Nuclear IL-33 restrains the early conversion of fibroblasts to an extracellular matrix-secreting phenotype.

Interleukin (IL)-33 is a cytokine that appears to mediate fibrosis by signaling via its receptor ST2 (IL-33R/IL1RL1). It is also, however, a protein that after synthesis is sorted to the cell nucleus, where it appears to affect chromatin folding. Here we describe a novel role for nuclear IL-33 in regulating the fibroblast phenotype in murine kidney fibrosis driven by unilateral ureteral obstruction. Transcriptional profiling of IL-33-deficient kidneys 24 h after ligation revealed enhanced expression of fibrogenic genes and enrichment of gene sets involved in extracellular matrix formation and remodeling. These changes relied on intracellular effects of IL-33, because they were not reproduced by treatment with a neutralizing antibody to IL-33 that prevents IL-33R/ST2L receptor signaling nor were they observed in IL-33R/ST2-deficient kidneys. To further explore the intracellular function of IL-33, we established transcription profiles of human fibroblasts, observing that knockdown of IL-33 skewed the transcription profile from an inflammatory towards a myofibroblast phenotype, reflected in higher levels of COL3A1, COL5A1 and transgelin protein, as well as lower expression levels of IL6, CXCL8, CLL7 and CCL8. In conclusion, our findings suggest that nuclear IL-33 in fibroblasts dampens the initial profibrotic response until persistent stimuli, as enforced by UUO, can override this protective mechanism.

Exploring the potential effect of paricalcitol on markers of inflammation in de novo renal transplant recipients.

Following a successful renal transplantation circulating markers of inflammation may remain elevated, and systemic inflammation is associated with worse clinical outcome in renal transplant recipients (RTRs). Vitamin D-receptor (VDR) activation is postulated to modulate inflammation and endothelial function. We aimed to explore if a synthetic vitamin D, paricalcitol, could influence systemic inflammation and immune activation in RTRs. Newly transplanted RTRs were included in an open-label randomized controlled trial on the effect of paricalcitol on top of standard care over the first post-transplant year. Fourteen pre-defined circulating biomarkers reflecting leukocyte activation, endothelial activation, fibrosis and general inflammatory burden were analyzed in 74 RTRs at 8 weeks (baseline) and 1 year post-engraftment. Mean changes in plasma biomarker concentrations were compared by t-test. The expression of genes coding for the same biomarkers were investigated in 1-year surveillance graft biopsies (n = 60). In patients treated with paricalcitol circulating osteoprotegerin levels increased by 0.19 ng/ml, compared with a 0.05 ng/ml increase in controls (p = 0.030). In graft tissue, a 21% higher median gene expression level of TNFRSF11B coding for osteoprotegerin was found in paricalcitol-treated patients compared with controls (p = 0.026). Paricalcitol treatment did not significantly affect the blood- or tissue levels of any other investigated inflammatory marker. In RTRs, paricalcitol treatment might increase both circulating and tissue levels of osteoprotegerin, a modulator of calcification, but potential anti-inflammatory treatment effects in RTRs are likely very modest. [NCT01694160 (2012/107D)]; [www.clinicaltrials.gov].

Inhibition of Endothelial NOTCH1 Signaling Attenuates Inflammation by Reducing Cytokine-Mediated Histone Acetylation at Inflammatory Enhancers.

OBJECTIVE: Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. APPROACH AND RESULTS: Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1ß stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers. CONCLUSIONS: Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.

Early introduction of oral paricalcitol in renal transplant recipients. An open-label randomized study.

In stable renal transplant recipients with hyperparathyroidism, previous studies have indicated that vitamin D agonist treatment might have anti-proteinuric effects. Animal studies indicate possible anti-fibrotic and anti-inflammatory effects. Early introduction of paricalcitol in de novo renal transplant recipients might reduce proteinuria and prevent progressive allograft fibrosis. We performed a single-center, prospective, randomized, open-label trial investigating effects of paricalcitol 2 µg/day added to standard care. Participants were included 8 weeks after engraftment and followed for 44 weeks. Primary end point was change in spot urine albumin/creatinine ratio. Exploratory microarray analyses of kidney biopsies at study end investigated potential effects on gene expression. Secondary end points included change in glomerular filtration rate (GFR), pulse wave velocity (PWV), and endothelial function measured by peripheral arterial tonometry as reactive hyperemia index (RHI). Seventy-seven de novo transplanted kidney allograft recipients were included, 37 receiving paricalcitol. Paricalcitol treatment lowered PTH levels (P = 0.01) but did not significantly reduce albuminuria (P = 0.76), change vascular parameters (PWV; P = 0.98, RHI; P = 0.33), or influence GFR (P = 0.57). Allograft gene expression was not influenced. To summarize, in newly transplanted renal allograft recipients, paricalcitol reduced PTH and was well tolerated without negatively affecting kidney function. Paricalcitol did not significantly reduce/prevent albuminuria, improve parameters of vascular health, or influence allograft gene expression.

Interleukin-33 drives a proinflammatory endothelial activation that selectively targets nonquiescent cells.

OBJECTIVE: Interleukin (IL)-33 is a nuclear protein that is released from stressed or damaged cells to act as an alarmin. We investigated the effects of IL-33 on endothelial cells, using the prototype IL-1 family member, IL-1ß, as a reference. METHODS AND RESULTS: Human umbilical vein endothelial cells were stimulated with IL-33 or IL-1ß, showing highly similar phosphorylation of signaling molecules, induction of adhesion molecules, and transcription profiles. However, intradermally injected IL-33 elicited significantly less proinflammatory endothelial activation when compared with IL-1ß and led us to observe that quiescent endothelial cells (ppRb(low)p27(high)) were strikingly resistant to IL-33. Accordingly, the IL-33 receptor was preferentially expressed in nonquiescent cells of low-density cultures, corresponding to selective induction of adhesion molecules and chemokines. Multiparameter phosphoflow cytometry confirmed that signaling driven by IL-33 was stronger in nonquiescent cells. Manipulation of nuclear IL-33 expression by siRNA or adenoviral transduction revealed no functional link between nuclear, endogenous IL-33, and exogenous IL-33 responsiveness. CONCLUSIONS: In contrast to other inflammatory cytokines, IL-33 selectively targets nonquiescent endothelial cells. By this novel concept, quiescent cells may remain nonresponsive to a proinflammatory stimulus that concomitantly triggers a powerful response in cells that have been released from contact inhibition.

The alarmin IL-33 is a notch target in quiescent endothelial cells.

The molecular mechanisms that drive expression of the alarmin interleukin-33 (IL-33) in endothelial cells are unknown. Because nuclear IL-33 is a marker of endothelial cell quiescence (corroborated in this study by coexpression of cyclin-dependent kinase inhibitor p27(Kip1)), we hypothesized that Notch signaling might be involved in regulating IL-33 expression. Activation of Notch1 by immobilized Notch ligands was sufficient to induce nuclear IL-33 expression in cultured endothelial cells. Conversely, IL-33 expression was inhibited by the γ-secretase inhibitor DAPT or by inhibiting the function of Dll4, Jagged1, Notch1, or the canonical Notch transcription factor RBP-Jκ. Insensitivity to cycloheximide indicated that IL-33 was a direct target of Notch signaling, well in line with the identification of several conserved RBP-Jκ binding sites in the IL33 gene. The in vivo expression of Dll4 but not of Jagged1 was well correlated with expression of IL-33 in quiescent vessels, and subcutaneous injection of DAPT in healthy skin reduced IL-33 expression, indicating that Notch signaling was involved. On the other hand, loss of IL-33 during angiogenesis occurred despite sustained Dll4 and Notch1 expression, suggesting that other signals may override the IL-33-driving signal in this context. Taken together, our data demonstrate that endothelial nuclear IL-33 is induced by Notch and that Dll4 may be the dominant ligand responsible for this signaling in vivo.

Statins affect the presentation of endothelial chemokines by targeting to multivesicular bodies.

BACKGROUND: In addition to lowering cholesterol, statins are thought to beneficially modulate inflammation. Several chemokines including CXCL1/growth-related oncogene (GRO)-α, CXCL8/interleukin (IL)-8 and CCL2/monocyte chemoattractant protein (MCP)-1 are important in the pathogenesis of atherosclerosis and can be influenced by statin-treatment. Recently, we observed that atorvastatin-treatment alters the intracellular content and subcellular distribution of GRO-α in cultured human umbilical vein endothelial cells (HUVECs). The objective of this study was to investigate the mechanisms involved in this phenomenon. METHODOLOGY/ PRINCIPAL FINDINGS: The effect of atorvastatin on secretion levels and subcellular distribution of GRO-α, IL-8 and MCP-1 in HUVECs activated by interleukin (IL)-1ß were evaluated by ELISA, confocal microscopy and immunoelectron microscopy. Atorvastatin increased the intracellular contents of GRO-α, IL-8, and MCP-1 and induced colocalization with E-selectin in multivesicular bodies. This effect was prevented by adding the isoprenylation substrate GGPP, but not the cholesterol precursor squalene, indicating that atorvastatin exerts these effects by inhibiting isoprenylation rather than depleting the cells of cholesterol. CONCLUSIONS/ SIGNIFICANCE: Atorvastatin targets inflammatory chemokines to the endocytic pathway and multivesicular bodies and may contribute to explain the anti-inflammatory effect of statins at the level of endothelial cell function.

Inflammatory bowel disease-associated interleukin-33 is preferentially expressed in ulceration-associated myofibroblasts.

Interleukin-33 (IL-33) is a novel member of the interleukin-1 family that induces mucosal pathology in vivo and may drive fibrosis development and angiogenesis. To address its potential role in inflammatory bowel disease, we explored its tissue expression in biopsy specimens from untreated ulcerative colitis patients, observing a 2.6-fold up-regulation of IL-33 mRNA levels, compared to controls. Immunohistochemical analyses of surgical specimens showed that a prominent source of IL-33 in ulcerative colitis lesions were ulceration-associated myofibroblasts that co-expressed the fibroblast marker heat shock protein 47, platelet-derived growth factor receptor (PDGFR)ß, and, in part, the myofibroblast marker α-smooth muscle actin (SMA). In contrast, IL-33-positive myofibroblasts were almost absent near the deep fissures seen in Crohn's disease. A screen of known and putative activators of IL-33 in cultured fibroblasts revealed that the Toll-like receptor-3 agonist poly (I:C) was among the strongest inducers of IL-33 and that it synergized with transforming growth factor-ß, a combination also known to boost myofibroblast differentiation. Experimental wound healing in rat skin revealed that the de novo induction of IL-33 in pericytes and the possible activation of scattered, tissue-resident IL-33(+)PDGFRß(+)αSMA(-) fibroblast-like cells were early events that preceded the later appearance of IL-33(+)PDGFRß(+)αSMA(+) cells. In conclusion, our data point to a novel role for IL-33 in mucosal healing and wound repair and to an interesting difference between ulcerative colitis and Crohn's disease.

Activation of mTORC1 signaling pathway in AIDS-related lymphomas.

Using immunohistochemistry with antibodies against the phosphoserine residues in both S6rp and 4E binding protein 1, we identified the activation of the mammalian target of rapamycin (mTORC)1 pathway in 29 cases of AIDS-related lymphoma. These cases represented a diverse spectrum of histological types of non-Hodgkin lymphoma (24 cases) and classic Hodgkin lymphoma (five cases). mTORC1 was also activated in the hyperplastic but not involuted follicles of HIV-associated lymphadenopathy in eight cases, supporting the notion that mTORC1 activation is a common feature of transformed lymphocytes irrespective of either their reactive or malignant phenotype. We also found that in B-cell lines that represent diffuse large B-cell lymphoma, Burkitt lymphoma, Epstein-Barr virus-infected lymphocytes, and human herpesvirus 8-positive primary effusion lymphoma, inhibitors of Syk, MEK, and, seemingly, phosphoinositide 3 kinases suppressed mTORC1 activation, in particular when these inhibitors were used in combination. These findings indicate that AIDS-related lymphoma and other histologically similar types of lymphomas that are derived from transformed B lymphocytes may display clinical responses to inhibitors that directly target mTORC1 or, possibly, upstream activators of the mTORC1 pathway.

Anaplastic lymphoma kinase (ALK)-induced malignancies: novel mechanisms of cell transformation and potential therapeutic approaches.

Among the many oncogenic variants of the anaplastic lymphoma kinase (ALK), nucleophosmin 1 (NPM)/ALK fusion protein expressed in the subset of T-cell lymphoma (ALK(+)TCL) is currently the best characterized. NPM/ALK activates several signal transduction pathways, including PI3K/AKT, MEK/ERK, mTORC1, STAT3, and STAT5b. In turn, the pathways modulate expression and function of many genes and proteins involved in the key cellular functions such as proliferation, growth, survival, metabolism, and angiogenesis. Recent data indicate that NPM/ALK also promotes immune evasion of the ALK(+)TCL by inducing through STAT3 activation the expression of immunosuppressive cytokines interleukin-10 (IL-10) and transforming growth factor-beta (TGFss) and cell surface protein CD274 (PD-L1, B7-H1). In addition, NPM/ALK protects its own expression by mediating via STAT3 and at least one member of the DNA methyltransferase family DNMT1 epigenetic silencing of the SHP-1 and STAT5a genes. In ALK+TCL cells, SHP-1 and STAT5a proteins act as potent tumor suppressors by promoting degradation of the NPM/ALK protein and inhibiting expression of the NPM/ALK gene, respectively. These findings provide further rationale to therapeutically target ALK and its effector proteins, foremost STAT3. They also suggest that immunotherapeutic approaches to ALK(+)TCL and, possibly, other ALK-driven malignancies may require inhibition of ALK and STAT3 to achieve the optimal clinical efficacy.

Gamma c-signaling cytokines induce a regulatory T cell phenotype in malignant CD4+ T lymphocytes.

In this study, we demonstrate that malignant mature CD4(+) T lymphocytes derived from cutaneous T cell lymphomas (CTCL) variably display some aspects of the T regulatory phenotype. Whereas seven cell lines representing a spectrum of primary cutaneous T cell lymphoproliferative disorders expressed CD25 and TGF-beta, the expression of FOXP3 and, to a lesser degree, IL-10 was restricted to two CTCL cell lines that are dependent on exogenous IL-2. IL-2, IL-15, and IL-21, all of which signals through receptors containing the common gamma chain, induced expression of IL-10 in the IL-2-dependent cell lines as well as primary leukemic CTCL cells. However, only IL-2 and IL-15, but not IL-21, induced expression of FOXP3. The IL-2-triggered induction of IL-10 and FOXP3 expression occurred by signaling through STAT3 and STAT5, respectively. Immunohistochemical analysis of the CTCL tissues revealed that FOXP3-expressing cells were common among the CD7-negative enlarged atypical and small lymphocytes at the early skin patch and plaque stages. Their frequency was profoundly diminished at the tumor stage and in the CTCL lymph node lesions with or without large cell transformation. These results indicate that the T regulatory cell features are induced in CTCL T cells by common gamma chain signaling cytokines such as IL-2 and do not represent a fully predetermined, constitutive phenotype independent of the local environmental stimuli to which these malignant mature CD4(+) T cells become exposed.

Differential effects of interleukin-2 and interleukin-15 versus interleukin-21 on CD4+ cutaneous T-cell lymphoma cells.

In this study, we compared the effects of interleukin-2 (IL-2), IL-15, and IL-21 on gene expression, activation of cell signaling pathways, and functional properties of cells derived from CD4+ cutaneous T-cell lymphoma (CTCL). Whereas both IL-2 and IL-15 modulated, in a CTCL cell line, the expression of >1,000 gene transcripts by at least 2-fold, IL-21 up-regulated <40 genes. All three cytokines induced tyrosine phosphorylation of Jak1 and Jak3 in CTCL cell lines and native leukemic (Sezary) cells. However, only IL-2 and IL-15 strongly activated signal transducers and activators of transcription 5, phosphoinositide 3-kinase/Akt, and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK signaling pathways in the cell lines and mitogen-primed native cells. In contrast, IL-21 selectively activated signal transducers and activators of transcription 3. Whereas all three cytokines protected CTCL cells from apoptosis, only IL-2 and IL-15 promoted their proliferation. The effects of the cytokine stimulation were Jak3 kinase- and Jak1 kinase- dependent. These findings document the vastly different effect of IL-2 and IL-15 versus IL-21 on CTCL cells. They also suggest two novel therapeutic approaches to CTCL and, possibly, other CD4+ T-cell lymphomas: inhibition of the Jak1/Jak3 kinase complex and, given the known strong immunostimulatory properties of IL-21 on CD8+ T, natural killer, and B cells, application of this cytokine to boost an immune response against malignant CD4+ T cells.

IL-21 enhances antitumor responses without stimulating proliferation of malignant T cells of patients with Sézary syndrome.

IL-21, a common gamma-chain cytokine secreted by activated CD4+ T cells, influences both humoral and cell-mediated immune responses through the regulation of T, B, dendritic, and natural killer (NK) cells. Sézary syndrome is an advanced form of cutaneous T-cell lymphoma, a clonally derived malignancy of CD4+ T cells that is characterized by profound defects in host cellular immune function. As a modulator of both innate and adaptive immune responses, IL-21 could play an important role in augmenting cell-mediated immunity in these patients. Normal donor and Sézary syndrome patient peripheral blood mononuclear cells were cultured with IL-21 and tested for CD8+ T- and NK-cell activation, NK-cell cytotoxicity, and tumor cell proliferation and apoptosis. IL-21 resulted in a modest increase in CD8+ T- and NK-cell activation, associated with a marked increase in cytolytic activity against both K562 and malignant CD4+ T-cell targets. Although IL-21 failed to demonstrate pro-apoptotic effects on the malignant CD4+ T cells, it is noteworthy that it had no demonstrable proliferative effects on these cells. Thus, IL-21 may play an important role in enhancing the host immune response of Sézary syndrome patients through the increased cytolytic activity of T and NK cells.

IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes.

We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as the PI3K/Akt and MEK/ERK pathways, the IL-2-dependent cell lines activated the pathways in response to IL-2 and IL-15 but not IL-21. Activation of mTORC1 and MEK/ERK was nutrient dependent. The mTORC1, PI3K/Akt, and MEK/ERK pathways could also be activated by IL-2 in the primary leukemic, mitogen-preactivated CTCL cells. mTORC1 activation was also detected in the CTCL tissues in the lymphoma stage-dependent manner with the highest percentage of positive cells present in the cases with a large cell transformation. Rapamycin inhibited mTORC1 signaling and suppressed CTCL cell proliferation but showed little effect on their apoptotic rate when used as a single agent. Activation of the mTORC1, PI3K/Akt, and MEK/ERK pathways was strictly dependent on the Jak3 and Jak1 kinases. Finally, mTORC1 activation was transduced preferentially through the PI3K/Akt pathway. These findings document the selective gammac-signaling cytokine-mediated activation of the mTORC1 pathway in the CTCL cells and suggest that the pathway represents a therapeutic target in CTCL and, possibly, other T-cell lymphomas.

PU.1 activates transcription of SHP-1 gene in hematopoietic cells.

Protein-tyrosine phosphatase SHP-1 is the key negative regulator of numerous signaling pathways. SHP-1 is expressed in the hematopietic and epithelial cells as two structurally similar mRNA transcripts controlled by two different promoters designated P2 and P1, respectively. Whereas the transcriptional regulation of the SHP-1 gene P1 promoter has been partially elucidated, the structure and functional control of the P2 promoter remain unknown despite the critical role played by SHP-1 in the normal and malignant lymphoid and other hematopoetic cells. Using luciferase reporter assays with the set of constructs that contained a gradually truncated intron 1 of the SHP-1 gene, we identified the minimal (<120 bp) fragment that is able to fully activate expression of the reporter gene. Furthermore, we found that PU.1 (a member of the Ets transcription factor family that plays a crucial role in differentiation and function of the lymphoid and myeloid cells) binds to the identified P2 promoter both in vitro and in vivo. PU.1 also activates the promoter in the sequence specific manner and is critical for its expression as evidenced by the profound supression of the SHP-1 gene transcription upon the siRNA-mediated depletion of PU.1. These findings provide an insight into the structure of the hematopoietic cell-specific P2 promoter of the SHP-1 gene and identify PU.1 as the transcriptional activator of the P2 promoter.

Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) oncoprotein induces the T regulatory cell phenotype by activating STAT3.

The mechanisms of malignant cell transformation mediated by the oncogenic, chimeric nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) tyrosine kinase remain only partially understood. Here we report that the NPM/ALK-carrying T cell lymphoma (ALK+TCL) cells secrete IL-10 and TGF-beta and express FoxP3, indicating their T regulatory (Treg) cell phenotype. The secreted IL-10 suppresses proliferation of normal immune, CD3/CD28-stimulated peripheral blood mononuclear cells and enhances viability of the ALK+TCL cells. The Treg phenotype of the affected cells is strictly dependent on NPM/ALK expression and function as demonstrated by transfection of the kinase into BaF3 cells and inhibition of its enzymatic activity and expression in ALK+TCL cells. NPM/ALK, in turn, induces the phenotype through activation of its key signal transmitter, signal transducer and activator of transcription 3 (STAT3). These findings identify a mechanism of NPM/ALK-mediated oncogenesis based on induction of the Treg phenotype of the transformed CD4(+) T cells. These results also provide an additional rationale to therapeutically target the chimeric kinase and/or STAT3 in ALK+TCL.

Expression and oncogenic role of Brk (PTK6/Sik) protein tyrosine kinase in lymphocytes.

Tyrosine kinases play a fundamental role in cell proliferation, survival, adhesion, and motility and have also been shown to mediate malignant cell transformation. Here we describe constitutive expression of the protein tyrosine kinase Brk in a large proportion of cutaneous T-cell lymphomas and other transformed T- and B-cell populations. The kinase is expressed in the nuclear localization and activated state. Brk expression was also induced in normal T cells on their activation. Introduced expression of the Brk gene resulted in markedly diminished cytokine and growth factor dependence of transfected BaF3 lymphocytes in regard to their in vitro proliferation and survival. Brk also conferred in vivo oncogenicity on the BaF3 cells. siRNA-mediated inhibition of the endogenous Brk in malignant T cells diminished their growth and survival capacity. These findings document inducible expression of Brk in normal T lymphocytes and persistent expression of the activated kinase in malignant T and B cells. Furthermore, our results indicate that Brk may play a key role in lymphomagenesis, hence identifying the kinase as a potential therapeutic target in lymphomas.

Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are highly sensitive to selective inhibition of CDK4 kinase activity.

The prognosis for patients with mantle cell lymphoma (MCL) is poor, and at present there is no truly effective therapy. Gene translocation-mediated constitutive expression of cyclin D1 seems to play the key role in the pathogenesis of MCL. Here we report that although 3 of 4 MCL cell lines expressed the recently identified, highly oncogenic cyclin D1b isoform, as well as the canonical cyclin D1a, 8 MCL patient samples expressed only the cyclin D1a protein despite expressing detectable cyclin D1b mRNA. Cell lines and tissue samples displayed constitutive activation of the cyclin D1 signaling cascade, as evidenced by strong expression of CDK4, Rb phosphorylation, and cyclin D1/CDK4 coassociation. All MCL cell lines and tissues examined displayed nondetectable to diminished expression of the cyclin D1 inhibitor p16. Novel small molecule CDK4/CDK6 inhibitor PD0332991 profoundly suppressed--at low nanomolar concentrations--Rb phosphorylation, proliferation, and cell cycle progression at the G0/G1 phase of MCL cells. These findings provide evidence that MCL should be very sensitive to targeted therapy aimed at functional inhibition of the cyclin D1/CDK4 complex.

TNF-alpha production-enhancing activity of 2-(1-adamantylamino)-6-methylpyridine (AdAMP) in cultures of human normal and neoplastic cells.

The purpose of this study was to determine the TNF-alpha-stimulatory effect of a novel immunomodulator 2-(1-adamantylamino)-6-methylpyridine (AdAMP) on normal and neoplastic human cells. In a panel of several human ovarian cancer cell lines, almost half of them spontaneously secreted significant amounts of TNF-alpha. When incubated with AdAMP, a 3-fold enhancement of TNF-alpha production by cells was observed. Furthermore, the phorbol myristic acetate ester (PMA)-induced release of TNF-alpha in cultures of U937 cells was increased in the presence of AdAMP. Primary monocytes isolated from peripheral blood did not respond to AdAMP. Although cytokine release was not triggered in human peripheral blood monocytes, AdAMP co-stimulated these cells to produce TNF-alpha and IL-8 during incubation with lipopolysaccharide (LPS). No effect of AdAMP was found on IL-1beta and IL-6 production by monocytes. In cultures of peripheral blood T lymphocytes, AdAMP significantly decreased the adhesion of these cells to matrix proteins in an in vitro assay. The results suggest that AdAMP, as a stimulator of cytokine secretion, may have potential application in tumor therapy.

Activation of mammalian target of rapamycin in transformed B lymphocytes is nutrient dependent but independent of Akt, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, insulin growth factor-I, and serum.

The study examines the preponderance and mechanism of mammalian target of rapamycin (mTOR) activation in three distinct types of transformed B lymphocytes that differ in expression of the EBV genome. All three types [EBV-immortalized cells that express a broad spectrum of the virus-encoded genes (type III latency; EBV+/III), EBV-positive cells that express only a subset of the EBV-encoded genes (EBV+/I), and EBV-negative, germinal center-derived cells (EBV-)] universally displayed activation of the mTOR signaling pathway. However, only the EBV+/III transformed B cells displayed also activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway that is considered to be the key activator of mTOR and of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway that coactivates one of the immediate targets of mTOR, p70 S6K1. Activation of the PI3K/Akt and MEK/ERK, but not of the mTOR pathway, was inhibited by serum withdrawal and restored by insulin growth factor-I. In contrast, activation of mTOR, but not PI3K/Akt and MEK/ERK, was sensitive to nutrient depletion. Both direct Akt (Akt inhibitors I-III) and a PI3K inhibitor (wortmannin at 1 nmol/L) suppressed Akt phosphorylation without significantly affecting mTOR activation. Furthermore, rapamycin, a potent and specific mTOR inhibitor, suppressed profoundly proliferation of cells from all three types of transformed B cells. U0126, a MEK inhibitor, had a moderate antiproliferative effect only on the EBV+/III cells. These results indicate that mTOR kinase activation is mediated in the transformed B cells by the mechanism(s) independent of the PI3K/Akt signaling pathway. They also suggest that inhibition of mTOR signaling might be effective in therapy of the large spectrum of B-cell lymphomas.