Gil Leibowitz

Telomerase Activity Is Sufficient To Allow Transformed Cells To Escape from Crisis

ABSTRACT The introduction of simian virus 40 large T antigen (SVLT) into human primary cells enables them to proliferate beyond their normal replicative life span. In most cases, this temporary escape from senescence eventually ends in a second proliferative block known as “crisis,” during which the cells cease growing or die. Rare immortalization events in which cells escape crisis are frequently correlated with the presence of telomerase activity. We tested the hypothesis that telomerase activation is the critical step in the immortalization process by studying the effects of telomerase activity in two mortal SVLT-Rasval12-transformed human pancreatic cell lines, TRM-6 and βlox5. The telomerase catalytic subunit, hTRT, was introduced into late-passage cells via retroviral gene transfer. Telomerase activity was successfully induced in infected cells, as demonstrated by a telomerase repeat amplification protocol assay. In each of nine independent infections, telomerase-positive cells formed rapidly dividing cell lines while control cells entered crisis. Telomere lengths initially increased, but telomeres were then maintained at their new lengths for at least 20 population doublings. These results demonstrate that telomerase activity is sufficient to enable transformed cells to escape crisis and that telomere elongation in these cells occurs in a tightly regulated manner.

Glucotoxicity and β-Cell Failure in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus is increasing worldwide with a trend of declining age of onset. It is characterized by insulin resistance and a progressive loss of beta-cell function. The ability to secrete adequate amounts of insulin is determined by the functional integrity of beta-cells and their overall mass. Glucose, the main regulator of insulin secretion and production, exerts negative effects on beta-cell function when present in excessive amounts over a prolonged period. The multiple metabolic aberrations induced by chronic hyperglycemia in the beta-cell include increased sensitivity to glucose, increased basal insulin release, reduced response to stimulus to secrete insulin, and a gradual depletion of insulin stores. Inadequate insulin production during chronic hyperglycemia results from decreased insulin gene transcription due to hyperglycemia-induced changes in the activity of beta-cell specific transcription factors. Hyperglycemia may negatively affect beta-cell mass by inducing apoptosis without a compensatory increase in beta-cell proliferation and neogenesis. The detrimental effect of excessive glucose concentrations is referred to as glucotoxicity. The present review discusses the role of glucotoxicity in beta-cell dysfunction in type 2 diabetes mellitus.

Towards gene therapy of diabetes mellitus

A definitive treatment for diabetes mellitus will be one that maintains a normal blood glucose concentration in the face of fluctuating dietary intake. To accomplish this, there must be mechanisms to sense the amount of blood glucose coupled to rapid release of the right amount of insulin. While mechanical devices to accomplish this are being developed, ultimately the best approach is likely to be based on genetic modification of cells. These could be pancreatic beta-cells, which are the only cells that produce insulin, or other cells that are involved in the pathogenesis of diabetes. Although definitive treatment of diabetes using genetically modified cells is a long-term goal, much progress is being made. This review discusses various approaches to modifying cells genetically, both in vitro and in vivo, for the treatment of diabetes.

Protection from cell death in cultured human fetal pancreatic cells.

Endocrine cells from the human fetal pancreas will proliferate in vitro on extracellular matrix but lose hormone expression, and redifferentiation requires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation and culture. The mechanism of cell death was examined at each stage throughout the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of trehalose to the media to prevent necrosis increased yield 17-fold, while during culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increased yield 1.8-fold. Following disruption of cell–matrix interactions and reaggregation, there was marked evidence of apoptotic bodies by the TUNEL assay. Addition of nicotinamide or Z-VAD, or removal of arginine from the media during reaggregation, reduced the number of apoptotic bodies and the effect was additive. However, a combination of treatments was necessary to significantly increase the yield of viable cells. We conclude that cell death of human fetal pancreatic tissue in culture results from both necrosis and apoptosis and that understanding the mechanisms at the cellular level will lead to protocols that will improve cell viability and promote β-cell growth.

Improvement of ER stress-induced diabetes by stimulating autophagy

Pancreatic β-cell dysfunction is central in diabetes. The diabetic milieu may impair proinsulin folding, leading to β-cell endoplasmic reticulum (ER) stress and apoptosis, and thus a worsening of the diabetes. Autophagy is crucial for the well-being of the β-cell; however, the impact of stimulating autophagy on β-cell adaptation to ER stress is unknown. We studied the crosstalk between ER stress and autophagy in a rodent model of diabetes, called Akita, in which proinsulin gene mutation leads to protein misfolding and β-cell demise. We found that proinsulin misfolding stimulates autophagy and, in symmetry, inhibition of autophagy induces β-cell stress and apoptosis. Under conditions of excessive proinsulin misfolding, stimulation of autophagy by inhibiting MTORC1 alleviates stress and prevents apoptosis. Moreover, treatment of diabetic Akita mice with the MTORC1 inhibitor rapamycin improves diabetes and prevents β-cell apoptosis. Thus, autophagy is a central adaptive mechanism in β-cell stress. Stimulation of autophagy may become a novel therapeutic strategy in diabetes.

Anti-inflammatory Agents in the Treatment of Diabetes and Its Vascular Complications.

The association between hyperglycemia and inflammation and vascular complications in diabetes is now well established. Antidiabetes drugs may alleviate inflammation by reducing hyperglycemia; however, the anti-inflammatory effects of these medications are inconsistent and it is unknown whether their beneficial metabolic effects are mediated via modulation of chronic inflammation. Recent data suggest that immunomodulatory treatments may have beneficial effects on glycemia, β-cell function, and insulin resistance. However, the mechanisms underlying their beneficial metabolic effects are not always clear, and there are concerns regarding the specificity, safety, and efficacy of immune-based therapies. Herein, we review the anti-inflammatory and metabolic effects of current antidiabetes drugs and of anti-inflammatory therapies that were studied in patients with type 2 diabetes. We discuss the potential benefit of using anti-inflammatory treatments in diabetes and important issues that should be addressed prior to implementation of such therapeutic approaches.

Abrogation of Autophagy by Chloroquine Alone or in Combination with mTOR Inhibitors Induces Apoptosis in Neuroendocrine Tumor Cells

Background: Everolimus (RAD001), an mTORC1 inhibitor, demonstrated promising, but limited, anticancer effects in neuroendocrine tumors (NETs). Torin1 (a global mTOR inhibitor) and NVP-BEZ235 (a PI3K/mTOR inhibitor) seem to be more effective than RAD001. Autophagy, a degradation pathway that may promote tumor growth, is regulated by mTOR; mTOR inhibition results in stimulation of autophagy. Chloroquine (CQ) inhibits autophagy. Aim: To explore the effect of CQ alone or in combination with RAD001, Torin1 or NVP-BEZ235 on autophagy and on NET cell viability, proliferation and apoptosis. Methods: The NET cell line BON1 was treated with CQ with or without different mTOR inhibitors. siRNA against ATG5/7 was used to genetically inhibit autophagy. Cellular viability was examined by XTT, proliferation by Ki-67 staining and cell cycles by flow cytometry. Apoptosis was analyzed by Western blotting for cleaved caspase 3 and staining for annexin V; autophagy was evaluated by Western blotting and immunostaining for LC3. Results: RAD001, Torin1, NVP-BEZ235 and CQ all decreased BON1 cell viability. The effect of RAD001 was smaller than that of the other mTOR inhibitors or CQ. Torin1 and NVP-BEZ235 markedly inhibited cell proliferation, without inducing apoptosis. CQ similarly decreased cell proliferation, while robustly increasing apoptosis. Treatment with Torin1 or NVP-BEZ235 together with CQ was additive on viability, without increasing CQ-induced apoptosis. Inhibition of autophagy by ATG5/7 knockdown increased apoptosis in the presence or absence of mTOR inhibitors, mimicking the CQ effects. Conclusion: CQ inhibits NET growth by inducing apoptosis and by inhibiting cell proliferation, probably via inhibition of autophagy. CQ may potentiate the antitumor effect of mTOR inhibitors.

Effects of proinsulin misfolding on β-cell dynamics, differentiation and function in diabetes

ER stress due to proinsulin misfolding has an important role in the pathophysiology of rare forms of permanent neonatal diabetes (PNDM) and probably also of common type 1 (T1D) and type 2 diabetes (T2D). Accumulation of misfolded proinsulin in the ER stimulates the unfolded protein response (UPR) that may eventually lead to apoptosis through a process called the terminal UPR. However, the β‐cell ER has an incredible ability to cope with accumulation of misfolded proteins; therefore, it is not clear whether in common forms of diabetes the accumulation of misfolded proinsulin exceeds the point of no return in which terminal UPR is activated. Many studies showed that the UPR is altered in both T1D and T2D; however, the observed changes in the expression of different UPR markers are inconsistent and it is not clear whether they reflect an adaptive response to stress or indeed mediate the β‐cell dysfunction of diabetes. Herein, we critically review the literature on the effects of proinsulin misfolding and ER stress on β‐cell dysfunction and loss in diabetes with emphasis on β‐cell dynamics, and discuss the gaps in understanding the role of proinsulin misfolding in the pathophysiology of diabetes.