Background: The antihyperglycemic drug metformin may have beneficial effects on the prevention and treatment of cancer. Metformin is known to activate AMP-activated protein kinase (AMPK). It has also been shown to inhibit cyclin D1 expression and proliferation of some cultured cancer cells. However, the mechanisms of action by which metformin mediates cell cycle arrest are not completely understood.

We describe a procedure for detecting highaffinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.DNA sequences required for the regulation of eukaryotic gene expression have now been identified, largely through in vitro mutagenesis and subsequent analysis of the transcriptional activity of the mutagenized genes. What remains undetermined in most of these cases is the identity of specific transcription factors that interact with these DNA sequences to direct gene expression. The interaction of specific proteins with DNA regulatory sequences probably represents a fundamental process in controlling gene activity, and thus, the overall complement of sequence-specific proteins in the cell nucleus may largely determine the growth or differentiation state of the cell.The existence of site-specific DNA-protein interactions in eukaryotes has been demonstrated by several approaches. These include nitrocellulose filter-binding assays (1-4), detection of discrete DNase-hypersensitive sites in chromatin (5-7), exonuclease III digestion of chromatin (8), affinity chromatography (9, 10), and in vitro transcription (11)(12)(13)(14)(15)(16)(17)(18)(19). A limitation of these techniques is that they identify the site of protein binding within the DNA but do not identify the specific proteins involved. Only after extensive purification procedures can information be obtained about the nature of the proteins involved. Bowen et al. (20) have introduced a procedure that allows detection of DNA-binding proteins by blotting electrophoretically separated proteins on nitrocellulose. This procedure, however, has not proven to be effective for directly identifying site-specific DNA-binding proteins without partial purification (21-23) or heat-inactivation (3) of crude nuclear extracts. We describe here a procedure that uses protein blotting and that requires no prior purification steps, which has the potential for directly identifying and studying the regulation of proteins that interact with a target DNA sequence. We have used this procedure to identify a group of proteins that appear to bind with specificity and high affinity to the promoter region of the gene encoding the transferrin receptor. MATERIALS AND METHODSPreparation of Nuclear Extract. Cells were grown, at 370C in a 10% CO2 atmosphere in roller bottles, in Dulbecco's modified Eagle's medium (GIBCO) containing 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 ,ug/ml), Confluent cultur...

Phenformin (phenethylbiguanide; an anti-diabetic agent) plus oxamate [lactate dehydrogenase (LDH) inhibitor] was tested as a potential anti-cancer therapeutic combination. In in vitro studies, phenformin was more potent than metformin, another biguanide, recently recognized to have anti-cancer effects, in promoting cancer cell death in the range of 25 times to 15 million times in various cancer cell lines. The anti-cancer effect of phenformin was related to complex I inhibition in the mitochondria and subsequent overproduction of reactive oxygen species (ROS). Addition of oxamate inhibited LDH activity and lactate production by cells, which is a major side effect of biguanides, and induced more rapid cancer cell death by decreasing ATP production and accelerating ROS production. Phenformin plus oxamate was more effective than phenformin combined with LDH knockdown. In a syngeneic mouse model, phenformin with oxamate increased tumor apoptosis, reduced tumor size and 18F-fluorodeoxyglucose (FDG) uptake on positron emission tomography/computed tomography compared to control. We conclude that phenformin is more cytotoxic towards cancer cells than metformin. Furthermore, phenformin and oxamate have synergistic anti-cancer effects through simultaneous inhibition of complex I in the mitochondria and LDH in the cytosol, respectively.