The Saccharomyces cerevisiae RAD9, RAD17 and RAD24 genes are required for suppression of mutagenic post-replicative repair during chronic DNA damage.

In Saccharomyces cerevisiae, a DNA damage checkpoint in the S-phase is responsible for delaying DNA replication in response to genotoxic stress. This pathway is partially regulated by the checkpoint proteins Rad9, Rad17 and Rad24. Here, we describe a novel hypermutable phenotype for rad9Delta, rad17Delta and rad24Delta cells in response to a chronic 0.01% dose of the DNA alkylating agent MMS. We report that this hypermutability results from DNA damage introduction during the S-phase and is dependent on a functional translesion synthesis pathway. In addition, we performed a genetic screen for interactions with rad9Delta that confer sensitivity to 0.01% MMS. We report and quantify 25 genetic interactions with rad9Delta, many of which involve the post-replication repair machinery. From these data, we conclude that defects in S-phase checkpoint regulation lead to increased reliance on mutagenic translesion synthesis, and we describe a novel role for members of the S-phase DNA damage checkpoint in suppressing mutagenic post-replicative repair in response to sublethal MMS treatment.

Quality control metrics for LC-MS feature detection tools demonstrated on Saccharomyces cerevisiae proteomic profiles.

Quantitative proteomic profiling using liquid chromatography-mass spectrometry is emerging as an important tool for biomarker discovery, prompting development of algorithms for high-throughput peptide feature detection in complex samples. However, neither annotated standard data sets nor quality control metrics currently exist for assessing the validity of feature detection algorithms. We propose a quality control metric, Mass Deviance, for assessing the accuracy of feature detection tools. Because the Mass Deviance metric is derived from the natural distribution of peptide masses, it is machine- and proteome-independent and enables assessment of feature detection tools in the absence of completely annotated data sets. We validate the use of Mass Deviance with a second, independent metric that is based on isotopic distributions, demonstrating that we can use Mass Deviance to identify aberrant features with high accuracy. We then demonstrate the use of independent metrics in tandem as a robust way to evaluate the performance of peptide feature detection algorithms. This work is done on complex LC-MS profiles of Saccharomyces cerevisiae which present a significant challenge to peptide feature detection algorithms.

Multigene real-time PCR detection of circulating tumor cells in peripheral blood of lung cancer patients.

BACKGROUND: CLCA2, HMGB3, L587S and ASH1 were identified in lung cancer tissues using genetic subtraction, microarray and quantitative PCR, and found to be specific and complementary for detection of non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). MATERIALS AND METHODS: A real-time RT-PCR assay, simultaneously detecting four genes, was developed and tested on lung cancer specimens. RESULTS: Twenty-two out of 24 adenocarcinomas, 18/18 squamous, 4/5 large cell, 2/2 small cell and 2/2 bronchoalveolar/neuroendocrine cancer tissue samples tested positive. Specificity was demonstrated by evaluation of 194 other tumor and corresponding normal tissues. Circulating tumor cells in the peripheral blood of 49/108 lung cancer patient samples tested positive, and general correlations of multigene expression signals to disease status were observed. Changes in multigene expression during treatment and disease recurrence in individual patients could be detected. CONCLUSION: These data indicate the diagnostic and prognostic utility of a multigene real-time RT-PCR assay to detect tumor cells in the peripheral blood of lung cancer patients.

Characterization of a proapoptotic antiganglioside GM2 monoclonal antibody and evaluation of its therapeutic effect on melanoma and small cell lung carcinoma xenografts.

Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.

Identification and characterization of L985P, a CD20 related family member over-expressed in small cell lung carcinoma.

We recently reported on the use of cDNA subtraction combined with microarray based expression analysis for identifying genes that are differentially over-expressed in small cell lung carcinoma. One of the several hundred genes identified using this approach was termed L985P and its molecular characterization is described in this report. The differential over-expression of L985P mRNA in SCLC, as determined by microarray analysis, was confirmed by real-time RT-PCR and Northern blot analysis. Immunohistochemical analyses show that L985P protein is highly expressed in SCLC with very restricted expression observed in normal lung, which was confined to the apical region of the ciliated bronchiolar epithelium. Flow cytometric and immunohistochemical analysis showed that L985P was localized to the cell surface. Sequence homology comparison indicated that L985P is identical to MS4A8B, a member of the recently described membrane-spanning 4-domain family, subfamily A (MS4A) gene family. The MS4A gene family currently consists of greater than 20 distinct human and mouse proteins that include CD20 and FcepsilonRIbeta. Both CD20 and FcepsilonRIbeta are involved in signaling events that regulate diverse cellular functions including cell growth regulation and differentiation. Collectively, the results presented herein demonstrate that L985P is differentially over-expressed in SCLC and may have potential clinical utility as an immunotherapeutic target for the treatment of SCLC.

Molecular and immunological evaluation of the transcription factor SOX-4 as a lung tumor vaccine antigen.

The developmental transcription factor SOX-4 has been shown to be highly and differentially overexpressed in primary small cell lung carcinomas (SCLC). To examine the potential of SOX-4 for broad use as a lung cancer vaccine, we have evaluated the expression of SOX-4 in a panel of primary adenocarcinoma, squamous, and large cell tumor samples as well as in a panel of established small cell and non-small cell lung carcinoma tumor cell lines. SOX-4 mRNA is shown to be overexpressed in a substantial fraction of each of these lung tumor types. To examine the immunological potential of SOX-4, we have evaluated the presence of SOX-4-specific CD4 and CD8 T cells in PBMC of healthy donors and the presence of SOX4-specific Abs in sera from SCLC patients. We demonstrate the presence of both CD4 and CD8 T cells that recognize naturally processed epitopes derived from SOX-4 as well as the presence of SOX-4-specific Abs in sera from SCLC patients, but not in sera from healthy donors. The lung tumor-specific overexpression and demonstration of a comprehensive Ag-specific immune response specific for SOX-4 support the use of this molecule in the development of whole gene-, peptide-, or protein-based vaccination strategies against lung cancer. Furthermore, the identification of naturally processed T cell and Ab epitopes from SOX-4 provides valuable tools for the development of peptide-based vaccination strategies against lung cancer as well as to monitor SOX-4-specific responses in vaccinated patients.

Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis.

To identify genes that are differentially over-expressed in Small Cell Lung Carcinoma (SCLC) we have used a combination of suppression subtractive hybridization and cDNA microarray to analyse the expression profiles of 2400 cDNAs clones. Genes that are over-expressed in SCLC were identified using 32 pairs of fluorescence-labeled cDNA samples representing various lung tumors and normal tissues. This comprehensive approach has resulted in the identification of 209 genes that are differentially over-expressed in SCLC. Quantitative real-time PCR was used to further validate the expression of 43 genes in SCLC tumors and various normal tissues. Discussed in this report are nine genes, which showed the most promising SCLC tumor to normal tissue differential expression profiles, including seven known and two novel genes. The large number of differentially expressed genes identified from this analysis and the characterization of these genes will provide valuable information in better understanding the biology of SCLC and help us in developing these gene products as potential targets for diagnostic as well as therapeutic usage.