Celastrol targets proteostasis and acts synergistically with a heat-shock protein 90 inhibitor to kill human glioblastoma cells.

Glioblastoma multiforme is a devastating disease of the central nervous system and, at present, no effective therapeutic interventions have been identified. Celastrol, a natural occurring triterpene, exhibits potent anti-tumor activity against gliomas in xenograft mouse models. In this study, we describe the cell death mechanism employed by celastrol and identify secondary targets for effective combination therapy against glioblastoma cell survival. In contrast to the previously proposed reactive oxygen species (ROS)-dependent mechanism, cell death in human glioblastoma cells is shown here to be mediated by alternate signal transduction pathways involving, but not fully dependent on, poly(ADP-ribose) polymerase-1 and caspase-3. Our studies indicate that celastrol promotes proteotoxic stress, supported by two feedback mechanisms: (i) impairment of protein quality control as revealed by accumulation of polyubiquitinated aggregates and the canonical autophagy substrate, p62, and (ii) the induction of heat-shock proteins, HSP72 and HSP90. The Michael adduct of celastrol and N-acetylcysteine, 6-N-acetylcysteinyldihydrocelastrol, had no effect on p62, nor on HSP72 expression, confirming a thiol-dependent mechanism. Restriction of protein folding stress with cycloheximide was protective, while combination with autophagy inhibitors did not sensitize cells to celastrol-mediated cytotoxicity. Collectively, these findings imply that celastrol targets proteostasis by disrupting sulfyhydryl homeostasis, independently of ROS, in human glioblastoma cells. This study further emphasizes that targeting proteotoxic stress responses by inhibiting HSP90 with 17-N-Allylamino-17-demethoxygeldanamycin sensitizes human glioblastoma to celastrol treatment, thereby serving as a novel synergism to overcome drug resistance.

Letrozole Potentiates Mitochondrial and Dendritic Spine Impairments Induced by ß Amyloid.

Reduced estrogens, either through aging or postsurgery breast cancer treatment with the oral nonsteroidal aromatase inhibitor letrozole, are linked with declined cognitive abilities. However, a direct link between letrozole and neuronal deficits induced by pathogenic insults associated with aging such as beta amyloid (Aß 1-42) has not been established. The objective of this study was to determine if letrozole aggravates synaptic deficits concurrent with Aß 1-42 insult. We examined the effects of letrozole and oligomeric Aß 1-42 treatment in dissociated and organotypic hippocampal slice cultures. Changes in glial cell morphology, neuronal mitochondria, and synaptic structures upon letrozole treatment were monitored by confocal microscopy, as they were shown to be affected by Aß 1-42 oligomers. Oligomeric Aß 1-42 or letrozole alone caused decreases in mitochondrial volume, dendritic spine density, synaptophysin (synaptic marker), and the postsynaptic protein, synaptopodin. Here, we demonstrated that mitochondrial and synaptic structural deficits were exacerbated when letrozole therapy was combined with Aß 1-42 treatment. Our novel findings suggest that letrozole may increase neuronal susceptibility to pathological insults, such as oligomeric Aß 1-42 in Alzheimer's disease (AD). These changes in dendritic spine number, synaptic protein expression, and mitochondrial morphology may, in part, explain the increased prevalence of cognitive decline associated with aromatase inhibitor use.

Autocrine insulin action activates Akt and increases survival of isolated human islets.

AIMS/HYPOTHESIS: The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt. METHODS: In order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period. RESULTS: We demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation. CONCLUSIONS/INTERPRETATION: Our findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.

Vesicular roundness and compound release in PC-12 cells.

The principal goals of this study were to establish a quantitative morphological analysis of spatial and regional properties of dense core vesicles, and to use this analysis to assess whether homotypic fusion is prominent in chronically treated PC-12 cells at elevated release levels. Simple computerized image processing of electron-micrographs provided the binary images of vesicular dense cores, whilst the artificial intelligence methods were needed to determine the vesicular membranes. As in the past, the presence of large, highly irregular vesicles, provided the morphological evidence of fused vesicles, but the irregularity of vesicular shape was assessed quantitatively-from its roundness. Free space of each vesicle was determined from the distance to its nearest-neighbor, or from the size of its Voronoi polygon. Within a Voronoi polygon, each point is closer to that vesicle than to any other vesicle. Large vesicles were not less round and did not have larger free space, as expected if they result from fusion of several smaller vesicles. In conclusion, we present a novel and rigorous morphological analysis of spatial and regional properties of dense core vesicles. The results demonstrate that the homotypic fusion is not prominent in PC-12 cells, before or following a chronic treatment that enhances release.

Rapid change of quantal size in PC-12 cells detected by neural networks.

The basic building block of synaptic transmission-the number of molecules released per vesicle (quantal size (QS)) often changes with stimulation, but there is no agreement about what factors regulate it. To throw more light on this problem spontaneous quantal release was recorded amperometrically in PC-12 cells. Amperometric current spikes, representing single vesicle release, were detected by thresholding and were separated from spurious events on the basis of their amplitude and time course using a pattern recognition system based on the principal component neural network methods. The frequency of current spikes, their amplitude, quantal size, rise time and decay time were typically non-stationary, even in the absence of stimulation. Their running values changed much more than those of memoryless stationary random data with the same probability density distribution. Irrespective of how much the quantal size, rise and decay times varied, their amplitude dependence remained constant, or changed with a very different time course. In conclusion, the quantal size is highly labile in PC-12 cells. The lability does not appear to result from the changes of fusion pore dynamics or the mechanism of release of vesicular content, but because of the preferential release of large vesicles.

Perspectives on reproductive senescence and biological aging: studies in genetically altered follitropin receptor knockout [FORKO] mice.

Increased life expectancy leads to increased age-associated health issues in both sexes. For menopausal women the most important of these appear to result from the severe estrogen deficiency caused by ovarian dysfunction. The consequences among others include hot flashes, osteoporosis, obesity, impaired memory, higher incidence of Alzheimer's disease and cardiovascular disease. Ovarian function and steroidogenesis are influenced by pituitary gonadotropins, including follicle-stimulating hormone (FSH), whose actions are mediated through ovarian receptors. This article highlights our recent data pertinent to aging as derived from a novel genetically modified animal model [the FORKO mouse (FOllitropin Receptor KnockOut) lacking the FSH receptor. FORKO female mice experience a chronic depletion of estrogen (E2) from early development, and have phenotypes similar to aging women, with ovarian failure, obesity, skeletal changes, and ovarian tumors. A variety of findings support the conclusion that E2 deficiency in FORKO mice is responsible for their neural impairments associated with glial cell hypertrophy, region-specific brain cells loss, and abnormal behavior. Findings from mice with FSH receptor haploinsufficiency mice ('menopausal mice') are also shedding light on the molecular basis of menopausal conditions that include degeneration of the hippocampus. Many phenotypes noted in the null condition also occur in +/- females but in an age related manner. Thus, the FORKO mouse becomes an excellent model to investigate mechanisms underlying age-related changes especially when these events are accelerated, as in menopausal women. Opportunities abound to assess the potential benefits/adverse effects of hormone replacement regimen on various targets.

Positive regulation of ERK activation and MKP-1 expression by peroxovanadium complex bpV (phen).

Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 micromol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.

Signals for death and differentiation: a two-step mechanism for in vitro transformation of adult islets of Langerhans to duct epithelial structures.

Phenotypic change of adult pancreatic islets has been implicated in the development of certain pancreatic cancers and in islet transplant failure. The aim of this study was to characterize intracellular events that mediate changes in adult islet phenotype. Using an in vitro islet-to-duct transformation model, canine islets were induced to undergo phenotypic transformation to duct-like epithelial structures through a two-stage process. Stage one was characterized by widespread islet cell apoptosis associated with the formation of cavitary spaces within the islets. During this stage, c-Jun N-terminal regulated kinase (JNK) and caspase-3 activities were elevated, while extracellular signal-regulated kinase (ERK) and Akt activities were decreased. The second stage of the process was characterized by an inversion in the balance in activity between these signal transduction pathways and by a concomitant decrease in apoptosis. The transformed islets were no longer immunoreactive for islet cell hormones, but expressed the duct epithelial cell marker CK-AE1/AE3. In contrast to islet cells, these duct epithelial cells were highly proliferative. To clarify the role of the identified changes in signal transduction events, we performed additional studies using pharmacological inhibitors of enzyme activity and demonstrated that inhibition of JNK and caspase-3 activity prevented cystic transformation. Our results indicate that the balance in signaling activity between ERK/Akt and JNK/caspase-3 appears to be an important regulator of islet cell death and differentiation.

Tyrosine phosphatase inhibition enhances neurotrophin potency and rescues nigrostriatal neurons in adult rats.

Neurotrophic factors regulate a variety of cellular processes, including neuronal survival during development and after injury. For instance, brain-derived neurotrophic factor (BDNF) can prevent the death of dopaminergic substantia nigra neurons in rats. Most neurotrophic factor receptors, such as TrkB for BDNF, are tyrosine kinases whose signaling is terminated by protein tyrosine phosphatases (PTPs). We tested the idea that inhibition of PTPs, and thus potentially enhancement of the efficiency of endogenous trophic factors and their receptors, would lead to increased neuronal survival. After a 2-week infusion of the small PTP inhibitor molecule peroxovanadium (pVa, pervanadate) close to the substantia nigra of adult rats, up to 66% of axotomized substantia nigra neurons had survived, compared to only 33% in control rats infused with PBS. PVa most likely affected TrkB and/or downstream signaling molecules, as ineffective doses of BDNF and pVa had a synergistic effect when given simultaneously, rescuing 82% of the neurons. PVa stimulated tyrosine hydroxylase (TH) expression in the noninjured substantia nigra but did not prevent axotomy-induced loss of TH. These results raise the possibility that PTP inhibition can prevent neuronal death by enhancing neurotrophic factor signaling pathways in the adult mammalian nervous system, identifies an important role for PTPs in neuronal functioning, and points to a novel small molecule treatment approach for neurologic disorders

Chronic estrogen deficiency leads to molecular aberrations related to neurodegenerative changes in follitropin receptor knockout female mice.

The follitropin receptor knockout (FORKO) mouse undergoes ovarian failure, thereby providing an animal model to investigate the consequences of the depletion of circulating estrogen in females. The estrogen deficiency causes marked defects in the female reproductive system, obesity, and skeletal abnormalities. In light of estrogen's known pleiotropic effects in the nervous system, our study examined the effects of genetically induced estrogen-testosterone imbalance on this system in female FORKO mice. Circulating concentrations of 17-beta-estradiol (E2) in FORKO mice are significantly decreased (FORKO -/-: 1.13+/-0.34 pg/ml; wild-type +/+: 17.6+/-3.5 pg/ml, P<0.0001, n=32-41); in contrast, testosterone levels are increased (-/-: 37.7+/-2.3 pg/ml; wild-type +/+: 3.9+/-1.7 pg/ml, P<0.005, n=25-33). The focus was on the activities of key enzymes in the central cholinergic and peripheral nervous systems, on dorsal root ganglia (DRGs) capacity for neurite outgrowth, and on the phosphorylation state of structural neurofilament (NF) proteins. Choline acetyltransferase activity was decreased in several central cholinergic structures (striatum 50+/-3%, hippocampus 24+/-2%, cortex 12+/-3%) and in DRGs (11+/-6%). Moreover, we observed aberrations in the enzymatic activities of mitogen-activated protein kinases (extracellular-regulated kinase and c-Jun N-terminal kinase) in the hippocampus, DRGs, and sciatic nerves. Hippocampal and sensory ganglia samples from FORKO mice contained hyper-phosphorylated NFs. Finally, explanted ganglia of FORKO mice displayed decreased neurite outgrowth (20-50%) under non-treated conditions and when treated with E2 (10 nM). Our results demonstrate that genetic depletion of circulating estrogen leads to biochemical and morphological changes in central and peripheral neurons, and underlie the importance of estrogen in the normal development and functioning of the nervous system. In particular, the findings suggest that an early and persisting absence of the steroid leads to neurodegenerative changes and identify several key enzymes that may contribute to the process. This model provides a system to explore the consequences of circulating estrogen deprivation and other hormonal imbalances in the nervous system.

Bisperoxovanadium complex promotes dopamine exocytosis in PC12 cells.

The effects of the peroxovanadium complex potassium bisperoxo(1,10-phenanthroline)-oxovanadate (bpV[phen]) have been studied on dopamine (DA) exocytosis in PC12 cells. Bisperoxo(1,10-phenanthroline)-oxovanadate does not elicit dopamine secretion in PC12 cells. However, treatment of PC12 cells with 30 microM bpV[phen] for 20 min significantly enhances the secretion induced by the Ca(2+)-ionophore A23187. The effects appear to be irreversible, and strikingly different from the transient and suppressing effects of orthovanadate, which, like bpV[phen], is also a protein tyrosine phosphatase inhibitor. Contrastingly, the short-lived peroxovanadates, formed in situ by the addition of hydrogen peroxide and orthovanadate, are relatively ineffective. The Ca(2+) chelating agent EGTA abolishes bpV[phen]-enhanced dopamine release. The extracellular-regulated protein kinases (ERK) and synaptophysin, proteins implicated in exocytosis, are both tyrosine-phosphorylated by bpV[phen] in a dose- and time-dependent manner, with a maximal effect at 30 microM. Pre-treatment of cells with PD98059 significantly reduced dopamine release (P<0.05). These results suggest that this peroxovanadium complex enhances dopamine exocytosis, at least in part, by ERK-mediated signaling pathway and synaptophysin-associated phosphatase(s).

Block copolymers modify the internalization of micelle-incorporated probes into neural cells.

An important therapeutic concern is rate and extent of internalization of drugs into cells. Hydrophilic agents often internalize poorly and slowly, and highly lipophilic ones too rapidly. The incorporation of drugs into micelles allows regulation of their internalization parameters, and newly-described block copolymers can be selectively tailored to suit specific drugs. This report compares internalization of Cell Tracker CM-DiI (DiI), a highly lipophilic non-cytotoxic fluorescent probe in common use in biology, from the freely-presented (non-micelle-incorporated) and micelle-incorporated states. DiI was effectively incorporated (>60%) into 25-50 nm diameter spherical micelles made from polycaprolactone-b-polyethylene oxide block copolymer. Confocal microscopy was used to evaluate the internalization of DiI into mixed neuron-glia cultures (2-14 days in vitro, 2DIV-14DIV). Incorporation of DiI into micelles strikingly reduced the rate and extent of its internalization in both 2DIV and 14DIV cultures. Both the age of the cultures and the block copolymer employed to construct the micelles significantly influence the internalization of micelle-incorporated probe.

MKP-1 as a target for pharmacological manipulations in PC12 cell survival.

Dual specificity mitogen activated protein kinase phosphatase-1 (MKP-1) inactivates extracellular signal-regulated kinase (ERK), p38 and/or c-jun N-terminal protein kinase (JNK) by dephosphorylation via a negative feed-back loop. The aim of the present study was to assess the role of expression of MKP-1 and phosphorylation status of mitogen-activated protein kinases (MAPKs) in promoting cell survival in PC12 cells. We used FK506 and three different monoperoxovanadium complexes (mpVs) as pharmacological tools for manipulation of MKP-1 expression. Peroxovanadium compounds, known to be insulinomimetic agents and protein tyrosine phosphatase inhibitors, are cytotoxic to the cells, they activate JNK and down-regulate MPK-1. On the other hand, FK 506 has transient effect on ERK activation. However, when the agents are used in combination, ERK phosphorylation is prolonged and intensified, MKP-1 expression is increased, and cell survival is enhanced. The concomitant alterations observed in intensities and duration of phospho-ERKs and phospho-JNKs signals suggest that monoperoxovanadium complexes in combination with FK 506 enhance survival of PC12 cells by an induction of MKP-1 expression.

Modulation of JNK and p38 stress activated protein kinases in isolated islets of Langerhans: insulin as an autocrine survival signal.

OBJECTIVE: The objective of this study was to determine the effects of islet isolation and cytokine exposure on e-JUN NH2 terminal kinase (JNK) and p38 activation and whether insulin or the p38 inhibitor PD169316 could modify the response. SUMMARY BACKGROUND DATA: Islet transplantation exposes the cells of the graft to a variety of stressful stimuli that could promote beta-cell death and lead to graft failure. METHODS: Islets from canine (n = 12) and cadaveric human (n = 6) pancreata were isolated and purified. Islets were cultured in CMRL 1066 with and without 100 ng/ml insulin. The response to cytokine stimulation with tumor necrosis factor (TNF)alpha and IL-1 beta and the p38 inhibitor PD169316 was also observed. Islet lysates were analyzed by Western blotting for total and phosphorylated JNK and p38 content. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL) assay and by a specific cell death enzyme-linked immunosorbant assay (ELISA). RESULTS: In unstimulated islets, JNK activity was highest immediately following isolation, declining over 3 days to a low baseline level. The activity of p38 was lowest immediately after isolation, increasing progressively with time. The addition of insulin resulted in a more rapid decline in JNK activity, as opposed to p38, which showed no decrease in phosphorylation in response to insulin. In the cytokine stimulation studies, IL-1 beta stimulated p38 activation in a dose dependent manner, while JNK was relatively unaffected. PD169316 (100 microg/ml) was able to inhibit p38 activation in response to the isolation procedure as well as cytokine stimulation. Apoptotic activity was highest 24 hours after isolation, and was significantly reduced when islets were maintained in insulin-supplemented medium. CONCLUSIONS: Inhibition of the stress-activated protein kinase (SAPK) pathways may be important for the maintenance of islet cell survival following islet isolation for transplantation. This study supports an autocrine role of insulin in this process.

Differential regulation of JNK activation and MKP-1 expression by peroxovanadium complexes.

Bisperoxovanadium complexes have been identified as insulinomimetic agents and protein tyrosine phosphatase inhibitors. The aim of the present study was to examine the effects of the most potent bisperoxovanadium complex, potassium bisperoxo (1,10-phenanthroline) oxovanadate (V) [bpV(phen)], on expression and activation of c-jun N-terminal protein kinases (JNK) and on expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cell lines. We compared the effects of bpV(phen) with the effects of tumor necrosis factor-alpha (TNF-alpha), a known regulator of JNK phosphorylation and inducer of MKP-1. Treatment with bpV(phen) causes significant and sustained down-regulation of MKP-1 expression both in PC12 and HeLa cells. In contrast, TNF-alpha induces MKP-1 expression in PC12 cells and does not alter MKP-1 expression in HeLa cells. Both bpV(phen) and TNF-alpha induce MKP-1 expression in OVCAR-3 cell line but with different dynamics: TNF-alpha causes transient and bpV(phen) sustained induction of MKP-1 expression. Temporal pattern of level of MKP-1 expression correlates with the regulation of JNK phosphorylation by bpV(phen) and TNF-alpha in PC12 cells. However, no detectable phospho-JNK signal is observed in either OVCAR-3 or HeLa cells treated with bpV(phen). In contrast, TNF-alpha causes strong and sustained JNK phosphorylation in OVCAR-3 cell line, and strong but transient JNK activation in HeLa cells. BpV(phen) and TNF-alpha does not alter JNK expression in any of the cell lines studied. We demonstrate that the effect of two stressors, bpV(phen) and TNF-alpha, on MKP-1 expression and JNK phosphorylation are strikingly different, depending on the cell type. These results suggest the possible role of MKP-1 in regulation of JNK phosphorylation in both PC12 and OVCAR-3 cell lines treated with bpV(phen).

Phosphatidylinositol 3-kinase signaling to Akt mediates survival in isolated canine islets of Langerhans.

The isolation of islet cells from the pancreas by enzymatic digestion causes many of these cells to undergo apoptosis. The aim of this work was to investigate the role of phosphatidylinositol 3-kinase (PI3-K)/Akt signaling in mediating the survival of isolated islets. Insulin-like growth factor-1 (IGF-I) was examined as a potential culture media supplement that could rescue isolated islets from their apoptotic fate. Western blot analysis demonstrated that Akt phosphorylation peaks 20 h after routine islet isolation. PI3-K inhibition with wortmannin abolished both basal and IGF-I-mediated Akt phosphorylation. IGF-I did not increase survival of isolated islets under normal conditions but it did have a protective effect against cytokine (TNF-alpha, IL-1beta, INF-gamma)-mediated cell death. The protective effect of IGF-I against cytokine-stimulated apoptosis was blocked by wortmannin. In addition, inhibition of basal levels of PI3-K activity caused a 31% decrease in islet survival, as shown by MTT assay. These results demonstrate that the PI3-K/Akt pathway mediates survival of isolated islets of Langerhans.

PCL-b-PEO micelles as a delivery vehicle for FK506: assessment of a functional recovery of crushed peripheral nerve.

The aim of this work was to test in vivo a new block copolymer-based delivery system containing lipophilic drug FK506, known as Tacrolimus. Tacrolimus is currently used in clinics as an immunosupressant agent, and more recently it has been shown that it can exert neurotrophic effects. We prepared, characterized, and assessed polycaprolactone-b-polyethylenoxyde (PCL-b-PEO) micelles containing FK506 in vitro and in vivo. By using well-established animal model of peripheral nerve injury (crushed sciatic nerve), we show that the rate of functional recovery of injured nerve is significantly enhanced in rats treated with micellar FK506. These findings support the notion that PCL-b-PEO is a suitable polymer material for FK506 and suggest its wider applicability as a delivery vehicle for other biologically active, poorly soluble therapeutic agents.

Cell loss in isolated human islets occurs by apoptosis.

Purified islet allografts have largely failed to maintain long-term glucose homeostasis in human recipients, and the reasons for this are unclear. It is noteworthy, however, that islet isolation destroys or removes cellular and noncellular elements of the pancreas that could play an important role in supporting islet survival. The purpose of this study was to determine whether human islet isolation leads to the induction of programmed cell death. Human islets were enzymatically isolated from cadaveric donor pancreata using Liberase or Collagenase P, purified over a discontinuous BSA gradient, then cultured in RPMI 1640 at 37 degrees C in 5% CO2 for < or = 7 days. Islets were examined daily by routine histology and immunocytochemistry for islet hormones, DNA fragmentation [cell death; enzyme-linked immunosorbent assay (ELISA) and TUNEL assay] and for transglutaminase (TG) activity, two indicators of apoptosis. TG activity and DNA fragmentation increased by 1,000% and 1,890%, respectively (p < 0.05) This corresponded to the appearance of pyknotic nuclei on light microscopy, the presence of apoptotic bodies on electron microscopy, and the demonstration of TUNEL-positive cells. These were present primarily in a distribution that corresponded to the insulin-immunoreactive cells. At 5 days, 31.4 +/- 2.2% of islet cells were TUNEL positive. In summary, apoptosis of islet cells appears soon after islet isolation, and involves primarily the beta cell. This is the first report of apoptosis of islet cells after human islet isolation. The loss of beta-cell mass could be implicated in the failure of islet transplantation and merits further investigation.

Polycaprolactone-b-poly(ethylene oxide) copolymer micelles as a delivery vehicle for dihydrotestosterone.

Block copolymer micelles formed from copolymers of poly(caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) were investigated as a drug delivery vehicle for dihydrotestosterone (DHT). The physical parameters of the PCL-b-PEO micelle-incorporated DHT were measured, including the loading capacity of the micelles for DHT, the apparent partition coefficient of DHT between the micelles and the external medium and the kinetics of the release of DHT from the micelle solution. The MTT survival assay was used to assess the in vitro biocompatibility of PCL-b-PEO micelles in HeLa cell cultures. The biological activity of the micelle-incorporated DHT was evaluated in HeLa cells which had been co-transfected with the expression vectors for the androgen receptor and the MMTV-LUC reporter gene. The PCL-b-PEO micelles were found to have a high loading capacity for DHT and the release profile of the drug from the micelle solution was found to be a slow steady release which continued over a 1-month period. The biological activity of the micelle-incorporated DHT was found to be fully retained.