[The Effects of the Hydrogen Sulfide Donor GYY4137 on the Proteasome Pool of Colorectal Cancer Cells].

Cancer cells are characterized by an increased level of metabolism and are highly dependent on the correct functioning of the processes that ensure homeostasis. Reactive sulfur species (RSS) are important molecular modulators of metabolic processes in both healthy and tumor cells. The effect of RSS and, in particular, H2S, on key cellular systems, including the ubiquitin-proteasome system (UPS), which provides the destruction of most intracellular proteins, has been shown. The main components of the UPS are proteasomes, multisubunit protein complexes, within which proteolysis occurs. At the same time, data on the effect of H2S directly on the pool of proteasomes in tumor cells are insufficient. Here, we studied the effect of incubation of SW620B8-mCherry colorectal adenocarcinoma cells expressing a fluorescently labeled proteasome subunit with 50, 100, and 200 µM of the hydrogen sulfide donor GYY4137. The effect of the substance on the proteasome pool was assessed 6, 24, 48, and 72 h after administration. It was shown that the chymotrypsin-like and caspase-like proteasome activity decreases in cells incubated with 200 µM of the GYY4137 for 24 h. This coincided with an increase in the expression of proteasome subunit genes. In lysates of cells incubated with 200 µM GYY4137 for 48 h an increase in the content of the constitutive ß5 subunit was observed and the activity of proteasomes leveled off. Following prolonged incubation with GYY4137 (72h), an increase in the expression levels of some proteasome genes was also observed, although this did not have a significant effect on the activity and subunit composition of proteasomes. Thus, the obtained data indicate the modulation of proteasome activity by the hydrogen sulfide donor and the effect of GYY4137 on transcription and translation of proteasome genes.

[Changes in the Activities and Contents of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice].

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer's disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune ß-subunits (by 2.8 folds, ß1i; 2.2 folds, ß2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.

[Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging].

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the ß5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the ß5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit ß5; increased expression of genes encoding immune proteasome subunits ß5i and ß1i. In tissue homogenates of aged mice, an increase in the content of immune subunits ß1i and ß2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the ß5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the ß5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

[Dynamics of the Functional Activity and Expression of Proteasome Subunits during Cellular Adaptation to Heat Shock].

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.

Effect of Neoadjuvant Chemoradiation Therapy on Proteasome Pool in Rectal Cancer.

In untreated rectal cancer patients, the chymotrypsin-like activity of proteasomes in tumor tissue was 3-fold higher than that in conventionally normal tissue, which is explained by up-regulation of expression of immunoproteasomes and total pool of proteasomes. After neoadjuvant chemoradiation therapy, expressions of the total pool of proteasomes and immunoproteasomes in the tumor as well as the relative ratios of these indices to those in conventionally normal tissue were smaller by 1.4-3.3 times in comparison with the untreated patients. These changes were paralleled with pronounced (4.5-fold) down-regulation of proteasome activity in the tumor and a 3.7-fold decrease of activity ratio for the proteasomes in tumor and in conventionally normal tissue. The number of immunoproteasome subunits and the chymotrypsin-like activity of proteasomes can be viewed as potential markers to prognosticate effectiveness of neoadjuvant chemoradiation therapy in rectal cancer patients.

[Diagnostics of thyroid cancer: limitations of the existing methods and perspectives for future developments].

A novel approach to the development of a precise method of intraoperative diagnostics of thyroid cancer has been proposed on the basis of fundamental study of proteasomes in malignant tumors of mammals and human. The method is based on estimation of proteasome activity in small fragments of the tumor and adjacent tissues.

Changes in proteasome chymotrypsin-like activity during the development of human mammary and thyroid carcinomas.

Changes in the proteasome chymotrypsin-like activity in mammary and thyroid carcinomas in comparison with the adjacent tissue were studied at stages T(1-4)N(0-3)M(0) and T(2-3)N(0-1)M(0), respectively. The activities changed in a wave-like manner over the course of mammary carcinoma growth in cases with and without metastases. The minimum increment of the activity in the tumor was recorded during the T(2)N(0) stage in the absence of local metastases. The increment of the activity reached the peak in N(1) tumors of the same size with metastases. The activities in the tumor and adjacent tissues virtually did not differ during the T(3-4)N(1-3) stages. The time course of proteasome activity changes in thyroid tumors of the studied stages was similar to that in mammary carcinoma. The results can be used for development of methods for evaluating the aggressiveness of mammary and thyroid tumors.

[Immune proteasomes in the development of rat immune system].

The dynamics of the expression of LMP7 and LMP2 proteasome subunits in embryonic and early postnatal development of rat spleen and liver is investigated in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The immune subunits LMP7 and LMP2 distribution in spleen and liver cells in the development process is also studied. A mutual for both organs tendency to the increase of the expression of both LMP7 subunit and LMP2 one on P21 (the 21st postnatal day) as compared to the embryonic period is discovered. However, the total proteasome level is shown to be constant. At definite development stages, the dynamics of immune subunits expression in the spleen and liver was different. In the spleen gradual enhancement of both immune subunits level being detected on P1, P18 and P21, in the liver gradual enhancement periods on E16 (the 16th embryonic day) and E18 changed to the stage of the shrink of immune subunits level on P5. This level did not reliably change till P18 and was augmented on P21. The alterations revealed were accompanied by chymotrypsin-like activity raise and caspase-like activity drop in spleen by P21 as compared with the embryonic period, which proves the enlargement of proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased by P21 in comparison with the embryonic period. Such dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Besides, it is discovered that the increment of immune subunits expression in the early spleen development is connected with the process of successive forming the white pulp by B- and T-lymphocytes enriched by immune subunits. In the liver, the growth of immune subunits level by P21 was accompanied by their expression expansion in hepatocytes, while their plunge by P5 may be related to the loss of liver function of a primary lymphoid organ of the immune system by this stage and disappearance of B-lymphocytes enriched by immune proteasomes in it. In the spleen and liver, MHC class I molecules were revealed at the periods of the raise of proteasome immune subunits level. On E21 , the liver was enriched by neuronal NO-synthase, its level decreased after birth and enhanced to P18. This fact indicates the possibility of the induction of the immune subunits LMP7 [character: see text] LMP2 expression in hepatocytes in signal way with neuronal NO-synthase participation. The results obtained prove that T-cell immune response with spleen participation as regards rat liver cells is possible starting with P19-P21 stage. First, at this period, white pulp T-area is formed in the spleen. Second, enhanced immune proteasomes and MHC class I molecules levels in hepatocytes can procure antigenic epitopes formation from foreign proteins and their delivery to cell surface for their subsequent presentation for cytotoxic T-lymphocytes.

[DNA vaccine encoding alpha-fetoprotein with fused ornithine decarboxylase degradation signal significantly suppresses hepatocellular carcinoma growth in mice].

Hepatocellular carcinoma (HCC) is among the most frequent malignancies in humans. HCC therapy is not efficient and the usual outcome is poor. In this regard, novel approaches to treat and prevent HCC are urgently needed. The Alpha-fetoprotein (AFP) is a serological marker of HCC. Recently it has been shown, that the DNA vaccines expressing AFP are capable in generating immune response against AFP. However, both, the immunization procedures and DNA vaccines used before were complex and not always very effective. We have shown that DNA vaccine encoding HIV-1 reverse transcriptase (RT) with fused ornithine decarboxylase (ODC) degradation signal induced a strong Th-1 immune response against RT in mice. Using this approach we designed a set of novel DNA vaccines bearing AFP and ODC degradation signal. Results obtained on transfected cells demonstrated efficient expression and fast proteasomal degradation of the recombinant AFP. The anti-tumor immune response stimulation was shown in immunized animals and most importantly a notable retardation of tumor growth was observed as a result of protective vaccination.

[Changes in the proteasome function after induction of donor-specific tolerance in rats with ovarian allograft].

Induction of donor-specific tolerance in a recipient is one of the methods for enhancing acceptance of the grafts of endocrine glands in the absence of immunodepressants, which interfere with hormone production. This paper describes changes in the proteasome pool in the rat liver, spleen, and graft during the development of donor-specific tolerance after intraportally infusing the recipient with donor splenocytes with subsequent allografting of ovarian tissue into the renal capsule. It has been demonstrated that the shift in the balance in the liver and graft proteasome pools towards the variants with the LMP2 subunit determines the development of immunological tolerance and graft retention. On the contrary, an increase in the forms with the LMP7 subunit induces the immune response and graft rejection.

Changes in the Proteasome Pool during Malignant Transformation of Mouse Liver Cells.

Multiple forms of proteasomes regulate cellular processes by destroying proteins or forming the peptides involved in those processes. Various pathologies, including carcinogenesis, are related to changes in the functioning of the proteasome forms. In this study, we looked at the changes in the pool of liver proteasomes during nodular regenerative hyperplasia and formation of adenoma and hepatocellular carcinoma in mice treated with Dipin, followed by partial liver resection. The relative content of various proteasome forms was determined using Western blot analysis. The chymotrypsin-like activity of proteasomes was assessed from the hydrolysis of the commercial Suc-LLVY-AMC substrate. It was found that changes in the proteasome pool appeared already during the formation of diffuse nodules, the changes being the increased expression of the X(ß5) constitutive subunit and the LMP7(ß5i) and LMP2(ß1i) immune subunits, accompanied by the increase of the total proteasome pool and the decrease in the chymotrypsin-like activity. These changes were more pronounced in hepatocellular carcinoma. The content of the total proteasome pool and the LMP2(ß1i) immune subunit and the chymotrypsin-like activity in adenoma were intermediate compared to those in the samples of liver with diffuse nodules and carcinoma. In addition, the level of the Rpt6 subunit present in the 19S proteasome activator was increased in carcinoma. Our results indicate that nodular regenerative hyperplasia and adenomatosis may be stages preceding carcinogenesis. We also conclude that there is a need to find signalling pathways that change the expression of various proteasome subunits during carcinogenesis. The 19S proteasome activator, which is overexpressed in malignant tumours, can be a promising target for the development of new anticancer drugs.

[Formation of immune proteasomes and development of the immune system in mammalian ontogenesis].

Current concepts of the structure of immune proteasomes and their role in immune response have been considered. The main attention has been paid to the formation of immune proteasomes in secondary lymphoid and nonlymphoid organs during ontogenesis of mammals. The causes of ineffective formation of immune system in early postnatal development have been discussed.

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development.

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.

[Exclusion of immune proteasomes from mouse ascitic carcinoma Krebs-II cells].

Pools of 26S and 20S proteasomes were studied in the spleen, liver, lung, and ascitic carcinoma Krebs-II of mouse. Western blotting demonstrated that the pool of 26S proteasomes in ascitic carcinoma Krebs-II was twice that in control lung cells and did not significantly differ by total 26S proteasome quantities from the spleen and liver. At the same time, the level of immune subunit LMP7 was 12 times lower in it compared to lung proteasomes and 4-5 times lower compared to spleen and liver proteasomes. Immune subunit LMP2 was undetectable by this technique in the ascitic carcinoma in contrast to the lung, spleen, and liver. All immune subunits in the studied organs and ascitic carcinoma Krebs-II are components of 26S but not 20S proteasomes.

[Changes in proteasome activity and subunit composition during postnatal development of rat].

The dynamics of the activities of 26S and 20S proteasomes in the rat liver and spleen have been studied during postnatal development from 1 to 90 days. The activities of proteasome forms both in spleen and in liver increased in adult animals as compared to one day rats. The activities of both proteasome forms in the liver did not differ significantly from those in the spleen at all stages of postnatal development. Using Western blot with monoclonal antibodies to Rpt6 subunit, we confirmed the presence of 26S proteasome in both organs at all stages of postnatal development. Studies with polyclonal antibodies to beta1i (LMP2) subunit showed the appearance of the immune subunit in the spleen by day 9 and in the liver only by day 23 of postnatal development. This result suggests the earlier formation of the spleen as an organ with immune functions.

[Multiple forms of the proteasomes and some approaches to their separation]. / Mnozhestvennost' form proteasomy i nekotorye podkhody k ikh razdeleniiu.

A technique for proteasome isolation from rat liver was developed; it allows isolation of 26S proteasome fraction free from 20S proteasome admixture. The technique includes consecutive stages of protein fractionation by different concentrations of ammonium sulfate, gel filtration on Sepharose 2B, and high-speed ultracentrifugation.

[Sex hormone receptors, prostaglandins and cAMP, in functional activity of the uterus]. / Rol retseptorov polovykh gormonov, prostaglandinov i TsAMF v funktsionalnoi aktivnosti matki.

The levels of progesterone and estrogen receptors, cAMP and prostaglandins E2 and F2 alpha were studied in the decidua of pregnant women, which was obtained during surgical, spontaneous or prostaglandin-induced abortion or by pregnancy termination with vacuum aspiration after RU-486 treatment (before the onset of abortion). Our data have allowed the authors to suggest that (i) for the maintenance of normal activity of decidua during pregnancy, the following points are considerably important: the constant ratio of progesterone and estrogen receptors in favor of progesterone receptors, low PG levels and high cAMP levels; (ii) both spontaneous and induced abortion are characterized by decreases in the levels of progesterone receptor (which in turn changes the ratio of progesterone and estrogen receptors in favor of the latter), by increased PG levels and decreasing cAMP concentrations.

[Prostaglandin content in decidual tissue of women in spontaneous abortion]. / Soderzhanie prostaglandinov v detsidual'noi tkani zhenshchin pri samoproizvol'nom aborte.

Prostaglandins E2 and F2 alpha were measured in decidual tissue of women after spontaneous abortions at pregnancy terms 8 to 10 weeks and after abortions induced by sulprostone, a synthetic prostaglandin E2 analog, at pregnancy term 4-5 weeks. Women in whom pregnancy was terminated surgically at relevant periods were controls. Prostaglandin E2 concentration was found to be 5 times higher and that of prostaglandin F2 alpha 40 times higher in women with spontaneous abortions than in surgical abortion. In sulprostone-induced abortion prostaglandin concentrations in decidual tissue were the same as in controls. These data indicate that a drastic increase of prostaglandin levels in spontaneous abortion is not only a result of myometrial contractions or decidual tissue rejection, but an important component in the chain of events leading to miscarriage.

The effect of RU486 on progesterone and oestrogen receptor concentration in human decidua on early pregnancy.

Concentrations of progesterone receptor (PR) and oestrogen receptor (ER) were measured by radioligand assay in decidual tissue of women undergoing termination of early pregnancy (amenorrhoea up to 49 days). Pregnancies were terminated by vacuum aspiration at 12 or 36 h after oral administration of placebo or antiprogestin RU486 in different doses. Treatment with RU486 decreased decidual PR content, the effect being observed at 12 h as well as at 36 h after 600 mg RU486 and at 36 h after 3 x 25 mg RU486 given at 12 h intervals. PR concentration 12 h after a single dose of 25 mg RU486 was not affected. ER content was unchanged at 12 h after RU486 but increased 36 h after 600 mg and 3 x 25 mg RU486. Our data suggest that apart from blocking progesterone action, RU486 may exert its abortifacient effect through decreasing the PR concentration. The simultaneous decrease of PR concentration and an increase of ER concentration changes the balance between them in favour of ER, which might also play a role in the abortifacient effect of RU486.