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The Human T-cell Lymphotropic Virus (HTLV-1) is a Deltaretrovírus that was first isolated in the 1970s, and associated with Adult T-cell Leucemia-Lymphoma (ATLL), and subsequently to Tropical Spastic Paraparesis-Myelopathy (TSP/HAM). The genetic diversity of the virus varies among geographic regions, although its mutation rate is very low (approximately 1% per thousand years) in comparison with other viruses. The present study determined the genetic diversity of HTLV-1 in the metropolitan region of Belém, in northern Brazil. Blood samples were obtained from patients at the UFPA Tropical Medicine Nucleus between January 2010 and December 2013. The DNA was extracted and the PX region of the HTLV was amplified using nested PCR. The positive samples were then digested using the Taq1 enzyme for the identification and differentiation of the HTLV-1 and HTLV-2. The 5'LTR region of the positive HTLV-1 samples were amplified by nested PCR, and then sequenced genetically. The phylogenetic analysis of the samples was based on the maximum likelihood method and the evolutionary profile was analyzed by the Bayesian approach. Overall, 78 samples tested positive for HTLV-1, and 44 were analyzed here. The aA (cosmopolitan-transcontinental) subtype was recorded in all the samples. The following evolutionary rates were recorded for the different subtypes-a: 2.10-3, b: 2.69. 10-2, c: 6.23. 10-2, d: 3.08. 10-2, e: 6. 10-2, f: 1.78. 10-3, g: 2.2. 10-2 mutations per site per year. The positive HTLV-1 samples tested in the present study were characterized by their low genetic diversity and high degree of stability.

Cyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)-transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1-transformed lines (n = 8), primary ATL cells (n = 11), and CD4 + cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572-treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572-treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL., (© 2017 by The American Society of Hematology.)

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Torin2 is a second-generation ATP-competitive inhibitor of the mammalian target of rapamycin (mTOR). Dysregulation of mTOR signaling pathway, consisting of mTOR complexes mTORC1 and mTORC2, is a promising therapeutic target in some human malignancies. We examined antitumor effects of Torin2 in adult T-cell leukemia/lymphoma (ATL)-related cell lines compared to those of rapamycin, a classical mTOR inhibitor., Materials and Methods: Cell growth was monitored by detecting viable cells with Cell Counting Kit-8 or trypan blue. Cell cycle was studied by flow cytometric analysis. The phosphorylation status of proteins in the mTOR signaling pathway was examined by western blot analysis., Results: Torin2 exhibited greater efficacy in cell growth inhibition than rapamycin, associated with a strong reduction of phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) (Ser 473), that is downstream of mTORC2., Conclusion: Since mTORC2 activates AKT, Torin2 might inhibit both mTORC1 and mTORC2, resulting in stronger growth inhibition of ATL cells., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL). This disease manifests after a long clinical latency period of up to 2-3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5) on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i) in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment.

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell lymphotropic virus type-I (HTLV-I) without curative treatment at present. To illuminate the pathogenesis of ATLL we performed whole transcriptome sequencing of purified ATLL patient samples and discovered recurrent somatic mutations in CCR4, encoding CC chemokine receptor 4. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330, or Y331 in the carboxy terminus. Functionally, the CCR4-Q330 nonsense isoform was gain-of-function because it increased cell migration toward the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation after receptor engagement by CCL22 in ATLL cells and conferred a growth advantage in long-term in vitro cultures. These findings implicate somatic gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition of CCR4 signaling might have therapeutic potential in this refractory malignancy.

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/β genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G(2)/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.

The retrovirus, human T-cell-lymphotrophic virus-1 (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-I-encoded protein tax constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems that in turn activate the Jak3 (Janus kinase 3)/STAT5 (signal transducers and activators of transcription 5) pathway, suggesting a therapeutic strategy that involves targeting Jak3. We evaluated the action of the Jak3 inhibitor CP-690,550 on cytokine dependent ex vivo proliferation that is characteristic of peripheral blood mononuclear cells (PBMCs) from select patients with smoldering or chronic subtypes of ATL, or from those with HAM/TSP whose PBMCs are associated with autocrine/paracrine pathways that involve the production of IL-2, IL-9, IL-15, and their receptors. CP-690,550 at 50 nM inhibited the 6-day ex vivo spontaneous proliferation of PBMCs from ATL and HAM/TSP patients by 67.1% and 86.4%, respectively. Furthermore, CP-690,550 inhibited STAT5 phosphorylation in isolated ATL T cells ex vivo. Finally, in an in vivo test of biological activity, CP-690,550 treatment of mice with a CD8 T-cell IL-15-transgenic leukemia that manifests an autocrine IL-15/IL-15Rα pathway prolonged the survival duration of these tumor-bearing mice. These studies support further evaluation of the Jak3 inhibitor CP-690,550 in the treatment of select patients with HTLV-I-associated ATL and HAM/TSP.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease in which the Jak2/Stat5 pathway is constitutively activated. This study found that AZ960, a novel inhibitor of Jak2 kinase, effectively induced growth arrest and apoptosis of human T-cell lymphotropic virus type 1, HTLV-1-infected T cells (MT-1 and MT-2) in parallel with downregulation of the phosphorylated forms of Jak2 and Bcl-2 family proteins including Bcl-2 and Mcl-1. Interestingly, AZ960 increased levels of Bcl-xL in MT-1 and MT-2 cells in association with accumulation of cAMP response element-binding protein bound to the Bcl-xL promoter as measured by chromatin immunoprecipitation assay. Importantly, genetic inhibition of Bcl-xL by a small interfering RNA potentiated antiproliferative effects of AZ960 in MT-1 cells. Taken together, Jak2 is an attractive molecular target for treatment of ATL. Concomitant blockade of Jak2 and Bcl-xL may be a promising treatment strategy for this lethal disease., (©2010 AACR.)

Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), transforms CD4(+) T cells to permanent growth through its transactivator Tax. HTLV-1-transformed cells share phenotypic properties with memory and regulatory T cells (T-reg). Murine T-reg-mediated suppression employs elevated cyclic AMP (cAMP) levels as a key regulator. This led us to determine cAMP levels in HTLV-1-transformed cells. We found elevated cAMP concentrations as a consistent feature of all HTLV-1-transformed cell lines, including in vitro-HTLV-1-transformed, Tax-transformed, and patient-derived cells. In transformed cells with conditional Tax expression, high cAMP levels coincided with the presence of Tax but were lost without it. However, transient ectopic expression of Tax alone was not sufficient to induce cAMP. We found specific downregulation of the cAMP-degrading phosphodiesterase 3B (PDE3B) in HTLV-1-transformed cells, which was independent of Tax in transient expression experiments. This is in line with the notion that PDE3B transcripts and cAMP levels are inversely correlated. Overexpression of PDE3B led to a decrease of cAMP in HTLV-1-transformed cells. Decreased expression of PDE3B was associated with inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In summary, Tax transformation and its continuous expression contribute to elevated cAMP levels, which may be regulated through PDE3B suppression. This shows that HTLV-1-transformed cells assume biological features of long-lived T-cell populations that potentially contribute to viral persistence.

Notch signaling requires a series of proteolytic cleavage events to release the Notch intracellular domain (NICD) that functions directly in signal transduction. The Notch receptor is locked down in a protease-resistant state by a negative regulatory region (NRR) that protects an ADAM (a disintegrin and metalloprotease) cleavage site. Engagement with ligand-bearing cells induces global conformational movements in Notch that unfold the NRR structure to expose the ADAM cleavage site and initiate proteolytic activation. Although both ADAM10 and ADAM17 have been reported to cleave Notch to facilitate NICD release by gamma-secretase, the relevant ADAM has remained controversial. Our study provides new insight into this conflict, as we find that although Notch1 (N1) is a substrate for both ADAM10 and ADAM17, the particular ADAM required for receptor activation is context dependent. Specifically, ADAM10 was absolutely required for N1 signaling induced by ligands, while signaling independent of ligands required ADAM17. In contrast to the strict and differential use of ADAM10 and ADAM17 in normal and dysregulated signaling, respectively, both proteases participated in signaling intrinsic to N1 mutations associated with leukemia. We propose that in addition to exposing the ADAM cleavage site, activating N1 conformational changes facilitate selective cleavage by specific proteases.

Amplification of the NUP214-ABL1 oncogene can be detected in patients with T cell acute lymphoblastic leukemia (T-ALL). We screened 29 patients with T cell malignancies for the expression of NUP214-ABL1 by reverse transcription-polymerase chain reaction (RT-PCR). NUP214-ABL1 was detected in three (10%) patients. These results were confirmed by fluorescence in situ hybridization techniques. We also studied the activity of imatinib, nilotinib and dasatinib against the human NUP214-ABL1-positive cell lines PEER and BE-13. All three tyrosine kinase inhibitors decreased the viability of PEER and BE-13 cells, but nilotinib and dasatinib had >1-log lower IC(50) values than imatinib (P<0.001). In contrast, the NUP214-ABL-negative T-ALL cell line Jurkat, was remarkably resistant to tyrosine kinase inhibition. The inhibition of cellular proliferation was associated with time-dependent induction of apoptosis and inhibition of ABL, CrKL and STAT5 phosphorylation. Moreover, dasatinib was active in a NUP214-ABL1-positive leukemia xenograft murine model and in marrow lymphoblasts from a patient with NUP214-ABL1-positive T-ALL. On the basis of these results, ABL1 kinase inhibitors warrant clinical investigation in patients with NUP214-ABL1-positive T-cell malignancies.

Common molecular machineries between hematopoietic stem cell (HSC) maintenance and leukemia prevention have been highlighted. The tumor suppressor Fbxw7 (F-box and WD-40 domain protein 7), a subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle. We demonstrate that inactivation of Fbxw7 in hematopoietic cells causes premature depletion of HSCs due to active cell cycling and p53-dependent apoptosis. Interestingly, Fbxw7 deletion also confers a selective advantage to cells with suppressed p53 function, eventually leading to development of T-cell acute lymphoblastic leukemia (T-ALL). Thus, Fbxw7 functions as a fail-safe mechanism against both premature HSC loss and leukemogenesis.

SPA-1 (signal-induced proliferation associated gene-1) functions as a suppressor of myeloid leukemia by negatively regulating Rap1 signaling in hematopoietic progenitor cells (HPCs). Herein, we showed that transplantation of HPCs expressing farnesylated C3G (C3G-F), a Rap1 guanine nucleotide exchange factor, resulted in a marked expansion of thymocytes bearing unique phenotypes (CD4/CD8 double positive [DP] CD3(-) TCRbeta(-)) in irradiated recipients. SPA-1(-/-) HPCs expressing C3G-F caused a more extensive expansion of DP thymocytes, resulting in lethal T-cell acute lymphoblastic leukemia (T-ALL) with massive invasion of clonal T-cell blasts into vital organs. The C3G-F(+) blastic thymocytes exhibited constitutive Rap1 activation and markedly enhanced expression of Notch1, 3 as well as the target genes, Hes1, pTalpha, and c-Myc. All the T-ALL cell lines from C3G-F(+) SPA-1(-/-) HPC recipients expressed high levels of Notch1 with characteristic mutations resulting in the C-terminal truncation. This proliferation was inhibited completely in the presence of a gamma-secretase inhibitor. Transplantation of Rag2(-/-) SPA-1(-/-) HPCs expressing C3G-F also resulted in a marked expansion and transformation of DP thymocytes. The results suggested that deregulated constitutive Rap1 activation caused abnormal expansion of DP thymocytes, bypassing the pre-T-cell receptor and eventually leading to Notch1 mutations and Notch-dependent T-ALL.

T-cell acute lymphoblastic leukemias (T-ALLs) are highly malignant tumors with 20% of patients continues to fail therapy, in part due to chemoresistance of T-ALL cells via largely unknown mechanisms. Here, we showed that lack of Bcl-2-interacting mediator of cell death (Bim)(EL) protein expression, a BH3-only member of the Bcl-2 family proteins, conferred resistance of a T-ALL cell line, Sup-T1, to etoposide-induced apoptosis. Overexpression of Bim(EL) significantly restored its sensitivity to etoposide-induced caspase activation and poly(ADP-ribose) polymerase cleavage. Surprisingly, we found that constitutive activation of the c-Jun N-terminal kinase (JNK) pathway in Sup-T1 cells promoted phosphorylation and degradation of Bim(EL) via the proteosome. Blocking with a proteosome inhibitor yielded an elevated level of Bim(EL) and accumulation of Bim(EL) species phosphorylated at Ser(69). Pretreatment of Sup-T1 cells with a specific JNK inhibitor, SP600125, also increased the Bim(EL) level and resensitized the cells to etoposide-induced apoptosis. Together, our findings suggest that the JNK activation status may correlate with the Bim(EL) level and in turn can control the sensitivity of T-ALL cells to chemotherapeutic agents.

Proteins of the ephrin-B group operate in nonlymphoid cells through the control of their migration and attachment, and are crucial for the development of the vascular, lymphatic, and nervous systems. Ephrin-B activity is deregulated in various nonlymphoid malignancies; however, their precise role in cancer has only started to be addressed. We show here that ephrin-B1, a member of the ephrin-B group, is expressed in pediatric T-cell leukemias, including leukemia cell line Jurkat. Treatment of Jurkat cells with ephrin-B-stimulating EphB3 enhances ephrin-B1 phosphorylation and induces its relocalization into lipid rafts. These events are mediated by the T lineage-specific kinase, Lck, as ephrin-B1 phosphorylation and lipid raft association are blocked in the Lck-deficient clone of Jurkat, JCAM1.6. Ephrin-B1 also induces colocalization of the CrkL and Rac1 cytoskeleton regulators and initiates in leukemic cells a strong repulsive response. The absence of Lck blocks ephrin-B1-induced signaling and repulsion, confirming the essential role for Lck in ephrin-B1-mediated responses. This shows a new role for ephrin-B1 in the regulation of leukemic cells through the Lck-dependent Rac1 colocalization with its signaling partner, CrkL, in lipid rafts. In agreement with its repulsive action, ephrin-B1 seems to support metastatic properties of leukemic cells, as suppression of ephrin-B1 signaling inhibits their invasiveness. Because ephrin-B1-activating EphB proteins are ubiquitously expressed, our findings suggest that ephrin-B1 is likely to play an important role in the regulation of malignant T lymphocytes through the control of lipid-raft-associated signaling, adhesion, and invasive activity, and therefore may represent a novel target for cancer treatment.

Background: Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells., Results: We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells., Conclusion: AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.

To isolate pharmacologically safe compounds that can induce apoptosis of tumor cells, leaves of an aromatic plant (Zanthoxylum schinifolium), which are widely used as a food flavor and herbal medicine in Korea and Japan, were sequentially extracted by organic solvents. An apoptogenic ingredient in the methylene chloride extract was further purified by silica gel column chromatography and identified as auraptene (AUR). The IC(50) value of AUR against Jurkat T cells was 16.5 microg/ml. After the treatment of Jurkat T cells with AUR, the endoplasmic reticulum (ER) stress-mediated activation of caspase-12 and -8 and subsequent apoptotic events including c-Jun N-terminal kinase (JNK) activation, cleavage of FLICE inhibitory protein and Bid, mitochondrial cytochrome c release, activation of caspase-9 and -3, degradation of poly (ADP-ribose) polymerase and apoptotic DNA fragmentation were induced in a dose-dependent manner. The cytotoxicity of AUR was not blocked by the anti-Fas neutralizing antibody ZB-4. The AUR-induced cytotoxicity and apoptotic events were abrogated by ectopic over-expression of Bcl-xL or addition of the pan-caspase inhibitor z-VAD-fmk. The individual or simultaneous addition of the m-calpain inhibitor (E64d), JNK inhibitor (SP600125) and mitochondrial permeability transition pore inhibitor (CsA) failed to prevent apoptotic events including caspase-8 activation and Bid cleavage, unless the caspase-8 inhibitor (z-IETD-fmk) was combined, whereas AUR-induced caspase-12 activation was sustained even in the concomitant presence of z-IETD-fmk. These results demonstrated that the apoptotic effect of AUR on Jurkat T cells was exerted by the ER stress-mediated activation of caspase-8, and the subsequent induction of mitochondria-dependent or -independent activation of caspase cascade, which could be suppressed by Bcl-xL.

Calcineurin is a calcium-activated serine/threonine phosphatase critical to a number of developmental processes in the cardiovascular, nervous and immune systems. In the T-cell lineage, calcineurin activation is important for pre-T-cell receptor (TCR) signaling, TCR-mediated positive selection of thymocytes into mature T cells, and many aspects of the immune response. The critical role of calcineurin in the immune response is underscored by the fact that calcineurin inhibitors, such as cyclosporin A (CsA) and FK506, are powerful immunosuppressants in wide clinical use. We observed sustained calcineurin activation in human B- and T-cell lymphomas and in all mouse models of lymphoid malignancies analyzed. In intracellular NOTCH1 (ICN1)- and TEL-JAK2-induced T-cell lymphoblastic leukemia, two mouse models relevant to human malignancies, in vivo inhibition of calcineurin activity by CsA or FK506 induced apoptosis of leukemic cells and rapid tumor clearance, and substantially prolonged mouse survival. In contrast, ectopic expression of a constitutively activated mutant of calcineurin favored leukemia progression. Moreover, CsA treatment induced apoptosis in human lymphoma and leukemia cell lines. Thus, calcineurin activation is critical for the maintenance of the leukemic phenotype in vivo, identifying this pathway as a relevant therapeutic target in lymphoid malignancies.

In this report, inhibitors of the gamma-secretase enzyme have been exploited to characterize the antiproliferative relationship between target inhibition and cellular responses in Notch-dependent human T cell acute lymphoblastic leukemia (T-ALL) cell lines. Inhibition of gamma-secretase led to decreased Notch signaling, measured by endogenous NOTCH intracellular domain (NICD) formation, and was associated with decreased cell viability. Flow cytometry revealed that decreased cell viability resulted from a G(0)/G(1) cell cycle block, which correlated strongly to the induction of apoptosis. These effects associated with inhibitor treatment were rescued by exogenous expression of NICD and were not mirrored when a markedly less active enantiomer was used, demonstrating the gamma-secretase dependency and specificity of these responses. Together, these data strengthen the rationale for using gamma-secretase inhibitors therapeutically and suggest that programmed cell death may contribute to reduction of tumor burden in the clinic.

One in seven types of human cancer is associated with an oncogenic virus infection. Most human tumors have high telomerase activity but very short telomeres, yet the maintenance of these short telomeres is critical to avoid telomere end fusion or senescence and to support active proliferation. Oncogenic viruses have evolved a wide repertoire of strategies to stimulate telomerase functions at the transcriptional and post-transcriptional levels. Since telomerase activity is absent in somatic cells, the inhibition of telomerase is an attractive target for cancer therapeutics.

For long, T-cell acute lymphoblastic leukemia (T-ALL) remained in the shadow of precursor B-ALL because it was more seldom, and showed a normal karyotype in more than 50% of cases. The last decennia, intense research has been carried out on different fronts. On one side, development of normal thymocyte and its regulation mechanisms have been studied in multiple mouse models and subsequently validated. On the other side, molecular cytogenetics (fluorescence in situ hybridization) and mutation analysis revealed cytogenetically cryptic aberrations in almost all cases of T-ALL. Also, expression microarray analysis disclosed gene expression signatures that recapitulate specific stages of thymocyte development. Investigations are still very much actual, fed by the discovery of new genetic aberrations. In this review, we present a summary of the current cytogenetic changes associated with T-ALL. The genes deregulated by translocations or mutations appear to encode proteins that are also implicated in T-cell development, which prompted us to review the 'normal' and 'leukemogenic' functions of these transcription regulators. To conclude, we show that the paradigm of multistep leukemogenesis is very much applicable to T-ALL and that the different genetic insults collaborate to maintain self-renewal capacity, and induce proliferation and differentiation arrest of T-lymphoblasts. They also open perspectives for targeted therapies.

Glucocorticoids (GC) are common components in chemotherapeutic protocols for lymphoid malignancies. GC-induced apoptosis requires the intrinsic, BCL-2 family-regulated pathway. Treatment of CCRF-CEM (T cell acute lymphoblastic leukemia) cells with the GC, dexamethasone (Dex), activates p38-mitogen activated protein kinase (p38-MAPK) and then induces mRNA transcription and synthesis levels of BIM, a BH3-only pro-apoptotic BCL-2 family member. Dex-induced apoptosis is dramatically inhibited by downregulation of BIM by shRNA or by pretreatment with a p38-MAPK inhibitor, SB203580, which also reduces BIM induction. These findings indicate that BIM induction through p38-MAPK activation is a critical pathway in GC-induced cell death.

The deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal cells, allows for selective therapy using L-alanosine, an inhibitor of de novo AMP synthesis. We demonstrate that MTAP- T-ALL cells obtained at relapse are as sensitive to L-alanosine toxicity as diagnosis samples. The therapeutic index of L-alanosine can be increased by the use of a MTAP substrate, which protects MTAP+ normal cells. Since MTAP substrates MTA and 5'deoxyadenosine are prone to toxicities associated with adenosine, we synthesized and evaluated a potentially nontoxic MTAP substrate, 9-beta-D-erythrofuranosyladenine (EFA). The cytotoxicity of EFA to hematopoietic progenitors erythroid burst-forming units (BFU-Es) and granulocyte-macrophage colony-forming units (CFU-GMs) was at least 26- to 41-fold less than that of MTA. In addition, EFA selectively rescued MTAP+ MOLT-4 cells from L-alanosine toxicity at 25 microM with negligible toxicity even at 100 microM. As for MTA, significant, albeit incomplete, rescue was achieved at 12.5 microM, but higher concentrations were toxic. EFA at 20 microM or less rescued primary MTAP+ T-ALL cells and normal lymphocytes from L-alanosine toxicity. Collectively, these data indicate that EFA is an effective agent for salvaging MTAP+ cells from L-alanosine toxicity and is superior to MTA due to lower cytotoxicity.

Adult T cell leukemia/lymphoma (ATLL) has been characterized as one of the most aggressive human neoplasias and its incidence is thought to be caused by both genetic and epigenetic alterations to the host cellular genes of T cells infected with human T cell leukemia virus type I (HTLV-I). A multilobulated nuclear appearance is an important diagnostic marker of ATLL, and we have now identified that the molecular mechanisms underlying these formations occur through microtubule rearrangement via phosphatidylinositol 3-kinase (PI3-kinase) activation by AILIM/ICOS signaling. We also show that PTEN and/or SHIP-1, which are PIP3 inositol phosphatases that inhibit the activation of downstream effectors of the PI3-kinase cascade, are disrupted in both ATLL neoplasias and in multilobulated nuclei-forming Jurkat cells. This down-regulation of PTEN was found to be essential for the formation of ATLL-type nuclear lobules. Furthermore, PI3-kinase and PTEN activities were observed to be closely associated with cellular proliferation. Thus, our results suggest that alteration of PI3-kinase signaling cascades, as a result of the down-regulation of inositol phosphatases, induces ATLL-type multilobulated nuclear formation and is also associated with the cellular proliferation of malignant T cell leukemias/lymphomas.

In immortal cells, the existence of a mechanism for the maintenance of telomere length is critical. In most cases this is achieved by the reactivation of telomerase, a cellular reverse transcriptase that prevents telomere shortening. Here we report that the telomerase gene (hTERT) promoter is up-regulated during transmission of human T-cell lymphotropic virus type-I (HTLV-I) to primary T cells in vitro and in ex vivo adult T-cell leukemia/lymphoma (ATLL) samples, but not asymptomatic carriers. Although Tax impaired induction of human telomerase reverse transcriptase (hTERT) mRNA in response to mitogenic stimulation, transduction of Tax into primary lymphocytes was sufficient to activate and maintain telomerase expression and telomere length when cultured in the absence of any exogenous stimulation. Transient transfection assays revealed that Tax stimulates the hTERT promoter through the nuclear factor kappaB (NF-kappaB) pathway. Consistently, Tax mutants inactive for NF-kappaB activation could not activate the hTERT or sustain telomere length in transduced primary lymphocytes. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays suggested that an increased binding of c-Myc and Sp1 is involved in the NF-kappaB-mediated activation of the hTERT promoter. This study establishes the role of Tax in regulation of telomerase expression, which may cooperate with other functions of Tax to promote HTLV-I-associated adult T-cell leukemia.

Adult T-cell leukemia (ATL) is a fatal T-cell malignancy that arises long after infection with human T-cell leukemia virus type I (HTLV-I). We reported previously that nuclear factor-kappaB (NF-kappaB) was constitutively activated in ATL cells, although expression of the viral proteins was barely detectable, including Tax, which was known to persistently activate NF-kappaB. Here we demonstrate that ATL cells that do not express detectable Tax protein exhibit constitutive IkappaB kinase (IKK) activity. Transfection studies revealed that a dominant-negative form of IKK1, and not of IKK2 or NF-kappaB essential modulator (NEMO), suppressed constitutive NF-kappaB activity in ATL cells. This IKK activity was accompanied by elevated expression of p52, suggesting that the recently described noncanonical pathway of NF-kappaB activation operates in ATL cells. We finally show that specific inhibition of NF-kappaB by a super-repressor form of IkappaBalpha (SR-IkappaBalpha) in HTLV-I-infected T cells results in cell death regardless of Tax expression, providing definitive evidence of an essential role for NF-kappaB in the survival of ATL cells. In conclusion, the IKK complex is constitutively activated in ATL cells through a cellular mechanism distinct from that of Tax-mediated IKK activation. Further elucidation of this cellular mechanism should contribute to establishing a rationale for treatment of ATL.

Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.

Death-associated protein (DAP) kinase is a pro-apoptotic serine/threonine kinase with a death domain, which is involved in apoptosis induced by interferon-gamma, tumor necrosis factor-alpha, and Fas ligand. Down-regulation of DAP kinase gene expression by hypermethylation of its promoter region might result in resistance to apoptotic cell death, and could provide a basis for tumor development. In the present study, we employed methylation-specific polymerase chain reaction to examine the methylation status of CpG islands in the DAP kinase gene in 19 cases of T-cell malignancies (including eight adult T-cell leukemia/lymphoma), 24 of natural killer (NK)/T-cell, and 34 of B-cell. Frequency of methylation was significantly higher in B-cell (27 of 34, 79.4%) than in T-cell malignancies (nine of 19, 47.4%) (P<0.05). Fifteen of 24 (62.5%) NK/T-cell lymphomas showed DNA methylation. One B-cell lymphoma cell line with DNA methylation was resistant to apoptotic stimuli, and treatment of the cells with a demethylating agent restored apoptotic cell death. These findings suggested that suppression of DAP kinase expression by DNA methylation might play a substantial role in the development of not only B-cell, but also T- and NK/T-cell lymphomas.

The novel prodrug of butyric acid, pivaloyloxymethyl butyrate (AN-9), a histone deacetylase inhibitor, shows great promise as an effective and relatively nontoxic anticancer agent for solid malignancies. However, little is known about its effects on hematopoietic malignancies. In this study, we show that 21 primary samples of acute leukemia were sensitive to the antiproliferative effects of AN-9, with a 50% inhibitory concentration (IC(50)) of 45.8 +/- 4.1 microM. In colony-forming assays, primary T-cell acute lymphoblastic leukemia (T-ALL) cells were 3-fold more sensitive to AN-9 than the normal hematopoietic progenitors, erythroid burst-forming units and granulocyte/monocyte colony-forming units. AN-9 induced apoptosis in the T-ALL cell line CEM. A common problem with cancer is chemoresistance, which is often typical of relapsed cancers. Remarkably, a T-ALL sample at diagnosis and an acute myeloid leukemia sample at relapse that were resistant to doxorubicin in vitro were sensitive to AN-9, with an IC(50) of 50 microM for both samples. More strikingly, samples from 2 infants with t(4;11) ALL obtained at diagnosis and relapse each were the most sensitive to AN-9, with IC(50) values of 25 microM and 17 microM, respectively. Furthermore, a doxorubicin-resistant clone of HL60, HL60/ADR, obtained by the transfection of the MDR-1 gene, was equally sensitive to AN-9 cytotoxicity as the parental cells. AN-9 induced the expression of p21 in an infant leukemia sample with 11q23 rearrangement, but not in T- or B-precursor ALL. Collectively, our results suggest that AN-9 is a selective agent for hematopoietic malignancies that can circumvent the mechanisms of chemoresistance limiting most conventional chemotherapy.

The present study examines the clinical significance of serum neuron-specific enolase (NSE) in patients with adult T cell-leukemia (ATL). Serum NSE values were measured using a radioimmunoassay in 35 patients (acute type, n = 15; lymphoma type, n = 10; chronic type, n = 10) and in 7 controls carrying T lymphotropic virus type-1 (HTLV-1). Serum NSE values >10 ng/mL were detected in 9 of 15 patients with acute type (60%), 5 of 10 with lymphoma type (50%), and in one of 10 patients with chronic type (10%) ATL, but in none of the HTLV-1 carriers. Contrary to previous findings demonstrating that 20% of patients with non-Hodgkin's lymphoma (NHL) had positive serum NSE, the frequency of a high NSE value in patients with acute and lymphoma type ATL was much higher (60% and 50%, respectively). The serum NSE value positively correlated with serum thymidine kinase activity (TK) and serum soluble interleukin-2 receptor (sIL-2R) levels (P < 0.04 and P < 0.01, respectively). Serum NSE values at the initial diagnosis were adversely related to overall survival time according to the log-rank test (P < 0.02). Pathological examinations demonstrated that both patients with anaplastic large cell lymphoma type ATL had cytoplasmic NSE and CD30 markers on cell membranes. These findings suggest that serum NSE is partially produced by ATL cells and that ATL tumor cells seem preferentially produce NSE compared with other NHL cells. Serum NSE may be a novel marker of disease aggressiveness as well as a prognostic factor for ATL., (Copyright 2002 Wiley-Liss, Inc.)

Methylthioadenosine phosphorylase (MTAP) is an important enzyme used for the salvage of adenine and methionine. Cells lacking this enzyme are expected to be sensitive to purine synthesis inhibitors and/or methionine starvation. We reported previously that the MTAP gene is deleted in adult T cell leukemia (ATL) cells. In the present study, we expanded our series and used a real-time quantitative PCR assay for accurate diagnosis of the deletion and nine of 65 primary ATL samples (13.8%) were MTAP negative. In spite of this low incidence, ATL cells showed significantly higher sensitivity to L-alanosine, an inhibitor of de novo adenosine monophosphate (AMP) synthesis, than normal lymphocytes, suggesting that the MTAP gene is inactivated not only by deletion but also by other mechanisms. Indeed, a real-time quantitative RT-PCR assay disclosed that primary ATL cells had significantly lower MTAP mRNA expression than normal lymphocytes. Since MTAP-negative ATL cell lines also showed much higher sensitivity to L-alanosine than MTAP-positive ATL cell lines, we used these cell lines to investigate whether it is possible to develop selective therapy targeting MTAP deficiency. A substrate of MTAP, methylthioadenosine (MTA) or its substitutes rescued concanavalin A (Con A)-activated normal lymphocyte proliferation from L-alanosine toxicity. All the compounds except 5'-deoxyadenosine, however, also caused the undesirable rescue of MTAP-negative ATL cell lines. 5'-Deoxyadenosine had the desired ability to rescue hematopoietic progenitor cells without rescuing ATL cell lines. These results support the rationale for a chemotherapy regimen of L-alanosine combined with 5'-deoxyadenosine rescue in MTAP-deficient ATL.

Nuclear factor kappaB (NF-kappaB) plays a major role in the pathogenesis of human T-cell lymphotrophic virus I-associated malignancy. Proteasome inhibitors provide a rational approach to control constitutively activated NF-kappaB in human T-cell lymphotrophic virus I-infected T cells. We report that the proteasome inhibitor PS-341 decreased NF-kappaB DNA binding activity by preventing degradation of IkappaB(alpha). In our murine model of adult T-cell leukemia, PS-341 used alone did not yield prolongation of the survival of tumor-bearing mice. However, when combined with the current clinically approved drug humanized anti-Tac, therapy with PS-341 was associated with a complete remission in a proportion of treated animals, whereas only a partial response was observed in animals treated with humanized anti-Tac alone.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and of tropical spastic paraparesis/HTLV-I-associated myelopathy. Infiltration of various tissues by circulating leukemic cells is a characteristic of ATL. Matrix metalloproteinases (MMPs), which mediate the degradation of the basement membrane and extracellular matrix, play an important role in metastasis and tumor cell dissemination. The aim of this study was to explore whether expression of MMP-2 and MMP-9 was deregulated by HTLV-I infection. The data showed that HTLV-I-infected T-cell lines expressed high levels of MMP-9 compared with uninfected T-cell lines. In contrast, the levels of the related MMP-2 were not significantly altered by HTLV-I infection. In addition, the elevated expression of MMP-9 in HTLV-I-infected cells was attributable to the action of the viral transactivator protein Tax. The results show that Tax can activate the MMP-9 promoter and induce MMP-9 expression in T cells, indicating that the constitutive expression of MMP-9 in virus-infected cell lines is at least in part mediated by Tax. Activation of the MMP-9 promoter by Tax occurs mainly through the action of NF-kappaB and SP-1. The biologic significance of these observations was validated by the following 2 findings: MMP-9 expression was increased in primary ATL cells, and plasma MMP-9 levels were elevated in ATL patients. In addition, plasma levels of MMP-9 correlated with organ involvement in ATL patients. Together these data suggest that overexpression of MMP-9 in HTLV-I- infected cells may be in part responsible for the invasiveness of ATL cells.

Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus 1.50 +/- 0.23 microM; P < 0.01). The lower sensitivity of TPMT+ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 +/- 282 versus 1515 +/- 183 pmol/5 x 10(6) cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 +/- 34 versus 27 +/- 10 pmol/5 x 10(6) cells; P = 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 +/- 0.6% versus 7.2 +/- 2.0%; P < 0.001). The higher sensitivity of TPMT+ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 +/- 1055 versus 174 +/- 77 pmol/5 x 10(6) cells; P = 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.

DNA topoisomerase II (topo II) is an essential nuclear enzyme and is the target for etoposide, which is used in the therapy of childhood acute lymphoblastic leukaemia (ALL). Topo II exists as two isoforms referred to as topo IIalpha and topo IIbeta. To determine whether cellular levels of topo IIalpha and beta are an important factor in determining drug sensitivity/resistance requires accurate, precise measurements of the two isoforms. We have developed a quantitative Western blotting method to accurately measure the absolute amounts of human topo IIalpha and beta, using recombinant human topo IIalpha and beta as standards. This quantitative method has been used to assess the efficiency of several commonly used topo II extraction protocols. The extractable amount of topo IIalpha and beta was found to be salt-dependent. However extraction using the optimal salt concentration was found to be as efficient as extraction with DNase I/Rnase A digestion and SDS solubilisation. Using the optimum extraction procedure and the quantitative immunoblotting method, topo IIalpha and beta was quantified in cell lines, peripheral blood lymphocytes and in lymphoblasts from children with newly diagnosed ALL.

After exposure to cytotoxic drugs at relatively low concentration, many cell types undergo G2-M arrest and then either mitotic cell death or, in the case of hematopoietic cells, apoptosis. We have sought to examine this phenomenon in two lymphoblastoid cell lines. After continuous or short-term exposure to etoposide (final concentration, 0.5 microM), up to 80% of cells accumulated at G2-M by 24 h, and subsequently either underwent apoptosis or re-entered the cell cycle. In this and the other studies undertaken, the CEM and MOLT-4 lines behaved similarly. Progressive accumulation of cells at G2-M was accompanied by increasing levels of cyclin B1. Commitment to apoptosis was assessed by evidence of caspase activation using a number of different criteria. A decreased amount of Mr 32,000 procaspase-3 was evident 24-48 h after drug treatment. However, cleavage of caspase substrates poly(ADP-ribose) polymerase and lamin B indicated caspase activation occurring within 3-6 h of drug treatment. Protease activity in corresponding cell extracts increased progressively from 6 h or earlier to 24 h after the addition of etoposide to the medium. Such increase was consequent on drug treatment and not attributable to cells being at G2-M. Treatment with 1.5 mM caffeine abrogated etoposide-induced G2-M arrest, and in cells so treated, the etoposide-induced increase in protease activity was also abrogated. However, there was no impact of caffeine on cytotoxicity under these conditions. Although mitotic cell death is precipitated subsequent to prolonged G2-M arrest in many cell types, the present data suggest that commitment to apoptosis occurs in parallel to G2-M arrest in leukemic cells.

We examined the expression of messenger RNA (mRNA) of the human inducible nitric oxide synthase (hiNOS) gene in a panel of human T-cell lines. Reverse transcriptase-polymerase chain reaction showed that human T-cell leukemia virus type-I (HTLV-I)-infected T-cell lines (MT-1, SLB-1, and C5/MJ) expressed mRNA for the hiNOS, but TL-Om1 or uninfected Jurkat, H9, and CCRF-CEM did not. The MT-1, SLB-1, and C5/MJ cell lines are infected with HTLV-I and express the viral transactivator Tax, whereas TL-Om1 cells, although derived from adult T-cell leukemia (ATL) leukemic cells, do not express Tax. There was, thus, a correlation between Tax and hiNOS mRNA expression. The transcriptional regulatory region of the hiNOS gene was activated by Tax in Jurkat, in which endogenous hiNOS is induced by Tax. Deletion analysis showed that the region of hiNOS encompassing nucleotides -159 to -111 contained the minimum Tax-responsive elements. Mutations in the NF-kappaB element at position -115 and -106 bp in the hiNOS promoter were still activated by Tax, and a Tax mutant defective for activation of the NF-kappaB pathway retained the ability to activate the hiNOS promoter. In addition, overexpression of the dominant-negative mutants of IkappaBalpha and I kappaBbeta failed to reduce Tax-induced activation of hiNOS gene. Furthermore, hiNOS mRNA was detected in leukemic cells from ATL patients. Our results show that the hiNOS promoter contains a minimum Tax-responsive element located between nucleotides -159 and -111, and imply that the expression of the hiNOS gene is involved in the pathogenesis of HTLV-I-associated diseases.

Retinoids, the analogues of vitamin A, have a broad range of effects on different cell types. One biologically active form of vitamin A is all-trans-retinoic acid (ATRA), which binds to retinoic acid receptors, as does its intracellular metabolite, 9-cis-RA. Earlier studies have documented G1 cell cycle arrest and the induction of apoptosis in human adult T-cell leukemia cells after ATRA treatment. Previous work exploring the growth-inhibitory activity of ATRA in human malignancies has implicated several mechanisms that can arrest cells in the G1 phase of the cell cycle, including activation of p21Waf1 and inhibition of cyclin D1 expression. Therefore, we decided to examine the effects of ATRA exposure on G1 cell cycle components in human adult T-cell leukemia cells. Our data demonstrate a correlation between cyclin/cyclin-dependent kinase activity and subunit complex formation with duration of drug exposure. We also observed an increase in p53 protein levels that were not associated with an increase in p21Waf1 levels. Furthermore, we observed a differential effect on cell cycle progression that was temporally related to length of ATRA exposure. These observations, consistent with a bimodal effect of ATRA on cell cycle progression, may have important implications for the clinical application of ATRA.

We have recently shown that the multiple tumor suppressor gene 1 (MTS1 ) encoding the p16(INK4a) and p19(ARF) cell-cycle inhibitors is inactivated by deletion or disruption in most human T-cell acute lymphoblastic leukemias (T-ALLs), representing the most frequent genetic event thus far described in this disease. To analyze the mechanism of these chromosomal events, we used cloning, sequencing, and/or polymerase chain reaction mapping to study 15 rearrangements occurring in the MTS1 locus. We found that these breakpoints occur in two clusters (MTS1(bcralpha) and MTS1(bcrbeta) ). The three rearrangements occurring in MTS1(bcralpha) correspond to a recurrent recombination juxtaposing 5' MTS2 exon 1 and 5' MTS1 exon 1alpha sequences. Breakpoints for 10 of 12 rearrangements within MTS1(bcrbeta) are located at a polymorphic (CA) repeat, suggesting that this sequence might play a role in the clustering. For both MTS1(bcralpha) and MTS1(bcrbeta), sequence analyses and PCR mapping experiments show that the tightly clustered breakpoints are located in the vicinity of heptamers whose sequence is similar to those involved in the V(D)J recombination. Moreover, short deletions, GC-rich random nucleotide additions, and clone-specific junctional sequences are present in all cases, further suggesting that the rearrangements are due to illegitimate V(D)J recombinase activity. These data are the first demonstration that a tumor suppressor gene can be inactivated by the V(D)J recombinational mechanism. Moreover, they reinforce the view that this process, normally required for T-cell differentiation, plays a crucial role in the pathogenesis of T-ALL.

Glucocorticoids (GC) induce programmed cell death (apoptosis) in immature lymphocytes and are an essential component in the therapy of acute lymphatic leukemia. The mechanism underlying GC-induced apoptosis particularly in leukemia cells is, however, not well understood. Most forms of apoptosis seem to employ a common final effector pathway characterized by specific proteolytic events mediated by interleukin 1beta-converting enzyme (ICE) and/or other ICE-like cysteine proteases. These events may result in the morphologic changes characteristic of apoptosis. To determine whether a similar proteolytic pathway is activated during GC-induced leukemia cell apoptosis, we investigated poly(ADP-ribose) polymerase (PARP), a typical target of ICE-like proteases, during GC-induced apoptosis of the human acute T-cell leukemic cell line CEM-C7H2. Our studies showed proteolytic PARP cleavage suggestive of activation of ICE-like proteases that preceeded morphologic signs of apoptosis. We further established stably transfected CEM-C7H2 sublines expressing the cowpox virus protein CrmA that inhibits some, but not all, ICE-like proteases. GC-induced PARP cleavage and apoptosis were neither inhibited nor delayed in crmA-expressing cell lines. In contrast, crmA expression rendered the same lines resistant to Apo1/Fas-induced PARP cleavage and apoptosis. Thus, different proteases might be activated during the effector phases of GC-and Apo1/Fas-induced apoptosis in human leukemia cells.

Folylpolyglutamate synthetase (FPGS) is responsible for the metabolism of natural folates and a broad range of folate antagonists to polyglutamate derivatives. Recent studies indicated increased accumulation of methotrexate (MTX) polyglutamates (MTX-PG) in blast cells as a predictor of favorable treatment outcome in childhood acute lymphoblastic leukemia (ALL). We determined the expression of FPGS activity in blasts from children with ALL at diagnosis and after treatment with MTX as a single agent, before conventional remission induction therapy. The levels of enzyme activity in ALL blasts at diagnosis (median of 689 pmol/h/mg protein) were significantly higher (P = .003) than those found in acute nonlymphoblastic leukemia (ANLL) blasts (median of 181 pmol/h/mg protein). Comparable lineage differences in normal lymphoid versus nonlymphoid cells suggest a lineage-specific control of FPGS expression, FPGS activity increased in ALL blasts after in vivo exposure to MTX. The median increase in FPGS activity was significantly higher (P = .003) in B-lineage ALL (188%) than in T-lineage ALL (37%). Likewise, the percentage of intracellular long chain MTX-PG (Glu3-6) was significantly higher (P = .02) in B-lineage ALL (92%) than in T-lineage ALL (65%), consistent with higher FPGS activity in B-lineage blasts. This finding could explain, at least in part, the superior outcome in children with B-lineage ALL treated with antimetabolite therapy.

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered rings containing either pyrrole or cyclopentene rings were characterized at the cellular and biochemical level. Five of these antifolates were more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell growth by 50% relative to untreated control (EC50), 12 nM], the antifolate used in the clinic, and two were more potent than 10-ethyl-10-deazaaminopterin (EC50, 2.7 nM); similar patterns of response were obtained in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects could be reversed by leucovorin, indicating an antifolate mechanism. These antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme activity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resistance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibition of human thymidylate synthase was generally weak. These 6,5-fused ring heterocyclic antifolates utilized the reduced folate/MTX transporter for uptake, based on the cross-resistance of MTX uptake-impaired CCRF-CEM cells, and were efficient substrates for this uptake system, based on inhibition of [3H]MTX uptake (IC50, 0.3-5.8 microM; aminopterin IC50, 2.6 microM). These analogues were substrates for CCRF-CEM folylpolyglutamate synthetase, with several being among the most active substrates now known (highest Vrel/Km 0.73; MTX and 10-ethyl-10-deazaaminopterin, 0.013 and 0.24, respectively). Substrate activity for murine intestinal folylpolyglutamate synthetase was also assayed, and a different specificity pattern was observed. These new antifolates are apparently not substrates for aldehyde oxidase. Analogues containing the fused cyclopentene ring are preferred to those containing the fused pyrrole ring based on growth inhibitory potency, effectiveness against decreased uptake mutants and apparent affinity for transport, and inhibition of DHFR. In addition, fused cyclopentene-containing analogues are efficiently polyglutamylated. The data indicate that antifolates with 2,4-diaminopyrimidine-fused five-membered rings, especially those containing the fused cyclopentene ring, are an important new class of antifolates which warrant further exploration at the synthetic and preclinical levels.

To determine the role of the enzyme CTP synthetase (EC 6.3.4.2) in the synthesis in situ of CTP in normal and in malignant lymphoblastic cells, the metabolism of radiolabeled pyrimidine ribonucleosides was studied in proliferating normal T lymphocytes and was compared with that of proliferating MOLT-3 cell-line cells and differentiated (non-proliferating) MOLT-3 cells. Both the incorporation of [14C]uridine into UTP and CTP and the incorporation of [14C]cytidine in CTP, as well as the fluxes of these labeled nucleosides through the nucleotide pools into nucleic acids, were elevated in proliferating MOLT-3 cells compared to proliferating T lymphocytes. Furthermore, the conversion of UTP into CTP was enhanced in proliferating MOLT-3 cells compared to proliferating T lymphocytes, indicating a higher activity of CTP synthetase in the leukemic cells. In non-proliferating MOLT-3 cells, the pyrimidine ribonucleotide fluxes were decreased compared to proliferating MOLT-3 cells and proliferating T lymphocytes. However, the decreased ratio of uracil/cytosine ribonucleotides that was found in proliferating T lymphocytes and proliferating MOLT-3 cells compared to non-proliferating blood lymphocytes, was preserved in the differentiated MOLT-3 cells. Moreover, although the fluxes had decreased, most CTP was still synthesized by CTP synthetase in the differentiated MOLT-3 cells. Thus, the elevated activity of CTP synthetase in MOLT-3 cells was independent of the cell growth and maturation stage. We conclude that the increased activity of CTP synthetase is associated with the process of malignant transformation in MOLT-3 cells. Therefore, CTP synthetase offers an attractive target for selective therapy in human acute T-lymphoid leukemia.

In 91 of 106 adult patients with acute lymphoblastic leukemia (ALL) enrolled in the treatment protocol ALL HOVON-5 between May 1988 and October 1991, the immunophenotype of the leukemia was determined and correlated with clinical characteristics at presentation. The immunological marker analysis was performed in ten laboratories, all members of the Dutch Study Group on Immunophenotyping of Leukemias and Lymphomas (SI-HON). Undifferentiated blasts were found in four patients, 67 had B-lineage ALL, 18 had T-lineage ALL, and two had biphenotypic ALL. The age of T-lineage ALL patients was lower (mean 29.3) than that of B-lineage ALL patients (mean 35.5). Tumor mass, as expressed by leukocyte count, organomegaly, and LDH, was more pronounced in T-lineage ALL. Hemoglobin and platelet count was similar in all (sub)types. CD34 was expressed in 58% of the leukemias, but most frequently in the common B-ALL (70%). Thirteen percent of the leukemias expressed one or more markers not associated with their lineage. In this prospective study immunological data were not evaluable for 15 patients. On four of them data were not available because of dry tap, for six patients the typing was technically insufficient, and for four patients the results were unclassifiable; with one patient the marker analysis was not performed.

Acid phosphatase (AP) reaction is a specific cytochemical marker for T-cell lymphoblastic leukaemia (T-ALL). Twenty six cases of ALL were diagnosed as T-ALL employing cytochemical profiles including AP and sheep red blood cell rosettes. Three district patterns of AP reaction were observed. On one end of the spectrum, 100% of the positive blasts showed the classical dot-like reaction while on the other 100% showed a scattered reaction. Between the two a mixed pattern was observed with 30-80% of the positive blasts showing the dot-like reaction while the rest showed scattered pattern. Dot-like pattern showed L1 morphology, high counts and low SRBC rosettes while scattered showed L2 morphology, low counts and high SRBC rosettes. The pattern observed in our series differs from the dot-like reaction observed in T-ALL in the western series. We recommend that this pattern must be borne in mind when employing AP reaction for T-ALL diagnosis (JPMA 42: 151, 1992).