Heidi Kiil Blomhoff

Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFA) may reduce cell multiplication in cultures of normal, as well as transformed, white blood cells. We assessed the sensitivity of 14 different leukemia cell lines to PUFA by measuring cell number after 3 days of incubation. Ten of the examined cell lines were sensitive to 30, 60 and/or 120 μM of arachidonic, eicosapentaenoic and docosahexaenoic acid, whereas four cell lines were resistant. The sensitivity to PUFA was not associated with any particular cell lineage, clinical origin or specific mRNA pattern of bcl-2 and c-myc. Effects on cell viability were assessed by studying cell membrane integrity, DNA fragmentation and cell morphology. The sensitive cell lines Raji and Ramos died by necrosis and apoptosis, respectively, during incubation with eicosapentaenoic acid, whereas the viability of the resistant U-698 cell line was unaffected. The effects of EPA on Raji cells, was counteracted by vitamin E, indicating that lipid peroxidation was involved. However, apoptosis induced by eicosa- pentaenoic acid in Ramos cells, was unaffected by vitamin E, as well as eicosanoid synthesis inhibitors. In conclusion, our results indicate that a majority of leukemia cell lines are sensitive to PUFA. This sensitivity may be caused by induction of apoptosis or necrosis by very long-chain polyunsaturated fatty acids.

Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34+ hematopoietic progenitor cells

OBJECTIVE Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells. MATERIALS AND METHODS CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed. RESULTS The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K. CONCLUSIONS Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.

Retinoic Acid Stimulates the Cell Cycle Machinery in Normal T Cells: Involvement of Retinoic Acid Receptor-Mediated IL-2 Secretion

The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell-stimulating agents. This was reflected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27Kip1, increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-affinity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs.

Activation of the CAMP signaling pathway increases apoptosis in human B‐precursor cells and is associated with downregulation of Mcl‐1 expression

During B‐ and T‐cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B‐precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10+ cells in a dose‐dependent manner (19%–94% with 0–1,000 μM forskolin after 48 hours incubation, IC50 = 150 μM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8‐chlorophenylthio‐cAMP (8‐CPT‐cAMP). Specific involvement of cAMP‐dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp‐isomer of 8‐bromo‐adenosine‐ 3′, 5′‐ monophosphorothioate (Rp‐8‐Br‐cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp‐8‐Br‐cAMPS. Furthermore, we investigated the expression of Bcl‐2 family proteins. We found that treatment of the cells with forskolin or 8‐CPT‐cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl‐1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl‐2, Bcl‐xl, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8‐CPT‐cAMP (1.3‐fold, n = 6, P = 0.045) compared to B‐precursor cells, and a smaller decrease in Mcl‐1 levels (1.5‐fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B‐progenitor and mature B cells and suggest that cAMP‐increased apoptosis could be mediated, at least in part, by a decrease in Mcl‐1 levels. J. Cell. Physiol. 180:71–80, 1999.

Activation of cAMP signaling inhibits DNA damage-induced apoptosis in BCP-ALL cells through abrogation of p53 accumulation

In lymphocytes, the second messenger cyclic adenosine monophosphate (cAMP) plays a well-established antiproliferative role through inhibition of G(1)/S transition and S-phase progression. We have previously demonstrated that, during S-phase arrest, cAMP inhibits the action of S phase-specific cytotoxic compounds, leading to reduction in their apoptotic response. In this report, we provide evidence that cAMP can also inhibit the action of DNA-damaging agents independently of its effect on S phase. Elevation of cAMP in B-cell precursor acute lymphoblastic leukemia cells is shown to profoundly inhibit the apoptotic response to ionizing radiation, anthracyclins, alkylating agents, and platinum compounds. We further demonstrate that this effect depends on the ability of elevated cAMP levels to quench DNA damage-induced p53 accumulation by increasing the p53 turnover, resulting in attenuated Puma and Bax induction, mitochondrial outer membrane depolarization, caspase activation, and poly(ADP-ribose) polymerase cleavage. On the basis of our findings, we suggest that cAMP levels may influence p53 function in malignant cells that retain wild-type p53, potentially affecting p53 both as a tumor suppressor during cancer initiation and maintenance, and as an effector of the apoptotic response to DNA-damaging agents during anticancer treatment.

Vitamin A status significantly alters nuclear factor-κB activity assessed by in vivo imaging

Our study aimed to investigate, in vivo, the relationship between vitamin A status and NF‐κ B activity, a transcription factor central in regulating inflammatory and immune responses. We used a novel transgenic murine NF‐κ B‐luciferase reporter model that enabled molecular imaging of NF‐κ B activity in live mice via an intensified image‐capture apparatus. Whole‐body luminescence, which reflects overall NF‐κ B activity, was elevated 2.2‐fold in vitamin A‐ deficient (VAD) mice compared with control mice. Specifically, NF‐κ B activity in VAD mice was increased 1.8‐fold in the lymph nodes and 1.4‐fold in the thymus and, NF‐κ B induction in UVB radiation‐exposed skin was also enhanced in VAD mice compared with control mice. The administration of all‐trans retinoic acid to VAD mice resulted in a transient reduction in NF‐κ B activity and, conversely, a single dose of the RAR‐pan‐antagonist, AGN 194310, administered to control mice, led to a marked, transient induction of whole‐body luminescence. Our results suggest that vitamin A status, and vitamin A itself, affects NF‐κ B activity in vivo and that the elevated NF‐κ B activity in VAD may be a mechanism underlying some of the features of VAD syndrome.

Regulation of B cell proliferation and differentiation by retinoic acid

Vitamin A protects against development of infectious diseases, and B cells are important players in this process. Keys to the protective role of retinoic acid (RA) against infections appear to be its ability to enhance antibody responses against T-cell dependent and independent type 2 antigens, as well as to locally stimulate IgA production in mucosal tissues. The elucidation of molecular mechanisms involved in RA-mediated regulation of proliferation and differentiation of B cells not only helps us to understand how RA differentially regulates subsets of B cells, but might also lead to more targeted treatment of selected immune disorders and B cell malignancies.

Combined Action of ERK and NFκB Mediates the Protective Effect of Phorbol Ester on Fas-induced Apoptosis in Jurkat Cells

The mechanisms whereby phorbol esters antagonize Fas-induced apoptosis in Jurkat T cells are poorly defined. In the present study, we report that protection from Fas-induced apoptosis by 12-O-tetradecanoylphorbol 13-acetate (TPA) is dependent on both ERK and NFκB activation. First, we showed that two specific mitogen-activated protein kinase/ERK kinase-inhibitors, PD98059 and U0126, both counteracted TPA-mediated suppression of Fas-induced apoptosis. Moreover, the dose-dependence of U0126-mediated inhibition of ERK phosphorylation correlated with that of reversion of the anti-apoptotic effect of TPA. Second, we observed an excellent correlation between repression of TPA-induced NFκB activation by an irreversible inhibitor of IκBα phosphorylation, BAY11–7082, and its ability to abrogate TPA-induced suppression of Fas-mediated apoptosis. Furthermore, we located the anti-apoptotic effect of both ERK and NFκB to lie upstream of the mitochondrial membrane potential depolarization event. Finally, although each inhibitor at optimal, non-toxic concentration by itself only partly reversed TPA-mediated repression of apoptosis, the combination of U0126 and BAY11–7082 completely abolished the anti-apoptotic effect of TPA. Together these findings suggest that TPA-induced activation of ERK and NFκB are parallel events that are both required for maximal inhibition of Fas-induced apoptosis in Jurkat T cells.

RAR-, not RXR, ligands inhibit cell activation and prevent apoptosis in B-Lymphocytes

We have previously shown that retinoids inhibit activation of human peripheral blood B‐lymphocytes. In the present paper, we wished to explore the involvement of nuclear retinoid‐specific receptors in this process by using ligands specific for the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We found that the RAR‐specific ligand TTAB reduced anti‐IgM‐induced B‐cell activation in a dose‐dependent manner. Thus, at 100 nM of TTAB, DNA synthesis was reduced by approximately 60%. In contrast, the RXR‐selective ligand SR11217 had no effect on DNA synthesis. Similar findings were obtained when the expression of the activation antigen CD71 (appears late in G1) was examined. The role of retinoids in apoptosis of resting peripheral blood B‐lymphocytes was examined using the same receptor‐selective ligands. Again, we found that the RAR‐selective ligands were more potent effectors than were the RXR‐selective ligands. In spite of the inhibitory effects of retinoids on B‐cell proliferation, the same retinoids significantly promoted the survival of the cells. Thus, 10 nM TTAB significantly reduced spontaneous apoptosis of in vitro cultured B‐cells at day 3 from 45% to 30%, as determined by vital dye staining and DNA end‐labeling. Again, the RXR‐specific ligand SR11217 had no effect. Interestingly, we found that CD40 ligand was able to potentiate the retinoid‐mediated inhibition of apoptosis. By reverse transcriptase polymerase chain reaction (PCR), we found that peripheral blood B‐lymphocytes expressed RARα, RARγ, and RXRα, but not RARβ, RXRβ, or RXRγ. Hence, the lack of effect of the RXR‐specific ligand SR11217 on growth and apoptosis was not due to absence of RXRs. In conclusion, the ability of retinoids to inhibit growth and prevent apoptosis of normal human B‐lymphocytes indicates a dual role of retinoids in this cell compartment, and it appears that both effects of retinoids are mediated via RARs and not RXRs. J. Cell. Physiol. 175:68–77, 1998.

Retinoic acid dampens LPS-induced NF-κB activity: results from human monoblasts and in vivo imaging of NF-κB reporter mice.

Bacterial lipopolysaccharide (LPS) is a major inducer of systemic inflammatory reactions and oxidative stress in response to microbial infections and may cause sepsis. In the present study, we demonstrate that retinoic acid inhibits LPS-induced activation in transgenic reporter mice and human monoblasts through inhibition of nuclear factor kappaB (NF-kappaB). By using noninvasive molecular imaging of NF-kappaB luciferase reporter mice, we showed that administration of retinoic acid repressed LPS-induced whole-body luminescence, demonstrating in vivo the dynamics of retinoic acids ability to repress physiologic response to LPS. Retinoic acid also inhibited LPS-induced NF-kappaB activity in the human myeloblastic cell line U937. Retinoic-acid-receptor-selective agonists mimicked - while specific antagonists inhibited - the effects of retinoic acid, suggesting the involvement of nuclear retinoic acid receptors. Retinoic acid also repressed LPS-induced transcription of NF-kappaB target genes such as IL-6, MCP-1 and COX-2. The effect of retinoic acid was dependent on new protein synthesis, was obstructed by a deacetylase inhibitor and was partly eliminated by a signal transducer and activator of transcription-1 (STAT1)/methyltransferase inhibitor, indicating that retinoic acid induces a new protein, possibly STAT1, that is involved in inhibiting NF-kappaB. This provides more evidence for retinoic acids anti-inflammatory potential, which may have clinical implications in terms of fighting microbial infections.