[Ontogenetic aspects of changing lysozyme activity under acute somatic pain].

OBJECTIVE: Ouraim was to study influence of acute somatic pain on lysozyme activity of rats of different age: newborn, rats after eye opening, rats at the age of a month, adult and old rats. METHODS: Lysozyme activity was checked before pain irritation and 2, 30, 60, 120, 180min afterwards using Dorofeychuk's method in our modification. Pain effect was modeling by electrical stimulation. RESULTS: activity of lysozyme was 0. 434±0. 01 units in intact newborn rats, It was higher than in adult rats - 0.260 ±0.01 units (p <0.001) and it was unchanged during the experiment. We found low lysozyme activity in rats after eye opening - 0.015±0.003 units and it was stable during the experiment. Rats at the age ofa month had diphasic reaction: lysozyme activity was 0.191±0.01 units in intact rats, it increased up to 0.378±0.01 units (p <0.001) in 2 min after painful irritation and it decreased up to 0.113±0.02 units (p <0.001) in 30 min. Lysozyme activity was 0.260±0.01 units. Single-phase reaction was determined after acute painful irritation: increase of lysozyme activity after acute somatic pain up to 0,450±0,014 units (p <0.001). Lysozyme activity was 0.246±0.02 units in blood plasma of old rats. It decreased up to 0.1701±0.01 units (p <0.01) after painful irritation and it was 0.387±0.01 (p <0.001) in the end of the experiment. CONCLUSION: Response on pain irritation has differences in different groups. The common vector of pain response was the increase of lysozyme activity in rats at the age of a month, adult rats, rats after eye opening and old rats. Reaction of increasing lysozyme activity was strongly detected in adult rats. The results demonstrate preventive lasozyme resistance to potential tissue damage or contamination.

[The role of different kinase pathways of signal transduction in proliferation of E1A + Ras transformants].

In this paper we have explored the role of different kinase pathways of signal transduction in proliferation control of E1A + Ras transformants, using specific inhibitors of MAP-kinases ERK, JNK, p38 and PI3-kinase. According to our data, suppression of signalling cascades driven by RI3K only arrested proliferation of E1A + Ras cells, while suppression of either MAP-kinase did not lead to noticeable antiproliferative effect. We have shown that suppression of RI3K with LY294002 gave rise to accumulation of cyclin-dependent kinase inhibitor p27(KiP1) but not p21(Waf1). Accumulation of p27(KiP1) in LY294002-treated E1A + Ras cells was accompanied by a decrease in Cyclin E-Cdk2 and Cyclin A-Cdk2 activity, which caused diminution of Rb phosphorylation and strengthening of E2F-Rb binding. Binding of E2F with hypophosphorylated Rb resulted in inhibition of E2F activity and reduction of E2F-regulated gene transcription, these genes being necessary for S-phase entry and DNA synthesis. Thus, RI3K--Akt cascade plays the key role in maintenance of autonomous proliferation of cells transformed with E1A and cHa-ras oncogenes. Inhibition of PI3K leads to p27(Kip1) accumulation and cell cycle arrest, consequently.

[Induction of premature senescence program by an inhibitor of histone deacetylase sodium butyrate in normal and transformed rat fibroblasts].

We investigated a possibility to induce the premature cell senescence in rat embryo fibroblasts and E1A + cHa-ras transformants. We found that after the treatment with sodium butyrate, an inhibitor of histone deacetylases, both normal and transformed cells completely stopped to proliferate and accumulated at G1/S and G2/M phases of the cell cycle. The cloning efficiency data show that the cell cycle arrest induced by sodium butyrate is irreversible and correlates with the accumulation of active phosphorylated form of stress kinase p38, and with the expression of marker of senescence--beta-galactosidase activity (SA beta-Gal). The program resembling the premature senescence after sodium butyrate treatment is supposed to develop both in normal and transformed cells. The irreversible block of proliferation in E1A + cHa-ras transformants may be regarded as an example of activation of anticancer program like that of premature senescence in the tumor rodent cells.

[Role of the TCF phosphorylation state and the chromatin structure in the negative transcription regulation of the c-fos proto-oncogene in E1A + c-Ha-ras transformed cells]. / Rol' statusa fosforilirovaniia belkov TCF i struktury khromatina v negativnoi reguliatsii transkriptsii protoonkogena c-fos v transformirovannykh kletkakh E1A + cHa-ras.

As compared with normal rat embryo fibroblasts (REF), c-fos transcription is suppressed in REF transformed with the E1A and cHa-ras complementing oncogenes. Negative regulation of the fos promoter is due to the serum-responsive element (SRE) constantly bound with the serum response factor (SRF) and with the ternary complex factor (TCF), which form the SRF/SRF/TCF complex. Possible mechanisms of the c-fos suppression were studied, including changes in the TCF content and phosphorylation and replacement of TCF by repressor proteins. In addition, the transcription factors were tested for DNA-binding activity, and c-fos transcription was analyzed by RT-PCR in various cell culture conditions and in the presence of phosphatase and histone deacetylase inhibitors. The c-fos suppression in transformed REF was explained by formation of a transcriptionally inactive chromatin structure in the fos promoter region as a result of histone deacetylation, rather than by insufficient TCF phosphorylation.

[Histone deacetylase inhibitor blocks proliferation of cells transformed with oncogenes E1A and cHa-ras]. / Ingibitor deatsetilaz gistonov ostanavlivaet proliferatsiiu kletok, transformirovannykh onkogenami E1A i cHa-ras.

Rat embryonic fibroblasts, transformed with E1A and cHa-ras oncogenes, are unable to stop in the cell cycle checkpoints under growth factor withdrawal and genotoxic stresses (Bulavin et al., 1999). In the present paper, we showed that sodium butyrate, an inhibitor of histone deacetyase activity, decreased the share of cells being in S-phase, and caused G1/S and G2/M blocks of the cell cycle in the transformants. By means of RT-PCR and immunoblotting, we found that NaB significantly changed the expression of genes involved in proliferation: cyclins D1, A, E and cyclin-dependent kinases Cdk2 and Cdk4, whereas the amount of p21Waf1 and p27Kip1 inhibitors greatly increased. Along with accumulation of p21Waf1 protein content, that of Cdk2-bound p21 increases. Taken together, these data allow to suggest that NaB treatment does evidently restore the capability of p21Waf1 to inhibit cyclin-kinase activity. One may suppose that inhibition of HDAC activity by sodium butyrate leads to activation of yet unknown HDAC-dependent genes, which is followed by restoration of p21Waf1 function in spite of the E1A oncogene expression.

[Constitutive activity of MAP kinase cascades in REF cells transformed by E1A and cHa-ras oncogenes]. / Konstitutivnaia aktivnost' MAP-kinaznykh kaskadov v kletkakh REF, transformirovannykh onkogenami E1A i cHa-ras.

Proteins of Ras family play an important role in regulation of cell growth and proliferation, and their mutations can lead to growth factor-independent proliferation due to constitutive activity of various signal transduction cascades. In the present work, we studied the activity of ERK, JNK and p38 MAP-kinase cascades in rat embryo fibroblast cells transformed with oncogenes E1A and cHa-ras. These transformed cells are characterized by a high and non-regulated activity of transcription factor AP-1 involved in the regulation of cell proliferation. Since phosphorylation of AP-1 depends on the activity of relevant MAP-kinase cascades (ERK, JNK and p38), we analysed the expression of non-phosphorylated forms of the kinases and their phosphorylated state in E1A + cHa-ras cells using antibodies specific to non-phosphorylated and phosphorylated proteins. It has been established that transformed cells contain higher amounts of non-phosphorylated ERK, JNK and p38 kinases, thus implying a reduced degradation of these and other proteins in the transformants. The content of phosphorylated (active) forms studied in Western blot-analysis with phosphoantibodies was shown to be also higher in exponentially growing E1A + cHa-ras cells. But serum stimulation of the starved cells gave insignificant rise to an increase of ERK, JNK and p38 phosphorylation. Nevertheless, an in vitro kinase assay performed with the kinases, either immunoprecipitated by antibody or bound to GST-fusion substrates, enabled us to show a certain level of stimulation of c-Jun-associated (JNK) and MEF2A-associated (p38) kinase activity in serum stimulated E1A + cHa-ras cells. Thus, the obtained results show that transformation of fibroblasts with E1A and ras oncogenes may contribute to constitutive activation of ERK, JNK and p38 kinase cascades responsible for a high and non-regulated activity of MAP-kinase-dependent transcription factors, in particular AP-1.

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells.

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α-/- cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α-/- cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α-/- cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.

HDAC inhibitor-induced activation of NF-κB prevents apoptotic response of E1A+Ras-transformed cells to proapoptotic stimuli.

HDAC inhibitors (HDACIs) are capable of suppressing the cell growth of tumour cells due to the induction of apoptosis and/or cell cycle arrest. This allows of considering HDACIs as promising agents for tumour therapy. The final outcome - apoptotic cell death or cell cycle arrest - depends on the type of tumour and cellular context. In this report, we addressed the issue by analysing effects produced in E1A+Ras-transformed MEF cells by HDAC inhibitors sodium butyrate (NaB), Trichostatin A (TSA) and some others. It has been shown that the HDACIs induced cell cycle arrest in E1A+Ras-transformed cells but not apoptosis. The antiapoptotic effect of HDACIs is likely to be a result of NF-κB-dependent signaling pathway activation. HDACI-induced activation of NF-κB takes place in spite of a deregulated PI3K/Akt pathway in E1A+Ras cells, suggesting an alternative mechanism for the activation of NF-κB based on acetylation. HDACI-dependent activation of NF-κB prevents the induction of apoptosis by cytostatic agent adriamycin and serum deprivation. Accordingly, suppression of NF-κB activity in HDACI-arrested cells by the chemical inhibitor CAPE or RelA-siRNA resulted in the induction of an apoptotic programme. Thus, our findings suggest that the activation of the NF-κB pathway in HDACI-treated E1A+Ras-transformed cells blocks apoptosis and may thereby play a role in triggering the programme of cell cycle arrest and cellular senescence.

p21(Waf1) is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate.

Cell senescence is characterized by senescent morphology and permanent loss of proliferative potential. HDAC inhibitors (HDACI) induce senescence and/or apoptosis in many types of tumor cells. Here, we studied the role of cyclin-kinase inhibitor p21(waf1) (Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-ßGal staining and accumulation of γH2AX foci) in p21(Waf1+/+) versus p21(Waf1-/-) mouse embryonic fibroblast cells transformed with E1A and cHa-Ras oncogenes (mERas). While short treatment with the HDACI sodium butyrate (NaB) induced a reversible G(1) cell cycle arrest in both parental and p21(Waf1-/-) cells, long-term treatment led to dramatic changes in p21(Waf1+/+) cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-ßGal-positive and accumulated γH2AX foci associated with mTORC1 activation. The p21(Waf1+/+) cells lost their ability to migrate into the wound and through a porous membrane. Suppression of migration was accompanied by accumulation of vinculin-staining focal adhesions and Ser3-phosphorylation of cofilin, incapable for F-actin depolymerization. In contrast, the knockout of the p21(Waf1) abolished most of the features of NaB-induced senescence, including irreversibility of cell cycle arrest, hypertrophy, additional focal adhesions and block of migration, γH2AX foci accumulation and SA-ßGal staining. Rapamycin, a specific inhibitor of mTORC1 kinase, decreased cellular hypertrophy, canceled coffilin phosphorylation and partially restored cell migration in p21(Waf1+/+) cells. Taken together, our data indicate a new role of p21(Waf1) in cell senescence, which may be connected not only with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.

Selective repression of c-fos gene transcription in rat embryo fibroblasts transformed by oncogenes E1A and cHa-ras.

Transformation of REF cells by oncogenes E1A and cHa-ras leads to activation of AP-1 factor concomitantly with down-regulation of c-fos gene transcription. Here we addressed two issues: (i) how does transcription of Fos/Jun-regulated genes change in the cells lacking Fos-Jun heterodimers; (ii) to which extent HDAC-mediated chromatin reorganization does affect, apart from c-fos, transcription of some other early and late-response genes. To this end, we studied the kinetics of serum-stimulated transcription of c-fos, c-jun, fra-1, egr-1, and cyclinD1 genes, as well as the effects of sodium butyrate, an inhibitor of histone deacetylase activity, on transcription of these genes in normal REF cells and transformants E1A+ras.