Described in Anticancer Res., 16, 3459-3466, (1996)

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METHOD

The effects of serum stimulation on gene expression were studied using MCF-7 breast cancer cells that were growth arrested by culturing in Dulbecco's modified Eagle's medium (DMEM) with 0.5% fetal bovine serum for 48 hr before adding 10% fetal bovine serum to stimulate cell proliferation. Human colorectal tumors and normal mucosa (5 cm from the tumor) were flash frozen in liquid N₂ within 10 minutes of removal from the patient. The tissue was confirmed normal or tumor by histographical analysis. For analysis of protein and enzyme activity, homogenized tissues and cells were centrifuged to remove particulate materials at 8000 x g for 5 minutes. Procedures for the isolation of total cellular RNA and the preparation of Northern blots have been described previously( Briehl MM et al ). A thioredoxin reductase probe was prepared as previously described ( Gasdaska JR et al ). A thiredoxin reductase probe was made by random labelling of purified, cloned human thioredoxin reductase cDNA fragments, bp 1 to 3695, ( Gasdaska JR et al (1995)) using a DNA labelling kit (Gibco BRL, Gaithersburg, MD). After hybridization with the probes for thioredoxin or thioredoxin reductase the blots were stripped and reprobed with 13-actin cDNA or 36B4 cDNA to normalize for unequal loading and transfer ( Laborda J). Autoradiograms of blots were quantified using a densitometer (Eagle Eye II, Stratagene, CA).

Rabbit affinity-purified polyclonal antibody was prepared by passing rabbit antiserum to human thiredoxin through a thiredoxin affinity column and eluting bound antibody with 100 mM glycine, pH 2.5. Rabbit polyclonal antiserum to human thioredoxin reductase was used using a synthetic peptide derived from protein aequencing of human placental thioredoxin reductase ( Gasdaska JR et al (1995)). Western blots of cell and tissue extracts unsing these antibodies were visulaized using the ECL system (Amersham Life Sciences, Rockford, IL). Thiredoxin reductase activity was measured by a modification of Holmgreen as previously described ( Mau and Powis).

Units

Desity relative to 36B4 housekeeping gene.

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References

Briehl MM, Cotgreave IA and Powis G: Downregulation of the antioxidant defense during glucocorticoid-mediated apoptosis. Cell Death Differ, 2: 41-46, 1995

Grever MR, Schepartz SA and CHabner BA: The National Cancer Institute: cancer drug discovery and development program. Semi. Oncol, 19: 622-638, 1992

Gasdaska PY, Oblong JE, Cotgreave IA and Powis G: The predicted amino acid sequence of human thioredoxin is identical to that of the autocrine growth factor human adult T-cell derived factor (ADF): Thioredoxin mRNA is elevated in some human tumors. Biophys. Acta, 1218: 292-296, 1994

Gasdaska PY, Gasdaska JR, Cochran S and Powis G: Cloning and sequencing of human thioredoxin reductase. FEBS Lett. , 373: 5-9, 1995

Laborda J: 36B4 cDNA used as an estradiol-independent mRNA control in the cDNA for human acidic ribosomal phosphoprotein PO. Nuc. Acids Res., 19: 3998, 1991

Holmgrem A: Bovine thioredoxin system: Purification of Thioredoxin reductase from calf liver and thymus and studies of its function in dissulfide reduction. J. Biol. Chem., 252: 4600-4606, 1977

Mau B-L and Powis G: Inhibition of cellular thioredoxin reductase by diaziquone and doxorubicin. Biochem. Pharmacol., 43: 1621-1626, 1992