Clonal conversion of B lymphoid leukemia reveals cross-lineage transfer of malignant states.

Even though leukemia is considered to be confined to one specific hematopoietic cell type, cases of acute leukemia of ambiguous lineage and patients relapsing in phenotypically altered disease suggest that a malignant state may be transferred between lineages. Because B-cell leukemia is associated with mutations in transcription factors of importance for stable preservation of lineage identity, we here investigated the potential lineage plasticity of leukemic cells. We report that primary pro-B leukemia cells from mice carrying heterozygous mutations in either or both the Pax5 and Ebf1 genes, commonly mutated in human leukemia, can be converted into T lineage leukemia cells. Even though the conversion process involved global changes in gene expression and lineage-restricted epigenetic reconfiguration, the malignant phenotype of the cells was preserved, enabling them to expand as T lineage leukemia cells in vivo. Furthermore, while the transformed pro-B cells displayed plasticity toward myeloid lineages, the converted cells failed to cause myeloid leukemia after transplantation. These data provide evidence that a malignant phenotype can be transferred between hematopoietic lineages. This has important implications for modern cancer medicine because lineage targeted treatment of leukemia patients can be predicted to provoke the emergence of phenotypically altered subclones, causing clinical relapse.

Mechanosensitive Ca²âº-permeable channels in human leukemic cells: pharmacological and molecular evidence for TRPV2.

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²âº and Mg²âº. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³âº (10 µM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 µM) and SKF 96365 (250 µM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 µM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 µM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²âº signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.

Silver nanoparticles exhibit size-dependent differential toxicity and induce expression of syncytin-1 in FA-AML1 and MOLT-4 leukaemia cell lines.

Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp's being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types.

Prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim reduces the association of Bcl-2 with Bak or Bim, provoking Bak activation and mitochondrial apoptosis in nocodazole-treated Jurkat T cells.

Treatment of Jurkat T cells with the microtubule-depolymerizing agent nocodazole (NOC) caused prometaphase arrest and apoptosis. NOC-induced mitochondrial apoptotic events including Bak activation, Δψm loss, cytochrome c release, and caspase cascade activation were blocked by Bcl-2 overexpression. However, mitotic arrest, Cdc25C activation, upregulation of cyclin B1 levels, Cdk1 activation, Bcl-2 phosphorylation at Thr-56 and Ser-70, and Bim phosphorylation were retained. The treatment of Jurkat T cells concomitantly with NOC and the G1/S-blocking agent hydroxyurea resulted in G1/S arrest and complete abrogation of all apoptotic events. The association of Bcl-2 with Bim or Bak declined after the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, whereas the association of Bcl-2 with Bax remained relatively constant. Although Bax was redistributed from the cytosol to the mitochondria, resulting in an increase in the mitochondrial level of Bax following NOC treatment, the subcellular localization of Bcl-2, Bim, Bak and apoptosis-inducing factor was confined to the mitochondrial fraction irrespective of NOC treatment. Experiments using selective caspase inhibitors showed that mitochondria-dependent activation of caspase-9 and -3 was crucial for NOC-induced apoptosis. NOC-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and mitochondria-dependent apoptotic events were significantly suppressed by a Cdk1 inhibitor roscovitine, but not by the JNK inhibitor SP600125 or the p38 MAPK inhibitor SB203580. These results show that the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, which was mediated by Cdk1, could reduce the association of Bcl-2 with Bak or Bim to allow Bak activation and mitochondrial apoptotic events in Jurkat T cells exposed to NOC.

An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced Bcl-xL deamidation in T cell transformation.

A transgenic mouse model of T cell lymphoma was used to investigate the transforming events mediated by an oncogenic tyrosine kinase in pretumorigenic CD4-CD8- (DN) thymocytes. Parental CD45(-/-) and p56(lck-F505Y) mice do not develop tumors, whereas their CD45(-/-)p56(lck-F505Y) progeny develop T lymphomas. Increased but nononcogenic p56lck kinase activity in p56(lck-F505Y) mice DN thymocytes causes cell-cycle progression, survival, and Bcl-XL upregulation. Additional unique oncogenic signals occur in pretumorigenic CD45(-/-)p56(lck-F505Y) thymocytes in which p56lck kinase activity is 2- to 3-fold higher relative to p56(lck-F505Y): inhibition of DNA repair, inhibition of DNA-damage-induced Bcl-XL deamidation, Bax conformational change and mitochondrial translocation, cytochrome c release, and the apoptotic caspase execution cascade. Inhibition of Bcl-XL deamidation may be a critical switch in oncogenic kinase-induced T cell transformation.

Proteasome inhibitor bortezomib targeted tumor-endothelial cell interaction in T-cell leukemia/lymphoma.

Endothelial cells have special relevance in tumor progression. Here, we investigated the effect of the proteasome inhibitor bortezomib on tumor-endothelial cell interaction in T-cell leukemia/lymphoma. In vitro, T-leukemia/lymphoma cell lines and primary T-leukemia/lymphoma cells were cultured with endothelial cells, either together or separately in Millicell Hanging Cell Culture system, the latter permits mutual cell exchange. At clinically achievable concentrations, in addition to a direct cytotoxicity on T-leukemia/lymphoma cells, bortezomib inhibited tumor cell adhesion to endothelial cells and endothelial cell migration toward tumor cells. In vivo, a murine tumor xenograft model was achieved by subcutaneous injection of Jurkat cells. Bortezomib also triggered an inhibition on tumor-endothelial cell contact and subsequent tumor cell infiltration. Cell adhesion molecule intracellular cell adhesion molecule-1 expression was significantly downregulated both on the tumor cells and on the endothelial cells. Taken together, bortezomib could not only act on tumor cells themselves but also abrogate tumor cell interaction with endothelial cells. This delineates another therapeutic mechanism of bortezomib in T-cell malignancies.

NK-like homeodomain proteins activate NOTCH3-signaling in leukemic T-cells.

BACKGROUND: Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs. METHODS: Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses. RESULTS: Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3 and HEY1 was detected in primary TLX1/3 positive T-ALL cells corresponding to the cell line data. CONCLUSION: Identification and analysis of MSX2 in hematopoietic cells implicates a modulatory role via NOTCH3-signaling in early T-cell differentiation. Our data suggest that reduction of NOTCH3-signaling by physiological downregulation of MSX2 expression during T-cell development is abrogated by ectopic expression of oncogenic NKLs, substituting MSX2 function.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)C theta is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-kappa B activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pT alpha/pre-TCR and constitutive activation of NF-kappa B characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKC theta is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-kappa B activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKC theta in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-kappa B activation. This paper therefore suggests that PKC theta mediates the activation of NF-kappa B by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

Pathogenesis of neutropenia in large granular lymphocyte leukemia and Felty syndrome.

T-cell large granular lymphocyte leukemia (TLGL) is an atypical chronic lymphoproliferative disorder derived from cytotoxic T-cells (CTL). Unlike most forms of leukemia, the pattern of bone marrow infiltration in TLGL may be subtle and the cytopenias are often lineage specific, with neutropenia dominating. Both granulocytic survival and proliferation defects are observed and are mediated by humoral and cell-mediated mechanisms respectively. Splenic production of immune complexes induces a neutrophil survival defect, where as Fas expression by leukemic CTL results in a marrow based proliferation defect. These humoral and cell-mediated pathways induce granulocytic apoptosis through independent intracellular mechanisms which are not mutually exclusive and may be observed concurrently in individual patients with either TLGL or FS. A variety of therapeutic interventions have been utilized in the management of TLGL and Felty syndrome, including methotrexate, cyclosporine A, cyclophosphamide, glucocorticoids, myeloid colony stimulating factors and splenectomy. Their efficacy and mechanisms of action are reviewed.

Metabolic profiling by 13C-NMR spectroscopy: [1,2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells.

Metabolic profiling is defined as the simultaneous assessment of substrate fluxes within and among the different pathways of metabolite synthesis and energy production under various physiological conditions. The use of stable-isotope tracers and the analysis of the distribution of labeled carbons in various intermediates, by both mass spectrometry and NMR spectroscopy, allow the role of several metabolic processes in cell growth and death to be defined. In the present paper we describe the metabolic profiling of Jurkat cells by isotopomer analysis using (13)C-NMR spectroscopy and [1,2-(13)C(2)]glucose as the stable-isotope tracer. The isotopomer analysis of the lactate, alanine, glutamate, proline, serine, glycine, malate and ribose-5-phosphate moiety of nucleotides has allowed original integrated information regarding the pentose phosphate pathway, TCA cycle, and amino acid metabolism in proliferating human leukemia T cells to be obtained. In particular, the contribution of the glucose-6-phosphate dehydrogenase and transketolase activities to phosphoribosyl-pyrophosphate synthesis was evaluated directly by the determination of isotopomers of the [1'-(13)C], [4',5'-(13)C(2)]ribosyl moiety of nucleotides. Furthermore, the relative contribution of the glycolysis and pentose cycle to lactate production was estimated via analysis of lactate isotopomers. Interestingly, pyruvate carboxylase and pyruvate dehydrogenase flux ratios measured by glutamate isotopomers and the production of isotopomers of several metabolites showed that the metabolic processes described could not take place simultaneously in the same macrocompartments (cells). Results revealed a heterogeneous metabolism in an asynchronous cell population that may be interpreted on the basis of different metabolic phenotypes of subpopulations in relation to different cell cycle phases.

Dichotomy of all-trans retinoic acid inducing signals for adult T-cell leukemia.

We previously reported that all-trans retinoic acid (ATRA) inhibits growth in human T-cell leukemia virus type 1 (HTLV-1)-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-kappaB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax, or pol mRNA) using the real-time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT inhibited proviral DNA but not NF-kappaB transcriptional activity, and sIL-2R on HTLV-1; however, ATRA inhibited of NF-kappaB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease in sIL-2R induced by ATRA may be caused by the actions of a NF-kappaB inhibitor acting on the NF-kappaB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-kappaB inhibitor and as an RT inhibitor.

Secretion of parathyroid hormone-like activity from human T-cell lymphotropic virus type I-infected lymphocytes.

Because many patients with adult T-cell leukemia/lymphoma (ATLL) develop hypercalcemia with similar characteristics to those of humoral hypercalcemia of malignancy (HHM) (Arch. Intern. Med., 148: 921-925, 1988), we investigated if ATLL cells produce parathyroid hormone (PTH)-like activity. Conditioned media from cultures of human T-cell lymphotropic virus type I-infected cell line (MT-2) as well as peripheral lymphocytes from a hypercalcemic ATLL patient stimulated cyclic AMP production in osteoblast-like rat osteogenic sarcoma cells (UMR 106) and bone resportion in organ cultures of fetal mouse calvaria. Furthermore, the stimulation of cyclic AMP production by conditioned medium of MT-2 cells was inhibited by human PTH(3-34), indicating that MT-2 cells secrete PTH-like activity. The PTH-like activity from MT-2 cells was chromatographically indistinguishable from the one extracted from a solid tumor causing HHM. The present results along with our previous observation that MT-2 cells constitutively express mRNA for PTH-related protein (Biochem. Biophys. Res. Commun., 154: 1182-1188, 1988) demonstrate that a PTH-like activity is synthesized and secreted by these cells, and are consistent with the hypothesis that elaboration of PTH-like activity by ATLL cells may be the mechanism by which hypercalcemia develops in ATLL patients as well as in solid cancer patients with HHM. However, these results do not rule out the possibility that other factors such as interleukin 1 are also involved and may act in concert with PTH-like activity in the development of hypercalcemia in ATLL.

A new classification of clinical stages of adult T-cell leukemia based on prognosis of the disease.

Adult T-cell leukemia (ATL) has been classified into three clinical subtypes: acute, chronic, and smoldering type. However, these clinical subtypes of ATL do not always correlate with prognosis. We propose a new criterion for classification of clinical stages of ATL, based on the expression of Ki-67 antigen in peripheral blood T lymphocytes. We analyzed Ki-67 antigen expression in 45 ATL patients, classified as 18 acute, 17 chronic, and ten smoldering type. We found that in 23 patients with more than 18% Ki-67 antigen-positive T cells, the disease progressed very aggressively, and all patients died within 1 year (mean survival, 105 days). This group included 15 of 16 patients classified as 'acute', seven of 15 patients classified as 'chronic', and one of nine patients classified as 'smoldering' type. In contrast, 15 out of 17 patients with less than 18% Ki-67 antigen-positive T cells were still alive at the end of the study, with a mean survival of 750 days. Only two patients died in this group, and after a very long survival (599 and 978 days, respectively). We therefore propose the following new classification for ATL: (i) Aggressive ATL; percentage of Ki-67 antigen-positive cells more than 18% in peripheral blood T lymphocytes, (ii) Stable ATL; percentage less than 18%. This classification strongly correlates with the prognosis of patients with ATL.

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling.

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.

T-small lymphocyte disorders.

Less than 2% of all lymphoproliferative diseases are indolent or small T-cell disorders, and include T-cell chronic lymphocytic leukemia (CLL)/prolymphocytic leukemia (PLL), large granular lymphocyte (LGL) leukemia, and mycosis fungoides (MF). T-PLL has an aggressive clinical course characterized by high lymphocyte counts, marked hepatosplenomegaly, anemia, thrombocytopenia, and median survival times less than 1 year. The majority of cases are associated with abnormalities of chromosome 14. T-CLL probably represents a small cell variant of T-PLL with a similar aggressive course and similar cytogenetics. T-LGL leukemia is a clonal disorder of CD3+, cytotoxic T lymphocytes. Common clinical features include neutropenia, anemia, splenomegaly, and recurrent bacterial infections. The prognosis is dictated by the severity of the neutropenia, with 10-year actuarial survival rates greater than 80%, and most deaths related to sepsis. A small subset of LGL leukemias have a natural killer (NK) phenotype, are refractory to treatment, and result in multiorgan failure and death in a few months. Mycosis fungoides (MF), the most common of the small T-cell disorders, is a cutaneous T-cell lymphoma with a chronic course, often extending over decades, with most patients eventually succumbing to infection. The small T-lymphocyte disorders represent a rare, diverse group of diseases, which generally have an indolent course, but are not curable.

Radiation resistance in a multidrug resistant human T-cell leukemia line.

In clinical practice, cancers refractory to chemotherapy commonly appear to be comparatively radioresistant. One mechanism by which cancer cells become resistant to chemotherapy is pleiotropic multidrug resistance, characterized by cross resistance to a number of otherwise unrelated heterocyclic antineoplastic agents, including vinca alkaloids, anthracyclines, dactinomycin, and others. We have studied a drug sensitive human leukemia cell line, CEM; a pleiotropic multidrug resistant subline of CEM, CEM/VLB100; VLB-1, a drug sensitive revertant subline arising during in vivo passage of CEM/VLB100; and a methotrexate resistant subline of CEM, CEM-MTX. Using soft-agar colony formation after graded doses of X rays as an endpoint, we found that CEM, CEM-MTX, and CEM/VLB100 had similar terminal slopes (D0 = 0.66 Gy). However, the CEM/VLB100 survival curve had a broader initial shoulder (n = 3.0, Dq = 0.75 Gy) than did CEM (n = 1.6, Dq = 0.25 Gy) or CEM/MTX (n = 1.0, Dq = 0 Gy), suggesting that CEM/VLB100 has an increased capacity to repair radiation-induced DNA damage. This was tested by comparing the cell lines' abilities to accumulate sublethal damage. In split dose recovery experiments, CEM/VLB100 demonstrated increased ability to repair sublethal radiation damage following fractionated irradiation compared with the CEM parental line. Although it no longer demonstrated multidrug resistance, VLB-1 still displayed diminished radiation sensitivity. On the basis of these and other investigators' results, we suggest that diminished radiation sensitivity is separate from, but can be closely associated with, the multidrug-resistant phenotype.

Unusual relapse of adult T-cell leukemia/lymphoma after spontaneous remission.

A 42-year-old man was diagnosed with acute adult T-cell leukemia/lymphoma (ATL/L). Abnormal peripheral blood cells (45% of white blood cells) (Fig. 1a), hypercalcemia, and systemic lymphadenopathy were observed. Flow cytometric analysis (FCM) using peripheral mononuclear cells (PMNC) revealed that the immunophenotype of tumor cells was CD4+ CD8- CD25+ CD45RA- CD45RO+. Nevertheless, he developed a spontaneous remission 6 months later. At remission, the number of CD4-, CD25-, and CD45RO-positive cells decreased, while CD8- and CD45RA-positive cells increased to normal levels as previously reported by Suzuki et al. [1]. He was then referred to the outpatient clinic where he was periodically evaluated and received no therapy. Because of a serious sense of fullness he was re-admitted 30 months after diagnosis. Physical examination revealed ascites and small lymphadenopathy in the right axilla. Atypical lymphoid cells were not observed on microscopic examination of the blood smear. FCM using PMNC revealed that CD4+ CD25+ cells (3%) were within the normal range. Serum calcium was also within the normal range. Abdominal ultrasound examination showed massive ascites. Paracentesis demonstrated that the ascitic fluid had a high white blood cell count (3.15 x 10(9)/l) with a marked increase in abnormal large cells (Fig. 1b). FCM using mononuclear cells in the fluid revealed that 87.3% of the cells were double-positive for CD4 and CD25. Southern blot analysis of the cells confirmed monoclonal integration of human T-lymphotropic virus type 1 (HTLV-1) proviral DNA. The integrated genome was considered to be identical with that detected at initial presentation (Fig. 2). A diagnosis of relapsed ATL/L, with the same clone as was detected at initial diagnosis, was made. Although he was treated with cytotoxic drugs, he did not respond and he died of renal failure 1 month after relapse. Autopsy revealed nodular invasive lesions at the rectovesical pouch, omentum, diaphragm, and pericardium with peritoneal dissemination.

Poor outcome of autologous stem cell transplantation for adult T cell leukemia/lymphoma: a case report and review of the literature.

A limited number of patients with adult T cell leukemia/lymphoma (ATL) who received autologous stem cell transplantation (ASCT) have been reported. We report here a case of fatal systemic Candida krusei infection in a female patient with ATL undergoing ASCT. All of the eight patients (including seven patients in the literature) with ATL who received ASCT developed relapse of ATL or death due to ASCT complication, irrespective of subtype or remission state of ATL, source or selection of SCT or conditioning regimen. At present, ASCT appears to provide little benefit for ATL in contrast to that for other types of aggressive non-Hodgkin's lymphoma.

Wortmannin-enhanced X-ray-induced apoptosis of human T-cell leukemia MOLT-4 cells possibly through the JNK/SAPK pathway.

We demonstrated that enhancement of X-ray-induced apoptosis/rapid cell death by wortmannin accompanied by increased activation of JNK/SAPK in human leukemia MOLT-4 cells. Rapid cell death/apoptosis was determined either by the dye exclusion test or by the appearance of Annexin V-positive cells and cleaved PARP fragments. Enhancement was observed only at higher concentrations of wortmannin, i.e. 1 microM or more. At these high concentrations, both DNA-PK and ATM were inhibited. X-ray-induced phosphorylation of Ser 15 of p53/TP53, accumulation of both p53/TP53 and p21/WAF1/CDKN1A, and phosphorylation of XRCC4 were all suppressed. The enhancement of apoptosis/rapid cell death by wortmannin was prevented by addition of caspase inhibitors, Z-VAD-FMK or Ac-DEVD-CHO, or by transfection and overexpression of mouse Bcl2, which is known as an anti-apoptosis protein. The requirement for a high concentration of wortmannin, i.e. 1 microM or more, indicates that inhibition of both DNA-PK and ATM was necessary for the enhanced apoptosis/rapid cell death. Phosphorylation of AKT/PKB was completely suppressed at a much lower concentration, i.e. 0.1 microM wortmannin, where no enhancement of X-ray-induced apoptosis/rapid cell death was observed. On the other hand, X-ray-induced phosphorylation of JNK and its kinase activity as well as apoptosis/rapid cell death were all significantly enhanced only at high concentrations of wortmannin, i.e. 1 microM or more. Furthermore, the extent of enhancement of both JNK phosphorylation and of apoptosis/rapid cell death by wortmannin was less in Rh1a cells, which are ceramide- and radiation-resistant variant cells compared to the parental MOLT-4 cells. Therefore, activation of the JNK pathway was considered important for the enhancement of X-ray-induced apoptosis/rapid cell death of MOLT-4 cells by wortmannin, because of the requirement for a higher concentration of wortmannin than that required for inhibition of AKT phosphorylation. The suppression of the AKT-dependent pathway by wortmannin may have some underlying role in activating the JNK pathway toward the enhancement of cell death in the current system.

Mechanisms of cachexia induced by T-cell leukemia in the rat.

Body wasting (cachexia) is a common feature of cancer and a major cause of morbidity and mortality. The mechanisms underlying cachexia are largely unknown, and studies in experimental animals have focused mainly on solid tumors. Therefore, the objective of the present study was to quantify and investigate cachexia in experimentally induced T-cell leukemia in the rat. Induction of leukemia by serial passage (injection of cervical lymph node suspension) resulted in a rapid increase in white blood cell (WBC count, hypertrophy of the spleen (by day 11), and severe morbidity within 17 to 18 days. Body weight gain and food intake declined steadily in leukemic animals from day 12, although weight loss was significantly greater in pair-fed, nonleukemic animals. However, leukemic rats had a lower body fat content and higher water content than pair-fed animals on day 18, so the measurement of body weight significantly underestimated the severity of cachexia. Resting oxygen consumption (VO2), measured during the light phase, declined in pair-fed animals from day 13, but was elevated in leukemic rats on days 12 to 18 by 25% (P < .05, one-way ANOVA) compared with pair-fed rats and by 7% (P < .05, one-way ANOVA) relative to free-feeding controls. Hypermetabolism was associated with an increase in brown adipose tissue (BAT) activity (74% and 89%, respectively, P < .05, one-way ANOVA) in leukemic rats compared with control and pair-fed groups. Effects of leukemia on VO2 and BAT were prevented by administration of the adrenergic antagonist, propranolol. These results indicate that T-cell leukemia in the rat results in rapid and severe cachexia, which is largely due to marked hypophagia, but is also accompanied by inappropriately high rates of energy expenditure that are mediated by sympathetic activation of BAT thermogenesis.