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We hypothesized that amplification or overexpression of HER-2 (c-erbB-2), the Ki-67 antigen (Mib1), cyclin D-1 (CD1), interleukin-6 (IL-6), or the transforming growth factor beta II receptor, (TGFbetaRII), would predict relapse in women with early stage, estrogen (ER) and/or progesterone receptor (PR) positive breast cancer treated with tamoxifen. Conditional logistic regression models and a new novel analytic method - support vector machines (SVM) were used to assess the effect of multiple variables on treatment outcome. All patients had stage I-IIIa breast cancer (AJCC version 5). We paired 63 patients who were disease-free on or after tamoxifen with 63 patients who had relapsed (total 126); both disease-free and relapsed patients were matched by duration of tamoxifen therapy and time to recurrence. These 126 patients also served as the training set for SVM analysis and 18 other patients used as a validation set for SVM. In a multivariate analysis, larger tumor size, increasing extent of lymph node involvement, and poorer tumor grade were significant predictors of relapse. When HER-2 or CD1 were added to the model both were borderline significant predictors of relapse. The SVM model, after including all of the clinical and marker variables in the 126 patients as a training set, correctly predicted relapse in 78% of the 18 patient validation samples. In this trial, HER-2 and CD1 proved of borderline significance as predictive factors for recurrence on tamoxifen. An SVM model that included all clinical and biologic variables correctly predicted relapse in >75% of patients. [ABSTRACT FROM AUTHOR]

Background: Recent studies have shown that minichromosome maintenance (MCM) proteins (Mcm2-7) may be useful proliferation markers in dysplasia and cancer in various tissues. Aims: To investigate the use of Mcm7 as a proliferation marker in 79 lymph node negative prostate cancers and compare it with Ki-67, a commonly used cell proliferation marker. Methods: The percentage of proliferating cells (proliferation index; Pl) was calculated for basal and luminal epithelial cells in benign prostate tissue, prostatic intraepithelial neoplasia (PIN), and epithelial cells in adenocarcinoma. The Pl for each biomarker was correlated with the preoperative prostate specific antigen concentration, the Gleason score, surgical resection margin status, and the AJCC pT stage for each patient. Results: The mean Pls for Ki-67 and Mcm7 were: benign luminal epithelium 0.7 and 1.2 and benign basal epithelium 0.8 and 8.2; PIN non-basal epithelium 4.9 and 10.6 and PIN basal epithelium 0.7 and 3.1; adenocarcinoma 9.8 and 22.7, respectively. Mcm7 had a significantly higher mean Pl (p<0.0001) than Ki-67 for all cell categories except benign luminal epithelial cells. Mcm7 was a better discriminatory marker of proliferation between benign epithelium, PIN, and invasive adenocarcinoma (p<0.0001) than Ki-67. The drop in Mcm7 mean basal cell Pl from benign epithelium to PIN epithelium was significantly larger than for Ki-67 (p<0.0001). Mcm7 had a significantly higher Pl than Ki-67 at each risk level. Conclusion: Mcm7 may be a useful proliferation marker in prostatic neoplasia and warrants further evaluation as a complementary tool in the diagnosis of PIN and prostate carcinoma. [ABSTRACT FROM AUTHOR]

Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation–DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50–90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3′-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery. [ABSTRACT FROM AUTHOR]

The mouse Nramp1 gene was initially identified by positional cloning as a candidate for the host resistance locus Bcg on mouse Chromosome (Chr) 1. Subsequent studies have shown that Nramp is a small gene family of at least three members in the mouse genome. We report the isolation and characterization of a cDNA clone corresponding to the second member of the human NRAMP family, NRAMP2. Predicted amino acid sequence analysis of the NRAMP2 polypeptide identifies a polytopic membrane protein highly homologous to NRAMP1, with 66% identical residues (80% overall homology), resulting in identical predicted secondary structures for the two proteins. Sequence conservation is particularly high in the predicted transmembrane domains (90%), suggesting that these regions play a key role in the structural and functional aspects common to both proteins. As opposed to its NRAMP1 counterpart, whose expression is restricted to phagocytic cells, Northern blotting analyses indicate that NRAMP2 mRNA transcripts are expressed at low levels in all tissues analyzed. Fluorescence in situ hybridization has identified 12q13 as the chromosomal location for human NRAMP2. [ABSTRACT FROM AUTHOR]