Radiation Induces Diffusible Feeder Cell Factor(s) That Cooperate with ROCK Inhibitor to Conditionally Reprogram and Immortalize Epithelial Cells

Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells

The Less We Eat, the Longer We Live: Can Caloric Restriction Help Us Become Centenarians?

Striving for longevity is neither a recent human desire nor a novel scientific field. The first article on this topic was published in 1838, when the average human life expectancy was approximately 40 years. Although nowadays people on average live almost as twice as long, we still (and perhaps more than ever) look for new ways to extend our lifespan. During this seemingly endless journey of discovering efficient methods to prolong life, humans were enthusiastic regarding several approaches, one of which is caloric restriction (CR). Where does CR, initially considered universally beneficial for extending both lifespan and health span, stand today? Does a lifelong decrease in food consumption represent one of the secrets of centenarians’ long and healthy life? Do we still believe that if we eat less, we will live longer? This review aims to summarize the current literature on CR as a potential life-prolonging intervention in humans and discusses metabolic pathways that underlie this effect

Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells

The roles of protection of telomeres 1 (POT1) in human ovarian cancer have not been fully elucidated. Here, we investigated the impact of POT1 knockdown (POT1-KD) on in vitro cell proliferation, tumorigenesis, and histone deacetylase inhibitor (HDACi) response in human ovarian cancer-derived SK-OV3 cells. The POT1 gene was knocked down by infection with POT1 lenti-shRNA. POT1, c-Myc, and hTERT mRNA levels and relative telomere length were determined by qRT-PCR; POT1 protein levels were determined by western blot. The relative telomerase activity levels were detected using qTRAP; cell proliferation was assessed using cumulative population doubling (cPD) experiments. Cell tumorigenicity was evaluated by anchorage-independent cell growth assays, and cell response to HDACi was determined by luminescence cell viability assays. Results indicate that lenti-shRNA-mediated POT1-KD significantly reduced POT1 mRNA and protein expression. POT1-KD immediately downregulated c-Myc expression, which led to the inhibition of cell proliferation, tumorigenesis, and HDACi response. However, after brief suppression, c-Myc expression increased in the medium term, which resulted in enhanced cell proliferation, tumorigenesis, and HDACi response in the POT1-KD cells. Furthermore, we discovered that c-Myc regulated cell proliferation and tumorigenesis via hTERT/telomerase/telomere pathway

The Expression of Insulin in the Central Nervous System: What Have We Learned So Far?

After being discovered over a century ago, insulin was long considered to be a hormone exclusively produced by the pancreas. Insulin presence was later discovered in the brain, which was originally accounted for by its transport across the blood-brain barrier. Considering that both insulin mRNA and insulin were detected in the central nervous system (CNS), it is now known that this hormone is also synthesized in several brain regions, including the hypothalamus, hippocampus, cerebral and cerebellar cortex, and olfactory bulb. Although many roles of insulin in the CNS have been described, it was initially unknown which of them could be attributed to brain-derived and which to pancreatic insulin or whether their actions in the brain overlap. However, more and more studies have been emerging lately, focusing solely on the roles of brain-derived insulin. The aim of this review was to present the latest findings on the roles of brain-derived insulin, including neuroprotection, control of growth hormone secretion, and regulation of appetite and neuronal glucose uptake. Lastly, the impairment of signaling initiated by brain-derived insulin was addressed in regard to memory decline in humans

HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes

<div><p>Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.</p> </div

Bmi1 cooperates with E7 in cell immortalization.

<p>HFKs were doubly infected with pX-Bmi1 and pLXSN-E7, pLXSN-E7 alone, or empty vector. (A) Growth curve. Cells were passaged as described in the Methods section to determine the growth rate and lifespan of the cell populations. Bmi1, in cooperation with E7, induced cell immortalization equivalent to E6 or hTERT and E7. (B) Telomere length. A quantitative PCR-based technique (see Methods) was used to quantify the average telomere length in the indicated cell cultures.</p

Bmi1 protein expression is increased in immortalized and tumorigenic cervical cell lines and positively correlates with disease stage in cervical dysplasia and neoplasia <i>in vivo</i>.

<p>(<b>A</b>) Bmi1 protein levels were quantified by Western blot in primary HFKs and primary human ectocervical cells (HECs) expressing E6 or immortalized by E6/E7 and the cervical cancer cell line HeLa. Lysates were separated by 4–20% gradient SDS-PAGE. Antibodies were used to detect hTERT (1∶1000, Origene), Bmi1 (1∶200, F6, Millipore) and GAPDH (1∶2000, FL-335, Santa Cruz). (<b>B</b>)Tissue from a case of invasive cervical cancer was acquired. Representative images are shown containing cancerous lesions and adjacent normal, intact epithelium. Tissue staining with hematoxylin and eosin (<b>i</b>) and immunohistochemical stain with Bmi1 (1∶200, F6, Millipore) (<b>ii</b>) are shown. (<b>C</b>)Tissues of Cervical Intraepithelial Neoplasia Stage 1 (CIN1), CIN2, CIN3 or carcinoma <i>in situ</i>, and invasive cervical carcinoma were acquired. To quantify Bmi1 expression, stained slides were subjected to a randomized, blinded review by a board-certified clinical pathologist. A subset of slides was scored multiple times to demonstrate reproducibility. For each sample, the case number and diagnosis is provided with the corresponding an intensity score, the percentage of positive cells, the corresponding positivity score, and the combined score. Each case received an intensity score from 0–3 (0 = negative, 1 = weak, 2 = moderate, 3 = intense) and the percentage of positive cells was recorded, which was converted to a positivity score (0 = less than 10%, 1 = 11–49%, 2 = 50–74%, 3 = 75–100%). Combined scores were calculated by adding the intensity score and positivity scores. (<b>D</b>) Immunohistochemical staining with hematoxylin and eosin (<b>i, iii, v, vii, ix</b>) and for Bmi1 protein (1∶100, F6, Millipore) (<b>ii, iv, vi, viii, x</b>) was performed. Representative images are shown. Relevant controls are shown, staining with hematoxylin and eosin (<b>i</b>) and for Bmi1 protein (1∶100, F6, Millipore) (<b>ii</b>). Scale bar = 50 µm. (<b>E</b>) Mean and standard deviation of combined scores are shown.</p

Catalytic-defective hTERT mutants cooperate with HPV E7 to immortalize HFKs.

<p><u>(A) Growth curves.</u> Primary HFKs were transduced with the indicated pBABE-puro based retroviruses containing wild-type hTERT, hTERT N+T, or hTERT-D868A and pLXSN-based retroviruses containing E7 or empty vector and then doubly selected with puromycin and G418 as previously described. Cultures were passed continuously in vitro and growth curves were plotted with population doubling over time in culture. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than three times with similar results. Wild-type hTERT, hTERT-D868A, and hTERT N+T are all able to immortalize HFKs in combination with E7. <u>(B) Telomerase activity in early passage of the transduced cells.</u> CHAP lysates were harvested from early (p5) and telomerase activity was measured by quantitative real-time TRAP. <u>(C) Telomerase activity in late passage of the transduced cells.</u> Telomerase activity in late passage of cells was measured by quantitative real- time TRAP.</p