Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL.

Acute promyelocytic leukaemia (APL), associated with chromosomal translocations involving the retinoic acid receptor alpha gene (RARA) and the PML gene, is sensitive to retinoic acid (RA) treatment, while APL patients harbouring translocations between RARA and the PLZF gene do not respond to RA. We have generated PML-RARA and PLZF-RARA transgenic mice and show here that these fusion proteins play a critical role in leukaemogenesis and in determining responses to RA in APL, because PLZF-RARA transgenic mice develop RA-resistant leukaemia, while PML-RARA mice are responsive to RA treatment. We demonstrate that both PML-RARalpha and PLZF-RARalpha fusion proteins can act as transcriptional repressors and are able to interact with nuclear receptor transcriptional co-repressors, such as SMRT. PLZF-RARalpha, but not PML-RARalpha, can form, via its PLZF moiety, co-repressor complexes which are insensitive to RA. Histone deacetylase inhibitors such as Trichostatin A (TSA), in combination with RA, can overcome the transcriptional repressor activity of PML-RARalpha and PLZF-RARalpha as well as the unresponsiveness of PLZF-RARalpha-expressing leukaemic cells to RA. Thus, our findings unravel a crucial role for transcriptional silencing in APL pathogenesis and resistance to RA in APL.

Intratumor RNA interference of cell cycle genes slows down tumor progression.

Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.

Role of promyelocytic leukemia (PML) protein in tumor suppression.

The promyelocytic leukemia (PML) gene encodes a putative tumor suppressor gene involved in the control of apoptosis, which is fused to the retinoic acid receptor alpha (RARalpha) gene in the vast majority of acute promyelocytic leukemia (APL) patients as a consequence of chromosomal translocations. The PMLRARalpha oncoprotein is thought to antagonize the function of PML through its ability to heterodimerize with and delocalize PML from the nuclear body. In APL, this may be facilitated by the reduction to heterozygosity of the normal PML allele. To determine whether PML acts as a tumor suppressor in vivo and what the consequences of deregulated programmed cell death in leukemia and epithelial cancer pathogenesis are, we crossed PML(-/-) mice with human cathepsin G (hCG)-PMLRARalpha or mammary tumor virus (MMTV)/neu transgenic mice (TM), models of leukemia and breast cancer, respectively. The progressive reduction of the dose of PML resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PMLRARalpha TM. By contrast, PML inactivation did not affect neu-induced tumorigenesis. In hemopoietic cells from PMLRARalpha TM, PML inactivation resulted in impaired response to differentiating agents such as RA and vitamin D3 as well as in a marked survival advantage upon proapoptotic stimuli. These results demonstrate that: (a) PML acts in vivo as a tumor suppressor by rendering the cells resistant to proapoptotic and differentiating stimuli; (b) PML haploinsufficiency and the functional impairment of PML by PMLRARalpha are critical events in APL pathogenesis; and (c) aberrant control of programmed cell death plays a differential role in solid tumor and leukemia pathogenesis.

Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells.

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.

Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C beta(1).

Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type beta(1) is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCbeta(1) under different stimuli implicate PLCbeta(1) as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCbeta(1) in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35-36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCbeta(1) cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCbeta(1). By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCbeta(1) on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35-36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCbeta(1) could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCbeta(1), which has been shown to behave as a key signalling intermediate in the control of the cell cycle.

Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase beta family in NIH 3T3 cells.

Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment.

Re-examination of the mechanisms regulating nuclear inositol lipid metabolism.

Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane. Very recently, intriguing new data have highlighted that some of the mechanisms regulating nuclear inositol lipid metabolism differ in a substantial way from those operating at the cell periphery. Here, we provide an overview of recent findings regarding the regulation of both nuclear phosphatidylinositol 3-kinase and phosphoinositide-specific phospholipase C-beta1.

Morphology of two classes of target-specific bullfrog sympathetic preganglionic neurons.

These experiments took advantage of the unique ability to define target-specific sympathetic preganglionic neurons in the bullfrog spinal cord in order to examine the morphologies of different classes of preganglionic neurons. Sympathetic preganglionic neurons were identified by retrograde transport of fast blue from the sympathetic chain. Subsequently, fast blue-labelled sympathetic preganglionic neurons in fixed spinal cord slices were filled with lucifer yellow and processed for visualization with lucifer yellow antiserum, biotinylated secondary antiserum, and avidin peroxidase. Target specificity of sympathetic preganglionic neurons was determined by anatomical position; sympathetic preganglionic neurons that control the vasculature (C-type sympathetic preganglionic neurons) lie in a position caudal to those that control nonvascular targets [B-type sympathetic preganglionic neurons; Horn and Stofer (1988) J. Comp. Neurol. 268:71]. These two classes of sympathetic preganglionic neurons have qualitatively similar morphologies. However, they exhibit significant quantitative differences in total dendritic length and the rostrocaudal extent of dendrites. These differences are likely to be associated with differences in the number of synapses received by these two classes of sympathetic preganglionic neurons. Moreover, the segmental control of sympathetic preganglionic neurons by descending brainstem projections is likely to be finer for those involved in vascular control than for those that influence other targets.

Identification of cell types in brain slices of the inferior colliculus.

Different type neurons in the inferior colliculus may have different functions. Recent intracellular studies of the inferior colliculus suggest that intrinsic electrical properties contribute to discharge patterns, but the intrinsic discharge patterns have not been fully characterized in the central nucleus, the main part of the inferior colliculus. Whether different types of neurons are related to different discharge patterns is unclear. We have used intracellular and whole-cell patch clamp-recording techniques in a brain slice preparation to better characterize discharge patterns and cell types in the central nucleus. Several types of discharge pattern were found in the inferior colliculus in response to long pulses of intracellular depolarizations. Rebound and buildup-pauser discharges, together, comprise neurons with a sustained response and are the majority of the neurons in the inferior colliculus. Both of these types of discharge pattern could be adapting or regular. Onset discharges distinguished another group of neurons. Onset neurons can also entrain to higher frequency stimuli than sustained neurons. Discharge patterns are correlated with distinctive current-voltage relationships and with some aspects of dendritic morphology. However, the morphological data demonstrates that the discharge patterns do not correspond simply to disc-shaped (flat) or stellate (less-flat) categories. This is the first extensive analysis of electrophysiological properties of the central nucleus of the inferior colliculus in vitro. We suggest that there may be at least three functional classes of neurons and have implications for signal processing in the inferior colliculus.

Nuclear phospholipase C: a novel aspect of phosphoinositide signalling.

The role of polyphosphoinositides in cellular signalling is well known and recently it has also been shown that the nucleus is a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. It has been demonstrated that phospholipase C specific for inositol lipids (PLC) is one of the main steps of the inositol lipid cycle. The PLC beta family, and especially type beta 1, has given rise to considerable interest since, due to their common COOH-terminus they show nuclear localisation in addition to that at the plasma membrane. It is well established that an autonomous intranuclear inositide cycle exists, and that this cycle is endowed with conventional lipid kinases, phosphatases and PLCs. Among this latter the beta 1 type undergoes stimulation or inhibition under different stimuli and this implicates the beta 1 isoform as a key enzyme for mitogen-activated cell growth as well as for differentiation. Indeed, both the overexpression and the down-regulation of PLC beta 1, by means of antisense mRNA, have demonstrated that PLC plays a role in the nuclear compartment.

Segmental restriction and target specificity of bullfrog preganglionic neurons that exhibit galanin-like immunoreactivity.

These experiments were designed to examine the distribution of galanin-like peptide immunoreactivity (GAL-IR) in bullfrog sympathetic preganglionic neurons and to identify the peripheral target organs affected by these neurons. Cells expressing GAL-IR were observed in the intermediolateral column of segments 7 and 8 only. Apparent GAL-IR innervation is present, but rare, in sympathetic chain ganglia. Double-labelling with retrogradely transported fast blue and galanin antiserum demonstrated that most GAL-IR neurons project via splanchnic nerves to innervate the adrenal gland, which receives a dense plexus of GAL-IR fibers surrounding chromaffin cells. The adrenal gland is also innervated by preganglionic neurons in segments 5 and 6 that do not express GAL-IR. Because nitric oxide is expressed in sympathoadrenal preganglionic neurons in mammals (Anderson, C.R., Neurosci. Lett., 139 (1992) 280), we examined whether it is expressed in bullfrog preganglionic neurons. Nicotinamide adenine dinucleotide phosphate-diaphorase positive neurons are present in bullfrog spinal grey at segments 5 through 8. These neurons were not double-labelled with fast blue retrogradely transported from the sympathetic chain, celiac ganglion, or adrenal gland; nor were they double-labelled with GAL-antiserum. Thus nitric oxide is apparently not expressed in bullfrog sympathetic preganglionic neurons.

Hypertrophy of stellate ganglion cells in hypertensive, but not hyperactive, rats.

The dendritic complexity of peripheral autonomic neurons is positively matched with the size of the target they innervate, apparently by trophic interactions with the target (D. Purves, W. D. Snider, and J. T. Voyvodic. Nature Lond. 336: 123-128, 1988). We have asked whether the vascular hypertrophy associated with hypertension is accompanied by dendritic hypertrophy of sympathetic ganglion cells. To do this, we examined the morphology of stellate ganglion cells in the spontaneously hypertensive rat (SHR), its normotensive control Wistar-Kyoto rat (WKY), and two new strains derived from the SHR that independently express the hypertensive phenotype of the SHR (WKHT) and the behavioral hyperactivity present in the SHR (WKHA). Cells were examined by intracellular staining with horseradish peroxidase in in vitro preparations of the ganglia. Carotid arterial wall size was also examined. Significant hypertrophy of both the carotid arterial wall and stellate ganglion cell dendrites was observed in the two hypertensive strains (SHR and WKHT) but not in either of the normotensive strains (WKY and WKHA). This increased total dendritic length of stellate ganglion cells associated with hypertension provides a greater target area for preganglionic innervation that may result in hyperinnervation of these cells.

A monosynaptic GABAergic input from the inferior colliculus to the medial geniculate body in rat.

The goal was to investigate possible monosynaptic GABAergic projections from the inferior colliculus (IC) to thalamocortical neurons of the medial geniculate body (MGB) in the rat. Although there is little evidence for such a projection in other sensory thalamic nuclei, a GABAergic, ascending auditory projection was reported recently in the cat. In the present study, immunohistochemical and tract-tracing methods were used to identify neurons in the IC that contain GABA and project to the MGB. GABA-positive projection neurons were most numerous in the central nucleus and less so in the dorsal and lateral cortex. They were rare in the lateral tegmental system and brachium of the IC. The dorsal nucleus of the lateral lemniscus also contained GABA-positive projection neurons. In brain slices, stimulation of the brachium produced monosynaptic inhibitory postsynaptic potentials in morphologically identified thalamocortical relay neurons. The inhibitory potentials cannot originate locally, because they persisted when ionotropic glutamatergic transmission was blocked. Typically, brachium stimulation elicited a GABAA-mediated inhibitory potential followed by an excitatory potential and a longer latency GABAB-mediated inhibitory potential. We conclude that the GABA-containing neurons of the IC make short-latency, monosynaptic inputs to the thalamocortical projection neurons in the MGB. Such inputs may distinguish the main auditory pathway from indirect or tegmental auditory pathways as well as from other sensory systems. Monosynaptic inhibitory inputs to the medial geniculate may be important for the regulation of firing patterns in thalamocortical neurons.

A role for nuclear phospholipase Cbeta 1 in cell cycle control.

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 PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(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 PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(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 PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4.

Acute leukemia with promyelocytic features in PML/RARalpha transgenic mice.

Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations involving the retinoic acid receptor alpha (RARalpha) locus on chromosome 17. In the majority of cases, RARalpha translocates and fuses with the promyelocytic leukemia (PML) gene located on chromosome 15. The resulting fusion genes encode the two structurally unique PML/RARalpha and RARalpha/PML fusion proteins as well as aberrant PML gene products, the respective pathogenetic roles of which have not been elucidated. We have generated transgenic mice in which the PML/RARalpha fusion protein is specifically expressed in the myeloid-promyelocytic lineage. During their first year of life, all the PML/RARalpha transgenic mice have an abnormal hematopoiesis that can best be described as a myeloproliferative disorder. Between 12 and 14 months of age, 10% of them develop a form of acute leukemia with a differentiation block at the promyelocytic stage that closely mimics human APL even in its response to retinoic acid. Our results are conclusive in vivo evidence that PML/RARalpha plays a crucial role in the pathogenesis of APL.

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G + C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

Gene rearrangements in the molecular pathogenesis of acute promyelocytic leukemia.

Acute Promyelocytic Leukemia (APL) is a distinct subtype of myeloid leukemia that in the USA alone affects more than 3,000 individuals every year. APL is characterized by three distinct and unique features: i) the accumulation in the bone marrow of tumor cells with promyelocytic features; ii) the invariable association with specific translocations which always involve chromosome 17 and the Retinoic Acid Receptor alpha (RAR alpha) locus; iii) the exquisite sensitivity of APL blasts to the differentiating action of Retinoic Acid (RA). These features have led APL to become the paradigm for therapeutic approaches utilizing differentiating agents. The last 5 years have provided crucial insights into the molecular basis of APL. RAR alpha translocates in 99% of cases to a gene located on chromosome 15 that we initially named myl and subsequently has been called PML. In a few cases, RAR alpha variably translocates to chromosome 11 where it fuses to the PLZF gene or to a newly described partner, NuMA. In addition, RAR alpha is also found translocated to chromosome 5 where it fuses to the NPM gene. The cloning of variant translocations in APL and the comparative analysis of their associated products is crucial for the understanding of the molecular etiopathogenesis of the disease. The generation of animal models, i.e., transgenic mice expressing the fusion genes, will be instrumental in determining the precise contribution of these fusion genes to leukemogenesis. In fact, mice harboring a PML/RAR alpha transgene whose expression is specifically targeted to the myeloid-promyelocytic lineage develop acute myeloid leukemia with promyelocytic features. Moreover, the functional analysis of the various fusion proteins, as well as RAR alpha partners, is revealing striking common features beneath a misleading structural heterogeneity which unravels a possible unifying molecular mechanism towards APL leukemogenesis.