Mutant Jurkat Research Articles

L-plastin associated syndrome of immune deficiency and hematologic cytopenia

BackgroundLCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematological and immune defects. ObjectiveTo determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1. MethodsWe performed genetic, protein and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1, which we evaluated by engineering the orthologous mutation in mice and Jurkat cells. ResultsA splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect results in at least two aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshifting deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells, and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T cell populations. Functional analysis revealed that LCP1c740-1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunological analysis revealed defective actin organisation in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12, impaired germinal centre B cell expansion after immunisation, and reduced cytokinesis during T cell proliferation. ConclusionWe describe a unique human hematopoietic defect affecting neutrophils, lymphocytes and platelets, arising from partial LCP1 deficiency.

Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis

Wild-type (wt) p53-induced phosphatase 1 (Wip1), encoded by the protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) gene, is a serine/threonine phosphatase induced upon genotoxic stress in a p53-dependent manner. Wip1/PPM1D is frequently overexpressed, amplified and mutated in human solid tumors harboring wt p53 and is thus currently recognized as an oncogene. Oncogenic Wip1 dampens cellular stress responses, such as cell cycle checkpoints, apoptosis and senescence, and consequently increases resistance to anticancer therapeutics. Targeting Wip1 has emerged as a therapeutic strategy for tumors harboring wt p53. However, little is known about the efficacy of Wip1-targeted therapies in tumors lacking p53. The present study aimed to investigate the potential role of oncogenic Wip1 in p53 mutant (mt) Jurkat cells. In the present study, it was demonstrated that p53 mt Jurkat cells exhibited PPM1D/Wip1 gene amplification and expressed relatively high levels of Wip1, as confirmed by gene copy number and RNA expression analysis. In addition, Jurkat cells underwent G2 cell cycle arrest, apoptotic cell death and senescence in response to etoposide and doxorubicin, although the phosphorylation levels of DNA damage response (DDR) elements, including ataxia-telangiectasia mutated, ataxia-telangiestasia and Rad3-related, checkpoint kinase (Chk)1 and Chk2 were significantly low. Accordingly, the targeting of Wip1 phosphatase by RNA interference increased the phosphorylation of DDR elements, but decreased the rate of apoptosis in response to etoposide or doxorubicin in Jurkat cells. The induction of senescence or cell cycle arrest was not affected by the knockdown of Wip1. The results suggest that increased Wip1 expression enhances the apoptotic sensitivity of Jurkat cells in response to chemotherapeutic agents by attenuating DDR signaling. The present study highlights the possible pro-apoptotic role of Wip1 in a p53 mt T-cell acute lymphoblastic leukemia cell line. The data suggest the careful consideration of future treatment strategies aiming to manipulate or target Wip1 in human cancers lacking p53.

Abstract 646: Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1

Abstract Acute (AML) and chronic (CML) myeloid leukemia together account for approximately 50% of newly diagnosed leukemias. Both AML and advanced CML have dismal survival outcomes but are susceptible to immunotherapy. We discovered PR1 (VLQELNVTV) as an effective human leukocyte antigen (HLA)-A2 restricted peptide and have targeted it in clinical trials using PR1 peptide vaccine. PR1 is derived from the serine proteases proteinase-3 and neutrophil elastase which are highly expressed in myeloid leukemia blasts. PR1 was shown to be immunogenic in myeloid malignancies, and we are currently developing several PR1 targeting therapies. We have completed the development and humanization of a T-cell receptor-like monoclonal antibody (h8F4) that targets PR1/HLA-A2 and eliminates primary human AML xenografts in NSG mice. To improve the efficacy of h8F4, we have developed a bi-specific T-cell engaging (BiTE) antibody that targets leukemia PR1/HLA-A2 and the T-cell receptor CD3. This would enhance the anti-leukemia immune response by bringing the T-cell in close proximity to the leukemia cell, and by further activating the T-cells to eliminate the target leukemia. Here we demonstrate successful construction and expression of the h8F4 BiTE in CHO cells. The antibody was purified from cell culture supernatant via Ni-NTA column chromatography, and identity was confirmed using western blot analysis. Next, flow cytometry was used to test the binding specificities of each targeting region of the BiTE antibody. The h8F4 BiTE antibody showed increased binding to T2 cells pulsed with PR1 peptide compared with T2 pulsed with irrelevant CMV peptide pp65 (MFI=627 and 72.4, respectively). In addition, the h8F4 BiTE demonstrated 6.8 fold higher binding to wild type Jurkat cells, which express CD3, in comparison with the CD3-lacking mutant Jurkat cell line J.RT3-T3.5. These results indicate that both regions of the h8F4 BiTE can efficiently recognize their intended targets. Next, we investigated whether h8F4 BiTE engagement with both PR1/HLA-A2 on T2 cells and CD3 on healthy donor PBMC could activate T-cells. We show with flow cytometry that upon 24h incubation in the presence of h8F4 BiTE and T2 cells pulsed with PR1 peptide, both CD4 and CD8 T-cells increase their surface expression of CD69 (21 fold in CD4; 10.3 fold in CD8), CD25 (35 fold in CD4; 3.5 fold in CD8) and CD71 (26.6 fold in CD4; 640 fold in CD8) compared to co-incubation in the absence of BiTE protein. In conclusion, this study shows that we have successfully developed a h8F4 BiTE which recognizes both the CD3 on T cells and PR1/HLA-A2 complexes on myeloid leukemia. Furthermore, the h8F4 BiTE can activate multiple T-cell subsets from healthy donor PBMC as early as 24h following co-incubation with PR1-pulsed T2 cells. This novel BiTE antibody could provide a safer and more potent treatment option for patients with aggressive myeloid leukemias that will improve upon currently available highly toxic standard therapies. Citation Format: Amanda Herrmann, Jin Im, Sijie Lu, Jeffrey Molldrem. Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2014-646

Hypoxia Activated Pro-Drug TH-302 Induces Hypoxia-Dependent Anti-leukemia Activity in vitro and in vivo

Caspase-8 mutant Jurkat cells (JurkatI9.2) were completely resistant to birinapant, further supporting the critical role of caspase-8 in birinapant-mediated apoptosis. Birinapant synergistically enhanced apoptosis when combined with various nucleoside analogues clinically used in AML therapy and demethylating agents even in the cells co-cultured with bone marrow-derived stromal cells (MSCs). Mechanistic studies showed that decitabine and 5-azacytidine increased and activated caspase-8; induced XAF-1, a XIAP antagonist known to be hypermethylated in various malignant cells; and inhibited canonical and non-canonical NF-kB signaling. In addition, birinapant was effective against blasts from primary AML samples cocultured with MSCs with no toxicity in normal CD34 cells. Importantly, birinapant not only induced apoptosis in bulk AML blast but also in CD3438 AML stem/progenitor cells and prolonged mouse survival which was further enhanced by demethylating agents in a xenograft human AML model. Collectively, we showed that cIAP1 and caspase-8 are overexpressed and SMAC underexpressed in AML stem/progenitor cells and that inhibition of IAPs by birinapant synergistically enhances drug induced-death of AML cells in vitro and in vivo and has the potential to eliminate AML stem/progenitor cells.

Static magnetic field of 6 mT induces apoptosis and alters cell cycle in p53 mutant Jurkat cells

This study aimed to investigate the effect of 6 milliTesla (mT) static magnetic field (SMF) on apoptosis induction and cell cycle alteration in T-lymphoblastoid Jurkat E6.1 cells. Exposure of human p53 mutant Jurkat cells to 6 mT SMF resulted in apoptosis, which was detected by luminometric and flow cytometric analysis also, phosphorylated ATM and E2F1 proteins were detected by western blot analysis.Based on luminescence detection data, apoptosis initiated 36 h after exposure to 6 mT SMF. Apoptosis also reached its maximum rate 48 h after treatment. Flow cytometric analysis revealed a temporary G2 arrest after exposure to 6 mT SMF. Indeed, cellular population of S and G2 phases was increased. Based on reports of other investigations on the effect of magnetic fields on Ca2+flux changes in cell membranes and the effect of MFs on free radical formation, it can be suggested that the magnetic fields may induce the apoptosis and alter the cell population in different cell cycle phases of Jurkat cells via changing the Ca2+fluxes through cell membranes and playing a role in free radical formation. Western blot analysis showed that the amount of phosphorylated ATM and E2F1 proteins were increased in treated cells. The results of luminometric and flow cytometric detection did not show a significant difference in the apoptosis rate between 6 h-treated and 24 h-treated cells by 6 mT SMF. Thus, 6 mT SMF can induce apoptosis and alter cell cycle in Jurkat cells via a p53-independent pathway.

Gamma radiation alters cell cycle and induces apoptosis in p53 mutant E6.1 Jurkat cells

This study aimed at investigating the effect of gamma radiation with 1.5, 3.0 and 7.5Gy doses on apoptosis induction, cell cycle alteration and increment of amount of p-ATM (phosphorylated ATM) and p-E2F1 (phosphorylated E2F1) proteins in Jurkat T-lymphoblastoid E6.1 cells. Exposure of human p53 mutant Jurkat cells to gamma radiation resulted in apoptosis, which was detected by luminometric and flow cytometric analysis. Also, phosphorylated ATM (ataxia telangiectasia mutated) and E2F1 (elongation factor) proteins were detected by western blot analysis.Based on luminescence detection data the lethal dose of 7.5Gy induced cell death 12h after exposure (p<0.05) while sub-lethal doses of 1.5 and 3.0Gy induced apoptosis 18h after exposure (p<0.05). Flow cytometric analysis revealed a G2 arrest 24h after exposure to 3.0 and 7.5Gy. This arrest was accompanied by cell death with an increasing rate of occurrence up to 72h after exposure. Western blot analysis showed that 1h after cell irradiation by 1.5, 3.0 and 7.5Gy, the amount of p-ATM increased to its maximum rate and remained constant up to 6h, and then it decreased. Moreover, the amount of phosphorylated E2F1 (Ser-31) increased 2h after exposure to the same doses and remained constant up to 12h after irradiation. Survival and cell division of treated Jurkat cells showed a decrease compared to the control group. We believe that ionizing radiation-induced DNA damage activates a p53-independent apoptosis pathway via back-up systems in which the phosphorylation of ATM and E2F1 proteins was involved. Thus, gamma radiation can induce apoptosis and cell cycle alteration in Jurkat cells via a P53-independent pathway.

Abstract 2023: The role of RASSF1A in FasL expression and function

Abstract RASSF1A is a tumor suppressor protein that is epigenetically silenced in a variety of tumor cells, including those of lymphocytic origin. Several previous studies have identified a role for RASSF1A in death receptor-dependent apoptosis in abnormal cells of epithelial origin downstream of tumor necrosis factor receptor 1 (TNF-R1) engagement; however relatively little is known about a possible role for Rassf1A in lymphocytic cells. Interaction of Fas ligand (FasL) on activated T lymphocytes with the Fas receptor on other immune cells plays a major role in down-modulating immune responses through activation-induced cell death (AICD). Apoptosis of T cells that have undergone clonal expansion in response to antigenic stimulation through the TCR is a key regulatory feature of the adaptive immune response that prevents the accumulation of excessive lymphocytes once the antigen has been cleared. We have examined the effect of RASSF1A overexpression on FasL transcriptional activation and function in the Jurkat human T cell line. We have demonstrated that over-expression of RASSF1A induced a significant increase in FasL promoter (FasLp) activity. Similar-fold increases in transcriptional activity were observed when the distal NFAT binding site (3X NFAT plus minimal promoter) within FasLp was used in co-transfection reporter assays; however, other transcription reporters (AP1, NFkB) were unaffected. Experiments with mutant Jurkat cell lines indicate that early signaling mediators, including tyrosine kinases and PLCγ, are critical in the RASSF1A-induced enhancement of FasL production. Poly (ADP-ribose) polymerase (PARP) cleavage is mediated by caspases, and is indicative of apoptosis. Supernatants from anti-CD3 stimulated Jurkat cells that stably overexpress RASSF1A show an increased ability to induce cleavage of PARP in apoptosis-sensitive J16 target cells when compared to anti-CD3 stimulated vector-transfected cells. Taken together, these results indicate that RASSF1A plays a role in expression of soluble FasL leading to increased apoptosis-inducing activity by these lymphocytic cells. The loss of this activity could contribute to outgrowth of malignant clones of lymphocytic origin in which RASSF1A is silenced. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2023. doi:1538-7445.AM2012-2023

The WD40 Repeat Protein WDR26 Binds Gβγ and Promotes Gβγ-dependent Signal Transduction and Leukocyte Migration

The Gβγ subunits of heterotrimeric G proteins transmit signals to control many cellular processes, including leukocyte migration. Gβγ signaling may regulate and be regulated by numerous signaling partners. Here, we reveal that WDR26, a member of the WD40 repeat protein family, directly bound free Gβγ in vitro, and formed a complex with endogenous Gβγ in Jurkat T cells stimulated by the chemokine SDF1α. Suppression of WDR26 by siRNAs selectively inhibited Gβγ-dependent phospholipase Cβ and PI3K activation, and attenuated chemotaxis in Jurkat T cells and differentiated HL60 cells in vitro and Jurkat T cell homing to lymphoid tissues in scid mice. Similarly, disruption of the WDR26/Gβγ interaction via expression of a WDR26 deletion mutant impaired Gβγ signaling and Jurkat T cell migration, indicating that the function of WDR26 depends on its binding to Gβγ. Additional data show that WDR26 also controlled RACK1, a negative regulator, in binding Gβγ and inhibiting leukocyte migration. Collectively, these experiments identify WDR26 as a novel Gβγ-binding protein that is required for the efficacy of Gβγ signaling and leukocyte migration.

Antagonizing IAPs by SMAC Mimetic TL32711 Induces Apoptosis in AML Cells Including AML Stem/Progenitor Cells Alone and in Combination with Chemotherapy

Abstract 66The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs), including cIAP1, cIAP2, and XIAP, is antagonized by SMAC (second mitochondrial-derived activator of caspases). XIAP directly binds and inhibits caspase-9 and caspase-3 and suppresses both mitochondrion-mediated intrinsic and death receptor-mediated extrinsic apoptosis pathways, while the cIAPs are components of the cytoplasmic signaling complex containing members of TNF receptor associated factors and suppress death receptor/caspase-8 mediated extrinsic pathway activation. SMAC mimetics are a new class of anti-cancer agents that induce rapid degradation of cIAP1, relieve XIAP-mediated caspase repression, and promote TRAIL or TNFa-dependent apoptosis in various malignant cell types.To assess the therapeutic potential of SMAC mimetics in AML, we determined the protein levels of cIAP1 and XIAP, which are targets of SMAC mimetics, and caspase-8, the initiator caspase of the death receptor pathway by reverse phase protein array in blasts obtained from 511 newly diagnosed AML patients and in CD34+38− stem/progenitor cells isolated from blasts of these patients. We found that all three proteins were expressed in AML blasts. Importantly, we observed that the protein levels of cIAP1 and caspase-8 in CD34+38− AML stem/progenitor cells were significantly higher than those in bulk AML cells (P < 0.001).TL32711 (TL) is a highly potent and well-tolerated SMAC mimetic in clinical development that promoted rapid degradation of cIAP1 at low nM concentrations and induced pronounced apoptosis in AML cell lines in the presence of TNFα. Cell death was enhanced in the presence of TRAIL, confirming activation of the death receptor pathway as a significant mechanism of apoptosis induction. Caspase-8 mutant Jurkat cells (JurkatI9.2) were completely resistant to TL, further supporting the critical role of caspase-8 in TL-mediated cell death. TL synergistically enhanced apoptosis when combined with various nucleoside analogues clinically used in AML therapy such as Ara-C, clofarabine, and demethylating agents decitabine and 5-azacytidine (5-AC). Mechanistic studies showed that decitabine and 5-AC increased and activated caspase-8, decreased cFLIP, and induced XAF-1, a XIAP antagonist known to be hypermethylated in various malignant cell types. In addition, TL had single agent activity against blasts from primary AML samples with no toxicity in CD34+ cells from normal bone marrows at doses effective against AML cells. Importantly, TL not only induced apoptosis in bulk AML blast but also in CD34+38− AML stem/progenitor cells.Collectively, we showed that cIAP1 and caspase-8 are overexpressed in AML stem/progenitor cells and that inhibition of IAPs by the novel SMAC mimetic TL32711 synergistically enhances drug induced-death of AML cells and also has the potential to eliminate AML stem/progenitor cells. Disclosures:Weng:Tetralogic Pharmaceuticals: Employment. McKinlay:Tetralogic Pharmaceuticals: Employment.

Identification of signaling pathways involved in the mechanism of action of the immunomodulatory peptide gamma-D-glutamyl-L-tryptophan.

5591 Background: Gamma-D-glutamyl-L-tryptophan (SCV-07) is being studied for attenuation of oral mucositis (OM) in head and neck cancer patients receiving chemoradiation therapy. In a pilot study SCV-07-treated subjects had delayed onset of ulcerative OM (Adkins 2009) which correlated with changes in circulating cytokines (Alterovitz 2010). In animal studies SCV-07 inhibits growth of different xenografted tumor types. SCV-07 acts via immune modulation; initial evaluation of SCV-07 signaling demonstrated inhibition of cytokine induced STAT3 transcriptional activity. In this study we explored further the molecular mechanisms by which SCV-07 interferes with mucositis pathobiology and concurrently exhibits anti-tumor activity. Methods: Tyrosine phosphorylated STAT3 (STAT3 PY) was evaluated by Western blot of Jurkat or CD45 mutant Jurkat cells with or without IL-10 or IFNα stimulation and SCV-07. Global phosphorylation profiles were analyzed using phosphoprotein detection array kits. C57/B6 mice inoculated with B16F0 tumor cells were treated with vehicle or SCV-07 and plasma cytokines analyzed by multiplex assay when tumor inhibition was evident. Results: SCV-07 significantly inhibited IL-10- and IFNα-induced STAT3 PY in Jurkat cells which was blocked by pervanadate treatment, implicating tyrosine phosphatase activity. Phosphatase CD45 was not required since STAT3 PY inhibition was seen in CD45 mutant Jurkat cells. However, SCV-07 induced activating phosphorylation of the phosphatase SHP-2 and altered phosphorylation of Src family kinases and STAT5a/b, known substrates for SHP-2. SCV-07 also decreased IFNα stimulated phosphorylation of SHP-2 associated immunoreceptors Siglec3 (CD33) and SIRPbeta1 (CD172B). SCV-07-treatment of B16 bearing mice slowed tumor growth and significantly decreased the STAT3-induced cytokines MCP-1 and IL-12p40 which are implicated in immune pathology. Conclusions: These data suggest a model where SCV-07, via SHP-2 activation and STAT3 inhibition, could shift macrophage subsets from M2 to M1 and T cells from Th2 to Th1, thereby promoting an immune regulatory environment that inhibits both mucositis and tumor progression.

Competition between SLP76 and LAT for PLCγ1 binding in resting T cells

The constitutive interaction between the P1 domain (a 67-amino-acid functional domain within the proline-rich region) of SLP76 and the SH3 domain of phospholipase Cγ1 (PLCγ1) has been shown. To determine the significance of the interaction between SLP76 and PLCγ1 in resting T cells, we examined molecules associated with PLCγ1 in the absence of both SLP76 and, more specifically, the P1 domain of SLP76. Using a mutant Jurkat T-cell line, we showed that PLCγ1 associated with LAT when the constitutive association with SLP76 was blocked. We also found that the PLCγ1 association with LAT occurred in the membranes of resting T cells. Further experiments demonstrated that LAT competed with SLP76 for PLCγ1 binding and that the LAT interaction with PLCγ1 was mediated by the SH3 domain of PLCγ1. Collectively, these results suggest that the constitutive association of SLP76 with PLCγ1 is required to prevent the association with LAT as well as the premature recruitment of PLCγ1 to the cell membrane.

RasGRP1 Is Essential for Ras Activation by the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate in Epidermal Keratinocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes and suggest JNK2 as one of the relevant downstream targets. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

In vitro evaluation of dichloro-bis(pyrazole)palladium(II) and dichloro-bis(pyrazole)platinum(II) complexes as anticancer agents

Cisplatin (cis-diamminedichloroplatinum) was first identified for its anti-bacterial activity, and was later also shown to be an efficient anticancer agent. However, the therapeutic use of this anticancer drug is somewhat limited by its toxic side effects, which include nephrotoxicity, nausea, and vomiting. Furthermore the development of drug-resistant tumours is commonly observed following therapy with cisplatin. Hence there is a need for improved platinum derived drugs to overcome these limitations. Apoptosis contributes significantly to the cytotoxic effects of anticancer agents such as cisplatin; therefore in this study the potential anticancer properties of a series of pyrazole palladium(II) and platinum(II) complexes, [(3,5-R(2)pz)(2)PdCl(2)] [R = H (1), R = Me (2)] and [(3,5-R(2)pz)(2)PtCl(2)] [R = H (3), R = Me (4)], were evaluated by assessment of their pro-apoptotic activity. The induction of apoptosis was measured in CHO cells by the detection of phosphatidylserine (PS) exposure using the annexin V and APOPercentage assays; DNA fragmentation using the Terminal deoxynucleotide transferase dUTP Nick End Labelling (TUNEL) assay; and the detection of activated caspase-3. The platinum complexes were shown to be considerably more active than the palladium complexes, with complex 3 demonstrating the highest level of cytotoxic and pro-apoptotic activity. The LD(50) values for complex 3 and cisplatin were 20 and 70 microM, respectively, demonstrating that the cytotoxic activity for complex 3 was three times higher than for cisplatin. Various human cancer cell lines, including CaSki, HeLa, as well as the p53 mutant Jurkat T cell line were also shown to be susceptible to complex 3. Collectively, this in vitro study provides insights into action of palladium and platinum complexes and demonstrates the potential use of these compounds, and in particular complex 3, in the development of new anticancer agents.

Renal Cell Carcinoma Tumors Induce T Cell Apoptosis through Receptor-Dependent and Receptor-Independent Pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.

Prostaglandin E2 Activates HPK1 Kinase Activity via a PKA-dependent Pathway

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that the immunosuppressive eicosanoid, prostaglandin E(2) (PGE(2)), is capable of activating HPK1 in T cells. In this report, we demonstrate that unlike the TCR-induced activation of HPK1 kinase activity, the induction of HPK1 catalytic activity by PGE(2) does not require the presence of phosphotyrosine-based signaling molecules such as Lck, ZAP-70, SLP-76, and Lat. Nor does the PGE(2)-induced HPK1 activation require the intermolecular interaction between its proline-rich regions and the SH3 domain-containing adaptor proteins, as required by the signaling from the TCR to HPK1. Instead, our study reveals that PGE(2) signal to HPK1 via a 3' -5 '-cyclic adenosine monophosphate-regulated, PKA-dependent pathway. Consistent with this observation, changing the serine 171 residue that forms the optimal PKA phosphorylation site within the "activation loop" of HPK1 to alanine completely prevents this mutant from responding to PGE(2)-generated stimulation signals. Moreover, the inability of HPK1 to respond to PGE(2) stimulation in PKA-deficient S49 cells further supports the importance of PKA in this signaling pathway. We speculate that this unique signaling pathway enables PGE(2) signals to engage a proven negative regulator of TCR signal transduction pathway and uses it to inhibit T cell activation.

Loss of Caspase-9 Reveals Its Essential Role for Caspase-2 Activation and Mitochondrial Membrane Depolarization

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.

GPCR-TCR Heterodimer

Stromal cell-derived factor 1α (SDF-1α) is a chemokine that signals through the G protein-coupled receptor (GPCR) CXCR4. In T cells, SDF-1α stimulates gene expression, chemotaxis, and cell adhesion. Kumar et al. provide evidence that SDF-1α stimulates an interaction between CXCR4 and the T cell receptor (TCR) that allows SDF-1α to signal through the TCR immunotyrosine-based activation motifs (ITAMs). Jurkat cells expressing a fluorescently tagged CXCR4 and CD3-ζ subunit of the TCR showed colocalization of the two proteins at the surface and in intracellular vesicles after SDF-1α stimulation. The interaction was confirmed by coimmunoprecipitation and two different fluorescence energy transfer (FRET) assays. The interaction between the two proteins was not blocked by pertussis toxin, which inhibits some of the SDF-1α-stimulated signals, but was blocked by inhibition of actin polymerization or phosphoinositide 3-kinase activity. Mutant Jurkat cell lines lacking either the β subunit of the TCR, which are deficient in cell surface TCRs, or the nonreceptor tyrosine kinase ZAP-70 exhibited decreased responsiveness to SDF-1α stimulation in terms of extracellular signal-regulated kinase (ERK) phosphorylation and stimulation of an AP-1-dependent reporter gene. SDF-1α signaling was restored in the TCR-β mutant cells if the cells were reconstituted with the β subunit or with a chimeric molecule consisting of the extracellular and transmembrane domains of CD8 and the cytoplasmic domain of CD3-ζ. Expression of various mutant versions of this CD8/CD3-ζ chimera was used to demonstrate that the CD3-ζ ITAM was essential for SDF-1α signaling. The AP-1-dependent gene expression, but not gene expression mediated by two other transcription factors NF-AT or NF-κB, in Jurkat cells was much greater in response to costimulation with SDF-1α and an antibody against CD3 to activate the TCR than in response to either TCR activation or SDF-1α alone. The authors propose a model whereby SDF-1α binding to the CXCR4 induces reorganization of the membrane, allowing CXCR4 and TCR to interact, which allows ZAP-70 to participate in signaling from both receptors, resulting in an enhanced response and increased ERK activation and AP-1-dependent gene activation. A. Kumar, T. D. Humphreys, K. N. Kremer, P. S. Bramati, L. Bradfield, C. E. Edgar, K. E. Hedin, CXCR4 physically associates with the T cell receptor to signal in T cells. Immunity 25 , 213-224 (2006). [PubMed]

Molecular Determinants of Kinase Pathway Activation by Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) mainly activates programmed cell death through caspases. By contrast, TNF primarily induces gene transcription through the inhibitor of kappaB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. Apo2L/TRAIL also can stimulate these kinases, albeit less strongly; however, the underlying mechanisms of this stimulation and its relation to apoptosis are not well understood. Here we show that Apo2L/TRAIL activates kinase pathways by promoting the association of a secondary signaling complex, subsequent to assembly of a primary, death-inducing signaling complex (DISC). The secondary complex retained the DISC components FADD and caspase-8, but recruited several factors involved in kinase activation by TNF, namely, RIP1, TRAF2, and NEMO/IKKgamma. Secondary complex formation required Fas-associated death domain (FADD), as well as caspase-8 activity. Apo2L/TRAIL stimulation of JNK and p38 further depended on RIP1 and TRAF2, whereas IKK activation required NEMO. Apo2L/TRAIL induced secretion of interleukin-8 and monocyte chemoattractant protein-1, augmenting macrophage migration. Thus, Apo2L/TRAIL and TNF organize common molecular determinants in distinct signaling complexes to stimulate similar kinase pathways. One function of kinase stimulation by Apo2L/TRAIL may be to promote phagocytic engulfment of apoptotic cells.

The Cathepsin B Death Pathway Contributes to TNF Plus IFN-γ-Mediated Human Endothelial Injury

Vascular endothelial cells are primary targets of cytokine-induced cell death leading to tissue injury. We previously reported that TNF in combination with LY294002, a PI3K inhibitor, activates caspase-independent cell death initiated by cathepsin B (Cat B) in HUVEC. We report that TNF in the presence of IFN-gamma activates Cat B as well as a caspase death pathway in both HUVEC and human dermal microvascular endothelial cells, but only activates caspase-mediated death in HeLa cells and human embryonic kidney (HEK)293 cells. Like LY294002, IFN-gamma triggers Cat B release from lysosomes in HUVEC. Cat B-triggered death involves mitochondria, indicated by release of cytochrome c, loss of mitochondrial membrane potential and inhibition of death by overexpressed Bcl-2. Cat B effects on mitochondria do not depend upon Bid cleavage. Unexpectedly, overexpression of a dominant negative mutated form of Fas-associated death domain protein (FADD), which blocks caspase activation by TNF, potentiates TNF activation of Cat B and cell death in HUVEC. Similarly, mutant Jurkat cells lacking FADD also show increased susceptibility to TNF-induced Cat B-dependent cell death. These observations suggest that the Cat B death pathway is cell type-specific and may contribute to cytokine-mediated human tissue injury and to the embryonic lethality of FADD gene disruption in mice.

Apoptosis by Low-Dose Methotrexate Occurs in Fully Activated Proliferating CD4+ and CD8+ Lymphocytes and in Leukemic LGL after Mitogenic Stimulation using a FADD-Dependent Pathway.

Low-dose methotrexate (MTX) is used as an immunosuppressive agent for the treatment of rheumatoid arthritis (RA), Large Granular Lymphocyte (LGL) leukemia, Cutaneous T Cell Lymphoma (CTCL), autoimmune diseases, and prevention of GvHD during bone marrow transplants. The mechanism for immunosuppression is not clearly understood but most data suggests that apoptosis of activated lymphocytes plays a critical role. In this study, we wanted to define the MTX-sensitive population and to determine the apoptotic pathway activated by MTX. Using a clinically relevant dosage range (8 nM- 1 μM), MTX-mediated apoptosis was first examined in a T lymphoblastic leukemia cell line (CEM). The apoptotic pathway induced by MTX included phosphotidylinositol externalization and caspase-3 activation along with a slight increase in mitochondrial membrane depolarization. We next examined a series of tumor cell lines and normal cells for evidence of MTX-induced apoptosis. Using the same clinically relevant dosage range, we found that MTX-induced apoptosis was primarily observed in the four T cell leukemia cell lines including CEM, Jurkat, MT-2, and HUT78 and in normal PBMCs activated with mitogens and IL-2. Less MTX-induced apoptosis was observed in two myeloid leukemia cell lines including HL-60 and K562 and in a B cell leukemia cell line Raji, and the multiple myeloma cell line 8226. Unactivated PBMCs were resistant to MTX-mediated apoptosis. T cells that are clonally expanded in patients with T-LGL leukemia have a CD8+ cytotoxic phenotype, whereas other diseases that are treated with low-dose MTX, such as CTCL and RA, are characterized by the expansion of CD4+ T cells. We found that both freshly sorted CD4+ and CD8+ cells were MTX resistant. In contrast, PHA plus IL-2 treatment induced MTX sensitivity in T cell with both immunophenotypes. We also examined clinical samples from patients with LGL leukemia. We found that freshly isolated PBMCs from T-LGL leukemia patients were resistant to MTX. Clonal cells from the peripheral blood of LGL leukemia patients are in G0/G1 phase of the cell cycle. Interestingly, we found that PHA plus IL-2 treatment induced the cells to enter S-phase and to become MTX sensitive. These results suggest that only fully activated, proliferating T cells from patients with LGL leukemia undergo apoptosis in response to low-dose MTX.Because there was only minor depolarization of mitochondria after MTX treatment in both CEM cells and normal activated PBMCs, we wanted to examine upstream apoptotic events after MTX treatment. We found that caspase-8 cleavage and enzymatic activity was induced by MTX in both CD95 Type I (HUT78) and Type II (CEM and Jurkat) cells but that there was a differential requirement for caspase-8 activity for apoptosis. We found that caspase-8 activation was independent of the Fas receptor as shown by immunoprecipitation experiments and MTX apoptotic assays in the JM3A5 Fas-receptor mutant Jurkat cell line. Using a Jurkat cell line with a homozygous deletion of the FADD gene, we found that caspase-8 activation, caspase-3 activation, and apoptosis in response to MTX were dependent on the adaptor protein FADD. These findings have important implications for understanding the mechanism of MTX for immunosuppressive therapy.