated antigens and commercial human albumin

We showed previously that soluble tumor-associated antigens (sTAA) isolated from breast cancer patients could suppress chemically-induced tumorigenesis in rats in comparison to the effect of commercial human albumin (CHA). Herein we analyze the possible mechanism of those findings. The following groups of mammary tumor-bearing rats were used in the studies: i) control rats treated with saline; ii) rats treated with CHA; and iii) rats treated with human sTAA. Different zones of the spleen, regional lymph nodes and tumors and their cellular content (B and T cells) were analyzed using the methods of morphometry and immunohistochemistry. Treatment of tumor-bearing rats with CHA resulted in a significant decrease in the size of the germinal center of the follicles. The number of B lymphocytes in the mantle layer of the follicles, the marginal zone and red pulp decreased significantly. The number of CD8+ T cells also decreased in the marginal zone and red pulp, whereas the number of CD4+ T cells increased in the periarterial lymph sheath (PALS) and the red pulp. Reaction of the spleen to vaccination with sTAA manifested in a significant increase in the size of most areas of the white pulp and in the number of B lymphocytes. In lymph nodes from control rats or those treated with CHA, CD8+ lymphocytes mainly accumulated in the paracortical zone. In rats treated with sTAA, CD8+ lymphocytes accumulated also in the medulla. The number of CD4+ T cells in these rats sharply increased and accumulated mainly in the medulla around the vessels. The total number of lymphocytes was changed differently in different areas of tumors (peripheral vs. at depth). The number of CD8+ cells significantly increased at depth of tumors, and also the ratio in the number of these cells at depth of tumors compared to a periphery increased. No difference was found in response of lymph cells to different types of treatment. All findings indicated a strict antitumor effect of vaccination with the sTAA, which prevents the development of insufficiency of the immune system when an intensive immune reaction takes place.     

Potentiation of antitumor immunity by antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy (ADEPT) has displayed antitumor activity in animal models and clinical trials. We examined whether antitumor immunity is generated during ADEPT by employing an immunoenzyme composed of the monoclonal antibody (MAb) RH1 conjugated to beta-glucuronidase to target rat AS-30D hepatocellular carcinoma tumors. A glucuronide prodrug of p-hydroxyaniline mustard was used to treat malignant ascites after immunoenzyme localization at the cancer cells. ADEPT cured more than 96% of Sprague-Dawley rats bearing advanced malignant ascites, and all cured rats were protected from a lethal challenge of AS-30D cells. Immunization with radiation-killed AS-30D cells or AS-30D cells coated with immunoenzyme did not provide tumor protection. Likewise, ex vivo treatment of tumor cells by ADEPT before injection into rats did not protect against a tumor challenge. AS-30D and N1-S1 hepatocellular carcinoma cells but not unrelated syngeneic tumor cells were lysed by peritoneal exudate cells isolated from ADEPT-cured rats. Depletion of CD8(+) but not CD4(+) T cells or natural killer (NK) cells reduced the cytolytic activity of peritoneal lymphocytes. ADEPT did not cure tumor-bearing rats depleted of CD4(+) and CD8(+) T cells even though it was curative when given 7 days after tumor transplantation in rats with an intact immune system, indicating that ADEPT can synergize with host immunity to increase therapeutic efficacy. These results have important implications for the clinical application of ADEPT.     

Response of T- and B-lymphocytes in the spleen of mammary tumor-bearing rats to treatment with tamoxifen and soluble tumor-associated antigens

We analyzed the role of T- and B-lymphocytes in the antitumor effects of the anticancer drug tamoxifen and soluble tumor-associated antigens (sTAA) on rat mammary carcinogenesis. Studies were performed on the spleen from the following groups of mammary tumor-bearing rats. i) Rats in group 1 were not exposed to DMBA and served as age-related controls. Rats in other groups were exposed to DMBA and received different types of treatment; ii) rats in group 2, received no additional treatment, and served as carcinogen-related controls; iii) rats in group 3 were treated with the commercial hormone-dependent anticancer drug tamoxifen by weekly subcutaneous (s.c.) injections of 10 mg dissolved in 0.5 ml distilled water per rat; iv) rats in group 4 were vaccinated s.c. weekly with a preparation of sTAA (50 micro l/rat) dissolved in 0.5 ml of phosphate-buffered saline; v) rats in group 5 were treated with tamoxifen and were also vaccinated with a preparation of sTAA. Different zones of the spleen were measured and their T- and B-cell contents were analyzed immunohistochemically. The treatment with tamoxifen significantly increased the total number of lymphocytes in the follicles, PALS (periarterial lymph sheath) and red pulp relative to all other groups. The combined treatment with tamoxifen and sTAA increased the areas of white pulp, the PALS, and marginal zone. The number of B-cells was higher in the marginal zone of spleens from age-related controls, as well as from rats treated with sTAA and those treated with tamoxifen and sTAA. The number of CD4+ lymphocytes in the PALS was higher in rats treated with sTAA and tamoxifen, and notably so in those treated with sTAA alone. The number of CD8+ lymphocytes was significantly lower in the PALS of spleens from all tumor-bearing rat groups compared to the unexposed age-related control rats. We suggest that the tumor-suppressive effect of sTAA and tamoxifen is accompanied by the activation of B- and T-lymphocyte production.     

Enhancing Effects of β-Estradiol 3–Benzoate but Not Methoxychlor on the Promotion/Progression Stage of Chemically-induced Mammary Carcinogenesis in Ovariectomized Rats

Modifying effects of β-estradiol 3–benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12–dimethylbenz( a )anthracene (DMBA)-induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague-Dawley rats. Twenty-eight weeks after a single DMBA (100 mg/kg body weight) initiation, when the incidence of mammary tumor-bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor-bearing, ovariectomized group; ii) tumor-bearing, intact group; iii) no-tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor-bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no-tumor, ovariectomized group. However, MXC did not show any effect in the tumor-bearing, or no-tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor-bearing, intact group. The results of our study suggest that MXC has no promotion/progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA-induced mammary tumorigenesis.     

Spontaneous mammary tumors in aging Noble rats

Noble (Nb) rat strain has been used for the study of hormonal carcinogenesis of mammary and prostate glands, for its susceptibility to develop premalignant lesions as well as carcinomas in these organs by sex hormones. However, background information on the spontaneously developed mammary tumors in this rat strain is scarce. We report on the incidence rate, latency period and histopathology of mammary tumors spontaneously developed in the senile intact and untreated Nb rats compared with those induced by either combined treatments with sex steroids or 7,12-dimethylbenz[a]anthracene (DMBA) in the same rat strain. We observed that the incidence rate of spontaneous mammary tumors was 45% in female Nb rats and 3% in the males. The average age of the female Nb rats to develop palpable tumors was 14 months and rarely detected in animals less than 12 months old. It was also noted that the incidence rate of the spontaneous mammary tumors was similar to those induced by combined treatments with sex steroids for 8-10 months (46.7% for T+E2 and 55.6% for T+DES) but less than those by DMBA treatment in 8 months (over 80%). Histologically, majority of the spontaneous mammary tumors were fibroadenomas, which comprised 70% of all collected tumors and about 20% were carcinomas whereas tumors induced by steroid hormones and DMBA were all carcinomas. Distant metastases of spontaneous mammary carcinomas to lung, liver and lymph nodes were also noted, but rarely.     

Enhancing effects of beta-estradiol 3-benzoate but not methoxychlor on the promotion/progression stage of chemically-induced mammary carcinogenesis in ovariectomized rats.

Modifying effects of beta-estradiol 3-benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague-Dawley rats. Twenty-eight weeks after a single DMBA (100 mg / kg body weight) initiation, when the incidence of mammary tumor-bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor-bearing, ovariectomized group; ii) tumor-bearing, intact group; iii) no-tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor-bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no-tumor, ovariectomized group. However, MXC did not show any effect in the tumor-bearing, or no-tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor-bearing, intact group. The results of our study suggest that MXC has no promotion / progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA-induced mammary tumorigenesis.     

Inhibitory action of aminoglutethimide on DMBA-induced mammary carcinogenesis

The present communication reports the inhibitory action of aminoglutethimide on 9,10-dimethyl-1,2-benzanthracene (DMBA) induced mammary carcinogenesis in virgin female Holtzman rats. When 20 mg of DMBA is given to the rats and maintained on normal diet for 32 weeks, approximately 70% of the animals develop mammary tumors. When animals similarly treated with DMBA are put on diets containing 0.025%, 0.05%, and 0.1% of aminoglutethimide for 32 weeks, there is a decline in the number of tumor-bearing animals as well as in the number of tumors per tumor-bearing animal, especially at higher diet doses of the drug. This inhibitory action on the induction of mammary tumors by DMBA could be mainly due to the suppressive action of the drug on endocrine functions.     

Rat soluble tumor-associated antigens inhibit chemically-induced mammary tumorigenesis in syngeneic rats

This study examined whether soluble 66 and 51 kDa tumor-associated antigens (sTAA), isolated from the serum of rats with mammary cancer, possess specific suppressive effects on chemically-induced mammary tumorigenesis in syngeneic counterparts. Dimethylbenzanthracene (DMBA, 10 mg/rat, two administrations) was used to induce mammary tumors in 8-week-old Sprague Dawley rats. After the appearance of numerous tumors, preparations of sTAA (50 to 60 microg/rat in 0.5 ml sterile PBS) obtained from breast cancer patients (heterologous sTAA) or from syngeneic mammary tumor-bearing rats (syngeneic sTAA) were administered weekly for 12 weeks. The following groups of mammary tumor-bearing rats were studied: groups 1 and 3, control rats treated with saline; group 2, rats treated with heterologous sTAA; and group 4, rats treated with syngeneic sTAA. The experiment was terminated when tumors in 50% of the rats became ulcerous. The treatment with both types of sTAA significantly decreased, compared to controls and initial values, the yield and total area of the tumors. We conclude that syngeneic sTAA have tumor-suppressive properties, which are very similar to those in heterologous sTAA.     

干酪乳杆菌对二甲基苯蒽诱发大鼠乳腺肿瘤的抑制作用及其免疫学机制

目的：探讨干酪乳杆菌对二甲基苯蒽（DMBA）诱发的大鼠乳腺肿瘤的抑制效果和对免疫功能的影响。方法：雌性SD大鼠按体质量随机分为正常对照组、模型组、低剂量和高剂量干酪乳杆菌干预组。模型组及低、高剂量干酪乳杆菌干预组大鼠右侧臀部皮下一次性注射100 mg/kg DMBA建立乳腺癌模型。低和高剂量干酪乳杆菌干预组分别灌胃给予4和8 mL/（kg·d）干酪乳杆菌（1×10⁸ CFU/mL）,正常对照组和模型组均给予5 mL/（kg·d）大豆油灌胃。每天1次,持续16周后处死大鼠,完整剥离肿瘤组织及脏器,计算各组大鼠乳腺癌发生率、抑瘤率及脏器指数。流式细胞术检测大鼠外周血中自然杀伤细胞（NK）活性和T淋巴细胞亚群分布。酶联免疫吸附实验（ELISA）检测血清中细胞因子IL-4、IL-6、IL-10、IL-12、IFN-γ和TNF-α水平。结果：正常对照组大鼠无肿瘤发生,模型组、低剂量和高剂量干酪乳杆菌干预组均有肿瘤发生。与模型组比较,高剂量干酪乳杆菌组大鼠肿瘤潜伏期延长,肿瘤发生率和平均瘤质量降低（P均 < 0.05）;抑瘤率达到41.2%;且该组胸腺指数显著升高（P < 0.05）;TCRαβ⁺CD161a⁺NK细胞百分比、CD3⁺CD8⁺T细胞百分比均显著升高（P均 < 0.05）;血中CD3⁺Foxp3⁺细胞百分比明显下降（P < 0.05）。ELISA结果显示,与正常对照组比较,模型组血清中IL-4水平明显降低,而IL-6、IL-12和TNF-α水平显著升高（P均 < 0.05）;与模型组比较,高剂量干酪乳杆菌干预组,血清IL-4、IL-10浓度显著增高,IL-6、IL-12浓度显著降低（P均 < 0.05）;低和高剂量干酪乳杆菌干预组血清中TNF-α浓度均显著下降（P均 < 0.05）。结论：干酪乳杆菌对乳腺癌大鼠肿瘤生长具有一定抑制作用,其作用机制可能与干酪乳杆菌调节CD4⁺、CD8⁺T细胞、NK细胞及调节性T细胞等免疫细胞分布,改善炎性相关细胞因子水平,从而提高机体免疫调节作用有关。     

Direct Activation of Human CD8+ Cytotoxic T Lymphocytes by Interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8⁺ T cells present after 28 days in the induction culture. When purified CD8⁺ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8⁺ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.     

The immune system response during development and progression of carcinogen-induced rat mammary tumors: prevention of tumor growth and restoration of immune system responsiveness by thymopentin

Summary A detailed analysis of the immune system response has been performed during the development and progression of dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors. For this aim, a number of immune parameters (thymocyte and splenocyte proliferative response to T-dependent mitogens, antibody production, lymphocyte subset phenotyping, interleukin 2 receptor expression in resting and activated lymphocytes, thymus morphology and morphometry), were correlated with tumor appearance and growth at different (- 7, 0, + 15, + 30, + 60, + 90, and + 120 days) time intervals after intragastric administration of DMBA, in the absence or the presence of a concomitant treatment with the thymic pentapeptide thymopentin (TP5). A profound and time-dependent immunosuppression characterized the treatment with the carcinogen. Both cell-mediated and humoral immune responses showed a 50% inhibition 2 weeks after DMBA administration, with a peak after 30 days, followed by a plateau until 120 days of observation. The mechanism responsible for reduced ability of thymocytes and splenocytes to respond to both Con-A and PHA was explained by the significant inhibition of one of the key steps of T cell activation, namely the expression of IL-2 receptor in lymphocytes from DMBA-treated animals. The flow cytometric analysis of lymphocyte subpopulations revealed an important reduction in the overall populations of thymocytes and splenocytes. At the thymus gland level, a dramatic reduction of double positive CD4+CD8+ and a decrease of CD4+CD8- and CD4-CD8+ were observed, together with a marked atrophy of the thymic cortex, and impairment of the thymic microenvironment. One hundred and twenty days after DMBA administration, approximately 60 to 70% of the animals developed tumors with a mean tumor surface area of 2.88 ± 0.86 cm², and a number of 2.44 ± 1.0. Treatment with TP5 (100 ng/animal, three times a week, starting a week before DMBA), produced specific effects on different immune compartments and tumoral growth, characterized by a significant reversal of immune depression with a stimulatory effect measured on lymphoproliferative assays, lymphocyte subset distribution, and IL-2 receptor expression. Moreover, thymic atrophy was almost completely prevented in TP5 treated animals. Of major interest, a significant delay in the appearance and growth of tumors was observed in TP5 treated rats. When DMBA-treated animals were followed for the entire observation period (0–120 days) and the immune responsiveness correlated according to tumor progression, stability, or regression, a positive correlation was calculated between the degree of immune system depression and the individual rate of tumor growth; in TP5-treated rats the majority of the tumors were static or regressing tumors.It is concluded that a profound and specific suppression of thymus-dependent immune functions follows the treatment with the carcinogen DMBA. Such immune deficits appear 2 weeks after the carcinogen administration, and may represent early indicators of tumor development and growth. A key role is suggested for the thymus gland and its peptide secretion, since the dramatic atrophy of this gland and the abnormal thymusdependent immune parameters studied are almost completely reversed by a synchronized treatment with thymopentin. Although from the present results it seems not possible to conclusively assign a specific causeeffect relationship, from the parallel delay in tumor appearance and growth in TP5-treated rats it seems tempting to speculate that the decline in thymus-dependent immune functions may play a major role in tumor development.     

Effect of age and sex on the development of neoplasms in wistar rats receiving a single intragastric instillation of 7,13-Dimethylbenz(a)anthracene

Wistar rats of different age and sex received a single intragastric instillation of 7,12-dimethylbenz(a)anthracene (DMBA). During the following 16 months, 136 of 284 rats developed 182 tumors, of which half were malignant. Among 98 untreated controls, 8 rats developed tumors, of which 4 were malignant. Among the youngest groups (8–15 days) exposed to DMBA, fibroadenoma of the breast in females and subcutaneous sarcoma in both sexes were the most frequent. Among young adult females (6–8 weeks), mammary carcinoma predominated; older females (26 weeks) had less breast tumors. Among adult males, epidermoid carcinoma and papilloma of the skin were most frequent. Castration suppressed development of breast cancer.     

Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25- lymphocytes is thymus and proliferation independent

The CD25- and CD25+ CD4 T-lymphocyte compartments are tightly regulated. We show here that tumors break such balance, increasing the number of CD4+CD25+ T cells in draining lymph node and spleen but not contralateral node of tumor-bearing mice. Tumor injection in thymectomized and CD25-depleted mice shows that CD4+CD25+ T-cell expansion occurs even in the absence of the thymus and independently from proliferation of preexisting CD25+ T cells. These newly generated cells are bona fide regulatory T cells (T reg) in terms of Foxp3 expression and suppression of CD3-stimulated or allogeneic effector cell proliferation. Transfer of congenic Thy1.1 CD4+CD25- T cells, from mice treated or not with vinblastine, into tumor-bearing or tumor-free mice and analysis of recovered donor lymphocytes indicate that conversion is the main mechanism for acquiring the expression of CD25 and Foxp3 through a process that does not require proliferation. Although conversion of CD4+CD25- T cells for generation of T regs has been described as a natural process that maintains peripheral T-reg population, this process is used by the tumor for immune escape. The prompt recovery of T regs from monoclonal antibody-mediated CD25 depletion in tumor-bearing mice suggests attempts able to inactivate rather than deplete them when treating existing tumors.     

Increase of auto anti-phosphatidylinositol antibodies in plasma of female rats during the appearance of DMBA-induced malignant mammary tumors

Using a lipid-adapted ELISA, antiphosphatidylinositol (PtdIns) antibodies (Ab) have been found in sera of cancer patients. With the same procedure, we have checked their possible raising in plasma of female rats during the appearance of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. Twenty days after DMBA administration, the mean anti-PtdIns Ab level was already higher than that of control rats (oil). Immunochemical analysis of the anti-PtdIns Ab site showed a rather high Ab avidity (8 x 10(-9) M, at half-displacement) and high specificity for glyceryl phosphate inositol residues. Other phospholipids (PL) were not recognized by the anti-PtdIns Ab induced by malignant cell transformation.     

Autologous mixed lymphocyte-tumor reaction and autologous mixed lymphocyte reaction. II. Generation of specific and non-specific killer T cells capable of lysing autologous tumor

The specific and non-specific nature of autotumor cytotoxicity induced in autologous mixed lymphocyte-tumor culture (AMLTC) and autologous mixed lymphocyte culture (AMLC) was studied in patients with carcinomatous pleural effusions. Small- and medium-sized blood lymphocytes that were isolated by centrifugation on discontinuous Percoll gradients did not lyse autologous, freshly isolated effusion tumor cells. In vitro activation of the small lymphocytes, but not of the medium lymphocytes, with autologous tumor cells generated cytotoxic potential restricted to autologous tumor. When stimulated with autologous non-malignant non-T cells, the medium lymphocytes, but not small lymphocytes, were triggered to cytotoxicity that acted not only on autologous tumor cells but also on allogeneic tumor cells, T blasts, and tumor cell lines. Experiments using monoclonal antibodies (MAb) and complement (C') showed that both types of killer cells were CD2+ CD3+ CD16- T cells. Autotumor cytotoxicity developed in AMLTC was mediated by the CD4- CD8+ T cell subset in 6 of 9 cases and the CD4+ CD8- subset in the other 3 cases. In contrast, cytotoxicity induced in AMLC was exerted exclusively by the CD8+ subset. The enrichment of blasts from cultured T cells on discontinuous density gradients enhanced autotumor killing activity, with no reactivity recorded for blast-depleted, resting T cells. Addition of mitomycin-C-treated large granular lymphocytes (LGL) to AMLTC abolished the induction of autotumor killer cells, whereas non-specific killer cells were generated in AMLC irrespective of the presence of LGL. These results indicate that stimulation of autoreactive T cells in AMLTC and in AMLC could induce 2 distinct types of autotumor killer cells.     

In vivo antitumor activity of tumor-infiltrating lymphocytes expanded in recombinant interleukin-2

A method was described for the generation of cells from tumor-bearing mice; these cells were capable of exhibiting significant antitumor reactivity when adoptively transferred into tumor-bearing hosts. Tumor cell suspensions from a variety of tumors were able to be separated using enzymatic techniques and they were cultured in medium containing recombinant interleukin-2. Activated infiltrating lymphocytes within these tumors expanded; and, by 6-8 days after initiation of culture, lymphocytes predominated and were able to grow to large numbers. The adoptive transfer of these tumor-infiltrating lymphocytes (TILs) made possible mediation of the reduction of established 3-day pulmonary micrometastases from 5 of 7 tumors tested, including two 3-methylcholanthrene (CAS: 56-49-5)-induced sarcomas, one 1,2-dimethylhydrazine (CAS: 540-73-8)-induced colon carcinoma, and the B16 melanoma, all in C57BL/6 mice, as well as the 1660 bladder carcinoma in BALB/c mice. Approximately 2-4 X 10(6) transferred cells were capable of totally eliminating 3-day established metastases. These cells were thus 50 to 100 times more effective than lymphokine-activated killer cells in reducing established metastases; however, they could not be generated from all tumors. The concomitant administration of recombinant interleukin-2 enhanced, by approximately fivefold, the in vivo activity of these cells. The specificity of action of TILs in vivo was different from that determined by classic amputation rechallenge experiments. The tumor-infiltrating lymphocytes that developed this antitumor reactivity appeared to be Thy-1+ and did not bear the asialo GM1 antigen. The potent antitumor effect of these TILs, when transferred in vivo to tumor-bearing hosts, raises the possibility of utilizing similar approaches for the isolation and therapeutic use of lymphocytes with antitumor reactivity from human tumors.     

Inhibition of rat mammary tumorigenesis by human chorionic gonadotropin associated with increased expression of inhibin.

This work was designed to test whether the suppression of 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinomas by human chorionic gonadotropin (hCG) is associated with the synthesis of inhibin. For this purpose, virgin rats received 8 mg DMBA/100 g body weight; 20 d later they were injected daily with 100 IU of hCG for 40 d (DMBA+hCG group). Age-matched untreated (control), hCG-, and DMBA+saline-treated rats were used for comparisons. Mammary tissues were collected for histopathological and mRNA analyses after 5, 10, 20, and 40 d of hCG injection and 20 d after treatment. None of the animals in the control and hCG-treated groups developed mammary tumors. DMBA-treated rats developed a high incidence of both microscopic lesions, i.e., intraductal proliferations and ductal carcinomas in situ, and palpable tumors. In DMBA+hCG-treated rats, the incidence of microscopic and palpable tumors was markedly reduced. In these animals, alpha- and beta-inhibin immunoreactivity was elevated in the non-tumoral mammary glands in association with lobule formation and in the tumors. Inhibin A and B mRNAs were also elevated in the mammary tissue, and c-myc and c-jun were induced by the hormonal treatment. DMBA alone did not modify the expression of these genes. Our findings indicate that inhibin production and gene activation are associated with both mammary gland differentiation and tumor regression.     

Antitumor activity of a Streptococcus pyogenes preparation (OK-432). II. Analysis of the cytotoxic lymphocytes induced by OK-432 injection into tumor-bearing F344 rats

In the present study, the antigenic phenotype and target cell specificity of the cytotoxic lymphocytes observed in F344 rats following the ip inoculation of a syngeneic MADB106 mammary carcinoma and the single injection of OK-432 were examined. When the cytotoxicity of peritoneal exudate cells (PECs) was measured in a 4-hour 51Cr release assay, appreciable cytotoxicity against MADB106 tumor cells was evident by day 7-14 following OK-432 injection. With the use of an antibody (R1-3B3) and complement depletion of cytotoxic PECs, the MADB106 killer cells appeared to consist of both R1-3B3- (non-T) and R1-3B3+ (T) cells, with most of the anti-MADB106 killing residing in the R1-3B3- cell population. The R1-3B3- killer cells were further defined as: a) phenotypically asialoGM1+, b) present in athymic nude rats, and c) accompanied by some augmentation of YAC-1 killing [the prototype rat natural killer (NK) target], suggesting that some of these R1-3B3- killer cells were typical NK cells. However, it was also observed that most of the R1-3B3- cells that killed MADB106 tumor cells were: 1) phenotypically or functionally different from either cytotoxic T-cells or typical NK cells; 2) observed only in MADB106 tumor-bearing rats challenged with OK-432; 3) not present on day 1-2 following OK-432 injection, the time when YAC-1 killing was maximally augmented; and 4) present in high numbers in a secondary response following reinoculation of the MADB106 tumor cells into cured rats. The in vivo relevance and possible derivation of these various cytotoxic lymphocyte populations in the syngeneic tumor-bearing hosts are discussed.     

Inhibition of rat mammary carcinogenesis by an arotinoid without a polar end group (Ro 15-0778)

The influence of an arotinoid without a polar end group (Ro 15-0778) on rat mammary carcinogenesis was investigated. Mammary tumors were induced by oral administration of 15 mg, 7,12-dimethylbenz-(a)anthracene (DMBA) to 50-day-old female Sprague-Dawley rats. Ro 15-0778 inhibited the development of mammary adenocarcinomas. The percentage of tumor-bearing rats, the mean number of tumors per rat as well as the mean total volume of tumors per rat were dose-dependently reduced by Ro 15-0778. The results are of particular interest, since this compound—probably because of the lack of a polar end group—does not induce the signs and symptoms of hypervitaminosis A. The inhibition of mammary cancer development by Ro 15-0778 compares favorably with that of N-(4-hydroxyphenyl) retinamide the hitherto most effective retinoid for prevention of chemically-induced mammary cancers in rats.