Biomedical subjects

J M Gallo

Publications and source records attributed to J M Gallo.

At least 19 recordsLinked to original sources

PubMed2006

Adenoviral modification of mouse brain derived endothelial cells, bEnd3, to induce apoptosis by vascular endothelial growth factor.

A second generation genetically-engineered cell-based drug delivery system, referred to as apoptotic-induced drug delivery (AIDD), was developed using endothelial cells (ECs) that undergo apoptosis upon binding of vascular endothelial growth factor (VEGF) to a Flk-1:Fas fusion protein (FF). This new AIDD was redesigned using mouse brain derived ECs, bEnd3 cells, and an adenovirus vector in order to enhance and control the expression of FF. The FF was tagged with a HA epitope (FFHA) and designed to be coexpressed with green fluorescence protein (GFP) by the regulation of cytomegalovirus promoters in the adenovirus vector. bEnd3 cells showed favorable coexpression of FFHA and GFP consistent with the multiplicity of infection of the adenovirus. Immunofluorescence analysis demonstrated that FFHA was localized at the plasma membrane, whereas GFP was predominantly located in the cytoplasm of ECs. Cell death was induced by VEGF, but not by platelet derived growth factor or fibroblast growth factor in a dose-dependent manner (range 2-20 ng/ml), and revealed caspase-dependent apoptotic profiles. The FFHA expressing bEnd3 cells underwent apoptosis when cocultured with a glioma cell (SF188V+) line able to overexpress VEGF. The combined data indicated that the FFHA adenovirus system can induce apoptotic signaling in ECs in response to VEGF, and thus, is an instrumental modification to the development of AIDD.

Adenoviridae↗

PubMed2001

Pharmacodynamic-mediated reduction of temozolomide tumor concentrations by the angiogenesis inhibitor TNP-470.

The angiogenic phenotype is associated with increased tumor neovascularization and a state of vascular hyperpermeability to macromolecules. Angiogenesis inhibitors could reverse these processes, resulting in tumor capillaries that have normal membrane permeability. It was proposed that the switch from a hyperpermeable to a normal permeable state could have the untoward effect of decreasing tumor concentrations of anticancer drugs coadministered with angiogenesis inhibitors. The current investigation evaluated a potential drug interaction between the angiogenesis inhibitor O-(N-chloroacetyl-carbamoyl)-fumagillol (TNP-470) and the alkylating agent temozolomide (TMZ), in xenograft models that differentially expressed vascular endothelial growth factor (VEGF), a driving force for angiogenesis. Nude rats bearing either s.c. low VEGF (V-) or high VEGF (V+) or intracerebral V+ gliomas were administered either a multiple-dose regimen of TNP-470 or vehicle control. One day after the last dose of vehicle or TNP-470, a steady-state dosing regimen of TMZ was administered with subsequent collection and high-performance liquid chromatography analysis of plasma and either tumor homogenate or tumor microdialysis steady-state TMZ concentrations, and in some cases [5-(3-methyltriazen-1-yl)imidazole-4-carboximide] MTIC, its active metabolite. Microvessel density (MVD) was quantitated by image analysis using an anti-CD31 method. Statistical analyses of pharmacokinetic and pharmacodynamic end points in the control and TNP-470 treatment groups were completed by nonparametric tests. In both the s.c. and intracerebral V+ models, TNP-470 treatment produced significant reductions in TMZ tumor concentrations and tumor:plasma concentration ratios compared with control, being reduced an average of 25% and 50% in the s.c. and intracerebral tumors, respectively. MTIC concentrations in V+ s.c. tumors also were reduced by 50% in the presence of TNP-470. Consistent with the lower extent of neovascularization in the V- tumors, TMZ and MTIC tumor concentrations were not different in TNP-470 and control treatment groups in s.c. tumors. MVD was reduced by TNP-470 compared with vehicle control in the V+ tumors, but was unaltered in V- tumors, attesting to the use of MVD as a pharmacodynamic end point and the effectiveness of TNP-470 as an angiogenesis inhibitor. Angiogenesis inhibitor's pharmacodynamic actions on tumor angiogenesis can produce a reduction in tumor concentrations of coadministered anticancer agents. It is increasingly important to understand the pharmacokinetic and pharmacodynamic behavior of each class of drug so that optimal dosing regimens can be designed.

Angiogenesis Inhibitors↗

PubMed2001

Phase I trial of multiple cycles of high-dose carboplatin, paclitaxel, and topotecan with peripheral-blood stem-cell support as front-line therapy.

PURPOSE: To determine the safety and feasibility of delivering multiple cycles of front-line high-dose carboplatin, paclitaxel, and topotecan with peripheral-blood stem-cell (PBSC) support. PATIENTS AND METHODS: Patients were required to have a malignant solid tumor for which they had received no prior chemotherapy. Mobilization of PBSC was achieved with either filgrastim alone or in combination with cyclophosphamide and paclitaxel. Patients then received three or four cycles of high-dose carboplatin (area under the concentration-time curve [AUC] 16), paclitaxel (250 mg/m(2)), and topotecan (10-15 mg/m(2)), with the latter two agents administered as 24-hour infusions and supported with PBSC and filgrastim. Cycles were repeated every 28 days. RESULTS: Twenty patients were enrolled onto the trial and were assessable for toxicity and clinical outcome. Dose-limiting toxicities were stomatitis and prolonged hematopoietic recovery. The maximum-tolerated dose of topotecan was 12.5 mg/m(2) when given with high-dose carboplatin and paclitaxel for three cycles. Four cycles were able to be given with a dose of topotecan of 10 mg/m(2). The pharmacokinetics of each compound were not affected by the other agents. Eleven (85%) of 13 patients with assessable disease responded. CONCLUSION: Multiple cycles of high-dose carboplatin, paclitaxel, and topotecan can be safely administered with filgrastim and PBSC support. The recommended doses for phase II study are carboplatin AUC 16, paclitaxel 250 mg/m(2), and topotecan 10 mg/m(2). Trials are currently being conducted with this regimen as front-line treatment in patients with advanced ovarian cancer and extensive small-cell carcinoma. This approach remains experimental and should be used only in the context of a clinical trial.

Adult↗

PubMed2001

Targeted delivery of a peripheral benzodiazepine receptor ligand-gemcitabine conjugate to brain tumors in a xenograft model.

PURPOSE: Peripheral benzodiazepine receptors (PBRs) are overexpressed in brain tumors compared to normal brain, and could serve as a target to selectively increase anticancer drug delivery through a PBR ligand-drug conjugate system. We have previously synthesized PBR ligand-gemcitabine conjugates based on the model PBR ligand, PK11195. The goal of the current study was to examine this new drug delivery strategy in an intracerebral xenograft model by measurement of steady-state drug distribution following administration of gemcitabine (GEM) and PK11195-GEM. METHODS: In vitro PBR receptor binding and cytotoxicity assays were used to screen three different PK11195-GEM conjugates (GG01, GG02, GG03) in human SF126 glioma cells. Based on these findings and the favorable chemical stability of GG01, here referred as PK11195-GEM, pharmacokinetic investigations of PK11195-GEM and GEM were conducted in male rats. These studies consisted of single-dose and steady-state dosing regimen studies, the latter to assess drug distribution in normal brain and brain tumors. PK11195-GEM and GEM were measured in blood and tissue samples by HPLC. RESULTS: All PBR-GEM conjugates demonstrated appreciable receptor binding affinity and cytotoxicity with mean IC50 values ranging from 248 to 376 nM and 5.6 to 29.1 nM, respectively. The cytotoxicity of GEM was comparable with a mean IC50 value of 5.9 nM. Following administration of single 8 mg/kg doses of PK11195-GEM to rats (n=4), PK11195-GEM had a mean total clearance of 126.3 +/- 29.6 ml/min per kg, and a volume of distribution at steady-state of 1,261.9 +/- 31.05 ml/kg that resulted in a very short elimination half-life of 16.1 +/- 5.8 min. In comparison GEM had a similar volume of distribution (993.8 +/- 131.6 ml/kg), reduced clearance (3.4 +/- 0.8 ml/ min per kg), and longer half-life (235.6 +/- 26.7 min). In nude rats bearing intracerebral tumors, mean steady-state tumor/plasma, tumor/right brain, and tumor/left brain PK 11195-GEM concentration ratios were 1.75 +/- 0.46, 5.49 +/- 5.2, and 9.96 +/- 3.2, respectively. The analogous values following GEM administrations were 0.81 0.5, 3.67 1.57, and 5.21 +/- 1.95, respectively. These values indicate a minimum twofold increase in tumor target selectivity for the conjugate delivery system compared to GEM treatment. CONCLUSION: Targeting intracellular PBRs is a new drug delivery strategy based on the use of low molecular weight drug conjugates that can be administered systemically. It was demonstrated under steady-state conditions that PK11195-GEM possessed a twofold enhancement in brain tumor selectivity compared to GEM alone. This type of target selectivity would allow higher tumor concentrations to be achieved in conjunction with lower drug concentrations in normal or non-target tissues.

Animals↗

PubMed2001

Sites of phosphorylation in tau and factors affecting their regulation.

The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.

Alzheimer Disease↗

PubMed2001

Androgen-induced up-regulation of tubulin isoforms in neuroblastoma cells.

Androgens regulate the physiology of motor neurones both during development and in adult life. In particular, androgens increase the rate of axonal regeneration after axotomy, an effect correlated with the up-regulation of tubulin. In order to determine whether this was the result of a direct hormone action on neurones, we examined the effect of testosterone on microtubular proteins in human neuroblastoma SH-SY5Y cells. Treatment of proliferating SH-SY5Y cells with testosterone resulted in an up-regulation of alpha- and beta-tubulin. By contrast, no change in tubulin was observed either in cells differentiated into a neuronal phenotype by retinoic acid or in adrenal SW13 cells. We also show that an up-regulation of the ubiquitous beta(II)-tubulin and of the neurone-specific beta(III)-tubulin isoforms contributes to the overall increase in tubulin in response to androgen treatment. The increase in tubulin levels following testosterone treatment was abolished by co-incubation with antiandrogens, indicating that this effect is mediated through a classical mechanism of steroid action. The two microtubule-associated proteins, tau and MAP2b, remained unchanged following testosterone exposure. Thus, these results demonstrate that tubulin is a direct neuronal target of androgen regulation and suggest that dysregulation of tubulin expression may contribute to the pathogenesis of some motor neuronopathies.

Androgen Antagonists↗

PubMed2000

Application of microdialysis to characterize drug disposition in tumors.

Microdialysis is an in vivo sampling technique that was initially developed to measure endogenous substances in the field of neurotransmitter research. In the past decade, microdialysis has been increasingly applied to study the pharmacokinetics and drug metabolism in the blood and various tissues of both animals and humans. This paper describes the general aspects of this in vivo sampling technique followed by the survey of the recent papers regarding the application of microdialysis to characterize anticancer drug disposition in solid tumors. It can be concluded that microdialysis is a very suitable method to obtain drug concentration-time profiles in the interstitial fluid of solid tumors as well as of other variety of tissues.

Animals↗

PubMed2000

Mutually exclusive expression of beta(III)-tubulin and vimentin in adrenal cortex carcinoma SW13 cells.

During embryogenesis, the maturation of neuroblasts into neurones is accompanied by the down-regulation of vimentin and by the expression of neuronal microtubular proteins. Here, we show that human adrenal cortex SW13 cells express beta(III)-tubulin, MAP2b and tau. Analysis of vimentin-positive and -negative subclones of SW13 cells revealed that, under defined cultured conditions, beta(III)-tubulin and MAP2b were present only in vimentin-deficient cells and that beta(III)-tubulin repression occurred at the transcriptional level in vimentin-positive cells. These results suggest that vimentin repression and beta(III)-tubulin expression are co-ordinated by an upstream mechanism relevant to the control of cytoskeletal protein expression during neuronal development.

Adenocarcinoma↗

PubMed2000

Population pharmacokinetic model for topotecan derived from phase I clinical trials.

PURPOSE: To characterize the pharmacokinetics of topotecan in a population model that would identify patient variables or covariates that appreciably impacted on its disposition. PATIENTS AND METHODS: All data were collected from 82 patients entered in four different phase I trials that were previously reported as separate studies from 1992 to 1996. All patients received topotecan as a 30-minute constant-rate infusion on a daily-times-five schedule and were selected for this study because their daily dose did not exceed 2.0 mg/m(2). Among the 82 patients were 30 patients classified as having renal insufficiency and 13 patients with hepatic dysfunction. The population pharmacokinetic model was built in sequential manner, starting with a covariate-free model and progressing to a covariate model with the aid of generalized additive modeling. RESULTS: A linear two-compartment model characterized total topotecan plasma concentrations (n = 899). Four primary pharmacokinetic parameters (total clearance, volume of the central compartment, distributional clearance, and volume of the peripheral compartment) were related to various combinations of covariates. The relationship for total clearance (TVCL [L/h] = 32.0 + [0.356(WT - 71) + 0.308(HT - 168.5) - 8.42(SCR - 1.1)] x [1 + 0.671 sex]) was dependent on the patients' weight (WT), height (HT), serum creatinine (SCR), and sex and had a moderate ability to predict (r(2) = 0.64) each patient's individual clearance value. The addition of covariates to the population model improved the prediction errors, particularly for clearance. Removal of 10 outlying patients from the analysis improved the ability of the model to predict individual clearance values (r(2) = 0.77). CONCLUSION: A population pharmacokinetic model for total topotecan has been developed that incorporates measures of body size and renal function to predict total clearance. The model can be used prospectively to obtain a revised and validated model that can then be used to design individualized dosing regimens.

Aged↗

PubMed2000

Phase I pharmacokinetic trial of perillyl alcohol (NSC 641066) in patients with refractory solid malignancies.

Perillyl alcohol (POH) is a monoterpene with anticarcinogenic and antitumor activity in murine tumor models. Putative mechanisms of action include activation of the transforming growth factor beta pathway and/or inhibition of p21ras signaling, leading to differentiation or apoptosis. In this Phase I trial, 17 patients took POH p.o. three times daily for 14 days of each 28-day cycle. The starting dose of POH was 1600 mg/m2/dose, with escalations to 2100 and 2800 mg/m2/dose in subsequent cohorts. Chronic nausea and fatigue were dose-limiting toxic effects at 2800 mg/m2. Grade 1-2 hypokalemia was common at 2100 and 2800 mg/m2. Although POH could not be detected in plasma, two of its metabolites, dihydroperillic acid (DHPA) and perillic acid (PA), were measured in plasma and urine on days 1 and 15 after the first and last doses of POH, respectively. Both area under the concentration versus time curve and peak plasma concentration (Cmax) values increased with dose and exhibited high intersubject variability. Day 15 DHPA Cmax values ranged from a mean +/- SD of 22.6+/-12 microM at 1600 mg/m2/dose to 42.4+/-15.24 microM at 2800 mg/m2/dose. Corresponding mean +/- SD Cmax values for PA were 433.2+/-245.8 and 774.1+/-439.6 microM. One patient treated at the 2800 mg/m2/dose had markedly prolonged plasma levels of both PA and DHPA and developed grade 3 mucositis. POH treatment did not consistently alter the expression of p21ras, rap1, or rhoA in peripheral blood mononuclear cells obtained from patients treated at the highest dose level. The metabolites PA and DHPA did not change expression or isoprenylation of p21ras in MCF-7 breast or DU145 prostate carcinoma cells at concentrations that exceeded those achieved in patient plasma after POH treatment. We conclude that POH at 1600-2100 mg/m2 p.o. three times daily is well tolerated on a 14-day on/14-day off dosing schedule. Inhibition of p21ras function in humans is not likely to occur after POH administration at safe doses of the present oral formulation.

Administration, Oral↗

PubMed2000

Pharmacokinetics of the chemopreventive agent oltipraz and of its metabolite M3 in human subjects after a single oral dose.

The dithiolethione oltipraz (OPZ) has activity as a chemopreventive agent in animal models and is in early clinical trials. OPZ undergoes metabolism by molecular rearrangement to yield a pyrrolopyrazine derivative, M3, which we have previously shown to be inactive in the induction of detoxication genes. M3 is metabolized further: at least 10 possible conjugates have been described in three species. We developed a new high-performance liquid chromatography method to simultaneously measure plasma concentrations of OPZ and of M3. This method was applied to serial plasma samples in a Phase I clinical trial, in which OPZ was administered at single doses varying from 125 to 1000 mg/m2. OPZ and M3 concentration-time profiles were highly variable among individuals, and the occurrence of secondary concentration peaks suggested substantial enterohepatic cycling. Absorption was rapid, and the mean time to peak was 2.2 h. Maximum plasma concentration values were proportional to the dose. Harmonic mean half-lives at these doses ranged from 9.3-22.7 h. There were indications of dose-dependent pharmacokinetic properties because apparent clearance and volume of distribution at steady state increased with dose, although these changes were not statistically significant as a result of high interpatient variability. Accordingly, there were less than proportional increases in the OPZ and M3 area under the curve and maximum plasma values. Interpretation of OPZ and M3 disposition is confounded by the unknown bioavailability factor; however, the most likely inferences are that bioavailability of OPZ decreases with increasing dose and that metabolism to M3 is saturable.

Administration, Oral↗

PubMed1999

Activation of the AT(2) receptor of angiotensin II induces neurite outgrowth and cell migration in microexplant cultures of the cerebellum.

Microexplant cultures from three-day-old rats were used to investigate whether angiotensin II (Ang II), through its AT(1) and AT(2) receptors, could be involved in the morphological differentiation of cerebellar cells. Specific activation of the AT(2) receptor during 4-day treatment induced two major morphological changes. The first was characterized by increased elongation of neurites. The second change was cell migration from the edge of the microexplant toward the periphery. Western blot analyses and indirect immunofluorescence studies revealed an increase in the expression of neuron-specific betaIII-tubulin, as well as an increase in expression of the microtubule-associated proteins tau and MAP2. These effects were demonstrated by co-incubation of Ang II with 1 microM DUP 753 (AT(1) receptor antagonist) or with 10 nM CGP 42112 (AT(2) receptor agonist) but abolished when Ang II was co-incubated with 1 microM PD 123319 (AT(2) receptor antagonist), indicating that differentiation occurs through AT(2) receptor activation and that the AT(1) receptor inhibits the AT(2) effect. Taken together, these results demonstrate that Ang II is involved in cerebellum development for both neurite outgrowth and cell migration, two important processes in the organization of the various layers of the cerebellum.

Angiotensin II↗

PubMed1999

Distribution of small magnetic particles in brain tumor-bearing rats.

Small (10-20 nm) uncharged magnetic particles (SMP) were evaluated for their ability to target intracerebral rat glioma-2 (RG-2) tumors in vivo. In an effort to determine the influence of particle size on blood-tumor barrier uptake, the tissue distribution of the injected particles was evaluated following intraarterial injection (4 mg/kg SMP) in male Fisher 344 rats bearing RG-2 tumors with a magnetic field of 0 G or 6000 G applied to the brain for 30 min. Animals were sacrificed at 30 min or 6 h post-injection after which tissues were collected and analyzed for magnetite content. In the presence of a magnetic field, SMP localized in brain tumor tissue at levels of 41-48% dose/g tissue after 30 min and 6 h respectively, significantly greater than non-target tissues. In the absence of a magnetic field only 31-23% dose/g tissue was achieved for the same time points. Tumor targeting of the SMP for brain tumor was demonstrated by large target selectivity indexes (ts) of 2-21 for normal brain tissue, indicating a 2-21 fold increase in concentrations compared to normal brain. In comparison with larger (1 micron) diameter magnetic particles, SMP concentrated in brain tumor at significantly higher levels than magnetic neutral dextran (p = 0.0003) and cationic aminodextran (p = 0.0496) microspheres previously studied. TEM analysis of brain tissue revealed SMP in the interstitial space of tumors, but only in the vasculature of normal brain tissue. These results suggest that changes in the vascular endothelium of tumor tissue promote the selective uptake of SMP and provide a basis for the design of new small drug-loaded particles as targeted drug delivery systems for brain tumors.

Animals↗

PubMed1998

Identification of novel in vitro PKA phosphorylation sites on the low and middle molecular mass neurofilament subunits by mass spectrometry.

Phosphorylation of the head domains of intermediate filament proteins by second messenger-dependent kinases is important in regulating filament assembly. In the case of neurofilaments, head domain phosphorylation is known to be important in assembly, but few sites have been identified. Using matrix-assisted laser desorption-ionization (MALDI) and nano-electrospray mass spectrometry, we report the identification of several novel in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites in the low (NF-L) and middle (NF-M) molecular mass neurofilament subunits. Neurofilament polypeptides were purified from adult rat brain, and fractions containing a mixture of NF-L and NF-M were nonradioisotopically phosphorylated with PKA prior to proteolytic digestion of the polypeptides in situ in polyacrylamide excised from SDS gels. Sites of phosphorylation were determined by mass spectrometric analysis of mixtures enriched in tryptic phosphopeptides. In NF-L, four novel sites were identified: serines 12, 41, and 49 in the head domain and serine 435 in the carboxyl-terminal tail domain, and data consistent with phosphorylation of serine 2 were obtained. Recombinant rat NF-L protein was also phosphorylated with PKA, and the same serines were identified as phosphorylation sites, with two additional sites, serine 43 and probable phosphorylation of serine 55. In NF-M, one novel site, serine 1 in the amino-terminal head domain, was found to be phosphorylated, and serine 46, also in the amino-terminal head domain, was confirmed as a PKA phosphorylation site.

Amino Acid Sequence↗

PubMed1998

Use of capillary electrophoresis methods to characterize the pharmacokinetics of antisense drugs.

As antisense drugs become mature for clinical trials, analytical techniques to analyze antisense DNA in biological media for characterization of their pharmacokinetics will be in demand. Due to the superior resolving power of capillary gel electrophoresis (CGE), CGE will likely be a preferred method in quantifying intact oligonucleotides as well as the putative metabolic products. Nonetheless, biological mediums can influence the stability of the gel column, making a CGE assay time-consuming. In one approach, high-performance liquid chromatography (HPLC) was used to quantify the total amount of antisense compounds to increase the sample throughput and CGE was used to determine the relative percentage of the intact and metabolic species on specific samples. Alternatively, extensive sample pretreatment procedures were performed and the samples were quantified and characterized directly by CGE alone with the use of an internal standard. Both methods have been used to characterize the pharmacokinetics of antisense compounds. This review focuses on the instrumental and technical aspects of analyzing antisense DNA in biological mediums using CGE either as a single or a combined method towards better understanding of the pharmacokinetics of antisense DNA. Moreover, the newer analytical technologies of capillary electrophoresis (CE), which hold great potential to be used for pharmacokinetic applications, such as the replenishable sieving matrix combined with an innovative coupling approach and microchip CE, will also be explored.

Animals↗

PubMed1998

Modulation of angiogenesis by human glioma xenograft models that differentially express vascular endothelial growth factor.

To evaluate the potential actions of vascular endothelial growth factor (VEGF) on capillary permeability and drug transport, tumorigenic human glioma cell lines were developed that expressed different levels of VEGF. Three human glioma cell lines (i.e. U373, SF126, SF188) were screened for VEGF under normoxic and hypoxic (i.e. induced by CoCl2) conditions by Western blot analysis. Subsequent to these results, sense and antisense VEGF164 cDNA transfections were conducted. It was found that parental SF188 (SF188/V-) and SF188/V+ (sense transfected) cells could serve as an appropriate in vivo model based on their divergent levels of VEGF expression. Media derived from SF188/V+ cells stimulated endothelial cell growth by 30-60%, and enhanced endothelial cell clonogenicity by 5-10-fold compared to SF188/V- or empty vector transfected cells. Nude rats implanted with either SF188/V- or SF188/V+ cells subcutaneously and intracerebrally formed tumors, with those derived from SF188/V+ cells growing at a faster rate. Immunohistochemistry analysis indicated that the expression of VEGF and number of capillaries (factor VIII assay) were approximately 3-fold greater in SF188/V+ tumors compared to SF188/V- tumors. Pharmacological assays, such as measurements of cytotoxicity and DNA adducts, in SF188/V- and SF188/V+ cells treated with carmustine or temozolomide were similar. Therefore, other than differences in VEGF expression and growth in vivo, SF188/V- and SF188/V+ cells possess a similar phenotype, and can serve as models to evaluate the influence of VEGF on drug transport.

Animals↗

PubMed1998

Delivery of cytotoxic drugs from carrier cells to tumour cells by apoptosis.

A new drug delivery approach, apoptotic-induced drug delivery (AIDD), is presented that is based on apoptosis as a mechanism to trigger delivery of drugs from carrier cells. It was investigated whether apoptotic drug-loaded carrier cells could deliver drugs to tumour cells by various mechanisms, including drug release through a more permeable apoptotic cell membrane, and by phagocytosis of drug-loaded apoptotic cells by tumour cells. The feasibility of this novel concept was evaluated in an in vitro carrier cell model that consisted of S49 mouse lymphoma cells that apoptose upon exposure to dexamethasone (DX). Membrane permeability was evaluated by measurement of release of a fluorescent dye (calcein-AM, C-AM) from C-AM-loaded S49 cells. Phagocytotsis of fluorescent PKH-26-labeled S49 cells was determined in co-culture studies with rat glioma (RG-2) cells using fluorescence microscopy and flow cytometry. Cytotoxicity of RG-2 cells due to temozolomide (TMZ)-loaded S49 cells was evaluated by a colony formation assay following co-culture of these cells for up to 8h. Calcein release from S49 cells was enhanced by approximately 30% at 48 h following treatment with DX compared to control S49 cells. Based on both flow cytometric and microscopic analyses, RG2 phagocytized apoptotic S49 cells to a four- to sevenfold greater extent than control S49 cells at co-incubation times from 4-48 h. The TMZ-loaded apoptotic S49 cells caused the largest degree of toxicity, about 50% cell kill, whereas TMZ-loaded control S49 caused 30% cell kill. The preliminary data suggest that AIDD should be further explored.

Journal Article↗

PubMed1998

Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA) is a rare form of motor neuron degeneration linked to a CAG repeat expansion in the first exon of the androgen receptor gene coding for a polyglutamine tract. In order to investigate the properties of the SBMA androgen receptor in neuronal cells, cDNAs coding for a wild-type (19 CAG repeats) and a SBMA mutant androgen receptor (52 CAG repeats) were transfected into mouse neuroblastoma NB2a/d1 cells. The full length androgen receptor proteins, of 110-112 kDa and 114-116 kDa for the wild-type and mutant protein, respectively, were detected by Western blotting in transfected cells. In addition, the presence of an expanded polyglutamine tract in the SBMA androgen receptor appears to enhance the production of C-terminally truncated fragments of the protein. A 74 kDa fragment was particularly prominent in cells expressing the SBMA androgen receptor. From its size, it can be deduced that the 74 kDa fragment lacks the hormone binding domain but retains the DNA binding domain. The 74 kDa fragment may therefore be toxic to motor neurons by initiating the transcription of specific genes in the absence of hormonal control. Immunofluorescence microscopy on transfected NB2a/d1 cells showed that, after hormone activation, the wild-type androgen receptor translocated to the nucleus whereas the SBMA androgen receptor was mainly localized in the cytoplasm in the form of dense aggregates with very little androgen receptor protein in the nucleus. This could explain the reduction in transcriptional activity of the SBMA mutant as compared with wild-type androgen receptor.

Animals↗