Thioredoxin 1 is responsible for antibody disulfide reduction in CHO cell culture.

During large-scale manufacturing of an IgG1 monoclonal antibody in Chinese hamster ovary (CHO) cells, reduction of the antibody's disulfide bonds was observed. We present evidence that mammalian thioredoxin 1 (TXN1) is the terminal enzyme responsible for this reduction event. We demonstrate a marked prevention of IgG1 disulfide bond reduction in a cell-density dependent manner by knocking down expression of TXN1 via lentivirus transduction of short hairpin RNA.

A switch between cytoprotective and cytotoxic autophagy in the radiosensitization of breast tumor cells by chloroquine and vitamin D.

Calcitriol or 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, has been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25-dihydroxyvitamin D3 with radiation appears to kill p53 wild-type, estrogen receptor-positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, annexin/PI analysis, caspase-3, and PARP cleavage as well as cell cycle analysis.

Detection of ERalpha-SRC-1 interactions using bioluminescent resonance energy transfer.

Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein-protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP(2), a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET(2) energy transfer relies on the distance and orientation generated by the fusion proteins.

Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET).

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence.

Expression of estrogen receptor coregulators in normal and malignant human endometrium.

Objectives: To compare the expression of nuclear receptor coregulators in normal and malignant human endometrium and to identify any relationship to grade, stage, age, depth of myometrial invasion, estrogen receptor alpha (ERalpha), or progesterone receptor (PR) expression.

Methods: Gene expression of SRC-1, SRC-2, SRC-3, N-CoR, SMRT, ERalpha, and PR was measured in 26 samples of normal endometrium and 30 primary endometrial carcinomas using real-time RT-PCR. ERalpha protein expression of each tissue was also measured by Western blot.

Reduced c-Met expression by an adenovirus expressing a c-Met ribozyme inhibits tumorigenic growth and lymph node metastases of PC3-LN4 prostate tumor cells in an orthotopic nude mouse model.

Purpose: The expression of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, frequently increases during prostate tumor progression. However, whether reduced c-Met expression inhibits tumor growth and metastasis has not been ascertained.

Experimental Design: c-Met expression was reduced by infection of an adenovirus expressing a c-Met ribozyme into the highly metastatic human prostate cancer cell line PC3-LN4.