Bid-induced release of AIF from mitochondria causes immediate neuronal cell death.

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.

Targeted gene therapy for breast cancer with truncated Bid.

We studied the efficiency of the proapoptotic factor tBid, targeted to tumor cells using the promoters of the hTERT, Survivin and Muc1 genes, in killing breast cancer cells. tBid is the active fragment of the proapoptotic protein Bid and is generated in response to death receptor activation. When placed under control of a strong CMV promoter, tBid was highly efficient in killing breast cancer cells. When expression of tBid was driven by tumor-specific promoters, the magnitude of killing was significant in cell lines with high levels of promoter activity. For successful gene therapy with targeted tBid, it is therefore crucial to be able to predict promoter activity prior to selection of the therapeutic construct. To test whether gene expression could serve as a predictor, we correlated expression of Survivin, hTERT and Muc1 genes with the activity of the corresponding promoters in a panel of breast cancer cell lines. Expression of the Muc1 gene correlated well with the activity of its promoter and the resultant tumor cell killing. For the hTERT and Survivin promoters, however, promoter activity did not correlate well with the expression of the corresponding genes. The implications and possible mechanism of these discrepancies are discussed.

Loosening of cell cycle controls of human lymphocytes under the action of tumour promoter TPA.

The effect of tumour promoter TPA (12-O-tetradecanoylphorbol-13-acetate) on the cell cycle of human peripheral blood lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro was studied and it was found that TPA caused cells to accumulate in all the cell cycle phases. This accumulation took place preferentially at later culture passages, when lymphocytes stimulated by PHA alone stopped mainly in G0/G1 phases. Other effects of TPA were cell induction to enter higher DNA ploidy and to survive and even synthesize DNA under colchicine block of mitosis or under cytochalasin block of cytokinesis. In addition, in experiments in which a transitory block through the G1 phase of cell cycle was applied with use of aminopterin, we could show that a fraction of TPA-treated cells still entered the active phase of DNA synthesis. These findings suggest that TPA causes cell cycle controls to become loose, thereby enhancing adaptability of human lymphocytes to various hindrances in the course of cell cycle and eventually causing them to acquire characteristics known to be common for tumour cells.

Importance of 1,25-dihydroxyvitamin D3 and the nonadherent cells of marrow for osteoblast differentiation from rat marrow stromal cells.

Although steroid hormones regulate mature osteoblast function, much less is known about their actions on osteoprogenitor cells. The possibility of steroid hormone regulation of early stages in osteoblast differentiation was investigated by measuring the growth and induction of the osteoblast marker enzyme alkaline phosphatase (AP) in rat bone marrow stromal cell cultures. Experiments were performed in charcoal-stripped serum; conditions which markedly impaired stromal cell growth. However, growth could be stimulated by nonadherent marrow cell-derived conditioned medium. 1,25(OH)2D3, but not dexamethasone, 17 beta-estradiol, or retinoic acid, increased both stromal cell proliferation and AP activity. The increased proliferation with 1,25(OH)2D3 was nonadherent cell-dependent. BMP-2 also increased AP levels and acted in synergy with 1,25(OH)2D3. These results suggest that (i) nonadherent marrow cells may support stromal cell development, and (ii) 1,25(OH)2D3 as well as glucocorticoids may regulate osteogenesis from the bone marrow but a similar role for estrogen is not supported.

[Possible mechanisms regulating the entry of normal and neoplastic cells into the DNA synthesis phase]. / Vozmozhnye mekhanizmy reguliatsii vstupleniia normal'nykh i neoplasticheskikh kletok v fazu sinteza DNK.

Literature data concerning some aspects in the regulation of the entry of normal and neoplastic cells into the phase of DNA synthesis are reviewed. Basing on the analysis of data reported by different authors, a hypothetical model is suggested for regulation of cell passing from G- to S-period. The main chain in the regulation is the constitutive synthesis of protein-repressor which blocks the expression of genes whose products are necessary for replication ("replication genes"). The repressor inactivation is achieved by growth factors through protein kinases. A comparison is made between the transcriptional mechanisms in eukaryotic and prokaryotic cells. Four hypothetic types of genes have been isolated responsible for regulation of progression of the cell to phase S. The role of quantitative and/or qualitative changes in the products of these genes in neoplastic transformation is discussed.

[Induction of the traits of the differentiated T-cell phenotype in the blast cells of patients with acute lymphoblastic leukemia. I. The expression of T-cell markers as affected by different inducers]. / Induktsiia nekotorykh priznakov differentsirovannogo T-kletochnogo fenotipa u blastnykh kletok bol'nykh ostrym limfoblastnym leikozom. I. Ekspressiia T-kletochnykh markerov pod vliianiem razlichnykh induktorov.

The expression of T-lymphocyte markers has been demonstrated by blast cells of patients with non-T, non-B and pre-T-cell acute lymphoblastic leukaemia (ALL). The main inducing agent is phytohemagglutinin (PHA). Its action was enhanced when combined with phorbol ether (TPA) or ouabain. Dimethylsulfoxide, ouabain and concanavalin A had no similar inducing effect. TPA caused the expression of some T-cell markers revealed by monoclonal antibodies but had only a little inducing effect to E-receptor.

[Induction of the traits of the T-cell differentiated phenotype in a blast cell population from patients with acute lymphoblastic leukemia. II. Parallel changes in the antigenic characteristics and functional properties of blast cells as affected by inducers]. / Induktsiia nekotorykh priznakov differentsirovannogo T-kletochnogo fenotipa v populiatsii blastnykh kletok bol'nykh ostrym limfoblastnym leikozom. II. Parallel'nye izmeneniia antigennykh kharakteristik i nekotorykh funktsional'nykh svoistv blastnykh kletok pod vliianiem induktorov.

Data are provided which allow to assume that the appearance of T-cell markers on blast cells of patients with non T-, non B- and pre-T-cell acute lymphoblastic leukaemia may reflect the process of differentiation in leukaemic lymphoblasts in T-cells under the action of phytohaemagglutinin (PHA) combined with phorbol ether (TPA) or ouabain. As result of the induction, in the lymphoblast population the number of cells carrying the markers of non-differentiated phenotype decreased, which was proportional to the increase in the number of cells with differentiated T-lymphocytic phenotype. The expression of T-cell markers was suppressed by the inhibitors of transcription and protein synthesis. As a result of induction the leukaemic cells acquire the ability to form T-cell colonies in semisolid agar which reflects their functional changes under the action of inducers. Possible mechanisms of PHA inducing action are discussed.

[Initial changes in ouabain-dependent and ouabain-resistant potassium fluxes in human peripheral blood lymphocytes exposed to phytohemagglutinin]. / Nachal'nye izmeneniia uabainzavisimykh i uabainrezistentnykh potokov kaliia u limfotsitov perifericheskoi krovi cheloveka pri deistvii fitogemaggliutinina.

The early changes in potassium fluxes in PHA-treated human lymphocytes were studied. The increase in both ouabain-sensitive potassium influx and ouabain-resistant potassium efflux at the optimal mitogenic concentration of PHA was observed. No change in potassium content was found.

[Effect of the tumor promoter TPA on the distribution of PHA-stimulated human lymphocytes by cell cycle phases]. / Vliianie opukholevogo promotora TFA na raspredelenie stimulirovannykh FGA limfotsitov chelovkea po fazam keltochnogo tsikla.

Patterns of the cell cycle distribution in human peripheral blood lymphocytes, stimulated by PHA alone and PHA plus 12-o-tetradecanoylphorbol-13-acetate (TPA), were studied using DNA cytometry in different times after PHA stimulation. In the first period (nearly 3 days after PHA stimulation) TPA induces no significant differences in the characters under consideration, but in the later period, when the proliferation of the cultures stimulated by PHA alone is reducing, in other cultures stimulated by PHA plus TPA the percentage of cells in S-phase does not reduce, whereas the percentage of cells in G2-phase is rising, which may suggest that this phase is blocked. Concurrently the tetraploid cells are appearing. Accumulation of cells in G2-phase can be overcome by the application of chlorpromazine, which is known to inhibit the membrane-associated protein kinase C.

[Changes in the content of potassium, sodium and potassium influx in human peripheral blood lymphocytes during long-term cultivation with phytohemagglutinin]. / Izmenenie soderzhaniia kaliia, natriia i vkhodnykh potokov kaliia v limfotsitakh perifericheskoi krovi cheloveka pri dlitel'nom kul'tivirovaniia s fitogemaggliutininom.

Intracellular potassium, sodium and potassium influx were examined in PHA-activated human lymphocytes within 6 days of cultivation. DNA flow cytometry was used to estimate the percentage of cells in G1, S and G2 + M phases. Potassium influx and content per g protein were found to be increased, whereas sodium content decreased with the progression of cells from G1 to S phases, being maximum on the 3rd day. Later on the percentage of cells in S phase was seen diminished, and the potassium content decreased just as sodium content increased. It is concluded that ionic changes may correlate with the entering of cells into S phase.

[Effect of mitogenic stimulation and heat shock on protein syntheses in human T cells]. / Vliianie mitogennoi stimuliatsii i teplovogo shoka na belkovye sintezy v T-kletkakh cheloveka.

Effects of moderate (42 degrees C, 1 hour) and strong (44 degrees C, 1 hour) heat shocks on resting (TR) and phytohemagglutinin stimulated human T-cells (TP) were studied. Both treatments were shown to cause in the latter considerable fall of the level of protein synthesis, as compared to resting cells. Mitogen-stimulated cells stopped their proliferation irreversibly and part of them (approx: 40%) died after even mild shock (at 42 degrees C). Following heat treatment in both the cell types the synthesis of heat shock 70 and 90 kDa proteins was induced which was much more pronounced in TR. These and earlier results of the authors allow a conclusion that involvement of cells in active proliferation may decrease their resistance to stress, and that this phenomenon coincides with the diminishing in synthesis and accumulation of stress proteins.

[Effect of interleukin 2 on blast proliferative activity and expression of T-cell markers in patients with acute lymphoblastic leukemia]. / Vliianie interleikina-a na proliferativnuiu aktivnost' blastov i ékspressiiu nekotorykh T-kletochnykh markerov u bol'nykh ostrym limfoblastnym leikozom.

The proliferative activity of blast cells from patients with acute lymphoblastic leukemia was compared with the activity of lymphoid cells of the thymus and blood of healthy subjects. The spontaneous proliferative activity in cells obtained extempore varied widely. On the basis of this index groups of patients with high proliferative activity (group 1) exceeding the level of thymocyte proliferation and with low proliferative activity (group 2) were distinguished. Direct correlation was revealed between the initial spontaneous proliferative activity and the number of leukocytes as well as with the absolute number of blasts in the blood. A decrease in the initial high proliferative activity in the time course of cultivation was noted in some patients of group 1 (group 1B). In the majority of these patients (91%) the administration of interleukin-2 into the cultural medium prevented a decrease in the proliferative activity. In the other patients of group 1 (group 1A) a high spontaneous proliferative activity was not decreased during cultivation. In group 1A a proliferative response to T-cell growth factor was observed in 36% of patients. The patients' blast cells in group 2 did not respond to this lymphokine. The expression de novo of T-cell markers was observed in some patients of group 1 as a result of interleukin-2 action. The role of interleukin-2 in the pathogenesis of acute lymphoblastic leukemia is discussed.

[Study of the characteristics of various hematologic diseases using monoclonal antibodies LT1, LT2 and LT7]. / Kharakteristika nekotorykh gematologicheskikh zabolevanii s pomoshch'iu monoklonal'nykh antitel LT-1, LT-2, LT-7.

Monoclonal antibodies (MCA) LT1, LT2 and LT7 were characterized. It was established that MCA LT1 and LT2 (IgG1 and IgM classes, respectively) interacted with antigen 67 kD, and MCA LT7 (IgGZa)--with antigen 40 kD. MCA LT1, LT2 and LT7 were shown to identify normal and neoplastic cells of T-lymphoid type. MCA LT1 and LT2 also reacted with B-lymphocytes of chronic lymphocytic leukemic patients. MCA LT1 and LT2 were referred to CD5 (Cluster of Differentiation) and MCA LT7--to CD7 by the molecular weight of an identified antigen and the spectrum of its expression. The importance of MCA studied in the diagnosis of subtypes of acute and chronic lymphocytic leukemia has been confirmed.

HLH transcription factor activity in osteogenic cells.

To examine possible mechanisms underlying osteoblast differentiation from mesenchymal stem cells, we investigated bHLH functional activity in cell lines representing different stages of osteoblast maturation. Interaction of nuclear proteins with oligonucleotides corresponding to various bHLH binding sequences (known as E-boxes) was determined in mobility shift assays. Both ADD-1 oligonucleotide, a binding site for transcription factor ADD-1, and OCE-1, an E-box from osteocalcin promoter, produced retarded bands after incubation with nuclear extracts from osteogenic cells. Cells at different stages of osteogenic maturation demonstrated similar patterns and intensity of binding, as did cells treated with different osteogenic inducers. Binding to ADD-1 and OCE-1 was not tissue-specific as it was also observed in fibroblastic 10T1/2 cells. MEF-1 oligonucleotide, the E-box sequence from the muscle creatine kinase enhancer, demonstrated no changes in binding with nuclear extracts from moderately differentiated (W-20) or relatively mature (ROS 17/2.8) cells under any conditions tested. However, in poorly differentiated RI-2J cells, which do not express osteogenic markers unless treated with dexamethasone, induction of differentiation was reflected in transient inhibition of binding to MEF-1. Inhibition of binding was not seen under differentiation-restrictive conditions. Promoter-reporter studies also demonstrated inhibition of MEF-1 driven CAT expression by dexamethasone under differentiation-permissive conditions in RI-2J cells. These data suggest that bHLH gene expression is not required for the early steps of osteogenesis; moreover, inhibition of bHLH protein binding to a MEF1-type E box might be an integral part of osteogenic commitment.

Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.

Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with 10(-8) M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well as osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10(-8) M 1,25-dihydroxyvitamin D3 (vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.