Cryo-nanoscale chromosome imaging—future prospects

The high-order structure of mitotic chromosomes remains to be fully elucidated. How nucleosomes compact at various structural levels into a condensed mitotic chromosome is unclear. Cryogenic preservation and imaging have been applied for over three decades, keeping biological structures close to the native in vivo state. Despite being extensively utilized, this field is still wide open for mitotic chromosome research. In this review, we focus specifically on cryogenic efforts for determining the mitotic nanoscale chromatin structures. We describe vitrification methods, current status, and applications of advanced cryo-microscopy including future tools required for resolving the native architecture of these fascinating structures that hold the instructions to life

PROTEIN EXTRACTION AND WESTERN BLOTTING FROM METHACARN-FIXED TISSUE

Polyacrylamide gel electrophoresis (PAGE) of proteins with subsequent western blotting has become a routine technique for the analysis of proteins from both cultured cells and fresh whole tissue. We have developed a method to extract proteins from methacarn‐fixed tissue which renders them suitable for SDS‐PAGE and western blotting. With a panel of antibodies to specific intermediate filaments, transforming growth factor‐α (TGF‐α), and albumin, immunohistochemistry was performed in parallel with western blotting on sections cut from methacarn‐fixed samples of normal rat liver and liver from rats treated under a regime which induces oval cell proliferation. Immunohistochemistry enabled the determination of changes in tissue distribution and abundance of the target proteins, which was mirrored by the corresponding western blot data. This technique can be especially effective when used in conjunction with immunohistochemistry. Tissue samples are easy to prepare, avoiding the precautions which need to be taken when handling fresh tissue (Abstract: J Pathol 1994; 173S: No. 41). Copyright © 1995 John Wiley & Sons, Ltd

A role for STEAP2 in prostate cancer progression

Prostate adenocarcinoma is the second most frequent cancer worldwide and is one of the leading causes of male cancer-related deaths. However, it varies greatly in its behaviour, from indolent non-progressive disease to metastatic cancers with high associated mortality. The aim of this study was to identify predictive biomarkers for patients with localised prostate tumours most likely to progress to aggressive disease, to facilitate future tailored clinical treatment and identify novel therapeutic targets. The expression of 602 genes was profiled using oligoarrays, across three prostate cancer cell lines: CA-HPV-10, LNCaP and PC3, qualitatively identifying several potential prognostic biomarkers. Of particular interest was six transmembrane epithelial antigen of the prostate (STEAP) 1 and STEAP 2 which was subsequently analysed further in prostate cancer tissue samples following optimisation of an RNA extraction method from laser captured cells isolated from formalin-fixed paraffin-embedded biopsy samples. Quantitative analysis of STEAP1 and 2 gene expression were statistically significantly associated with the metastatic cell lines DU145 and PC3 as compared to the normal prostate epithelial cell line, PNT2. This expression pattern was also mirrored at the protein level in the cells. Furthermore, STEAP2 up-regulation was observed within a small patient cohort and was associated with those that had locally advanced disease. Subsequent mechanistic studies in the PNT2 cell line demonstrated that an over-expression of STEAP2 resulted in these normal prostate cells gaining an ability to migrate and invade, suggesting that STEAP2 expression may be a crucial molecule in driving the invasive ability of prostate cancer cells