Anti-tumor activity of 5-aminoimidazole-4-carboxamide riboside with AMPK-independent cell death in human adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma caused by human T-cell lymphotropic virus type I (HTLV-1). Acadesine or 5-aminoimidazole-4-carboxamide riboside (AICAR) is an AMP-activated protein kinase (AMPK) activator that was recently shown to have tumor suppressive effects on B cell chronic lymphocytic leukemia, but not ATL. This study evaluated the cytotoxic effects of AICAR on ATL-related cell lines and its anti-tumor activity. Here, we demonstrated that AICAR induced cell death via apoptosis and the mitochondrial membrane depolarization of ATL-related cell lines (S1T, MT-1, and MT-2) but not non-HTLV-1-infected Jurkat cells. However, AICAR did not increase the phosphorylation levels of AMPKα. In addition, AICAR increased the expression of the death receptors (DR) DR4 and DR5, and necroptosis-related proteins including phosphorylated receptor-interacting protein family members and the mixed lineage kinase domain-like protein. Interestingly, HTLV-1 Tax, an HTLV-1-encoded oncogenic factor, did not affect AICAR-induced apoptosis. Furthermore, AICAR inhibited the growth of human ATL tumor xenografts in NOD/SCID/gamma mice in vivo. Together, these results suggest that AICAR induces AMPK-independent cell death in ATL-related cell lines and has anti-tumor activity, indicating that it might be a therapeutic agent for ATL.

CDK9 Inhibitor Induces Apoptosis, Autophagy, and Suppression of Tumor Growth in Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a hematopoietic malignancy with a poor prognosis that develops in approximately 5% of human T-cell leukemia virus type 1 (HTLV-1) carriers. Cyclin-dependent kinase 9 (CDK9), together with Cyclin T, forms a transcription elongation factor, positive transcription elongation factor b (P-TEFb). P-TEFb promotes transcriptional elongation by phosphorylating the second serine (Ser2) of the seven amino acid repeat sequence in the C-terminal domain of RNA polymerase II (RNAP II). CDK9 inhibitors suppress cell proliferation by inducing apoptosis in chronic lymphocytic leukemia and breast cancer but there are no reports on autophagy of CDK9 inhibitors. Here, we investigated the effect of LY2857785, a novel CDK9 selective inhibitor, on cell death in ATL-related cell lines in vitro, freshly isolated cells from ATL patients ex vivo, and on ATL tumor xenografts in NOD/SCID mice in vivo. LY2857785 significantly reduced cell viability and induced apoptosis, as shown by annexin V-positive cells, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-3, and suppressed the levels of anti-apoptotic protein myeloid cell leukemia-1 (MCL-1). LY2857785 decreased RNAP II Ser2 phosphorylation and downstream c-Myc protein levels. Interestingly, LY2857785 also increased microtubule-associated proteins 1A/1B light chain 3B (LC3)-II binding to autophagosome membranes. Furthermore, LY2857785 decreased the viability of freshly isolated ATL cells and induced apoptosis. Finally, LY2857785 significantly decreased the growth of ATL tumor xenografts. These results suggest that LY2857785 induces cell death of ATL cells by MCL-1-dependent apoptosis and autophagy and has anti-tumor activity.

The unique activity of saponin: Induction of cytotoxicity in HTLV-1 infected cells.

Infection with the retrovirus human T-cell leukemia virus type 1 (HTLV-1) sometimes causes diseases that are difficult to cure. To find anti-HTLV-1 natural compounds, we opted to screen using the HTLV-1-infected T-cell line, MT-2. Based on our results, an extract of the pulp/seeds of Akebia quinata Decaisne fruit killed MT-2 cells but did not affect the Jurkat cell line that was not infected with virus. To determine the active ingredients, seven saponins with one-six sugar moieties were isolated from A. quinata seeds, and their activities against the two cell lines were examined. Both cell lines were killed in a similar manner by Akebia saponins A and B. Further, Akebia saponins D, E, PK and G did not exhibit cytotoxicity. Akebia saponin C had a similar activity to the extract found in the screening. This compound was found to enhance Gag aggregation, induce the abnormal cleavage of Gag, suppress virion release, and preferentially kill HTLV-1 infected cells; however, their relationship remains elusive. Our findings may lead to the development of new therapies for infectious diseases based on the removal of whole-virus-infected cells.

Elucidation of the Mechanism of Host NMD Suppression by HTLV-1 Rex: Dissection of Rex to Identify the NMD Inhibitory Domain.

The human retrovirus human T-cell leukemia virus type I (HTLV-1) infects human T cells by vertical transmission from mother to child through breast milk or horizontal transmission through blood transfusion or sexual contact. Approximately 5% of infected individuals develop adult T-cell leukemia/lymphoma (ATL) with a poor prognosis, while 95% of infected individuals remain asymptomatic for the rest of their lives, during which time the infected cells maintain a stable immortalized latent state in the body. It is not known why such a long latent state is maintained. We hypothesize that the role of functional proteins of HTLV-1 during early infection influences the phenotype of infected cells in latency. In eukaryotic cells, a mRNA quality control mechanism called nonsense-mediated mRNA decay (NMD) functions not only to eliminate abnormal mRNAs with nonsense codons but also to target virus-derived RNAs. We have reported that HTLV-1 genomic RNA is a potential target of NMD, and that Rex suppresses NMD and stabilizes viral RNA against it. In this study, we aimed to elucidate the molecular mechanism of NMD suppression by Rex using various Rex mutant proteins. We found that region X (aa20-57) of Rex, the function of which has not been clarified, is required for NMD repression. We showed that Rex binds to Upf1, which is the host key regulator to detect abnormal mRNA and initiate NMD, through this region. Rex also interacts with SMG5 and SMG7, which play essential roles for the completion of the NMD pathway. Moreover, Rex selectively binds to Upf3B, which is involved in the normal NMD complex, and replaces it with a less active form, Upf3A, to reduce NMD activity. These results revealed that Rex invades the NMD cascade from its initiation to completion and suppresses host NMD activity to protect the viral genomic mRNA.

SRT1720 induces SIRT1-independent cell death in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops after a long period of human T-cell leukemia virus (HTLV)-1 infection and is associated with host aging in addition to genetic abnormalities in HTLV-1 infected cells. SIRT1 is a histone deacetylase involved in cell cycle and apoptosis. We previously showed the high expression of SIRT1 protein in peripheral blood mononuclear cells from patients with ATL. There have been many reports that SIRT1 inhibitors show tumor-suppressive effects. On the other hand, SIRT1 activator SRT1720 induces the cell death of multiple myeloma and breast cancer cells. However, the effect of SRT1720 on ATL is unknown. This study aimed to evaluate the effect of SRT1720 on cell death in leukemic cell lines in vitro and freshly isolated ATL cells ex vivo and in an ATL in vivo mouse model. SRT1720 reduced cell viability in vitro and ex vivo. Additionally, SRT1720 increased the number of apoptotic cells, as shown by annexin V positive cells, cleaved poly (ADP-ribose) polymerase 1, cleaved caspase-3, and fragmented DNA. SRT1720 also induced mitochondrial outer membrane permeabilization with the generation of mitochondrial reactive oxygen species and autophagy. However, SIRT1 knockdown did not attenuate SRT1720-induced cell death in leukemic cell lines. Finally, SRT1720 treatment decreased the growth of human ATL tumor xenografts in immunodeficient mice. Our study shows that while SRT1720 does not target SIRT1, it induces cell death in ATL cells via apoptosis and autophagy and has antitumor activity.

Cell death induced by dorsomorphin in adult T-cell leukemia/lymphoma is AMPK-independent.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasm with poor prognosis that develops after chronic infection with human T-cell leukemia virus type 1 (HTLV-1). Although AMP-activated protein kinase (AMPK) is a critical cellular energy sensor, it has recently become clear that AMPK can act as a tumor regulator. Here, we assessed the expression of AMPK in primary ATL cells and the effects of dorsomorphin, an AMPK inhibitor, on primary ATL cells and HTLV-1-infected T-cell lines. AMPK expression in acute and chronic ATL patients was significantly higher than in asymptomatic HTLV-1 carriers and healthy donors. Dorsomorphin induced apoptosis in peripheral blood mononuclear cells from ATL patients. Dorsomorphin also induced dose- and time-dependent apoptosis in HTLV-1-infected T-cell lines. Dorsomorphin increased the production of intracellular reactive oxygen species (ROS) and induced ataxia telangiectasia-mutated Ser1981 phosphorylation and p53 accumulation. These results indicated that dorsomorphin induces apoptosis via ROS-mediated DNA damage in HTLV-1-infected T-cell lines. Furthermore, dorsomorphin suppressed the growth of human ATL tumor xenografts in NOD/SCID mice. Together, these data suggest that AMPK could be a candidate therapeutic target for ATL and that dorsomorphin could be a therapeutic agent for ATL.

Targeting Excessive EZH1 and EZH2 Activities for Abnormal Histone Methylation and Transcription Network in Malignant Lymphomas.

Although global H3K27me3 reprogramming is a hallmark of cancer, no effective therapeutic strategy for H3K27me3-high malignancies harboring EZH2WT/WT has yet been established. We explore epigenome and transcriptome in EZH2WT/WT and EZH2WT/Mu aggressive lymphomas and show that mutual interference and compensatory function of co-expressed EZH1 and EZH2 rearrange their own genome-wide distribution, thereby establishing restricted chromatin and gene expression signatures. Direct comparison of leading compounds introduces potency and a mechanism of action of the EZH1/2 dual inhibitor (valemetostat). The synthetic lethality is observed in all lymphoma models and primary adult T cell leukemia-lymphoma (ATL) cells. Opposing actions of EZH1/2-polycomb and SWI/SNF complexes are required for facultative heterochromatin formation. Inactivation of chromatin-associated genes (ARID1A, SMARCA4/BRG1, SMARCB1/SNF5, KDM6A/UTX, BAP1, KMT2D/MLL2) and oncovirus infection (HTLV-1, EBV) trigger EZH1/2 perturbation and H3K27me3 deposition. Our study provides the mechanism-based rationale for chemical dual targeting of EZH1/2 in cancer epigenome.

High expression of NAMPT in adult T-cell leukemia/lymphoma and anti-tumor activity of a NAMPT inhibitor.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes induced by human T-cell leukemia virus-1 and has a poor outcome. New molecular targets for the prevention and treatment of ATL are needed urgently. We previously reported high expression of Sirtuin 1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, in primary acute-type ATL cells. NAD+ biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) modulates Sirtuin 1 activity. Here, we examined the expression and effects of inhibiting NAMPT, a rate-limiting enzyme in NAD+ biosynthesis, in ATL cells. We found that peripheral blood mononuclear cells from patients with acute-type ATL expressed significantly higher levels of NAMPT protein than cells from healthy subjects. FK866, a NAMPT inhibitor, induced apoptosis of freshly isolated ATL cells ex vivo and HTLV-1-infected T-cell lines in vitro, which was accompanied by activation of caspases, DNA fragmentation, and disruption of mitochondrial transmembrane potential. However, a pan-caspase inhibitor failed to prevent this FK866-induced cell death, while FK866 increased the caspase-independent cell death mediator endonuclease G. Intriguingly, FK866 also activated autophagy, as demonstrated by increases in protein levels of autophagosome marker LC3-II. Thus, FK866 simultaneously activated apoptosis and autophagy. Finally, FK866 treatment markedly decreased the growth of human ATL tumor xenografts in immunodeficient mice. We showed that NAMPT is highly expressed in primary ATL cells ex vivo, and that FK866 induces autophagy and caspase-dependent and -independent cell death pathways in vitro and has an anti-tumor activity in vivo. These results suggest a novel therapeutic strategy for patients with this fatal disease.

Anti-cancer activity of the cell membrane-permeable phytic acid prodrug.

Phytic acid (IP6) is an ingredient in cereals and legumes, and limited amounts of this compound are considered to enter the cell and exert anti-cancer effects. These effects have been seen by studying cells treated with around 1-5 mM IP6. However, such a large amount of IP6 chelates metals and changes the pH in cell culture medium. To overcome this problem, we synthesized a prodrug of IP6 (Pro-IP6) and elucidated generation of IP6 from Pro-IP6 in cells. Cellular experiments using Pro-IP6 demonstrated selective anti-cancer effects including apoptosis and inhibition of Akt activation. Furthermore, an in vivo study using mice with adult T-cell leukemia also showed that Pro-IP6 reduced the size of the cancer. Taken together, Pro-IP6 is a useful biological tool and may lead to development of new anti-cancer drugs.

Anesthetic effect of a mixture of alfaxalone, medetomidine, and butorphanol for inducing surgical anesthesia in ICR, BALB/c, and C57BL/6 mouse strains.

The anesthetic effects of alfaxalone combined with medetomidine and butorphanol were investigated for ICR, BALB/c, and C57BL/6 mice. Mice were administered a combination of 0.5 or 0.75 mg/kg medetomidine and 5 mg/kg butorphanol with 30 or 40 mg/kg alfaxalone (0.5MBA30, 0.75MBA30 and 0.75MBA40, respectively). The drug combinations were administered subcutaneously and were compared with a widely used combination of 0.3 mg/kg medetomidine, 4 mg/kg midazolam, and 5 mg/kg butorphanol (MMB). All three MBA combinations achieved surgical anesthesia, although the recovery time was longer with 0.75MBA30 and 0.75MBA40 compared with 0.5MBA30. Furthermore, several mice exhibited a considerable jumping reaction immediately after injection with 0.75MBA30 and 0.75MBA40. Therefore, 0.5MBA30 may be suitable for inducing surgical anesthesia in the mouse strains tested. The anesthetic scores for 0.5MBA30 were improved compared with those of MMB in all three mouse strains; however, the body temperature drop in C57BL/6 mice was greater with 0.5MBA30. Our results show that the alfaxalone combination, 0.5MBA30, should allow surgical operations that are more stable in more strains of mice than MMB, although the combination may cause hypothermia, especially in C57BL/6 mice.

A novel mouse model of adult T-cell leukemia cell invasion into the spinal cord.

Adult T-cell leukemia (ATL) is a mature T-cell malignancy caused by human T-cell leukemia virus type I infection, and 10%-25% of patients show central nervous system (CNS) involvement. CNS involvement significantly reduces survival and there are no effective treatments for CNS involvement. Therefore, an appropriate animal model is required to evaluate the inhibitory effects of novel drugs on the progression of ATL with CNS involvement. Here, we established a mouse model of ATL with CNS involvement using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice inoculated with ATL cells intramuscularly in the postauricular region, and these mice showed paraparesis. Of the 10 mice inoculated with ATL cells intramuscularly (I.M.) at 5 weeks of age, 8 (80%) showed paraparesis, whereas none of the 10 mice inoculated with ATL cells subcutaneously (S.C.) showed paraparesis. In the I.M. group, PCR detected HTLV-1-specific genes in the thoracic and lumbar vertebrae; however, in the S.C. group, the vertebrae were negative for HTLV-1 genes. Histological analysis revealed a particularly high incidence of tumors, characterized by accumulation of the injected cells, in the thoracic vertebrae of mice in the I.M. group. Tumor cell infiltration was relatively high in the bone marrow. Spinal cord compression caused by invasion of the tumor mass outside the pia mater was observed in the thoracic vertebrae of the spinal cord. In conclusion, we have reported a mouse model of tumor growth with paraparesis that may be used to assess novel therapeutic agents for ATL with CNS involvement.

New SIRT2 inhibitors: Histidine-based bleomycin spin-off.

Bleomycin is considered to exert its antitumor activity via DNA cleavage mediated by activated oxygen generated from the iron complex in its chelator moiety. Spin-offs from this moiety, HPH-1Trt and HPH-2Trt, with anti-cancer activities were recently synthesized. In this paper, we developed inhibitors of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of Sirtuin protein (SIRT2), based on HPH-1Trt/HPH-2Trt, and aimed to generate new anti-cancer drugs. HPH-1Trt and HPH-2Trt had in vitro anti-SIRT2 inhibitory activity with 50% inhibitory concentration (IC50) values of 5.5 and 8.8 µM, respectively. A structural portion of HPH-1Trt/HPH-2Trt, a tritylhistidine derivative TH-1, had stronger activity (IC50 = 1.7 µM), and thus, fourteen derivatives of TH-1 were synthesized. Among them, TH-3 had the strongest activity (IC50 = 1.3 µM). Selective binding of TH-3 in the pocket of SIRT2 protein was confirmed with a molecular docking study. Furthermore, TH-3 strongly lowered viability of the breast cancer cell line MCF7 with an IC50 of 0.71 µM. A structure-activity relationship study using cell lines suggested that the mechanism of TH-3 to suppress MCF7 cells involves not only SIRT2 inhibition, but also another function. This compound may be a new candidate anti-cancer drug.

Novel small molecule SIRT2 inhibitors induce cell death in leukemic cell lines.

BACKGROUND: Sirtuin 2 (SIRT2) is a member of the sirtuin family, nicotinamide adenine dinucleotide+-dependent deacylases, which participates in modulation of cell cycle control, neurodegeneration, and tumorigenesis. SIRT2 expression increases in acute myeloid leukemia blasts. Downregulation of SIRT2 using siRNA causes apoptosis of HeLa cells. Therefore, selective inhibitors of SIRT2 are candidate therapeutic agents for cancer. Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy that has a poor prognosis and develops after long-term infection with human T-cell leukemia virus (HTLV)-1. Sirtuin 1 inhibition has been shown to induce apoptosis and autophagy in HTLV-1-infected cell lines, whereas the effects of SIRT2 inhibition alone have not been elucidated. METHODS: We assessed the efficacy of our small molecule selective SIRT2 inhibitors NCO-90/141 to induce leukemic cell death. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF. Apoptotic cells were detected by annexin V-FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling assays by flow cytometry. Caspase activity was detected using an APOPCYTO Intracellular Caspase Activity Detection Kit. The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit. RESULTS: Our novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells. NCO-90/141 induced apoptosis via caspase activation and mitochondrial superoxide generation in leukemic cell lines. However, a caspase inhibitor did not prevent this caspase-associated cell death. Interestingly, NCO-90/141 increased the LC3-II level together with autophagosome accumulation, indicating autophagic cell death. Thus, NCO-90/141 simultaneously caused apoptosis and autophagy. CONCLUSIONS: These results suggest that NCO-90/141 are highly effective against leukemic cells in caspase-dependent or -independent manners via autophagy, and they may have a novel therapeutic potential for treatment of leukemias including ATL.

Effects of expressing human T-cell leukemia virus type 1 (HTLV-I) oncoprotein Tax on DOK1, DOK2 and DOK3 gene expression in mice.

Transgenic mice expressing the tax gene from human T-cell leukemia virus type 1 (HTLV-I) genome developed T-cell leukemia or histiocytic sarcoma after at least 12 months. The transgenic mice showed low expression of the downstream of tyrosine kinase (DOK) family members, DOK1, DOK2 and DOK3, which were recently reported to be tumor suppressor genes. Mice showed low DOK2 expression at 5-6 months of age, before disease onset. The expression of DOK1 and DOK3 was not significantly reduced at any age tested. These results suggest that downregulation of DOK2 by the expression of the viral tax gene is the first step in the development of T-cell leukemia or histiocytic sarcoma.

Evaluation of a combination of alfaxalone with medetomidine and butorphanol for inducing surgical anesthesia in laboratory mice.

The anesthetic effects of alfaxalone were investigated in mice. Mice were administered alfaxalone (100 mg/kg) alone or the combinations of 0.3 mg/kg of medetomidine and 5 mg/kg of butorphanol with alfaxalone at doses of 20 mg/kg (M/B/A20), 40 mg/kg (M/B/A40), 60 mg/kg (M/B/A60), or 80 mg/kg (M/B/A80). Control mice received 0.3 mg/kg of medetomidine, 4 mg/kg of midazolam, and 5 mg/kg of butorphanol (M/M/B). Each drug was administrated by intraperitoneal (IP) or subcutaneous (SC) routes. M/M/B IP did not achieve surgical anesthesia but M/M/B SC achieved surgical anesthesia within 10 min after administration and maintained anesthesia for 45 min. The anesthetic scores were very low after IP or SC administration of alfaxalone alone. M/B/A20 IP and SC did not achieve surgical anesthesia. M/B/A40 IP did not achieve surgical anesthesia but M/B/A40 SC achieved surgical anesthesia within 10 min after administration and maintained anesthesia for 35 min. M/B/A60 SC achieved surgical anesthesia within 5 min after administration and maintained anesthesia for 75 min. By contrast, M/B/A60 IP did not achieve surgical anesthesia. M/B/A80 SC achieved surgical anesthesia within 5 min after administration and maintained anesthesia for 85 min. By contrast, M/B/A80 IP did not achieve surgical anesthesia and one mouse died about 10 min after drug administration. Administration of atipamezole rapidly reversed anesthesia induced by M/B/A60 in mice. These results suggest that M/B/A60 SC, an alfaxalone-based combination, is suitable for inducing surgical anesthesia in laboratory mice.

Invasion of histiocytic sarcoma into the spinal cord of HTLV-1 tax transgenic mice with HTLV-1-associated myelopathy/tropical spastic paraparesis-like disease.

Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Transgenic (Tg) mice expressing HTLV-1 Tax also develop T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. We found that 8 of 297 Tax-Tg mice developed HAM/TSP-like disease with symmetrical paraparesis of the hind limbs, but these symptoms were absent in non-Tg littermates and in other mice strains at our animal facilities. We could perform detailed evaluations for five of these mice. These evaluations showed that the disease was not inflammatory, unlike that in HAM/TSP patients, but instead involved the invasion of histiocytic sarcoma cells into the lumbar spinal cord from the bone marrow where they had undergone extensive proliferation.

p53 dysfunction precedes the activation of nuclear factor-κB during disease progression in mice expressing Tax, a human T-cell leukemia virus type 1 oncoprotein.

Transgenic (Tg) mice expressing Tax, a human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, develop mature T-cell leukemia/lymphoma. The leukemic cells in Tg mice expressing Tax show p53 dysfunction and nuclear factor-κB (NF-κB) activation, similar to that seen in adult T-cell leukemia/lymphoma (ATLL) cells from patients infected with HTLV-1. However, it is unclear when these effects occur in HTLV-1 carriers during the development of ATLL. Here, we examined p53 function and NF-κB activity before the onset of leukemia in Tax-expressing Tg (Tax-Tg) mice between 4 and 25 months of age. At 4-10 months of age, 71% of mice showed p53 inactivation, without evidence for NF-κB activation, even though tax expression was consistent from 4 to 25 months of age. The decline in p53 function resulted from decreased p53 accumulation after DNA damage. From 11 months of age onward, 75% of mice showed p53 dysfunction and 37.5% showed constitutive NF-κB activation with the components of p50 and RelB. An NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced NF-κB activity (i.e. p50/RelB) but did not restore p53 function. In vivo, treatment with DHMEQ until 24 months of age prevented the onset of T-cell leukemia in Tax-Tg mice. These results suggest that the Tax-induced decline in p53 function, which is independent of NF-κB activation in the early stage, might be the first stage in the onset of ATLL. NF-κB activity is involved in the later stages of ATLL onset.

Viral interference with host mRNA surveillance, the nonsense-mediated mRNA decay (NMD) pathway, through a new function of HTLV-1 Rex: implications for retroviral replication.

Nonsense-mediated mRNA decay (NMD) is an essential and conserved cellular mRNA quality control mechanism. RNA signals to express viral genes from overlapping open reading frames potentially initiate NMD, nevertheless it is not clear whether viral RNAs are sensitive to NMD or if viruses have evolved mechanisms to evade NMD. Here we demonstrate that the genomic and full-length mRNAs of Human-T-cell Leukemia Virus type-I (HTLV-1), a retrovirus responsible for Adult T-cell Leukemia (ATL), are sensitive to NMD. They exhibit accelerated turnover in NMD-activated cells, while siRNA-mediated knockdown of NMD-master-regulator, UPF1, promotes enhanced stability of them. These effects on RNA stability were recapitulated by a reporter construct encoding the HTLV-1 translational frameshift signal of gag-pol. In agreement with the RNA stability, viral protein expression from the integrated provirus was inversely correlated with cellular NMD activity. We further demonstrated that the viral RNA-binding protein, Rex, approves the stability of viral RNA by inhibiting NMD. Significantly, Rex establishes a general block to NMD, as both NMD-responsive reporter transcripts and natural host-encoded NMD substrates were stabilized in the presence of Rex. Thus, we suggest that Rex not only stabilizes viral transcripts, but also perturbs cellular mRNA metabolism and host cell homeostasis via inhibition of NMD.

A transgenic mouse model of human T cell leukemia virus type 1-associated diseases.

Human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia/lymphoma (ATLL) and several inflammatory diseases. Tax, the protein encoded by HTLV-1, may be responsible for the development of the diseases caused by this virus. To investigate the pathogenic role of Tax, several transgenic mouse strains expressing Tax have been developed in recent years. These mice develop various tumors including large granular lymphocytic leukemia, as well as inflammatory diseases such as arthritis. These results suggest that Tax expression alone is sufficient to cause both malignant neoplastic diseases and inflammatory diseases. However, until recently, there were no tax transgenic mice that develop T cell leukemia and lymphoma resembling ATLL. The first successful induction of leukemia in T cells was pre-T cell leukemia generated in transgenic mice in which a mouse lymphocyte-specific protein tyrosine kinase p56 (lck) (lck)-proximal promoter was used to express the tax gene in immature T cells. Subsequently, transgenic mice were established in which the lck-distal promoter was used to express Tax in mature T cells; these mice developed mature T cell leukemia and lymphoma that more closely resembled ATLL than did earlier mouse models.

Natural infection of murine norovirus in conventional and specific pathogen-free laboratory mice.

Noroviruses cause most cases of acute viral gastroenteritis worldwide. The lack of a cell culture infection model for human norovirus necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict norovirus inactivation. Murine norovirus (MNV) may be used to construct a small animal model for studying the biology and pathogenesis of noroviruses because MNV is the only norovirus that replicates in cell culture and a small animal model. However, recent studies have shown that natural MNV infection is widespread in laboratory mouse colonies. We investigated MNV infection in both conventional and specific pathogen-free (SPF) genetically modified mice from Japan and the US, and commercial mice from several animal breeders in Japan, using serological and molecular techniques. MNV antibodies were detected in 67.3% of conventional mice and 39.1% of SPF mice from Japan and 62.5% of conventional mice from the US. MNV antibodies were also found in 20% of commercial SPF C57BL/6 mice from one of three breeders. Partial gene amplification of fecal isolates from infected animals showed that the isolates were homologous to reported MNV sequences. These results suggest that both conventional and SPF laboratory mice, including commercial mice, are widely infected with MNV, which might require considerable attention as an animal model of human disease.