Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

Dysregulation of Wnt pathway components in human salivary gland tumors.

OBJECTIVES: To determine the expression level of the Wnt components-WIF1 (Wnt inhibitory factor 1), WNT1, and beta-catenin-in salivary gland tumor cells and to investigate the mechanisms that contribute to activation of the Wnt pathway in human salivary gland tumors. DESIGN: The expression of WIF1, WNT1, and beta-catenin in salivary gland normal tissue and tumor cell lines was analyzed by reverse transcription-polymerase chain reaction and Western blot analysis. A relationship between the expression of distinct genes was determined by Pearson correlation. The presence of rearrangements involving WIF1 was evaluated by reverse transcription-polymerase chain reaction analysis. SUBJECTS: Samples were obtained from 6 normal salivary glands and 10 cell lines established from primary benign and malignant salivary gland tumors. RESULTS: The expression of WIF1 was high in normal salivary gland tissue but was significantly down-regulated in all salivary gland tumor cell lines analyzed (P < .001). The WIF1 rearrangements were recurrent but rare in salivary gland tumors. Expression of WNT1 protein was undetectable in normal tissue but readily detectable by Western blot analysis in all salivary gland tumor cell lines. beta-Catenin messenger RNA expression was significantly up-regulated in salivary gland tumor cells. A positive linear correlation between beta-catenin and PLAG1 (pleomorphic adenoma gene 1) gene expression was observed. CONCLUSIONS: This is the first report (to our knowledge) showing down-regulation of an antagonist of the Wnt pathway, WIF1, and up-regulation of a Wnt agonist, WNT1, in salivary gland tumor cells. This dysregulation of WNT1 and WIF1 expression, coupled with the observed increase in beta-catenin transcription, may consequently promote salivary gland oncogenesis. Our data support the study of the Wnt pathway as a putative therapeutic target for salivary gland cancer.

WIF1, an inhibitor of the Wnt pathway, is rearranged in salivary gland tumors.

Chromosome rearrangements involving 12q13-15 are frequent among several tumors, including pleomorphic adenomas. The common molecular target for these aberrations is the HMGA2 gene, but various fusion partners of HMGA2 have been reported in tumors. Here we report the identification of the WNT inhibitory factor 1 (WIF1) gene as a novel HMGA2 fusion partner in a salivary gland pleomorphic adenoma. In normal salivary gland tissue WIF1 is expressed at a high level and HMGA2 is not expressed. However, in the pleomorphic adenoma expressing the HMGA2/WIF1 fusion transcript, we observed re-expression of HMGA2 wild-type transcripts and very low levels of WIF1 expression. These data suggest a possible synergistic effect between upregulation of HMGA2 and downregulation of WIF1. We screened 13 additional benign and malignant salivary gland tumors and detected WIF1 rearrangement in one out of two carcinomas ex-pleomorphic adenoma analyzed. In this malignant tumor, the rearrangement of one WIF1 allele coexists with loss of the other allele, a classic signature of a tumor suppressor gene. WIF1 is an antagonist of the Wnt signaling pathway, which plays a critical role in human cancer. In transgenic mouse models, Wnt activation leads to a high frequency of benign and malignant salivary gland tumors. To our knowledge, this is the first report suggesting that WIF1 is a recurrent target in human salivary gland oncogenesis and that downregulation of WIF1 plays a role in the development and/or progression of pleomorphic adenomas.

Identification of MMS19 domains with distinct functions in NER and transcription.

Nucleotide excision repair (NER) and RNA polymerase II (Pol II) transcription are essential cellular processes which are intimately intertwined. They share an indispensable multiprotein complex, TFIIH, and impairments in either process can impact the efficiency of the other. Like TFIIH, MMS19 is required for NER and Pol II transcription, but its precise role in each process is unknown. We showed previously that the human MMS19 gene originates multiple splice variants, some of which may encode distinct MMS19 protein isoforms. Here we characterize a novel MMS19 transcript and demonstrate for the first time that MMS19 splice variants are conserved across species and are functionally distinct. Expression of human MMS19 splice variants in mms19-deleted yeast cells produced unique patterns of thermosensitivity and ultraviolet radiation-sensitivity that point to three MMS19 structural domains with distinct in vivo functions. MMS19 polypeptides lacking domain A are able to fulfill the role of full-length MMS19 in NER but not in transcription. MMS19 polypeptides lacking part of domain B are efficient in transcription but not in NER. MMS19 polypeptides lacking domain C (HEAT repeats) are unable to fulfill either function. Our data suggest that the MMS19 HEAT repeat domain is essential for MMS19 function in NER and transcription, while domains A and B, within MMS19 N-terminus, modulate the balance between DNA repair and transcription. Our results highlight the functional significance of MMS19 transcripts and the possible contribution of MMS19 isoforms to regulate the switch between NER and transcription. Furthermore, our work associates for the first time specific protein domains with MMS19's role in NER and transcription.