Using a cryo scanning transmission X-ray microscope (Maser, et al. (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc. 197, 68-79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.
Previous results showed that overexpression of the WTH3 gene in multidrug resistance (MDR) cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. The WTH3 gene promoter was found to be differentially regulated in paired MDR vs non-MDR MCF7 cells owing to epigenetic modifications and transcription factor modulations. To understand further the mechanisms that govern WTH3's differential expression, we uncovered a p53-binding site in its promoter, which indicated that WTH3 could be regulated by the p53 gene. This hypothesis was then tested by different strategies. The resulting data revealed that (1) the WTH3 promoter was upregulated by the p53 transgene in diverse host cells; (2) there was a correlation between WTH3 expression levels and p53 gene status in a cell line panel; (3) a WTH3 promoter region was directly targeted by the p53 protein in vitro and in vivo. In addition, overexpression of the WTH3 gene promoted the apoptotic phenotype in host cells. On the basis of these findings, we believe that the negative role played by the WTH3 gene in MDR development is through its proapoptotic potential that is regulated by multiple mechanisms at the transcription level, and one of these mechanisms is linked to the p53 gene.
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