Molecular modifications of the androgen receptor (AR) can cause resistance to androgen deprivation therapy (ADT) in prostate cancer patients. Since lack of representative tumor samples hinders therapy adjustments according to emerging AR-modifications, we evaluated simultaneous detection of the two most common AR modifications (AR-V7 splice variant and AR point mutations) in circulating tumor cells (CTCs). We devised a single-tube assay to detect AR-V7 splice variants and AR point mutations in CTCs using immunomagnetic cell isolation, followed by quantitative real-time PCR and DNA pyrosequencing. We prospectively investigated 47 patients with PSA progression awaiting therapy switch. Comparison of response to newly administered therapy and CTC-AR-status allowed effect size estimation. Nineteen (51%) of 37 patients with detectable CTCs carried AR-modifications. Seventeen patients carried the AR-V7 splice variant, one harbored a p.T878A point mutation and one harbored both AR-V7 and a p.H875Y mutation. We estimated a positive predictive value for response and non-response to therapy by AR status in CTCs of ~94%. Based on a conservative calculation, we estimated the effect size for molecularly-informed therapy switches for prospective clinical trial planning to ~27%. In summary, the ability to determine key resistance-mediating AR modifications in CTCs has the potential to considerably improve prostate cancer treatment.
Corynebacterium glutamicum has recently been shown to grow on ethanol as a carbon and energy source and to possess high alcohol dehydrogenase (ADH) activity when growing on this substrate and low ADH activity when growing on ethanol plus glucose or glucose alone. Here we identify the C. glutamicum ADH gene (adhA), analyze its transcriptional organization, and investigate the relevance of the transcriptional regulators of acetate metabolism RamA and RamB for adhA expression. Sequence analysis of adhA predicts a polypeptide of 345 amino acids showing up to 57% identity with zinc-dependent ADH enzymes of group I. Inactivation of the chromosomal adhA gene led to the inability to grow on ethanol and to the absence of ADH activity, indicating that only a single ethanol-oxidizing ADH enzyme is present in C. glutamicum. Transcriptional analysis revealed that the C. glutamicum adhA gene is monocistronic and that its expression is repressed in the presence of glucose and of acetate in the growth medium, i.e., that adhA expression is subject to catabolite repression. Further analyses revealed that RamA and RamB directly bind to the adhA promoter region, that RamA is essential for the expression of adhA, and that RamB exerts a negative control on adhA expression in the presence of glucose or acetate in the growth medium. However, since the glucose-and acetate-dependent down-regulation of adhA expression was only partially released in a RamB-deficient mutant, there might be an additional regulator involved in the catabolite repression of adhA.
Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h–1 and biomass yields up to 0.47 g dry weight (g ethanol)–1. Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.
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