I. I. Khegai

Walker 256 tumor growth in rats with hereditary defect of vasopressin synthesis

Stable deceleration of Walker 256 tumor growth was detected in Brattleboro rats with vasopressin synthesis defect in comparison with normal WAG rats. In contrast to continuous tumor growth typical of rats, the growth of this tumor in Brattleboro rats was negligible and was observed during the first 15–18 days after transplantation, after which the tumor regressed and disappeared. The effect was age-dependent and was more pronounced in old animals. Repeated injection of Walker 256 cells does not lead to tumor development, which attested to direct involvement of the immune system in the detected phenomenon.

Humoral Immune Response in Ontogeny of the Brattleboro Rats with a Hereditary Defect of Vasopressin Synthesis

In the past decade, the regulatory interactions between the hypothalamic–pituitary–adrenal (HPA) and immune systems were established by numerous studies [1, 2]. These interactions are mediated by neuromtransmitters, cytokines, and their receptors, which have already been identified [3, 4]. One of these neuroendocrine modulators is a nanopeptide arginine– vasopressin (AVP) synthesized in hypothalamus. In immunocompetent organs (the thymus, spleen, and lymphatic nodes), syntheses of both AVP and AVP receptors were also detected [5, 6]. The level of AVP in the rat thymus decreases significantly with aging [7]. The AVP concentration is increased in the hypothalamus and blood plasma of athymic nude mice [8], after administration of bacterial lipopolysaccharide endotoxin [9], and under the influence of interleukins (IL-1 and IL-2) [10, 11]. AVP affects thymocyte development and differentiation [10, 11], and, like IL-2, stimulates T-lymphocyte proliferation. Along with corticotropinreleasing hormone, AVP is involved in the stressinduced suppression of immune functions [6]. The AVP antagonists inhibit the effects associated not only with the neuroendocrine, but also with the immune system.

The effect of vasopressin on the Zajdela hepatocellular carcinoma growth rate

222 In mammals, water balance is maintained by a complex neurohormonal system, in which vasoo pressin, a peptide from neurohypophysis that controls reabsorption of water in the tubules, plays the most important role [1]. The impairment of synthesis and secretion of vasopressin leads to pathologically increased excretion of urine with low osmolarity and, as a result, increased water consumption [2]. Brattlee boro rats have an impaired vasopressin expression in the hypothalamus due to deletion of guanine in the coding region leading to the reading frame shift and loss of its single stop codon [3]. Homozygous mutant animals cannot produce the endogenous hormone, so they daily consume and excrete the amount of water equal to 100% of their body weight [4]. The absence of vasopressin in blood causes other functional disorders that are not directly related to water balance control. Significant alterations are found in the immune syss tem [5, 6]. We previously showed that the growth rate of the transplanted Walker 256 carcinosarcoma changed depending on the lack of vasopressin in Bratt tleboro rats. Two after the inoculation of tumor cells, large mature nodules started to reduce down to comm plete disappearance. On the contrary, WAG (Wistar Albino Glaxo) rats with actively expressed vasopressin gene and physiologically normal vasopressin blood level suffered from progressive growth of the tumor, which then caused their death [7].To confirm the putative role of vasopressin in neoplastic processes, we conducted a study in which rats were transplanted with Zajdela ascitic hepatocellular carcinoma with the orii gin and types of tumor cells different from those of the Walker 256 carcinosarcoma. Adult male Brattleboro and WAG rats weiging 260–300 g (7 animals in each group) were used in the study. The rats were obtained from the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. The rats were kept under the standard conditions with free access to food and water and natural lighting. The study was carried out according to the regulations for handling experii mental animals (the Addendum to the Order of the Ministry of Health no. 755 of August 12, 1977) and international regulations on the protection of animals used for research purposes. Experimental protocols were approved by the Bioethics Committee of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. Ascitic tumors are widely used models for in vivo studies and …

Pattern RT1A and proteasome expression in cellular fractions of Zajdela ascitic hepatoma.

152 The key problem of experimental and clinical oncology today is the identification of possible mech anisms affecting the progression and regression of malignant neoplasms. The realization of these mecha nisms includes both innate and adaptive immunity: natural killer cells (NK cells), macrophages, neutro phils, eosinophils, dendritic cells, various cytokines, and specific cytotoxic T cells [1]. One of the molecular mechanisms underlying tumor development may be a reduced effectiveness of presentation and recognition of oncoantigens by the immune system. An important role in the generation of antigenic peptide epitopes, forming complexes with the major histocompatibility complex (MHC) class I molecules for their presenta tion to cytotoxic T cells of the CD8 phenotype, is played by immune proteasomes [2, 3]. We have previ ously shown that an increase in the level of immune proteasomes and a decrease in the level of constitutive proteasomes are accompanied by regression of Walker 256 carcinosarcoma, transplanted to Brattleboro rats with a genetically defective synthesis of arginine vaso pressin [4]. At the same time, the change in the ratio of multiple forms of proteasomes towards the decrease in the content of immune proteasomes causes an active growth of Krebs II ascitic carcinoma in mice [5]. The expression of immune proteasomes is correlated with the expression of MHC class I antigens. Low expres sion of MHC class I antigens promotes the escape of tumor growth from the immune control and is charac teristic of such tumors as melanomas, prostate adeno carcinoma, and breast cancer [3, 6]. Cases when a low expression of MHC class I antigens was caused by dis turbances in the system of antigen preparation and pre sentation (in particular, at low expression of the immune proteasome subunits) were described [3, 4, 7]. However, studies providing evidence in favor of this assumption are rare.

Concordance between vasopressin gene expression and growth of walker 256 carcinosarcoma in rats

The growth features of Walker 256 carcinosarcoma in rats of different genotypes were investigated. The experiments has been carried out on rats of the inbred Brattleboro and WAG lines, as well as on their hybrids segregated during congenic translocation of the normal vasopressin gene to the genotype of the Brattleboro rats. Brattleboro rats do not express the vasopressin gene. It has been found that there are only two types of tumor growth dynamics. In rats of the inbred Brattleboro line and in homozygotes di/di, that were segregated by backcrossings of heterozygous offsprings from the original crossbreeding between (WAG × Brattleboro) F1 × Brattleboro and the individuals with parental Brattleboro genotype, having grown to some extent the tumor regresses and disappears. In hybrid heterozygous siblings of di/+ genotype tumor grows linearly with time and always leads to fatal outcome. It has been found that, in the congenic procedure, the tumor regression trait is stably maintained and persistently inherited in lineage concordantly with the di/di genotype and, in rats with at least one allele of a normally expressed vasopressin gene, continuous and lethal tumor growth is always observed.

Neurohormonal Regulation of Tumor Growth

Neurohormones vasopressin and oxytocin are synthesized in the hypothalamus and are transported along the axons to the neurohypophysis as a part of equimolar complexes with hormone-specific neurophysins. The tumors of epithelial origin synthesize ectopic vasopressin and have an ability to express all types of receptors of neurohypophysis hormones. Vasopressin and oxytocin receptors provide the transduction of signals to protein kinases A, B, and C and activate intracellular cascades of the CREB, MDM2, and TORC1/2 proteins and mitogen-activated protein kinases. Central endocrine and autocrine neurohormonal contours are involved in the regulation of proliferative, migration, and angiogenic processes accompanied by tumor progression. Tumor growth and development occur in close cooperation with the supporting stroma. The interstitial tissue is involved in signal communication of tumor cells by integrins and integral CD44 glycoproteins formulating hyaluronic acid. Hyaluronic acid metabolites modulate the effect of neurohormones and peptide growth factors; intermediate hyaluronan fragments with molecular weight of approximately 20 kDa elicit the most significant angiogenic effect. Platelets expressing AVPR1 vasopressin receptors are an important source of hyaluronidase 2 hydrolyzing macromolecular hyaluronan to fragments of intermediate length. The AVPR2 receptors localized in endothelium and AVPR1-AVPR2 vasopressin receptors expressing themselves in the tumor cells are involved in the mechanisms controlling local hemostasis. Neurohormonal regulatory contours are involved in optimization of the balance of inducing and inhibiting signals generated by the tumor and stroma in the process of progressive growth.

[Features of the immune proteasome expression in ascite Zajdela hepatoma after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis].

The expression of the total proteasome pool, immune subunits LMP2 and LMP7, TAP1 and TAP2 transporters, and RT1A molecules of the major histocompatibility complex (MHC) class I in ascite Zajdela hepatoma cells was studied on the 10th day after implantation into Brattleboro rats with the hereditary defect in the synthesis of arginine-vasopressin (AVP) in the hypothalamus and WAG rats with normal AVP expression. Western-blot analysis revealed a threefold increase in the total number of proteasomes and immune subunit LMP2 and an eightfold increase in the immune subunits LMP7 in Zajdela hepatoma after its implantation in Brattleboro rats as compared with WAG rats. Differences in the expression of immune subunits LMP2 and LMP7 in Zajdela hepatoma in Brattleboro rats may contribute to different functions of these proteasomes, namely, the important role of the subunit LMP7 in antitumor immunity. Zajdela hepatoma growth in WAG rats was accompanied by a fall in both the total proteasome pool and immune proteasomes as compared with their content in Brattleboro rats, whose tumors regressed. The analysis of the content of peptide transporters TAP1 and TAP2 in Zajdela hepatoma implanted into Brattleboro and WAG rats showed their pronounced expression in tumor cells of both rat strains. In Zajdela hepatoma implanted into Brattleboro rats, a threefold increase in the basic molecule of MHC class I-RT1A was identified as compared with its expression in the tumor implanted to WAG rats. Furthermore, the content of CD8 and CD4 T-lymphocytes in the spleen of WAG and Brattleboro rats on the 10th day after implantation of Zajdela hepatoma was analyzed with flow cytometry. An increase in T-lymphocytes expressing the CD8 and CD4 antigens in the spleen of Brattleboro rats after implantation of the tumor as compared with WAG rats was shown. Increased numbers of both cytotoxic T lymphocytes and helper T-cells may facilitate tumor regression in Brattleboro rats. At the same time, a reduced number of subpopulations of T-lymphocytes in the spleen of WAG rats after implantation of hepatoma was accompanied by splenomegaly and growth of the tumor. Based on analysis of the data obtained it can be concluded that the deficiency of AVP in Brattleboro rats in Zajdela hepatoma leads to an increased expression of immune subunit LMP7 and basic molecules of MHC class I resulting in tumor immunogenicity and its elimination by the adaptive immune system.

Changes in lipid metabolism during Walker 256 tumor growth and the therapeutic effect of hyperthermia.

The dynamics of lipoprotein content during Walker 256 tumor growth in rats was studied. Moderate changes in HDL and LDL were paralleled by signifi cant changes in VLDL level. A 2-fold increase of VLDL in comparison with the intact control was recorded on day 10 of tumor growth. Exposure to total hyperthermia additionally stimulated VLDL synthesis and this parameter increased by 4 times and more in rats with tumors in comparison with controls. This effect of hyperthermia correlated with signifi cant subsequent decrease of rat mortality caused by the lethal effect of the tumor.

Restoration of expression of MHC class I molecule in Walker 256 tumor in growth dynamics in Brattleboro rats

11 Establishing possible mechanisms influencing the growth and regression of malignant neoplasias is the key problem of modern experimental and clinical oncology. It is known that realization of these mecha� nisms includes both innate and adaptive immunity; natural killer cells (NK cells), macrophages, dendritic cells, various cytokines, and specific cytotoxic T cells. However, spontaneous regression of malignant tumors occurs very rarely, which is associated primarily with the “avoidance” of their recognition by the immune system. One of the causes of this phenomenon may be a low level of expression of the major histocompatibil� ity complex (MHC) class I antigens, which are required for presenting oncoantigens to cytotoxic T cells and/or disturbance of the regulatory mechanisms of their expression [1]. Our earlier study demonstrated complete regression of Walker 256 carcinosarcoma, a tumor that causes death of control WAG rats on day 25–27 after transplantation, in Brattleboro rats with genetically deficient synthesis of arginine–vasopressin (AVP) in the hypothalamus [2]. AVP deficiency is obviously a key condition that determines the regression of this tumor in Brattleboro rats. Apparently, the observed effect was not deter� mined by the rapid hormonal effect of AVP. According to published data, chronic systemic administration of AVP to Brattleboro rats normalizes the disturbed water–salt balance in them but has no effect on the increased activity of NK cells observed in these rats [3]. Since innate and adaptive immunity are related, it can be assumed that the enhancement of innate anti� tumor immunity in Brattleboro rats induces the devel� opment of specific immune reactions. This may be expressed as an increase in the efficiency of recogni� tion of transformed cells. The fact that a repeated transplantation of Walker 256 to Brattleboro rats did not result in tumor development allowed us to assume the involvement of adaptive immunity in the realiza� tion of this phenomenon.

Effect of vasopressin gene expression on the growth of walker 256 carcinosarcoma in rats

The growth pattern of the Walker 256 solid tumor has been studied in rats with different doses of the mutant vasopressin gene. In contrast to the vasopressin gene of normal WAG rats, that of mutant Brattleboro rats has a deletion in the coding region that blocks expression at the translation level. The mutation is inherited as a recessive character and is expressed in homozygous Brattleboro rats as diabetes insipidus with an increased water consumption because of the absence of vasopressin in the blood. (WAG × Brattleboro) F1 hybrids have the same normal phenotype as WAG rats, including a low water consumption. Walker 256 carcinosarcoma, which is not strain-specific, intensely grows only in WAG and (WAG × Brattleboro) F1 rats. In these groups, the growth of the tumor leads to the animal death within approximately 30 days after the inoculation of tumor cells. In Brattleboro rats, the carcinosarcoma grows less intensely: the tumor node somewhat increases only within the first two weeks, after which the tumor began to decrease and eventually disappears altogether. Both characters exhibit a 100% concordance at the individual level.