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We analyzed the morphological changes accompanying the development of cancer in the mouse forestomach. The main aim of the study was to evaluate whether cancer in this area of the stomach arises de novo or undergoes a series of precancerous changes. Tumors were induced by the 1,2-dimethylbenz(a)antracene (DMBA) at a total dose of 4 mg/mouse. The suspected areas of the stomach were studied morphologically in 79 mice. Benign tumors (squamous-cell papillomas) and malignant tumors (squamous-cell carcinomas) were found in 40 mice. Tumors arose in all cases together with differential changes in the forestomach epithelium. These changes were seen as irregular diffuse hyperplasia or focal proliferation, with or without differential signs of dysplasia. Destruction of the basal epithelial membrane indicated transformation of the process into malignant invasive carcinoma. Thus, in chemically induced cancer of the forestomach, squamous-cell carcinoma develops as the final stage of morphologically recognizable precancerous changes in the epithelial layer. De novo formation of such tumors in the forestomach was not observed.

In our previous studies, we showed that soluble tumor-associated antigens (sTAA) of 66 kDa and 51 kDa have distinct tumor-preventive effects on chemically induced mammary cancer in rats and are able to repair the damage caused by tumorigenesis in its early stages. In the present study, we investigated whether these proteins can prevent the development of chemically induced tumors in mice. The study was performed on C3H/He mice which have the ability to develop many spontaneous tumors with age. Forty-four, 6-week-old mice were exposed twice at a 2-week interval to the carcinogen 9,10-dimethyl-1,2-benz(alpha)anthracene (DMBA), at a dose of 2 mg/mouse administered intragastrically. Two months later, the mice were divided into two groups. One group received sterile saline twice a week at a dose of 0.2 ml/mouse, intraperitoneally (i.p.). The other group received sTAA twice a week at a dose of about 10 microl in 0.2 ml of sterile saline/mouse, i.p. Periodically, all mice were checked for the presence of tumors. The experiment was terminated at week 35. Vaccination with sTAA increased the time of involvement of mice in the experiment, prevented the tumorigenic effect of DMBA, and inhibited further development of existing tumors.

Previously, we showed that the 66 and 51 kDa soluble tumor-associated antigens (sTAAs) have distinct suppressive effects on chemically induced mammary cancer in rats, both alone and in combination with the hormone-related anticancer drug tamoxifen. Here, we describe the effects of both sTAA and tamoxifen on the histological structure of ovaries in mammary tumor-bearing 30- to 34-week-old rats. Central ovary sections were pooled, the number of the healthy and degenerated follicles were counted, and the size of the corpora lutea was estimated. In follicular development primordial, primary, preantral and antral stages were recognized. Only healthy follicles with visible nuclei were counted. Follicular degeneration was estimated as the number of atretic follicles with follicular remnants. Treatment with tamoxifen alone or in combination with sTAA significantly increased the number of primordial follicles and atretic follicles in the ovaries, and promoted the formation of small follicular cysts. Total area of the corpora lutea decreased. sTAA participated in this process by increasing apoptosis in degenerated follicles.

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.

This study examined whether the soluble 66 and 51 kDa tumor-associated antigens (sTAA) could promote suppression by the anticancer drug 5-fluorouracil (5-Fu) of chemically induced mammary tumorigenesis, and which, if any, morphological changes in the immune organs accompany this treatment. Dimethylbenzanthracene (DMBA, 8 mg/rat, twice) was used to induce mammary tumors. After the appearance of many large tumors, the preparations of sTAA and 5-Fu, alone or in combination, were administered in weekly doses, for 4 weeks. The following groups of mammary tumor-bearing rats were studied: 1) control non treated rats, 2) rats treated with sTAA, 3) rats treated with 5-Fu, 4) rats treated with 5-Fu and sTAA. The experiment was terminated when tumors in 70% of control rats became ulcerous. Treatment with sTAA alone significantly decreased tumor yield and their total area relative to controls. Both of these parameters showed an even larger significant decrease after treatment with 5-Fu, and the most marked decrease was obtained after the combined treatment with 5-Fu and sTAA. Results demonstrated that not only do sTAA have tumor-suppressive properties, they also enhance the anticancer effects of 5-Fu and prevent its toxic side effects. Morphologically, the treatment with sTAA was manifested in a significant increase in the size of the spleen follicles and mantle layer compared to control rats with large tumors. The treatment with 5-Fu decreased the sizes of almost all areas of the spleen compared to control rats, whereas the combined treatment with 5-Fu and sTAA increased all these parameters to the levels found in rats treated with sTAA alone. The total areas of the cortex and paracortex in the lymph nodes increased after treatment with sTAA. Treatment with 5-Fu alone resulted in a significant decrease of these areas which, as seen in the spleen, increased after combined treatment with 5-Fu and sTAA. Similar changes were seen in the areas of the separate lymph node zones. We concluded that the addition of sTAA to conventional tumor chemotherapy regimens has a remarkable synergistic effect on mammary tumors leading to curative antitumor responses of the host's immune organs.

The effect of the synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on proliferative activity in colon tumors, and in mucosal epithelial cells adjacent to and located far from tumors was studied in rats. To evaluate the effect of Epitalon on different stages of carcinogenesis, different treatment regimens were used: during the tumor initiation stage, during the tumor-promotion stage, or during the entire process of tumor development. Eighty 2-month-old male LIO rats were exposed weekly to five subcutaneous injections of 1,2-dimethylhydrazine (DMH) at a single dose of 21 mg/kg body weight. Rats were divided into four groups. Control rats (group 1) received saline at a dose of 0.1 ml during the entire experiment. Rats in group 2 were treated with Epitalon at a dose of 1 micro g, five times a week, for 6 months, from the first injection of DMH till the end of the experiment. Rats in group 3 were treated with Epitalon after termination of the carcinogen injections. Rats in group 4 were treated with Epitalon only during the period of DMH exposure (for the first 5 weeks of the experiment). DMH induced proliferation of the secretory epithelium, and this phenomenon was accompanied by a decrease in the size of the stromal area and the area of lymph infiltration in colon tumors and in the colon mucosa adjacent to the tumors (group 1). Epitalon attenuated this effect, especially when the treatment was continued throughout the experiment (group 2). It increased the stromal areas, as well as that of lymphoid infiltration in the colon mucosa adjacent to the tumors. The intensity of lymphoid infiltration was activated in both the colon mucosa adjacent to a tumor and in the tumor. Mitotic activity of tumor cells was significantly inhibited by Epitalon when the treatment was given throughout the experiment (group 2). In parallel, a high level of apoptosis was seen in the same group. Thus, the strongest inhibitory effect of Epitalon on carcinogenesis in the colon mucosa was manifested when the treatment was continued throughout the experiment.

We studied whether feeding pregnant female rats different high fat diets affects structural zones in the spleen and lymph nodes, involved in production of T and B cells, as well as cell kinetics and apoptosis in some offspring with mammary glands tumors. Rat mothers were fed either a 7% or 15% corn-oil or a 7% or 15% olive-oil diet. At four weeks of age, female offspring (n=10-15 per group) were transferred to 7% corn oil diet. Five-week old offspring were exposed twice to the carcinogen, dimethylbenz(a)antracene (10 mg/rat/week). Three months later, tumors were counted and sized, and samples from the spleen, axillary lymph nodes and tumors collected for immunohistochemical analyses. Feeding the mothers with both the 7% and 15% olive-oil diets significantly increased the number of tumor-free rats in offspring. Tumors were characterized with active mitosis, intensive lymphoid infiltration inside a knot and high rates of apoptosis, particularly in tumors obtained from rats whose mothers were fed the 15% olive-oil diet. In the spleen, the 15% olive-oil diet significantly increased the areas of the follicles and germinal centers but only in tumor-free rats. In tumor-bearing rats, areas of germinal centers increased compared to the 7% olive-oil diet. The 15% olive-oil diet increased all areas of the lymph nodes in tumor-free rats, while in tumor-bearing rats, this diet increased the areas of the cortex and mantle layer. We conclude that exposure to various diets in utero and during lactation affects the immune system. In addition, the promotion of apoptosis may play a key role in the mechanisms involved in the transplacental effects on mammary tumor development as seen using a 15% olive-oil diet, similar to the high fat diets of Mediterranean countries.

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.

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 evaluated whether the feeding of rats with a 15% orange-pulp diet affects the lymphatic system and the tumorigenic response in rats exposed to a high dose of carcinogen. Five-week-old Sprague Dawley rats were divided into 2 groups fed a control chow diet or the same diet with 15% orange pulp. All rats were injected with 1,2-dimethylhydrazine (DMH) (20 mg/kg) weekly for 6 weeks. At 8 months, tumors, spleens and descending colon were taken from each group for analyses. Feeding rats the 15% orange-pulp diet did not reduce the tumor number but modified the number of adenocarcinomas found in the orange-pulp group compared to controls: 66.7% vs. 93.7%. The number of endophytic tumors was also significantly lower in the experimental group: 6.3% vs. 32.3% in controls. DMH affected the size of the splenic structures. The size of follicles and germinal centers decreased significantly in tumor-bearing rats compared to tumor-free rats. This effect was changed in rats fed the orange-pulp diet. In tumor-bearing rats from this group, only the area of the marginal zone decreased and the red pulp increased compared to tumor-free rats. The size of germinal centers significantly increased compared to tumor-bearing rats in controls. The total number of lymphoid cells decreased in germinal centers of spleens obtained from control tumor-bearing rats compared to tumor-free rats. DMH alone significantly increased the total number of cells in the colon mucosa of the rats fed the control diet. In tumor-bearing rats exposed to the carcinogen and fed the 15% orange-pulp diet, the total number of cells and the number of Ki-67+ cells increased in the depth of tumors whereas the number of CD8+ T cells increased in the colon mucosa, at the border of tumors and its depth. The caspase-3 protein a cysteine protease was elevated in tumors from rats fed the orange-pulp diet. Although the 15% orange-pulp diet did not change the number of tumors in the tumor-bearing rats, feeding rats orange pulp significantly decreased the number of endophytic tumors and increased the number of exophytic tumors. Increased activity of T cell killers in tumors and higher level of proteins involved with apoptosis following consumption of the orange pulp indicate a clear tumor suppressor effect of these dietary fibers.

We studied whether feeding pregnant female rats a 15% olive-oil diet affects the activity of lymph cells in the spleen and tumors in offspring with chemically-induced colon tumors. Rat mothers were fed either a 7% corn-oil or a 15% olive-oil diet. Five-week-old male offspring were divided into 3 groups. A control group was fed the 7% corn-oil diet similar to their mothers. The experimental group I was fed the 7% corn-oil diet whereas their mothers were fed the 15% olive-oil diet. The experimental group II was fed the same 15% olive-oil diet as their mothers. Experimental rats were injected weekly for 8 weeks with the carcinogen, 1,2-dimethylhydrazine (DMH), 20 mg/kg b.w. Results of experiments were studied 6 months later. The area of zones in the spleen responsible for producing B and T lymphocytes were measured and the number of cells counted. The activity of lymphoid elements of the spleen and of tumors were studied using immunohistochemical methods for evaluating the synthesis of CD8(+) lymphocytes and proliferative activity of lymphocytes in spleens and tumors. Feeding pregnant or lactating mothers with the 15% olive-oil diet had no marked tumor-protective effect on chemically-induced colon cancer in offspring. Diet-dependent changes were found at the cellular level. In the spleen of control offspring, the presence of a tumor was accompanied by an increase in the number of Ki-67(+) cells and CD8(+) lymphocytes in the red pulp. In experimental group I, DMH significantly increased the total cell number and the number of CD8(+) lymphocytes in the red pulp of the spleen in both tumor-bearing and tumor-free rats. In experimental group II, the total number of lymph cells and the number of CD8(+) lymphocytes increased compared to offspring fed a control diet. Tumor formation activated the proliferative activity of lymph elements. The total number of cells in infiltrates of the colon mucosa decreased in tumor-bearing rats compared to tumor-free counterparts, and this was seen in all three dietary groups of rats. In tumors from offspring of experimental group II, only the number of CD8(+) lymphocytes increased compared to those in offspring of experimental group I. The findings indicate that feeding mothers the 15% olive-oil diet had a cancer-inhibiting role in offspring, predominantly changes at the cellular level.

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 showed previously that soluble low-molecular-mass tumor-associated antigens (TAA) could suppress chemically-induced tumorigenesis. In this study, we analyzed the mechanism of those findings. Studies were performed on the spleen and mammary gland tumors obtained from the following groups of rats: i) control rats treated with dimethyl-benz(alpha)antracene (DMBA), ii) vaccinated and carcinogen-treated rats with non regressed tumors, iii) vaccinated and carcinogen-treated rats with regressed tumors. Different zones of the spleen and tumors and their cellular content (Ki67+ and CD8+ cells, and macrophages) were analyzed morphometrically and immunohistochemically. Reaction of the spleen to vaccination was manifested in a significant increase in all areas of the white pulp and in a decrease in the size of the red pulp. The total number of cells in the white pulp (germinal center and PALS) and in the marginal zone was significantly higher in the spleen of rats with regressed tumors. The number of Ki67+ cells decreased significantly in both groups of vaccinated rats, but most prominently in the marginal zone and the red pulp in rats with regressed tumors. An increased number of CD8+ lymphocytes and macrophages was also seen in the red pulp. Different areas of the tumors (peripheral vs. inside at depth) showed different responses to vaccination and this difference was related to conditions of carcinogenesis, i.e. non-regressed vs. regressed tumors. In regressed tumors, all parameters studied were easily distinguishable in both areas of the tumors, while in non-regressed tumors, a marked distinction was seen only at their periphery. In regressed tumors, a negative correlation was seen at depth tumors between the number of Ki67+ cells and the number of CD8+ lymphocytes (r=-0.48). The findings indicated a strict antitumor effect of vaccination with the soluble low-molecular-mass TAA, which prevents the development of insufficiency of the immune system when an intensive immune reaction takes place.

The suppression of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis by melatonin was previously demonstrated. The objective of the present work was to evaluate histologically and immunohistochemically the splenic immune response to the induced cancer and to melatonin. Spleens from rats, either untreated, injected with DMH, fed with melatonin or treated with both carcinogen and melatonin, were studied. The exposure to the carcinogen and the consequential carcinogenesis resulted in splenic changes that reflected the insufficiency of the immune response, as manifested in significant reduction of the white pulp and the simultaneous expansion of the red pulp. The effects of melatonin on most splenic components were inverse to those of DMH. The anti-carcinogenic properties of melatonin were evidenced from the reversal of the inhibitory effects of DMH, especially when the densities of lymphocytes in different parts of the spleen were compared. The combined treatment of the rats with DMH and melatonin resulted in the expansion of the splenic zones by 106% to 125%, compared to those from DMH-treated rats, and the numbers of CD8+ lymphocytes and Fas-positive cells increased sharply. Therefore we conclude that anti-carcinogenic effects of melatonin are related to activation of several elements of the host's lymphatic system.

We evaluated whether feeding pregnant female rats a diet high in olive-oil, that showed a tumor-preventive effect in adults, has a similar preventive effect on chemically-induced cancer in offspring (i.e. mammary glands and colon cancer in rats). The control group was fed the same 7% corn-oil diet as their mothers. Experimental group I was fed a 7% corn-oil diet while their mothers received a 15% olive-oil diet. Experimental group II was fed the same 15% olive-oil diet as their mothers. Female offspring were twice administered 7,12-dimethylbenz(a)antracene (DMBA) in doses of 10 mg/rat. Male offspring were injected 6 times with 1, 2-dimethylhydrazine (DMH) in doses of 20 mg/kg body weight. Effect of DMBA was manifested in a high rate of tumorigenesis: the number of tumor-bearing rats in control offspring reached 52.0%. This effect increased to 60.6% among offspring of experimental group II and to 67.7% in offspring of experimental group I. The mean tumor size increased significantly in control offspring. Following administration of DMH number of tumor-bearing rats was similar in all groups of offspring: 36.7%, 40.7% and 42.8%. Tumor types differed: the majority of tumors in the control group were benign polyps and adenomas (72.1%) and the number of adenocarcinomas was low (27.9%). The number of malignant tumors increased to 37.5% in offspring of experimental group II and to 45.5% in offspring of experimental group I. In control group offspring, a distinct tendency to increased body weight and a significant increase in spleen weight were seen. The findings indicate that feeding mothers a diet high in fat concentrations, even those with known tumor preventive significance in adults, lose this cancer-inhibiting role in offspring.