Dora Višnjić - Academia.edu (original) (raw)

Papers by Dora Višnjić

Research paper thumbnail of deficiency Hematopoiesis is severely altered in mice with an induced osteoblast

Research paper thumbnail of Inositol pyrophosphates modulate cell cycle independently of alteration in telomere length

Advances in biological regulation, Jan 26, 2015

Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the si... more Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the signalling response required for cell cycle progression after mating pheromone arrest. Overexpression of Kcs1 doubled the level of inositol pyrophosphates when compared to wild type cells and 30 min following the release from α-factor block further increase in inositol pyrophosphates was observed, which resulted that cells overexpressing Kcs1 reached G2/M phase earlier than wild type cells. Similar effect was observed in ipk1Δ cells, which are unable to synthesize IP6-derived inositol pyrophosphates (IP7 and IP8) but will synthesize IP5-derived inositol pyrophosphates (PP-IP4 and (PP)2-IP3). Although ipk1Δ cells have shorter telomeres than wild type cells, overexpression of Kcs1 in both strains have similar effect on cell cycle progression. As it is known that PP-IP4 regulates telomere length through Tel1, inositol polyphosphates, cell cycle and telomere length were determined in tel1Δ cel...

Research paper thumbnail of The mechanism of synergistic effects of arsenic trioxide and rapamycin in acute myeloid leukemia cell lines lacking typical t(15;17) translocation

International journal of hematology, Jan 11, 2015

Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promy... more Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promyelocytic leukemia (APL), but is much less efficacious in acute myeloid leukemia (AML) lacking t(15;17) translocation. Recent studies have indicated that the addition of mammalian target of rapamycin (mTOR) inhibitors may increase the sensitivity of malignant cells to ATO. The aim of the present study was to test for possible synergistic effects of ATO and rapamycin at therapeutically achievable doses in non-APL AML cells. In HL-60 and U937 cell lines, the inhibitory effects of low concentrations of ATO and rapamycin were synergistic and more pronounced in U937 cells. The combination of drugs increased apoptosis in HL-60 cells and increased the percentage of cells in G0/G1 phase in both cell lines. In U937 cells, rapamycin alone increased the activity of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the addition of ATO decreased the level of phospho...

Research paper thumbnail of Combined inhibition of PI3K and mTOR exerts synergistic antiproliferative effect, but diminishes differentiative properties of rapamycin in acute myeloid leukemia cells

Pathology oncology research : POR, 2011

A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining m... more A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining mammalian target of rapamycin (mTOR)-inhibitors with an inhibitor of the phosphatydilinositol 3-kinase PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. However, recent study demonstrated the potentiating effect of rapamycin on all-trans-retinoic acid (ATRA)-mediated differentiation of acute myelogenous leukemia (AML) cells, prompting us to investigate the effects of longitudinal inhibition of PI3K/Akt/mTOR signaling pathway on both proliferation and differentiative capacity of AML. In NB4, HL-60, U937 and K562 cell lines, rapamycin exerted minimal antiproliferative effects, and combining PI3K inhibitor LY 294002 and rapamycin inhibited proliferation more than LY 294002 alone. Rapamycin potentiated differentiation of ATRA-treated NB4 cells, but the combination of rapamycin and LY 294002 inhibited the expression of CD11b in both ATRA- an...

Research paper thumbnail of Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells

Biochimica et biophysica acta, Jan 20, 2003

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 ... more The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-...

Research paper thumbnail of Phorbol 12-myristate 13-acetate-mediated signalling in murine bone marrow cells

The Biochemical journal, Jan 15, 1995

Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the abi... more Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the ability of murine (CBA) bone marrow cells to form colonies in vitro. Treatment of marrow cells with PMA did not influence the 1,2-diacylglycerol or cyclic AMP concentrations, the intracellular Ca2+ concentration or phospholipase D activity. PMA increased particulate phospholipase A2 (PLA2) activity, lysophosphatidylcholine formation and arachidonic acid release from bone marrow cells; these effects were abolished when cells were pretreated with the putative PLA2 inhibitors heparin and mepacrine. While indomethacin and nordihydroguaiaretic acid inhibited either the cyclo-oxygenase or lipoxygenase pathway of arachidonic acid metabolism, as measured by their products prostaglandin E2 and leukotriene B4, they did not influence PMA-mediated PLA2 activation or translocation of protein kinase C (PKC) from the soluble to the particulate fraction. Treatment of cells with PMA increased the amounts of...

Research paper thumbnail of The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells

Research paper thumbnail of Conditional Ablation of the Osteoblast Lineage in Col2.3Δtk Transgenic Mice

Journal of Bone and Mineral Research, 2001

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragme... more Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.

Research paper thumbnail of Col1a1-Driven Transgenic Markers of Osteoblast Lineage Progression

Journal of Bone and Mineral Research, 2001

The modular organization of the type I collagen promoter allows creation of promoter-reporter con... more The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.

Research paper thumbnail of Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2013

Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Pl... more Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP(5), InsP(6), and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G(1) block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP(7) and InsP(8). Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [(3)H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest.

Research paper thumbnail of The activity of extracellular signal-regulated kinase is required during G2/M phase before metaphase–anaphase transition in synchronized leukemia cell lines

International Journal of Hematology, 2009

The pharmacological inhibitors of extracellular signal-regulated kinase (ERK) have been suggested... more The pharmacological inhibitors of extracellular signal-regulated kinase (ERK) have been suggested as a novel molecular target-based therapy for acute myeloid leukemia. Several studies have established the role of ERK in cell cycle progression from G 1 to S phase in response to mitogen, but the role of ERK after the restriction point is less clarified. In this study, we used models of aphidicolin and nocodazole-synchronized HL-60 and NB4 leukemia cell lines to determine the kinetics of ERK activity during the progression of the cell cycle and to test the effects of commercially available inhibitors on G 2 /M progression of synchronized leukemia cells. In aphidicolin-synchronized cells, the activity of ERK was low during early S phase and increased at late S and G 2 /M phase of the cell cycle. The presence of MEK inhibitors PD 98059 and U0126 caused a delay in G 2 /M phase. In nocodazole-synchronized cells, the activity of ERK was low during M/G 1 transition and MEK inhibitors had no effects on return of the cells to G 1 phase. These results demonstrate that the activity of ERK is required during G 2 /M phase of leukemia cell cycle before the cells reach metaphase-anaphase transition.

Research paper thumbnail of Presence of different phospholipase C isoforms in the nucleus and their activation during compensatory liver growth

FEBS Letters, 2004

Phospholipase C (PLC) was purified from the membrane-depleted rat liver nuclei. About 60% of the ... more Phospholipase C (PLC) was purified from the membrane-depleted rat liver nuclei. About 60% of the total PLCactivity corresponded to b 1b isoform, 30% to PLC-c 1 and less than 10% to PLC-d 1 . PLC-b 1b and -c 1 were found in the nuclear matrix, while PLC-d 1 was detected in the chromatin. Two peaks of an increase in the total PLC-activity were detected occurring at 6 and 20 h after partial hepatectomy. An early increase in PLC-b 1b activity in the nuclear matrix was associated with serine phosphorylation of the enzyme, while the later increase paralleled the increase in the amount of protein. The increase in the PLC-c 1 activity measured at 6 and 20 h after partial hepatectomy was associated with tyrosine phosphorylation of the enzyme. The activity of PLC-d 1 and the amount of the protein found in the chromatin was increased only at 20 h after partial hepatectomy.

Research paper thumbnail of The activation of nuclear phosphoinositide 3-kinase C2β in all-trans-retinoic acid-differentiated HL-60 cells

FEBS Letters, 2002

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 ... more The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.

Research paper thumbnail of Hematopoiesis is severely altered in mice with an induced osteoblast deficiency

Blood, 2004

We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene... more We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen alpha1 type I promoter (Col2.3 Delta TK). This construct confers lineage-specific expression in developing osteoblasts, allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition, treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3 Delta TK mice, and we show that after treatment transgenic animals lose lymphoid, erythroid, and myeloid progenitors in the bone marrow, followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis, active extramedullary hematopoiesis was observed in the spleen and liver, as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV, osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow.

Research paper thumbnail of Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2β to the nucleus

Biochemical Journal, 2009

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2 and PI3K-C2 act acutely downstrea... more Although the class II phosphoinositide 3-kinase enzymes PI3K-C2 and PI3K-C2 act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pool of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2 translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2 resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme in the cytosol and then in nuclei. Using detergent treated nuclei, PI3K-C2 co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically and phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2 levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutation of PI3K-C2 demonstrated that epidermal growth factor driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2 in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2 into the nucleus.

Research paper thumbnail of Different roles of protein kinase C α and δ isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells

Biochemical Journal, 1999

Research paper thumbnail of Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2003

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells ... more The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G1/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G1/S block, which correlates with G2/M phase of the cell cycle.

Research paper thumbnail of Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G1 phase of the cell cycle

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2007

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cel... more Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 minutes and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCβ 1 was detected with no change in the amount of PI-PLCβ 1b in nuclei isolated at 30 min and 11h after the addition of serum. PI-PLC inhibitor ET-18-OCH 3 and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCβ 1b activity occur in serum-stimulated cells during G 1 phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.

Research paper thumbnail of Activation of phosphoinositide 3-kinase C2β in the nuclear matrix during compensatory liver growth

Advances in Enzyme Regulation, 2006

In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable... more In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable PI3K-C2b activity is observed, which is sensitive to wortmannin (10 Mm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On western blots PI3K-C2b revealed a single immunoreactive band of 180 kD, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed in the nuclear matrix, suggesting that observed activation of enzyme is achieved by proteolysis. As it is know that PI3K-C2a is associated with nuclear speckles [Didichenko SA, Thelen M. Phosphatidylinositol 3-kinase C2a contains a nuclear localization sequence and associates with nuclear speckles. J Biol Chem 2001;276:48135-42.], the data presented in this report show that in the nuclear matrix PI3K-C2b is activated during the compensatory liver growth, which clearly demonstrates that different class II PI3K enzymes have different subnuclear localization and therefore might have different intranuclear functions. r

Research paper thumbnail of Short-Term and Long-Term-Effects of Phorbol 12Myristate 13Acetate and Different Inhibitors on the Ability of Bone Marrow Cells to Form Colonies In Vitro

Clinical Chemistry and Laboratory Medicine, 1995

The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated i... more The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated increase in the colony-forming potential of murine (CBA) bone marrow cells was studied using known modulators of the mitogenic signal. Pretreatment of cells for 60 minutes with staurosporine (1 μηιοΙ/1), an inhibitor of protein kinase C, completely prevented colony formation in the control group of cells and significantly reduced the number of colonies formed in the phorbol ester-treated group. Brief exposure (60 min) of cells to the phospholipase A 2 inhibitors, mepacrine (500 μιηοΐ/ΐ) and heparin (1 g/1), reduced the number of colonies formed in the control group and completely abolished the increase in the number of colonies formed after treatment of the cells with phorbol ester. When inhibitors of protein kinase C or phospholipase A 2 were present during the entire period of the colony forming assay (7 days), no colonies could be scored in either the control or phorbol ester-treated groups of bone marrow cells. Long-term treatment or temporary exposure (60 min) of cells to indomethacin (50 μπιοΙ/1), an inhibitor of cyclooxygenase, or nordihydroguaiaretic acid (50 μπιοΐ/ΐ), an inhibitor of lipoxygenase, had no effect on colony formation in both groups. Pretreatment of cells for 45 min with calcium ionophore A23187 (10 μιηοΐ/ΐ) failed to increase the number of colonies, compared with the control group. Moreover, simultaneous treatment of cells for 45 min with phorbol ester (500 nmol/1) and A23187 (10 μιηοΐ/ΐ) did not produce any further increase in the number of colonies, compared with the phorbol ester-treated group, suggesting that elevation of intracellular calcium is unimportant in the phorbol ester-mediated response. Dibutyryl cyclic adenosine monophosphate (50 μιηοΐ/ΐ) in the presence or absence of phorbol ester, failed to stimulate colony formation, indicating that cyclic AMP-dependent protein kinases are not involved in the signalling process. Temporary exposure (75 min) of bone marrow cells to okadaic acid (1 μπιοΐ/l), a potent inhibitor of serine/ threonine phosphatases, or to tyrphostine AG-115 (20 μηιοΙ/1), a tyrosine kinase inhibitor, did not effect colony growth in the control or phorbol ester-treated group. The results indicate that phospholipase A 2 activation is involved in the phorbol ester-mediated increase in colony formation, since, of the different agents applied, only staurosporine, an inhibitor of protein kinase C, and mepacrine and heparin, putative inhibitors of phospholipase A 2 , were capable of abolishing phorbol ester-mediated effects.

Research paper thumbnail of deficiency Hematopoiesis is severely altered in mice with an induced osteoblast

Research paper thumbnail of Inositol pyrophosphates modulate cell cycle independently of alteration in telomere length

Advances in biological regulation, Jan 26, 2015

Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the si... more Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the signalling response required for cell cycle progression after mating pheromone arrest. Overexpression of Kcs1 doubled the level of inositol pyrophosphates when compared to wild type cells and 30 min following the release from α-factor block further increase in inositol pyrophosphates was observed, which resulted that cells overexpressing Kcs1 reached G2/M phase earlier than wild type cells. Similar effect was observed in ipk1Δ cells, which are unable to synthesize IP6-derived inositol pyrophosphates (IP7 and IP8) but will synthesize IP5-derived inositol pyrophosphates (PP-IP4 and (PP)2-IP3). Although ipk1Δ cells have shorter telomeres than wild type cells, overexpression of Kcs1 in both strains have similar effect on cell cycle progression. As it is known that PP-IP4 regulates telomere length through Tel1, inositol polyphosphates, cell cycle and telomere length were determined in tel1Δ cel...

Research paper thumbnail of The mechanism of synergistic effects of arsenic trioxide and rapamycin in acute myeloid leukemia cell lines lacking typical t(15;17) translocation

International journal of hematology, Jan 11, 2015

Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promy... more Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promyelocytic leukemia (APL), but is much less efficacious in acute myeloid leukemia (AML) lacking t(15;17) translocation. Recent studies have indicated that the addition of mammalian target of rapamycin (mTOR) inhibitors may increase the sensitivity of malignant cells to ATO. The aim of the present study was to test for possible synergistic effects of ATO and rapamycin at therapeutically achievable doses in non-APL AML cells. In HL-60 and U937 cell lines, the inhibitory effects of low concentrations of ATO and rapamycin were synergistic and more pronounced in U937 cells. The combination of drugs increased apoptosis in HL-60 cells and increased the percentage of cells in G0/G1 phase in both cell lines. In U937 cells, rapamycin alone increased the activity of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the addition of ATO decreased the level of phospho...

Research paper thumbnail of Combined inhibition of PI3K and mTOR exerts synergistic antiproliferative effect, but diminishes differentiative properties of rapamycin in acute myeloid leukemia cells

Pathology oncology research : POR, 2011

A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining m... more A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining mammalian target of rapamycin (mTOR)-inhibitors with an inhibitor of the phosphatydilinositol 3-kinase PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. However, recent study demonstrated the potentiating effect of rapamycin on all-trans-retinoic acid (ATRA)-mediated differentiation of acute myelogenous leukemia (AML) cells, prompting us to investigate the effects of longitudinal inhibition of PI3K/Akt/mTOR signaling pathway on both proliferation and differentiative capacity of AML. In NB4, HL-60, U937 and K562 cell lines, rapamycin exerted minimal antiproliferative effects, and combining PI3K inhibitor LY 294002 and rapamycin inhibited proliferation more than LY 294002 alone. Rapamycin potentiated differentiation of ATRA-treated NB4 cells, but the combination of rapamycin and LY 294002 inhibited the expression of CD11b in both ATRA- an...

Research paper thumbnail of Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells

Biochimica et biophysica acta, Jan 20, 2003

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 ... more The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-...

Research paper thumbnail of Phorbol 12-myristate 13-acetate-mediated signalling in murine bone marrow cells

The Biochemical journal, Jan 15, 1995

Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the abi... more Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the ability of murine (CBA) bone marrow cells to form colonies in vitro. Treatment of marrow cells with PMA did not influence the 1,2-diacylglycerol or cyclic AMP concentrations, the intracellular Ca2+ concentration or phospholipase D activity. PMA increased particulate phospholipase A2 (PLA2) activity, lysophosphatidylcholine formation and arachidonic acid release from bone marrow cells; these effects were abolished when cells were pretreated with the putative PLA2 inhibitors heparin and mepacrine. While indomethacin and nordihydroguaiaretic acid inhibited either the cyclo-oxygenase or lipoxygenase pathway of arachidonic acid metabolism, as measured by their products prostaglandin E2 and leukotriene B4, they did not influence PMA-mediated PLA2 activation or translocation of protein kinase C (PKC) from the soluble to the particulate fraction. Treatment of cells with PMA increased the amounts of...

Research paper thumbnail of The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells

Research paper thumbnail of Conditional Ablation of the Osteoblast Lineage in Col2.3Δtk Transgenic Mice

Journal of Bone and Mineral Research, 2001

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragme... more Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.

Research paper thumbnail of Col1a1-Driven Transgenic Markers of Osteoblast Lineage Progression

Journal of Bone and Mineral Research, 2001

The modular organization of the type I collagen promoter allows creation of promoter-reporter con... more The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.

Research paper thumbnail of Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae

Journal of Biological Chemistry, 2013

Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Pl... more Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP(5), InsP(6), and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G(1) block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP(7) and InsP(8). Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [(3)H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest.

Research paper thumbnail of The activity of extracellular signal-regulated kinase is required during G2/M phase before metaphase–anaphase transition in synchronized leukemia cell lines

International Journal of Hematology, 2009

The pharmacological inhibitors of extracellular signal-regulated kinase (ERK) have been suggested... more The pharmacological inhibitors of extracellular signal-regulated kinase (ERK) have been suggested as a novel molecular target-based therapy for acute myeloid leukemia. Several studies have established the role of ERK in cell cycle progression from G 1 to S phase in response to mitogen, but the role of ERK after the restriction point is less clarified. In this study, we used models of aphidicolin and nocodazole-synchronized HL-60 and NB4 leukemia cell lines to determine the kinetics of ERK activity during the progression of the cell cycle and to test the effects of commercially available inhibitors on G 2 /M progression of synchronized leukemia cells. In aphidicolin-synchronized cells, the activity of ERK was low during early S phase and increased at late S and G 2 /M phase of the cell cycle. The presence of MEK inhibitors PD 98059 and U0126 caused a delay in G 2 /M phase. In nocodazole-synchronized cells, the activity of ERK was low during M/G 1 transition and MEK inhibitors had no effects on return of the cells to G 1 phase. These results demonstrate that the activity of ERK is required during G 2 /M phase of leukemia cell cycle before the cells reach metaphase-anaphase transition.

Research paper thumbnail of Presence of different phospholipase C isoforms in the nucleus and their activation during compensatory liver growth

FEBS Letters, 2004

Phospholipase C (PLC) was purified from the membrane-depleted rat liver nuclei. About 60% of the ... more Phospholipase C (PLC) was purified from the membrane-depleted rat liver nuclei. About 60% of the total PLCactivity corresponded to b 1b isoform, 30% to PLC-c 1 and less than 10% to PLC-d 1 . PLC-b 1b and -c 1 were found in the nuclear matrix, while PLC-d 1 was detected in the chromatin. Two peaks of an increase in the total PLC-activity were detected occurring at 6 and 20 h after partial hepatectomy. An early increase in PLC-b 1b activity in the nuclear matrix was associated with serine phosphorylation of the enzyme, while the later increase paralleled the increase in the amount of protein. The increase in the PLC-c 1 activity measured at 6 and 20 h after partial hepatectomy was associated with tyrosine phosphorylation of the enzyme. The activity of PLC-d 1 and the amount of the protein found in the chromatin was increased only at 20 h after partial hepatectomy.

Research paper thumbnail of The activation of nuclear phosphoinositide 3-kinase C2β in all-trans-retinoic acid-differentiated HL-60 cells

FEBS Letters, 2002

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 ... more The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.

Research paper thumbnail of Hematopoiesis is severely altered in mice with an induced osteoblast deficiency

Blood, 2004

We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene... more We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen alpha1 type I promoter (Col2.3 Delta TK). This construct confers lineage-specific expression in developing osteoblasts, allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition, treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3 Delta TK mice, and we show that after treatment transgenic animals lose lymphoid, erythroid, and myeloid progenitors in the bone marrow, followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis, active extramedullary hematopoiesis was observed in the spleen and liver, as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV, osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow.

Research paper thumbnail of Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2β to the nucleus

Biochemical Journal, 2009

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2 and PI3K-C2 act acutely downstrea... more Although the class II phosphoinositide 3-kinase enzymes PI3K-C2 and PI3K-C2 act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pool of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2 translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2 resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme in the cytosol and then in nuclei. Using detergent treated nuclei, PI3K-C2 co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically and phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2 levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutation of PI3K-C2 demonstrated that epidermal growth factor driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2 in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2 into the nucleus.

Research paper thumbnail of Different roles of protein kinase C α and δ isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells

Biochemical Journal, 1999

Research paper thumbnail of Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2003

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells ... more The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G1/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G1/S block, which correlates with G2/M phase of the cell cycle.

Research paper thumbnail of Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G1 phase of the cell cycle

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2007

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cel... more Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 minutes and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCβ 1 was detected with no change in the amount of PI-PLCβ 1b in nuclei isolated at 30 min and 11h after the addition of serum. PI-PLC inhibitor ET-18-OCH 3 and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCβ 1b activity occur in serum-stimulated cells during G 1 phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.

Research paper thumbnail of Activation of phosphoinositide 3-kinase C2β in the nuclear matrix during compensatory liver growth

Advances in Enzyme Regulation, 2006

In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable... more In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable PI3K-C2b activity is observed, which is sensitive to wortmannin (10 Mm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On western blots PI3K-C2b revealed a single immunoreactive band of 180 kD, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed in the nuclear matrix, suggesting that observed activation of enzyme is achieved by proteolysis. As it is know that PI3K-C2a is associated with nuclear speckles [Didichenko SA, Thelen M. Phosphatidylinositol 3-kinase C2a contains a nuclear localization sequence and associates with nuclear speckles. J Biol Chem 2001;276:48135-42.], the data presented in this report show that in the nuclear matrix PI3K-C2b is activated during the compensatory liver growth, which clearly demonstrates that different class II PI3K enzymes have different subnuclear localization and therefore might have different intranuclear functions. r

Research paper thumbnail of Short-Term and Long-Term-Effects of Phorbol 12Myristate 13Acetate and Different Inhibitors on the Ability of Bone Marrow Cells to Form Colonies In Vitro

Clinical Chemistry and Laboratory Medicine, 1995

The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated i... more The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated increase in the colony-forming potential of murine (CBA) bone marrow cells was studied using known modulators of the mitogenic signal. Pretreatment of cells for 60 minutes with staurosporine (1 μηιοΙ/1), an inhibitor of protein kinase C, completely prevented colony formation in the control group of cells and significantly reduced the number of colonies formed in the phorbol ester-treated group. Brief exposure (60 min) of cells to the phospholipase A 2 inhibitors, mepacrine (500 μιηοΐ/ΐ) and heparin (1 g/1), reduced the number of colonies formed in the control group and completely abolished the increase in the number of colonies formed after treatment of the cells with phorbol ester. When inhibitors of protein kinase C or phospholipase A 2 were present during the entire period of the colony forming assay (7 days), no colonies could be scored in either the control or phorbol ester-treated groups of bone marrow cells. Long-term treatment or temporary exposure (60 min) of cells to indomethacin (50 μπιοΙ/1), an inhibitor of cyclooxygenase, or nordihydroguaiaretic acid (50 μπιοΐ/ΐ), an inhibitor of lipoxygenase, had no effect on colony formation in both groups. Pretreatment of cells for 45 min with calcium ionophore A23187 (10 μιηοΐ/ΐ) failed to increase the number of colonies, compared with the control group. Moreover, simultaneous treatment of cells for 45 min with phorbol ester (500 nmol/1) and A23187 (10 μιηοΐ/ΐ) did not produce any further increase in the number of colonies, compared with the phorbol ester-treated group, suggesting that elevation of intracellular calcium is unimportant in the phorbol ester-mediated response. Dibutyryl cyclic adenosine monophosphate (50 μιηοΐ/ΐ) in the presence or absence of phorbol ester, failed to stimulate colony formation, indicating that cyclic AMP-dependent protein kinases are not involved in the signalling process. Temporary exposure (75 min) of bone marrow cells to okadaic acid (1 μπιοΐ/l), a potent inhibitor of serine/ threonine phosphatases, or to tyrphostine AG-115 (20 μηιοΙ/1), a tyrosine kinase inhibitor, did not effect colony growth in the control or phorbol ester-treated group. The results indicate that phospholipase A 2 activation is involved in the phorbol ester-mediated increase in colony formation, since, of the different agents applied, only staurosporine, an inhibitor of protein kinase C, and mepacrine and heparin, putative inhibitors of phospholipase A 2 , were capable of abolishing phorbol ester-mediated effects.