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ABSTRACT: Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer in sheep. A unique feature is that JSRV envelope protein is also the oncogene for this virus. Previous studies have identified the cytoplasmic tail (CT) of the envelope transmembrane (TM) protein as critical for transformation although other regions of Env have also been implicated. In this study, the roles of other Env regions in transformation were investigated. Chimeras between JSRV Env and the Env of a related non-oncogenic endogenous retrovirus (enJSRV, 5F16) were used. A chimera containing the membrane-spanning region (MSR) of enJSRV inserted into JSRV Env showed substantially reduced transformation, indicating that the MSR plays a role in transformation. Transformation by this chimera was highly dependent on both Ras/Raf/MEK/MAPK and PI3K/Akt/mTOR signaling. A chimera containing the two amino acids in the TM ectodomain that distinguish JSRV and enJSRV showed modestly reduced transformation. Chimeras in the SU protein indicated that the amino terminal region of SU contributes to transformation, while the C-terminal part is not important. To test if Env trimerization is important for transformation, we mutated a leucine-rich sequence in the putative trimerization domain in the ectodomain of TM (Tri-M). This mutant could not transform cells and it did not oligomerize. However, Tri-M could complement a non-transforming mutant CT mutant (Y590F) so oligomerization is not necessary for at least some aspects of transformation. These experiments provide new insight into the regions and residues of JSRV Env protein necessary for oncogenic transformation.
Virus Genes 08/2012; · 1.85 Impact Factor
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ABSTRACT: Murine leukemia viruses (MuLVs) encode two forms of Gag polyprotein: the precursor for the viral core proteins (Pr65(gag) for Moloney MuLV [M-MuLV]) and a longer glycosylated form (glyco-gag, or gPr80(gag)). gPr80(gag) is translated from the same unspliced viral RNA as Pr65(gag), from an upstream in-frame CUG initiation codon. As a result, gPr80(gag) contains 88 unique N-terminal amino acids that include a signal peptide that conducts gPr80(gag) into the rough endoplasmic reticulum, where it is glycosylated, exported to the cell surface, and cleaved into two proteins of 55 and 40 kDa. The amino-terminal 55-kDa protein remains cell associated with the 88 unique amino acids exposed to the cytosol. We previously showed that gPr80(gag) facilitates efficient M-MuLV release through lipid rafts. In this report, we found that the unique N-terminal domain of gPr80(gag) is sufficient to facilitate enhanced M-MuLV particle release from transfected 293T cells. A search for cellular proteins involved in gPr80(gag) function led to cellular La protein. Overexpression of mouse or human La enhanced M-MuLV particle release in the absence of glyco-gag, and the released virus had a reduced buoyant density characteristic of increased cholesterol content. Moreover, small interfering RNA (siRNA) knockdown of human La abolished glyco-gag enhancement of M-MuLV release. These results implicate La as a cellular protein involved in M-MuLV glyco-gag function. We also found that overexpression of mouse or human La could enhance HIV-1 release in the absence of gPr80(gag). Therefore, M-MuLV and HIV-1 may share a pathway for release through lipid rafts involving La.
mBio 01/2011; 2(1):e00341-10. · 5.31 Impact Factor
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ABSTRACT: Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia and HTLV-1-associated myelo-pathy/tropical spastic paraparesis. HTLV-1 is mainly transmitted through blood transfusion and breastfeeding, but viral proliferation in the body in vivo shortly after transmission is not well understood. To investigate whether the route of infection influences the early stages of viral proliferation, we inoculated BALB/c mice with MT-2 cells, an HTLV-1-producing human T-cell line, via different routes, and evaluated the proviral load and humoral immune responses. One month after infection, the provirus was detected in most organs of the mice infected intraperitoneally, and substantial proviral loads were detected in the peripheral blood and secondary lymphoid organs. In contrast, the mice infected intravenously and orally showed low proviral loads, and the provirus distribution was limited to the spinal cord among the intravenously inoculated mice and to the liver among the perorally inoculated mice. Mice infected intraperitoneally also exhibited higher interleukin-2 production than the mice infected intravenously or orally, or than the uninfected control mice, while anti-HTLV-1 antibody titers were comparable between the mice infected intraperitoneally and intravenously. These results demonstrate that the route of primary HTLV-1 infection influences the establishment of HTLV-1-infected cell proliferation and the cell reservoir in mice.
Experimental and therapeutic medicine 01/2011; 2(1):89-93.
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ABSTRACT: Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65(gag), the precursor for internal structural (Gag) proteins. Whereas gPr80gag is evolutionarily conserved among gammaretroviruses, its mechanism of action has been unclear, although it facilitates virus production at a late assembly or release step. Here, it is shown that gPr80gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular, gPr80gag-facilitated release occurs through lipid rafts, because gPr80gag-negative M-MuLV has a lower cholesterol content, is less sensitive to inhibition of release by the cholesterol-depleting agent MbetaCD, and there is less Pr65gag associated with detergent-resistant membranes in mutant-infected cells. gPr80gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.
Proceedings of the National Academy of Sciences 01/2010; 107(3):1190-5. · 9.68 Impact Factor
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ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is characterized by monoclonal proliferation of tumor cells that harbor integrated human T-cell leukemia virus type-1 (HTLV-1). These malignant cells accumulate in various organs including the liver, spleen and skin in addition to blood and lymph nodes. Although there have been several reports of animal models of HTLV-1 infection in which proviral distribution has been examined, clonal expansion of the experimentally infected host cells has not been extensively analyzed. Here we provide experimental evidence that clonal proliferation of the infected host cells occurs in the spleen for more than one year. During a 15 month period of persistent infection, two out of ten mice developed spontaneous tumors. Although the tumors were not ATL-like, cells exhibiting mono- or oligoclonal proliferation and having the same site of HTLV-1 integration were identified in tumor tissues as well as in the spleen. Quantitative analysis of the cells belonging to each cell clone suggested that these proliferating cell clones were associated with the tumors and that spontaneous tumor tissues might provide a suitable microenvironment for proliferation and accumulation of infected cell clones at the late stage of infection.
International Journal of Oncology 11/2009; 35(4):701-7. · 2.40 Impact Factor
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ABSTRACT: Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (DeltaGP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Reverse transcriptase-PCR cloning and sequencing identified alternate internal splicing events in the 5' end of JSRV env that could signify analogous doubly spliced Rej mRNAs, and cDNA clones expressing two of them also showed Rej activity. The predicted Rej proteins contain motifs similar to those found in MMTV Rem and other analogous retroviral regulatory proteins. Interestingly, in most cell lines, JSRV expression plasmids with Rej deleted showed normal transport of unspliced JSRV RNA to the cytoplasm; however, in 293T cells Rej modestly enhanced export of unspliced viral RNA (2.8-fold). Metabolic labeling experiments with [(35)S]methionine indicated that JSRV Rej is required for the synthesis of viral Gag polyprotein. Thus, in most cell lines, the predominant function of Rej is to facilitate translation of unspliced viral mRNA.
Journal of Virology 09/2009; 83(23):12483-98. · 5.40 Impact Factor
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ABSTRACT: Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5' end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3' end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.
Journal of Virology 09/2009; 83(23):12499-511. · 5.40 Impact Factor
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Takayuki Nitta,
Masakazu Tanaka,
Binlian Sun,
Eiji Sugihara,
Mako Kimura,
Yuhei Kamada,
Hideto Takahashi,
Shuji Hanai,
Shi-Wen Jiang,
Jun – Ichi Fujisawa,
Masanao Miwa
Cancer Science 05/2008; 99(5):872-878. · 3.33 Impact Factor
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ABSTRACT: 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.
Cancer Science 09/2006; · 3.33 Impact Factor
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Eiji Sugihara,
Masayuki Kanai,
Soichiro Saito, Takayuki Nitta,
Hideo Toyoshima,
Keiko Nakayama,
Keiichi I Nakayama,
Kenji Fukasawa,
Manfred Schwab,
Hideyuki Saya,
Masanao Miwa
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ABSTRACT: The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21Cip1 and p27Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after gamma-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27-/- and p27+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability.
Cancer Research 05/2006; 66(8):4020-9. · 7.86 Impact Factor
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ABSTRACT: To establish and characterize a new cholangiocarcinoma cell line from a patient living in the Opisthorchis viverrini (O. viverrini) endemic area of Northeast Thailand.
Fresh liver biopsy and bile specimens were obtained from a 65-year-old Thai woman with cholangiocarcinoma of the porta hepatis. After digestion, the cells were cultured in Ham's F12 media. The established cell line was then characterized for growth kinetics, cell morphology, imm-unocytochemistry and cytogenetics. Tumorigenicity of the cell line was determined by heterotransplanting in nude mice.
The primary tumor was a poorly differentiated tubular adenocarcinoma. Examination of the bile revealed malignant cells with O. viverrini eggs. The cholangioc-arcinoma cell line KKU-100 was established 4 mo after the primary culture-population doubling time was 72 h. KKU-100 possesses compact and polygonal-shaped epithelial cells. Immunocytochemically, this cell line exhibited cytokeratin, EMA, CEA, and CA125, but not alpha-fetoprotein (AFP), CA19-9, desmin, c-met, or p53. Such protein expressions parallel those of the primary tumor. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 78 and marked chromosomal structural changes. Inoculation of KKU-100 cells into nude mice produced a transplantable, poorly differentiated aden-ocarcinoma, similar to the original tumor.
KKU-100 is the first egg-proven, Opisthorchis-associated cholangiocarcinoma cell line, which should prove useful for further investigations of the tumor biology of this cancer.
World Journal of Gastroenterology 07/2005; 11(22):3392-7. · 2.47 Impact Factor
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Shuji Hanai, Takayuki Nitta,
Momoko Shoda,
Masakazu Tanaka,
Naomi Iso,
Izuru Mizoguchi,
Shinji Yashiki,
Shunro Sonoda,
Yuichi Hasegawa,
Toshiro Nagasawa,
Masanao Miwa
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ABSTRACT: Adult T-cell leukemia (ATL) occurs after a long latent period of persistent infection by human T-cell leukemia virus type 1 (HTLV-1). However, the mechanism of oncogenesis by HTLV-1 remains to be clarified. It was reported that the incidence curve of ATL versus age was consistent with a multistage carcinogenesis model. Although HTLV-1 is an oncogenic retrovirus, a mechanism of carcinogenesis in ATL by insertional mutagenesis as one step during multistage carcinogenesis has not been considered thus far, because the exact integration sites on the chromosome have not been analyzed. Here we determined the precise HTLV-1 integration sites on the human chromosome, by taking advantage of the recently available human genome database. We isolated 25 integration sites of HTLV-1 from 23 cases of ATL. Interestingly, 13 (52%) of the integration sites were within genes, a rate significantly higher than that expected in the case of random integration (P = 0.043, chi(2) test). These results suggest that preferential integration into genes at the first infection is a characteristic of HTLV-1. However considering that some of the genes are related to the regulation of cell growth, the integration of HTLV-1 into or near growth-related genes might contribute to the clonal selection of HTLV-1-infected cells during multistage carcinogenesis of ATL.
Cancer Science 05/2004; 95(4):306-10. · 3.33 Impact Factor
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Upama Liengswangwong, Takayuki Nitta,
Hironobu Kashiwagi,
Hiroko Kikukawa,
Toru Kawamoto,
Takeshi Todoroki,
Kazuhiko Uchida,
Thiravud Khuhaprema,
Anant Karalak,
Petcharin Srivatanakul,
Masanao Miwa
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ABSTRACT: The liver fluke infection-associated intrahepatic cholangiocarcinoma (ICC) is a major liver cancer in Northeast Thailand. The molecular basis of this ICC is poorly understood. To address possible roles of the DNA mismatch repair (MMR) system in ICC carcinogenesis, a fluorescence-labeling PCR/laser scanning technique with high sensitivity was employed to analyze genomic instability in the nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in 24 fresh and 13 formalin-fixed, paraffin-embedded tissues of ICC and their corresponding normal parts. Microsatellite instability (MSI) was assessed in nDNA, using 12 highly polymorphic loci including 5 Bethesda markers. These loci were mainly related to major MMR genes, hMSH2 and hMLH1. Also 3 (C)n and/or (C)n(A)n repeat instability at 1 noncoding region in the displacement-loop (D-loop) and 2 coding sequences in NADH dehydrogenase subunit 1 and subunit 5 gene in mtDNA were analyzed. MSI was only detected in 1 (2.7%), 6 (16.7%), 1 (2.9%), 1 (2.9%) or 2 (6.3%) out of 37, 36, 35, 35 or 32 cases at BAT-25, D2S123, D3S1611, D11S904 or D17S250, respectively. LOH was found at D3S1298, D3S1561, D5S346 and TP53 in 4 (18.2%) out of 22, 2 (18.2%) out of 11, 6 (33.3%) out of 18 and 3 (12.5%) out of 24 informative cases, respectively. In mtDNA, none except a single case out of the 37 (2.7%) exhibited repeat sequence instability in the D-loop. We conclude that the liver fluke infection-associated ICC in Thailand is classified as low frequency MSI or microsatellite stable type and that DNA MMR system, through hMSH2 and hMLH1 gene mutations, does not play a major role in its carcinogenesis.
International Journal of Cancer 12/2003; 107(3):375-80. · 5.44 Impact Factor
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ABSTRACT: Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked with HTLV-1-associated diseases. HTLV-1 proviral load is higher in persons with adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers. However there are little data available on the factors controlling HTLV-1 proviral load in carriers. To study the effect of genetic background on HTLV-1 proviral load, we employed a mouse model of HTLV-1 infection that we had established. Here we analyzed nine strains of mice and found there is a great variation of proviral load among mouse strains that is not necessarily dependent on major histocompatibility complex. The antibody response is also different among these strains. To our knowledge, this is the first demonstration of the importance of the genetic background other than major histocompatibility complex controlling the HTLV-1 proviral load.
Biochemical and Biophysical Research Communications 10/2003; 309(1):161-5. · 2.48 Impact Factor
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ABSTRACT: Human T-cell leukemia virus type 1 (HTLV-1) is an etiologic agent of adult T-cell leukemia / lymphoma and other HTLV-1-associated diseases. However, the interaction between HTLV-1 and T cells in the pathogenesis of these diseases is poorly understood. Mouse cells have been reported to be resistant to cell-free HTLV-1 infection. However, we recently reported that HTLV-1 DNA could be observed 24 h after cell-free HTLV-1 infection of mouse cell lines. To understand HTLV-1 replication in these cells in detail, we concentrated the virus produced from c77 feline kidney cell line and established an efficient infection system. The amounts of adsorption of HTLV-1 are larger in mouse T cell lines, EL4 and RLm1, than those in human T cell lines, Molt4 and HUT78, and are similar to that in human kidney cell line, 293T. Unexpectedly, however, the amounts of entry of HTLV-1 are about 10-fold larger in the two mouse cell lines than those in the three human cell lines employed. Moreover, viral DNA was detectable from 1 h in EL4 and RLm1 cells, but only from 2 - 3 h in 293T, Molt4 and HUT78 cells. However, the amount of viral DNA in EL4 cells became smaller than that in Molt4 cells. HTLV-1 expression could be detected until day 1 - 2 in RLm1 and EL4 cells, and until day 4 in Molt4 cells. Our results suggest that mouse cell experiments would give useful information to dissect the early steps of cell-free HTLV-1 infection.
Japanese journal of cancer research: Gann 08/2002; 93(7):760-6.
