Jon Lømo

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.

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.

Differential Expression of Bcl‐2 Homologs in Human CD34+ Hematopoietic Progenitor Cells Induced to Differentiate into Erythroid or Granulocytic Cells

The Bcl‐2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl‐2 homologs upon stimulation of CD34+ human hematopoietic progenitor cells. CD34+ cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G‐CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo‐ and SCF‐stimulated cells, we found a marked increase in the level of Bcl‐xL protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl‐xL protein expression was downregulated in G‐CSF‐ and SCF‐stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl‐1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl‐1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl‐2 expression was significantly downregulated in both Epo and SCF and G‐CSF‐ and SCF‐stimulated cells.

Low-potency glucocorticoid hydrocortisone has similar neurotoxic effects as high-potency glucocorticoid dexamethasone on neurons in the immature chicken cerebellum

High-potency glucocorticoids (GC) are used in the prophylaxis and treatment of neonatal bronchopulmonal dysplasia, but there is concern about side effects on the developing brain. Recently, the low-potency GC hydrocortisone (HC) has been suggested as an alternative to high-potency GC. We compared the neurotoxic effects of HC with the high-potency GC dexamethasone (DEX) in chicken cerebellum. A single dose of GC was injected into the egg at embryonic day 16 and the death of granule neurons in histologic sections of the cerebellar cortex was examined 24 h later. DEX and HC showed a similar dose-dependent induction of morphological apoptosis and caspase-3 activation in the internal granular layer. A doubling of the apoptosis rate compared to the basal rate was seen for the highest dose of DEX (5 mg/kg) and medium dose of HC (1 mg/kg). In cultures of embryonic chicken cerebellar granule cells, DEX and HC increased cell death and induced rapid caspase-3 activation in a similar dose-dependent manner. Transfection of granule cells with a luciferase reporter gene revealed that the dose needed for the activation of gene transcription (classical signalling pathway) with DEX was much lower than for HC. In conclusion, HC does not present itself as a safer drug than DEX in this model. In addition, it appears that DEX and HC induce apoptosis in immature granule neurons via a non-genomic (non-classical) mechanism.

Dexamethasone induces cell death which may be blocked by NMDA receptor antagonists but is insensitive to Mg2+ in cerebellar granule neurons.

Since dexamethasone may elevate the Ca2+ influx through NMDA receptors, we have investigated mechanisms of dexamethasone toxicity in rat cerebellar granule neurons. Dexamethasone concentrations over 0.1 microM induced cell death that reached about 20% of the death induced by glutamate. Dexamethasone-induced cell death was reduced by more than 80% by the mineralocorticoid antagonist RU 28318 or the NMDA receptor antagonists MK 801 and CGP 39551, whereas RU 28318 rescued only approximately 30% of cells treated with glutamate, indicating that dexamethasone requires NMDA receptors to induce acute neuronal toxicity and that a fraction of the neurons showed this toxicity. Mg2+ reduced the cell death induced by glutamate at potassium concentrations of 1 mM and 5 mM, but not at 25 mM. In contrast, cell death induced by dexamethasone was not significantly reduced by Mg2+ in any of the potassium concentrations. Both glutamate and dexamethasone induced toxicity with translocation of the apoptosis inducer NGFI-B to the mitochondria seen after 30 min-2 h concomitant with activation of apoptosis inducing factor (AIF) and caspase-3. In conclusion, dexamethasone induces a rapid toxicity which is blocked by NMDA receptor antagonists other than Mg2+, and involves mitochondrial apoptosis inducer NGFI-B.

Chicken cerebellar granule neurons rapidly develop excitotoxicity in culture

Rat cerebellar granule cell culture is widely used as a model to study factors that control neuronal differentiation and death (e.g. excitotoxicity). However, a main drawback of this model is its dependence on depolarizing culture condition (25 mM potassium). In addition, it is quite expensive to maintain and requires animal facilities. Here we report that cerebellar granule neuron cultures from chicken may be used as an alternative model to study excitotoxicity. Surprisingly, fetal chicken cells may be grown in a physiological potassium concentration (5 mM potassium). They develop excitotoxicity rapidly in culture (fully developed at 3 days in vitro), and respond to glutamate excitotoxicity similar to rat cultures (ROS production and activation of caspase-3).

Involvement of ICE (Caspase) Family in γ‐Radiation‐Induced Apoptosis of Normal B Lymphocytes

Normal lymphocytes are highly sensitive to the damaging effects of ionizing radiation, and undergo cell death by apoptosis. We have investigated the possible involvement of the Interleukin‐1β‐converting enzyme (ICE) (Caspase) protease family, which appears to play an important role as intracellular mediator of apoptosis. Resting B lymphocytes isolated from human peripheral blood were irradiated (6 Gy) and cultured for 24 h, resulting in 25 ± 5.1% apoptotic cells, as measured by the TUNEL assay (mean ± SD, n = 6). Addition of the ICE family inhibitor Z‐VAD.fmk (50 μM) completely inhibited apoptosis (2.0 ± 1.5% at 24 h). By using fluorogenic substrates containing the peptide recognition sequences DEVD and YVAD, the type of ICE family protease involved was examined more closely. A marked transient increase in DEVD‐, and absent YVAD‐cleavage activity indicated the involvement of a CPP32‐like protease, not an ICE‐like protease. Western blot analysis demonstrated that untreated B lymphocytes expressed the proform of the ICE family members CPP32 and ICH1L, but no detectable ICE. The induction of cell death by radiation was accompanied by the activation of CPP32 as shown by the cleavage of the proform to the active subunit p17, and the cleavage of poly(ADP‐ribose) polymerase (PARP), one of the known substrates of CPP32. In contrast, no activation of ICH1L could be detected. These results indicate the involvement of CPP32 and possibly other CPP32‐like proteases in radiation‐induced apoptosis of resting B lymphocytes.

Fine-needle aspiration cytology in nonpalpable mammographic abnormalities in breast cancer screening: results from the breast cancer screening programme in Oslo 1996-2001

Fine-needle aspiration cytology (FNAC) of nonpalpable mammographic lesions has been under attack from two sides for some years. There has been much discussion and controversy as to the ability to differentiate between in situ and invasive carcinomas in cytological material. A further issue is that of optimal sampling to obtain adequate cell material in sufficient quantity. We present the results of FNAC from 832 nonpalpable mammographic abnormalities detected in the course of the breast cancer screening programme in Oslo during 1996-2001. In 11.6% of cases the smears were inadequate, and there were 7% false negatives (FN) and 1.3% false positives. Of the FN, 64% represented microcalcifications and 86% were due to sampling errors. Absolute sensitivity was 74%, complete sensitivity 88% and specificity 88%. In 255 carcinomas a cytological diagnosis of them as in situ or invasive was made. In 93% of the invasive cases (190/205) these had been correctly identified as invasive on FNAC. In 78% of cases proper follow-up could be resolved by cytology/radiology alone. Suboptimal sampling and localization remains the main cause of FN FNAC results. Problems in differentiating between in situ and invasive breast carcinomas can be significantly reduced by applying strict criteria for in situ lesions.

Retinoic acid induces apoptosis of human CD34+ hematopoietic progenitor cells: Involvement of retinoic acid receptors and retinoid X receptors depends on lineage commitment of the hematopoietic progenitor cells

Retinoids are bifunctional regulators of growth and differentiation of hematopoietic cells. In this study we explored the effects of retinoic acid (RA) on apoptosis of human CD34+ hematopoietic progenitor cells isolated from normal bone marrow. RA (100 nM) induced an increase in the percentage of dead cells from 24% to 44% at day 6 (p < 0.05, n = 6) as compared to control cells cultured in medium alone. The effect was dose dependent and appeared relatively late. Significant differences were observed from day 4 onward. Apoptosis, or programmed cell death, was demonstrated as the mode of cell death by using the TUNEL assay, which detects single strand nicks in DNA, or by the Nicoletti technique demonstrating a subdiploid population by DNA staining. RA previously was found to inhibit granulocyte colony-stimulating factor--and not granulocyte-macrophage colony-stimulating factor--stimulated proliferation of CD34+ cells. However, we found that RA opposed anti-apoptotic effects of G-CSF and GM-CSF on CD34+ cells (G-CSF: 8% dead cells at day 6; G-CSF + RA: 20%; GM-CSF: 12%; GM-CSF + RA: 27%). Moreover, RA induced apoptosis of CD34+ cells and CD34+CD71+ cells stimulated with erythropoietin. To explore the receptor signaling pathways involved in RA-induced apoptosis, we used selective ligands for retinoic acid receptors (RARs; RO13-7410) and retinoid X receptors (RXRs; RO 25-6603). We found that RARs were involved in RA-mediated apoptosis of myeloid progenitor cells, whereas RARs as well as RXRs were involved in RA-mediated apoptosis of erythroid progenitor cells.

Glucocorticoids dexamethasone and hydrocortisone inhibit proliferation and accelerate maturation of chicken cerebellar granule neurons.

Glucocorticoid (GC) treatment in premature infants may have detrimental effects on the immature brain. Here we show that GCs dexamethasone (Dex) and hydrocortisone (HC) reduce proliferation and induce differentiation of chicken embryo cerebellar neurons in vivo and in vitro. Granule neurons incorporating bromodeoxyuridine were reduced in the internal granular layer (IGL) after 24-h exposure to both substances on embryonic day 17, with Dex about 100-fold more potent than HC. The effects were blocked by GR antagonist RU 38486. Both GCs also increased the expression of neuronal differentiation markers microtubule-associated protein 2 (Map2) and neuronal nuclei protein (NeuN), measured by western blotting of whole cerebellar lysates and immunohistochemistry, respectively. Treatment of cerebellar granule neuron cultures with both GCs significantly reduced the percentage of proliferating-cell nuclear antigen (PCNA) positive neurons and increased NeuN positive neurons, with similar dose-response relationship as in vivo. The cytostatic agent cytosine arabinoside showed comparable effects both on proliferation and differentiation. In conclusion, the effects of Dex and HC on chicken cerebellar granule neuron proliferation are GR mediated and reflect their pharmacological potency. In addition, the effects on differentiation may be related to a cell cycle block per se, since cytosine arabinoside mimicked the effect of the GCs.