Isolation of simian retroviruses closely related to human T-cell leukemia virus by establishment of lymphoid cell lines from various non-human primates

Simian retroviruses closely related to human T-cell leukemia virus (HTLV) were isolated by establishing virus-producing lymphoid cell lines from 7 species of non-human primates. By co-cultivation with human umbilical cord-blood cells and/or in the presence of interleukin-2, lymphoid cell lines were successfully established from the chimpanzee. African green monkey, pig-tailed macaque, red-faced macaque, Formosan monkey, Japanese monkey and bonnet monkey that had antibodies against HTLV antigens. These cell lines reacted with human sera of ATL patients and monoclonal antibodies against p19 and p24 of HTLV antigens. Cellular DNAs contained the provirus sequences homologous to HTLV-I by Southern blot hybridization. Moreover, they produced extracellular type-C virus particles and RNA-dependent DNA polymerase. All of these lymphoid line cells had Tac antigen, interleukin-2 receptor, and those of chimpanzee and red-faced macaque had helper/induced T-cell markers, while those derived from African green monkey had suppressor/cytotoxic T-cell markers. Furthermore, simian HTLV-related viruses of pig-tailed macaque, red-faced macaque and Japanese monkey were transmitted to human lymphocytes on co-cultivation.     

Adult T-cell leukemia-like disease in monkey naturally infected with simian retrovirus related to human T-cell leukemia virus type I

Spontaneous T-cell leukemia similar to human adult T-cell leukemia (ATL) was found in an African green monkey naturally infected with simian retrovirus closely related to human T-cell leukemia virus type I (HTLV-I). Monoclonal integration of the simian retrovirus was detected in the primary leukemic cells, suggesting an association of the retrovirus with ATL-like leukemia in the monkey.     

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.     

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.     

Antibodies to human immuno-deficiency virus and human T-cell leukemia virus type I in Japanese patients with hematologic malignancies

One thousand six hundred and seventy-four blood samples drawn between January 1980 and April 1986 from 1454 Japanese, including 251 leukemia, 409 lymphoma, 76 adult T-cell leukemia and 25 benign lymphadenitis patients, were tested for antibodies to HIV and HTLV-I. No patient with lymphadenitis or lymphoma associated with HIV infection was found. In 87 patients with acute and chronic leukemias who had received multiple transfusions, 8 were positive for anti-HTLV-I antibody after blood transfusions amounting to 115 units, on average, while no patient was positive for anti-HIV antibody. Overall, no sample was positive for anti-HIV antibody, whereas 153 (10.5%) were positive for anti-HTLV-I antibody. These results indicate that the transmission of HIV by blood transfusions is far less prevalent than that of HTLV-I in Tokyo at present.     

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.     

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.     

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.     

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.     

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.     

Development of adult T-cell leukemia-like disease in African green monkey associated with clonal integration of simian T-cell leukemia virus type I

Proviral integration of a simian retrovirus highly homologous to human T-cell leukemia virus type I was examined in cellular DNAs extracted from primary peripheral blood lymphocytes of 31 adult African green monkeys (Cercopithecus aethiops) that were seropositive for simian T-cell leukemia virus type I (STLV-I). Among these monkeys, one case with overt leukemia, showing pleomorphic leukemia cells similar to those in human adult T-cell leukemia (ATL), and five cases in a preleukemic state of ATL-like disease were found. Judging from the integration site of the provirus genome, primary lymphocytes of these leukemic or preleukemic cases contained monoclonally proliferated STLV-I-infected cells, whereas lymphocytes of other seropositive monkeys without hematological abnormalities were polyclonal, and those of seronegative monkeys did not contain the provirus. The restriction patterns with PstI ans SstI of most STLV-I proviruses were identical to those of the previous isolate from this species, but in three monkeys there was a deletion of one PstI site. From the correlation of the development of simian ATL-like disease with the monoclonal integration of the STLV-I provirus genome, it should be indicated that STLV-I has similar leukemogenicity to human T-cell leukemia virus type I, and so STLV-I infection in African green monkeys will be useful as an animal model of human ATL.     

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.     

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.     

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.     

Human T-lymphotropic virus type I provirus and T-cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL) is a rare type of post-thymic T-cell neoplasm, the etiology of which is unknown. Patients with T-PLL have been found to be seronegative for human T-lymphotropic virus type-I (HTLV-I), and their leukemia cells do not retain monoclonally integrated HTLV-I provirus. Recently, we have demonstrated the presence of defective HTLV-I provirus by polymerase chain reaction in the DNA extracted from peripheral blood cells or affected lymph nodes of T-PLL patients. Although there is a possibility, from our observation, that an alternative mechanism is operating in HTLV-associated leukemogenesis, it is still unknown whether and how HTLV-I can contribute to the leukemogenesis of T-PLL. In this review, we describe controversial issues and discuss a role of HTLV-I in the leukemogenesis of T-PLL.     

A gag-env hybrid protein of human T-cell leukemia virus type I and its application to serum diagnosis

A fused gene of the gag and env sequences of human T-cell leukemia virus type I (HTLV-I), the causative agent of adult T-cell leukemia, was constructed in vitro and expressed in Escherichia coli. The gag-env hybrid protein accumulated as insoluble granules with a yield of approximately 12% of the total proteins. In an enzyme-linked immunosorbent assay done with the use of the gag-env hybrid protein, all 57 seropositive sera gave positive signals, and none of the sera from normal persons did. This system can produce large quantities of the gag-env hybrid protein, which can be used for mass screening of human sera for HTLV-I infection.     

Centrosome amplification in adult T-cell leukemia and human T-cell leukemia virus type 1 Tax-induced human T cells

Centrosomes play pivotal roles in cell polarity, regulation of the cell cycle and chromosomal segregation. Centrosome amplification was recently described as a possible cause of aneuploidy in certain solid tumors and leukemias. ATL is a T-cell malignancy caused by HTLV-1. Although the precise mechanism of cell transformation is unclear, the HTLV-1-encoded protein, Tax, is thought to play a crucial role in leukemogenesis. Here we demonstrate that lymphocytes isolated from patients with ATL show centrosome amplification and that a human T cell line shows centrosome amplification after induction of Tax, which was suppressed by CDK inhibitors. Micronuclei formation was also observed after centrosome amplification in Tax-induced human T cells. These findings suggest that Tax deregulates CDK activity and induces centrosome amplification, which might be associated with cellular transformation by HTLV-1 and chromosomal instability in HTLV-1-infected human T cells.     

Sex-specific mortality from adult T-cell leukemia among carriers of human T-lymphotropic virus type I

Perinatal infection with human T-lymphotropic virus type I (HTLV-I) is considered a risk factor for adult T-cell leukemia (ATL). Incidence of ATL in Japan is generally higher in males compared with females, perhaps partly due to an earlier average age of infection among males. We estimated sex-specific ATL mortality among perinatally-infected HTLV-I carriers in the prospective Miyazaki Cohort Study in Japan. Based on the approximated proportion of perinatally-infected carriers, the relative risk (RR) of ATL for males compared with females was calculated. Six ATL deaths (4 males, 2 females) occurred among the 550 HTLV-I carriers in the cohort during 13 years of follow-up. The overall ATL mortality was 190.5 (95% CI 51.9-487.7) per 10(5) person-years for males and 51.7 (6.3-186.8) per 10(5) person-years for females (age-standardized RR = 3.9, p=0.02). By approximating the number of persons who acquired infection perinatally, the estimated mortality among those perinatally-infected HTLV-I carriers was 209.1 (57.0-535.2) per 10(5) person-years for males and 60.9 (7.4-219.9) per 10(5) person-years for females (age-standardized RR = 3.7, p=0.02). The adjusted RR changed minimally from the unadjusted RR, suggesting that earlier age of infection alone is unlikely the explanation for the male predominance in ATL. Based on the small number of cases available for analysis, aspects of gender itself appear to play a role in the development of this malignancy.     

Human T-cell leukemia virus type I induces adult T-cell leukemia: from clinical aspects to molecular mechanisms.

BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) is a causative virus of adult T-cell leukemia (ATL), HTLV-I-associated myelopathy/tropical spastic paraparesis, and HTLV-I-associated uveitis. ATL is a neoplastic disease of CD4-positive T lymphocytes that is characterized by pleomorphic tumor cells with hypersegmented nuclei, termed "flower cells." The mechanisms of leukemogenesis have not been fully clarified. METHODS: The authors reviewed the virological, clinical, and immunological features of HTLV-I and ATL and summarized recent findings on the oncogenic mechanisms of ATL and therapeutic advances. RESULTS: Multiple factors, such as viral genes, genetic and epigenetic alterations, and the host immune system, may be implicated in the leukemogenesis of ATL. Among them, viral genes, tax, and HBZ have been thought to play important roles. The prognosis of aggressive-type ATL remains poor, regardless of intensive chemotherapy. Effectiveness of allogeneic stem cell transplantation for ATL has been recently reported. CONCLUSIONS: Although the precise mechanism of leukemogenesis of ATL remains unclear, recent progress provides important clues in oncogenesis by HTLV-I. Future research should focus on the composition of novel therapeutic strategies, including prevention, based on the evidence in the leukemogenic mechanisms.