HTLV-I Tax Increases Genetic Instability by Inducing DNA Double Strand Breaks during DNA Replication and Switching Repair to NHEJ

Background

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.

Results

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.

Conclusions

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.     

Evidence for cooperative transforming activity of the human pituitary tumor transforming gene and human T-cell leukemia virus type 1 Tax

Aneuploidy is frequent in cancers. Recently it was found that pituitary tumor transforming gene (PTTG; also called Pds1p or securin) is overexpressed in many different tumors. Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that primarily infects CD4+ T lymphocytes and causes adult T-cell leukemia. Here, we report that overexpression of human PTTG cooperated with the HTLV-I Tax oncoprotein in cellular transformation. Coexpression of Tax and PTTG enhanced chromosomal instability and neoplastic changes to levels greater than overexpression of either factor singularly. Cells that overexpressed both PTTG and Tax induced tumors more robustly in nude mice than cells that expressed either PTTG alone or Tax alone.     

Human T-cell leukemia virus type 1 tax attenuates the ATM-mediated cellular DNA damage response

Genomic instability, a hallmark of leukemic cells, is associated with malfunctioning cellular responses to DNA damage caused by defective cell cycle checkpoints and/or DNA repair. Adult T-cell leukemia, which can result from infection with human T-cell leukemia virus type 1 (HTLV-1), is associated with extensive genomic instability that has been attributed to the viral oncoprotein Tax. How Tax influences cellular responses to DNA damage to mediate genomic instability, however, remains unclear. Therefore, we investigated the effect of Tax on cellular pathways involved in recognition and repair of DNA double-strand breaks. Premature attenuation of ATM kinase activity and reduced association of MDC1 with repair foci were observed in Tax-expressing cells. Following ionizing radiation-induced S-phase checkpoint activation, Tax-expressing cells progressed more rapidly than non-Tax-expressing cells toward DNA replication. These results demonstrate that Tax expression may allow premature DNA replication in the presence of genomic lesions. Attempts to replicate in the presence of these lesions would result in gradual accumulation of mutations, leading to genome instability and cellular transformation.     

Clastogenic effect of the human T-cell leukemia virus type I Tax oncoprotein correlates with unstabilized DNA breaks

Expression of the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein rapidly engenders DNA damage as reflected in a significant increase of micronuclei (MN) in cells. To understand better this phenomenon, we have investigated the DNA content of MN induced by Tax. Using an approach that we termed FISHI, fluorescent in situ hybridization and incorporation, we attempted to characterize MN with centric or acentric DNA fragments for the presence or absence of free 3'-OH ends. Free 3'-OH ends were defined as those ends accessible to in situ addition of digoxigenin-dUTP using terminal deoxynucleotidyl transferase. MN were also assessed for centromeric sequences using standard fluorescent in situ hybridization (FISH). Combining these results, we determined that Tax oncoprotein increased the frequency of MN containing centric DNA with free 3'-OH and decreased the frequency of MN containing DNA fragments that had incorporation-inaccessible 3'-ends. Recently, it has been suggested that intracellular DNA breaks without detectable 3'-OH ends are stabilized by the protective addition of telomeric caps, while breaks with freely detectable 3'-OH are uncapped and are labile to degradation, incomplete replication, and loss during cell division. Accordingly, based on increased detection of free 3'-OH-containing DNA fragments, we concluded that HTLV-I Tax interferes with protective cellular mechanism(s) used normally for stabilizing DNA breaks.     

The HTLV-I tax protein transcriptionally modulates OX40 antigen expression

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.     

Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Na?ve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23(ts); a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC(Cdc20p). This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G(2)/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APC(Cdc20p), leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.     

The retroviral oncoprotein Tax targets the coiled-coil centrosomal protein TAX1BP2 to induce centrosome overduplication

Emerging evidence suggests that supernumerary centrosomes drive genome instability and oncogenesis. Human T-cell leukaemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukaemia (ATL). ATL cells are aneuploid, but the causes of aneuploidy are incompletely understood. Here, we show that centrosome amplification is frequent in HTLV-I-transformed cells and that this phenotype is caused by the viral Tax oncoprotein. We also show that the fraction of Tax protein that localizes to centrosomes interacts with TAX1BP2, a novel centrosomal protein composed almost entirely of coiled-coil domains. Overexpression of TAX1BP2 inhibited centrosome duplication, whereas depletion of TAX1BP2 by RNAi resulted in centrosome hyperamplification. Our findings suggest that the HTLV-I Tax oncoprotein targets TAX1BP2 causing genomic instability and aneuploidy.     

Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.     

Unique and important consequences of RECQ1 deficiency in mammalian cells

Five members of the RecQ subfamily of DEx-H-containing DNA helicases have been identified in both human and mouse, and mutations in BLM, WRN, and RECQ4 are associated with human diseases of premature aging, cancer, and chromosomal instability. Although a genetic disease has not been linked to RECQ1 mutations, RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage.(1) Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous γ-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks.(2) Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin.(2) RECQ1 catalytic activities(3) and interactions with DNA repair proteins(2,4,5) are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis.     

XRCC3 is required for efficient repair of chromosome breaks by homologous recombination

XRCC3 was originally identified as a human gene able to complement the DNA damage sensitivity, chromosomal instability and impaired growth of the mutant hamster cell line irs1SF. More recently, it has been cloned, sequenced and found to bear sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51. The phenotype of irs1SF and the identification of XRCC3 as a member of the RAD51 gene family have suggested a role for XRCC3 in repair of DNA damage by homologous recombination. Homologous recombinational repair (HRR) of a specifically induced chromosomal double-strand break (DSB) was assayed in irs1SF cells with and without transient complementation by human XRCC3. Complementation with XRCC3 increased the frequencies of repair by 34- to 260-fold. The results confirm a role for XRCC3 in HRR of DNA DSB, and the importance of this repair pathway for the maintenance of chromosomal integrity in mammalian cells.     

Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells.

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sj?gren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.     

Role of Mammalian Rad9 in Genomic Stability and Ionizing Radiation Response

Eukaryotic cells have evolved DNA damage response mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. The Schizosaccharomyces pombe Rad9 gene was initially identified as encoding a cell cycle checkpoint protein. When the mammalian homologue of S. pombe Rad9 was inactivated, however, chromosomal instability was observed even in the absence of DNA damaging agents. Both an increase in chromosome end-to-end associations and telomere loss were observed in cells with inactivated mammalian Rad9. This telomere instability correlated with enhanced S- and G2-phase specific cell killing, delayed kinetics of γ-H2AX foci appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase specific DNA damage repair. Inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S- and G2-phase of the cell cycle. These newly defined functions of mammalian Rad9 are discussed in relation to telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.     

Cell-type-specific trans-activation of herpes simplex virus thymidine kinase promoter by the human T-cell leukemia virus type I Tax protein.

The human T-cell leukemia virus type I Tax protein (HTLV-I Tax) is known as a trans-activating factor for a variety of genes, including those of cytokines. Here, we show that Tax is capable of activating the herpes simplex virus thymidine kinase (HSV-TK) promoter in certain mammalian cell lines. In murine NIH 3T3 fibroblasts and human HeLa cells, trans-activation by Tax was remarkably strong, whereas in human chondrocytic HCS-2/8 and monkey kidney Cos-7 cells, the responsiveness of the TK promoter to Tax was poor. Deletion analysis revealed that one of the two previously described Sp1 sites is required for the Tax responsiveness, whereas the CTF binding site is not. The results suggest possible interactions between the oncogenic Tax protein and the viral TK in coinfected cells in vivo. Care should be taken in the context of HTLV-I research, as the HSV-TK promoter has been widely used in molecular biology and gene therapeutics.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.