Transgenic mice expressing Tel-FLT3, a constitutively activated form of FLT3, develop myeloproliferative disease.

Evidence is continuing to accumulate that the FMS-like tyrosine kinase 3 (FLT3) receptor plays an important role in acute leukemias. Acute myeloid leukemia patients often express constitutive active mutant forms of the receptor in their leukemic cells. A t(12;13)(p13;q12) translocation between Tel and the FLT3 receptor was recently described in a patient with myeloproliferative disease (MPD). Here a Tel-FLT3 construct mimicking this fusion protein was used to generate transgenic mice. The fusion protein was previously found to constitutively activate FLT3 signaling and transform Ba/F3 cells. Expression of the fusion protein in the transgenic mice was found in all tissues assayed including spleen, bone marrow (BM), thymus and liver. These mice developed splenomegaly and had a high incidence of MPD with extramedullary hematopoiesis in the liver and lymph nodes. Spleens also had increased dendritic and natural killer cell populations. In vitro analysis of the hematopoietic progenitor cells derived from Tel-FLT3 transgenic mice showed a significant increase in the number of CFU-GM in the BM, and CFU-GM, BFU-E and CFU-GEMM in the spleen. BM also showed significant increases of in vivo CFU-S colonies. Thus, transgenic mice expressing constitutively activated Tel-FLT3 develop MPD with a long latency and also result in the expansion of the hematopoietic stem/progenitor cells.

Central nervous system lymphoma associated with mycophenolate mofetil in lupus nephritis.

Lymphomas, both within and outside the central nervous system, are uncommon among patients with systemic lupus erythematosus (SLE). We describe a 58-year old Korean woman with SLE who presented with acute headache and confusion in the setting of prednisone and mycophenolate mofetil (MMF) therapy used to treat focal proliferative and membranous lupus nephritis. Three-dimensional brain magnetic resonance imaging (MRI) showed two peripherally ('ring') enhancing lesions within the basal ganglia, bilaterally, with associated mass effect and subfalcine herniation. A brain biopsy revealed an Epstein-Barr virus (EBV)-positive diffuse large B cell lymphoma. This is the first description of CNS lymphoma in a patient treated with MMF for lupus nephritis. While intracerebral lymphoma in the immunocompromised patient with lupus is rare, this disorder should be considered in the differential diagnosis of new-onset neurological symptoms among such patients.

Stromal inhibition of megakaryocytic differentiation correlates with blockade of signaling by protein kinase C-epsilon and ERK/MAPK.

Contact with bone marrow stromal cells maintains normal and leukemic hematopoietic progenitors in an undifferentiated state. Recently, stromal contact has been shown to diminish the yield of megakaryocytes in cultures of primary human hematopoietic stem cells. This inhibition may explain the poor megakaryocytic engraftment frequently observed after bone marrow transplantation. In the current study, stromal co-culture is shown to render K562 cells refractory to megakaryocytic induction. This stromal inhibition correlated with the selective down-regulation in K562 cells of protein kinase C-epsilon (PKC-epsilon), which has recently been implicated in regulation of megakaryocytic lineage commitment. In addition, the stromal inhibition correlated with inactivation of the ERK/MAPK pathway, which has also been implicated in promoting megakaryocytic development. Forced expression of PKC-epsilon by retroviral transduction was insufficient to reverse the stromal blockade of ERK/MAPK signaling or of megakaryocytic induction. Thus stromal interruption of ERK/MAPK signaling occurred independently of PKC-epsilon levels and correlated more closely with megakaryocytic blockade. These findings provide potential mechanisms for stromal inhibition of hematopoietic differentiation and possibly for the poor megakaryocytic engraftment seen after bone marrow transplantation.

Induction of megakaryocytic differentiation in primary human erythroblasts: a physiological basis for leukemic lineage plasticity.

In myelodysplasias and acute myeloid leukemias, abnormalities in erythroid development often parallel abnormalities in megakaryocytic development. Erythroleukemic cells in particular have been shown to possess the potential to undergo megakaryocytic differentiation in response to a variety of stimuli. Whether or not such lineage plasticity occurs as a consequence of the leukemic phenotype has not previously been addressed. In this study, highly purified primary human erythroid progenitors were subjected to stimuli known to induce megakaryocytic differentiation in erythroleukemic cells. Remarkably, the primary erythroid progenitors rapidly responded with morphological and immunophenotypic evidence of megakaryocytic differentiation, equivalent to that seen in erythroleukemic cells. Even erythroblasts expressing high levels of hemoglobin manifested partial megakaryocytic differentiation. These results indicate that the lineage plasticity observed in erythroleukemic cells reflects an intrinsic property of cells in the erythroid lineage rather than an epiphenomenon of leukemic transformation.

A potential role for protein kinase C-epsilon in regulating megakaryocytic lineage commitment.

Multiple studies have shown that intracellular signal transduction by the protein kinase C (PKC) family participates in the initiation of megakaryocyte differentiation. In this study, multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel PKCs, in particular PKC-epsilon. In transfection studies, the isolated variable domain of PKC-epsilon selectively blocked exogenous activation of the megakaryocyte-specific alpha IIb promoter. Constitutively active mutants of PKC-epsilon, but not of other PKC isozymes, cooperated with the transcription factor GATA-1 in the activation of the alpha IIb promoter. The functional cooperation between GATA-1 and PKC-epsilon displayed dependence on cellular milieu, as well as on the promoter context of GATA binding sites. In aggregate, the data suggest that PKC-epsilon specifically participates in megakaryocytic lineage commitment through functional cooperation with GATA-1 in the activation of megakaryocytic promoters.

CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolytic activity of V alpha 24J alpha Q T cells: a feedback mechanism for immune regulation.

The precise immunologic functions of CD1d-restricted, CD161+ AV24AJ18 (Valpha24JalphaQ) T cells are not well defined, although production of IL-4 has been suggested as important for priming Th2 responses. However, activation of human Valpha24JalphaQ T cell clones by anti-CD3 resulted in the secretion of multiple cytokines notably important for the recruitment and differentiation of myeloid dendritic cells. Specific activation of Valpha24JalphaQ T cells was CD1d restricted. Expression of CD1d was found on monocyte-derived dendritic cells in vitro, and immunohistochemical staining directly revealed CD1d preferentially expressed on dendritic cells in the paracortical T cell zones of lymph nodes. Moreover, myeloid dendritic cells both activated Valpha24JalphaQ T cells and were susceptible to lysis by these same regulatory T cells. Because myeloid dendritic cells are a major source of IL-12 and control Th1 cell differentiation, their elimination by lysis is a mechanism for limiting the generation of Th1 cells and thus regulating Th1/Th2 responses.

Pseudo-spikes are common in histologically benign lymphoid tissues.

T cell receptor gene rearrangement is a classic marker of T cell clonality and is a useful adjunct in the diagnosis of T cell lymphomas and leukemias. Rearranged V-J gene segments amplified by polymerase chain reaction (PCR) are traditionally analyzed by polyacrylamide gel electrophoresis. We and others have analyzed TCR-gamma PCR products using capillary gel electrophoresis, which produces single nucleotide resolution and provides improved diagnostic sensitivity over conventional methods. However, with this marked increase in resolution and sensitivity, it is necessary to re-define normal variation of TCR-gamma gene rearrangement in control tissues to allow appropriate interpretation of monoclonality if present. Using DNA capillary gel electrophoresis, we examined the spectrum of normal patterns for TCR-gamma in a variety of T-cell-rich, histologically benign tissue types, including spleen, lymph node, tonsil, and blood, and compared this with the patterns in T cell lymphoma samples. We defined relative peak heights as h1/h2, where h1 represents the peak height of the largest peak above the normally distributed population, and h2 represents the peak height of the normally distributed curve. We found spikes in almost 20% of histologically benign samples with relative peak heights that were more than 0.5 and up to 1.5. We designated these as pseudo-spikes, because they may be mistaken for monoclonal spikes. In contrast, the relative peak height of the T cell lymphoma samples that showed clonal rearrangement was much higher than that of the pseudo-spikes, being at least 2 in 11/11 and at least 3 in 10/11 cases. Our data suggest that peaks with relative height of at least 3 represent a true clonal population in diagnostic samples. Peaks with relative heights of less than 1.5 may be insignificant, while peaks with relative heights between 1.5 to 3 may warrant further evaluation. Although capillary gel electrophoresis is superior in assessing T cell clonality, caution must be exercised when interpreting results, because pseudo-spikes appear to be common in benign tissues with lymphoid populations and are not necessarily indicative of clonal malignant T cell population.

Sustained activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway is required for megakaryocytic differentiation of K562 cells.

The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/MAP kinase pathway in megakaryocytic differentiation of K562 cells, the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on ERK activation were determined. Both TPA and bryostatin are known to activate PKC but paradoxically have opposing effects on megakaryocytic differentiation. TPA, a differentiation inducer, caused sustained activation of ERK (>24 h), whereas bryostatin, a differentiation blocker, only transiently activated ERK ( approximately 6 h) and attenuated the activation of ERK by TPA. To confirm a requirement for sustained ERK activation for megakaryocytic differentiation, PD098059, a synthetic inhibitor of the MAP kinase kinase 1 (MEK1) was employed. Introduction of PD098059 at any time during the first 18 h of TPA treatment completely abrogated megakaryocytic differentiation of K562 cells. After 24 h of TPA treatment, introduction of PD098059 failed to block differentiation. Differentiation blockade by PD098059 occurred via inhibition of MEK because transfection of a constitutively active mutant of MEK2 could override the PD098059 blockade. Experiments with conditioned media suggested that sustained activation of the ERK/MAP kinase pathway promoted the autocrine secretion of megakaryocytic lineage determination factors.

Investigation of calcium-induced hydrolysis of phosphoinositides in normal and lithium-treated parathyroid cells.

BACKGROUND: Lithium-induced hyperparathyroidism is characterized by a reduction in parathyroid sensitivity to changes in extracellular calcium (Ca2+). Ca(2+)-induced transmembrane signal transduction in the parathyroid cell is known to result in the hydrolysis of phosphatidylinositol bisphosphate (PIP2), generating increases in intracellular inositol phosphates, a process which is mediated by a calcium receptor. MATERIALS AND METHODS: To determine if lithium's effect on parathyroid cell function is mediated by an alteration in Ca(2+)-induced hydrolysis of PIP2, inositol 4-monophosphate (IP1), and inositol 1,4,5-trisphosphate (IP3) were measured using anion-exchange chromatography in normal and lithium chloride (LiCl)-treated bovine parathyroid cells at Ca2+ concentrations varying from 0.5 mmol/L to 5.0 mmol/L. IP1 and IP3 concentrations were determined in terms of percent control, defined as the IP1 or IP3 concentration at an [Ca2+] of 0.5 mmol/L. RESULTS: Increases in [IP1]/10(6) cells (mean +/- standard error of the mean [SEM]) in response to progressive increases in Ca2+ from 0.5 mmol/L to 5.0 mmol/L varied from 825 +/- 228 to 4,474 +/- 382 in control cells versus 1,139 +/- 243 to 4,689 +/- 630 in cells pretreated with LiCl (P > 0.05). The increases in [IP3]/10(6) cells (mean +/- SEM) in response to increases in Ca2+ from 0.5 mmol/L to 5.0 mmol/L, varied from 146 +/- 14 to 385 +/- 35 in control cells versus 134 +/- 16 to 327 +/- 55 in cells pretreated with LiCl (P > 0.05). CONCLUSIONS: Our results demonstrate that LiCl does not effect Ca(2+)-induced hydrolysis of PIP2, suggesting that the desensitizing effect of LiCl on the parathyroid cell is not the result of a Ca2+ receptor-mediated phenomenon.

Lithium-induced alterations in parathyroid cell function: insight into the pathogenesis of lithium-associated hyperparathyroidism.

BACKGROUND: Reduced parathyroid sensitivity to changes in calcium (Ca2+) has been observed in patients treated with lithium (Li+). In order to investigate this desensitization phenomenon, the effect of Li+ on cytosolic calcium (Cai2+) regulation was examined. METHODS: Transmembrane signal transduction and Ca2+ sequestration were investigated in bovine parathyroid cells by measuring changes in [Cai2+] in response to 5 mmol/L magnesium (Mg2+), 0.5 to 2.5 mmol/L Ca2+, 25 mumol/L adenosine triphosphate (ATP), and 1 mumol/L ionomycin in cells pretreated with 1 to 10 mmol/L lithium chloride (LiCl) and control cells. Measurement of Cai2+ was made using fura-2. RESULTS: Increases in [Cai2+] in response to Ca2+ and Mg2+ were blunted following overnight culture with as low as 1 mmol/L LiCl. In normocalcemic medium, 1 mmol/L Ca2+ produced an 81% increase in [Cai2+] in control cells compared with a 58% increase in cells pretreated with LiCl (P < 0.01), whereas in hypocalcemic medium, increases in [Cai2+] were similar in lithium-treated and control cells (78% versus 82%, P > 0.1). The ATP produced increases in [Cai2+] from 225 +/- 9 nmol/L to 366 +/- 10 nmol/L in control cells, compared with 221 +/- 7 nmol/L to 308 +/- 10 nmol/L in cells pretreated with 5 mmol/L LiCl (P < 0.01). Ionomycin-induced increases in [Cai2+] were unaffected by Li+. CONCLUSIONS: We concluded that the in vitro desensitizing effects of Li+ occur at therapeutic concentrations, but only in the presence of Ca2+ in concentrations that induce transmembrane signaling; and that Li+ blunts increases in [Cai2+] related to cation and ATP-induced transmembrane signal transduction without affecting ionomycin-releasable Ca2+ stores.

Stimulus-secretion coupling in parathyroid cells deficient in protein kinase C activity.

The role of protein kinase C (PKC) in regulating cytosolic Ca2+ concentrations ([Ca2+]i) and parathyroid hormone (PTH) secretion was studied in bovine parathyroid cells rendered deficient in PKC activity by incubation with phorbol 12-myristate 13-acetate (PMA). Pretreatment with PMA caused a time- and concentration-dependent loss of functional PKC activity as assessed by the failure of [Ca2+]i and PTH secretion to respond to the subsequent addition of PKC activators or the inhibitor staurosporine. Pretreatment for 24 h with 1 microM PMA caused a loss of 85% of the total and 98% of the cytosolic PKC activity. Cells so pretreated were considered to be "PKC downregulated." Increasing the concentration of extracellular Ca2+ or Mg2+ caused corresponding increases in [Ca2+]i that were similar in control and in PKC-downregulated cells. PTH secretion regulated by extracellular Ca2+ or Mg2+ was likewise similar in control and PKC-downregulated cells. Stimulus-secretion coupling is thus unimpaired in parathyroid cells deficient in PKC activity. Cytosolic Ca2+ responses remained depressed in cells incubated for 24 h with low concentrations of PMA (30 or 100 nM). However, under these conditions, extracellular Ca2+ still suppressed PTH secretion similarly to control cells. These results reveal a dissociation between cytosolic Ca2+ and PTH secretion and suggest that signals other than cytosolic Ca2+ are involved in the regulation of PTH secretion.

Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes.

Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca2+. To test this hypothesis, Xenopus laevis oocytes were injected with poly(A)(+)-enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca2+ or other polycations. Cytosolic Ca2+ concentrations were measured indirectly by recording Cl- currents through the endogenous, cytosolic Ca(2+)-activated Cl- channel. Increasing the concentration of extracellular Ca2+ (from 0.7 to 5 mM) or Mg2+ (from 0.8 to 10 mM) elicited oscillatory increases in the Cl- current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL-60 cells or rat liver. Responses elicited by extracellular Mg2+ persisted when extracellular Ca2+ was reduced to low micromolar levels. La3+, Gd3+, or neomycin B also evoked oscillatory increases in the Cl- current in oocytes under conditions of low extracellular Ca2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca2+ receptor protein(s) on parathyroid cells that regulates cellular function.

Cytosolic calcium homeostasis in bovine parathyroid cells and its modulation by protein kinase C.

1. The effects of protein kinase C (PKC) activators and inhibitors on the mechanisms regulating cytosolic Ca2+ homeostasis in dissociated bovine parathyroid cells loaded with fura-2 were examined. 2. Stepwise increases in the concentration of extracellular Ca2+ (from 0.5 to 2 or 3 mM) elicited transient followed by sustained increases in the concentration of intracellular free Ca2+ ([Ca2+]i). Cytosolic Ca2+ transients reflected the mobilization of intracellular Ca2+ and influx of extracellular Ca2+ whereas sustained increases in [Ca2+]i resulted from the influx of extracellular Ca2+. Brief (1-2 min) pretreatment with phorbol myristate acetate (PMA) shifted the concentration-response curve for extracellular Ca(2+)-induced cytosolic Ca2+ transients to the right without affecting the maximal response. Cytosolic Ca2+ transients elicited by extracellular Mg2+ were similarly affected by PMA. 3. These effects of PMA were mimicked by various other activators of PKC with the rank order of potency PMA > phorbol dibutyrate > bryostatin , > (-)indolactam V > mezerein. Isomers or analogues of these compounds that do not alter PKC activity (4 alpha-phorbols and (+)indolactam V) did not alter [Ca2+]i. 4. PKC activators depressed evoked increases in [Ca2+]i when influx of extracellular Ca2+ was blocked with Gd3+. Cytosolic Ca2+ transients elicited by extracellular Mg2+ in the absence of extracellular Ca2+ were similarly inhibited by PKC activators. Activation of PKC thus inhibits the mobilization of intracellular Ca2+ elicited by extracellular divalent cations. 5. Increases in the concentration of extracellular Ca2+ caused corresponding increases in the formation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3). Pretreatment with PMA shifted the concentration-response curve for extracellular Ca(2+)-induced [3H]InsP3 formation to the right without affecting the maximal response. 6. PKC activators also caused some depression of steady-state increases in [Ca2+]i elicited by extracellular Ca2+. In contrast, PMA did not affect increases in [Ca2+]i elicited by ionomycin or thapsigargin. 7. Ba2+ was used to monitor divalent cation influx. PMA decreased the rate of rise of the fluorescent signal elicited by extracellular Ba2+. 8. All these effects of PKC activators on [Ca2+]i were blocked or reversed by staurosporine at concentrations (30-100 nM) that inhibited PKC activity in parathyroid cells. Staurosporine alone potentiated cytosolic Ca2+ responses evoked by submaximal concentrations of extracellular divalent cations. 9. PKC thus depresses both the mobilization of intracellular Ca2+ and the influx of extracellular Ca2+ in parathyroid cells. The effects on [Ca2+]i provide evidence for a Ca2+ receptor on the surface of parathyroid cells that uses transmembrane signalling mechanisms common to some other Ca(2+)-mobilizing receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein kinase C modulates hormone secretion regulated by extracellular polycations in bovine parathyroid cells.

1. The role of protein kinase C (PKC) in the regulation of parathyroid hormone (PTH) secretion was examined in dissociated bovine parathyroid cells. 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2 mM inhibited PTH secretion by 60-80%. Similar depressive effects on secretion were obtained by increasing the concentration of extracellular Mg2+ from 1 to 7 mM or by adding La3+ (to 40 microM). The PKC activator phorbol myristate acetate (PMA) depressed PTH secretion at the lower and potentiated secretion at the higher concentrations of extracellular Ca2+, Mg2+ or La3+. The inhibitory effect of PKC on secretion correlated positively with the magnitude of the inhibitory effect elicited by elevated extracellular Ca2+. 3. The stimulatory effects of PKC activators on PTH secretion were reversed completely and the inhibitory effects were reversed partially by the PKC inhibitor staurosporine. Staurosporine alone did not affect secretion at low (0.5 mM) or high (2 mM) concentrations of extracellular Ca2+ but it did depress secretion at intermediate concentrations (around 1 mM) of extracellular Ca2+. 4. The stimulatory effects of PKC activators on secretion were overcome by increases in the concentration of extracellular Ca2+ (to 5 or 10 mM) or La3+ (to 100 microM). In contrast, increasing the concentration of extracellular Mg2+ to 11 or 19 mM did not alleviate the potentiating effects of PKC activators. The different results obtained with Ca2+ and Mg2+ could not be explained by their different effects on cytosolic Ca2+ and suggests that different cations can have varying degrees of efficacy to activate functional responses linked to the Ca2+ receptor on bovine parathyroid cells. 5. PTH secretion stimulated by isoprenaline was not affected by PKC activators or staurosporine. Similarly, the inhibitory effects of extracellular ATP gamma S on secretion were unaffected by PKC activators. These results show that PKC activators affect specifically PTH secretion regulated by extracellular polycations. 6. The stimulatory effect of PKC activators on secretion parallels its inhibitory effects on [Ca2+]i and inositol trisphosphate formation, showing that PKC blunts the mechanisms associated with extracellular Ca(2+)-induced inhibition of secretion. The specificity of these actions suggests that PKC acts at a very early step of stimulus-secretion coupling in parathyroid cells, specific to that used by extracellular polycations and perhaps involving the Ca2+ receptor.

Lithium effects on dispersed bovine parathyroid cells grown in tissue culture.

It is not clear whether hypercalcemia and hyperparathyroidism associated with lithium therapy are the result of an unmasking of preexisting disease or a direct effect of lithium on the parathyroid glands. To investigate this phenomenon, parathyroid hormone (PTH) secretion and cytosolic calcium concentrations [( Ca]i) were measured in normal and lithium-treated dispersed bovine parathyroid cells grown in tissue culture and incubated with varying concentrations of extracellular calcium [( Ca]e) (0.5 to 2.5 mmol/L). Results indicate that lithium has two effects on parathyroid secretory response: (1) a decrease in low calcium-stimulated PTH release and (2) a potentiation of PTH release at physiologic concentrations of extracellular calcium. [Ca]i was assessed by use of fura-2, a calcium-sensitive fluorescent indicator. Resting [Ca]i levels were unaffected by lithium (103 +/- 13 nmol/L in controls vs 101 +/- 5 nmol/l in lithium-treated cells, mean +/- SE). Subsequent increases in [Ca]i in response to increases in [Ca]e were significantly less in lithium-treated cells, with no difference at maximal [Ca]e. Increases in [Ca]i in response to a submaximal concentration of extracellular magnesium were also blunted in cells pretreated with lithium. In conclusion, our data suggest that, at physiologic calcium concentrations, lithium decreases parathyroid cell sensitivity to changes in [Ca]e, reducing [Ca]i levels and increasing PTH secretory response.