No abstract

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLC␤ 1 , which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLC␤ 1 exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLC␤ 1 subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLC␤ 1 that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLC␤ 1 signaling in G 1 progression by means of a specific target, i.e. cyclin D3/cdk4.It is demonstrated that an autonomous intranuclear inositide cycle exists and that nuclear PLC␤ 1 1 is a key enzyme for cell proliferation and differentiation (1). The enzymes of polyphosphoinositide turnover occur in the nucleus of mammalian cells and yeast as well (Ref. references therein),and there is evidence for phosphatidylinositol bisphosphate (PIP 2 ) synthesis and degradation in the nuclear matrix (3). The evidence obtained with confocal and electron microscope analysis indicates that enzymes required for the synthesis and hydrolysis of phosphoinositides are localized at ribonucleoprotein structures of the inner nuclear matrix involved in transcript processing within the interchromatin domains (4). Although phosphatidylinositol cycle is activated only in nuclei from HeLa cells in S phase (5), striking changes occur mainly in PLC␤ 1 activity a few minutes after growth factor stimulation (1). PLC␤ 1 is composed of two subtypes, 150-kDa PLC␤ 1 a and 140-kDa PLC␤ 1 b, that are derived from a single gene by alternative RNA splicing (6). The two forms of the PLC␤ 1 are detectable both in cytosolic and nuclear fractions although PLC␤ 1 b exists almost entirely in the nucleus (7), and the ␤ 1 a form localizes in equal amount in nuclei and plasma membrane (8). Previous investigations from our group have demonstrated that the nucleus-confined PLC␤ 1 is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells even in the presence of inducers of erythroid differentiation, possibly due to a continuous stimulation of the cell cycle (9). With the above in min...

TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL. We found that parental (PT) HL60 cells were very sensitive to a 6 h incubation in the presence of TRAIL and died by apoptosis. In contrast, AR cells were resistant to TRAIL concentrations as high as 2 g/ml for 24 h.

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents, which target plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV 4-11 cell lines, as well as primary leukemia cells. Perifosine treatment induced cell death by apoptosis in AML cell lines. Perifosine caused Akt and ERK 1/2 dephosphorylation as well as caspase activation. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on the Fas/FasL system and c-jun-N-kinase activation. In MV 4-11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine reduced the clonogenic activity of AML, but not normal, CD34 þ cells, and markedly increased blast cell sensitivity to etoposide. Our findings indicate that perifosine, either alone or in combination with existing drugs, might be a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K-Akt survival pathway.

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB)/mammalian Target Of Rapamycin (mTOR) signaling pathway plays a critical role in many cellular functions which are elicited by extracellular stimuli. However, constitutively active PI3K/Akt/mTOR signaling has also been firmly established as a major determinant for cell growth, proliferation, and survival in an wide array of human cancers. Thus, blocking the PI3K/AKT/mTOR signal transduction network could be an effective new strategy for targeted anticancer therapy. Pharmacological inhibitors of this signaling cascade are powerful antineoplastic agents in vitro and in xenografted models of tumors, and some of them are now being tested in clinical trials. Recent studies showed that PI3K/Akt/mTOR axis is frequently activated in acute myelogenous leukemia (AML) patient blasts and strongly contributes to proliferation, survival, and drug-resistance of these cells. Both the disease-free survival and overall survival are significantly shorter in AML cases with PI3K/Akt/mTOR upregulation. Therefore, this signal transduction cascade may represent a target for innovative therapeutic treatments of AML patients. In this review, we discuss the possible mechanisms of activation of this pathway in AML cells and the downstream molecular targets of the PI3K/Akt/mTOR signaling network which are important for blocking apoptosis, enhancing proliferation, and promoting drug-resistance of leukemic cells. We also highlight several pharmacological inhibitors which have been used to block this pathway for targeted therapy of AML. These small molecules induce apoptosis or sensitize AML cells to existing drugs, and might be used in the future for improving the outcome of this hematological disorder.

No abstract

Data emerging from preclinical settings suggest that the LKB1/AMPK pathway is critically involved in regulating proliferation and survival of malignant hematopoietic cells. Thus, it is proposed that drugs activating the LKB1/AMPK axis may offer a novel and less toxic treatment option for some types of hematological malignancies.

Inositide-specific phospholipase Cbeta1 (PLCbeta1) signaling in cell proliferation has been investigated thoroughly in the G(1) cell cycle phase. However, little is known about its involvement in G(2)/M progression. We used murine erythroleukemia cells to investigate the role of PLCbeta1 in G(2)/M cell cycle progression and screened a number of candidate intermediate players, particularly mitogen-activated protein kinase (MAPK) and protein kinase C (PKC), which can, potentially, transduce serum mitogenic stimulus and induce lamin B1 phosphorylation, leading to G(2)/M progression. We report that PLCbeta1 colocalizes and physically interacts with lamin B1. Studies of the effects of inhibitors and selective si-RNA mediated silencing showed a role of JNK, PKCalpha, PKCbetaI, and the beta1 isoform of PI-PLC in cell accumulation in G(2)/M [as observed by fluorescence-activated cell sorter (FACS)]. To shed light on the mechanism, we considered that the final signaling target was lamin B1 phosphorylation. When JNK, PKCalpha, or PLCbeta1 were silenced, lamin B1 exhibited a lower extent of phosphorylation, as compared to control. The salient features to emerge from these studies are a common pathway in which JNK is likely to represent a link between mitogenic stimulus and activation of PLCbeta1, and, foremost, the finding that the PLCbeta1-mediated pathway represents a functional nuclear inositide signaling in the G(2)/M transition.