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DNA-dependent and DNA-independent associations of DNA-binding proteins are important in transcriptional regulation. The analysis of DNA-independent associations frequently relies on assaying protein interaction in the absence of target DNA sequences. We have found that contaminating DNA in protein preparations can stabilize DNAdependent associations that may appear DNA-independent. Three cellular proteins of 70, 85, and 110 kDa coimmunoprecipitated with the octamer motif-binding protein Oct-2 because of the presence of contaminating DNA in the cell extracts. In addition, heterodimer formation between Oct-i (or Oct-2) and Pit-i during protein-affinity chromatography was stabilized by the contaminating DNA. In both instances, these DNAdependent protein associations were selectively inhibited by ethidlum bromide in the precipitation reaction without any evident effect on DNA-independent protein associations. Thus, ethidlum bromide may serve as a simple and general indicator of DNA-dependent and DNA-independent protein associations.Transcriptional regulation depends not only on the interactions between sequence-specific DNA-binding proteins and their respective cis-regulatory elements but also on the interactions among these proteins and with other components of the transcriptional machinery (reviewed in refs. 1-3). The availability of tools for studying sequence-specific transcription factors (e.g., cDNA clones and antibodies) has allowed more detailed analysis of the mechanisms by which proteinprotein interactions, both DNA-dependent and DNAindependent, regulate transcription. Current methods for determining DNA-independent protein-protein interactions include coimmunoprecipitation and protein-affinity chromatography. We have used coimmunoprecipitation to identify proteins that associate with the octamer motif-binding proteins Oct-1 (OTF-1, NFIII) and Oct-2 (OTF-2). These transcription factors are particularly interesting because they display the same DNA-binding specificity (4) and share very similar DNA-binding POU domains (5, 6) but display qualitatively different RNA polymerase II transcriptional activation properties. Oct-1 is an effective activator of small nuclear RNA-type promoters, whereas Oct-2 is an effective activator of mRNA-type promoters (7). Therefore, it is likely that Oct-1 and Oct-2 interact preferentially with different components of the transcriptional machinery.We identified four cellular proteins of 68, 70, 85, and 110 kDa that coimmunoprecipitated with Oct-2 from labeled cell extracts in what appeared to be a DNA-independent manner. We noticed, however, that the 70-, 85-, and 110-kDa proteins could bind DNA on their own. We subsequently identified the 70-and 85-kDa proteins as the two heterologous subunits of the human autoantigen Ku, which possesses strong nonspecific DNA-binding properties (ref. 8 and references therein). This observation suggested that the association of these three proteins with Oct-2 might be mediated by contaminating DNA in the labeled cell extracts. Consisten...

Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20–200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K–AKT–mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics.Research in contextFatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers.

Background Patients with bladder cancer are apt to develop multiple recurrences that require intervention. We examined the recurrence, progression and bladder cancer-related mortality rates in a cohort of individuals with high-grade non-muscle-invasive bladder cancer. Methods Using linked SEER-Medicare data, we identified subjects with a diagnosis of high-grade, non-muscle-invasive disease in 1992–2002 and were followed until 2007. We then used multivariate competing-risks regression analyses to examine recurrence, progression, and bladder cancer-related mortality rates. Results Of 7,410 subjects, 2,897 (39.1%) experienced a recurrence without progression, 2,449 (33.0%) experienced disease progression, of whom 981 succumbed to bladder cancer. Using competing-risks regression analysis, we found the 10-year recurrence, progression, and bladder cancer-related mortality rates to be 74.3%, 33.3%, and 12.3%, respectively. Stage T1 was the only variable associated with a higher rate of recurrence. Women, black race, undifferentiated grade, stage Tis and T1 were associated with a higher risk of progression and mortality. Advanced age (≥70) was associated with a higher risk of bladder cancer-related mortality. Conclusions Nearly three-fourths of patients diagnosed with high-risk bladder cancer will recur, progress, or die within ten years of their diagnosis. Even though most patients do not die of bladder cancer, the vast majority endures the morbidity of recurrence and progression of their cancer. Increasing efforts should be made to offer patients intravesical therapy with the goal of minimizing the incidence of recurrences. Furthermore, the high recurrence rate seen during the first two years of diagnosis warrants an intense surveillance schedule.

Guideline-recommended care with radical cystectomy is underused for patients with muscle-invasive bladder cancer. Many bladder cancer patients whose survival outcomes might benefit with surgery are receiving alternative less salubrious treatments.