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Introduction: Genistein (plant isoflavone) is a well-known anti-cancer drug with estrogenic-like properties. Genistein also regulates sugar and lipid metabolism; thus, it has anti-diabetic properties. The aim of the study was to evaluate in vitro effects of genistein on glucose transport, fatty acids oxidation, activation of PKB, and expression of genes related to insulin pathway in differentiated myoblast C2C12 mouse cell line., Material and Methods: Differentiated myoblast C2C12 mouse cell line was used to assess the effects of different genistein concentrations on glucose transport and fatty acids oxidation measured by radioactivity technique, activation of PKB, and expression of selected genes related to insulin signaling pathway (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A, SH-PTP2) at the mRNA and protein levels. Cells were incubated with various concentrations of genistein under standard conditions for 0-48 hours., Results: Genistein in low concentrations (0.1-1 μM) significantly increased glucose transport and decreased fatty acids oxidation in C2C12 cells after 48 h of incubation. High concentration of genistein (50 μM) had the opposite effect. Genistein stimulated PKB phosphorylation during the first 5-10 minutes of incubation. There was no significant impact on the protein expression of selected genes (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A-Ca, ER-a and ER-b) after 48 h treatment. We observed inverse correlation between genistein concentration and the expression of SH-PTP2 protein. Genistein affected the expression pattern of mRNAs for genes related to the insulin pathway, however, not the expression of the encoded proteins., Conclusions: The results of this study showed that depending on the concentration and time of incubation genistein significantly affects glucose and lipid metabolism and at low concentration modifies expression pattern of a few genes in C2C12 cells.

The translationally controlled tumor protein (TCTP) plays a role in cell growth, cell cycle and cancer progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,a key regulator of cell cycle, controls actin organization and negatively regulates cell motility via regulation of RhoA expression. We studied the organization of actin and cytokeratin cytoskeleton and the expression of TCTP, p53,cyclin A, RhoA and actin in HIO180 non-transformed ovarian epithelial cells, and OVCAR3 and SKOV3 (expressing low level of inducible p53) ovarian epithelial cancer cells with different metastatic potential. Immunostaining and ultrastructural analyses illustrated a dramatic difference in the organization of the cytokeratin and actin filaments in non-transformed versus cancer cell lines. We also determined that there is an inverse relationship between the level of TCTP/RhoA and actin/p53/cyclin A expression in ovarian cancer cell lines. This previously unidentified negative relationship between TCTP/RhoA and actin/p53/cyclin A may suggest that this interaction is linked with the high aggressiveness of ovarian cancers.

MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells, and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular, vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division.

The understanding of cell cycle regulation benefited greatly from omic approaches. Because the cell cycle engine relies heavily on proteins, proteomic methods play a key role in identification of cell cycle players. The proteomic approach delivers an enormous volume of data, but it often lacks comprehensiveness. To ensure the comprehensiveness of results the discovery of novel proteins must be followed by functional analysis. Using Xenopus laevis oocytes in two different proteomic screens, we have recently identified a number of proteins whose behavior suggested specific and unexpected roles in M-phase entry. Functional analysis of EP45 identified in one of these screens has shown that M-phase entry is stimulated by Oocyte-Maturation-ENhancer ('Omen') activity. The second screen suggests the presence of an antagonistic activity, which we call 'Omre' (Oocyte- -Maturation-REpressor). The equilibrium between Omen and Omre activities may determine the quality of oocytes and further embryo development via participation in making the decision whether to enter oocyte maturation. It remains an open question whether similar activities operate during mitotic divisions in embryonic and adult cells. Identifying such activities in somatic cells might impact on cancer treatments.

Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis), the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein), a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit). We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of other candidates is in progress.