Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.     

Spindle checkpoint protein Bub1 corrects mitotic aberrancy induced by human T-cell leukemia virus type I Tax

Bub1 is a component of the mitotic spindle checkpoint apparatus. Abnormality of this apparatus is known to cause multinuclei formation, a hallmark of chromosomal instability (CIN). A549, aneuploid cell line, aberrantly passed through the mitotic phase and became multinuclei morphology in the presence of nocodazole. Time-lapse videomicroscopy showed unreported bizarre morphology, which we named 'mitotic lobulation' in A549 cells just before the exit from mitosis and multinuclei formation. External expression of wild-type Bub1-EGFP clearly suppressed the multinuclei formation by retaining A549 cells at the mitotic phase during 48 h of time-lapse observation. This suppressive effect on mitotic aberrancy should not be mere restoration of normal Bub1 function, because A549 cells express proper amount of Bub1, which distributed cytoplasm during interphase and concentrated at kinetochore in metaphase. Furthermore, external expression of wild-type Bub1-EGFP suppressed multinuclei formation induced by Tax both in A549 and HeLa cells. Tax is known to induce mitotic abnormality by binding and inactivating Mad1. These observations, therefore, suggest functional redundancy between Bub1 and other mitotic checkpoint protein(s) and a possibility of correction of mitotic aberrancy by external Bub1 expression.     

Genomic instability driven by the human T-cell leukemia virus type I (HTLV-I) oncoprotein,Tax

The importance of maintaining genomic stability is evidenced by the fact that transformed cells often contain a variety of chromosomal abnormalities such as euploidy, translocations, and inversions. Gene amplification is a well-characterized hallmark of genomic instability thought to result from recombination events following the formation of double-strand, chromosomal breaks. Therefore, gene amplification frequency serves as an indicator of genomic stability. The PALA assay is designed to measure directly the frequency with which a specific gene, CAD, is amplified within a cell's genome. We have used the PALA assay to analyse the effects of the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax, on genomic amplification. We demonstrate that Tax-expressing cells are five-times more likely to undergo gene amplification than control cells. Additionally, we show that Tax alters the ability of cells to undergo the typical PALA-mediated G(1) phase cell cycle arrest, thereby allowing cells to replicate DNA in the absence of appropriate nucleotide pools. This effect is likely the mechanism by which Tax induces gene amplification. These data suggest that HTLV-I Tax alters the genomic stability of cells, an effect that may play an important role in Tax-mediated, HTLV-I associated cellular transformation.     

Transformation of hamster spleen lymphocytes by human T-cell leukemia virus type I

Co-cultivation of spleen cells of Syrian golden hamsters with lethally irradiated MT-2 cells harboring human T-cell leukemia virus type I (HTLV-I) resulted in the establishment of lymphoid cell lines, HCT-1 and HCT-2, which exhibited the normal karyotype of golden hamsters. Cells of both the HCT-1 and HCT-2 lines lacked surface immunoglobulins and reacted with a monoclonal antibody (MAb) specific for hamster T cells. Some were positive for OKIa1. None of them expressed HTLV structural antigens (p19 and p24) or virus particles, but they contained HTLV-I proviral DNA monoclonally. By immunochemical analysis of the labelled cell antigens, sera from adult T-cell leukemia (ATL) patients reacted with the two polypeptides, p37 and p40, which may not be viral structural proteins and still remain to be characterized. HCT-1 and HCT-2 cells were transplantable into newborn hamsters, pre-treated with anti-hamster thymocyte serum and non-treated, respectively, producing diffuse malignant lymphoma. These findings indicated that HTLV-I not only immortalized but also transformed hamster T cells non-productively.     

Human T-cell leukemia virus type I and adult T-cell leukemia

Human T-cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia (ATL) in about 5% of carriers after a long latent period. After its infection, HTLV-I promotes the clonal proliferation of HTLV-I infected cells in vivo by actions of encoded viral proteins, including Tax. However, leukemic cells frequently lack the expression of Tax by the genetic and epigenetic changes of HTLV-I provirus, suggesting that Tax is not always necessary after transformation. Alternatively, ATL cells without Tax protein could escape from the host immune system since Tax is the major target of cytotoxic lymphocytes. During the latent period, alterations of host genome accumulate, finally leading to onset of ATL.     

Tax protein of human T-cell leukemia virus type I is required for maintenance of the transformed phenotype.

We have isolated and characterized revertants of a clonal cell line (40MRatcl-1) of human T-cell leukemia virus type I Tax-transformed Rat1 cells. The 40MRatcl-1 cells contain a single copy of tax gene, form large colonies in soft agar, elicit tumors rapidly in nude mice and revert to the normal phenotype at low frequency. From one of its subclones (B7) bearing pSV2gpt DNA as a marker gene, four morphologically reverse-transformed cell lines were isolated. They display contact inhibition at confluency, lose the ability to form colonies in soft agar, fail to form tumors in nude mice and restore the transformed phenotype similar to that of 40MRatcl-1 cells by transfection with the tax-expression plasmid. Southern blot analysis revealed that they have lost the tax gene. Our results indicate that transformation of Rat1 cells by Tax is not the consequence of secondary mutations of cellular genes and that tax functions are directly required for establishment and maintenance of the transformed phenotype.     

Inactivation of lymphocyte-transforming activity of human T-cell leukemia virus type I by heat

Peripheral blood lymphocytes from 2 normal individuals seronegative for human T-cell leukemia virus type I (HTLV-I) were co-cultured with HTLV-I-producing MT-2 cells that had been heated at 56 degrees for 30 min or exposed to 10,000 rad of X-irradiation. HTLV-I-induced lymphocyte transformation was consistently achieved by co-culture with irradiated MT-2 cells but not by co-culture with heated MT-2 cells. The heat treatment was found to be lethal to both MT-2 cells and the virus. These findings are discussed in terms of their potential clinical application for preventing the transmission of HTLV-I.     

Oral transmission of human T-cell leukemia virus type I in the rabbit

Four female rabbits were given twice-weekly oral inoculation of 2-4 X 10(7) cells from a male rabbit lymphoid cell line persistently infected with human T-cell leukemia virus type I (HTLV-I). After 8 weeks, one of them was found to be seroconverted for HTLV-I. Peripheral lymphocytes from the 4 rabbits were cultured in the presence of T-cell growth factor, and a lymphoid cell line with a normal female karyotype was established only from the seroconverted rabbit. This cell line was reactive with a monoclonal antibody to rabbit T-cells and expressed HTLV-I antigens and virus particles.     

Experimental infection of rabbits with human T-cell leukemia virus type I

Rabbits were successfully infected with human T-cell leukemia virus type I (HTLV-I) and produced antibodies to adult T-cell leukemia-associated antigens (ATLA) on intravenous inoculation of the HTLV-producing human cord T-cell line MT-2, or autologous cell lines established by cocultivation with MT-2 cells. Lymphocytes taken from the rabbits between 4 and 14 days after the inoculation of MT-2 cells, but not lymphocytes obtained in earlier or later periods, could be immortalized in vitro and expressed ATLA and type C virus particles. However, lymphocytes harvested during an early culture period (5 to 8 days) were found to be negative for ATLA. The transformed cells had the karyotype of a normal male rabbit. Two rabbits inoculated with the autologous HTLV-producing cell lines did not allow their growth in vivo, but some peripheral blood lymphocytes could be immortalized in vitro. One of these transformed cell lines was examined for the integration of HTLV-I provirus genome. The transformed cells were found to contain the provirus genome and also to be monoclonal with respect to the integration site of the provirus genome, unlike the inoculated cells.     

Arsenic trioxide and the growth of human T-cell leukemia virus type I infected T-cell lines

A novel therapeutic potential for acute promyelocytic leukemia using arsenic trioxide (As(2) O(3) ) has been reported. Recent in vitro studies demonstrated that As(2) O(3) effectively inhibits the growth of some cell lines derived from patients with malignant lymphoma, chronic lymphocytic leukemia and multiple myeloma. Adult T-cell leukemia (ATL) is an aggressive neoplasm of mature T-cell origin caused by human T-cell leukemia virus type-I (HTLV-I) the prognosis of which still remains very poor. A possible role of As(2) O(3) for the treatment of ATL is demonstrated from evidence that As(2) O(3) significantly inhibits the growth of HTLV-I infected T-cell lines and induces apoptosis in fresh ATL cells at clinically achievable concentration of the agent. The growth inhibition of As(2) O(3) treated HTLV-I infected T-cell lines was induced by both apoptosis and G(1) phase accumulation. Cleaved bcl-2 protein and an enhanced expression of bak protein in the cells were coincidentally observed during As(2) O(3) treatment. A broad spectrum caspase inhibitor, z-Val-Ala-DL-Asp-fluoromethylketone inhibited the apoptosis induced by As(2) O(3). Increased expression of p53, Cip1/p21 and Kip1/p27, and dephosphorylation of retinoblastoma protein (pRb) were detected in the As(2) O(3) treated cells. In conclusion, As(2) O(3) might become a new therapeutic tool in the treatment of ATL as As(2) O(3) induces apoptosis by destruction of the bcl-2 protein and enhancement of the bak protein production proceeding to activate caspases, and also induces G(1) phase accumulation by enhancement of p53, Cip1/p21, Kip1/p27 and dephosphorylation of pRb to HTLV-I infected T-cell lines.     

Mode of transmission of human T-cell leukemia virus type I (HTLV I) in a human promyelocytic leukemia HL60 cell

HTLV-I propagated in IMR90 human diploid fibroblasts was transmitted to human myeloid leukemia HL60 cells at a low efficiency. After co-cultivation for 3 months, the viral genome was detected in 14/48 HL60 cell clones. Among the 14 HTLV-I-infected clones, 8 contained subgenomic fragments alone or in addition to the complete HTLV-I genome. The frequency of deleted proviruses (9/24 total proviruses) was unexpectedly high. Hirt's supernatant of some of the clones harboring complete HTLV-I genome(s) in the chromosome contained both linear and circular HTLV-I proviral DNAs. The circular DNAs were composed of one LTR and 2 LTR closed circular proviruses. These clones produced infectious HTLV-I constitutively, which was proved by transmission of the viral genome into fresh IMR90 cells by co-cultivation. However, in these clones, re-integration of extrachromosomal provirus into their own chromosomes was not observed.     

Pathological features of diseases associated with human T-cell leukemia virus type I

In the early 1980s, the first human retrovirus, human T-cell leukemia virus type I (HTLV-I), was isolated and its characterization opened up the new field of human viral oncology. Adult T-cell leukemia/lymphoma (ATLL), which is associated with HTLV-I, is characterized clinically by the appearance of characteristic flower cells, a rapid clinical course, occasional skin lesions, lymphadenopathy and hepatosplenomegaly. Severe opportunistic infections are occasionally accompanied. In addition, HTLV-I infection is associated with autoimmune and reactive disorders, such as HTLV-I-associated myelopathy and uveitis, and is also related to immunodeficient infectious diseases. Pathological findings of ATLL in the lymph nodes, skin, liver and other organs have been described. Common histological features are a diffuse proliferation of atypical lymphoid cells that vary in size and shape. In addition to ATLL, non-neoplastic organopathies have been documented in many organs, such as the central nerve system, lung, skin, lymph nodes and gastrointestinal tract. To clarify the HTLV-I-associated diseases, it is important to understand the pathological variations.     

Transcriptional activation of survivin through the NF-kappaB pathway by human T-cell leukemia virus type I tax

Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. We previously reported the survivin expression profile in ATL, a CD4-positive T-cell malignancy caused by HTLV-I. HTLV-I Tax is thought to play an important role in immortalization of T cells. We have shown also that the expression of Tax protected the mouse T-cell line CTLL-2 against apoptosis induced by deprivation of IL-2 and converted its growth from being IL-2 dependent to being IL-2 independent through the NF-kappaB pathway. In our study, we demonstrate that constitutive expression of survivin was associated with resistance to apoptosis after IL-2 deprivation in Tax-expressing CTLL-2 cells. Transient transfection assays showed that survivin promoter was transactivated by Tax, via the activation of NF-kappaB. Pharmacological NF-kappaB inhibition resulted in suppression of survivin expression and caused apoptosis of Tax-expressing CTLL-2 cells. Our findings suggest that activated NF-kappaB signaling contributes directly to malignant progression of ATL by preventing apoptosis, acting through the prosurvival protein survivin.     

Simulation of dynamic changes of human T-cell leukemia virus type I carriage rates

The human T-cell leukemia virus type I (HTLV-I) is transmitted via breast milk, semen, or blood transfusion. The last route was not responsible for HTLV-I infection before the advent of modern medicine, nor will it be a major route in the future because anti HTLV-I antibody-positive blood is now screened out. Thus, the carriage rates in various areas of Japan have to be explained by the former two transmission methods. Based on the relationship between the two modes of transmission and carriage rates, several simulation experiments were performed. These experiments revealed that: (a) No population with a vertical transmission rate lower than 50% can be maintained as endemic for the virus. (b) Slight differences in horizontal transmission rates can cause a large change of the carriage rates. (c) A 1,000-fold carriage rate difference would become indistinguishable within a hundred generations if both modes of transmission were operating at nearly the same rate. (d) The probability of a formerly non-endemic population becoming endemic due to a single female carrier is not negligible. (e) Prevention of vertical transmission is much more effective in lessening the carriage rate within a short period of time than is prevention of horizontal transmission. A simulation for a real population is also presented.     

Human T-cell leukemia virus type I associated lymphadenitis.

Histopathologic changes in lymph nodes were examined from ten patients with mild lymphadenopathy, a few atypical lymphocytes in their peripheral blood, skin lesions, and proviral DNA of human T-cell leukemia virus type I (HTLV-I) in their nodes. The proviral DNA of HTLV-I was detected by southern blot analysis, in situ hybridization, and/or polymerase chain reaction techniques. The lymph nodes showed preserved nodal architecture with diffuse infiltration of small to intermediate-sized lymphocytes in association with scattered transformed lymphocytes and a few immunoblast-like cells in the enlarged paracortex. The infiltrating lymphocytes were positive for CD4, but neither rearrangement nor deletion of T-cell receptors and immunoglobulin heavy chain genes was detected. Eight of ten patients received no therapy, and all patients were alive and healthy more than 5 months after the biopsies. The histologic findings resembled those of a viral infection and could be distinguished from HTLV-I associated lymphomas.     

Seroepidemiologic studies of human T-cell leukemia/lymphoma virus type I in Jamaica

The prevalence of HTLV-I antibodies was evaluated in Jamaica among persons with various malignant, infectious, autoimmune and hematologic disorders and in clinically normal persons. Results document that: (1) the prevalence of HTLV-I antibodies in this population increases with age; (2) overall, there is no significant difference in the antibody prevalence between males and females; (3) antibody-positive individuals are born in all major regions of the island and geographical variance in antibody prevalence by place of birth was not prominent; (4) there is further confirmation of the high prevalence of HTLV-I antibody-positive lymphomas in Jamaica; and (5) the prevalence of HTLV-I antibodies in hemophiliacs, patients with chronic lymphocytic leukemia (CLL), myelogenous leukemias, and patients with breast cancer is higher than in the age-matched population without malignancies, although none of these differences were statistically significant. The increased prevalence in hemophiliacs is most likely related to their frequent transfusion with blood products, but it has not yet been determined whether the prevalence in patients with other diseases is related to their diseases or other as yet undefined factors in common.     

Defective provirus form of human T-cell leukemia virus type I in adult T-cell leukemia/lymphoma: clinicopathological features

Human T-cell leukemia virus type I (HTLV-I) is associated with adult T-cell leukemia/lymphoma (ATLL). To examine the relationship between defective HTLV-I proviruses and clinicopathological features, we examined 95 patients with ATLL showing clonal integration of HTLV-I proviral DNA; 77 patients (81%) showed 1 clonal band, 15 (16%) showed 2 clonal bands, and 3 (3%) showed 3 clonal bands. In addition, the defective proviral form was detected in 28 patients (29%): 23 (30%) of the 77 with 1 clonal band, 4(27%) of the 15 with 2 clonal bands, and 1(33%) of the 3 with 3 clonal bands. The numbers of clonal bands had no association with the presence of defective proviruses. We classified the 95 patients with ATLL into four types according to clinicopathological features (smoldering leukemia, chronic leukemia, acute leukemia, and lymphoma types). The distribution of patients with the defective form was not different among these four types. The HTLV-I genomes must have integrated into the human genome DNA and been deleted partially in the cells. The defective form was kept during the clinical stage. All patients with the defective form showed defect of the gag or/and env region. No patient had a defect of the pX region. These data suggest that the pX region of HTLV-I must have played an important role in ATLL genesis.