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Background: Due to its unique efficacy in treatment-resistant schizophrenia, discontinuation of treatment with clozapine is frequently associated with a significant worsening of symptoms, but also with an increased risk of suicide. Based on the literature, this review aims at summarizing different monitoring recommendations in order to be able to continue this therapy despite the occurrence of side effects. In addition, we provide recommendations when rechallenge of a previously stopped treatment with clozapine can be considered and when a definite discontinuation must take place., Material and Methods: Medline, the Guideline for the use of clozapine 2013 of the Netherlands Clozapine Collaboration Group, and the S3 Guideline for Schizophrenia of the German Association of Psychiatry, Psychotherapy and Psychosomatics were searched for relevant literature, the last query dating from April 28th, 2023., Results: If agranulocytosis or cardiomyopathy develops, treatment with clozapine must be discontinued and should not be resumed thereafter. In contrast, treatment with clozapine which had to be discontinued due to myocarditis or prolongation of the QTc interval may be continued if left ventricular function is normal or after normalization of the QTc interval. Other side effects are usually not absolute contraindications for rechallenge but often require the adjunctive use of additional pharmacologic and non-pharmacologic measures., Conclusion: Taking into consideration various monitoring recommendations, cessation of treatment with clozapine can often be prevented or treatment with clozapine that has been discontinued due to side effects can be resumed., (© 2023. The Author(s).)

Background: According to current guidelines, clozapine should be used as a third step in treatment resistant schizophrenia (TRS). In everyday clinical practice, however, it is frequently used at a much later stage, which leads to a significant deterioration of prognosis. The first part of this narrative overview focuses on the most frequent side effects of clozapine, on the relevance of slow titration, and on specific aspects of therapeutic drug monitoring (TDM)., Material and Methods: Medline, the Guideline for the use of clozapine 2013 of the Netherlands Clozapine Collaboration Group, and the S3 Guideline for Schizophrenia of the German Association for Psychiatry, Psychotherapy and Psychosomatics were searched for relevant literature, the last query dating from April 28th, 2023., Results: Despite its unique efficacy clozapine is underused in clinical practice and prescription varies between and within countries. Next to hematological, metabolic, and vegetative side effects, clozapine induced inflammation manifesting in the form of pneumonia or myocarditis, which is mainly associated with rapid titration, represents a major clinical challenge with CRP monitoring being of particular relevance. In this context, it also has to be noted that sex, smoking behavior, and ethnic origin impact clozapine metabolism, thus requiring personalized dosing., Conclusion: Slow titration when possible, TDM, and CYP diagnostics when appropriate increase patient safety during treatment with clozapine and thus the likelihood of early prescription of this compound in TRS., (© 2023. The Author(s).)

Objectives: Demand for nursing home (NH) care is soaring due to gains in life expectancy and people living longer with chronic illness and disability. This is dovetailing with workforce shortages across the healthcare profession. Access to timely and appropriate medical care for NH residents is becoming increasingly challenging and can result in potentially avoidable hospitalisations (PAHs). In light of these factors, we analysed PAHs comparing NH patients with non-NH patients., Design: Cross-sectional study with claims data from 2015 supplied by a large German health insurance company within the federal state of Baden-Wuerttemberg., Setting: One-year observation of hospitalisation patterns for NH and non-NH patients., Participants: 3 872 245 of the 10.5million inhabitants of Baden-Wuerttemberg were covered., Methods: Patient data about hospitalisation date, sex, age, nationality, level of care and diagnoses were available. PAHs were defined based on international classification of diseases (ICD-10) diagnoses belonging to ambulatory care sensitive conditions (ACSCs). Adjusted ORs for PAHs for NH patients in comparison with non-NH patients were calculated with multivariable regression models., Results: Of the 933 242 hospitalisations in 2015, there were 23 982 for 13 478 NH patients and 909 260 for 560 998 non-NH patients. Mean age of hospitalised NH patients and level of care were significantly higher than those of non-NH patients. 6449 PAHs (29.6%) for NH patients and 136 543 PAHs (15.02%) for non-NH patients were identified. The adjusted OR for PAHs was significantly heightened for NH patients in comparison with non-NH patients (OR: 1.22, CI (1.18 to 1.26), p<0.0001). Moreover, we could observe that more than 90% of PAHs with ACSCs were unplanned hospitalisations (UHs)., Conclusions: Large numbers of PAHs for NH patients calls for improved coordination of medical care, especially general practitioner service provision. Introduction of targeted training programmes for physicians and NH staff on health problem management for NH patients could perhaps contribute to reduction of PAHs, predominantly UHs., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Laws regulating education of most health professional groups in Germany today mean that curricula re-profiling in response to changing priorities in the practice environment is a significant challenge. Legally dictated theoretical and clinical requirements for the vocational training of health professionals leaves little room for re-profiling in response to movements such as interprofessional education. An educational innovation was needed that worked within existing structures in Germany. The result was a formal collaboration between the Academy for Health Professionals and the University of Heidelberg allowing students undertaking vocational training to also complete a university degree in parallel. The aim of this article is to describe the curriculum development for the Bachelor of Science - Interprofessional Health Care. This article outlines an evidence-based approach to the process to curriculum development that resulted in a competency-based degree offering comprehensive interprofessional education at undergraduate level for healthcare students based in Germany.

Human saliva is finding increasing interest for proteomic and biomarker-discovery studies, due to the ease of collection and potential for simpler processing workflows compared to serum or plasma. However, it is known that salivary protein composition can vary with physiological and environmental factors. In this work, we have examined intra- and inter-person variability of saliva protein composition using an LC/MS methodology to profile low molecular weight human salivary proteins. Whole saliva was analyzed from four individuals over three consecutive days. Additional samples were used to determine baseline analytical and sample processing variation and to identify phosphoproteins. Individuals were observed to have a similar salivary protein pattern over multiple days, although the expression levels of particular proteins were variable. Significant differences in protein profiles were observed between subjects, allowing for delineation of individuals based on their protein profile. Comparison with alkaline phosphatase treated saliva revealed that several identified proteins were singly, doubly, or triply phosphorylated.

Liquid chromatography/mass spectrometry (LC/MS) peptide maps have become a basic tool for characterizing proteins of biological and pharmaceutical interest. The ability to generate reproducible maps with high protein sequence coverage is a central goal of methods development. We have applied a recently developed analytical approach (termed LC/MS(E)) to LC/MS peptide mapping. Using the LC/MS(E) approach, the mass detector alternates between a low-energy scanning mode (MS) for accurate mass peptide precursor identification, and an elevated-energy mode (MS(E)) for generation of accurate mass multiplex peptide fragmentation data. In this paper, we evaluate this analytical approach against a tryptic digest of yeast enolase. From the low-energy data, high peptide map coverage (98% of sequence from peptides >3 amino acids) was reproducibly obtained. The MS signal for essentially equimolar peptides varied over 2 orders of magnitude in intensity, and peptide intensities could be precisely and reproducibly measured. Using the temporal constraint that MS(E) peptide fragment ions exhibit chromatographic profiles that parallel the precursor ions that generated them, we were able to produce accurate mass time-resolved MS/MS information for all enolase peptides with sufficient abundance to produce a detectable fragment ion., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

In this paper, we present a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein fractionation (multidimensional chromatographic separation) with parallel online ESI-TOF-MS intact protein analysis, and fraction collection. Collected fractions were digested and protein identifications were produced using MALDI Q-TOF-MS analysis. These identifications were then linked with corresponding ESI-TOF-MS intact protein mass data to permit full protein characterization. This methodology was applied to an E. coli cytosolic protein fraction, and enabled the identification and characterization of proteins exhibiting co-translational processing, post-translational modification, and proteolytic processing events. The approach also provided the ability to distinguish between closely related protein isoforms. The summary of results from this study indicated that roughly one-third of all detected components generated corresponding data from both top-down and bottom-up analyses, and that significant and novel information can be derived from this application of the hybrid analytical methodology.

The balance between chromatographic performance and mass spectrometric response has been evaluated using an automated series of experiments where separations are produced by the real-time automated blending of water with organic and acidic modifiers. In this work, the concentration effects of two acidic modifiers (formic acid and trifluoroacetic acid) were studied on the separation selectivity, ultraviolet, and mass spectrometry detector response, using a complex peptide mixture. Peptide retention selectivity differences were apparent between the two modifiers, and under the conditions studied, trifluoroacetic acid produced slightly narrower (more concentrated) peaks, but significantly higher electrospray mass spectrometry suppression. Trifluoroacetic acid suppression of electrospray signal and influence on peptide retention and selectivity was dominant when mixtures of the two modifiers were analyzed. Our experimental results indicate that in analyses where the analyzed components are roughly equimolar (e.g., a peptide map of a recombinant protein), the selectivity of peptide separations can be optimized by choice and concentration of acidic modifier, without compromising the ability to obtain effective sequence coverage of a protein. In some cases, these selectivity differences were explored further, and a rational basis for differentiating acidic modifier effects from the underlying peptide sequences is described.

The performance characteristics of multidimensional liquid chromatographic protein separations were evaluated using on-line electrospray mass detection, and a novel workflow for automated LC/MS data processing. Two-dimensional ion exchange/reversed-phase LC separations of Escherichia coli cytosol were conducted using either a continuous linear or discontinuous step gradient in the first dimension. Chromatographic profiles of the top 100 most abundant components were characterized to assess overall separation reproducibility within each mode, and to characterize differences in component distribution between the two modes of operation. Analysis of the resulting data indicates that multidimensional separations of complex protein mixtures can be done reproducibly. Furthermore, under the conditions employed within this study, a linear first dimension gradient was more effective at fractionating the protein mixture, distributing fewer major components to multiple second dimension cycles than an equivalent step gradient. The application of on line mass spectrometry, and automated processing of the resulting data, proved valuable for producing component level analysis of multidimensional protein separations.

We designed a pharmacokinetic and pharmacodynamic phase I study of sequential topotecan (2.55-6.3mg/m2) by 72h infusion followed by five daily doses of etoposide for patients with refractory acute leukemia based upon synergistic anti-tumor activity of topoisomerase I and II inhibitors in vitro. Eight of the 29 patients achieved bone marrow aplasia and two patients achieved clinical remission. Common grade 3-4 toxicities included hepatic and gastrointestinal dysfunction, and correlated with increased steady-state plasma topotecan concentration. The predicted up-regulation of topoisomerase II activity by topoisomerase I inhibition was not observed at this dose and schedule and may provide insight into the modest anti-leukemia activity of the regimen.

The limitations of 2-D gels for global proteomics have encouraged the development of alternative approaches for identifying proteins in complicated mixtures, and determining their modification state. In this work, we describe the application of multidimensional liquid chromatography (SCX-RPLC) coupled with electrospray time-of-flight mass spectrometry and off-line fraction collection to analyze complex intact protein mixtures. Methods were developed using both standard proteins and an enriched yeast ribosomal fraction sample containing approximately 100 proteins, which permitted assessment of the effectiveness of the individual separation dimensions, as well as investigation of the interplay between separation capacity and electrospray MS performance.

In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of 13C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by "mass locking" with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated., (Copyright 2001 Academic Press.)

Purpose: In the setting of target-based anticancer drug development, it is critical to establish that the observed preclinical activity can be attributed to modulation of the intended target in early phase trials in human subjects. This paradigm of target modulation allows us to determine a Phase II or III dose (optimal biochemical/biological modulatory dose) that may not necessarily be the maximum tolerated dose. A major obstacle to target-based (often cytostatic) drug development has been obtaining relevant tumor tissue during clinical trials of these novel agents for laboratory analysis of the putative marker of drug effect., Experimental Design: From 1989 to present, we have completed seven clinical trials in which the end point was a biochemical or biological modulatory dose in human tumor tissues (not surrogate tissue). Eligibility enrollment required that patients have a biopsiable lesion either with computerized tomography (CT) guidance or direct visualization and consent to sequential (pre and posttreatment) biopsies., Results: A total of 192 biopsies were performed in 107 patients. All but 8 patients had sequential pre and posttreatment biopsies. Seventy-eight (73%) of the 107 patients had liver lesion biopsies. In eight patients, either one or both biopsies contained insufficient viable tumor tissue or no tumor tissue at all for analysis. Of a total of 99 patients in whom we attempted to obtain paired biopsies, a total of 87 (88%) were successful. Reasons for failure included patient refusal for a second biopsy (n = 2), vasovagal reaction with first biopsy precluding a second biopsy (n = 1), subcapsular hepatic bleeding (n = 1), and most commonly obtaining necrotic tumor, fibrous, or normal tissue in one of the two sequential biopsies (n = 8)., Conclusions: This is the first and largest reported series demonstrating that with adequate precautions and experience, sequential tumor biopsies are feasible and safe during early phase clinical trials.

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

High-efficiency capillary isoelectric focusing (cIEF) separations of protein complexes obtained from soluble protein fractions are demonstrated. Size-exclusion chromatography was used as a first dimension separation to fractionate putative protein complexes with apparent molecular masses of up to 1,500,000 from an Escherichia coli cytosolic fraction. Non-denaturing cIEF separations using highly hydrophilic polymer-coated capillaries constituted the second dimension. The conditions developed produced reproducible and high-efficiency separations, corresponding to approximately 2 x 10(6) theoretical plates and peak capacities of approximately 10(3) for pH 3-10 cIEF separations in 65 cm long capillaries. Combination of the two non-denaturing separation dimensions permitted isolation and analysis of individual protein complexes from complicated biological samples. Studies indicated that many E. coli complexes were stable on the time scale of the cIEF separations, but were degraded upon more extended periods of storage on ice, necessitating rapid sample processing and fast analysis techniques.

Purpose: In vitro and in vivo preclinical models have demonstrated synergistic activity when topoisomerase I and II inhibitors are administered sequentially. Topoisomerase I inhibitors increase topoisomerase II levels and increase cell kill induced by topoisomerase II poisons. We evaluated this hypothesis in a cohort of patients with advanced non-small-cell lung cancer (NSCLC)., Methods: A group of 19 patients with advanced NSCLC (70% adenocarcinoma) received topotecan at a dose of 0.85 mg/m2 per day as a continuous 72-h infusion from days 1 to 3. Etoposide was administered orally at a dose of 100 mg twice daily for 3 days on days 7-9 (schedule and dose derived from prior phase I trials). Total and lactone topotecan concentrations were measured at the end of the 72-h infusion. Blood samples were obtained immediately after each 72-h topotecan infusion in order to measure the mutational frequency at the hypoxanthine phosphoribosyl transferase (HPRT) locus in peripheral lymphocytes., Results: A total of 55 cycles were administered. Toxicity was mainly hematologic with grade 4 neutropenia occurring in 7% of courses. Only one partial response and two stable diseases were observed. The 1-year survival rate was 33%. There was a statistically significant difference between steady-state lactone concentrations between cycle 1 and cycle 2 with decreasing concentrations with cycle 2 (P = 0.02). This was explained by a statistically significant increase in the clearance of topotecan lactone during cycle 2 (P = 0.03). Total but not lactone concentrations correlated with nadir WBC, ANC and platelet levels. Steady-state plasma lactone levels correlated with the mutational frequency at the HPRT locus (P = 0.06). In the one patient with a partial response a sixfold increase in HPRT mutational frequency was observed, which was not seen in patients with progressive disease., Conclusion: The combination of topotecan and etoposide in this schedule of administration has minimal activity in adenocarcinoma of the lung. This lack of activity may be due to the delay in administration of etoposide after the topotecan as studies have shown that the compensatory increase in topoisomerase II levels after treatment with topoisomerase I inhibitors is shortlived (<24 h). The HPRT mutational frequency results suggest that the lack of clinical response may be associated with failure to achieve sufficient cytotoxic dose as indicated by a lack of increase in mutational frequency in those patients with progressive disease. HPRT mutational frequency may correlate with plasma steady-state topotecan lactone levels. Future studies should be directed toward earlier administration of topoisomerase II inhibitors after topoisomerase I inhibition.

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.

In the present work, capillary isoelectric focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (approximately 4-microm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-microm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (<3 cells/microL). CIEF experiments showed that yeast cell populations at early log, mid log, and stationary growth phases differ in isoelectric point, with values ranging from 5.2 to 6.4. The broader application of CIEF are projected for microorganism identification and separation based upon growth conditions.

Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ microL were detectable using conventional UV detection. CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels. Very small differences in peptide isoelectric points (deltapI approximately 0.01) could be resolved, High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.

Purpose: We conducted a phase I and pharmacologic study of a weekly 96-hour infusion of irinotecan to determine the maximum-tolerated dose, define the toxicity profile, and characterize the clinical pharmacology of irinotecan and its metabolites., Patients and Methods: In 26 adult patients with solid tumors, the duration and dose rate of infusion were escalated in new patients until toxicity was observed., Results: In 11 patients who were treated with irinotecan at 12.5 mg/m(2)/d for 4 days weekly for 2 of 3 weeks, dose-limiting grade 3 diarrhea occurred in three patients and grade 3 thrombocytopenia occurred in two patients. The recommended phase II dose is 10 mg/m(2)/d for 4 days given weekly for 2 of 3 weeks. At this dose, the steady-state plasma concentration (Css) of total SN-38 (the active metabolite of irinotecan) was 6.42 +/- 1.10 nmol/L, and the Css of total irinotecan was 28.60 +/- 17.78 nmol/L. No patient experienced grade 3 or 4 neutropenia during any cycle. All other toxicities were mild to moderate. The systemic exposure to SN-38 relative to irinotecan was greater than anticipated, with a molar ratio of the area under the concentration curve (AUC) of SN-38 to irinotecan of 0.24 +/- 0.08. One objective response lasting 12 months in duration was observed in a patient with metastatic colon cancer., Conclusion: The recommended phase II dose of irinotecan of 10 mg/m(2)/d for 4 days weekly for 2 of 3 weeks was extremely well tolerated. Further efficacy testing of this pharmacologic strategy of administering intermittent low doses of irinotecan is warranted.

Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr 78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phenomena also occur in V79-derived cell lines that are deficient in poly(ADP-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitivity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have also previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearing L1210 tumors. These observations prompted us to examine whether 6-AN pre-treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitization to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6-AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immunoblotting. To determine sensitivity after GRP78 up-regulation, the cells were washed and grown for 412 h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412 h time period allowed p(ADPR) metabolism to return to normal while GRP78 levels remained elevated, thus allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, cell sensitivity was determined by clonogenic survival assay or a fluorescence-based cytotoxicity assay. Based on changes in IC50 values, 6-AN caused an increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-expression of GRP78 in human tumor cell lines is associated with increased sensitivity to clinically useful chemotherapy agents. This sensitization occurred in three different tumor cell lines, each bearing a separate genetic defect associated with altered sensitivity., (Copyright 1999 Academic Press.)

We have studied the clonogenic survival response to X-rays and MNNG of V79 Chinese hamster cells and two derivative cell lines, ADPRT54 and ADPRT351, deficient in poly(ADP-ribose) polymerase (PARP) activity. Under conditions of exponential growth, both PARP-deficient cell lines are hypersensitive to X-rays and MNNG compared to their parental V79 cells. In contrast, under growth-arrested, confluent conditions, V79 and PARP-deficient cells become similarly sensitive to X-rays and MNNG suggesting that PARP may be involved in the repair of X-ray or MNNG-induced DNA damage in logarithmically growing cells but not in growth-arrested confluent cells. This suggestion, however, creates a dilemma as to how PARP can be involved in DNA repair in only selected growth phases while it is functionally active in all growth phases. To explain these paradoxical results and resolve this dilemma we propose a hypothesis based on the consistent observation that inhibition of PARP results in a significant increase in sister chromatid exchange (SCEs). Thus, we propose that PARP is a guardian of the genome that protects against DNA recombination. We have extended this theme to provide an explanation for our results and the studies done by many others.

Two complementary approaches utilizing reverse-phase high-performance liquid chromatography and liquid chromatography/mass spectrometry were developed to analyze recombinantly produced Group I and Group II human ADP-ribosylation factors (ARFs). We observe that the NH2 termini of Group II ARFs (ARF4 and ARF5) are efficiently processed by removal of the initiating methionine. In contrast, the NH2 termini of Group I ARFs (ARF1 and ARF3), although fully deformylated, undergo only partial methionine cleavage. This result is unexpected as ARFs are canonical substrates for methionine processing in both bacterial and eukaryotic systems, but it may explain the difficulties encountered by many researchers attempting to produce myristoylated ARFs in Escherichia coli. Additionally, we observe that a significant fraction of purified ARF4 contains a modification which we demonstrate to be consistent with mono-glutathionation. Both methionine retention and glutathione modification may impact ARF function and the methods presented here should be employed to determine the quality of recombinant ARFs., (Copyright 1998 Academic Press.)

We have shown previously that NAD/poly(ADP-ribose) polymerase-deficient cells that overexpress Mr 78,000 glucose-regulated stress protein (GRP78) are resistant to topoisomerase II inhibitors, such as etoposide, m-amsacrine, and doxorubicin. However, these cells have been found to be hypersensitive to DNA cross-linking agents, including melphalan, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These observations prompted us to examine whether overexpression of GRP78 is associated with modulation of cytotoxicity of clinically useful DNA-cross-linking agents such as melphalan, BCNU, and cisplatin. We up-regulated GRP78 in V79 Chinese hamster cells by 2-5-fold using two independent approaches that include exposure to 6-aminonicotinamide, or 2-deoxyglucose. Subsequently, these GRP78-overexpressing cells were trypsinized, plated in regular medium without GRP78-inducing agents, and allowed a 5-h attachment time before being treated with melphalan, BCNU, or cisplatin for 1 h to determine clonogenic survivals. In addition, repair of DNA cross-links induced by those agents were determined by alkaline elution assay. Our results show that the GRP78-overexpressing V79 cells are hypersensitive to DNA cross-linking agents compared to the control V79 cells. Furthermore, repair of drug-induced DNA cross-links appears to be considerably slower in these cells relative to that found in control V79 cells. Thus, our results suggest that (a) up-regulation of GRP78 is associated with an impairment of DNA cross-link repair, (b) up-regulation of GRP78 is associated with potentiation of cytotoxicity induced by alkylating and platinating agents, and (c) up-regulation of GRP78 can be considered as a potentially useful tool to modulate the cytotoxicity of clinically useful alkylating and platinating agents.

The GTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor). We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that "GSF activity" was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second-most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion after pretreatment with GTP gamma S.

In recent preclinical and clinical trials, topoisomerase I inhibitors have shown great promise as antitumor agents. These agents are most effective against tumors with high topoisomerase I activity. Therefore, determining topoisomerase I activity in advance may predict response to topoisomerase I inhibitors. Squamous cell carcinoma of the head and neck and normal tissue samples were obtained from 12 patients. Cellular extracts were prepared, and topoisomerase I activity assays were performed. The results suggest that topoisomerase I activity in squamous cell carcinoma of the head and neck is increased approximately sixtyfold compared to normal tissue. Increased activity often correlates with clinical responsiveness; these results predict that topoisomerase I inhibitors should be effective and selective against squamous cell carcinoma of the head and neck.

The two proteins from bovine brain previously shown to be required for the guanosine 5'-[gamma-thio]triphosphate-dependent inhibition of a well-characterized intra-Golgi transport assay, termed GGBF and GGBF, have been definitively identified as members of the ADP-ribosylation factor (ARF) family by electrospray MS analysis of the intact proteins, and of their tryptic fragments. Extensive protein-sequence information obtained from this analysis identified GGBF and GGBF as bovine ARF1 and ARF3 respectively. The sequence of bovine ARF3, which had not previously been determined, appears identical to that predicted from the rat and human ARF3 cDNAs. Further analysis of the N-terminal tryptic fragments of both bovine ARFs demonstrates N-terminal acylation solely with the C14:0 fatty acid (myristate). This finding establishes that the previously reported specific-activity difference between ARF1 and ARF3 in the intra-Golgi transport assay is not due to lipid heterogeneity at the N-terminus. This finding also indicates that the heterogeneity of N-terminal fatty-acyl groups previously observed on other myristoylated proteins is not universal.

We have used two different approaches to study the consequences of NAD/poly(ADP-ribose) deficiency on p53 expression and its activity in V79-derived cell lines. In the first approach, we have used two cell lines that are deficient in poly(ADP-ribose) (pADPR) synthesis because of deficiency in the enzyme poly(ADP-ribose) polymerase (PARP). In a second approach, we have used a cell line that is deficient in NAD/pADPR metabolism due to unavailability of NAD, the substrate for PARP. These NAD/PARP-deficient cell lines exhibit a significant reduction in both baseline p53 expression and its activity compared to their parental V79 cells. Furthermore, etoposide, a topoisomerase II inhibitor that was shown to cause an increase in p53 expression and subsequent apoptosis in V79 cells, failed to produce any significant increase in p53 expression or apoptotic DNA fragmentation in NAD/PARP-deficient cell lines. Thus, our studies suggest that NAD/pADPR synthesis may be involved in the regulation of p53 and its dependent pathways.

We have recently demonstrated that cell lines deficient in poly(ADP-ribose) synthesis due to deficiency in the enzyme poly(ADP-ribose) polymerase (PADPRP) or depletion of its substrate NAD+ overexpress GRP78. Furthermore, this overexpression of GRP78 is associated with the acquisition of resistance to topoisomerase II-directed drugs such as etoposide (VP-16); (S. Chatterjee et al., Cancer Res., 54: 4405-4411, 1994). Thus, our studies suggest that interference with NAD+-PADPRP metabolism could provide an important approach to (a) define pathways of GRP78 induction, (b) study the effect of GRP78 on other cellular processes, (c) elucidate the mechanism of GRP78-dependent resistance to topoisomerase II targeted drugs, and (d) modulate responses to chemotherapy in normal and tumor tissues. However, in the in vivo situation, it is impractical to interfere with NAD+-PADPRP metabolism by mutational inactivation of PADPRP or by depletion of its substrate NAD+. Therefore, we have examined several inhibitors of NAD+-PADPRP metabolism including 3-aminobenzamide, PD128763, and 6-aminonicotinamide for their ability to reproduce the results obtained with cell lines deficient in NAD+-PADPRP metabolism relative to the induction of GRP78 and subsequent development of resistance to VP-16. Our studies show that 6-aminoicotinamide treatment is highly effective in the induction of GRP78 and subsequent development of resistance to VP-16, whereas treatment with 3-aminobenzamide or PD128763 does not induce GRP78 and thus does not result in VP-16 resistance.

V511 and V513 cell lines, derived from Chinese hamster V79 cells following alkylating agent mutagenesis and subsequent selection with VP-16, showed resistance to cytotoxicity and DNA strand breaks induced by topoisomerase (topo) II inhibitors and were resistant to VP-16-induced sister chromatid exchanges. They showed no amplification of the multidrug-resistant p-glycoprotein. In a kinetoplast-DNA decatenation assay, V511 and V513 showed 51% and 49% topo II activity relative to parental V79 cells, respectively. By western-blot analysis all three logarithmically growing cell lines showed similar levels of topo II beta (M(r) 180,000), which increased as cells progressed to quiescence. In contrast, immunoreactive levels of topo II alpha (M(r) 170,000) were 6.8% in V511 and 62.4% in V513 relative to V79. V511 showed drastically decreased topo II alpha in both log growth and quiescence. In a second approach, immunoreactive topo II was analyzed in different phases of the cell cycle in logarithmically growing cells fractionated by fluorescence-activated cell sorting. All cell lines demonstrated relatively stable topo II beta throughout the cell cycle. Topo II alpha showed little cell cycle variation in V79 or V513. However, in V511, it was only detectable at low levels in G2/M phase. When cell growth parameters were measured, V511 and V513 showed a 17% increase in cell doubling time relative to V79. These studies indicate that cells with a drastic reduction in topo II alpha (V511) or mutant topo II alpha (V513) but with normal levels of topo II beta show only minor perturbations of cell growth.

Cell lines deficient in poly(ADP-ribose) synthesis due to enzyme deficiency (ADPRT54 and ADPRT351) or substrate deficiency (N2, N3, and N4) are resistant to topoisomerase II-directed agents, including etoposide (VP-16), N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide, and Adriamycin, relative to the effect of these agents on parental V79 Chinese hamster cells. Resistance is stable in the ADPRT54 and ADPRT351 cell lines, whereas resistance in the N2, N3, and N4 cell lines occurs when the cells are grown in nicotinamide-deficient medium to produce a state of NAD deficiency. However, sensitivity to VP-16 reverts to normal when cellular NAD levels return to control levels during growth in nicotinamide-containing complete medium. Poly(ADP-ribose) polymerase-deficient cell lines show constitutively increased levels of a protein at M(r) 78,000 on Coomassie blue-stained, sodium dodecyl sulfate-polyacrylamide gels that was subsequently confirmed with monoclonal antibodies to be M(r) 78,000 glucose-regulated stress protein (GRP78). Similarly, N2, N3, and N4 cells show induction of GRP78 under nicotinamide-deficient conditions. Induction of GRP78 is associated with elevated levels of GRP78 mRNA and appears to be regulated at the transcriptional level. When N3 cells with deficiency of poly(ADP-ribose) synthesis due to NAD deficiency are shifted to complete, nicotinamide-containing medium, they restore their NAD content, undergo a decrease in GRP78 levels, and regain sensitivity to VP-16. When V79 cells are shifted to nicotinamide-deficient medium they undergo a reduction in NAD content, followed by a progressive elevation in GRP78 levels, and they subsequently become increasingly resistant to VP-16. These studies demonstrate a clear association between deficiency of the NAD-poly(ADP-ribose) synthesis system, induction of GRP78 synthesis, and resistance to VP-16.

MCF-7 human breast cancer cells possess high levels of O6-alkylguanine-DNA alkyltransferase and moderate levels of glutathione, and are more resistant to chloroethylnitrosoureas (CNUs) than cells with low levels of either molecule. The role of each as a component of CNU resistance was assessed using O6-benzylguanine (O6-bG) or O6-methylguanine (O6-mG) to deplete the alkyltransferase and L-buthionine sulfoxamine (L-BSO) to deplete glutathione. O6-bG and O6-mG potentiated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity, resulting in a dose modification factor of 5.4 and 2.3, respectively, which reflected the more potent inhibitory effect of O6-bG. L-BSO alone had little effect on the survival of MCF-7 cells following BCNU exposure, but when combined with O6-mG, BCNU cytotoxicity was additive, yielding a dose modification factor of 3.2. O6-bG or O6-mG and L-BSO acted independently, as neither class of inhibitor affected the other's mechanism of CNU resistance. Furthermore, MCF-7 cells overexpressing GST mu were not more resistant to BCNU than the parent cell line in either the presence or absence of O6-bG or L-BSO. These results indicate that on a relative basis in MCF-7 cells, the alkyltransferase is the cell's first line of defense against CNUs. This suggests that therapeutic trials based on O6-bG-induced biochemical modulation of CNU resistance may increase the efficacy of these chemotherapeutic agents against human malignant cells and that L-BSO may have little additive effect when used with these agents.

We have measured glutathione content in small tissue samples derived from biopsies of primary and metastatic human colon tumors and from colon cancer cell lines in tissue culture and xenografts in athymic mice. Measurements were performed using an enzymatic cycling assay designed to quantitate extremely low levels of glutathione (GSH) (down to 10(-14) mol) from perchlorate extracts of tissue samples weighing less than 1 mg wet weight. Glutathione was stable in these acid extracts for at least 6 months when stored at -80 degrees C. A survey of normal tissues in mice, rats, and some human tissues showed considerable variation in GSH content of different tissues but generally similar levels were identifiable for the same tissues from different species. The highest GSH level was 56.9 nmol/mg protein in rat liver and the lowest was 1.8 nmol/mg protein in rat skeletal muscle. High GSH levels were also determined in mouse and human liver, while low GSH levels were detected in mouse muscle. Human colon cancer cell lines showed slightly higher GSH levels than did colon cancer tumor samples obtained from biopsies. These studies revealed a marked inter-individual difference in tumor GSH content, as well as a difference in GSH content between tumor deposits at different metastatic sites in the same individual. These results indicate the importance of direct tumor measurements of GSH content in clinical trials designed to modulate tumor glutathione content to try to increase sensitivity to chemotherapy or radiation therapy. Buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase, was shown to produce almost complete depletion of GSH in four different human colon cancer cell lines in 24 h. Buthionine sulfoximine was also shown to be capable of producing drastic depletion of GSH in human colon cancer grown as xenografts in athymic animals.

Starting with the V79 cell line, two poly(ADP-ribose) polymerase deficient mutants, designated ADPRT 54 and ADPRT 351, had been shown to be hypersensitive to x- and UV-irradiation and to topoisomerase I inhibitors but to be resistant to topoisomerase II inhibitors (Chatterjee, S.; Cheng, M. F.; Berger, N. A. Hypersensitivity to clinically useful alkylating agents and radiation in poly(ADP-ribose) polymerase-deficient cell lines. Cancer Commun. 2:401-407;1990). We now report that these mutants were hypersensitive to a series of different alkylating agents, including alkylsufonates, alkylnitrosoureas, and nitrosoguanidine. In addition, they were hypersensitive to the UV-mimetic agent 4-nitroquinoline-1-oxide. Our findings provide strong evidence that poly(ADP-ribose) polymerase was involved in the repair of alkylating agent induced DNA damage as well as in the damage induced by UV- and x-irradiation and radiomimetic agents. The poly(ADP-ribose) polymerase deficient cell lines showed a marked decrease in the shoulder region of their survival curves, suggesting that poly(ADP-ribose) polymerase was involved in the repair of alkylating agent induced sublethal damage.

CHO cells and cs-4-D3 cells were used to investigate the association between poly(ADP-rib) synthesis and the cessation of DNA synthesis and DNA fragmentation. The cs4-D3 cells are cold-sensitive DNA synthesis arrest mutants of CHO cells. Upon incubation at 33 degrees C, DNA synthesis in the cs4-D3 cells stops and the cells enter a prolonged G1 or G0 phase. The events that occurred when cs4 cells were incubated at 33 degrees C were similar to those that occurred when wild-type CHO cells grew to high density. (1) In both cases, DNA synthesis and cell growth stopped. (2) The NAD+ concentration/cell was 20-25% lower in growth-arrested cells than in logarithmically growing cells. (3) Poly(ADP-rib) synthesis was 3-4 fold higher in growth-arrested cells than in logarithmically growing cells. (4) The growth-inhibited cells developed DNA strand breaks which resulted in large percentages of their DNA appearing in the low molecular weight range of alkaline sucrose gradients. (5) Both the increased rate of poly(ADP-rib) synthesis and the development of DNA strand breaks appears to be characteristic of the G1 phase of the cell cycle. (6) When growth-inhibited cells were restored to conditions favorable for DNA synthesis and cell growth, the DNA strand breaks were repaired. (7) Prolonged incubation under growth-restrictive conditions resulted in the accumulation of more DNA strand breaks than the cells could repair. This was followed by cell death when the cells were restored to conditions favorable for cell growth.

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD+ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treatment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD+ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.

Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01-0.25 microgram dry weight, were dissected from freeze-dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose-6-phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and beta-hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose-6-phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6-phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol-1-phosphate, and malate averages ranged from 104% to 127% of brain; and fructose-1,6-bisphosphate and glucose-1,6-bisphosphate levels were on the average 50% and 70% those of brain, respectively.

Guanine deaminase was measured in nearly 100 different areas of mouse brain. The levels are relatively high in all parts of the telencephalon, both gray and white. It is especially active in parts of the olfactory tubercle and amygdala. Levels in the diencephalon range from low to as high as in the telencephalon. Brain areas caudal to the diencephalon, including all parts of the cerebellum, are almost uniformly below the level of detection. The enzyme is also virtually absent from the retina. The extreme range of concentration suggests that guanine deaminase might play a role in the metabolism of a neuroeffector.

Treatment of cells with DNA damaging agents results in a dose dependent decrease in NAD+ and ATP pool sizes. The decrease in NAD+ is associated with the activation of poly(ADP-ribose) polymerase and the decrease in ATP is consequent to the fall in NAD+. Depletion of both NAD+ and ATP can be blocked or retarded by inhibitors of poly(ADP-ribose) polymerase. Both the stimulation of poly(ADP-ribose) synthesis and the effect of enzyme inhibitors have been confirmed in intact cells by using enzymatic cycling techniques to measure the disappearance of NAD+ and high pressure liquid chromatography (HPLC) to measure fluctuations in polymer levels. As a consequence of the depletion of NAD+ and ATP pools, cells exhibit a marked impairment in their ability to conduct all energy dependent functions. Thus cells treated with high levels of DNA damaging agents exhibit severe suppression of DNA replication and repair, RNA synthesis and protein synthesis. The use of inhibitors of poly(ADP-ribose) polymerase to prevent the depletion of NAD+ and ATP partially restores the cells' ability to conduct DNA, RNA, and protein synthesis. This preservation of the NAD+ and ATP pools accounts for the recent observations that inhibitors of poly(ADP-ribose) stimulate the level of DNA repair synthesis in cells treated with high levels of DNA damaging agents. We have also examined cells from patients with several of the disorders of DNA repair and have found that cells from patients with Fanconi's anemia have lower than normal NAD+ levels.(ABSTRACT TRUNCATED AT 250 WORDS)

We used two different approaches to develop cell lines deficient in poly(ADP-ribose) synthesis to help determine the role of this reaction in cellular functions. One approach to this problem was to develop cell lines deficient in enzyme activity; the other approach was to develop cell lines capable of growing with such low nicotinamide adenine dinucleotide (NAD) levels so as to effectively limit substrate availability for poly(ADP-ribose) synthesis. The selection strategy for obtaining cells deficient in activity of poly(ADP-ribose) polymerase was based on the ability of this enzyme to deplete cellular NAD in response to high levels of DNA damage. Using this approach, we first obtained cell lines having 37-82% enzyme activity compared to their parental cells. We now report the development and characterization of two cell lines which were obtained from cells having 37% enzyme activity by two additional rounds of further mutagenization and selection procedures. These new cell lines contain 5-11% enzyme activity compared to the parental V79 cells. In pursuit of the second strategy, to obtain cells which limit poly(ADP-ribose) synthesis by substrate restriction, we have now isolated spontaneous mutants from V79 cells which can grow stably in the absence of free nicotinamide or any of its analogs. These cell lines maintain NAD levels in the range of 1.5-3% of that found in their parental V79 cells grown in complete medium. The pathway of NAD biosynthesis in these NAD-deficient cells is not yet known. Further characterization of these lines showed that under conditions that restricted poly(ADP-ribose) synthesis, they all had prolonged doubling times and increased frequencies of sister chromatid exchanges.

Frozen sections of retinas from rabbit (mostly rods), ground squirrel (mostly cones), and monkey (mixed rods and cones) were freeze dried, and samples from all the discrete layers analyzed for the enzymes which form cyclic GMP and subsequently convert it back to GTP. The distribution of cyclic GMP was also measured in monkey retina, and the retinal layers of both monkey and rabbit were analyzed for GTP, GTP plus GDP, ATP, ATP plus ADP, and UTP plus CTP. The ratio of guanylates to adenylates was found to be about 1:1 in photoreceptor cell layers, but only 1:4 or 5 in deeper layers. In all species, guanylate cyclase (EC 4.6.1.2) and cyclic GMP phosphodiesterase were highest in the outer segment layer. Other layers were lower by factors of 10 to 500. Guanylate kinase (EC 2.7.4.8) was extremely high in all photoreceptor cell layers except the outer segments, but was much lower elsewhere. Nucleoside diphosphokinase (EC 2.7.4.6) paralleled guanylate kinase throughout the photoreceptor cell layers, but did not fall to such low levels in the deeper layers of the retina. Although there were significant differences among the three species, they all displayed the same general enzyme pattern.

The possibility that corticosteroid cytotoxicity could be mediated by activation of poly(ADP-ribose) polymerase and consequent depletion of NAD and ATP was evaluated in steroid-sensitive S49.1 and steroid-resistant S49.143R mouse lymphoma cells and in lymphocytes from a patient with chronic lymphocytic leukemia. All cell types were shown to have the enzyme poly(ADP-ribose) polymerase and to increase activity in response to DNA strand breaks. Incubation of susceptible cells with 1 microM dexamethasone resulted in DNA strand breaks. Susceptible cells also showed a dose-dependent decrease in NAD and ATP that preceded loss of cell viability. These studies suggest that steroid-induced cytotoxicity in susceptible lymphocytes is due to the presence of DNA strand breaks that activate poly(ADP-ribose) polymerase to a sufficient degree to consume cellular pools of NAD with a consequent depletion of ATP and loss of cell viability.