We have identi®ed a novel protein, BAP1, which binds to the RING ®nger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclearlocalized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING ®nger, but not to germline mutants of the BRCA1-RING ®nger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially coexpressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozgyous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the ®rst nuclearlocalized ubiquitin carboxy-terminal hydrolase to be identi®ed. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.
TSG101 is a candidate tumor suppressor gene whose deletion in NIH3T3 cells leads to spontaneous lung metastases in nude mice. Aberrant transcripts of TSG101 have been identi®ed in 47% of primary breast carcinomas, without evidence of intragenic deletions at the TSG101 locus on 11p15. To investigate the possible role of TSG101 in lung cancer, which often shows 11p allele loss, we performed transcript analysis and mutational analysis of TSG101 in lung cancer cell lines. Reverse transcriptase RT ± PCR and Northern analysis detected a common TSG101 transcript, shortened because of an internal deletion, which was expressed simultaneously with the wild-type transcript in 89% of small cell lung cancer (SCLC) lines. In contrast, the wild-type transcript was expressed alone in normal tissues, primary non-small cell lung cancer (NSCLC) specimens, and the majority of NSCLC cell lines. Sequence of the shortened SCLC transcript was identical to that of the most common aberrant transcript identi®ed in breast cancer, consisting of a deletion of exons 2 ± 4 and part of 1 and 5. Southern analysis of SCLC lines expressing the shortened transcript did not detect any intragenic deletions. Single strand conformational polymorphism (SSCP) analysis and direct sequencing of TSG101 cDNAs also identi®ed no mutations or deletions. These results suggest that TSG101 is not mutated in lung cancer but that aberrant splicing of TSG101 occurs in SCLC.
Only Listeria monocytogenes that produce listeriolysin O (LLO) elicit protective immunity. Given the importance of tumor necrosis factor alpha (TNF-alpha) in anti-Listeria immunity, we have investigated TNF-alpha production by macrophages after they ingested live LLO-producing compared to LLO-non-producing bacteria. We used two genetically engineered strains of Listeria that differed only in their ability (Ly+) or inability (Ly-) to produce LLO. Ly+ and Ly- caused the same kinetics of increased mRNA abundance for TNF-alpha during the first 90 min after phagocytosis. However, only Ly+ caused sustained transcription of TNF-alpha mRNA, and this may account for the increased release of TNF-alpha. The transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) prevented the sustained abundance of cytokine mRNA 20 h after ingestion of Ly+. In addition, nuclear run-on assays indicated sustained transcription of TNF-alpha genes only after ingestion of Ly+. LLO itself was not responsible for the ability of Ly+ to stimulate the sustained transcription of the TNF-alpha genes. Instead, LLO may allow Listeria to survive within macrophages so that other bacterial products cause sustained TNF-alpha gene transcription. Both Ly+ and Ly- produced molecules, isolated by 50% ammonium sulfate, that induced cytokine production. In conclusion, we now report that Ly+ causes sustained transcription of the TNF-alpha gene and production of TNF-alpha by macrophages in vitro. We speculate that the TNF-alpha may activate endothelium and thus allow the recruitment of T cells to sites of infection. This may contribute to the ability of only LLO-producing Listeria to induce protective immunity.
Listeria monocytogenes is an intracellular bacterial pathogen. A single gene product, listeriolysin (LLO), is critical for the induction of protective immunity. We now show that listeria that produce functional LLO augment Ia expression by macrophages and are better presented to a Th1, CD4+ anti-listeria T cell line. We used two genetically engineered strains of listeria which differed only in their ability (Ly+) or inability (Ly-) to produce functional LLO. Ia-negative murine macrophages ingested either Ly+ or Ly-, and then were stimulated by interferon-gamma (IFN-gamma). Increasing numbers of live Ly+, but not Ly-, augmented IFN-gamma-induced Ia expression. Ly+ by itself did not induce Ia expression. Heat-killed Ly+ and Ly- did not augment IFN-gamma-induced Ia expression. The abundance of Ia on the macrophage cell surface is one major determinant of antigen presentation to CD4+ T cells. Consistent with their ability to augment la expression, Ly+ were better presented than Ly- to a CD4+, Th1, anti-listeria T cell line. When macrophages and T cells were from different inbred mouse strains, antigen presentation required identity at the class II region of the MHC gene complex. This indicated that antigen presentation occurred via Ia molecules. The increased ability of macrophages to present Ly+ is a product of the macrophage-listeria interaction, not a property of the T cell tine 86. If Ia-negative macrophages ingested Listeria and were then stimulated by IFN-gamma, Ly+ was presented more efficiently than Ly-. On the other hand, if Ia-positive macrophages ingested Listeria, then Ly+ and Ly- were presented equally well to T cells. Altogether our data is consistent with the hypothesis that macrophages interact differently with Ly+, and that this contributes to the ability of only live Ly+ to induce protective immunity.
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