AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

Study of the B cell memory compartment in common variable immunodeficiency.

We evaluated the B cell memory pool among blood B cells from 20 patients with common variable immunodeficiency (CVID). CD27+ B cell number was normal or increased in 6 patients (with 95% CD27+ B cells in 1 patient) and decreased in 14 patients. In 13 or 15 patients studied, the CD27 molecule was detectable on less than 50% IgG or IgA B cells, indicating a defect in the maturation of these memory cells. Within the group of patients with a low number of CD27+ B cells, no up-regulation of this molecule was observed after in vitro stimulation of purified B cells from 3 of 5 patients studied, suggesting an intrinsic B cell defect. In addition, ligation of the CD27 molecule was unable to trigger terminal differentiation of purified B cells in 1 of 2 cases with a large number of CD27+ B cells. Finally, the CD27 ligand was normally expressed on activated T cells in only 5 of 14 patients studied. These data confirm the heterogeneity of immunological defects in patients with CVID. Abnormal expression and/or function of the CD27-CD70 members of the TNF/TNF receptor family contribute to the immunological defect.

Functional interactions between bile acids, all-trans retinoic acid, and 1,25-dihydroxy-vitamin D3 on monocytic differentiation and myeloblastin gene down-regulation in HL60 and THP-1 human leukemia cells.

Bile acids were shown previously to inhibit proliferation and to induce monocytic differentiation in HL60 human acute promyelocytic leukemia cells (A. Zimber et al., Int. J. Cancer, 59: 71-77, 1994). In this report, we hypothesized that bile acids may exert a positive cooperativity with two known inducers of leukemic cell differentiation, all-trans retinoic acid and 1,25(OH)2-vitamin D3. Our results provide evidence that bile acids induced the monocytic differentiation of HL60 and THP-1 human leukemia cells exposed to ineffective concentrations of these inducers. The protein kinase C (PKC) inhibitors H-7 (10 and 20 microM) and staurosporine (5 and 20 nM) modulated the effects of bile acids on HL60 cell differentiation. Most interestingly, bile acids are shown herein to down-regulate the expression of the serine protease myeloblastin gene involved in the differentiation of myeloid hematopoietic cells. In agreement with the recent identification of nuclear receptors for bile acids, our data suggest that functional interactions between nuclear bile acid signaling pathways, PKC, and nuclear receptors for retinoic acid and vitamin D3 are involved in the down-regulation of the myeloblastin gene and the induction of cell differentiation in human leukemic cells.

Protein synthesis is involved in the modulation of the level of the phosphoenolpyruvate carboxykinase mRNA by changes in cell volume in isolated rat hepatocytes.

The mechanism of action of hydration state was studied on phosphoenolpyruvate carboxykinase (PCK) gene expression in isolated rat hepatocytes. Hypoosmolarity decreased the level of the PCK mRNA after a lag period of about 60 min. The decreasing effect of hypoosmolarity was totally blocked by inhibitors of both protein synthesis and gene transcription. Moreover, hypoosmolarity specifically increased the synthesis of a 45000 Mr protein, which decreased in the presence of inhibitors of transcription. A close relationship between the synthesis of the 45000 Mr protein and the decrease in the PCK mRNA level was observed, suggesting that this protein might potentially be involved in the regulation of the level of the PCK mRNA by cell swelling.

Identification of a novel Ca2+-stimulated S6-kinase in rat liver.

Extracellular calcium addition transiently stimulated two S6 peptide kinase activities in isolated rat hepatocytes. Mono Q chromatography revealed that the activities eluting at 0.15 M NaCl and 0.18 M NaCl were stimulated 4-fold and 2-fold, respectively. The kinase stimulated by calcium was a 40000-Mr S6 peptide kinase, as demonstrated by partial purification from whole liver. The protein kinase did not crossreact with antibodies directed against the N- or C-terminal part of p70 ribosomal S6 kinase (p70(S6K)) and the C-terminal part of p90 ribosomal S6 kinase (p90(rsk)). Following digestion of 40000-Mr S6 peptide kinase with trypsin, six peptides were sequenced. There was no similarity with the sequences of p70(S6K) and p90(rsk). Moreover, the obtained sequences could not be identified in the SwissProt or EMBL-genebank databases, suggesting that 40000-Mr S6 peptide kinase probably represents a novel protein kinase.

Glutamine inhibits the lowering effect of glucose on the level of phosphoenolpyruvate carboxykinase mRNA in isolated rat hepatocytes.

The expression of phosphoenolpyruvate carboxykinase (P-pyruvate CK) was shown to be decreased by hypoosmolarity and increased by glutamine in perfused liver from fed rats [Newsome, W. P., Warskulat, U., Noe, B., Wettstein, M., Stoll, B., Gerok, W. & Häussinger, D. (1994) Biochem, J. 304, 555-560]. This work was undertaken to specify the mechanisms of glutamine action, using isolated hepatocytes from rats that had been starved for 24 h. At low concentrations (up to 5 mM), glutamine elicited a decrease in the level of P-pyruvate CK mRNA through cell swelling and, at higher concentrations, an increase in the mRNA level was observed. Experiments with combinations of glucose and glutamine or glucose and various amino acids demonstrated that glutamine counteracted the inhibitory effect of glucose on P-pyruvate CK mRNA at a transcriptional, level, and strongly suggested that the amide group of glutamine was involved in this effect. The metabolism of glucose was required for the reinforcement of the apparent stimulatory effect of glutamine, as demonstrated by the use of various sugars. Glucosamine, but not mannosamine, increased the level of P-pyruvate CK mRNA, as did glucose plus glutamine. These results suggest that the pathway leading from glucosamine-6-phosphate production might be responsible, at least partly, for the effect observed on P-pyruvate CK mRNA.

Hypoosmolarity and glutamine increased the beta-actin gene transcription in isolated rat hepatocytes.

The mechanism of action of hydration state was studied on beta-actin gene expression in isolated hepatocytes. Results obtained with Northern blot analysis and run on transcription assays show that hypoosmolarity increased and hyperosmolarity decreased the beta-actin mRNA level through a corresponding modulation of the rate of the gene transcription. Glutamine, which is known to induce cell swelling, also increased the beta-actin mRNA level in a dose-dependent manner and induced a stimulation of the beta-actin gene transcription. Thus, cell hydration state regulates gene expression in the liver through a transcriptional mechanism.

Effects of interferon-alpha and -gamma on B cell differentiation in macroglobulinemia.

We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells via an IL-6 autocrine pathway. Here we investigate whether interferon-alpha or -gamma would interfere with B cell differentiation either in patients with IgM gammopathy of undetermined significance (IgM-MGUS) or Waldenström's macroglobulinemia (WM). A 65% inhibition of in vitro B cell differentiation was noted in 8 of 10 patients in the presence of either interferon-alpha or -gamma. Cells from 4 patients (3 IgM-MGUS and 1 WM) were susceptible to both types of interferon while B cell differentiation from 4 patients (3 IgM-MGUS and 1 WM) was inhibited only by one type of interferon. During in vitro culture, IL6 synthesis was unaffected by the presence of interferon alpha or gamma in the 8 cases studied. Likewise, no modulation of the constitutive B cell IL6-R expression from 6 patients studied (4 WM and 2 IgM-MGUS) was observed. These data indicate that interferons did not modify the differentiation of B cells in macroglobulinemia via modulation of the IL6-IL6-R pathway. This is in contrast with the mode of action of interferons in other lymphoid malignancies such as multiple myeloma or chronic lymphocytic leukemia where they directly modulate IL6-production and/or IL6-R expression.

Microcystin-LR induced an inhibition of protein synthesis in isolated rat hepatocytes.

The effect of microcystin-LR, an inhibitor of protein phosphatases PP1 and PP2A, was studied on protein synthesis by measuring the incorporation of labelled amino acid into protein in isolated rat hepatocytes. Microcystin-LR inhibited protein synthesis in the first minutes of the incubation period, and half-maximum effect was obtained at about 60 nM. Such an inhibition was also observed in the presence of different protein phosphatase inhibitors, i.e. okadaic acid, calyculin A and microcystin-RR. This effect was observed in whole hepatocytes, in the supernatant of the post-mitochondrial fraction and in the microsomal fraction. It was independent of a substrate supply and of the labelled amino acid used. Furthermore, this inhibition preceded the previously reported glucose-6-phosphatase activation induced by microcystin-LR [Claeyssens, Chédeville and Lavoinne (1993) FEBS Lett. 315, 7-10].

Inhibition of proliferation and induction of monocytic differentiation on HL60 human promyelocytic leukemia cells treated with bile acids in vitro.

We have tested the effect of several bile acids on the proliferation and differentiation of the HL60 human promyelocytic leukemia cell line in vitro. Deoxycholate, chenodeoxycholate and lithocholic acid caused dose-dependent inhibition of cell proliferation and induction of differentiation along the monocyte/macrophage pathway as determined by morphology, NBT test, non-specific esterase, and staining by monoclonal antibodies against specific cell-surface antigens. Optimal effects were obtained at 100, 75, and 60 microM of the 3 bile acids respectively. Cell-cycle flow-cytometric analysis showed that a substantial fraction of HL60 cells accumulated at the G0/G1 transition. Protein-kinase-C inhibitors such as sphinganine and H-7 inhibited the differentiation-inducing effect of bile acids, suggesting a possible role for PKC in this regulation. When bile acids were combined with non-effective concentrations of all-trans retinoic acid, enhancement of the monocytic differentiation of THP-1 human leukemia cells was observed. Our findings demonstrate induction of tumor-cell differentiation by bile acids, compounds that present minimal undesirable effects in humans.

Interaction of VIP, PACAP and related peptides in normal and leukemic human monocytes and macrophages.

The activation of the cAMP signaling pathway by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP) and related peptides was studied (i) in normal peripheral human monocytes and THP-1 leukemic human monocytes, (ii) in their derived macrophage counterparts respectively obtained after spontaneous differentiation or retinoic acid (RA) treatment, and (iii) in human bronchoalveolar macrophages. In THP-1 monocytes, PACAP increased basal adenylate cyclase activity 5.3-fold, with an affinity 50-times greater than that of VIP or helodermin (Ka = 3.2 x 10(-11) M VIP), whereas in normal peripheral monocytes, PACAP and VIP exhibited similar affinities and only increased cAMP generation 2-fold (EC50 = 10(-9) M). Spontaneous and RA-induced differentiation into normal and leukemic macrophages induced a progressive loss of cAMP production and regulation of superoxide anion production by VIP and related peptides. The neoplastic transformation in THP-1 monocytes and the deficiencies in the cAMP cascade observed during the terminal differentiation of normal and leukemic human macrophages may relate to a differential genetic expression of the VIP/PACAP receptor subtypes, and alterations in the functional activity of the stimulatory and inhibitory Gs/Gi subunits of adenylate cyclase.

Inhibition of protein phosphatases activates glucose-6-phosphatase in isolated rat hepatocytes.

Incubation of hepatocytes in the presence of microcystin-LR, okadaic acid, calyculin A (inhibitors of protein phosphatases PP1 and PP2A) or microcystin-RR (a specific inhibitor of PP2A) activated glucose-6-phosphatase both in the supernatant and in intact or disrupted microsomes. Puromycin, an inhibitor of protein synthesis, totally suppressed this activating effect, suggesting the involvement of protein phosphatases in the regulation of glucose-6-phosphatase synthesis.

Glutamine is a good substrate for glycogen synthesis in isolated hepatocytes from 72 h-starved rats, but not from 24 h- or 48 h-starved rats.

In isolated hepatocytes from 24 h-starved rats, no glycogen synthesis was observed in the presence of glutamine. By contrast, glutamine was the best gluconeogenic substrate to induce glycogen synthesis in isolated hepatocytes from 72 h-starved rats. The effect of glutamine on glycogen synthesis was not accompanied by parallel changes in glucose or lactate production. Glutamine activated glycogen synthase independently of the starvation period; however, the extent of synthase activation was 2-fold higher in isolated hepatocytes from 72 h-starved rats than in hepatocytes from 24 h-starved rats. This increase in synthase activation was associated with increased cell swelling. The rate of glutamine transport was not significantly different in hepatocytes from 24 h- and 72 h-starved rats. By contrast, the intracellular glutamate concentration was 1.5-fold higher after 3 days of starvation in hepatocytes incubated with 5 mM-glutamine. We propose that glutamine may play a key role in the glycogen synthesis observed in vivo after 3 days of starvation.

Metabolism of adenosine through adenosine kinase inhibits gluconeogenesis in isolated rat hepatocytes.

In isolated hepatocytes from fasted rats, 0.5 mM adenosine inhibited gluconeogenesis from glutamine, lactate and pyruvate. This inhibition was due to adenosine conversion through adenosine kinase. An increase in ketone body release was only observed in the presence of lactate or pyruvate, and the two phenomena (i.e. inhibition of gluconeogenesis and increased ketone-body release) were linked. With alanine, dihydroxyacetone or serine as substrates, adenosine did not change gluconeogenesis; however, its conversion through adenosine kinase also inhibited gluconeogenesis. With asparagine as substrate, 0.5 mM adenosine increased gluconeogenesis; this increase was due to adenosine conversion through adenosine deaminase. However, adenosine conversion through adenosine kinase inhibited gluconeogenesis from asparagine. Thus, whatever the substrate used, adenosine conversion through adenosine kinase inhibited gluconeogenesis. The inhibitory effect of adenosine on gluconeogenesis cannot be related to the decrease in Pi concentration and to the increase in ATP pool. Beside its effect on gluconeogenesis, adenosine inhibited ketogenesis measured without added substrate; adenosine conversion through adenosine kinase was also involved in the inhibition of ketogenesis.

Influence of 2-chloroadenosine on the nucleotide content of isolated rat hepatocytes.

2-Chloroadenosine is presumably a non-metabolizable analogue of adenosine; however, this compound induced an increase in the enzymatically measured nucleotide content of isolated rat hepatocytes. HPLC separation and spectral analysis of the peaks showed that this increase may be related to the formation of 2-chloro nucleotides and that the 2-chloro nucleotides appeared in the first minutes of the incubation period. These results demonstrate that 2-chloroadenosine may be metabolized by phosphorylation in rat liver cells.

Discrepancy between in vitro and in vivo passaged U-937 human leukemic cells: tumorigenicity and sensitivity to differentiating drugs.

The treatment of nude mice bearing tumors of transplanted human leukemic cells with drugs known to induce differentiation of the same leukemic cells in vitro does not always affect tumor yield, tumor cell differentiation or nude mice survival. We have transplanted human monoblastic leukemic cells of the U-937 cell line into newborn Swiss nu/nu mice. Priming with cyclophosphamide, followed by subcutaneous injections of at least 10 x 10(6) cells allowed us to obtain solid tumors. The cytology, HLA phenotype and in vitro proliferation characteristics of the U-937 tumor cells were conserved. However, these tumor cells were more tumorigenic when reinjected into nude mice and showed a modified response to differentiation induction. A decreased capacity to differentiate with retinoic acid (RA) and a resistance to 1-beta-D arabinofuranosyl cytosine (Ara-C) and 1-25 dihydroxy vitamin D3 (1-25 (OH)2 D3) were noted in three tumor cell lines tested. With regard to the latter, the resistance was not due to a modification of the number of cell receptors. The study shows that though in vivo transplantation of human leukemic cells in nude mice may lead to a selection of resistant cells, systematic checking of in vitro differentiation characteristics of the tumor cells permits the nude mouse model to be maintained for the in vivo screening of new differentiating agents.

A study of the efficacy of 5 alpha- and 5 beta-androstanes in chronic experimental aplastic anemia in mice.

The efficacy of in vivo administration of a 5 alpha-androstane and two 5 beta-androstanes (3 alpha and 3 beta) on CFU/GM and CFU/E mouse bone marrow stem cells was compared. The subjects were two groups of Swiss Webster mice: one normal group and one group in whom chronic aplastic anemia was induced by irradiation followed by mesenteric node lymphocyte grafting from C57/Bl donor mice. In normal animals, the two types of androgens (5 alpha and 5 beta) had the same efficacy, and the injection of 5 beta-androstane, before that of 5 alpha, significantly increased its efficacy. In aplastic mice, 5 alpha and 5 beta-androstanes had the same efficacy on CFU/E but 5 beta-androstanes were more efficient on the granulopoietic committed stem cells. The efficacy of the association of the two compounds in aplastic mice was not superior to that of each drug alone, which can be explained by the depletion of the stem cell compartment induced by the stimulating effect of the first androgen.

Kinetic studies of the reaction mechanism of rat liver phosphoglycerate kinase in the direction of ADP utilization.

The kinetic properties of rat liver phosphoglycerate kinase were investigated in the forward direction of the reaction (utilization of ADP). The kinetic studies were performed in an assay system using combined hexokinase/glucose-6-phosphate dehydrogenase as an ATP trap. The Km values for Mg ADP1- and 1,3-diphospho-D-glycerate were approximately 0.11 and 0.006 mM, respectively. Reciprocal plots of 1/v versus 1/ (Mg ADP1-) at different fixed concentrations of 1,3-diphospho-D-glycerate and 1/v versus 1/ (1,3-diphospho-D-glycerate) at different fixed concentrations of Mg ADP1- were apparently parallel. However, product inhibition studies (3-phospho-D-glycerate), dead-end inhibition studies (2,3-diphospho-D-glycerate), and adenosine and AMP inhibition patterns yielded results consistent with a rapid equilibrium random mechanism in which the binding of one substrate greatly decreases the affinity of the enzyme for the second substrate. Existence of two sites for 3-phospho-D-glycerate is suggested.

Two different in vitro growth patterns for erythroid precursors in 18 patients with pure erythrocytosis.

Growth patterns of marrow and blood erythroid progenitors were studied in 18 cases of pure erythrocytosis using different doses of erythropoietin. 8 cases demonstrated 'spontaneous' growth of CFU-E and blood BFU-E as observed in myeloproliferative disorders, but without an excess of circulating CFU-GM. 3 of these patients also had other symptoms of a pan-myelopathy. All these cases showed good sensitivity to 32P myelo-suppression. 10 cases demonstrated growth patterns of erythroid progenitors similar to those observed in normal subjects, except for an excess of blood BFU-E, which suggests an abnormality of homeostatic regulation. In 5 of these cases, myelo-suppression was not effective. It is suggested that a stem cell study could differentiate patients with pure erythrocytosis due to 'autonomous' abnormal stem cell growth from cases due to abnormal regulation factors, and that such a discrimination might be useful for the choice of therapy.