No abstract

Human T lymphocytes express a Ca2؉ -activated K ؉ current (IK), whose roles and regulation are poorly understood. We amplified hSK4 cDNA from human T lymphoblasts, and we showed that its biophysical and pharmacological properties when stably expressed in Chinese hamster ovary cells were essentially identical to the native IK current. In activated lymphoblasts, hSK4 mRNA increased 14.6-fold (Kv1.3 mRNA increased 1.3-fold), with functional consequences. Proliferation was inhibited when Kv1.3 and IK were blocked in naive T cells, but IK block alone inhibited re-stimulated lymphoblasts. IK and Kv1.3 were involved in volume regulation, but IK was more important, particularly in lymphoblasts. hSK4 lacks known Ca 2؉ -binding sites; however, we mapped a Ca 2؉ -dependent calmodulin (CaM)-binding site to the proximal C terminus (Ct1) of hSK4. Full-length hSK4 produced a highly negative membrane potential (V m ) in Chinese hamster ovary cells, whereas the channels did not function when either Ct1 or the distal C terminus was deleted (V m ϳ0 mV). Native IK (but not expressed hSK4) current was inhibited by CaM and CaM kinase antagonists at physiological V m values, suggesting modulation by an accessory molecule in native cells. Our results provide evidence for increased roles for IK/hSK4 in activated T cell functions; thus hSK4 may be a promising therapeutic target for disorders involving the secondary immune response.

Metformin may exert anti-cancer effects through indirect (insulin-mediated) or direct (insulin-independent) mechanisms. We report results of a neoadjuvant "window of opportunity" study of metformin in women with operable breast cancer. Newly diagnosed, untreated, non-diabetic breast cancer patients received metformin 500 mg tid after diagnostic core biopsy until definitive surgery. Clinical (weight, symptoms, and quality of life) and blood [fasting serum insulin, glucose, homeostasis model assessment (HOMA), C-reactive protein (CRP), and leptin] attributes were compared pre- and post-metformin as were terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Ki67 scores (our primary endpoint) in tumor tissue. Thirty-nine patients completed the study. Mean age was 51 years, and metformin was administered for a median of 18 days (range 13-40) up to the evening prior to surgery. 51 % had T1 cancers, 38 % had positive nodes, 85 % had ER and/or PgR positive tumors, and 13 % had HER2 overexpressing or amplified tumors. Mild, self-limiting nausea, diarrhea, anorexia, and abdominal bloating were present in 50, 50, 41, and 32 % of patients, respectively, but no significant decreases were seen on the EORTC30-QLQ function scales. Body mass index (BMI) (-0.5 kg/m(2), p < 0.0001), weight (-1.2 kg, p < 0.0001), and HOMA (-0.21, p = 0.047) decreased significantly while non-significant decreases were seen in insulin (-4.7 pmol/L, p = 0.07), leptin (-1.3 ng/mL, p = 0.15) and CRP (-0.2 mg/L, p = 0.35). Ki67 staining in invasive tumor tissue decreased (from 36.5 to 33.5 %, p = 0.016) and TUNEL staining increased (from 0.56 to 1.05, p = 0.004). Short-term preoperative metformin was well tolerated and resulted in clinical and cellular changes consistent with beneficial anti-cancer effects; evaluation of the clinical relevance of these findings in adequately powered clinical trials using clinical endpoints such as survival is needed.

Recent studies suggest that some serous ovarian carcinomas arise from the fallopian tube epithelium rather than the ovarian surface epithelium. This hypothesis places emphasis on the fallopian tube secretory epithelial cell as a cell-of-origin. Herein we report the development of a novel ‘ex-vivo’ primary human fallopian tube epithelium culture system that faithfully recapitulates the in-vivo epithelium, as demonstrated by morphological, ultrastructural, and immunophenotypic analyses. Mass spectrometry-based proteomics reveals that these cultures secrete proteins previously identified as biomarkers for ovarian cancer. We also utilize this culture system to study the response of the fallopian tube epithelium to genotoxic stress and find that the secretory cells exhibit a distinct response to DNA damage when compared to neighboring ciliated cells. The secretory cells demonstrate a limited ability to resolve the damage over time, potentially leaving them more susceptible accumulation of additional mutagenic injury. This divergent response is confirmed with in-situ studies using tissue samples, further supporting the use of this ex-vivo culture system to investigate fallopian tube epithelial pathobiology. We anticipate that this novel culture system will facilitate the study of serous ovarian carcinoma pathogenesis, and propose that similar culture systems could be developed for other organ-site specific epithelia.