Adult T-cell Leukemia Cells Research Articles

Apoptosis Induced by the Histone Deacetylase Inhibitor FR901228 in Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines and Primary Adult T-Cell Leukemia Cells

Apoptosis Induced by the Histone Deacetylase Inhibitor FR901228 in Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines and Primary Adult T-Cell Leukemia Cells

Retraction Note: Inhibition of constitutively active Jak-Stat pathway suppresses cell growth of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells

Retraction Note: Inhibition of constitutively active Jak-Stat pathway suppresses cell growth of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells

Advantage of higher-avidity CTL specific for Tax against human T-lymphotropic virus-1 infected cells and tumors

Strong CTL response can be observed and associated with the control of proviral load in human T-lymphotropic virus type 1 (HTLV-1) infection. However, there are few details with regard to how HTLV-1 specific CTLs work against HTLV-1 infected cells and adult T-cell leukemia cells (ATLs). In this study, using Tax-specific CTL lines with high- and low-functional avidity developed from HLA-A2-transgenic mice, we showed that higher avidity CTLs specific for Tax expressing larger numbers of TCRs and better binding strength to the antigen-HLA-A2 complex are much more efficient at eliminating HTLV-1 infected cells and, in particular, ATL tumor cells with the ability of recognizing a latent level of Tax product detected only with a real-time PCR. These findings suggest that such higher avidity CTLs specific for Tax in HTLV-1 could be responsible for preventing the development of HTLV-1 infection by detecting trace amount of antigens.

Thioredoxin-binding protein-2 (TBP-2/VDUP1/TXNIP) regulates T-cell sensitivity to glucocorticoid during HTLV-I-induced transformation

Although glucocorticoid (GC) is widely used for treating hematopoietic malignancies including adult T-cell leukemia (ATL), the mechanism by which leukemic cells become resistant to GC in the clinical course remains unclear. Using a series of T-cell lines infected with human T lymphotropic virus type-I (HTLV-I), the causative virus of ATL, we have dissected the transformation from interleukin (IL)-2-dependent to -independent growth stage. The transformation associates the loss of thioredoxin-binding protein-2 (TBP-2), a tumor suppressor and regulator of lipid metabolism. Here we show that TBP-2 is responsible for GC-induced apoptosis in ATL cells. In the IL-2-dependent stage, dexamethasone induced TBP-2 expression and apoptosis, both of which were blocked by GC receptor (GR) antagonist RU486. Knockdown of TBP-2 consistently reduced the amount of GC-induced apoptosis. In IL-2-independent stage, however, expression of GR and TBP-2 was suppressed and GC failed to induce apoptosis. Forced expression of GR led the cells to mild sensitivity to GC, which was also accomplished by treatment with suberoylanilide hydroxamic acid, a TBP-2 inducer. A transfection experiment showed that TBP-2 expression induced apoptosis in IL-2-independent ATL cells. Thus, TBP-2 is likely to be one of the key molecules for GC-induced apoptosis and a potential target for treating the advanced stage of ATL.

Leukemic T Cells Are Specifically Enriched In a Unique CD3dimCD7low Subpopulation of CD4+ T Cells In Acute-Type Adult T Cell Leukemia

AbstractAbstract 4144 [Background]Adult T-cell leukemia (ATL) is a malignant disorder caused by human T-cell leukemia virus type I (HTLV-I). Morphological discrimination of leukemic cells from non-leukemic T cells is often difficult in ATL since ATL cells reveal morphological diversity except for typical “flower cells”. Although a study using CD3 gating in flow cytometry reported that ATL cells were distinguishable as a CD3low population from normal lymphocytes, these cells were not well characterised as ATL cells. Considering that defective expression of CD7 as well as CD3 is common in ATL cells, we applied multi-color flow cytometry to detect a putative leukemia-specific cell population in the peripheral blood from ATL patients. [Methods and Results](1) In flow cytometry, after dead-cell and monocyte removal, CD4+ T lymphocytes were gated on the CD3 versus CD4 plot. Based on cell density and fluorescence intensity of CD3 and CD7 in this population, we designated three subpopulations on this plot: CD3highCD7high, CD3dimCD7dim and CD3dimCD7low(Results of a representative ATL and a control sample are shown in Figure). The proportion of the CD3dim/CD7low subpopulation was significantly higher in acute-type ATL CD4+ lymphocytes than in normal controls(Figure). (2) To extensively characterise this subpopulation, we next estimated the HTLV-I proviral load by quantitative real-time PCR after FACS sorting based on this CD3 versus CD7 plot. In all patient samples, HTLV-I proviral integration was detected in all subpopulations. However, the proviral load was significantly higher in the CD3dim/CD7low subpopulation compared to the CD3high/CD7high subpopulation. Almost all of the cells in the CD3dim/CD7low subpopulation were HTLV-I infected. (3) We next examined CCR4 and CD25 expression in each subpopulation. Both CCR4 and CD25 expression levels were maintained at very low and similar levels throughout all subpopulations in normal control cells and in the CD3high/CD7high subpopulation of patients with ATL as well. In contrast, CCR4 expression was significantly up-regulated in CD3dim/CD7low subpopulation of patients with ATL compared to the CD3high/CD7high subpopulation (MFI: 36.5±17.2 vs. 3.8±1.1). The expression of CD25 was also up-regulated in the subpopulation (MFI: 7.8±8.0 vs. 2.7±1.6). (4) Monoclonal expansion of HTLV-I-infected cells in the CD3dim/CD7low subpopulation was indicated by the genomic integration site analysis using a long inverse polymerase chain reaction (PCR) method. (5) We reviewed the glass-slide specimens of FACS-sorted samples to evaluate the morphology of each subpopulation on the CD3 versus CD7 plot. Atypical lymphocytes with morphology such as a notch in the nucleus were observed in all subpopulations. The majority of sorted cells from CD3dim/CD7low subpopulation showed “flower cell”-like morphology. (6) We also detected a small CD3dim/CD7dim subpopulation other than the CD3dim/CD7low and CD3high/CD7high subpopulations in all patients with acute-type ATL who were analysed(Figure). This subpopulation contained the same clone as the CD3dim/CD7low subpopulation, although a phenotypical difference existed between these subpopulations. [Display omitted] [Conclusion](1) Above findings indicate that leukemic T cells are specifically enriched in a unique CD3dim/CD7low subpopulation of CD4+ T cells in acute-type ATL. This multi-color FACS system may be useful for precisely monitoring disease during chemotherapy, detecting minimal residual disease and analysing ATL cells. (2) Previous reports have revealed that HTLV-I-infected cells transform through multi-step oncogenesis. Detailed analysis of these three subpopulations (CD3high/CD7high, CD3dim/CD7dim and CD3dim/CD7low) may give some insight into oncogenesis of HTLV-I-infected cells. Disclosures:No relevant conflicts of interest to declare.

Overexpression of SIRT1, a Longevity Gene-Encoded Protein, and Induction of Apoptosis by Its Inhibition In Adult T-Cell Leukemia Cells

AbstractAbstract 2768Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm developing after a long-term infection with human T-cell leukemia virus (HTLV-1), in which NF-kB is also implicated as an exacerbation factor. Despite recent progress in both chemotherapy and supportive care for hematological malignancies, the prognosis of ATL is still poor; overall survival at 3 years is only 24%. New strategies for the therapy and prophylaxis of ATL (e.g., vaccines and novel molecular target agents) are still required. SIRT1, an NAD+-dependent histone/protein deacetylase, plays a crucial role in various physiological processes, such as aging, metabolism, neurogenesis and apoptosis, due to its ability to deacetylate numerous substrates, such as histone and NF-kB. Here, we assessed how SIRT1 is regulated in primary ATL cells and leukemic cell lines. SIRT1 expression in ATL patients was significantly higher than that in healthy controls, especially in the acute type. Sirtinol, a SIRT1 inhibitor, induced significant growth inhibition or apoptosis in cells from ATL patients and leukemic cell lines, especially HTLV-1-related cell lines (S1T and MT-2). Sirtinol-induced apoptosis was mediated by activation of the caspase family, and inactivation of NF-kB, reducing IkBα phosphorylation. Interestingly, NAD+ augmented sirtinol-induced apoptosis following deacetylation of NF-kB via NAD+-dependent deacetylase. Thus, the SIRT1 inhibitor acted as a tumor suppressor, where NAD+ accelerated the SIRT1 inhibitor-induced apoptosis. These results suggest that SIRT1 is a crucial antiapoptotic molecule in ATL cells, and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Activation of AID by human T-cell leukemia virus Tax oncoprotein and the possible role of its constitutive expression in ATL genesis

Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1). Mutations of tumor suppressor genes have been described in ATL. Although Tax, a product of HTLV-1, is associated with cellular genetic aberrations, the mechanisms of such association are not fully clear. Activation-induced cytidine deaminase (AID) is involved in somatic DNA alterations of the immunoglobulin gene for amplification of immune diversity. However, inappropriate expression of AID acts as a genomic mutator that contributes to tumorigenesis. To gain insight into the molecular mechanism underlying the emergence of somatic mutations in various genes during leukemogenesis, we examined the expression of AID. HTLV-1-infected T-cell lines and ATL cells expressed high levels of AID compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Immunohistochemistry showed AID-positive ATL cells in lymph nodes and skin lesions. Infection of a human T-cell line and normal PBMCs with HTLV-1 induced AID expression. Tax transcriptionally activated AID gene through both the nuclear factor-kappaB subunit p50 and cyclic adenosine 3',5'-monophosphate response element-binding protein signaling pathways. p50, which lacks a transactivation domain, interacted with the transcriptional coactivator Bcl-3 in HTLV-1-infected T cells. Thus, activation of p50/Bcl-3 complexes in T cells in response to Tax might explain the constitutive expression of AID in HTLV-1-infected T cells. The constitutive expression of AID in ATL cells can be speculated to result from mutations induced by the Tax-activated AID and/or other Tax-associated mutagenic mechanisms during the pre-leukemic stage, which cause functional modification within the AID promoter or in any of its cellular regulatory activator proteins.

Genistein Induced Apoptotic Cell Death in Adult T-Cell Leukemia Cells through Estrogen Receptors

Adult T-cell leukemia (ATL) occurs in human T-lymphotropic virus type I-infected individuals and is endemic to the southwestern area of Kyushu in Japan. Here, we found that nM levels of genistein and 17β-estradiol had cytotoxic effects on ATL cells and activated caspase-3. The estrogen receptor antagonist ICI182780 negated the cytotoxic effect of genistein. In addition, G protein-coupled estrogen receptor agonist G-1 also had a cytotoxic effect on ATL cells. This is the first report suggesting that estrogen receptors are a molecular target for ATL therapy.

Human T cell leukemia virus type 1 (HTLV-1) and oncogene or oncomiR addiction?

The mechanism of HTLV-1 transformation of cells to Adult T cell leukemia (ATL) remains not fully understood. Currently, the viral Tax oncoprotein is known to be required to initiate transformation. Emerging evidence suggests that Tax is not needed to maintain the transformed ATL phenotype. Recent studies have shown that HTLV-1 transformed cells show deregulated expression of cellular microRNAs (miRNAs). Here we discuss the possibility that early ATL cells are Tax-oncogene-addicted while late ATL cells are oncogenic microRNA (oncomiR) - addicted. The potential utility of interrupting oncomiR addiction as a cancer treatment is broached.

Human T cell leukemia virus type 1 (HTLV-1) and oncogene or oncomiR addiction?

The mechanism of HTLV-1 transformation of cells to Adult T cell leukemia (ATL) remains not fully understood. Currently, the viral Tax oncoprotein is known to be required to initiate transformation. Emerging evidence suggests that Tax is not needed to maintain the transformed ATL phenotype. Recent studies have shown that HTLV-1 transformed cells show deregulated expression of cellular microRNAs (miRNAs). Here we discuss the possibility that early ATL cells are Tax-oncogene-addicted while late ATL cells are oncogenic microRNA (oncomiR) - addicted. The potential utility of interrupting oncomiR addiction as a cancer treatment is broached.

APOBEC3G Generates Nonsense Mutations in Human T-Cell Leukemia Virus Type 1 Proviral Genomes In Vivo

Human T-cell leukemia virus type 1 (HTLV-1) induces cell proliferation after infection, leading to efficient transmission by cell-to-cell contact. After a long latent period, a fraction of carriers develop adult T-cell leukemia (ATL). Genetic changes in the tax gene in ATL cells were reported in about 10% of ATL cases. To determine genetic changes that may occur throughout the provirus, we determined the entire sequence of the HTLV-1 provirus in 60 ATL cases. Abortive genetic changes, including deletions, insertions, and nonsense mutations, were frequent in all viral genes except the HBZ gene, which is transcribed from the minus strand of the virus. G-to-A base substitutions were the most frequent mutations in ATL cells. The sequence context of G-to-A mutations was in accordance with the preferred target sequence of human APOBEC3G (hA3G). The target sequences of hA3G were less frequent in the plus strand of the HBZ coding region than in other coding regions of the HTLV-1 provirus. Nonsense mutations in viral genes including tax were also observed in proviruses from asymptomatic carriers, indicating that these mutations were generated during reverse transcription and prior to oncogenesis. The fact that hA3G targets the minus strand during reverse transcription explains why the HBZ gene is not susceptible to such nonsense mutations. HTLV-1-infected cells likely take advantage of hA3G to escape from the host immune system by losing expression of viral proteins.

CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

Aurora A selective inhibitor MLN8237 suppresses the growth and survival of HTLV‐1‐infected T‐cells in vitro

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle. We have shown previously that Aurora A expression is increased aberrantly in human T-cell leukemia virus type 1 (HTLV-1)-infected T-cell lines and primary adult T-cell leukemia cells, and a pan-Aurora kinase inhibitor, which inhibits both Aurora A and Aurora B kinases, reduces viability and induces apoptosis in these cells. However, the specific effects of Aurora A inhibition on HTLV-1-infected T-cells are poorly understood. In this study, we addressed this question by comparing the effects of MLN8237, a selective inhibitor of Aurora A, on cell viability, cell cycle progression, and induction of apoptosis in HTLV-1-infected and -uninfected T-cell lines. MLN8237 reduced the viability of HTLV-1-infected T-cell lines within 24 h, but its effects on that of HTLV-1-uninfected T-cell lines were moderate. MLN8237 induced early apoptosis of HTLV-1-infected T-cell lines without induction of polyploidy. It induced p53 and p21 expression in HTLV-1-infected but not in -uninfected T-cell lines, suggesting that MLN8237-treated HTLV-1-infected T-cell lines exit from mitosis and activate a p53-dependent postmitotic G(1) checkpoint, leading to G(1) arrest followed by the induction of apoptosis. Our results suggest that specific inhibition of Aurora A kinase is a potentially useful therapeutic strategy in the treatment of adult T-cell leukemia and that further in vivo exploration is warranted.

Abstract 403: Deletion of HBZ alters lymphomagenesis in HTLV-1 infected humanized SCID mice

Abstract Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of Adult T-cell Leukemia (ATL), an aggressive, mature CD4+/CD25+ T-cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+ HPCs). Humanized NOD/SCID (HU-SCID) mice generated by injection with HTLV-1-infected CD34+ HPCs demonstrate persistent infection and reproducibly develop CD4+ T-cell lymphomas (∼80%) with clinical characteristics associated with ATL. The HTLV-1 bZIP protein (HBZ) is consistently expressed in all ATL cells and has been implicated in the maintenance of leukemogenesis. HBZ has previously been demonstrated to modulate Tax activity and to interact with the cellular factors CREB, Jun and p53. Transduction of HBZ into CD34+ HP/HSCs using a lentivirus vector suppresses clonogenic colony formation in vitro. Moreover, infection of CD34+ HP/HSCs with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ) results in elevated clonogenic colony formation in vitro, suggesting that HBZ suppresses hematopoiesis. To establish the role of HBZ in HTLV-1 replication and leukemogenesis in vivo, HU-SCID mice were infected with an infectious proviral clone lacking functional HBZ (HTLV-1ΔHBZ). HTLV-1ΔHBZ-infected HU-SCID mice developed lymphoproliferations that were phenotypically characterized as a mixture of immature lymphoid progenitors (CD90+) and mature B-cells (CD19+) starting at ∼10 weeks post-reconstitution, in contrast to wild-type HTLV-1 infection. Lymphoproliferations induced by HTLV-1ΔHBZ successfully engraft and expand in naïve NOD/SCID mice when injected into the peritoneal cavity and leukemic cells and lymphoma cells express HTLV-1 gp46env and p19gag. FACS-purified CD90+CD19− HTLV-1ΔHBZ infected cells also successfully engraft in naïve NOD/SCID mice and mature into CD19+ cells suggesting that these cells may be leukemic progenitors with the capability of maturing in vivo. In addition, a majority of HTLV-1ΔHBZ-infected HU-SCID mice also develop a progressive, flaccid hind limb paralysis with features similar to the neurological disorder, HAM/TSP. These results suggest that HBZ is dispensable for HTLV-1 replication and modulates lymphomagenesis in the HU-SCID model. The humanized SCID mouse provides a novel in vivo platform for analysis of the role of HTLV-1 genes in viral replication and pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 403.

Abstract 3674: Inhibition of noncanonical NF-kappa B activation by (−)-DHMEQ in vitro and in adult T-cell leukemia cells

Abstract [Objective and Rationale]Noncanonical NF-kappa B consisting of RelB and p52 is especially important for the maturation of B-lymphocytes and autoimmune diseases. In one hand, we have designed and synthesized a novel NF-kappa B inhibitor (−)-DHMEQ. It showed potent anti-inflammatory and anti cancer effects in animal models without any toxicity. We have reported (−)-DHMEQ covalently binds to the specific Cys residue of each NF-kappa B component and that it inhibits the DNA binding of canonical NF-kappa B components. However, the effects on noncanonical NF-kappa B has not been studied at the molecular level. In the present research we have studied theinhibitory effect of (−)-DHMEQ on noncanonical NF-kappa B in vitro and in cultured lymphoma cells in which noncanonical NF-kappa B is constitutively activated. [Experimental Design and Results]We prepared recombinant noncanonical NF-kappa B proteins, RelB and p52. (−)-DHMEQ was shown to covalently bind to Cys144 of RelB but not to p52 in MLADI-TOF/MS and mutation analysis. Either RelB or p52 does not form homodimer. Then, we prepared HeLa cells over–expressing full length RelB and p52. (−)-DHMEQ inhibited the NF-kappa B activity in RelB(wild type)/p52 cells but not in RelB(C144S)/p52 expressing cells in EMSA. Thus (−)-DHMEQ was shown to target Cys 144 of RelB in cultured cells to inhibit noncanonical NF-kappa B-DNA binding. It also inhibited RelB/p52 binding to DNA in vitro. Then, we looked for the cell line in which noncanonical NF-kappa B is constitutively activated using anti-RelB in EMSA. MT-1 cells are HTLV-1 transformed T-cells and often used for the adult T-cell leukemia (ATL) model. We found that the highly activated NF-kappa B is mainly consisting with noncanonical one in MT-1 cells. (−)-DHMEQ effectively inhibited the noncanonical NF-kappa B activity in MT-1 cells. [Conclusion] (−)-DHMEQ binds to RelB and effectively inhibited RelB/p52-DNA binding. We have found noncanonical NF-kappa B is highly activated ATL-like MT-1 cells, which was inhibited by (−)-DHMEQ. (−)-DHMEQ may be useful for the treatment of ATL and other diseases induced by noncanonical NF-kappaB activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3674.

Abstract 5606: Preclinical and clinical development of anti-CD25 recombinant immunotoxin LMB-2 in combination with chemotherapy for the treatment of adult T-cell leukemia

Abstract LMB-2, previously called anti-Tac(Fv)-PE38, is a recombinant immunotoxin containing the variable domains of the anti-CD25 Mab and a truncated form of Pseudomonas exotoxin. LMB-2 binds and internalizes via CD25, and a processed toxin fragment translocates to the cytosol where catalytic ADP-ribosylation leads to cell death. LMB-2 induces complete and partial responses (CR + PR) in patients with CD25+ hairy cell leukemia, and partial responses were reported in other hematologic malignancies including adult T-cell leukemia (ATL). While nearly all ATL cells express CD25 at high levels, clinical efficacy was limited by rapid progression between cycles and immunogenicity. Recently, preclinical studies using the anti-mesothelin recombinant immunotoxin SS1P indicated that soluble receptor is ∼10-fold higher in the interstitial space of the tumor xenograft than in the serum (PNAS, 104:17099, 2007) and that gemcitabine was synergistic against the xenografts when used prior to SS1P to deplete interstitial soluble receptor (Clin Cancer Res, 13:7166, 2007). To determine if soluble CD25 (sCD25) in the interstitial space of tumors could be a limiting factor in the antitumor activity of LMB-2 in ATL, xenografts of CD25+ human ATAC-4 cells were grown subcutaneously in nude mice and harvested so that the serum and intratumoral sCD25 concentrations could be compared. Intratumoral sCD25 concentrations were 21-159 (median 43) ng/ml, compared to between undetectable and 0.33 (median 0.093) ng/ml in serum (p=0.0008, n=8). Thus, intratumoral sCD25 was 86-2200 (median 610)-fold higher than serum sCD25 in this model. To determine if chemotherapy would improve the efficacy of LMB-2 towards these tumors, groups of ATAC-4-bearing mice (tumor implantation day −3) were treated with gemcitabine on days 1 and 3, and then LMB-2 on days 3, 5 and 8. Mice treated on this schedule with gemcitabine 40 mg/Kg x2 and LMB-2 50 ug/Kg x3 had smaller tumors than mice treated with twice the dose of either agent alone, confirming in vivo synergy, with the mechanisms currently under study. A clinical trial in ATL is now underway with fludarabine and cyclophosphamide on days 1, 2, and 3 followed by LMB-2 on days 3, 5 and 7, to determine if chemotherapy can prevent immunogenicity, enhance the antitumor activity of the immunotoxin, and when possible, determine if intratumoral sCD25 is depleted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5606.

Overexpression of caveolin-1 in adult T-cell leukemia

AbstractCaveolin-1 is implicated in the regulation of signal pathways. Adult T-cell leukemia (ATL) is a T-cell malignancy causatively associated with human T-cell leukemia virus type 1 (HTLV-1). To determine the role of caveolin-1 in leukemogenesis, we examined caveolin-1 expression levels in HTLV-1–infected T-cell lines and ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Caveolin-1–positive ATL cells were detected in ATL lymph nodes and skin lesions, and caveolin-1 was also detected in the plasma of patients with ATL. Infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 expression. The viral protein Tax transcriptionally activated caveolin-1 gene through nuclear factor-κB and cAMP response element binding protein signal pathways. HTLV-1–infected T-cell lines, and ATL cells are known to be resistant to transforming growth factor β (TGF-β)–induced growth inhibition. Caveolin-1 was colocalized with TGF-β type I receptor in HTLV-1–infected T-cell lines and suppressed TGF-β signaling. Caveolin-1 knockdown in an HTLV-1–infected T-cell line exhibited susceptibility to TGF-β. Thus, we describe a new function for Tax, repression of TGF-β signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

Antiproliferative Activity of Purine Nucleoside Phosphorylase Multisubstrate Analogue Inhibitors Containing Difluoromethylene Phosphonic Acid against Leukaemia and Lymphoma Cells

Potent inhibitors of purine nucleoside phosphorylase (PNP) are expected to act as selective agents against T-cell tumours. Five compounds with guanine, three with hypoxanthine, and five with 9-deazaguanine, all connected by a linker with difluoromethylene phosphonic acid, were studied on their inhibitory potential against human and calf PNPs. Antiproliferative activity of these analogues against lymphocytes as well as lymphoma and leukaemia cells has been also investigated. All tested compounds act as multisubstrate analogue inhibitors of PNP with the apparent inhibition constants in the range 5-100 nm, and also show a slight antiproliferative activity. Analogues with 9-deazaguanine aglycone have better anti-leukaemic and anti-lymphoma activities compared to the guanine and hypoxanthine analogues, and applied in the concentration of 100 mum, caused a statistically significant decrease in the cell viability in all human leukaemia and lymphoma cells used. Despite the high PNP inhibitory potential of tested analogues, no differences were observed between the effects on the growth of tumour cells sensible to the inhibition of PNP, such as human adult T-cell leukaemia and lymphoma cells, and other investigated cells. Obtained poor effects on cell proliferation could be explained probably by a poor ability of tested compounds to penetrate cell membranes.

A new high-performance liquid chromatography method determines low production of 9-β-D-arabinofuranosylguanine triphosphate, an active metabolite of nelarabine, in adult T-cell leukemia cells

The 9-beta-D-arabinofuranosylguanine (ara-G), an active compound of nelarabine, demonstrates potent cytotoxicity specifically on T-cell malignancies. In cells, ara-G is phosphorylated to ara-G triphosphate (ara-GTP), which is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Because ara-GTP is crucial to ara-G's cytotoxicity, the determination of ara-GTP production in cancer cells is informative for optimizing nelarabine administration. Here, we developed a new, sensitive isocratic-elution HPLC method for quantifying ara-GTP. Samples were eluted isocratically by using phosphate buffer at a constant flow rate. Ara-GTP was clearly separated from other nucleotides by using an anion-exchange column and it was quantitated by its peak area at 254 nm. The standard curve was linear with low variability and a sensitive detection limit (10 pmol). Furthermore, due to ara-G's specificity to T-cells we hypothesized that nelarabine might be effective against adult T-cell leukemia (ATL). The ara-GTP production was compared between T-lymphoblastic leukemia CCRF-CEM and ATL cell lines in vitro. When CEM cells were incubated with ara-G, the ara-GTP production increased in a concentration- and time-dependent manner. In contrast, 5 ATL cell lines accumulated lower ara-GTP in the same condition. While ara-G inhibited the growth of CEM cells with a 50% growth inhibition concentration of 2 microM, the inhibitory-concentration values were >1 mM in 8 of the 12 ATL cell lines. This ineffectiveness appeared to correspond with the low ara-GTP production. The present study is the first to evaluate the potential of ara-G against ATL cells; our results suggest that nelarabine would not be effective against ATL.

Deguelin Induces Cell Growth Arrest and Cell Death by Destabilizing Phosphorylated STAT3 and Survivin in Adult T-Cell Leukemia Cells.

Abstract 4784Adult T-cell leukemia (ATL) cells are remarkably resistant to conventional chemotherapy. Therefore, new agents that effectively kill these cells are needed. Deguelin is a rotenoid from African plant Mundulea sericea which has been recently shown to suppress the lung tumorigenesis via inhibiting the activation of PI3kinase-Akt signaling. More recently, deguelin has been shown to bind to ATP-binding pocket of heat shock protein (Hsp) 90 and inhibit functions of Hsp90 client proteins. However, the effects of deguelin on ATL cells are poorly understood. To investigate the effects, we examined the cell growth and cell death of human T-cell leukemia virus type 1 (HTLV-1)- transformed cells, KUT-1 and MT-2 (kindly provided by Dr. Hanada, Kagoshima Medical Center, Kagoshima and Dr. Harashima, Hayashibara Institute Inc., Okayama, Japan, respectively) by trypan-blue exclusion and Annexin V/PI stain. Deguelin inhibited the proliferation of KUT-1 and MT-2 cells in a time- and dose-dependent manner (IC50=0.5μM). In addition, flow cytometric analysis revealed a higher frequency of Annexin V(+) cells in deguelin-treated cells compared to that in untreated cells (KUT-1 P< 0.01, MT-2 P< 0.01, respectively). These results indicate that deguelin induces the cell growth arrest and cell death of ATL cells. To better understand the mechanism of these effects, we examined the intracellular signaling and the expression of apoptosis associated proteins by immunoblot analysis. We found that deguelin prevented the tyrosine phosphorylation of the transcription factor signal transducer and activator of transcription (STAT) 3, which is constitutively activated in these cells. In addition, the inhibitor of apoptosis protein, survivin expression was decreased 24 hours after deguelin treatment, indicating that deguelin induces the cell growth arrest and apoptosis via at least in part the inhibition of STAT3 phosphorylation and the down-regulation of survivin expression. To determine the mechanism of inhibition of STAT3 phosphorylation and survivin expression, MT-2 cells were treated with proteasome inhibitor MG132 and/or deguelin. MG132 restored STAT3 dephosphorylation and down-regulation of survivin expression by deguelin. In addition, quantitative RT-PCR assay showed that deguelin treatment did not alter survivin gene expression. These results suggested that deguelin induces the degradation of phosphorylated STAT3 and survivin via the ubiquitination/proteasome pathway in ATL cells. Since deguelin has been shown to inhibit the function of Hsp90 client proteins, deguelin may destabilize phosphorylated STAT3 and survivin by inhibiting Hsp90 function. Taken together, deguelin appears to induce cell growth arrest and apoptosis of ATL cells via the suppression of the tyrosine phosphorylation of STAT3 and down-regulation of survivin. Thus, deguelin merits further investigation as a potential therapeutic agent for this incurable disease. Disclosures:No relevant conflicts of interest to declare.