Enforced expression of the tumor suppressor p53 renders human leukemia cells (U937) more sensitive to 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis

The effects of enforced expression of p53 on the sensitivity of p53(-/-) human monocytic leukemia cells (U937) to apoptosis following exposure to the S-phase-specific antimetabolite 1-[beta-D-arabinofuranosyl]cytosine (ara-C) were examined. Cells were stably transfected with a plasmid containing a chimeric DNA construct encoding a temperature-sensitive p53 variant (135(ala-->val)), which transactivates at 32 degrees but is non-functional at 37 degrees. A significant reduction in the S-phase population was observed in ptsp53 mutants incubated at 32 degrees. Nevertheless, while vector controls did not exhibit differential sensitivity to ara-C at 32 degrees versus 37 degrees, temperature-sensitive p53 mutants displayed a significant increase in apoptosis at the permissive temperature. This was not accompanied by increased ara-CTP formation, DNA incorporation of [3H]ara-C, or altered expression of Bcl-2 or Bax. Enhanced sensitivity was associated with increased mitochondrial injury (e.g. cytochrome c release), caspase activation, and loss of clonogenic survival. Significantly, ptsp53 cells synchronized in S phase were markedly more sensitive to ara-C-mediated mitochondrial injury and apoptosis at 32 degrees, indicating that wild-type p53 specifically enhances the susceptibility of this subpopulation to ara-C lethality. Consistent with these results, transient transfection of human wild-type p53 cDNA rendered parental U937 cells more sensitive to ara-C-mediated cell death. Collectively, these findings indicate that p53 expression renders S-phase U937 cells more susceptible to ara-C-mediated mitochondrial dysfunction, cytochrome c release, apoptosis, and loss of clonogenic survival without enhancing ara-C metabolism. Such findings raise the possibility that loss of functional p53 activity allows leukemia cells to circumvent ara-C lethality.     

Analgesic duration and kinetics of liposomal bupivacaine after subcutaneous injection in mice

1. The objective of the present study was to assess the time-course profile of analgesia and bupivacaine concentrations at the site of injection after subcutaneous administration of a single dose of standard bupivacaine or a novel controlled-release liposomal bupivacaine formulation. 2. Groups of mice were injected subcutaneously with 0.2 mL of 0.5% standard bupivacaine or 0.5, 1 or 2% liposomal bupivacaine. 3. A prolonged duration of analgesia occurred in mice receiving liposomal bupivacaine. In the liposomal groups, the bupivacaine remained at the injection site for more than 96 h, compared with approximately 8 h in groups injected with standard bupivacaine. 4. These results confirm that the prolonged analgesia observed after injection of the liposomal formulation is associated with sustained higher levels of bupivacaine at the site of injection.     

mda-7 (IL-24) Inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling

Despite therapeutic interventions including surgery, chemotherapy and radiotherapy, glioblastoma multiforme (GBM) has a very poor prognosis and novel therapies are required. MDA-7 (IL-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound anti-proliferative and cytotoxic effects in a variety of tumor cells, but not in non-transformed cells. The present studies examined the combined impact of Ad.mda-7 and ionizing radiation on the proliferation and survival of GBM cells. Ad.mda-7 reduced the proliferation of rodent and human glioma cells in MTT assays and in colony formation assays. The anti-proliferative effects of Admda-7 were enhanced by radiation in a greater than additive fashion. In vitro, this cellular change correlated with enhanced cell numbers in G1/G0 and G2/M phases of the cell cycle, implying Ad.mda-7 radiosensitizes tumor cells in a cell cycle-independent manner. The radiosensitizing effects were not observed in cultures of non-transformed primary astrocytes. The enhanced reduction in growth correlated with increased necrosis and DNA degradation. Ad.mda-7 enhanced p38 and ERK1/2 activity but did not alter JNK or Akt activity. Irradiation of cells expressing MDA-7 suppressed ERK1/2 activity and dramatically enhanced JNK1/2 activity without altering either Akt or p38 activity. Inhibition of JNK1/2, but not p38, signaling abolished the radiosensitizing properties of MDA-7. Inhibition of neither ERK1/2 nor PI3K signaling enhanced the anti-proliferative effects of Ad.mda-7, whereas combined inhibition of both pathways enhanced cell killing, suggesting that ERK and PI3K signaling can be protective against MDA-7 lethality.     

Histone deacetylase inhibitors in cancer therapy

Histone deacetylase inhibitors (HDAC inhibitors) represent a novel class of antineoplastic agents that act by promoting acetylation of histones, leading in turn to uncoiling of chromatin and activation of a variety of genes implicated in the regulation of cell surivival, proliferation, differentiation, and apoptosis. The major classes of HDIs include shortchain fatty acids, hydroxamic acid derivatives, synthetic benzamide derivatives, and cyclic tetrapeptides. Members of each of these classes have now entered clinical trials in humans. Despite their shared capacity to trigger histone deacetylation, individual HDIs exert diverse actions on cell cycle regulatory, signal transduction, and survival-related proteins which in all probability accounts for their disparate actions. Major areas of investigation surrounding HDIs include elucidating the mechanisms by which they induce apoptosis in neoplastic cells, and characterizing the factors responsible for the decision of such cells to undergo maturation versus cell death in the response to these agents. In this context, attention has recently focused on the ability of HDIs to induce perturbations in cell cycle regulatory proteins (e.g., p21(CIP1)), downregulation of survival signaling pathways (e.g., Raf/MEK/ERK), and disruption of cellular redox state (e.g., induction of reactive oxygen species; ROS). Aside from efforts to combine HDIs with established cytotoxic drugs, attempts are underway to establish a rational basis for combining HDIs with differentiation- inducing agents (e.g., ATRA, hypomethylating agents such as 5'-deoxyazacytine) with the goal of triggering re-expression of turn or suppressor and/or differentiation-associated genes. Finally, the results of recent preclinical studies provide a strong rationale for combining HDIs with other novel, molecularly targeted agents, including inhibitors of survival signaling pathways or cell cycle progression. Collectively, these findings should provide a fertile environment for the development of novel HDI-containing regimens in the treatment of cancer for many years to come.     

Biogel Super-Sensitive and Biogel Indicator glove systems

Surgical gloves have been in use since the late 19th century. Initially, gloves were introduced to protect clinical staff from the carbolic acid used to sterilize equipment and prepare skin before surgery. During the mid-20th century emphasis was more towards protecting the patient from the clinician's skin flora and now, at the beginning of the 21st century, the balance lies between protecting the patient from the clinician and the clinician from the patient. The three main types of glove are latex, vinyl and polythene and they can be used singly or as part of a double gloving system. This article discusses how one hospital evaluated the glove systems Biogel Super-Sensitive and Biogel indicator underglove from Regent Medical (a division of SSL International). The results indicate that both systems are acceptable to clinical staff in a variety of specialty areas.     

Characterization of discriminative stimulus effects of the neuroactive steroid pregnanolone

Reduced pregnane neurosteroids such as allopregnanolone and pregnanolone are potent neuromodulators able to affect a number of membrane receptors, including gamma-aminobutyric acid (GABA)(A), N-methyl-D-aspartate (NMDA), 5-hydroxytryptamine (5-HT)(3), and sigma(1) receptors. The present study used a drug discrimination procedure to assess further the receptor effects of pregnanolone in vivo. Rats were trained to discriminate 5 mg/kg pregnanolone from saline in a two-lever operant task maintained by food reinforcement. The opiate agonist morphine and the negative GABA(A) modulator dehydroepiandrosterone sulfate did not substitute for pregnanolone. All of the GABA(A) positive modulators tested (allopregnanolone, epipregnanolone, androsterone, pentobarbital, midazolam, and zolpidem) dose dependently substituted for pregnanolone. The direct GABA-site agonists 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol and muscimol failed to substitute for pregnanolone. Ethanol and the sigma(1) receptor agonist SKF 10047 fully substituted for pregnanolone, and the NMDA antagonist MK-801 partially substituted for pregnanolone. The 5-HT(3) antagonist tropisetron did not substitute at any dose tested. The 5-HT(3) agonist SR 57227A reached full substitution, whereas the other 5-HT(3) agonist tested, m-chlorophenylbiguanide, produced partial substitution. These results suggest that positive GABA(A) modulation, but not direct agonism, confers a discriminative stimulus effect similar to pregnanolone. Additionally, antagonism of NMDA receptors and activation of 5-HT(3) and sigma(1) receptors modulate stimulus effects similar to the pregnanolone cue. Overall, the data suggest that pregnanolone produces discriminative stimulus effects representative of a wide-spectrum sedative hypnotic.     

Molecular biology of sodium channels and their role in cardiac arrhythmias

The sodium channel is an integral membrane protein that plays a central role in conduction of the cardiac impulse in working cardiac myocytes and cells of the His-Purkinje system. The channel has two fundamental properties, ion conduction and gating. Specific domains of the channel protein control each of these functions. Ion conduction describes the mechanisms of the selective movement of sodium ion across the pore in the cell membrane. The selectivity of the channel for sodium ions is at least 10 times greater than that for other monovalent cations; the channel does not normally conduct divalent cations. Gating describes the opening and closing of the sodium channel pore. Sodium channels open transiently during membrane depolarization and close by a process termed inactivation. The cardiac sodium channel protein is a multimeric complex consisting of an alpha and an auxiliary beta-subunit. The genes encoding the sodium channel have been cloned and sequenced. The alpha subunit gene, SCN5A is sufficient to express a functional channel. However, beta subunit co-expression increases the level of channel expression and alters the voltage dependence of inactivation. Mutations of the sodium channel may result in incomplete inactivation during maintained depolarization, a decrease in the level of channel expression or acceleration of inactivation. The resulting clinical phenotypes include long QT syndrome, type III (LQT III), Brugada syndrome, and heart block. LQT III and Brugada syndromes have a high case fatality rate and are best treated with an implantable defibrillator.     

Real-time quantitation of HIV-1 p24 and SIV p27 using fluorescence-linked antigen quantification assays

We developed fluorescence-linked antigen quantification (FLAQ) assays for HIV-1 and SIV antigen quantitation. The assays utilize polystyrene microspheres coated with monoclonal antibodies against HIV-1 Gag p24 or SIV Gag p27, which are incubated with unknown samples, flourochrome-conjugated detector antibody, and lysing agent. The fluorescence of individual microspheres is measured using flow cytometry. The speed, simplicity, and wide dynamic range of FIAQ assays makes them superior to enzyme-linked immunosorbent assays for many applications performed in research laboratories.