Identification of nonproliferating but viable hypoxic tumor cells in vivo

We have used the combination of pimonidazole labeling of hypoxic cells, bromodeoxyuridine labeling of proliferating cells, and cell sorting based on Hoechst 33342 perfusion to directly study hypoxia and proliferation in human tumor xenografts and transplantable murine tumors in vivo. Hypoxia was largely confined to cells in regions with the least perfusion, although in tumors exhibiting transient blood flow, hypoxic cells were not as highly localized. Similarly, proliferation and hypoxia were mutually exclusive except in areas of a tumor subjected to transient changes in perfusion. By determining the clonogenic potential, pimonidazole labeling intensity, and radiosensitivity of sorted tumor cell subpopulations, we have provided direct evidence that pimonidazole identifies hypoxic tumor cells of therapeutic relevance in vivo. Given that pimonidazole exhibits few diffusion or delivery problems and no apparent cytotoxicity, it appears to be a versatile and useful label for hypoxic cells in solid tumors.     

Inhibition of caspase proteases by CrmA enhances the resistance of human leukemic cells to multiple chemotherapeutic agents

Proteases that are members of the caspase (or interleukin-1beta converting enzyme (ICE)) protease family have been shown to be important mediators of apoptosis induced by Fas activation, neurotrophic factor withdrawal, and detachment from extracellular matrix. In this report we have investigated the potential importance of caspase proteases in apoptosis induced by multiple chemotherapeutic agents. Human T leukemic cells engineered to overexpress the cowpox virus CrmA protein, a direct and specific inhibitor of caspase proteases, were studied for their resistance to 1-beta-D-arabinofurasosyl-cytosine (Ara-C), etoposide (VP-16), doxorubicin (DOX), and cis-dichlorodiammine platinum (CP). Overexpression of CrmA dramatically inhibited drug-induced activation of caspases, as measured by processing of the inactive precursor form of caspase-3 and cleavage of caspase substrate proteins poly(ADP-ribose) polymerase (PARP) and lamin B. CrmA also significantly inhibited the kinetics of cell death induced by each of the four drugs. Moreover, when examined several days or weeks after initial exposure to drug, cultures of CrmA-expressing cells were found to have recovered and repopulated, whereas vector-transfected control cells did not. These studies demonstrate that caspase proteases play an important role in conferring sensitivity to multiple chemotherapy drugs, and that constitutive downmodulation of caspase activities can enhance chemoresistance.     

Retrovirus-mediated gene transfer of the multidrug resistance-associated protein (MRP) cDNA protects cells from chemotherapeutic agents

Transduction of hematopoietic progenitors with a multidrug resistance gene like mdr-1 or mrp aims to protect bone marrow from toxicity of chemotherapeutic agents. The interest in the use of mrp as an alternative to mdr-1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in the clinic to decrease mdr-1 tumor resistance, have little or no effect on MRP function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by multidrug resistance-associated protein (MRP). As a first step, we have constructed and tested two different mrp-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or PGK as promoters and generated ecotropic producer cells. We have shown by Southern blot analysis that retroviral supernatant from these producer cells can efficiently transmit the mrp gene to target cells. Mrp expression could be detected by fluorescence-activated cell sorting (FACS) analysis in the producer cells. The transduced cells have increased resistance to doxorubicin, vincristine, and etoposide. Furthermore, chemoprotection of the transduced cells was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP function. These data indicate that mrp retroviral vectors may be useful for chemoprotection and selection.     

Mutation of the p53 gene in human astrocytic tumours correlates with increased resistance to DNA-damaging agents but not to anti-microtubule anti-cancer agents

Human brain cancers (gliomas) overexpress large numbers of a receptor for interleukin 13 (IL13), making this receptor an attractive target for anti-glioma therapies. We have recently proposed that the glioma-associated IL13 receptor is different from the one expressed on some hemopoietic and somatic cells. In an attempt to identify an even more glioma-specific target, we have used an antagonist of a related cytokine, IL4, which neutralizes the physiological effects of both IL13 and IL4 on normal cells. Here we demonstrate that the IL4 antagonist also counteracts the action of cytotoxins targeted to the IL13 receptor on normal human cells. Importantly, the IL4 antagonist does not inhibit IL13-based cytotoxins on glioma cells at all. Thus, the IL13 receptor on glioma cells can be categorized as tumor-specific in the presence of an IL4 antagonist. We conclude that IL13 receptor-directed cytotoxins can be delivered to glioma cells without being cytotoxic to normal cells.     

Modulation of ras p21 oncoprotein levels and DNA strand breakage in human cells with chemotherapeutic agents and/or deferoxamine

Oncogenes are involved with the regulation of cellular proliferation. Ras oncogenes can be activated by chemical treatment and any increased activity could be modulated by further chemical treatment. In the present study, therefore, ras p21 protein expression was examined in in vitro cultures of human lymphocytes treated with mitomycin C and in the human colon adenocarcinoma Caco-2 cell line treated with doxorubicin with and without deferoxamine. Both chemotherapeutic agents act partially through oxygen radical mechanisms. Increases in p21 protein levels were seen with mitomycin C but no clear response was seen with doxorubicin. However, deferoxamine, with and without doxorubicin, altered p21 expression. Deferoxamine is an iron chelator so these results support the hypothesis that oxygen radicals were responsible for the altered p21 protein levels. Modulating responses were confirmed by measuring DNA strand-breakage in the Comet assay after treatment with doxorubicin and deferoxamine. Alterations of ras p21 protein expression in vitro might prove a suitable system for examining modulating effects on chemical carcinogens.     

Effects of WR-2721 (amifostine) and its metabolite WR-1065 on the antiproliferative activity of chemotherapeutic agents on neuroblastoma cells in vitro

Approaches through the middle cranial fossa directed at reaching the internal auditory canal (IAC) invariably employ exposure of the geniculate ganglion, the superior semicircular canal (SSC) or the epitympanum. This involves risk to the facial nerve and hearing apparatus. To minimize this risk, we conducted a laboratory study on 9 cadaver temporal bones by using an image-interactive guidance system (StealthStation) to provide topographic orientation in the middle fossa approach. Surface anatomic fiducials such as the umbo of the tympanic membrane, Henle's spine, the root of the zygoma and various sutures were used as fiducials for registration of CT-images of the temporal bone. Accurate localization of the IAC was achieved in every specimen. Mean target localization error varied from 1.20 to 1.38 mm for critical structures in the temporal bone such as the apex of the cochlea, crus commune, ampula of the SSC and facial hiatus. Our results suggest that frameless stereotaxy may be used as an alternative to current methods in localizing the IAC in patients with small vestibular schwannomas or intractable vertigo undergoing middle fossa surgery.     

Incorporation of ethidium bromide in the Drosophila salivary gland approached by microspectrofluorometry: evidence for the presence of both free and bound dye in the nuclei of cells in viable conditions

The incorporation of 10(-6) M ethidium bromide (EB) was studied in viable Drosophila melanogaster salivary glands with a spatial resolution reaching a few microns3, using a confocal laser microspectrofluorometer designed for spectral analysis. Spectra were recorded with the 514 nm Argon laser line during excitation times of 1 second (20 microW on the preparation) at 5 min intervals for 30 or 60 min, either at points in determined cell sites or serially throughout the cells. The fluorescence intensity time-course indicated that the EB intake was not an all-or-none process, but rather a graded, sensitive indicator of the functional state of the cell. On the micrometer scale, the cytoplasm behaved as an homogeneous substrate with the fluorescence intensity depending on EB intake and intracellular diffusion. In the nucleus, however, localized enhancement of the emission intensity was observed. Spectral analysis allowed us to characterize the interactions. The mean values of lambda max in the cytoplasm (600 nm), in the nucleus (601 nm) and outside the glands (602 nm) were less than for free EB in aqueous solution (630 nm); values of full width at half maximum were between 92 and 96 nm, which is much lower than the 120 nm observed for free EB. The recorded spectra were analyzed using a linear combination of two spectral models, namely free and DNA intercalated EB. In the nucleus, the free EB model spectra was found to represent up to 10% of the recorded spectra whereas it was near zero in the cytoplasm. The present data suggest that the intranuclear concentration of free EB (allowing for its lower fluorescence quantum yield) might be at least equal to that of the bound EB.     

In vitro and in vivo studies on the role of dimethylsulfoxide (DMSO) in resensibilization of bacterial strains resistant to antibiotics and chemotherapeutic agents

Resensibilization in vitro to seven antibiotics under the influence of DMSO was studied in 624 resistant strains of five species of bacteria (E. coli, S. typhi, S. pyogenes, S. viridans, S. aureus), 61 strains of tubercle bacilli resistant to isonicotinic acid hydrazide (INH) and 19 strains of tubercle bacilli resistant to rifampicin (RMP). DMSO in concentrations of 0.1-10.0% caused reversion of sensitivity in strains of E. coli, S. pyogenes and S. viridans. Reversion in vivo of sensitivity to INH of tubercle bacilli was studied in experimental tuberculosis of guinea pigs. Tubercle bacilli previously resistant to INH recovered complete sensitivity to the drug, enabling animals infected with the INH-resistant strain of bacilli to be treated with INH.     

The chemosensitizing potential of GF120918 is independent of the magnitude of P-glycoprotein-mediated resistance to conventional chemotherapeutic agents in a small cell lung cancer line

GF120918, at 250 ng/ml, increased the sensitivity of a P-glycoprotein (P-gp)-mediated multidrug resistant (MDR) small cell lung cancer cell line (H69/LX4) to the P-gp substrates, paclitaxel, taxotere, vinblastine, vinorelbine, daunorubicin and etoposide to levels which were either greater (in the case of etoposide) or close to that of the parent cell line (H69/P). This was achieved in spite of the great variation in the levels of resistance of the MDR cell line for the various anti-cancer drugs tested. These data suggest that GF120918 is a potent antagonist of P-gp mediated multidrug resistance, even in the case of high levels of resistance, as was the case with paclitaxel and taxotere (2560 and 2215 fold more than the sensitive parent cell line respectively).     

Contribution to the determination of the chemotherapeutic drug G1 in biological fluids

A sensitive gas chromatographic method for the quantitative determination of the new antibacterial and antifungal drug G1, 1-(5-bromofuran-2-yl)-2-bromo-2-nitroethene, has been optimized. The method involves a fast and single extraction step from spiked serum and urine samples. The G1 drug was quantified using an internal standard method and by means of a nitrogen-selective detector. The results are statistically significant and show that mean levels of G1 as low as 1 microg ml(-1) can be measured accurately.     

Study of glomeruli in the frog olfactory bulb using a fluorescent potential-sensitive dye

Localized irradiation of the skin and subcutaneous tissues with large single doses of gamma rays can induce immediate effects characterized by erythema, desquamation, and necrosis. Correlations between the evolution of the lesions and dosimetry studies have to be established by biophysical methods. NMR studies of the effects of an irradiated Fricke solution might be a means of controlling the delivered irradiation doses. After exposition to ionizing radiations, ferrous ions are transformed into ferric ions. Both are paramagnetic ions, and proton spin-lattice relaxation is accelerated depending on the oxidation reaction. In this study, solution of ammonium ferrous sulfate in an acid environment was incorporated into a gelling substance made with agarose, so that T1 weighted image contrast could be used to detect ferric ion formation. Experiments with 192Ir and 60Co gamma rays with doses in the 0 to 100 Gy range were conducted with Fe2+ concentrations of 0.5, 1, 1.5, and 2 mM in a gelling substance containing 4% agarose. A relationship was established between the amount of Fe3+ created and the spin-lattice proton relaxation rate, which led to a straightforward dose-effect relation. The use of such high doses allowed us to reproduce realistic conditions of accidental overexposure. A linear relationship was obtained between the doses absorbed and the NMR parameters measured (T1 and relative image intensity).     

Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells

The beta-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes are injected into the cytoplasm. This latter effect was independent of the polymer vector/cDNA ionic charge ratio, suggesting that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking. Cell division was not required for nuclear entry. Finally, comparative transfection and microinjection experiments with various cell lines confirm that barriers to gene transfer vary with cell type. We conclude that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.     

Identification of glutamate receptor subtypes mediating inputs to bipolar cells and ganglion cells in the tiger salamander retina

1. The effects of glutamate receptor agonists and antagonists on bipolar cells and ganglion cells were studied with the use of intracellular and extracellular recording in the superfused, isolated, flat-mounted tiger salamander retina. The goal of the experiments was to correlate glutamate receptor subtypes with their localization at specific synaptic sites in the tiger salamander retina. The drugs tested were the kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the N-methyl-D-aspartate (NMDA) receptor antagonist 3-(C+/-)-2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP) and L-2-amino-4-phosphonobutyrate (L-AP4). 2. The light responses of hyperpolarizing bipolar cells were suppressed by 20 microM CNQX, whereas L-AP4 had no effect on their light responses. In contrast, 20 microM CNQX had no effect on depolarizing bipolar cells, whereas L-AP4 abolished the light responses of these cells. 3. The light offset responses of OFF and ON-OFF ganglion cells were completely blocked by concentrations of CNQX as low as 5 microM. The light onset responses of ON-OFF ganglion cells were blocked when the concentration of CNQX was raised to 20 microM. In addition, 30 microM CPP partially blocked the light onset responses of ON-OFF ganglion cells but had a lesser effect on the light offset responses. 4. Twenty micromolars of CNQX blocked a transient component, and 20 microM CPP blocked a sustained component of the light response of sustained-ON ganglion cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.     

L-selectin-dependent leukocyte adhesion to microvascular but not to macrovascular endothelial cells of the human coronary system

To characterize L-selectin-dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-alpha (TNF-alpha)-stimulated HCMEC at shear stresses >2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti-L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3, which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-alpha-inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.     

Dendritic cells but not B cells present antigenic complexes to class II-restricted T cells after administration of protein in adjuvant

We have analyzed the relative contribution of dendritic cells (DC) and B cells in the presentation of peptide-class II complexes in an inflammatory situation in vivo. Draining lymph node cells from mice immunized subcutaneously with hen egg-white lysozyme (HEL) in adjuvant display HEL peptide-major histocompatibility complex class II complexes able to stimulate, in the absence of any further antigen addition, specific T hybridoma cells. The antigen-presenting capacity of three different antigen-presenting cell (APC) populations recruited in lymph nodes, DC (N418+, class II+, B220-, low buoyant density), large B cells (B220+, low buoyant density), and small B cells (B220+, high buoyant density), was analyzed. After immunization with HEL in adjuvant, DC are the only lymph node APC population expressing detectable HEL peptide-class II complexes. These results indicate that lymph node DC and not B cells are the APC initiating the immune response in vivo after administration of antigen in adjuvant.     

Function activity of blood polynuclears according to results of the nitroblue tetrazolium reaction in inhabitants of the Far North

The functional activity of polynuclear cells was studied in clinically healthy males aged 20-45 years constantly living in medium latitudes (the control group) and high latitudes (the experimental group). In persons who had lived in the North for a period of 6 months to 10 years and more the results of the spontaneous test with the use of nitrotetrazolium blue differed slightly from those in the control group, whereas after the stimulation of blood neutrophils with bacterial endotoxin these results proved to be significantly lower than those in the control group. With the increase of the time of residence in the North a tendency to greater individual differences in the results of the test with the use of nitrotetrazolium blue appeared, very low results being observed alongside very high ones. These facts indicate that under the extreme conditions prevailing in the arctic areas the reactivity of human neutrophils to bacterial stimulators is inhibited.     

The effect of the chemotherapeutic drug VP-16 on poly(ADP-ribosylation) in apoptotic HeLa cells

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters; in particular we have focused on changes in cellular morphology that are considered as markers of apoptosis. By immunofluorescence experiments we have shown that VP-16 causes the complete disruption of nucleoli and induces chromatin margination and fragmentation. Agarose gel electrophoresis of DNA from cells treated with 10-100 microM VP-16 showed the appearance of a characteristic ladder due to the internucleosomal DNA cleavage. The effect of etoposide on DNA integrity was not prevented by preincubation of cells with the protein synthesis inhibitor cycloheximide. These results provide experimental evidence indicating that the typical features of apoptosis are visible in HeLa cells exposed to VP-16. In this experimental system we have investigated whether the ADP-ribosylation process could be regulated by the presence of DNA fragments. By means of the activity gel technique, which allows the direct evaluation of automodified poly(ADP-ribose)polymerase, we have observed that in extracts from cells where etoposide-induced DNA fragmentation occurred, the autoribosylated form of the enzyme is greatly increased. Ribosylated poly(ADP-ribose)polymerase has been isolated by affinity chromatography on boronate column from cells permeabilized and labelled with [32P]NAD. Drug exposure caused a strong augmentation of modified enzyme. These observations suggest that activation of ADP-ribosylation process occurs in cells that show the typical features of apoptosis.     

Identification of specific relaxin-binding cells in the human female

Relaxin is secreted during pregnancy, but it has no verified effects in humans. The objective of the present study was to identify the cells containing specific relaxin-binding sites in the uterine cervix, vagina, uterus, mammary glands, mammary nipples, and term placenta in the human. The uterine cervix, vagina, and uterus were obtained from hysterectomy specimens. Mammary glands and nipples were obtained after modified radical mastectomy. Placenta was obtained after normal delivery. Tissue samples were cut into slices (0.5-3 cm3), frozen in liquid nitrogen, and cryosectioned (8 microm). Cells that bind relaxin were identified by sequential application of biotinylated porcine relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver enhancement for signal amplification. Relaxin bound with specificity to epithelial cells, smooth muscle cells, and blood vessels in the cervix, vagina, uterus, and mammary nipples; to epithelial cells and blood vessels in the mammary glands; and to skin of the mammary nipples. In addition, relaxin bound to individual cell types within the term placenta (amnion epithelium, syncytiotrophoblasts, blood vessels), and to sebaceous glands within the nipples. We conclude that the specific relaxin-binding cells probably contain relaxin receptors. Identification of putative relaxin receptors may provide insight into physiological and/or therapeutic roles of relaxin in the human.     

Identification of retinal ganglion cells projecting to the lateral hypothalamic area of the rat

Previous gain-of-function assays in Xenopus have demonstrated that Xwnt-3a can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. We report that expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. We further demonstrate that the ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants is dependent upon a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants was found to be dependent on endogenous Wnt signaling. We conclude that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways.