Leptin Reverts Pro-Apoptotic and Antiproliferative Effects of α-Linolenic Acids in BCR-ABL Positive Leukemic Cells: Involvement of PI3K Pathway

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced

Characterization of Spontaneous Bone Marrow Recovery after Sublethal Total Body Irradiation: Importance of the Osteoblastic/Adipocytic Balance

Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units – fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursor's proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions

Influence in vitro des adipocytes et de la leptine sur des cellules de leucémie myéloïde chronique

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukemia (CML). For this study, we postulated that adipocytes and lipids could be involved. To test this hypothesis we realized co-cultures of adipocytes and two BCR-ABL positive cell lines (PCMDS and K562) using T cell (Jurkat) and stroma cell (HS-5) lines as controls. We also treated leukemic cell lines with 50 to 250 µM of stearic, oleic, linoleic or alpha-linolenic acids with and without leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression were tested after 24h, 48h and 72h. In vitro, adipocytes induced a decrease in chronic myeloid leukemic cell (CML) proliferation and an increase in lipid accumulation in leukemic cells. On the one hand, CML cell lines induced adipocytes but not HS-5 cell death. Chromatography analysis showed that cells of BM microenvironment were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased the expression of Bcl-2, whereas oleic and linoleic acids had no effects. In contrast, alpha-linolenic acid decreased the proliferation and the survival of CML cell lines; it also decreased BCL-2 and OB-R expression in these cell lines. The effect of alpha-linolenic acids seemed to be related to PI3K pathway and Bcl-2 inhibition. Leptin production (344.68±59.7 pg/ml) was also detected in the co-culture medium. Leptin alone had no effect. But in the presence of leptin, the effect of alpha-linolenic acid on proliferation, survival, OB-R and BCl-2 was reduced

SDF-1 expression in irradiated bone marrows and CFU-Fs.

<p><b>A:</b> Labelling with anti-sdf1 antibodies is more intense in bone marrows from day-1 irradiated mice (<b>Ab</b>) compared to control ones (<b>Aa</b>) (original magnification ×200). <b>B:</b> Elisa quantification of sdf-1 in the supernatant of irradiated CFU-Fs showed an increase in sdf-1 until 4 hours after irradiation. <b>C:</b> The level of mRNA expression is not significantly different in control and irradiated CFU-Fs. Data are presented as mean values ± SEM of at least three independent experiments. ***, p<.001 as assessed by one way analysis of variance (ANOVA).</p

Differentiation of hematopoietic precursors in direct contact with CFU-Fs from irradiated (<i>black</i>) and non-irradiated (<i>grey</i>) bone marrows.

<p><b>A:</b> Immunophenotyping shows a myelo-monocytic (CD11b), a granulocytic (Ly-6G) and a B-lymphoid (CD45R/B220) engagement of hematopoietic precursors in lineages. <b>B:</b> The differentiation of mature neutrophils during the co-culture assay occurs earlier when precursors are co-cultured on “day 3” CFU-Fs. <b>C:</b> Inhibition of the contact in transwell conditions induces a significant reduction in the differentiation of the Gr1+ (<b>Ca</b>) and CD11b+ (<b>Cb</b>) lineages. Data are presented as mean values ± SEM of at least three independent experiments. **, p<.01; *, p<.05 as assessed by one way analysis of variance (ANOVA).</p

Expression of osteogenic and adipocytic markers in CFU-Fs after irradiation.

<p><b>A</b>: Kinetics of expression of osteoblastic markers mRNAs (<b>Aa:</b> Runx2, <b>Ab:</b> Osteopontin and <b>Ac:</b> Osteocalcin) after irradiation. <b>B:</b> Kinetics of expression of the adipocytic marker PPARγ mRNA after irradiation. <b>C:</b> Kinetics of expression of the adipocytic protein PPARγafter irradiation. <b>D:</b> Kinetics of expression of the osteoblastic protein Runx2after irradiation. Data are presented as mean values ± SEM of at least three independent experiments. ***, p<.001; **, p<.01; *, p<.05 as assessed by one way analysis of variance (ANOVA).</p

Proliferation and survival of hematopoietic precursors in co-culture with CFU-Fs from irradiated (<i>grey</i>) and non-irradiated (<i>black</i>) bone marrows.

<p><b>A:</b> Proliferation of hematopoietic precursors tested by ³H incorporation. <b>B:</b> Survival of hematopoietic precursors tested by Annexin-PI labeling. Data are presented as mean values ± SEM of at least three independent experiments. **, p<.01 as assessed by one way analysis of variance (ANOVA).</p

Expression of neuropilin-1 <i>in</i> CFU-Fs.

<p><b>A:</b> Immunohistochemistry for Neuropilin-1 expression in irradiated mice CFU-Fs (magnification ×200). <b>B:</b> Evolution of neuropilin-1 mRNAs in <i>in vitro</i>-irradiated CFU-Fs. Data are presented as mean values ± SEM of at least there independent experiments. **, p<.01; *, p<.05 as assessed by one way analysis of variance (ANOVA).</p