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Prostate cancer (PCa) is the most common invasive malignancy for men in the USA. The incidence and mortality rates of PCa are significantly higher among African American men, as compared to those in Caucasian men. Betulinic acid (BA) is a penta-cyclic triterpenoid that is often found in the bark of several species of plants. It possesses a variety of biological activities, including anti-cancer activities. We examined the cytotoxic effects and endoplasmic reticulum (ER) stress induced by BA and its ionic derivatives with PCa cells derived from African Americans and Caucasian men. The viability of all PCa cells was reduced by the BA compounds, and the cytotoxicity of these BA compounds was independent of ethnicity and androgen dependency. The BA compounds induced modest effects on ER stress proteins when compared with ER stress inducers, tunicamycin and thapsigargin. The induction of glucose regulated protein 78 (GRP78) was largely correlated with the expression of C/EBP homologous protein (CHOP) and cleaved poly [ADP-ribose] polymerase (PARP)/caspase-3 in the PCa cells. In summary, our data demonstrated that BA compounds impaired the growth of PCa cells regardless of ethnicity – through GRP78- and CHOP-independent pathways.

BackgroundKoala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag.ResultsIn this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results.ConclusionsThese results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction.

AbstractBackgroundOne of the unique features of gammaretroviruses is that they contain an additional extended form of Gag, glyco-gag, which initiates in the leader sequence. MuLV glyco-gag, gPr80Gag, promotes retrovirus replication and disease progression. Although virtually all infectious MuLVs encode glyco-gag, XMRV (xenotropic murine leukemia virus-related virus) lacks the classical gPr80Gag sequence. We examined XMRV to determine if its leader sequence contains glyco-gag activity, whether the presence of conventional gPr80Gag affects replication of XMRV, and we describe the evolution of glyco-gag-deficient MuLVs in Mus.ResultsWe introduced several mutations disrupting two putative but noncanonical glyco-gag proteins in the leader sequence region in XMRV and found that those mutations did not affect virus release nor susceptibility to the antiviral activity of hA3G (human APOBEC3G). A chimeric XMRV encoding the Moloney MuLV (M-MuLV) leader sequence (MXMRV) demonstrated that M-MuLV glyco-gag facilitated MXMRV release and increased infectivity. Infectivity assays with several cell lines showed that glyco-gag increases XMRV infectivity in all cell lines tested, but the level of this increase varies in different cell lines. Because MuLV glyco-gag counteracts mouse APOBEC3, we investigated whether M-MuLV glyco-gag enhances XMRV infection by counteracting human APOBEC3. Comparison of hAPOBEC3 isoforms expressed in different cell lines indicated that hA3B was the most likely candidate for a restrictive hA3. However over-expression of hA3B showed no enhanced restriction of infection by XMRV compared to MXMRV. Endogenous MuLVs in the sequenced mouse genome were screened for canonical glyco-gag, which was identified in two clades of xenotropic MuLVs (X-MuLVs) and ecotropic MuLVs, but not in other X-MuLVs or in any polytropic MuLVs.ConclusionsM-MuLV glyco-gag facilitates XMRV replication, and the leader sequence region in XMRV does not encode proteins equivalent to M-MuLV glyco-gag. The fact that the ability of glyco-gag to enhance XMRV infection varies in different cell lines suggests a glyco-gag sensitive restrictive factor that further reduces XMRV infectivity. The M-MuLV glyco-gag enhancement for XMRV replication is through a hAPOBEC3 independent mechanism. The absence of glyco-gag in MuLVs carried by western European mice suggests that loss of this sequence is a relatively recent event with limited subspecies distribution.

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)). gPr80gag 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. IMPORTANCE Retroviruses cause diseases such as leukemia and AIDS. An important aspect of viral replication is how viruses are released from infected cells. We previously found that a unique protein encoded by murine leukemia viruses (MuLVs), glyco-gag (or gPr80(gag)), enhances efficient virus release through cholesterol-rich membrane subdomains called lipid rafts. In this study, we found that the N-terminal domain of gPr80(gag) is sufficient to enhance viral release. A search for cellular proteins that participate in gPr80(gag) function led to cellular La protein. Overexpression of La phenocopied glyco-gag in enhancing M-MuLV release, and knockdown of La abolished glyco-gag function. M-MuLV glyco-gag also enhanced release of HIV-1, as did overexpression La in the absence of glyco-gag. Thus, M-MuLV and HIV-1 may share a cellular pathway for release through lipid rafts involving La. These results may also be relevant for other viruses that are released through lipid rafts.

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.

Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80gag or glyco-gag. Translation of this protein is initiated from fulllength viral mRNA at an upstream initiation site in the same reading frame as pr65gag, 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 MβCD, 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. [ABSTRACT FROM AUTHOR]

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.

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.

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory disorders. Aberrant activation of nuclear factor-kappaB (NF-kappaB) has been linked to HTLV-1 pathogenesis and to various kinds of cancers, including adult T-cell leukemia. NF-kappaB-inducing kinase (NIK) is critical for non-canonical activation of NF-kappaB and for the development of lymphoid organs. HTLV-1 activates NF-kappaB by the non-canonical pathway, but examination of the role of NIK in proliferation of HTLV-1-infected cells in vivo has been hindered by lack of a suitable animal model. Alymphoplasia (aly/aly) mice bear a mutation of NIK, resulting in defects in the development of lymphoid organs and severe deficiencies in both humoral and cell-mediated immunity. In the present study we therefore used a mouse model of HTLV-1 infection with aly/aly mice. The number of HTLV-1-infected cells in the reservoir organs in aly/aly mice was significantly smaller than in the control group 1 month after infection. In addition, aly/aly mice did not maintain provirus for 1 year and antibodies against HTLV-1 were undetectable. These results demonstrate that the absence of functional NIK impairs primary HTLV-1 proliferation and abolishes the maintenance of provirus. Interestingly, clonal proliferation of HTLV-1-infected mouse cells was not detected in aly/aly mice, which is consistent with the lack of HTLV-1 persistence. These observations imply that the clonal proliferation of HTLV-1-infected cells in secondary lymphoid organs might be important for HTLV-1 persistence.

Aim: To establish and characterize a new cholangiocarcinoma cell line from a patient living in the Opisthorchis viverrini (O. viverrini) endemic area of Northeast Thailand., Methods: 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., Results: 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., Conclusion: 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.