Ionizing radiation produces clustered lesions in DNA. Since the orientation of bi-stranded lesions affects their recognition by DNA repair enzymes, clustered damages are more difficult to process, and thus more toxic, than single oxidative lesions. In order to understand the structural determinants that leads to differential recognition, we use NMR spectroscopy and restrained molecular dynamics to solve the structure of two DNA duplexes, each containing two stable abasic site analogs positioned on opposite strands of the duplex and staggered in the 3’ (−1 duplex, (AP)2−1 duplex) or 5’ (+1 duplex, (AP)2+1 duplex) direction. Cross-peak connectivities observed in the non-exchangeable NOESY spectra indicate compression of the helix at the lesion site of the duplexes, resulting in the formation of two abasic bulges. The exchangeable proton spectra show the AP site partner nucleotides forming inter-strand hydrogen bonds that are characteristic of a Watson-Crick G•C base pairs, confirming the extra helical nature of the AP residues. Restrained molecular dynamics simulations generate a set of converging structures in full agreement with the spectroscopic data. In the (AP)2−1 duplex, the extra helical abasic site residues reside in the minor groove of the helix, while they appear in the major groove in the (AP)2+1 duplex. These structural differences are consistent with the differential recognition of bi-stranded abasic site lesions by human AP endonuclease.

The WTH3 gene's biological characteristics and relationship to multidrug resistance (MDR) were investigated further. Results showed that WTH3 was mainly located in the cytosol and capable of binding to GTP. In addition, WTH3's promoter function was significantly attenuated in MDR (MFC7/AdrR) relative to non-MDR (MCF7/WT) cells. Advanced analyses indicated that two mechanisms could be involved in WTH3's down-regulation: DNA methylation and trans-element modulations. It was found that the 5Vend portion of a CpG island in WTH3's promoter was hypermethylated in MCF7/AdrR but not MCF7/WT cells, which could have a negative effect on the WTH3 promoter. This idea was supported by the observation that a 45-bp sequence (DMR45) in this differentially methylated region positively influenced promoter activity. We also discovered that different nuclear proteins in MCF7/AdrR and MCF7/WT cells bound to methylated or nonmethylated DMR45. Moreover, a sequence containing a unique repeat that was also a positive cis-element for the promoter was attached by different transcription factors depending on whether they were prepared from MCF7/AdrR or MCF7/WT cells. These molecular changes, apparently induced by drug treatment, resulted in WTH3's down regulation in MDR cells. Therefore, present studies support previous observations that WTH3, as a negative regulator, participates in MDR development in MCF7/AdrR cells. (Cancer Res 2005; 65(16): 7421-8)

Earlier studies suggested that TSP50 is a testis-specific gene that encodes a protein, which is homologous to serine proteases but differs in that threonine replaces serine in its catalytic triad. Most importantly, it was abnormally reactivated in many breast cancer biopsies tested. While further investigating its biochemical and cell biological natures, we found that TSP50 exhibited enzyme activity and was located in the endoplasmic reticulum and cytosol membrane. During our studies to elucidate the regulatory mechanisms related to its differential expression, we discovered a putative p53-binding site and several Sp1-binding sites in the TSP50 promoter, which led us to test if it was regulated by the p53 gene. We found that the p53 transgene negatively regulated the TSP50 promoter in diverse types of cell lines. This result was consistent with other observations: (a) p53 overexpression reduced endogenous TSP50 expression; and (b) breast cancer cell lines containing mutated p53, such as MCF7/Adr, or normal p53, such as MCF7, produced high or low levels of TSP50 transcripts, which was consistent with the fact that TSP50 promoter activity was much higher in MCF7/Adr than that in MCF7 cells. We also found that the quantity of Sp1 transcription factor was lower in MCF7/Adr than in MCF7 cells, which suggested that another mechanism (i.e., transcription factor modulation) was also involved in TSP50 differential expression. [Cancer Res 2007;67(3):1239-45]