Lipoprotein internalisation induced by oncogenic AMPK activation is essential to maintain glioblastoma cell growth.

AIM OF THE STUDY: Metabolic adaptations are essential during tumour growth to maintain the high proliferation levels exhibited by cancer cells. In this study, we examined the transformations that occurred in the lipid metabolism in astrocytic tumours, and the possible role of the fuel-sensing enzyme AMPK. Metabolic targets might help design new and effective drugs for cancer. METHODS: To accomplish this objective, we studied both mice and human astrocytic tumours. We first used a mouse model of astrocytoma driven by oncogenic H-RasV12 and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We then confirmed the results in human glioblastoma cell lines and in glioblastoma tissue samples from patients. RESULTS: We show that the high levels of activated AMPK, observed in astrocytic tumours, increase extracellular lipid internalisation and reduce energy expenditure by inhibiting 'de novo' fatty acid (FA) synthesis, which allows tumour cells to obtain building blocks and energy to be able to create new organelles and new cells. CONCLUSIONS: Our findings demonstrate that AMPK plays a crucial role in glioblastoma cell growth and suggest that blocking lipoprotein receptors could potentially be used as a plausible therapeutic approach for these and other type of tumours with high levels of AMPK.

NIR-light active hybrid nanoparticles for combined imaging and bimodal therapy of cancerous cells.

We report the synthesis of a multifunctional biocompatible theranostic nanoplatform consisting of a biodegradable PLGA matrix surface-functionalized with indocyanine green (ICG), a near-IR fluorescent dye, and co-loaded with superparamagnetic iron oxide nanoparticles (SPIONs) and the anticancer drug doxorubicin (DOXO). Combination of chemo- and photothermal therapeutic efficacy as well as magnetic resonance and optical fluorescence imaging performance were successfully tested in vitro on a tumoral cervical HeLa cell line. Magnetic in vitro guided targeting of these nanoplatforms was also proven. These nanoconstructs also enabled to monitor their in vivo biodistribution by fluorescence imaging in a mice model, which revealed their effective accumulation in the tumor and, unexpectedly, in the brain area. A lower presence of nanoplatforms was noted in the reticulo-endothelial system. The present observations suggest the nanoplatforms ability to possibly overcome the blood brain barrier. These results open up new possibilities to use our multifunctional nanoplatforms to treat brain-located diseases.

Cyclin-dependent kinase antagonizes promyelocytic leukemia zinc-finger through phosphorylation.

Acute promyelocytic leukemia is associated with chromosomal translocations that involve the RARalpha gene and several distinct loci producing a variety of fusion proteins. One such fusion partner is promyelocytic leukemia zinc-finger gene (PLZF), a member of the POK (POZ and Krüppel) family of transcriptional repressors that is a key developmental regulator, stem cell maintenance factor and tumor suppressor. Overexpression of PLZF has been shown to induce cell cycle arrest at the G(1) to S transition and repress the expression of key pro-proliferative genes such as CCNA2 and MYC. However, given this data suggesting an important growth inhibitory role for PLZF, relatively little is known regarding regulation of its activity. Here we show that the main cyclin-dependent kinase involved at the G(1) to S transition (CDK2) phosphorylates PLZF at two consensus sites found within PEST domains present in the hinge region of the protein. This phosphorylation triggers the ubiquitination and subsequent degradation of PLZF, which impairs PLZF transcriptional repression ability and antagonizes its growth inhibitory effects. This critical mechanism of PLZF regulation may thus be relevant for cell cycle progression during the development and the pathogenesis of human cancer.

The role of promyelocytic leukemia zinc finger and promyelocytic leukemia in leukemogenesis and development.

Acute promyelocytic leukemia (APL) was originally distinguished by an extremely poor clinical outcome. In the past few years, however, important progress has been made in defining the molecular basis of APL pathogenesis and in optimizing its treatment to an extent that this leukemia is now considered curable. Two features are unique to this leukemia: its remission after retinoic acid (RA) treatment through induction of blast differentiation, and the presence in the leukemic blast of fusion proteins in which the retinoic acid receptor alpha (RARalpha) fuses to distinct partners. Here we review how a detailed analysis of the functions of two of these RARalpha partners, the promyelocytic leukemia (PML) and promyelocytic leukemia zinc finger (PLZF) proteins, has allowed a greater understanding of the molecular mechanisms implicated in APL pathogenesis.

N-glycosylated variants of growth hormone in human pituitary extracts.

This study was designed to investigate the existence, in human pituitary extracts, of growth hormone (GH) variants not encoded by the hGH-N gene. Using anion exchange-fast protein liquid chromatography followed by SDS-PAGE, we isolated several basic forms of pituitary GH. Incubation of these basic forms with endoglycosidase F/N-glycosidase F revealed that two of them (about 34 and 12 kD) were N-glycosylated. In contrast, no changes were found when samples were incubated with the O-linked glycosylation-specific O-glycosidase. Since the GH-N molecule lacks consensus sequences for N-linked glycosylation, our findings suggest that GH genes other than hGH-N are expressed in the human pituitary gland.

Expression of growth hormone receptor in the human brain.

This study was designed to investigate the presence of growth hormone receptor (GHR) expression in the human brain tissue, both normal and tumoral, as well as in the human glioblastoma cell line U87MG. Reverse transcription-polymerase chain reaction revealed the presence of GHR mRNA in all brain samples investigated and in U87MG cells. GHR immunoreactivity was also detected in this cell line using both immunocytochemistry and western blotting. All together, our data demonstrate the existence of GHR expression within the central nervous system (CNS), thus supporting a possible role for GH in the CNS physiology.

Role of growth hormone receptor in HL-60 cell survival.

Although it is presently well established that locally produced growth hormone (GH) plays a major role in the regulation of survival mechanisms in hemopoietic cells, the responsible mechanisms are poorly understood, and the involvement of the GH receptor (GHR) has not even been demonstrated to date. In this work we investigated the presence of GHR in the human promyelocytic leukemia cell line HL-60, as well as the ability of GH treatment to stimulate both GHR and survival signaling pathways downstream GHR. Our results demonstrate that (1) both GHR mRNA and GHR immunoreactivity are present in HL-60 cells; (2) GH treatment results in an increase in the phosphorylation of the GHR-associated Jak2 and Stat3 proteins, indicating the ability of the hormone to induce receptor activation; and (3) activation of GHR increases the activity of Akt, a serine/threonine kinase that plays a prominent role in the regulation of cell survival. Taken together, these results demonstrate that GHR activation promotes survival of HL-60 cells, thus suggesting that GH plays a major role in the regulation of cell survival in the hemopoietic system, via an autocrine/paracrine mechanism.

Activation of growth hormone receptor delivers an antiapoptotic signal: evidence for a role of Akt in this pathway.

A signaling pathway was delineated by which GH promotes cell survival. Experiments were performed in human leukemic cells (HL-60) and Chinese hamster ovary (CHO) cells. In HL-60 cells, GH treatment reduced starvation-induced cell death. In contrast, when HL-60 cells were treated with an anti-GH antibody, cell survival was sharply reduced. In CHO cells stably expressing either the wild-type (wtGHR) or a truncated form (delta454GHR) of the GH receptor in which GH induces a sustained activation of the receptor-associated tyrosine kinase JAK2, we found that GH stimulation inhibited programmed cell death induced by withdrawal of survival factors. This effect was enhanced in cells expressing the truncated form. In contrast, GH did not affect cell survival in CHO cells transfected with either the empty vector or a mutated GHR unable to transduce the signal (4P/AGHR). We also showed that the inhibitory action of GH on apoptosis is probably mediated via stimulation of the serine-threonine kinase Akt, as 1) GH treatment induces a prompt phosphorylation of Akt; and 2) GH effects on cell survival are abolished by transfection of an Akt mutant that exhibits dominant negative function. Experiments with pharmacological inhibitors demonstrated that GH-induced Akt phosphorylation is dependent on phosphoinositide 3-kinase activation. In contrast, we found no changes in Bcl-2 levels secondary to GHR activation.

Salivary gland is capable of GH synthesis under GHRH stimulation.

Twelve female rats weighing approximately 150 g received in the submaxillary gland a pellet capable of releasing 3.5 microg GHRH/h for 60 days. Another eight sex- and weight-matched animals received placebo pellets in the same place. After two months the animals were killed, heart blood was collected and pituitary and submaxillary glands were carefully dissected. Pituitary GH content in both placebo- and GHRH-treated animals showed similar values, but plasma GH and IGF-I levels were significantly lower in the animals carrying GHRH pellets (P<0.03); these animals also had a significantly higher GH content in the submaxillary gland (19.2+/-8 ng/mg protein) compared with the placebo-treated group (1.1+/-0.3 ng/mg protein). GH mRNA was present only in the submaxillary gland of GHRH-treated rats as determined by PCR-Southern blot and by in situ hybridization methods. It is concluded that high local GHRH levels are capable of inducing transdifferentiation in submaxillary gland cells to synthesize GH.

Pattern of presentation of the human growth hormone variant (hGH-V) gene in the normal population.

The human growth hormone variant (hGH-V) gene is a member of the GH gene family, expressed by the syncytiotrophoblast. Although its physiological role is poorly understood, certain data suggest that it may be involved in the control of fetal growth and development. As a first step to asses its physiological relevance, we investigated its degree of polymorphism in the normal population. Genetic studies have been difficult to carry out due to the high sequence identity among GH-family members. We overcame this problem by selectively amplifying a fragment of the hGH-V gene by PCR. DSCP analysis of the amplimers revealed a heterozygous pattern in one of the 64 subjects studied. Investigation of the subject's relatives showed a similar pattern in his father. In all, our results indicate that the hGH-V gene is highly conserved in the normal population.

Correlation of Pit-1 gene expression and Pit-1 content with proliferation and differentiation in human myeloid leukemic cells.

The transcription factor pituitary-1 (Pit-1) is a homeodomain-containing protein that is expressed mainly in the pituitary, where it drives the expression of growth hormone, prolactin, and thyroid-stimulating hormone beta chain genes. In addition, Pit-1 is required for adequate pituitary cell growth and may be involved in the pathogenesis of pituitary adenomas. Pit-1 expression has been also reported in nonpituitary tissues, where it might be involved in the control of cell proliferation. In order to elucidate such a possibility, we have investigated the changes in both Pit-1 mRNA and Pit-1 immunoreactivity in HL-60 cells following the addition of several differentiating agents. Our results show that while high Pit-1 levels are found in exponentially growing HL-60 cells, a significant decrease occurs after induction of cells to differentiate along the macrophage lineage with 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast no changes were observed when cells were treated with interferon-alpha, which also induces differentiation of HL-60 cells that, at odds with TPA, is not accompanied with growth arrest. In all, these findings suggest that Pit-1 expression is specifically associated with proliferation in HL-60 cells, thus supporting the idea that one of the functions of nonpituitary Pit-1 may be the control of cell proliferation.

Expression of the human growth hormone normal gene (hGH-N) in proliferating and differentiated HL-60 cells.

The possibility that human leukemic cells could synthesize growth hormone (GH) was investigated in the HL-60 cell line. Western blot analysis of protein extracts obtained from these cells revealed the existence of a major immunoreactive GH (irGH) band, with an approximate molecular weight of 22 kDa, together with lower amounts of 20- and 44-kDa bands. Stimulating proliferating HL-60 cells with KCl clearly increased GH concentration in the incubation medium as compared to basal values. RT-PCR amplification of HL-60 RNA and restriction assay of the amplimers demonstrated that those proteins were the result of the expression of the GH-N (normal) gene in this cell line. These results were confirmed by Northern blot, which also showed that the rate of GH-N gene expression was clearly dependent upon the proliferative state of the cells: while GH transcripts were easily detectable in actively proliferating cells, only minute amounts were observed when cells were induced to differentiate with dimethyl sulphoxide (DMSO). Similar differences were observed by Western blot. In all, these findings demonstrate that HL-60 cells are capable to produce and secrete a GH identical to pituitary GH. Interestingly, the rate of synthesis of the hormone dramatically increases when cells are actively proliferating. Therefore, it is likely that locally produced GH might be involved in the control of leukemic cell proliferation. Further studies are now in course to establish whether this mechanism occurs via an autocrine and/or paracrine way.