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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.

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.

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.

This study examined whether the soluble 66- and 51 kDa tumor-associated antigens (sTAA), isolated from the serum of breast cancer patients, possess specific suppressive effects on chemically-induced rat mammary tumorigenesis in comparison to commercial human albumin. Dimethylbenzanthracene (DMBA, 10 mg/rat, 2 administrations) was used to induce mammary tumors in 8-week-old Sprague Dawley rats. After the appearance of many large tumors, preparations of sTAA (50-60 micro g/rat in 0.5 ml sterile PBS) or commercial human albumin (HA, in the same doses as sTAA) were administered weekly, for 10-14 more weeks. The following groups of mammary tumor-bearing rats were studied: i) control non-treated rats, ii) rats treated with HA, iii) rats treated with sTAA. The experiment was terminated when tumors in 70% of the rats became ulcerous. The treatment with sTAA significantly decreased, compared to controls, the yield and total area of the tumors. In rats treated with sTAA, the appearance of new tumors stopped at week 5 as compared to week 7 in rats treated with HA and week 10 in control rats. In rats treated with sTAA, the time of appearance of ulcerous tumors increased to 8 weeks, as compared to 6 weeks in controls and in rats treated with HA. Duration of the experiment increased from 11 weeks in controls to 12 weeks in rats treated with HA and to 14 weeks in rats treated with sTAA. We conclude that sTAA have tumor-suppressive properties, which are well-defined if the treatment is begun on small tumors.

The aim of this review was to analyze the role of cell kinetics and apoptosis in the cellular mechanism underlying the effects of soluble tumor-associated antigens (sTAA) on chemically-induced mammary cancer in rats treated with the anticancer drug cyclophosphamide (CPA). Mammary gland cancer was induced with dimethylbenz(a)anthracene (DMBA), and tumors, the spleen, thymus, bone marrow and lymph nodes were studied by morphometric and immunohistochemical methods. We suggest that inhibition of the functional activity of the immune system is one of the main reasons for the toxic effects of CPA, and that the tumor-suppressive antitoxic effects of sTAA result from their activation of T-lymphocyte production in this system, particularly in the spleen and lymph nodes. The results of our experiments have shown that vaccination with sTAA actively promotes generation of the host's antitumor immune response. This is manifested in inhibited proliferative activity of the tumor cells, stimulated production of T lymphocytes and an increased rate of apoptosis among tumor cells.

We showed in a previous study that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this report, we analyzed the possible mechanism underlying this phenomenon. Studies were performed on the bone marrow and thymus from the following groups of rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, iv) rats treated with CPA and sTAA. The cellular content of the bone marrow and thymus (CD4+ and CD8+ lymphocytes) was analyzed morphometrically and immunohistochemically. In the bone marrow, CPA caused significant substitution of cellular components with fatty tissue whereas sTAA repaired this process. We found that CPA affects mainly the process of myelogenesis whereas sTAA protect the production of lymphocytes. In the thymus, CPA alone or in combination with sTAA repaired the inhibition effect of DMBA on synthesis of CD4+ and CD8+ thymocytes. sTAA further increased the amount of CD8+ T lymphocytes in the medulla of the thymus. Data in the literature as well as the findings presented here demonstrate that the tested treatment, including vaccination with sTAA, actively promotes the generation of the host's antitumor immune response.

The aim of this study was to analyze the roles of proliferative activity, lymphoid infiltration and apoptotic rate in the cellular mechanism underlying the promotion effects of soluble tumor-associated antigens (sTAA) in mammary cancer in rats treated with the anticancer drug cyclophosphamide (CPA). Studies were performed on the following groups of rats: i) control tumor-bearing rats, ii) tumor-bearing rats treated with sTAA, iii) tumor-bearing rats treated with CPA, iv) tumor-bearing rats treated with CPA and sTAA. Mammary gland cancer was induced with dimethylbenz(a)anthracene (DMBA), and the rate of lymphoid infiltration, T cell content (CD4+ and CD8+ cells), and mitotic and apoptotic indices in tumors were evaluated. In control tumor-bearing rats, high lymphoid proliferation, as well as a high number of CD8+ cells, was found in tumors. Treatment with sTAA further significantly increased the total number of lymphoid cells and the number of CD8+ lymphocytes. CPA sharply decreased the production of all lymphoid cells studied, especially of CD4+ lymphocytes. The combined treatment of CPA and sTAA increased the number of lymphoid cells, although they did not reach control levels. The mitotic index significantly decreased as a result of the treatment with CPA alone and especially after the combined treatment with CPA and sTAA. The results of our experiments demonstrate that vaccination with sTAA actively promotes the generation of the host's antitumor immune response. This is manifested in inhibited proliferative activity of tumor cells, stimulated production of T lymphocytes and increased rate of apoptosis among tumor cells.

The objective of this study was to examine whether splenectomy alters the tumorigenic response of rats to chemically-induced mammary tumors and to determine the role played by the parathymic lymph nodes (PTLN) in the antitumor immune response. Female rats were splenectomized and then exposed to 9,10-dimethyl-1,2-benz(a)anthracene (DMBA) to induce mammary tumors. Splenectomy decreased the rate of tumor appearance. The latent period for appearance of the first tumors in splenectomized rats was 12.0 +/- 0.9 week compared to 9.7 +/- 0.5 week in intact controls. The latent periods for appearance of all tumors including those with malignancy were similar for both rat groups. Splenectomy caused a significant increase in the size of the PTLN as well as changes in their structure. The cortex became thick, better formed and enriched with lymph elements of follicles and a wide venous net. CD8+ cells infiltrated the paracortex whereas CD4+ cells were located around the blood vessels. In conclusion, splenectomy inhibits the early stages of tumorigenesis, but does not prevent the progress of carcinogenesis. Different responses of splenectomized (operated) and intact rats to the effect of DMBA can be explained by an increase in the nonspecific resistance of splenectomized rats as a result of the operation. PTLN actively participate in this compensatory reaction of the immune system of animals.

We have shown previously that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this study, we analyzed the possible mechanism underlying this phenomenon. Studies were performed on tumors obtained from the following groups of mammary tumor-bearing rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, iv) rats treated with CPA and sTAA. All analyzed tumors represented different types of invasive duct carcinomas. The rate of lymphoid infiltration and T cell content (CD4+ and CD8+ cells) of tumors were analyzed immunohistochemically. In parallel, mitotic index was evaluated in tumor cells. In tumor-bearing rats, high lymphoid proliferation was found at the periphery of tumors, and to a lesser extent deep inside the tumors. In control tumors, CD4+ T cell content was very low whereas CD8+ cells were highly abundant, especially at the tumor periphery. Treatment with sTAA significantly increased the total number of lymph cells and the number of CD8+ lymphocytes inside the tumors. Cytoplasmic vacuolization, decreased mitotic index and various degrees of fibrosis were the most distinct changes in tumors treated with CPA alone. CPA also sharply decreased the activity of all lymph cells studied, especially of CD4+ lymphocytes which could no longer be observed following this treatment. The combined treatment of CPA and sTAA increased the number of lymph cells, although they did not reach control levels. Inhibition of mainly CD4+ lymphocyte synthesis of CPA was confirmed by the low CD4/CD8 ratio, which increased slightly after the combined treatment with CPA and sTAA. Findings in the present study demonstrate that vaccination with sTAA actively promotes the generation of the host's antitumor immune response.

In a previous study, we showed that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this report, we analyzed the underlaying mechanisms. Studies were performed on the spleen and lymph nodes from the following groups of mammary tumor-bearing rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, i.v.) rats treated with CPA and sTAA. Different zones of the spleen and lymph nodes were measured and their T cell content (CD4(+) and CD8(+) cells) was analyzed immunohistochemically. CPA decreased the size and cell content of follicles, splenic areas related to the production of B cells, of the marginal zone and to a lesser extent of the periarterial lymph sheath, and decreased the number of CD4(+) and, at a lower rate, of CD8(+ )T cells in the spleen. Addition of sTAA restored activity in the splenic zones producing these cells. Similar effects of CPA and sTAA were found in lymph nodes with accumulation of B lymphocytes in the primary and secondary follicles and of T lymphocytes, including both CD4(+) and CD8(+) cells, in the paracortical zone. We suggest that inhibition of the functional activity of the immune system is one of the main reasons for the toxic effects of chemotherapeutic drugs such as CPA and that the tumor-suppressive antitoxic effects of sTAA result from their activation of B- and T-lymphocyte production in this system, particularly in the spleen and lymph nodes.

This study examined whether the soluble tumor-associated antigens (sTAA) of 66 kDa and 51 kDa could promote suppression of chemically-induced rat mammary tumorigenesis by the hormone-related anticancer drug tamoxifen and prevent the drug's toxic side-effects. Dimethylbenzanthracene (DMBA, 10 mg/rat, 3 administrations) was used to induce mammary tumors in 8-week-old Wistar rats. Then, for 13-17 more weeks, preparations of sTAA (50 microg/rat) and tamoxifen (10 mg/rat) were administered, separately or in combination, on a weekly basis. The experiment was continued for 18 weeks and was terminated when the number of dead rats reached 50% in each group. Treatment with tamoxifen inhibited tumor growth and their malignance: the number of rats without malignant tumors significantly increased compared to controls, 27.3% and 5.6%, respectively. Treatment with sTAA resulted in a significant increase in the number of regressed tumors to 10.1% compared to 0% and 1.4% in control and tamoxifen-treated rats, respectively. Moreover, the period of 50% survival increased from 13 weeks in tamoxifen-treated rats to 17 weeks, and as a result, rats treated with sTAA were involved in the experiment for an average 14.3 weeks compared to 10 and 10.4 weeks in control and tamoxifen-treated groups. In rats treated simultaneously with tamoxifen and sTAA, the time of appearance of each new tumor increased from 4.5 weeks to 6.6 weeks with a significant increase to 14.3% in the number of regressed tumors. The period to 50% survival increased to 18 weeks, and these rats were involved in the experiment for up to 16.4 weeks. The number of rats without malignant tumors increased to 22.2% and the time of appearance of malignancy increased to 9.6 weeks, as compared to 7.3 weeks in controls. The results demonstrated that sTAA have tumor-suppressive properties, and also enhance the anticancer effects of tamoxifen and prevent its toxic side-effects.

The objective of this study was to examine how splenectomy affects the immune response, particularly T cells, in chemically-induced mammary tumors. Female rats were splenectomized and then exposed to 9,10-dimethyl-1,2-benz(a)anthracene (DMBA) to induce mammary tumors. Splenectomy significantly decreased the rate of tumor appearance and their malignant transformation. The tumor latency period in splenectomized rats was 12.0+/-0.9 weeks compared to 9.7+/-0.5 wk in intact controls, and malignancy appeared in 45% of splenectomized rats, compared to 70% in controls. By the end of the experiment, the total number of tumors and their size were similar in both groups. Blood CD4+ and CD8+ T cell concentrations were similar in tumor-bearing and tumor-free splenectomized animals, but in both groups CD4- and CD8- lymphocytes decreased sharply compared to control animals. In tumor-bearing rats, splenectomy also resulted in significantly more circulating natural killer cells. The spleens of tumor-bearing control rats had significantly fewer CD4+ and CD8+ lymphocytes and more CD4- and CD8- lymphocytes and natural killer cells than did their blood. In conclusion, splenectomy inhibits the early stages of tumorigenesis and reduces the rate of malignant transformation of benign tumors, but does not prevent the progress of carcinogenesis. Differences between splenectomized (operated) and intact rats to the effect of DMBA can be explained by an increase in non-specific resistance of splenectomized rats as a result of operation.

This study examined whether the soluble tumor-associated-antigens (TAA), of 66 kDa and 51 kDa, could promote suppression by anticancer drugs of chemically-induced mammary tumorigenesis. Dimethylbenzanthracene (DMBA, 10 mg/rat, twice) was used to induce mammary tumors. Then, for nine more weeks, the preparation of TAA and cyclophosphamide (CPA), alone or in combination with TAA, were administered in weekly doses. Twenty weeks after DMBA exposure, the mammary tumor yield was 2.4, 2.8 and 2.9 in the experimental groups compared to 3.5 in the controls. Seventy-five percent of the rats in the control group, but only 37% of TAA, 50% of the CPA, and 30% of the CPA and TAA treated animals had malignant tumors. In the experimental groups, 6.5%, 25% and 38%, respectively, of the tumors regressed, compared to 3% in controls. In the groups receiving CPA or TAA, regression was observed in the fifth week of treatment, and in the group receiving combined treatment, already in the first week. The size of the tumors in control rats increased during the last 10 weeks 3.6 times, in the CPA treated rats 1.15 times, but in those receiving CPA plus TAA it decreased by 0.7 times. The results of our experiment demonstrated that TAA have distinct tumor-suppressive properties, and can enhance the anticancer effects of CPA.

We compared effects of a high fat diet and a carcinogen on cellular elements of the spleen and mammary gland tumors in rats. Animals were fed a 15% olive-oil diet and a group of them were exposed to a carcinogen, dimethylbenz(a)anthacene (DMBA), in two doses of 10 mg/rat. Results of the experiments were evaluated after 4 months. We studied changes in the areas of different zones of the spleen related to production of B and T lymphocytes and also the number of cells in the spleen and tumors with positive reaction to receptors related to manifestation of apoptosis (FasL and p53) and receptors related to inhibition of apoptosis (bcl-2). In the spleen, dietary fats as well as DMBA alone decreased the zones related to production of B lymphocytes and increased the number of T lymphocytes. The combined effect of a carcinogen and a high fat diet manifested in an increase in the number of lymphoid cells and macrophages. In tumors from rats fed a low-fat diet, an extremely high number of lymphoid cells was seen in the border of tumors with T cell killers as a main component of these infiltrates. In tumors from rats fed a 15% olive-oil diet, the main component of the infiltrates were macrophages. High levels of p53+ and bcl-2+ cells were found in the spleen of rats exposed to a carcinogen. The combined effect of a carcinogen and the 15% olive-oil diet inhibited production of FasL and p53 receptors and stimulated synthesis of bcl-2 protein. In tumors, a carcinogen alone stimulated the high expression of FasL and p53 proteins, but in combination with the 15% olive-oil diet synthesis of these receptors decreased while production of bcl-2 protein increased sharply. This observation may serve as an additional proof of tumor-promoter effects of a high fat diet.

We studied whether feeding pregnant female mice with different fats affects lipid exchange and activity of the splenic lymphoid system in offspring exposed to low doses of carcinogen. Female mice were fed diets with either 7% or 15% corn oil or olive oil. The 4-week-old offspring of these mice were transferred to a chow diet, and exposed to a low dose of the carcinogen, dimethylbenz(a)antracene (2 mg/kg). Results of experiments were studied 5 months later. Concentrations of polyunsaturated linoleic and oleic acids were determined in the blood and liver of mothers and offspring. The activity of the splenic immune system in offspring was studied using immunohistochemical methods for evaluating the number of different types of lymphocytes (B and T cells), mitotic and apoptotic indexes and the number of Fas-positive lymphocytes. Serum concentrations of the fatty acids examined were unchanged in the blood of the mothers and their offspring. Concentration of both linoleic and oleic acids was significantly higher in the liver of mothers fed the 15% olive-oil or corn-oil diets. This high level was maintained in linoleic acid in offspring of mothers fed the 15% olive-oil diet. Spleen weight was higher in offspring of mothers fed a 15% corn-oil diet compared to those fed the 7% corn-oil diet. The 15% olive-oil diet slightly decreased the weight of the spleen compared to counterparts fed the 15% corn-oil diet. Immunohistochemical studies showed that the olive diet, partially of 15%, significantly stimulated B-cell blast transformation. The finding reflects the reaction of B lymphocyte-producing splenic zones to the carcinogenic effect, though to a weak extent. T lymphocyte-producing zones did not respond to the diets studied, probably due to the weak carcinogenic effect and lack of tumor appearance. The Fas activity of both B and T cells in the spleen was stimulated by the carcinogen and enhanced by feeding the mothers on the olive-oil diet. Maternal feeding with a diet rich in olive oil before pregnancy results in stimulation of morphological and functional attributes of the splenic immune system of the offspring, particularly related to producing of B lymphocytes.

This study attempted to elucidate whether the soluble tumour-associated proteins (TAA) of 66 kDa and 51 kDa molecular weight could suppress chemically induced mammary tumorigenesis. An intragastric dose of dimethylbenzanthracene (DMBA) was administered to rats and some were simultaneously immunised with the TAA. A single dose of DMBA resulted in 38% of the rats developing mammary tumours. However, simultaneous vaccination with the TAA preparation was significantly tumour-suppressive: mortality declined from 50% to 0% (p < 0.05); survival was extended from 9.4 weeks to 13.0 (p < 0.05), and 83% of the animals remained tumour free, compared to 13% of the control animals (p < 0.05). In 33% of the immunised animals the malignant tumours regressed completely. Such vaccination was also effective, although to a lesser extent, when the carcinogen dose was doubled. Then, 33% of the immunised and 22% of the control animals remained tumour-free, the latent period of malignant transformation was extended from 10.0 to 11.7 weeks, the initial tumour-free period lasted 9.3 weeks instead of 8.3 weeks and 10% survived compared to 50% of the controls. Vaccination with the soluble low molecular-weight TAA had distinct tumour-suppressive effects on mammary gland tumorigenesis.