LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro.

BACKGROUND: Relapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML. RESULTS: About 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75. CONCLUSION: Our results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.

Effect of continuous light on daily levels of plasma melatonin and cortisol and expression of clock genes in pineal gland, brain, and liver in atlantic salmon postsmolts.

Continuous light is a common practice in salmon farming, where it is used to enhance growth, induce smoltification, and regulate puberty. However, knowledge about how different tissues receive information about daylength is limited. The aim of the present study was to evaluate the daily expression of clock (Per1-like, Cry2, and Clock), the nuclear transcription factor (peroxisome proliferator-activated receptor, PPAR; CCAAT/enhancer binding protein, C/EBP), and the endoplasmic reticulum (ER) stress (protein disulfide isomerase associated 3, PDIA3) genes in the pineal gland, brain, and liver of Atlantic salmon postsmolts reared under 12-h light:12-h dark (LD) regimes or under continuous light (LL) for 6 wks following transfer to seawater. All measured clock mRNAs displayed daily variations in one or more organs under LD, as well as plasma levels of melatonin. Similar variations were noted in the liver c/ebpα, pineal c/ebpδ, and pdia3 mRNAs. Under LL, the clock and nuclear transcription factor mRNAs did not show any daily variation in the studied organs, with the exception of pineal pdia3. Furthermore, LL had the opposite effect on the levels of melatonin and cortisol, as observed by the increase in pineal Clock, Per2, pparα, and c/ebpα and c/ebpδ mRNAs and decrease in liver Clock, Per2, and pparα mRNAs compared to those under LD. The present findings show that the expression of clock genes is affected by the light across organs and that there is a relation between PPAR, C/EBP, and clock mRNAs; however, the functional role of the individual nuclear transcription factors related to this observation remains to be established in the pineal gland and liver. (Author correspondence: Tihu@nifes.no ).

Diurnal expression of clock genes in pineal gland and brain and plasma levels of melatonin and cortisol in Atlantic salmon parr and smolts.

In Atlantic salmon, the preadaptation to a marine life, i.e., parr-smolt transformation, and melatonin production in the pineal gland are regulated by the photoperiod. However, the clock genes have never been studied in the pineal gland of this species. The aim of the present study was to describe the diurnal expression of clock genes (Per1-like, Cry2, and Clock) in the pineal gland and brain of Atlantic salmon parr and smolts in freshwater, as well as plasma levels of melatonin and cortisol. By employing an out-of-season smolt production model, the parr-smolt transformation was induced by subjecting triplicate groups of parr to 6 wks (wks 0 to 6) under a 12 h:12 h light-dark (LD) regime followed by 6 wks (wks 6 to 12) of continuous light (LL). The measured clock genes in both pineal gland and brain and the plasma levels of melatonin and cortisol showed significant daily variations in parr under LD in wk 6, whereas these rhythms were abolished in smolts under LL in wk 12. In parr, the pineal Per1-like and Cry2 expression peaked in the dark phase, whereas the pineal Clock expression was elevated during the light phase. Although this study presents novel findings on the clock gene system in the teleost pineal gland, the role of this system in the regulation of smoltification needs to be studied in more detail.

Induction of circadian rhythm in cultured human mesenchymal stem cells by serum shock and cAMP analogs in vitro.

Circadian clocks have been shown to operate developmentally in mouse and human hematopoietic stem and progenitor cells in vivo, but little is known about their possible oscillations in vitro. Here, we show that repeated circadian oscillations could be induced in both cultured bone marrow-derived mesenchymal- and adipose-derived stem cells (MSCs and ASCs, respectively) by serum shock. In particular, the novel finding of rhythmic clock gene expression induced by cAMP analogs showed similarities as well as differences to serum-induced oscillations. Rhythmic PER1 expression was found in serum-shocked MSCs, suggesting the phosphorylation status of PER1 is important for its activity in circadian rhythms. Furthermore, immunofluoresent staining showed that the localization of PER1 was dependent on the level of PER1 expression. These inducible self-sustained circadian clocks in primary cultures of human MSCs in vitro with rhythmic changes in expression levels, phosphorylation, and localization of clock protein, PER1, may be of importance for maintaining the induced oscillations in stem cells. Therefore, the established cell models described here appear to be valuable for studying the molecular mechanism driving and coordinating the circadian network between stem and stromal cells.

Stress-induced expression of protein disulfide isomerase associated 3 (PDIA3) in Atlantic salmon (Salmo salar L.).

In mammals, disulfide isomerase associated 3, PDIA3, is a member of the endoplasmic reticulum (ER) stress proteins, which can be induced by oxidative stress; however, its role in relation to stress regulation is still unknown in fish. Here, we report the cloning of a coding region of PDIA3 from the Atlantic salmon. PDIA3 mRNA expression was evaluated in the liver of Atlantic salmon exposed to environmental hyperoxia stress and toxic perfluorooctane sulfonate (PFOS) exposure stress. The PDIA3 sequence contained two PDI-typical thioredoxin active sites of WCGHC and shared approximately 70% identity with mammalian PDIA3, and its mRNA was primarily expressed in the liver. PDIA3 was significantly increased in the liver of Atlantic salmon exposed to hyperoxic water during smoltification. Also Mn superoxide dismutase (Mn-SOD) and CCAAT/enhancer binding protein (C/EBP), other markers of oxidative stress, were upregulated by hyperoxia. Furthermore, PFOS exposure of hepatocytes resulted in elevated mRNA expression of PDIA3, Mn-SOD and C/EBPdelta as well as peroxisome proliferator-activated receptor gamma (PPARgamma). These results indicate a signaling connection between oxidative stress and ER stress. PDIA3 and C/EBPdelta may be valuable markers in fish for exposure and effect to environmental stress.

Leptin in human acute myelogenous leukemia: studies of in vivo levels and in vitro effects on native functional leukemia blasts.

BACKGROUND AND OBJECTIVES: Leptin receptors can be expressed by acute myelogenous leukemia (AML) cells, but the functional effects of leptin on native AML blasts have not been characterized in detail. We investigated systemic leptin levels in AML patients and in vitro effects of leptin on cultured AML blasts. DESIGN AND METHODS: Serum leptin levels were compared for patients with untreated AML and healthy controls. Native AML blasts were derived from a large group of consecutive patients, and effects of leptin on proliferation (suspension cultures and colony formation), constitutive cytokine secretion, differentiation and apoptosis regulation were assayed in vitro. RESULTS: Systemic leptin levels were decreased in patients with untreated AML, and leptin levels in acute leukemia patients were not altered during severe chemotherapy-induced cytopenia and complicating febrile neutropenia. In vitro studies demonstrated that leptin increased AML blast release of interleukin (IL) 1beta, IL6, tumor necrosis factor (TNF) alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). This enhancing effect showed no correlation with CD34 expression and was not dependent on the presence of serum, induction of differentiation or alteration of caspase 3 activity with decreased in vitro apoptosis. Leptin also increased spontaneous AML blast proliferation, whereas divergent effects on blast proliferation were observed in the presence of exogenous cytokines. The in vitro effects were usually observed at concentrations exceeding the systemic levels. INTERPRETATION AND CONCLUSIONS: Our results suggest that systemic leptin levels alone do not have a major influence on native AML blasts, but the systemic levels in combination with local leptin release in the bone marrow may affect the functional characteristics of these cells.

Functional inactivation of the SR family of splicing factors during a vaccinia virus infection.

SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.

Flt3-mediated signaling in human acute myelogenous leukemia (AML) blasts: a functional characterization of Flt3-ligand effects in AML cell populations with and without genetic Flt3 abnormalities.

BACKGROUND AND OBJECTIVES: Intracellular signaling initiated via Flt3 seems important in both leukemogenesis and chemosensitivity in acute myelogenous leukemia (AML). Flt3 is activated by binding of its natural Flt3-ligand (Flt3-L), but Flt3 genes with internal tandem duplications (Flt3-ITD) or Asp(D)-835 point mutations encode molecules with constitutive activation. The aim of this study was to compare functional effects of exogenous Flt3-L on AML blast populations with and without genetic Flt3 abnormalities. DESIGN AND METHODS: Native AML blasts were derived from 64 consecutive patients with high blast counts in peripheral blood, and in vitro models were used to characterize the Flt3-L effects. RESULTS: The Flt3 protein levels showed a similar wide variation between AML blast populations with and without genetic Flt3 abnormalities. Flt3-L was an autocrine growth factor only for 2 patients. Flt3-ITD+ AML cells had lower responsiveness to exogenous cytokines than cell populations without Flt3 abnormalities, but exogenous Flt3-L increased blast proliferation both for patients without Flt3 abnormalities and patients with Flt3-ITD as well as D835 mutations. This enhancement was observed even in the presence of other exogenous cytokines and included clonogenic AML progenitors. Flt3-L inhibited proliferation only for 1 patient, but had divergent effects on AML blast cytokine release. Flt3-L affected AML blast differentiation (inhibition of erythroid colonies, increased neutrophil granulation) only in a minority of patients, whereas it had an anti-apoptotic effect for a larger subset of patients. INTERPRETATION AND CONCLUSIONS: Intracellular signaling initiated by Flt3 ligation modulates the functional phenotype for native human AML blasts both with and without genetic Flt3 abnormalities.