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— Synthesis of the tumor-localizing preparation HPD (hematoporphyrin derivative) results in the formation of dimers and oligomers of hematoporphyrin joined by labile linkages. Studies with HPD and an HPD analog containing the chlorin analog of mesoporphyrin suggest the presence of two different linkages, either of which yields a tumor-localizing product. One such linkage is apparently derived from a hematoporphyrin-based oligomer present in commercial preparations of hematoporphyrin as an impurity. The other linkage is formed during the chemical steps leading to the conversion of hematoporphyrin to “HPD”.

Interactions between the drug camptothecin and DNA were studied using intact cells, DNA released after cell lysis, and purified DNA. The data show the following: 1. 1. The apparent molecular weight of DNA on alkaline sucrose or formamide gradients is lowered after cells are treated with the drug. 2. 2. The degradation of DNA detected on alkaline sucrose is related to the high pH of the gradients. 3. 3. Sedimentation of cell-free DNA through alkaline (but not neutral) sucrose gradients containing camptothecin, in the presence of light, results in degradation of DNA. 4. 4. Treatment of cells with high concentrations of camptothecin causes double strand breaks detectable on neutral sucrose gradients. 5. 5. An interaction between purified DNA and comptothecin lactone can be demonstrated by difference spectra. We conclude that camptothecin treatment produces alkaline labilization of cell DNA; this phenomenon can be observed using DNA from lysed cells. Additional cell DNA degradation not involving alkaline labilization can also be demonstrated. Damage of DNA may result from a drug-DNA interaction, or may follow dissociation of a DNA-protein complex.

Partitioning behavior of murine lymphoblasts was examined in biphasic aqueous systems containing dextran and poly(ethylene glycol) with no potential difference across the interface. Persistence of cells in the upper phase of the system could be promoted by addition of the palmitate ester of poly(ethylene glycol). Partitioning effects seen here may derive from affinity of membrane sialic acid residues for dextran-rich lower-phase droplets, and antagonism of these interactions by poly(ethylene glycol) palmitate. Several determinants of partitioning behavior were examined: 1. Membrane glycosylation. Partitioning behavior of P388 and P388/ADR lymphoblasts were compared. The latter has an inherently higher degree of membrane glycosylation, resulting in a lower partition coefficient in poly(ethylene glycol) palmitate-supplemented systems. 2. Degree of esterification. Singly-esterified poly(ethylene glycol) was substantially more effective at promoting cell partition into the upper phase than was doubly-esterified glycol. Use of the latter was associated with cell-cell aggregation. 3. Relative numbers of lower-phase droplets present. Decreasing the numbers of such droplets initially present markedly increased the partition coefficient. 4. Time-dependence. Even in the presence of poly(ethylene glycol) palmitate, the partition coefficient fell with time, indicating an interaction between cells and lower-phase droplets not antagonized by the ester.

This article provides a brief overview of mechanisms whereby acute leukemias become drug resistant. Modes of resistance to methotrexate and cytosine arabinoside are briefly summarized. Recent information on resistance to the natural products (vinca alkaloids, anthracyclines) is described in more detail.

In transplantable tumor cell lines, a common mode of resistance to several natural products, including the anthracyclines, the Vinca alkaloids and actinomycin D, involves an outward transport process which limits drug accumulation to sub-lethal levels (1–5). This transport system can be inhibited, and drug responsiveness promoted, by calcium antagonists of two structural classes: papaverine analogs, e.g. verapamil, and dihydropyrimidines, e.g. nitrendipine (6–9). This report describes studies on the two classes of calcium antagonists named above. The data indicate that verapamil is a substrate for the outward transport system and promotes anthracycline accumulation via competition for exodus. Nitrendipine is not a substrate for this outward transport system and may act via a ‘chaotropic’ mechanism.

Effects of ruthenium red on accumulation and cytotoxicity of m-AMSA, vinblastine and daunorubicin were examined in the P388 murine leukemia cell line and in an adriamycin-resistant subline, P388/ADR, which is cross-resistant to all three agents. Ruthenium red increased m-AMSA accumulation by both P388 and P388/ADR cells; the extent of this effect was a function of the concentration of both agents. Uptake enhancement occurred within 5 min of exposure of cells to ruthenium red and was readily reversed when cells were suspended in fresh medium. A 24-hr exposure to ruthenium red was needed to affect vinblastine or daunorubicin accumulation, and the effect was substantially less than that observed with m-AMSA. Ruthenium red protected P388 cells from m-AMSA toxicity. These data, together with reports indicating a protective effect of ruthenium red against vinblastine and anthracycline toxicity, suggest that the dye promotes tight binding of m-AMSA and other agents to cellular loci on which toxic effects are not exerted.

The present study evaluates the utility of the dihydropyridine in equilibrium pyridinium salt redox system for the specific delivery and sustained release of a model 2',3'-dideoxynucleoside to the brain of mice as the initial effort in a search for agents that may prove effective in reversing the complicating neurological disorders of AIDS. The unsaturated nucleoside 2',3'-didehydro-2',3'-dideoxythymidine (1), which is effective in protecting ATH8 cells against the cytopathogenicity of HIV-1, was converted to the corresponding N-methyl-1,4-dihydronicotinate derivative, 4, in three steps. The 5'-O-nicotinate ester, 2, obtained by reaction of 1 with nicotinyl chloride, was converted in quantitative yield to the N-methylpyridinium salt 3 on treatment with MeI in acetone. Reduction of the latter with Na2S2O4 gave 4 in 50% yield. Pseudo-first-order rate constants for the oxidation of 4 to 3 were observed in plasma (k = 3.54 x 10(-5) s-1) and in homogenates of mouse liver (k = 9.2 x 10(-5) s-1) and brain (k = 8.85 x 10(-5) s-1). None of the chemical delivery system 4 could be detected in the brain of female BDF/1 mice at 1 h postinjection. The peak level of 3 in the brain occurred at 3 h with a half-life of 25 h. Both 1 and N-methylnicotinic acid (trigonelline, 5) were readily identified by HPLC in a brain homogenate derived from mice injected (25 mg/kg) with 4. TLC showed a low level penetration of mouse brain by 1 (0.44 microgram/g wet tissue) following injection of the corresponding labeled [methyl-3H]-2',3'-unsaturated nucleoside (25 mg/kg). The data indicate that 4 crosses the blood-brain barrier to be oxidized by cerebral tissue to the ionic structure 3, which is "locked therein". The sustained local release of a 2',3'-dideoxynucleoside, such as 1, from a chemical delivery system (4) represents a potentially useful approach to the treatment of AIDS dementia complex.

Some properties of sialyltransferase activity in plasma and lymphocytes from patients with chronic lymphocytic leukemia were compared. Three distinct enzyme fractions were identified in plasma: (1) cation independent, irreversibly bound to agarose; (2) cation dependent, weakly bound to agarose; (3) strongly bound to agarose, lost upon dialysis. Lowering of the peripheral lymphocyte count by leukapheresis markedly decreased the level of serum sialyltransferase, suggesting the circulating lymphocyte is a source of the serum enzyme. The enzyme solubilized by detergent from lymphocytes showed a substantially lower Km for CMP-sialic acid than did the serum enzyme, was less sensitive to several inhibitors, was not irreversible bound to Agarose, and had a substantial cation requirement. The enzyme solubilized from the lymphocyte therefore generally resembles fraction 2 of serum.

Mutants of Escherichia coli , blocked in proline biosynthesis, and requiring very high external proline supplements for growth, have been studied. The mutants show normal permeability for inward and outward proline diffusion, and have no apparent defect in biosynthetic systems involved in protein biosynthesis. The high proline requirement has been related to the lack of the specific process responsible for concentrative uptake of proline at 37°. The mutant strain also lacks the ability to carry out rapid exchange at 0° between an intracellular pool and a low level of extracellular [ 14 C]proline. This suggests that concentrative uptake at 37° and exchange at 0° are mediated by closely related processes.

The effect of persantin on several transport systems was studied using murine leukemia cells. Low concentrations of persantin inhibited inward transport of phosphate, fucose, deoxycytidine and adenosine. At higher concentrations, inhibition of exodus of these substances from pre-loaded cells could be shown. In contrast, persantin preferentially inhibited efflux of O- methylglucose , uridine, uracil, and cycloleucine at levels which did not significantly slow influx. As the persantin level was elevated, entry of the latter group of compounds was also inhibited. These data have implications concerning the multiplicity of transport sites on the cell membrane.

Survival of mice bearing different transplantable leukemias and treated with 5-fluorouracil was compared with accumulation of drug nucleotides in vitro. There was significant correlation,suggesting that cellular capacity for conversion of the drug to nucleotides is a major determinant of inherent drug sensitivity of these leukemias.

Triamterene (2,4,7-triamino-6-phenylpteridine) and certain other pteridines stimulate the uptake of amethopterin by human small lymphocytes, apparently by removing a barrier to amethopterin transport. This stimulation did not extend appreciably to other cell types or to other lymphocyte transport systems tested.

Much interest of late has centered around RNAdirected DNA synthesis. Initially, with the finding of an RNA-dependent DNA polymerase in oncogenic RNA viruses [l-S] and what was described as the exclusive presence of the polymerase in acute leukemia cells as opposed to ‘normal’ cells [6] , speculation as to a molecular basis for leukemogenesis and a predictive test for leukemia abounded. However, with the identification [7, S] of the polymerase in ‘normal’ cells (cells not known to be RNA virally transformed) the hope of an absolute predictive index for leukemia or neoplasia has lost credibility, and the RNA-dependent DNA polymerase may be of more interest from the standpoint of gene amplification as opposed to a neoplastic predictor. In our studies of this polymerase we have undertaken an extensive, systematic experiment designed to note the effects of a number of potential inhibitors (primarily antibiotics) on the RNAdependent DNA polymerase in vitro. The present brief report gives the results of these experiments. Several compounds were shown to be potent inhibitors of an RNA-dependent DNA from mouse leukemia cells.

— Localization and photodynamic efficacy of a monocationic porphyrin (MCP) were assessed using murine leukemia cells in culture. This sensitizer localized at surface membrane loci and catalyzed selective photodamage to membrane structures. Although both cationic and hydrophobic, this porphyrin was not recognized by the multidrug transporter, which excludes many cationic agents from cells that express multidrug resistance. Photodynamic studies with the murine radiation-induced fibrosarcoma tumor model indicated moderate photosensitization of neoplastic lesions in vivo at 3 h, but not at 24 h after sensitizer administration. Pharmacokinetic studies indicate that plasma levels, not tissue levels were the major determinant of photodynamic therapy (PDT) response. Consistent with this observation, vascular damage and disturbances of tissue perfusion followed PDT. These effects were more pronounced in tumor-bearing skin than in normal skin. The therapeutic response to MCP appeared to be related mainly to secondary, probably vascular, effects.

International audience; The aim of this project is to develop Interventional Radiology (IR) simulator models for core skills training. Background.There is a shortage of radiologists trained in performance of IR procedures. Visceral and vascular IR techniques almost universally commence with a needle puncture, usually to a specific target for biopsy, or to introduce wires and catheters for diagnosis or treatment. These skills are learnt in an apprenticeship in simple diagnostic procedures in patients, though there are drawbacks to this training method. In addition, certification depends partly on a record of the number of procedures performed, with no current method of objective IR skills assessment. Computer based simulation is just one of a range of alternatives to apprenticeship training, which include physical and animal models, and rapid prototyping models to train catheterisation skills. Fixed models are expensive, lack physiology, have fixed anatomy and are destroyed by repeated needle puncture. Animal models have anatomical differences, lack pathology and, in the UK, political acceptability. Realistic Virtual Environments (VEs) can be derived from imaging data, with potential to introduce physiological processes,tissue deformation and haptics(touch).Patient anatomical variability and pathology can be obtained from multimodality (MR, CT) imaging studies using a series of semi-automated processing steps to segment ('label' in 3D) the anatomical data. Accurate simulation requires incorporation of data from procedural Task Analysis into the development phase, including critical procedure steps and their metrics for objective measures of operator competence. Novel, semiautomatic segmentation of patient specific imaging data have been developed at Leeds and Imperial to generate anatomically realistic three dimensional VEs that reflect variability across patients. Validation:Few, if any, simulations have been convincingly validated for training IR skills, though this should be achievable where content (replication of procedure steps) and fidelity (faithfulness of that replication) are appropriate to real world tasks. Indeed correct reproduction of the task, and appropriate fidelity are the cornerstone of producing a simulation that has relevance to a particular training objective. In our projects, validation draws on the team's occupational psychology expertise (at University of Hull) with evaluation by clinical radiologists at Liverpool, Leeds and Manchester. The simulator has been designed for use exclusively within radiology training curricula.

Research in autophagy continues to accelerate,1and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response. ©2008 Landes Bioscience.

Survival of mice bearing different transplantable leukemias and treated with cytosine arabinoside was compared with uptake and subsequent phosphorylation of the drug in vitro. Capacity for nucleotide formation was correlated with response and is apparently an important determinant of drug sensitivity. Drug uptake, although apparently mediated, was similar in all cell lines.

L-ASPARAGINASE (EC 3.5.1.1), which hydrolyses the amide group of asparagine, inhibits certain neoplasms1–4 and is used for treating human acute leukaemias5,6. Although asparaginase sequentially inhibits protein, RNA and DNA synthesis7, its cellular action is undefined. We have investigated the effects of asparaginase on the synthesis of carbohydrate–amino-acid linkages in glycoproteins which involve the amide group of asparagine and N-acetyl-glucosamine residues8. Our preparation of L-asparaginase (from Escherichia coli, 321–363 U/mg protein) was free of contaminating enzymatic activity, although it was slightly active (2.1 per cent of the activity towards asparagine) with glutamine as substrate. The preparation was free of glycosidase and proteolytic activities9,10.