The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3-5 exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3 singlestrand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time-and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner. INTRODUCTIONThe MRN complex, consisting of Mre11, Rad50 and NBS1, has diverse functions in DNA damage recognition (Petrini and Stracker, 2003), DNA replication (Costanzo et al., 2001), cell cycle checkpoint activation (Grenon et al., 2001), nonhomolgous end joining (Paull and Gellert, 2000), and telomere maintenance (Wu et al., 2007). The Mre11 complex binds DNA double-strand breaks (DSBs) soon after they are formed implicating it in DNA damage detection . Furthermore, the complex can tether linear duplex molecules (de Jager et al., 2001), and it is able to bridge broken DNA ends or sister chromatids (van den Bosch et al., 2003). Mre11 has 3Ј-5Ј exonuclease activity and endonuclease activity (Paull and Gellert, 1999), suggesting a role in the processing of DNA ends into forms that can be recognized by cell cycle checkpoint and DNA repair proteins (Paull and Gellert, 1999;Lee and Paull, 2005;Jazayeri et al., 2006). However, precise cellular roles of the Mre11 complex have been difficult to establish, because null mutations of all components of the complex are lethal to vertebrate cells (Luo et al., 1999;Yamaguchi-Iwai et al., 1999;Zhu et al., 2001).There are several lines of evidence implicating the MRN complex in DNA replication. The complex associates with chromatin and colocalizes with proliferating cell nuclear antigen (PCNA) throughout S phase (Maser et al., 2001). In addition, chromatin loading of Mre11 is enhanced by fork stalling, suggesting that the complex is loaded at the replication fork (Mirzoeva and Petrini, 2003). Depletion of Mre11 from DT40 or Xenopus leads to increased chromosomal breaks and accumulation of DSBs during DNA replication (Yamaguchi-Iwai et al., 1999;Costanzo et...

Summary For the presentation of peptide antigens to cytotoxic CD8+ T lymphocytes of the immune system, the expression of human leukocyte antigen (HLA) class I molecules on the cell surface is necessary. There is increasing evidence that surface HLA class antigen expression is altered in a variety of human tumours by either loss or down-regulation of these molecules, which may be a strategy for evasion of immunosurveillance by malignant cells. This study has examined the expression of HLA class molecules in head and neck squamous cell carcinoma (HNSCC) specimens by immunohistochemistry, using a wide panel of antibodies directed against allele-specific as well as monomorphic determinants of these molecules. The expression of TAP proteins, HLA-DR and the co-stimulatory molecule ICAM-1 were also studied. In addition, the expression of the tumour-associated antigens (TAA) p53 and MAGE genes was determined. Aberrant allelic expression of HLA class antigens was detected in 17 out of 34 (50%) of the specimens stained, whereas HLA class I expression determined by W6/32 staining was found to be heterogeneous in only 2 out of 34 (6%) cases. Decreased expression of ICAM-1 was observed in 12 out of 34 (35%) tumour specimens and de novo expression of HLA-DR (HLA class 11) by carcinoma cells in 13 out of 34 (38%) cases. Aberrant expression of HLA class I antigens was frequently observed in cases in which MAGE genes and p53 overexpression were detected. The altered expression of these immunomodulatory molecules in HNSCC may affect prognosis and has important implications for peptide-based immunotherapy strategies for these patients.

We vaccinated metastatic melanoma patients with irradiated, autologous melanoma cells genetically engineered to secrete interleukin 2 (IL-2) to investigate whether an anti-tumor immune response would be induced. Melanoma cell cultures were established from surgical specimens and were engineered to secrete IL-2 by infection with recombinant retrovirus. Twelve patients were vaccinated subcutaneously one, two, or three times with approximately 10(7) irradiated, autologous, IL-2-secreting tumor cells. Treatment was well tolerated, with local reactions at 11 of 24 injection sites and minor systemic symptoms of fever and headache after 6 injections. One patient developed anti-tumor DTH after the first vaccination and showed an increased response after the second vaccination. Anti-autologous tumor CTLs could be detected prevaccination in the peripheral blood of seven patients and their activity increased after vaccination in four patients. No UICC-defined clinical responses were seen, but three patients had stable disease for 7-15 months, one of whom has not yet progressed (15+ months). Thus, patient vaccination with autologous, genetically engineered tumor cells is feasible and safe. Anti-tumor DTH and CTLs can be induced in some patients with such a vaccine.

It has previously been reported that MACE-/, -2, -3 and -4 genes are expressed in human cancers including cutaneous melanoma. MAGE-/ and MACE-3 represent targets for specific immunotherapy because they encode peptide antigens which are recognised by cytotoxic T lymphocytes (CTL) when presented by HLA class I molecules, and pilot clinical trials with these peptides are currently in progress. It The MAGE gene family consists of 12 closely related human genes, of which 6, MAGE-I, -2, -3, -4, -6 and -12, are expressed in tumours of different histological types; with the exception of testis and placenta, they are not expressed in normal tissues . Short peptides derived from the processing of MAGE-1 and MAGE-3 gene products are recognised by cytotoxic T lymphocytes (CTL) in the context of HLA class I molecules and therefore represent potential targets for specific immunotherapy of tumours (van der Bruggen et al., 1991Bruggen et al., , 1994a Traversari et al., 1992; Gaugler et al., 1994; Celis et al., 1994).The frequency of expression of MAGE-1, -2, -3 and -4 in cutaneous melanoma is 35, 58,60 and 18%, respectively, with higher proportions of MAGE-positive tumours among metastatic lesions (48, 70, 76 and 22%, respectively) than among primary lesions (16,41,36 and 11%, respectively), suggesting a correlation between tumour progression and incidence of MAGE gene expression . Members of the MAGE gene family are also expressed in breast carcinoma (Brasseur et al., 1992; Russo et al., 1995), non-small cell lung carcinoma (Weynants et ab, 1994; Shichijo et a f , 1995), bladder carcinoma (Patard et al., 1995) and gastric carcinoma (Inoue et al., 1995).The expression of MAGE genes in uveal melanoma tissue has not previously been reported. Uveal melanomas occur with an overall incidence of 5-7/million/year (Shields, 1983); they constitute over 80% of all eye malignancies and are the most common fatal intraocular disease in adults (Egan et al., 1988). Our study has determined the frequency of expression of MAGE-I, -2, -3 and -4 genes in both primary and metastatic uveal melanoma tissue.Peptides processed from the melanocyte lineage-specific proteins tyrosinase, gp100/Pme117 and Melan-A/MART-1, which are present in cutaneous melanoma, have been shown to induce CTLs (Brichard et al., 1993;Bakker et al., 1994;Coulie et al., 1994;Kawakami et al., 1994). Since these antigens may represent additional candidates for peptide immunotherapy of melanoma, the expression of the genes encoding these antigens in uveal melanoma was also assessed in our study. MATERIAL AND METHODS Sample collectionPrimary uveal melanoma tissue was frozen in the vapour phase of liquid nitrogen immediately following enucleation. Metastatic tumour biopsies were frozen in liquid nitrogen immediately after removal (in Sheffield and Brussels) or immediately following pathological dissection (in Lausanne). All samples were subsequently stored at -80°C until use. Samples were collected in the UK (n = 28), Switzerland (n = 16), Belgium (n = 5 ) and France (n = 4). R...

Summary To determine if ovarian cancer patients would be suitable for MAGE-peptide vaccine-based immunotherapy, the frequency of expression of the MAGE-1-4 genes in ovarian tumours was assessed using reverse transcription polymerase chain reaction (RT-PCR) and product verification with digoxigenin-labelled oligonucleotide probes specific for each MAGEgene. In addition, the frequency of expression of more recently discovered tumour antigens (BAGE, GAGE -1, -2 and GAGE -3, -6) was established using RT-PCR and ethidium bromide staining. In this study 1/16 normal ovarian tissue specimens and 11/25 benign lesions expressed MAGE-1. In non-malignant tissue there was preferential expression of MAGE-1 in premenopausal women. A total of 15/27 malignant specimens expressed MAGE-1, including 10/14 serous cystadenocarcinomas. Expression of other tumour antigens was infrequent. The finding of MAGE-1 expression in both benign and malignant tissue questions previous assumptions regarding the role of MAGE genes in carcinogenesis. In addition, preferential MAGE-1 gene expression in non-malignant premenopausal tissue suggests that the MAGE genes may be involved in cellular proliferation as opposed to carcinogenesis or possibly that MAGE gene expression is under cyclical hormonal control. Finally, this study indicates that serous cystadenocarcinomas may be suitable tumours for MAGE-1 peptide immunotherapy.