Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

Abstract. 3-Methyladenine (3-MA) inhibits class III phosphoinositide 3-kinase (PI3K) and is widely used as an inhibitor of autophagy. 3-MA has also been shown to stimulate cell death of tumor cells under nutrient-starved conditions by inhibiting autophagy. To explore the possibility of this type of autophagy inhibitors as anticancer drugs, we examined the effects of 3-MA on the phenotypes of highly metastatic human fibrosarcoma HT1080 cells. We report here that although 3-MA did not markedly affect cell survival of the cells under either normal or amino acid-starved conditions, it strongly inhibited the invasiveness of the cells. 3-MA rapidly suppressed actin rich membrane ruffle and/or lamellipodia formation under normal conditions, leading to inhibition of cell migration and invasion of the cells without substantial inhibitions of small GTPase Rac activity and the production of matrix metalloproteinases MMP-2 and MMP-9. 3-MA abolished class I and class II PI3Ks in in vitro lipid kinase assays, and suppressed cell motility of the cells more strongly than the other PI3K inhibitors wortmannin and LY294002. Downregulation of Beclin 1, a protein required for autophagic body formation, by transfection of Beclin 1 siRNA did not inhibit membrane ruffle formation and cell migration. These results suggest that 3-MA suppresses the invasion of HT1080 cells, independently of autophagy inhibition, through inhibition of type I and II PI3Ks and possibly other molecules.

By using an assay system based on a human hepatoblastoma cell line (HB611) that continuously synthesizes hepatitis B virus DNA, the following compounds were found to inhibit hepatitis B virus DNA synthesis at concentrations that were significantly lower than their minimum cytotoxic concentrations: 9-(2-phosphonylmethoxyethyl)-2,6-dianiinopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, 9-(phosphonylmethoxyethyl)adenine, 2',3'-dideoxy-2',3'-didehydrocytidine, and 2',3'-dideoxycytidine. The most potent compound was PMEDAP (50% effective concentration, 0.02 pg/ml). The Recently, a number of assay systems have been described which seem suitable for evaluation of the inhibitory effects of antiviral compounds against hepadnaviruses, including HBV and duck hepatitis B virus (DHBV) (5, 9, 11, 13, 15). DHBV is able to propagate in primary duck hepatocytes in vitro (12). Selective inhibition of the synthesis of DHBV DNA and core antigen in primary duck hepatocytes can be used as a marker for anti-DHBV activity. Using this assay system, we examined a variety of purine and pyrimidine nucleoside analogs for their inhibitory effects on DHBV replication (15).Ueda et al. (13) have recently described a human hepatoblastoma cell line (HB611) that contains an integrated HBV genome and that accumulates a significant amount of HBV DNA replicative intermediates. Some of these DNA molecules are packed and released as Dane particles into the cell culture medium. Selective inhibition of the synthesis of these replicative intermediates could serve as a marker for anti-HBV activity. Using the HB611 cell line, we examined the inhibitory effects of selected antiviral compounds on HBV DNA synthesis. The compounds which we evaluated for their inhibitory effect on HBV DNA synthesis were previously examined for their inhibitory effects on DHBV DNA and core antigen synthesis in primary duck hepatocytes (15).The compounds fell into two categories: phosphonylmethoxyalkylpurines and -pyrimidines and 2',3'-dideoxynucleoside analogs. Their origins were as follows. Test compounds were assayed by a modification of the method described by Ueda et al. (13). The HB611 cell line was generously provided by K. Matsubara, Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan. HB611 cells were seeded in tissue culture trays (diameter, 35 mm) at a density of 5 x 104 cells per well by using Dulbecco modified minimum essential medium supplemented with 10% fetal bovine serum, 100 ,ug of streptomycin per ml, 100 IU of penicillin G per ml, and 0.2 mg of Geneticin R (GIBCO) per ml. After 2 days of incubation, when the cells were grown to confluency, the growth medium was withdrawn. Then, the cells were incubated for 12 days, during which time the medium containing the compounds was exchanged every 3 days. The cells were then harvested and total DNA was prepared upon lysis of the cells in 0.5 ml of 10 mM Tris hydrochloride (pH 7.8)-S5 mM EDTA-150 mM NaCI-1% sodium dodecyl sulfate-0.1 mg of proteinase K (Sigma) per ml. After...

Duck hepatitis B virus (DHBV) replication in primary duck hepatocytes was monitored by examining the synthesis of both DHBV DNA and DHBV core antigen. Several nucleoside analogs which were previously shown to inhibit the replication of DNA viruses (i.e., herpesviruses) and retroviruses were examined for their inhibitory effects on the synthesis of DHBV core antigen in primary duck hepatocytes. (S)-9-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine, 2',3'-dideoxyadenosine, and 2',3'-dideoxycytidine inhibited DHBV core antigen synthesis at concentrations that were significantly lower than those found to be toxic to the primary hepatocytes. Of all the compounds tested, (S)-HPMPA showed the lowest 50% effective concentration (0.5 ,ug/ml). The selectivity index or ratio of the 50% cytotoxic concentration to the 50% effective concentration of (S)-HPMPA was greater than 300. (S)-HPMPA not only inhibited DHBV core antigen but also DHBV DNA synthesis in DHBV-infected hepatocytes.Human hepatitis B virus (HBV) is a causative agent of both an acute and chronic form of hepatitis. More than 200 million people worldwide are estimated to be chronic carriers of HBV. HBV belongs to the family Hepadnaviridae, which also includes woodchuck hepatitis virus, ground squirrel hepatitis virus, tree squirrel hepatitis virus, and duck hepatitis B virus (DHBV) (3,(7)(8)(9)(10)(11)(12)(13)(14)19). All members of the hepadnavirus family have a number of characteristics in common such as morphological appearance, antigenic makeup, and DNA size and structure (3,(7)(8)(9)(10)(11)(12)(13)(14)19). Also, the pathological findings following infection with these viruses are quite similar (8,20). The replication of the hepadnaviruses (4, 10) is dependent on the reverse transcription of an RNA intermediate. This results in the formation of a minusstranded DNA, which then serves as a template for the synthesis of plus-stranded DNA.DHBV is able to propagate in primary duck hepatocytes in vitro. Tuttleman et al. (21) reported that upon infection of primary duck hepatocytes with DHBV, viral DNA is synthesized, virus-specific proteins are produced, and infectious virus is released into the culture medium. Using immunofluorescence, they demonstrated the production of both core and surface DHBV antigens in primary duck hepatocytes that were infected with DHBV. This in vitro system should prove suitable to evaluate the effects of antiviral drugs against DHBV. In this study, we examined a wide variety of such drugs that belong to the class of the purine and pyrimidine nucleoside analogs for their inhibitory effects on replication of DHBV in primary duck hepatocytes in vitro. was from ICN Nutritional Biochemicals, Cleveland, Ohio; 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), 2',3'-dideoxyuridine (DDU), 2',3'-dideoxyadenosine (DDA), 2',3'-dideoxycytidine (DDC), 3'-deoxyarabinofuranosylcytidine (D-ara-C), 3'-azido-2',3'-dideoxythymidine (AZT), 3'-azido-2',3'-dideoxyuridine (AZU), acyclovir [9-(2-hydroxyeth...

DNA polymerase θ θ θ θ (Polθ θ θ θ ) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Polθ θ θ θ in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Polθ θ θ θ polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, γ γ γ γ -irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Polθ θ θ θ is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Polθ θ θ θ in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross-links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes.

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HTLV-I seroprevalences of 3.63% (02/55), 12.19% (10/82) and 13.88% (10/72) were demonstrated among Tiryio, Mekranoiti and Xicrin Amazonian Indians, respectively, by the Western blotting enzyme assay (WBEI). By indirect immuno electron microscopy (IIEM), 2 Tiriyo, 9 Mekranoiti and 6 Xicrin Amerindians were reactive. Of 44 serum samples from Japanese immigrants, none reacted by any of the techniques before mentioned. One, 8 and 6 serum samples from Tiryio, Mekranoiti and Xicrin Indians, respectively, were both WBEI and IIEM positive. Our results strongly suggest that HTLV-I and/or an HTLV-I antigenic variant circulate (s) among populations living in the Amazon region of Brazil.

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