T-leukemia Cells Research Articles

Regulation of invariant natural killer T cell anti-tumor responses by the adaptor molecule Signaling Lymphocytic Activation Molecule-associated protein (SAP). (162.47)

Abstract Invariant natural killer T (iNKT) cells comprise a unique sub-lineage of innate-like T lymphocytes that participate in anti-tumor immune surveillance. However, several questions remain regarding the mechanisms by which these cells mediate killing of tumor cells. We observe that primary murine iNKT cell cytotoxicity against tumor cells is T cell receptor (TCR)-dependent and requires target cell presentation of iNKT cell-directed agonistic lipid antigens. Interestingly, expression of the cytoplasmic adaptor molecule SAP in primary murine iNKT cells is required for optimal TCR-mediated iNKT cell cytolytic responses towards B- and T-leukemia cells in vitro as well as iNKT cell-mediated control of leukemia cell growth in mice lacking CD8+ T and NK cells. iNKT cell cytotoxicity relies on expression of the Src family tyrosine kinase Fyn, a known SAP binding protein, and on the engagement of SLAM family receptors on iNKT and target cells. Of note, SAP- or Fyn- silenced human iNKT cells exhibit significantly impaired killing of T-leukemia cells in vitro, suggesting that SAP-associated signals are also critical for human iNKT cell anti-tumor cytotoxicity. These studies provide the first mechanistic insights into the signaling mechanisms underlying TCR-induced iNKT cell killing of tumor cells. Studies are ongoing to further elucidate the specific SLAM receptor(s) and downstream signaling mechanisms by which SAP regulates murine and human iNKT cell anti-tumor responses.

Study of molecular mechanisms of proapoptotic action of novel heterocyclic 4-thiazolidone derivatives

Aim. Mechanisms of induction of apoptosis signaling pathways in mammalian tumor cells treated by novel heterocyclic 4-thiazolidones with different side groups were studied. Methods. Annexin V/propidium iodide and DAPI (4',6-diamidino-2-phenylindole) staining of cells, Western-blot analysis of specific proteins. Results. 4-Thiazolidone derivatives of various structure possess similar cytotoxic activity in vitro (²N50 = 5 µM), and induce apoptosis in both leukemia (Jurkat, CCRF-CEM) and carcinoma (MCF-7, MDA-MD-231) cells. Western-blot analysis of the expression of several proteins of apoptosis signaling showed that the structure of lateral groups of 4thiazolidones may directly affect biological activity of these proteins in leukemia cells. In particular, compounds Les-3120 (pyrazoline-substituted thiazolidinone) and Les-3166 (thiazolidinone-benzothiazole conjugate) induced receptor-mediated apoptosis in Jurkat T-leukemia cells. 4-Iminothiazolidinone Les-3372 caused mitochondrial type apoptosis, mediated by AIF protein. Conclusions. Structure-functional relationships between the presence of specific side groups in novel 4-thiazolidones and the signaling apoptotic pathways induced by these compounds have been established. The obtained results allow designing new, «hybrid» compounds which can simultaneously induce more than one apoptotic pathway in tumor cells.

ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia cells

We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

Tagged and untagged TRAIL show different activity against tumor cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a novel cytotoxic ligand belonging to the TNF superfamily which is currently being developed as a cancer therapeutic drug. Here, we observed the different functions of recombinant TRAIL protein with a foreign protein label and non-labeled TRAIL. We used a prokaryotic expression system to prepare two different versions of the extracellular TRAIL 114-281aa protein: TRAIL-HS, a protein modified with 6xHis-Tag and S-Tag; and TRAIL-FT, which had no foreign protein. The proteins were purified using Ni-NTA chromatography (TRAIL-HS) and cation ion-exchange column chromatography (TRAIL-FT) and identified by SDS-PAGE and western blot analysis. We compared the abilities of the proteins to bind to death receptor 5 (DR5) by ELISA and to induce apoptosis in a normal liver cell line (Chang liver) and a human T-lymphocyte leukemia cell line (Jurkat) by MTT assay, GR staining and FACS. The results indicate that the biological functions of TRAIL-FT were superior to those of TRAIL-HS in binding and the induction of apoptosis, and may be useful to further the development and applications of TRAIL.

An oncogenic role of miR-142-3p in human T-cell acute lymphoblastic leukemia (T-ALL) by targeting glucocorticoid receptor-α and cAMP/PKA pathways

MicroRNAs (miRNAs) are a family of 19-24 nucleotide non-coding RNAs with posttranscriptional regulatory functions. The involvement of miRNAs in normal hematopoiesis implies that deregulated miRNAs might contribute to leukemogenesis. To date, although certain miRNAs have been established a clear oncogenic role in hematological malignancies, other individual miRNAs potentially involved in human leukemogenesis still remain elusive. In this report, we showed that miR-142-3p was upregulated in human T-leukemic cell lines and primary T-leukemic cells isolated from T-cell acute lymphoblastic leukemia (T-ALL) patients and its expressive levels were correlated with patients' prognosis. Such an oncogenic role of miR-142-3p could be explained by its targeting cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and glucocorticoid receptor alpha (GRα). High levels of miR-142-3p resulted in low levels of cAMP and weak activity of PKA, thus relieving the inhibitory effect of PKA on T-leukemic cell proliferation. Meanwhile, miR-142-3p decreased GRα protein expression by directly targeting the 3'-untranslational region of GRα mRNA, leading to glucocorticoid resistance. Transfection of the miR-142-3p inhibitor effectively converted glucocorticoid resistance, because of the resultant increase of GRα expression and PKA activity. These findings suggest that miR-142-3p is critical in T-cell leukemogenesis and may serve as a potential therapeutic target in T-ALL patients.

Differential requirements for c-Myc in chronic hematopoietic hyperplasia and acute hematopoietic malignancies in Pten-null mice

Myeloproliferative disorders (MPDs), lymphoproliferative disorders (LPDs), acute T-lymphocytic or myeloid leukemia and T-lymphocytic lymphoma were developed in inducible Pten (phosphatase and tensin homolog, deleted on chromosome ten)-knockout mice (Pten(-/-)). The appearance of these multiple diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether Myc function is required for the development of these hematopoietic disorders in Pten(-/-) mice, we generated inducible Pten/Myc double-knockout mice (Pten(-/-)/Myc(-/-)). By comparing the hematopoietic phenotypes of these double-knockout mice with those of Pten(-/-) mice, we found that both sets of animals developed MPDs and LPDs. However, none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPDs that developed in Pten(-/-) mice, which are dominated by granulocytes, megakaryocytes predominate in the MPDs of Pten(-/-)/Myc(-/-) mice. Our study suggests that the deregulation of phosphoinositide 3-kinase/Akt signaling in Pten(-/-) hematopoietic cells protects these cells from apoptotic cell death, resulting in chronic proliferative disorders. However, owing to the differential requirement for Myc in granulocyte as compared to megakaryocyte proliferation, Myc deletion converts Pten(-/-) MPDs from granulocyte- to megakaryocyte-dominated conditions. Myc is absolutely required for the development of acute hematopoietic malignancies.

Identification of T-lymphocytic leukemia–initiating stem cells residing in a small subset of patients with acute myeloid leukemic disease

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs, substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples, 39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However, 33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients, but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction, and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function, and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies.

Epigenetic regulation of CIITA expression in human T-cells

In humans, T-cells accomplish expression of MHC-II molecules through induction of CIITA upon activation. Here we show that CIITA promoter accessibility in T-cells is epigenetically regulated. In unstimulated T-cells, CIITA-PIII chromatin displays relative high levels of repressive histone methylation marks (3Me-K27-H3 and 3Me-K20-H4) and low levels of acetylated histones H3 (Ac-H3) and H4 (Ac-H4). These repressive histone marks are replaced by histone methylation marks associated with transcriptional active genes (3Me-K4-H3) and high levels of Ac-H3 and Ac-H4 in activated T-cells. This is associated with concomitant recruitment of RNA polymerase II. In T-leukemia cells, devoid of CIITA expression, similar repressive histone methylation marks and low levels of acetylated histone H3 correlated with lack of CIITA expression. This in contrast to CIITA expressing T-lymphoma cells, which display high levels of Ac-H3 and 3Me-K4-H3, and relative low levels of the 3Me-K27-H3 and 3Me-K20-H4 marks. Of interest was the observation that the levels of histone acetylation and methylation modifications in histones H3 and H4 were also noted in chromatin of the downstream CIITA-PIV promoter as well as the upstream CIITA-PI and CIITA-PII promoters both in normal T-cells and in malignant T-cells. Together our data show that CIITA chromatin in T-cells expressing CIITA display similar histone acetylation and methylation characteristics associated with an open chromatin structure. The opposite is true for T-cells lacking CIITA expression, which display histone modifications characteristic of condensed chromatin.

Camalexin induces apoptosis in T-leukemia Jurkat cells by increased concentration of reactive oxygen species and activation of caspase-8 and caspase-9

Camalexin, a major indole phytoalexin of Arabidopsis thaliana, accumulates in various cruciferous plants in response to environmental stress and reportedly displays antimicrobial activities against various plant pathogens. However, its cytotoxicity against eukaryotic cells and potential as a prospective drug for human diseases has been examined only in a limited context. Our data demonstrate the time- and concentration-dependent cytotoxicity of camalexin on human T-leukemia Jurkat cells in the micromolar range, and the lower potency of cytotoxic effects on human lymphoblasts and primary fibroblasts. Cytotoxicity of camalexin is enhanced by the glutathione-depleting agent buthionine sulfoximine and completely blocked by pan-caspase inhibitor Z-VAD-FMK. Treatment of Jurkat cells with camalexin resulted in activation of caspase-8, caspase-9, caspases-3/7, and apoptosis that was detected by the presence of a sub-G1 population of cells, externalization of phosphatidyl serine and decreased mitochondrial membrane potential. Staining with 2',7'-dichlorodihydrofluorescein diacetate and dihydroethidium bromide displayed increased concentration of reactive oxygen species (ROS) early in camalexin-treated Jurkat cells, prior to the onset of apoptosis, while staining with MitoSOX(™) dye identified mitochondria as a source of increased ROS. Our data suggest that this phytochemical, which has a wide range of predicted pharmacological activities, induces apoptosis in Jurkat leukemia cells through increased ROS followed by dissipation of mitochondrial membrane potential and execution of caspase-9- and caspase-8-initiated apoptosis. This is, to the best of our knowledge, the first report on antileukemic activity and mode of action of this unique indole phytoalexin.

Specific killing of CCR9 high-expressing acute T lymphocytic leukemia cells by CCL25 fused with PE38 toxin

We have previously demonstrated that CCR9 plays a pivotal role in drug resistance and invasion in human acute T-lymphocytic leukemia (T-ALL). In this study, we investigated whether the MOLT4 cells, which naturally express CCR9 at high levels, can be successfully killed by the specific ligand, CCL25 fused to Pseudomonas exotoxin 38 (PE38) toxin. Our results demonstrated that CCL25-PE38 was able to specifically kill MOLT4 cells via apoptosis induction, and suppress the growth of CCR9 + tumors. This work shows that CCR9 high-expressing human T-ALL cells can be successfully killed by delivering PE38 toxin fused to the ligand CCL25.

Detection of doxorubicin-induced apoptosis of leukemic T-lymphocytes by laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) was used to acquire the Raman spectra of leukemic T lymphocytes exposed to the chemotherapy drug doxorubicin at different time points over 72 hours. Changes observed in the Raman spectra were dependent on drug exposure time and concentration. The sequence of spectral changes includes an intensity increase in lipid Raman peaks, followed by an intensity increase in DNA Raman peaks, and finally changes in DNA and protein (phenylalanine) Raman vibrations. These Raman signatures are consistent with vesicle formation, cell membrane blebbing, chromatin condensation, and the cytoplasm of dead cells during the different stages of drug-induced apoptosis. These results suggest the potential of LTRS as a real-time single cell tool for monitoring apoptosis, evaluating the efficacy of chemotherapeutic treatments, or pharmaceutical testing.

Effects of paraoxon on neuronal and lymphocytic cholinergic systems

The cholinergic system in lymphocytes is hypothesized to be a key target for neurotoxic organophosphates (OPs). The present study determined the comparative effects of paraoxon, the active metabolite of OP-parathion, which is detected in the human neuroblastoma line, SH-SY5Y, and leukemic T-lymphocytes, MOLT-3, in vitro. Paraoxon induced cytotoxic effects in a dose- and time-dependent manner in both cells. Further, the paraoxon-induced modulatory effects were comparable despite different cell types, including over-expression of N-terminus acetylcholinesterase (N-AChE) protein, a marker of apoptosis, down-regulations of mRNA encoding M1, M2, and M3 muscarinic acetylcholine receptors (mAChRs), and induction in expression of c-Fos gene, an indication of certain mAChR subtype(s) activation. Furthermore, the non-selective cholinergic antagonist atropine partially attenuated the paraoxon-induced N-AChE and c-Fos activations in both types of cells. These results provide initial and additional information that OPs may similarly induce neuro- and immuno-toxic effects through mAChRs activation, and they underline the potential of using lymphocytes for assessing OPs-induced neurotoxicity.

The C2-streptavidin delivery system promotes the uptake of biotinylated molecules in macrophages and T-leukemia cells

Macrophages are tightly associated with inflammatory diseases as well as carcinogenesis, and therefore represent promising targets for drug delivery and gene transfer. We have recently established a novel protein delivery system based on the binary C2 toxin of Clostridium botulinum and streptavidin, allowing the uptake of exogenous biotinylated molecules into mammalian cells. Here, we applied this C2-streptavidin delivery system to macrophages and other leukocytes. First, the effect of wild-type C2 toxin on different leukocyte cell lines was tested, indicating no differences in sensitivity. Next, the uptake and stability of the engineered C2-streptavidin was analyzed in macrophages and Jurkat T-cells, showing internalization into the cytosol of both cell types with similar kinetics. The transporter did not exhibit any cytotoxic effect and did not interfere with phagocytosis in primary human macrophages. The C2-streptavidin system promoted specific uptake of biotinylated fluorophores into the cytosol of macrophages as revealed by confocal microscopy. In addition, flow cytometry analysis showed a significantly enhanced uptake of biotinylated fluorescent tracers in Jurkat leukemia cells mediated by the C2-streptavidin transporter. Our results demonstrate that C2-streptavidin is a functional delivery system for transport of biotinylated molecules into macrophages and other leukocytes without compromising cell viability and intrinsic functions such as phagocytosis.

Effect of bovine lactoferricin on DNA methyltransferase 1 levels in Jurkat T-leukemia cells

Abnormal methylation of the promoter of several genes is common in patients with acute lymphoblastic leukemia. Methylation of DNA is brought about by DNA methyltransferases (DNMT). Bovine lactoferricin (Lfcin B) is a cationic peptide that possesses potent in vitro and in vivo anticancer activity and might affect the expression of DNMT1. In the current study, we determined the mRNA and protein expression of DNMT1 in Jurkat T-leukemia cells, after incubation with Lfcin B, by real-time quantitative reverse transcription PCR and Western blot analysis. The results of real-time quantitative reverse transcription PCR showed that DNMT1 expression in Jurkat T-leukemia cells was reduced after treatment with Lfcin B, and Lfcin B reduced the half-life of DNMT1 mRNA from approximately 8 to 2h. The results of Western blot analysis showed that the expression of DNMT1 protein was down-modulated by Lfcin B in Jurkat T-leukemia cells. Moreover, we found that protein biosynthesis in Jurkat T-leukemia cells was essential for Lfcin B to down-modulate the expression of DNMT1.

Proteomic analysis revealed association of aberrant ROS signaling with suberoylanilide hydroxamic acid-induced autophagy in Jurkat T-leukemia cells

Suberoylanilide hydroxamic acid (SAHA) is a newly emerging histone deacetylase inhibitor (HDACi) and has been approved in phase II clinical trials for treating patients with cutaneous T-cell lymphoma. Autophagy is a conserved self-digestion process that degrades cytoplasmic materials and recycles long-lived proteins and organelles within cells. In this study, we demonstrate that SAHA stimulates autophagy in Jurkat T-leukemia cells, which was evidenced by the appearance of autophagic vacuoles, formation of acidic vesicular organelles, recruitment of LC3-II to the autophagosomes and conversion of LC3-I to LC3-II. Moreover, SAHA treatment upregulated expression of Beclin 1 and Atg7 and promoted formation of the Atg12-Atg5 conjugate. Furthermore, inhibition of autophagy by chloroquine (CQ) enhanced SAHA-induced apoptosis. To determine the underlying mechanism of SAHA-induced autophagy, two complementary proteomic approaches (2-DE and SILAC), coupled with ESI-Q-TOF MS/MS analysis are utilized to profile differentially expressed proteins between control and SAHA-treated Jurkat T-leukemia cells. In total, 72 proteins were identified with significant alterations. Cluster analysis of the changed proteins reveal several groups of enzymes associated with energy metabolism, anti-oxidative stress and cellular redox control, which suggested an abnormal reactive oxygen species (ROS) production in SAHA-treated Jurkat T-leukemia cells. These observations were further confirmed by ROS chemiluminescence assay. Mechanistic studies revealed that SAHA-triggered autophagy was mediated by ROS production, which could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we illustrated that Akt-mTOR signaling, a major suppressive cascade of autophagy, was inactivated by SAHA treatment. Taken together, our study identifies autophagy as a reaction to counter increased ROS and is thus involved as a cellular prosurvival mechanism in response to SAHA treatment.

α4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)‐binding protein

Mammalian alpha4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. alpha4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using alpha4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of alpha4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with alpha4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between alpha4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. alpha4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD-alpha4 immunocomplex. Furthermore, deletion mutants of alpha4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of alpha4, independent of the alpha4-PP2Ac binding site. Therefore, in addition to PP2Ac, alpha4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression.

Caspase cleavage of Ets-1 p51 generates fragments with transcriptional dominant-negative function

Ets-1 is a transcription factor that plays an important role in various physiological and pathological processes, such as development, angiogenesis, apoptosis and tumour invasion. In the present study, we have demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed in a caspase-dependent manner in Jurkat T-leukaemia cells undergoing apoptosis, resulting in three C-terminal fragments Cp20, Cp17 and Cp14 and a N-terminal fragment, Np36. In vitro cleavage of Ets-1 p51 by caspase 3 produces fragments consistent with those observed in cells undergoing apoptosis. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p51. This region is absent from the Ets-1 p42 isoform, which therefore cannot be cleaved by caspases. In Ets-1 p51, cleavage generates C-terminal fragments containing the DNA-binding domain, but lacking the transactivation domain. The Cp17 fragment, the major cleavage product generated during apoptosis, is devoid of transcriptional activity and inhibits Ets-1 p51-mediated transactivation of target genes by competing with Ets-1 p51 for binding to Ets-binding sites present in the target promoters. In the present study, we have demonstrated that caspase cleavage of Ets-1 within the exon VII-encoded region leads to specific down-regulation of the Ets-1 p51 isoform during apoptosis. Furthermore, our results establish that caspase cleavage generates a stable C-terminal fragment that acts as a natural dominant-negative form of the full-length Ets-1 p51 protein.

The Ca2+ channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells

Flunarizine is a Ca(2+) channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.

Effects of pluronic and doxorubicin on drug uptake, cellular metabolism, apoptosis and tumor inhibition in animal models of MDR cancers

Cancer chemotherapy is believed to be impeded by multidrug resistance (MDR). Pluronic (triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), PEO- b-PPO- b-PEO) were previously shown to sensitize MDR tumors to antineoplastic agents. This study uses animal models of Lewis lung carcinoma (3LL-M27) and T-lymphocytic leukemia (P388/ADR and P388) derived solid tumors to delineate mechanisms of sensitization of MDR tumors by Pluronic P85 (P85) in vivo. First, non-invasive single photon emission computed tomography (SPECT) and tumor tissue radioactivity sampling demonstrate that intravenous co-administration of P85 with a Pgp substrate, 99Tc-sestamibi, greatly increases the tumor uptake of this substrate in the MDR tumors. Second, 31P magnetic resonance spectroscopy ( 31P-MRS) in live animals and tumor tissue sampling for ATP suggest that P85 and doxorubicin (Dox) formulations induce pronounced ATP depletion in MDR tumors. Third, these formulations are shown to increase tumor apoptosis in vivo by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and reverse transcription polymerase chain reaction (RT-PCR) for caspases 8 and 9. Altogether, formulation of Dox with P85 results in increased inhibition of the growth solid tumors in mice and represents novel and promising strategy for therapy of drug resistant cancers.

Diagnosis and Treatment of Chronic T-Lymphocytic Leukemia in a Green Tree Monitor (Varanus prasinus)

A 13-year-old male green tree monitor (Varanus prasinus) presented with lethargy and chronic weight loss. Complete blood count (CBC) revealed marked leukocytosis (total leukocyte count = 278.1 × 103/μl) and peripheral blood films were characterized by marked lymphocytosis comprised of small, well-differentiated lymphocytes. Bone marrow aspiration revealed increased cellularity with a predominance of well-differentiated lymphocytes. Using immunocytochemistry, neoplastic lymphocytes in peripheral blood films were positive for CD3 epsilon and negative for CD79a and BLA.36, consistent with T-cell chronic lymphocytic leukemia (CLL). Distal tail necrosis was noted 17 days post-presentation and treated with partial caudectomy. Treatment was initiated for CLL with 0.8 mg/kg prednisone q 48 h 22 days post-presentation. CBC on days 94 to 151 post-presentation showed marked reduction in leukocytosis (counts remaining below 40.0 × 103/μL). The animal displayed improvement in activity, appetite, and gained weight during the initial treatment period. Marked leukocytosis (253.4 × 103/μL) returned on day 203 post-presentation, and on day 206 post-presentation the monitor received 0.08 mg/kg chlorambucil PO once in addition to ongoing prednisone administration. The animal died 210 days post-presentation. Histopathologic examination revealed neoplastic lymphocyte infiltration in the bone marrow at multiple sites as well as in liver, spleen, gall bladder, pancreas, kidneys, esophagus, and the oral cavity. Lymphocyte infiltration in multiple organs was primarily associated with areas of dense lymphocytic infiltrates surrounding blood vessels. This report describes the first published attempt of CLL treatment in a saurian species.