Cadherin switching dictates the biology of transitional cell carcinoma of the bladder: ex vivo and in vitro studies

Bladder cancer is the fifth most common malignancy in the UK. Clinically, the most important process in determining prognosis is the development of invasion, initially of the lamina propria and then beyond as these transitional cell carcinomas (TCCs) progress from stage pT1 to stages T2+. Cadherins and catenins are the main mediators of cell-cell interactions in epithelial tissues, and loss of membranous E-cadherin immunoreactivity is strongly correlated with high grade, advanced stage and poor prognosis in bladder cancer and other malignancies. However, the role of P-cadherin is yet to be fully elucidated in bladder TCC. The objectives of this study were to establish how the expression of cadherins and catenins determines clinical and in vitro behaviour in bladder TCC. Utilizing immunohistochemistry, immunofluorescence and western blotting, we demonstrated a significant reduction in the expression of E-cadherin and beta-catenin as grade and stage of bladder TCC progress, accompanied by a significant increase in P-cadherin expression (all p < 0.05, Pearson's chi2 test). Increased P-cadherin expression was also associated with a significantly worse bladder cancer-specific survival (log rank p = 0.008), with Cox regression showing P-cadherin to be an independent prognostic factor. Utilizing a variety of tissue culture models in a range of functional studies, we demonstrated that P-cadherin mediates defective cell-cell adhesion and enhances anchorage-independent growth. The results provide evidence that increased P-cadherin expression promotes a more malignant and invasive phenotype of bladder cancer, and appears to have a novel role late in the disease.     

Correlation between in vivo and in vitro toxic effects of foreign compounds

Studies in rats in vivo and in isolated hepatocytes from the same species and strain of animal in vitro with the hepatotoxicant hydrazine have shown that despite measuring the same parameters in each system, the effects do not always show a quantitative or qualitative correlation. For example depletion of glutathione and ATP occurred in both systems but required a much higher concentration of hydrazine in vitro. The effects on triglyceride levels, citrulline synthesis and taurine levels in vivo were not observed in vitro and the inhibition of urea synthesis and cytotoxicity in vitro were not observed in vivo.Inhibition of protein synthesis proved to be the marker which showed the best correlation, occurring at a similar hydrazine concentration in vitro and in vivo although not to the same extent.The situation with other toxicants is variable, in some cases correlation is good, in others modification of conditions in vitro are required.     

Ribozyme mediated destruction of influenza A virus in vitro and in vivo

Short catalytic RNAs with inherent, specific endoribonuclease activity, called ribozymes, have recently been shown to exist in nature. According to the structural models artificial ribozymes have been designed that can potentially hydrolyse any chosen target RNA sequence in trans at a specific site. We have constructed and characterized in vitro hammerhead and hairpin ribozymes designed to cleave viral RNA segment 5 of influenza A virus. Both ribozymes were functional under optimal in vitro conditions, but quantitative measurements indicate that the hammerhead ribozyme is considerably more efficient at this target site than the hairpin ribozyme. Mg²⁺ dependent hammerhead ribozyme-mediated cleavage reactions were enhanced at higher temperature and in presence of spermidine, but catalytic activities were retained also in cellular extract S-100 or nuclear extracts at physiological temperatures. Recombinant plasmids derived from transfection vector pSV2-neo were engineered to allow the expression of specific ribozymes under the control of SV40 early promoter or SV40 early+ late promoters. These plasmids were introduced by transfection into COS cells, and their expression and enzymatic activities were analyzed in stable cell lines after selection of neomycin-re-sistance. Several permanent ribozyme-express-ing clones were established and characterized: ribozyme coding DNA sequences and synthesis of ribozyme RNA molecules in the transfected cells were determined and monitored by polymerase chain reactions. It was found that the highest levels (up to 70-80%) of resistance to influenza A virus strain X-31 super-infection was observed in COS cells transfected with plasmids containing SV40 early or SV40 early+late promoters coinciding with relatively high and constitutive rates of ribozyme expression. These results suggest the feasibility of developing ribozymes designed against influenza virus to achieve therapeutic value. © 1994 Wiley-Liss, inc.     

Effects of liver ischemia on hepatic protein synthesis in vitro and in vivo

When an in vitro system is used to study the influence of ischemia on hepatic protein synthesis, an important question is whether alterations observed in vitro reflect changes in vivo. In the present study the effects of liver ischemia on protein synthesis were investigated in rats both in vitro and in vivo. Liver ischemia was induced by hepatic artery ligation. Protein synthesis in vitro was determined from leucine incorporation into proteins in liver slices incubated in a medium containing ¹⁴C-leucine (0.5 mmol/l) and in vivo from leucine incorporation into hepatic proteins after intraportal injection of a tracer dose of ¹⁴C-leucine. Leucine incorporation rate in non-ischemic liver was 0.16 pmol * g pror¹ h-¹ in vitro and 19.6 μmol g prot-¹. h^-1 in vivo. After hepatic artery ligation protein synthesis in vitro was reduced by about 60% and in vivo by about 80%. Thus, the relative changes were of the same magnitude in vitro and in vivo. This indicates that an in vitro system can be used to evaluate the effects of liver ischemia on hepatic protein synthesis.     

Meiotic spindle morphogenesis in in vivo and in vitro matured mouse oocytes: insights into the relationship between nuclear and cytoplasmic quality

BACKGROUND: This work addresses the hypothesis that events occurring within the follicle soon after the LH surge are essential for coordinating morphogenesis of the spindle and cytoplasm in mouse oocytes matured in vivo (IVO); we further tested whether in vitro maturation (IVM) fails to support these events. METHODS: Oocytes collected at 1, 2, 3, 4 and 5 h post-hCG or after IVM were analyzed for chromatin, nuclear lamina, microtubules (MTs) and centrosomal proteins by conventional fluorescence and confocal microscopy. In addition, these parameters were monitored in oocytes maintained in 50 microM roscovitine, followed by IVM, or in oocytes retrieved at 1.5 and 5 h post-hCG in vivo and cultured up to 16 h. RESULTS: A G2/M delay was observed in IVO oocytes based upon persistence of cytoplasmic MTs, nuclear lamina and centrosomes at the cortex; rapid meiotic progression in IVM oocytes was related to loss of these markers, indicating that a global activation of MPF occurred in culture. Also, maturating-promoting factor (MPF) inactivation resulted in cultured oocytes that exhibited IVO characteristics after drug removal. IVO-like characteristics were also exhibited by oocytes retrieved at 5 but not at 1.5 h after hCG treatment, even though these oocytes were subsequently cultured. CONCLUSIONS: The results emphasize the importance of coupling MT remodeling and cell cycle components during oocyte maturation to achieve a balanced coordination of nuclear and cytoplasmic maturation that under physiological conditions occurs within the first 5 h of LH stimulation.     

1H and 13C HR-MAS spectroscopy of intact biopsy samples ex vivo and in vivo 1H MRS study of human high grade gliomas

High-resolution magic angle spinning (HR-MAS) one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy has been used to study intact glioblastoma (GBM) brain tumour tissue. The results were compared with in vitro chemical extract and in vivo spectra. The resolution of 1H one-dimensional, 1H TOCSY and 13C HSQC HR-MAS spectra is comparable to that obtained on perchloric extracts. 13C HSQC HR-MAS spectra have been particularly useful for the identification of 37 different metabolites in intact biopsy tumours, excluding water and DSS components. To our knowledge, this is the most detailed assignment of biochemical compounds obtained in intact human tissue, in particular in brain tumour tissue. Tissue degradation during the recording of the NMR experiment was avoided by keeping the sample at a temperature of 4 degrees C. Detailed metabolical compositions of 10 GBM (six primary, two secondary and two unclassified) were obtained. A good correlation between ex vivo and in vivo MRS has been found.     

Validation studies with Muta Mouse: a transgenic mouse model for detecting mutations in vivo.

MutaMouse is a transgenic mouse engineered to detect mutations in vivo in any tissue of choice by using simple laboratory methods. The target is a bacterial lacZ gene incorporated via lambda phage into the genome of each mouse cell such that a concatamer of approximately 40 copies exists at a single site on both chromosomes of a homologous pair. In order to assess the potential usefulness of MutaMouse in detecting in vivo mutagenesis, several known mutagens/carcinogens were applied to male animals of 8-10 weeks in age. Intraperitoneal injections (single or 5 daily doses) of N-ethyl-N-nitrosourea (ENU), chlorambucil, procarbazine, cyclophosphamide, and acrylamide were investigated for mutagenic effects in bone marrow, liver, and testes. In addition, skin painting studies (single application) were performed with dimethylbenzanthracene (DMBA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and acetic acid. Increases in mutant frequency were clearly induced by all eight chemicals, the magnitudes of which were dependent on the chemical, dose, method of dosing, tissue analyzed, and the time lapse between treatment and isolation of DNA. Data on variability in mutant frequency was presented relative to the analyzed population of lacZ genes and number of animals per treatment group. Application of the MutaMouse model to the detection of heritable mutations was discussed.     

The mutagenic activity of 5-bromo-2′-deoxyuridine (BrdU) in vivo in rats

BrdU tablets were implanted subcutaneously in rats, and BrdU concentrations were determined in the serum. Within 5 hr peak concentrations of 10 μg BrdU/ml blood were reached. The influence of BrdU in vivo on cell cycling, DNA synthesis, spontaneous sister chromatid exchange (SCE) frequencies, and gene-mutation frequencies (6-TG^r) was determined in freshly isolated cells from a subcutaneous granulation tissue. The most significant effect of BrdU in vivo was a doubling of the spontaneous 6-TG^r frequency. In reconstruction experiments in vitro the mutagenic activity of BrdU applied in concentrations found in vivo was 2.5–6-fold higher. With the use of agar-coated tablets, BrdU concentrations in the blood were reduced by half, and no peak concentration was found. The differential staining of chromatids was still sufficient. Since the mutagenic effect of BrdU in vitro was found to be strongly concentration dependent, the use of agar-coated tablets is recommended in experiments in which the compound is used to demonstrate SCE in vivo.     

Establishment and characterization of an in vivo model for Epstein-Barr virus positive gastric carcinoma

Research regarding the role of the Epstein-Barr virus (EBV) in gastric carcinogenesis has been hampered by the absence of a suitable model system. SNU-719 is a gastric carcinoma cell line naturally infected with EBV. This cell line developed tumors in nude mice approximately 40-56 days after inoculation. SNU-719 also showed low serum dependency and anchorage independent growth in vitro. The developed tumors expressed EBERs, EBNA1, and LMP2A but not other EBV latent genes. Additionally, Qp was active and either mono- or bi-clonal EBV genome was observed in the tumor tissues. Because the developed tumors retained characteristics of EBV-associated gastric cancer, this cell line could serve as a useful in vivo system to investigate the tumorigenesis mechanism and treatment methods for this type of tumor.     

In vitro and in vivo mammalian mutation assays support a nonmutagenic mechanism of carcinogenicity for hydrazine

Hydrazine has been described as a mutagenic, probable human carcinogen. It is mutagenic in in vitro systems such as bacterial reverse mutation (Ames) tests and some yeast systems, as well as in in vivo systems with drosophila. It was shown to cause chromosome damage both in vitro and in vivo but was negative in some well‐validated mammalian mutation systems such as CHO HPRT assays. Importantly, there is only one in vivo gene mutation test reported, which was negative. Our objective was to determine if hydrazine is mutagenic in mammalian test systems. Thus, we conducted an in vitro gene mutation test in Muta?Mouse lung epithelial cells (FE1 cell assay) and a regulatory‐compliant in vivo Big Blue® mouse test. Consistent with previous reports, an additional six‐well Ames assay showed that hydrazine was mutagenic to bacteria. The FE1 cell assay was negative in conditions with and without metabolic activation when tested to cytotoxicity limits. In the Big Blue® mouse study, female mice received dosages of hydrazine up to 10.9 mg/kg via drinking water for 28 days. This dose is comparable to a dose used in a carcinogenicity study where female mice had significant increases in hepatocellular adenoma at 11.5 mg/kg. There were no increases in mutant frequency in liver and lung, two tissues sensitive to the carcinogenic effects of hydrazine in mice. Our research shows that hydrazine is not mutagenic in mammalian cells either in vitro or in vivo, indicating mutagenicity may not play a role in the carcinogenicity of hydrazine.     

Microstructural models for diffusion MRI in breast cancer and surrounding stroma: an ex vivo study

The diffusion signal in breast tissue has primarily been modelled using apparent diffusion coefficient (ADC), intravoxel incoherent motion (IVIM) and diffusion tensor (DT) models, which may be too simplistic to describe the underlying tissue microstructure. Formalin‐fixed breast cancer samples were scanned using a wide range of gradient strengths, durations, separations and orientations. A variety of one‐ and two‐compartment models were tested to determine which best described the data. Models with restricted diffusion components and anisotropy were selected in most cancerous regions and there were no regions in which conventional ADC or DT models were selected. Maps of ADC generally related to cellularity on histology, but maps of parameters from more complex models suggest that both overall cell volume fraction and individual cell size can contribute to the diffusion signal, affecting the specificity of ADC to the tissue microstructure. The areas of coherence in diffusion anisotropy images were small, approximately 1 mm, but the orientation corresponded to stromal orientation patterns on histology.     

Development and evaluation of olanzapine-loaded PLGA nanoparticles for nose-to-brain delivery: in vitro and in vivo studies

Olanzapine (OZ) is a second-generation or atypical antipsychotic which selectively binds to central dopamine D? and serotonin (5-HT(2c)) receptors. It has poor bioavailability due to hepatic first-pass metabolism and low permeability into the brain due to efflux by P-glycoproteins. The present investigation aimed to prepare a nanoparticulate drug delivery system of OZ using poly(lactic-co-glycolic acid) (PLGA) for direct nose-to-brain delivery to provide brain targeting and sustained release. PLGA nanoparticles (NP) were prepared by the nanoprecipitation technique and characterized by entrapment efficiency, particle size, zeta potential, modulated temperature differential scanning calorimetry (MTDSC) and X-ray diffraction (XRD) studies. The NP were evaluated for in vitro release, ex vivo diffusion, toxicity and pharmacokinetic studies. The NP were 91.2±5.2 nm in diameter and had entrapment efficiency 68.91±2.31%. MTDSC studies indicated broadening of the drug peak and a shift in the polymer peak, possibly due to physical interaction or H-bonding between the carbonyl groups of PLGA and the NH groups of OZ, and also due to the plasticization effect of OZ on PLGA. XRD studies indicated a decrease in the crystallinity of OZ or amorphization. In vitro drug release showed a biphasic pattern with initial burst release and, later, sustained release (43.26±0.156% after 120 h), following the Fickian diffusion-based release mechanism. Ex vivo diffusion through sheep nasal mucosa showed 13.21±1.59% of drug diffusion in 210 min from NP. Histopathological study of sheep nasal mucosa showed no significant adverse effect of OZ-loaded NP. In vivo pharmacokinetic studies showed 6.35 and 10.86 times higher uptake of intranasally delivered NP than OZ solution delivered through intravenous (IV) and intranasal (IN) route, respectively. These results proved that OZ could be transported directly to the brain after IN delivery of PLGA NP, enhanced drug concentration in the brain and would therefore be effective in improving the treatment of central nervous system disorders.     

Evaluation of the in vivo mutagenicity of isopropyl methanesulfonate in acute and 28‐day studies

Understanding the mutagenic dose response could prove beneficial in the management of pharmaceutically relevant impurities. For most alkyl ester impurities, such as isopropyl methanesulfonate (IPMS), little in vivo mutagenicity data exist for dose analysis. The likelihood of a sublinear dose response for IPMS was assessed by comparing the Swain Scott constant, the S_N1/S_N2 reaction mechanism and the O⁶:N⁷ guanine adduct ratio to that of more well‐known alkyl esters. Based on available information, IPMS was predicted to have a mutagenic profile most like ethyl nitrosourea. To test this hypothesis, mature male Wistar Han rats were administered IPMS using acute (single administration at 3.5 to 56 mg/kg) or subchronic (28 days at 0.125 to 2 mg/kg/day) exposures. The in vivo Pig‐a mutation assay was used to identify mutant phenotype reticulocyte (Ret) and red blood cell (RBC) populations. The maximum mutant response occurred approximately 15 and 28 days after the last dose administration in the mutant Ret and RBC populations respectively in the acute study and on Day 29 and 56 in the mutant Ret and RBC populations, respectively, in the subchronic study. A comparison of RBC mutant frequencies from acute and subchronic protocols suggests a sublinear response; however, this was not substantiated by statistical analysis. A No Observed Effect Level (NOEL) of 0.25 mg/kg/day resulted in a Permitted Daily Exposure equivalent to the Threshold of Toxicological Concern. An estimate of the NOEL based on the previously mentioned factors, in practice, would have pre‐empted further investigation of the potent mutagen IPMS. Environ. Mol. Mutagen. 56:322–332, 2015. © 2014 Wiley Periodicals, Inc.     

Low oxygen tension, as found in tissues in vivo, alters the mutagenic activity of aristolochic acid I and II in primary fibroblast-like rat cells in vitro

The mutagenic activities of aristolochic acid I (AAI) and II (AAII), the two main components of aristolochic acid (AA), were tested for mutagenicity in vivo in a subcutaneous granulation tissue in rats and in vitro in the corresponding freshly isolated and cultured target cells. In vivo at equimolar dose, AAI induced 16 times more 6-thioguanine resistant cells than AAII. Oxygen tension in vitro was adjusted to that found in vivo: in the subcutaneous connective tissue, the pO2 was determined to be 40 +/- 9 mm Hg, which corresponds in vitro to an O2 concentration of 5% in the incubator atmosphere. In vitro, AAI was 19 times more mutagenic than AAII at this low oxygen tension but exhibited only 4 times greater activity than AAII under standard culture conditions. These results indicate that the genotoxic activity of AA in mammals is mainly caused by AAI and that the exposure of cells to AAI and AAII in vitro at low pO2 corresponds more closely to the metabolic situation in vivo. Therefore, the mutagenic potency of the two chemicals can only be estimated correctly at tissue oxygen tension. The influence of pO2 on the mutation frequencies seems to arise from a modulation of the activation/detoxification pathways.     

Determination of hprt mutant and mutation frequencies and the molecular characterization of human derived in vivo T-lymphocyte mutants

Using a T-lymphocyte clonal assay, 73 6-thiogua-nine resistant T-lymphocytes were isolated from two blood samples obtained 4 months apart from a 50-year-old male subject. Sixty-six of these mutants were characterized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T-cell receptors (TCR) of these mutants all share a common γ-TCR rearrangement, and thus likely represent a single mutational event that underwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also recovered more than once. In two of the three cases, the mutants were also found to be clonally related, while in one case they were not. A number of identical exon loss events were also recovered, yet none of these were clonally related. This probably reflects the multiple pathways by which these mutations can arise. The TCR data was used to correct the observed mutant frequency to produce an estimate of the actual mutation frequency. The two mutant frequencies, 18 × 10^?6 and 19 × 10^?6, obtained from the first and second sampling periods, respectively, can thus be corrected to yield true mutation frequency's of 12 × 10^?6 each © 1995 Wiley-Liss, Inc.     

Regulation of T-cell migration and effector functions: insights from in vivo imaging studies

Summary: Studies of the immune system are providing us with ever more detailed information on the cellular and molecular mechanisms that underlie our evolutionarily conserved ability to fend off infectious pathogens. Progress has probably been fastest at two levels: the various basic biological functions of isolated cells on one side and the significance of individual molecules or cells to the organism as a whole on the other. In both cases, direct phenomenological observation has been an invaluable methodological approach. Where we know least is the middle ground, i.e. how immune functions are integrated through the dynamic interplay of immune cell subsets within the organism. Most of our knowledge in this area has been obtained through inference from static snapshots of dynamic processes, such as histological sections, or from surrogate cell co-culture models. The latter are employed under the assumption that an in vivo equivalent exists for each type of cellular contact artificially enforced in absence of anatomical compartmentalization. In this review, we summarize recent insights on migration and effector functions of T cells, focusing on observations gained from their dynamic microscopic visualization in physiological tissue environments.     

Development and in vivo evaluation of an oral drug delivery system for paclitaxel

The aim of the present study was to investigate the effect of poly(acrylic acid)-cysteine (PAA-cysteine) exhibiting a molecular mass of 100 and 250 kDa and reduced glutathione (GSH) on the absorption of the P-glycoprotein (P-gp) and cytochrome P450 (CYP450) substrate paclitaxel in vitro and in vivo. In vitro transport studies were performed with Caco-2 monolayers. Furthermore, the delivery system based on PAA-cysteine, GSH and paclitaxel was evaluated in vivo in rats. In vitro, the formulation comprising 0.5% (m/v) PAA-cysteine (100 kDa)/0.5% (m/v) GSH improved the transport of paclitaxel 6.7-fold (P(app) = 8.7 ± 1.3 × 10(-6) cm/s) in comparison to paclitaxel itself serving as buffer only control (P(app) = 1.3 ± 0.4 × 10(-6) cm/s). Moreover, in the presence of the formulation containing 0.5% (m/v) PAA-cysteine (250 kDa)/0.5% (m/v) GSH paclitaxel absorption was even 7.4-fold (P(app) = 9.7 ± 0.3 × 10(-6) cm/s) improved in comparison to the buffer only control. In vivo, the oral administration of formulations containing 1 mg of paclitaxel, 1 mg of GSH and 8 mg of PAA-cysteine (100 kDa or 250 kDa) resulted in an improved paclitaxel plasma concentration and bioavailability. The area under the plasma concentration-time curve (AUC(0-8)) of paclitaxel was 4.7-fold and 5.7-fold improved in comparison to the oral formulation containing paclitaxel alone, respectively. Moreover, c(max) was improved by 6.3-fold and even 7.3-fold in comparison to the oral formulation containing paclitaxel alone, respectively. Thus, according to the achieved results it is suggested that PAA-cysteine in combination with GSH would be a potentially valuable tool for improving the oral bioavailability of P-gp and CYP450 substrates such as paclitaxel.     

Quantitative susceptibility mapping and R1 measurement: Determination of the myelin volume fraction in the aging ex vivo rat corpus callosum

In studies of the white matter (WM) in aging brains, both quantitative susceptibility mapping (QSM) and direct R₁ measurement offer potentially useful ex vivo MRI tools that allow volumetric characterization of myelin content changes. Despite the technical importance of such MRI methods in numerous age-related diseases, the supposed linear relationship between the estimates of either the QSM or R₁ method and age-affected myelin contents has not been validated. In this study, the absolute myelin volume fraction (MVF) was determined by transmission electron microscopy (TEM) as a gold standard measure for comparison with the values obtained by the aforementioned MR methods. To theoretically evaluate and understand the MR signal characteristics, QSM simulations were performed using the finite perturber method (FPM). Specifically, the simulation geometry modeling was based on TEM-derived structures aligned orthogonally to the main magnetic field, the construct of which was used to estimate the magnetic field shift (ΔB) changes arising from the conjectured myelin structures. Experimentally, ex vivo corpus callosum (CC) samples from rat brains obtained at 6 weeks (n = 3), 4 months (n = 3), and 20 months (n = 3) after birth were used to establish the relationship between changes quantified by either QSM or R₁ with the absolute MVF by TEM. From the ex vivo brain samples, the scatterplot of mean MVF versus R₁ was fitted to a linear equation, where R_1mean = 0.7948 × MVF_mean + 0.8118 (Pearson's correlation coefficient r = 0.9138; p < 0.01), while the scatterplot of mean MVF versus MRI-derived magnetic susceptibility (χ) was also fitted to a line where χ_{measured,mean} = −0.1218 × MVF_mean − 0.006345 (r = −0.8435; p < 0.01). As a result of the FPM-based QSM simulations, a linearly proportional relationship between the simulated magnetic susceptibility, χ_{simulated,mean}, and MVF (r = −0.9648; p < 0.01) was established. Such a statistically significant linear correlation between MRI-derived values by the QSM (or R₁) method and MVF demonstrated that variable myelin contents in the WM (i.e., CC) can be quantified across multiple stages of aging. These findings further support that both techniques based on QSM and R₁ provide an efficient means of studying the brain-aging process with accurate volumetric quantification of the myelin content in WM.