Differential activity of GSK-3 isoforms regulates NF-κB and TRAIL- or TNFα induced apoptosis in pancreatic cancer cells.

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3α or GSK-3ß. In contrast, depletion of GSK-3ß, but not GSK-3α, sensitized PDA cell lines to TNFα-induced cell death. Further experiments demonstrated that TNFα-stimulated IκBα phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3ß-deficient MEFs. Nonetheless, inhibition of GSK-3ß function in MEFs or PDA cell lines impaired the expression of the NF-κB target genes Bcl-xL and cIAP2, but not IκBα. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3ß targeted to the nucleus but not GSK-3ß targeted to the cytoplasm, suggesting that GSK-3ß regulates NF-κB function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3ß overexpression and nuclear localization contribute to TNFα and TRAIL resistance via anti-apoptotic NF-κB genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.

The mitotic kinase Aurora--a promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα(+) breast cancer cells.

In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα(+) breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα(+) breast cancer patients resistant to conventional endocrine therapy.

SOX2 promotes dedifferentiation and imparts stem cell-like features to pancreatic cancer cells.

SOX2 (Sex-determining region Y (SRY)-Box2) has important functions during embryonic development and is involved in cancer stem cell (CSC) maintenance, in which it impairs cell growth and tumorigenicity. However, the function of SOX2 in pancreatic cancer cells is unclear. The objective of this study was to analyze SOX2 expression in human pancreatic tumors and determine the role of SOX2 in pancreatic cancer cells regulating CSC properties. In this report, we show that SOX2 is not expressed in normal pancreatic acinar or ductal cells. However, ectopic expression of SOX2 is observed in 19.3% of human pancreatic tumors. SOX2 knockdown in pancreatic cancer cells results in cell growth inhibition via cell cycle arrest associated with p21(Cip1) and p27(Kip1) induction, whereas SOX2 overexpression promotes S-phase entry and cell proliferation associated with cyclin D3 induction. SOX2 expression is associated with increased levels of the pancreatic CSC markers ALDH1, ESA and CD44. Importantly, we show that SOX2 is enriched in the ESA(+)/CD44(+) CSC population from two different patient samples. Moreover, we show that SOX2 directly binds to the Snail, Slug and Twist promoters, leading to a loss of E-Cadherin and ZO-1 expression. Taken together, our findings show that SOX2 is aberrantly expressed in pancreatic cancer and contributes to cell proliferation and stemness/dedifferentiation through the regulation of a set of genes controlling G1/S transition and epithelial-to-mesenchymal transition (EMT) phenotype, suggesting that targeting SOX2-positive cancer cells could be a promising therapeutic strategy.

Mutant K-Ras increases GSK-3ß gene expression via an ETS-p300 transcriptional complex in pancreatic cancer.

Glycogen synthase kinase-3 beta (GSK-3ß) is overexpressed in a number of human malignancies and has been shown to contribute to tumor cell proliferation and survival. Although regulation of GSK-3ß activity has been extensively studied, the mechanisms governing GSK-3ß gene expression are still unknown. Using pancreatic cancer as a model, we find that constitutively active Ras signaling increases GSK-3ß gene expression via the canonical mitogen-activated protein kinase signaling pathway. Analysis of the mechanism revealed that K-Ras regulates the expression of this kinase through two highly conserved E-twenty six (ETS) binding elements within the proximal region. Furthermore, we demonstrate that mutant K-Ras enhances ETS2 loading onto the promoter, and ETS requires its transcriptional activity to increase GSK-3ß gene transcription in pancreatic cancer cells. Lastly, we show that ETS2 cooperates with p300 histone acetyltransferase to remodel chromatin and promote GSK-3ß expression. Taken together, these results provide a general mechanism for increased expression of GSK-3ß in pancreatic cancer and perhaps other cancers, where Ras signaling is deregulated.

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3beta positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-kappaB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3beta and to assess the anti-cancer effect of GSK-3beta inhibition in RCC. METHODS: Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3beta in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT-PCR, BrDU incorporation and MTS assays to study the effect of GSK-3beta inactivation on renal cancer cell proliferation and survival. RESULTS: We detected aberrant nuclear accumulation of GSK-3beta in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-kappaB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells. CONCLUSIONS: Our results show nuclear accumulation of GSK-3beta as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.

Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas.

The majority of anaplastic large cell lymphomas (ALCLs) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is oncogenic due to its constitutive tyrosine kinase activity. Transformation by NPM-ALK not only increases proliferation, but also modifies cell shape and motility in both lymphoid and fibroblastic cells. We report that the Rac1 GTPase, a known cytoskeletal regulator, is activated by NPM-ALK in ALCL cell lines (Karpas 299 and Cost) and transfected cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We have identified Vav3 as one of the exchange factors involved in Rac1 activation. Stimulation of Vav3 and Rac1 by NPM-ALK is under the control of Src kinases. It involves formation of a signaling complex between NPM-ALK, pp60(c-src), Lyn and Vav3, in which Vav3 associates with tyrosine 343 of NPM-ALK via its SH2 domain. Moreover, Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant demonstrates the central role of GTPases in NPM-ALK elicited motility and invasion.

A role for a RhoA/ROCK/LIM-kinase pathway in the regulation of cytotoxic lymphocytes.

Polarization of lipid rafts and granules to the site of target contact is required for the development of cell-mediated killing by cytotoxic lymphocytes. We have previously shown that these events require the activation of proximal protein tyrosine kinases. However, the downstream intracellular signaling molecules involved in the development of cell-mediated cytotoxicity remain poorly defined. We report here that a RhoA/ROCK/LIM-kinase axis couples the receptor-initiated protein tyrosine kinase activation to the reorganization of the actin cytoskeleton required for the polarization of lipid rafts and the subsequent generation of cell-mediated cytotoxicity. Pharmacologic and genetic interruption of any element of this RhoA/ROCK/LIM-kinase pathway inhibits both the accumulation of F-actin and lipid raft polarization to the site of target contact and the subsequent delivery of the lethal hit. These data define a specialized role for a RhoA-->ROCK-->LIM-kinase pathway in cytotoxic lymphocyte activation.

Regulation of NK cell-mediated cytotoxicity by the adaptor protein 3BP2.

Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95(vav) and phospholipase C-gamma isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.

Vav-2 controls NFAT-dependent transcription in B- but not T-lymphocytes.

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

The Rho family guanine nucleotide exchange factor Vav-2 regulates the development of cell-mediated cytotoxicity.

Previous pharmacologic and genetic studies have demonstrated a critical role for the low molecular weight GTP-binding protein RhoA in the regulation of cell-mediated killing by cytotoxic lymphocytes. However, a specific Rho family guanine nucleotide exchange factor (GEF) that activates this critical regulator of cellular cytotoxicity has not been identified. In this study, we provide evidence that the Rho family GEF, Vav-2, is present in cytotoxic lymphocytes, and becomes tyrosine phosphorylated after the cross-linking of activating receptors on cytotoxic lymphocytes and during the generation of cell-mediated killing. In addition, we show that overexpression of Vav-2 in cytotoxic lymphocytes enhances cellular cytotoxicity, and this enhancement requires a functional Dbl homology and Src homology 2 domain. Interestingly, the pleckstrin homology domain of Vav-2 was found to be required for enhancement of killing through some, but not all activating receptors on cytotoxic lymphocytes. Lastly, although Vav and Vav-2 share significant structural homology, only Vav is able to enhance nuclear factor of activated T cells-activator protein 1-mediated gene transcription downstream of the T cell receptor. These data demonstrate that Vav-2, a Rho family GEF, differs from Vav in the control of certain lymphocyte functions and participates in the control of cell-mediated killing by cytotoxic lymphocytes.

Cutting edge: WIP, a binding partner for Wiskott-Aldrich syndrome protein, cooperates with Vav in the regulation of T cell activation.

Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP), specifically binds to a region of WASp that is frequently mutated in Wiskott-Aldrich syndrome. Due to the similar phenotypes of WASp- and Vav-deficient T cells, and the putative importance of the WIP/WASp complex in mediating normal signals from the TCR, we investigated the role of WIP in regulating NF-AT/AP-1-mediated gene transcription. We show that WIP has the ability to enhance Vav-mediated activation of NF-AT/AP-1 gene transcription. In addition, we provide evidence that the interaction of WIP with WASp is necessary, but not sufficient for the ability of WIP to regulate NF-AT/AP-1 activity. Finally, we have identified a region in WIP required for its regulation of NF-AT/AP-1 activity. Our data suggests that the WIP-WASp interaction is important for NF-AT/AP-1-mediated gene transcription, and that defects seen in the activation of T cells from WAS patients may be due to the inability of these cells to form a functional WIP/WASp-signaling complex.

A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing.

Plasma membrane microdomains containing sphingolipids and cholesterol (lipid rafts) are enriched in signaling molecules. The cross-linking of certain types of cell surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes. However, little is known about the regulation of the initial raft redistribution and whether negative regulatory signaling pathways target this phase of cellular activation. We used natural killer (NK) cells as a model to investigate the regulation of raft redistribution, as both positive and negative signals have been implicated in the development of their cellular function. Here we show that after NK cells form conjugates with sensitive tumor cells, rafts become polarized to the site of target recognition. This redistribution of lipid rafts requires the activation of both Src and Syk family protein tyrosine kinases. In contrast, engagement of major histocompatibility complex (MHC)-recognizing killer cell inhibitory receptors (KIRs) on NK cells by resistant, MHC-bearing tumor targets blocks raft redistribution. This inhibition is dependent on the catalytic activity of KIR-associated SHP-1, a Src homology 2 (SH2) domain containing tyrosine phosphatase. These results suggest that the influence of integrated positive and negative signals on raft redistribution critically influences the development of cell-mediated cytotoxicity.

Specific subdomains of Vav differentially affect T cell and NK cell activation.

The Vav protooncogene is a multidomain protein involved in the regulation of IL-2 gene transcription in T cells and the development of cell-mediated killing by cytotoxic lymphocytes. We have investigated the differential roles that specific protein subdomains within the Vav protooncogene have in the development of these two distinct cellular processes. Interestingly, a calponin homology (CH) domain mutant of Vav (CH-) fails to enhance NF-AT/AP-1-mediated gene transcription but is still able to regulate the development of cell-mediated killing. The inability of the CH- mutant to enhance NF-AT/AP-1-mediated transcription appears to be secondary to defective intracellular calcium, because 1) the CH- mutant has significantly reduced TCR-initiated calcium signaling, and 2) treatment with the calcium ionophore ionomycin or cotransfection with activated calcineurin restores NF-AT/AP-1-mediated gene transcription. The pleckstrin homology (PH) domain of Vav has also been implicated in regulating Vav activation. We found that deletion of the PH domain of Vav yields a protein that can neither enhance gene transcription from the NF-AT/AP-1 reporter nor enhance TCR- or FcR-mediated killing. In contrast, the PH deletion mutant of Vav is able to regulate the development of natural cytotoxicity, indicating a functional dichotomy for the PH domain in the regulation of these two distinct forms of killing. Lastly, mutation of three tyrosines (Y142, Y160, and Y174) within the acidic domain of Vav has revealed a potential negative regulatory site. Replacement of all three tyrosines with phenylalanine results in a hyperactive protein that increases NF-AT/AP-1-mediated gene transcription and enhances cell-mediated cytotoxicity. Taken together, these data highlight the differential roles that specific subdomains of Vav have in controlling distinct cellular functions. More broadly, the data suggest that separate lymphocyte functions can potentially be modulated by domain-specific targeting of Vav and other critical intracellular signaling molecules.

Cutting edge: a role for the adaptor protein LAT in human NK cell-mediated cytotoxicity.

Stimulation of NK cell-mediated cytotoxicity involves the coupling of proximal Src and Syk family protein tyrosine kinases to downstream effectors. However, the mechanisms linking these second messenger pathways are incompletely understood. Here, we describe a key role for the LAT (p36) adaptor protein in human NK cell activation. LAT is tyrosine phosphorylated upon stimulation of NK cells through FcgammaRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with several phosphotyrosine-containing proteins. In addition to the biochemical evidence showing LAT involvement in NK cell activation, a genetic model shows that LAT is required for FcR-dependent phosphorylation of phospholipase C-gamma. Furthermore, overexpression of LAT in NK cells leads to increased Ab-dependent cell-mediated cytotoxicity and "natural cytotoxicity," thus demonstrating a functional role for LAT in NK cells. These data suggest that LAT is an important adaptor protein for the regulation of human NK cell-mediated cytotoxicity.

SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors.

Activation of immune system cells via antigen-, Fc-, or natural killer cell-triggering-receptor stimulation is aborted by co-engagement of inhibitory receptors. Negative signaling by killer cell inhibitory receptors and related receptors depends on the Src homology 2 (SH2)-containing protein tyrosine phosphatase SHP-1. Using a combination of direct binding and functional assays, we demonstrated that the SH2 domain-containing leukocyte protein 76 (SLP-76) is a specific target for dephosphorylation by SHP-1 in T cells and natural killer cells. Furthermore, we showed that tyrosine-phosphorylated SLP-76 is required for optimal activation of cytotoxic lymphocytes, suggesting that the targeted dephosphorylation of SLP-76 by SHP-1 is an important mechanism for the negative regulation of immune cell activation by inhibitory receptors.

The Vav-Rac1 pathway in cytotoxic lymphocytes regulates the generation of cell-mediated killing.

The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell-mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.

Sequential analysis of bone marrow and peripheral blood after stem cell transplant for myeloma shows disparate tumor involvement.

Treatment with combination chemotherapy has not resulted in long-term remissions in multiple myeloma (MM) despite advances in drug discovery and protocol improvement over the last 25 years. Increasingly, peripheral blood (PB) stem cell transplants (PBSCT) are being used along with chemotherapy and total body irradiation as treatment for multiple myeloma. Although the majority of tumor cells are found within the bone marrow (BM), tumor cells circulate in the PB in patients with MM. Therefore, one potential problem with PBSCT is contamination of the stem cell harvests with tumor cells. Although substantial reduction in BM tumor load is achieved after chemotherapy and autologous transplantation, most patients still relapse. In an attempt to identify and quantitate the residual tumor within sequential BM and PB samples of patients with MM following autologous PB stem cell transplants we have used a tumor-specific detection assay, allele-specific oligonucleotide-PCR (ASO-PCR). We found that while the BM tumor burden may fluctuate in some patients by as much as 4-logs after transplant, the PB tumor remains quite stable, and does not reflect the tumor burden in the BM. Moreover, analysis of PB involvement over time was not predictive of marrow involvement or of potential relapse. These results suggest that the PB is frequently involved in MM and further indicate that it represents a compartment that is only minimally altered by intensive therapy.

Circulating blood B cells in multiple myeloma: analysis and relationship to circulating clonal cells and clinical parameters in a cohort of patients entered on the Eastern Cooperative Oncology Group phase III E9486 clinical trial.

Recent analyses of circulating blood B cells in myeloma have generated controversy concerning the exact levels of these cells and whether they may represent circulating clonal tumor B cells. Previous reports suggested that CD19+ B cells are markedly increased in myeloma patients and that this population shares clonotypic rearrangements with the malignant plasma cell. We studied the numbers of CD19+ B cells by flow cytometry in previously untreated newly diagnosed myeloma patients in Eastern Cooperative Oncology Group (ECOG) phase III trial E9486. There were 628 patients who were eligible for the clinical protocol E9486, but of these 521 were also entered on the companion laboratory study (E9487) and had CD19 data. In comparison with normal controls, the myeloma patients exhibited a marked heterogeneity in the number of circulating CD19+ B cells as detected by flow cytometry. Approximately 20% of patients had significantly increased levels of circulating CD19+ B cells. However, the total CD19+ blood population from myeloma was not significantly different from the median of age-matched, normal controls. Analysis of CD19+ blood cells in relationship to circulating clonal cells was done in 13 myeloma patients using a clonotypic, quantitative allele-specific oligonucleotide-polymerase chain reaction (PCR) assay. No correlation was found between the numbers of CD19+ B cells (range, 5% to 51%) and PCR estimates of the number of clonal cells in the peripheral blood (range, .009% to 3.6%). Low CD19+ B-cell level (<125 microL) was associated with clinical stage III (P = .033). A significant relationship exists between higher levels (> or = 125/microL) of CD19 cells and longer overall survival (P < .0001). In addition, high CD19 levels also predicted a clinical response and longer event-free survival. There was a strong inverse association between the level of CD19 values at diagnosis and infections within the first 2 months of diagnosis. Importantly, the number of deaths related to infections was significantly greater in the low versus high CD19 group (P < .0202). Also, CD19 is an independent prognostic factor in addition to plasma cell labeling indices, beta2-microglobulin, hemoglobin, and plasmablastic morphology. Patients with infections were more likely to have low levels of CD19+ cells. In summary, higher CD19+ cell levels are a favorable prognostic sign with no apparent relationship to circulating tumor cells. In addition, this analysis strongly suggests that low peripheral blood levels of CD19+ cells are an adverse prognostic sign in myeloma. The CD19+ cell levels in myeloma patients is an important parameter in the overall assessment of these patients.

Activating mutations in the N- and K-ras oncogenes differentially affect the growth properties of the IL-6-dependent myeloma cell line ANBL6.

Although the underlying genetic defect in multiple myeloma is unknown, activating mutations in the N- and K-ras oncogenes are common. Recent studies have suggested that ras mutations are associated with disease progression. We have introduced an activated N-ras12, N-ras61, or K-ras12 cDNA into the interleukin 6 (IL-6)-dependent multiple myeloma cell line ANBL6 to determine the effect of N- and K-ras on the growth/death properties of ANBL6. All three transduced cell populations demonstrate a growth advantage over the parent ANBL6 when propagated on normal human bone marrow stromal cells. In the absence of bone marrow stromal cells, augmentation of growth was observed in all three mutant ras-expressing populations at optimal and suboptimal concentrations of IL-6. Furthermore, in the absence of IL-6, all mutant ras populations demonstrated an augmentation in DNA synthesis when compared to the parent ANBL6. However, growth of the K-ras12 population in the absence of IL-6 was significantly inhibited when compared to the mutant N-ras populations. This could be explained by the observation that in the absence of IL-6, N-ras12 and N-ras61 suppress apoptosis, whereas K-ras12 does not. We also found that mutant ras expression could result in early protection from glucocorticoid-induced apoptosis similar to that observed by the addition of IL-6. However, the combination of mutant ras and IL-6 could completely block the glucocorticoid induction of apoptosis in long-term cultures. These data suggest that mutations in different ras family members may have similar or distinct effects on myeloma tumor growth and death and may alter the response to glucocorticoid treatment.

Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity.

Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.