HTLV-I p30 inhibits multiple S phase entry checkpoints, decreases cyclin E-CDK2 interactions and delays cell cycle progression

<p>Abstract</p> <p>Background</p> <p>Human T-cell leukemia virus type I (HTLV-I) has efficiently adapted to its host and establishes a persistent infection characterized by low levels of viral gene expression and slow proliferation of HTLV-I infected cells over decades. We have previously found that HTLV-I p30 is a negative regulator of virus expression.</p> <p>Results</p> <p>In this study we show that p30 targets multiple cell cycle checkpoints resulting in a delayed entry into S phase. We found that p30 binds to cyclin E and CDK2 and prevents the formation of active cyclin E-CDK2 complexes. In turn, this decreases the phosphorylation levels of Rb and prevents the release of E2F and its transcriptional activation of genes required for G1/S transition. Our studies also show that HTLV-II p28 does not bind cyclin E and does not affect cell cycle progression.</p> <p>Conclusions</p> <p>In contrast to HTLV-I, the HTLV-II-related retrovirus is not oncogenic in humans. Here we report that the HTLV-I p30 delays cell cycle progression while its homologue, HTLV-II p28, does not, providing evidence for important differences between these two related retrovirus proteins.</p

Inducible nitric oxide synthase mediates DNA double strand breaks in Human T-Cell Leukemia Virus Type 1-induced leukemia/lymphoma

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive and fatal malignancy of CD4(+) T-lymphocytes infected by the Human T-Cell Virus Type 1 (HTLV-1). The molecular mechanisms of transformation in ATLL have not been fully elucidated. However, genomic instability and cumulative DNA damage during the long period of latency is believed to be essential for HTLV-1 induced leukemogenesis. In addition, constitutive activation of the NF-κB pathway was found to be a critical determinant for transformation. Whether a connection exists between NF-κB activation and accumulation of DNA damage is not clear. We recently found that the HTLV-1 viral oncoprotein, Tax, the activator of the NF-κB pathway, induces DNA double strand breaks (DSBs). RESULTS: Here, we investigated whether any of the NF-κB target genes are critical in inducing DSBs. Of note, we found that inducible nitric oxide synthase (iNOS) that catalyzes the production of nitric oxide (NO) in macrophages, neutrophils and T-cells is over expressed in HTLV-1 infected and Tax-expressing cells. Interestingly, we show that in HTLV-1 infected cells, iNOS expression is Tax-dependent and specifically requires the activation of the classical NF-κB and JAK/STAT pathways. A dramatic reduction of DSBs was observed when NO production was inhibited, indicating that Tax induces DSBs through the activation of NO synthesis. CONCLUSIONS: Determination of the impact of NO on HTLV-1-induced leukemogenesis opens a new area for treatment or prevention of ATLL and perhaps other cancers in which NO is produced

HTLV-I tax increases genetic instability by inducing DNA double strand breaks during DNA replication and switching repair to NHEJ.

Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood.Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway.This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation

Tax expression switch DDSB repair from HR to error-prone NHEJ.

<p>A) Colocalization of Ku80 (specific to NHEJ DNA repair) and γ-H2AX in MT-2 and WT4 cells 30 min after gamma-irradiation. B) Colocalization of γH2AX foci and Ku80 in Tax-inducible JPX9 after induction of Tax expression by CdCl. Non-induced JPX9 and CdCl treated Jurkat control cells were used to demonstrate specificity to Tax expression. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with Nikon's NIS-Elements software. C) Colocalization of γ-H2AX foci and Rad51 (specific to HR DNA repair) after Tax induction in JPX9 and in Jurkat cells that were used as control. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with Nikon's NIS-Elements software.</p

HTLV-I-transformed cells and normal PBMC are equally sensitive to DNA damaging agents and have an overall similar rate of DNA repair.

<p>HTLV-I-transformed cells (MT-2, MT-4, C8166, 1185) and Tax-immortalized cells (WT4, WT4B, WT4I) and PBMC were exposed to 10 nM of Doxorubicin, washed and rate of repair was evaluated by immunostaining of γ-H2AX -revealed DDSB foci and quantified by microscopy at 0 h, 2.5 h, 5 h and 10 h after treatment. Because of various numbers of breaks in the absence of treatment, DNA breaks were normalized at 0 for time T = 0. Data were generated from counting 100 cells. Rate of repair is represented by the decreasing number of γ-H2AX foci at 2.5, 5 and 10 hours after doxorubicin treatment. Data were generated from counting 100 cells.</p

Tax inhibits the DDSB HR DNA repair through activation of NF-kB.

<p>A) Representation of in vivo HR assays using the DR-GFP HR reporter and the pI-SceI vectors. B) Jurkat T cells were cotransfected with DR-GFP vector either with the control vector or with the pI-SceI, and along with Tax or Tax mutants M47 or M22 using Amaxa electroporation. Forty-eight hours later the expression of GFP was assessed by FACS analysis. Relative percentage of GFP-expressing cells was represented by histograms corresponding to the average of 3 independent experiments. C) Activation of NF-kB reporter luciferase vector by HTLV-I Tax and Tax mutants M47 and M22. D and E) In vivo HR assay was performed in the presence of Tax and along with coexpression of a phosphorylation-defective dominant negative IkBα mutant that efficiently blocks NF-kB activation by Tax. F) Inhibition of HR by Tax was investigated using HTLV-I molecular clones expressing Tax or Tax mutants M47 and M22 at physiological levels.</p

Tax-associated DDSB are not repaired by SSA or MMEJ.

<p>A and B) Tax-inducible cell line JPX9 and Jurkat control cells were induced with CdCl, γ-irradiated with a dose of 2Gy and treated with or without DNA-PK inhibitor NU7026. γH2AX foci were detected by microscopy at time t = 0, 2 h, 4 h, 6 h and 24 h. Image results provided are representative of the experiment performed in duplicate. Average number of γ-H2AX foci per cell was quantified by microscopy. The error bars in the figures represent the standard error of the mean (SEM) for 30 to 50 cells per sample. The images were obtained using epifluorescence Nikon Ti-s and have been treated with the deconvolution feature provided with the Nikon's NIS-Element software. C) HTLV-I Tax-expressing cells MT-4 were untreated (left) or incubated with DNA-PK inhibitor for 2 days (middle panel) or 6 days (inhibitor was replenished every 2 days). Cell cycle was analyzed by FACS after propidium iodide staining. Arrows indicate cell cycle arrest in G2/M and aneuploidy.</p

Tax-associated DDSB occur in S phase during DNA replication.

<p>A) HTLV-I-transformed MT-2 cells were pulse-labeled with BrDU to identify cells in S phase of the cell cycle. Immunofluorescence experiments indicated that γ-H2AX foci are detected in BrDU positive cells only. Cells were counter stained with DAPI. B) HTLV-I-transformed MT-2 cells were dual stained with Cyclin A, a marker of S phase, and γ-H2AX, a marker of DDSB foci. Cells were counter-stained with DAPI. C) HTLV-I-transformed MT-2 cells were dual stained with PCNA, which appear as a punctuated pattern in S phase, and γ-H2AX, to reveal DDSB foci. Cells were counter-stained with DAPI. D) Accumulation of DDSB foci in S phase was confirmed in Tax-only expressing cells using Tax-inducible JPX9 cells and Jurkat control, both treated with CdCl and dual-stained with Cyclin A and γ-H2AX antibodies. E) JPX9 cells were synchronized in G0/G1 by exposure to hydroxyurea (HU) overnight. G0/G1 JPX9 cells and non-synchronized JPX9 cells were exposed to CdCl to induce Tax expression and analyzed for presence of γ-H2AX DDSB foci. F) Efficacy of HU treatment was demonstrated by cell cycle analyses by FACS after PI staining. From top to bottom, untreated cells, HU G1-arrested cells, HU G1-arrested cells released by washing out HU and culturing 20 hours. G) Western blot analyses confirmed that HU treatment did not affect Tax expression.</p