Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a -negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT ؉ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a -dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.

The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.

With increasing frequency during serial passage in culture, primary human keratinocytes express p16 INK4A(p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4 R24C ), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4 R24C or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16؉/؊ individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4 R24C and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.The replicative life span of normal human fibroblasts is limited by a senescence mechanism that responds to partial telomere shortening by triggering a p53-and p21 cip1 (p21)-dependent growth arrest (5,8,12). Expression of hTERT, the telomerase catalytic subunit, in presenescent fibroblasts and several other cell types, including retinal pigmented epithelial cells, vascular endothelial cells, and mesothelial cells, is sufficient to permanently evade this senescence mechanism and immortalize these cell types (7,17,78). Recent studies have indicated, however, that telomere shortening alone cannot completely explain the replicative life span limit and immortalization barrier exhibited by a diverse set of epithelial cell types.For keratinocytes (17, 31), mammary epithelial cells (11,21,31,56), bladder urothelial cells (51), and prostate epithelial cells (28, 57)...

Quantitative expression of ER-related genes, proliferation genes, and immune-related genes are strong predictors of pCR in women with locally advanced breast cancer receiving neoadjuvant anthracyclines and paclitaxel.

Purpose: This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal-tolerated dose (MTD), doselimiting toxicities (DLT), pharmacokinetics, pharmacodynamics, and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent, and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K).Patients and Methods: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily, initially on days 1 to 21 every 28 days and later, using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated 18 F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue.Results: Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea, rash, and fatigue, whereas the DLT was grade 3 maculopapular rash (450 mg, 2 of 3 patients; 330 mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC >20 hÁmmol/L. Significant increase in plasma insulin and glucose levels, and >25% decrease in 18 F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinumrefractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively.Conclusion: Pictilisib was safely administered with a doseproportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels !100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily.

Objective: To estimate the magnitude of serious eye disorders and of visual impairment in a defined elderly population of a typical metropolitan area in England, and to assess the frequency they were in touch with, or known to, the eye care services. Design: Cross sectional survey using two stage cluster random sampling. Setting: General practices in north London. Subjects: Random sample of people aged 65 and older, drawn from a defined population of elderly people registered with 17 general practice groups. Main outcome measures: Proportions and population prevalence estimates were determined for visual acuity, assessed with the person's own spectacles (if any), classified into four categories: prevalence of cataract, age related macular degeneration, and refractive error causing visual impairment and of definite primary open angle glaucoma; and status of contact with eye services. Results: 1547 of 1840 (84%) eligible people were examined. The population prevalence of bilateral visual impairment (visual acuity < 6/12) was 30%, of which 72% was potentially remediable. 92 of these 448 cases (21%) had visual acuity < 6/60 ("blindness") in one or both eyes. Prevalence of cataract causing visual impairment was 30%; 88% of these people were not in touch with the eye services. The prevalence of vision impairing, age related macular degeneration was 8% and of glaucoma (definite cases) was 3%. Three quarters of the people with definite glaucoma were not known to the eye services. Conclusions: Untreated visual impairment and eye disorders affect a substantial proportion of people aged 65 years and older. These findings should contribute to the setting up of future strategies for preservation of sight and eye health services in general.

Background: Oncotype DX™ is a clinically validated, high-complexity, multianalyte reverse transcription-PCR genomic test that predicts the likelihood of breast cancer recurrence in early-stage, node-negative, estrogen receptor-positive breast cancer. The Recurrence Score™ (RS) provides a more accurate, reproducible measure of breast cancer aggressiveness and therapeutic responsiveness than standard measures. Individualized patient management requires strict performance criteria for clinical laboratory tests. We therefore investigated the analytical performance of the assay. Methods: Assays used a pooled RNA sample from fixed paraffin-embedded tissues to evaluate the analytical performance of a 21-gene panel with respect to amplification efficiency, precision, linearity, and dynamic range, as well as limits of detection and quantification. Performance variables were estimated from assays carried out with sample dilutions. In addition, individual patient samples were used to test the optimized assay for reproducibility and sources of imprecision. Results: Assay results defined acceptable operational performance ranges, including an estimated maximum deviation from linearity of <1 cycle threshold (C T ) units over a >2000-fold range of RNA concentrations, with a mean quantification bias of 0.3% and CVs of 3.2%-5.7%. An analysis of study design showed that assay imprecision

Aims-Scanning laser polarimetry is a new technique allowing quantitative analysis of the retinal nerve fibre layer in vivo. This technique was employed to investigate the variation of the retinal nerve fibre layer thickness in a group of normal subjects of diVerent ages and ethnic groups. Methods-150 normal volunteers of different ages and ethnic groups were recruited for this study. Three consecutive 15 degree polarimetric maps were acquired for each subjects. Nerve fibre layer thickness measurements were obtained at 1.5 disc diameters from the optic nerve. Four 90 degree quadrants were identified. Results-The mean nerve fibre layer thickness varied from a minimum of 55.4 µm to a maximum of 105.3 µm, with a mean thickness value of 78.2 (SD 10.6) µm. Superior and inferior quadrants showed a comparatively thicker nerve fibre layer than nasal and temporal quadrants. Retinal nerve fibre layer thickness is inversely correlated with age (p < 0.001). White people showed thicker nerve fibre layers than Afro-Caribbeans (p = 0.002). Conclusion-The results indicate a progressive reduction of the nerve fibre layer thickness with increasing age. This may be due to a progressive loss of ganglion axons with age as suggested in postmortem studies. A racial diVerence in nerve fibre layer thickness is present between whites and Afro-Caribbeans. (Br J Ophthalmol 1997;81:350-354)