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The killing of tumor cells by CD8(+) T cells is suppressed by the tumor microenvironment, and increased expression of inhibitory receptors, including programed cell death protein-1 (PD-1), is associated with tumor-mediated suppression of T cells. To discover cellular defects triggered by tumor exposure and associated PD-1 signaling, we established an ex vivo imaging approach to investigate the response of antigen-specific, activated effector CD8(+) tumor infiltrating lymphocytes (TILs) after interaction with target tumor cells. Although TIL–tumor cell couples readily formed, couple stability deteriorated within minutes. This was associated with impaired F-actin clearing from the center of the cellular interface, reduced Ca(2+) signaling, increased TIL locomotion, and impaired tumor cell killing. The interaction of CD8(+) T lymphocytes with tumor cell spheroids in vitro induced a similar phenotype, supporting a critical role of direct T cell–tumor cell contact. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus contributes to the suppression of TIL function by inducing a state of impaired subcellular organization.

Purpose: Our preclinical studies showed that the PARP inhibitor, olaparib, prior to carboplatin attenuated carboplatin cytotoxicity. We evaluated sequence-specific pharmacokinetic and pharmacodynamic effects, safety, and activity of the combination. Experimental Design: Eligible patients had metastatic or recurrent women's cancer. Olaparib tablets were introduced (100 or 200 mg twice daily, days 1–7) in a 3 + 3 dose escalation with carboplatin AUC4 or 5 every 21 days, up to eight cycles, followed by olaparib 300 mg twice daily maintenance. Patients were randomly assigned to starting schedule: cohort A (olaparib days 1–7, carboplatin on day 8) or B (carboplatin on day 1, olaparib days 2–8) during cycle 1. Patients received the reversed scheme in cycle 2. Blood was collected for olaparib pharmacokinetics, platinum–DNA adducts, comet assay, and PAR concentrations. The primary objectives were to examine schedule-dependent effects on olaparib pharmacokinetics and platinum–DNA adducts. Results: A total of 77 (60 ovarian, 14 breast, and 3 uterine cancer) patients were treated. Dose-limiting toxicity was thrombocytopenia and neutropenia, defining olaparib 200 mg twice daily + carboplatin AUC4 as the MTD. Olaparib clearance was increased approximately 50% when carboplatin was given 24 hours before olaparib. In vitro experiments demonstrated carboplatin preexposure increased olaparib clearance due to intracellular olaparib uptake. Quantities of platinum–DNA adducts were not different as a function of the order of drug administration. Responses included 2 CRs and 31 PRs (46%) with a higher RR in BRCA mutation carriers compared with nonmutation carriers (68% vs. 19%). Conclusions: Tablet olaparib with carboplatin is a safe and active combination. Carboplatin preexposure causes intracellular olaparib accumulation reducing bioavailable olaparib, suggesting carboplatin should be administered prior to olaparib. Clin Cancer Res; 23(6); 1397–406. ©2016 AACR.

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates., eLife digest Cells receive dozens of signals at different times and in different places. Integrating incoming information and deciding how to respond is no easy task. Signaling molecules on the cell surface pass messages inwards using chemical messengers that interact in complicated networks within the cell. One way to unravel the complexity of these networks is to look at specific groups of signaling molecules in test tubes to see how they interact. But the interior of a living cell is a very different environment. Molecules inside cells are tightly packed and, under certain conditions, they interact with each other by the thousands. They form structures known as ‘supramolecular complexes’, which changes their behavior. One such supramolecular complex is the ‘central supramolecular activation cluster’, or cSMAC for short. It forms under the surface of immune cells called T cells when they are getting ready to fight an infection. Under the microscope, the cSMAC looks like the bullseye of a dartboard, forming a crowd of signaling molecules at the center of the interface between the T cell and another cell. Its exact role is not clear, but evidence suggests it helps to start and stop the signals that switch T cells on. The cSMAC contains two key protein adaptors called LAT and SLP-76 that help to hold the structure together. So, to find out what the cSMAC does, Clark et al. genetically modified these adaptors to gain control over when the cSMAC forms. Clark et al. examined mouse T cells using super-resolution microscopy and electron microscopy, watching as other immune cells delivered the signal to switch on. As the T cells started to activate, the composition of the cSMAC changed. In the first two minutes after the cells started activating, the cSMAC included a large number of different components. This made T cell activation more efficient, possibly because the supramolecular complex was helping the network of signals to interact. Later, the cSMAC started to lose many of these components. Separating components may have helped to stop the activation signals. Understanding how T cells activate could lead to the possibility of turning them on or off in immune-related diseases. But these findings are not just relevant to immune cells. Other cells also use supramolecular complexes to control their signaling. Investigating how these complexes change over time could help us to understand how other cell types make decisions.

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.

Background Based on pre-clinical and clinical activity in adult refractory tumors, and absence of significant neuro-, nephro-, or oto-toxicity, we conducted a pediatric phase 1 trial to determine the toxicities, maximum tolerated dose (MTD), and pharmacokinetics of satraplatin, an oral platinum analogue, in children and young adults with refractory solid tumors. Procedure Satraplatin was administered orally once daily on days 1–5 of a 28-day cycle at dose level (DL) 1 (60 mg/m2/dose), and DL2 (80 mg/m2/dose). Toxicities, responses, satraplatin pharmacokinetics, and pharmacogenomic expression of specific DNA repair genes were evaluated. Results Nine patients received 1–15 cycles (median = 2). The MTD was exceeded at DL2 with delayed prolonged myelosuppression as dose-limiting toxicity (DLT) in 2/4 patients. At DL1, 0/5 patients had DLTs. Common non-DLTs included myelosuppression, gastrointestinal toxicities, fatigue, headache, liver enzyme elevation, and electrolyte abnormalities. No significant neuro-, nephro-, or oto-toxicity was observed. No objective responses were observed but 2 patients experienced prolonged disease stabilization (­­­6–15 cycles). Satraplatin exposure (day 1 plasma ultrafiltrate area under the curve) was similar at DL1 and DL2. A strong correlation between estimated creatinine clearance and satraplatin pharmacokinetic parameters (clearance, area under the curve, and peak concentration) was observed. Conclusions The MTD of oral satraplatin in children with solid tumors was 60 mg/m2/dose daily ×5 days every 28 days, which is lower than the adult recommended dose of 80–120 mg/m2/dose. The toxicity profile was similar to adults and delayed myelosuppression was the DLT. No significant neuro-, nephro- or oto-toxicities were observed. Pediatr Blood Cancer 2015;62:603–610. © 2015 Wiley Periodicals, Inc.

Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.

Three‐dimensional live cell imaging of the interaction of T cells with antigen presenting cells (APC) visualises the subcellular distributions of signalling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signalling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signalling as it occurs inside live cells with resolution in time and space across thousands of cells.

Small cell lung cancer (SCLC) is an aggressive lung cancer subtype in need of better therapies. Histone deacetylase inhibitors (HDIs) promote increased lysine acetylation in nucleosomal histones and are thought to relax chromatin, thereby allowing increased access of transcription factors and DNA damaging agents alike to DNA. We studied whether two HDIs, belinostat and romidepsin, could be effectively combined with cisplatin or etoposide (VP-16) for SCLC cells. Analysis of cell survival and synergy was performed using CalcuSyn mathematical modeling to calculate a combination index. Immunostaining of γH2AX was performed to evaluate persistence of DNA damage following simultaneous or sequential exposure. Based on CalcuSyn modeling, HDIs synergized with DNA damaging agents only when added simultaneously. An additive-to-antagonistic effect was seen with HDI pretreatment for 24 h or with addition after cisplatin or etoposide. Furthermore, pretreatment with HDIs resulted in normalization of cell cycle and reduced PARP degradation as compared with simultaneous treatment. The increase in γH2AX phosphorylation confirmed that simultaneous but not sequential treatment enhanced double-stranded DNA breaks. These results suggest that DNA relaxation is not required for synergy of HDIs with DNA damaging agents, and that scheduling of drug administration will be critical for rational development of clinical protocols.

PURPOSE: Cisplatin, carboplatin, and oxaliplatin are chemically reactive anticancer drugs with modest activity in brain tumors. Previously, we have demonstrated that drug exposure in cerebrospinal fluid (CSF) for these platinum analogs is

The human mannose 6-phosphate uncovering enzyme participates in the uncovering of the mannose 6-phosphate recognition tag on lysosomal enzymes, a process that facilitates recognition of those enzymes by mannose 6-phosphate receptors to ensure delivery to lysosomes. Uncovering enzyme has been identified on the trans-Golgi network at steady state. It has been shown to traffic to the plasma membrane from where it is rapidly internalized via endosomal structures, the process being mediated by a tyrosine-based internalization motif, Y488HPL, in its cytoplasmic tail. Using immunogold electron microscopy a GFP-uncovering enzyme fusion construct was found to be colocalized with the cation-dependent mannose 6-phosophate receptor in regions of the trans-Golgi network, suggesting that uncovering enzyme might follow a similar pathway of exit from the trans-Golgi network as that of the cation-dependent mannose 6-phosohate receptor. In this study, we identified the signal sequence in the cytoplasmic tail of uncovering enzyme responsible for its exit from the trans-Golgi network. Using GFP fusion constructs of the transmembrane and cytoplasmic domains of uncovering enzyme, we could show, by automated analysis of confocal immunofluorescence images, that residues Q492EMN in the cytoplasmic tail of uncovering enzyme are involved in its exit from the trans-Golgi network. Detailed characterization of the exit signal revealed that residue Q492 is the most important to the exit function while M494 and N495 also contribute. The cytoplasmic tail of the uncovering enzyme does not possess any of the known canonical signal sequences for interaction with Golgi-associated gamma ear-containing adaptor proteins. The identification of a trans-Golgi network exit signal in its cytoplasmic tail elucidates the trafficking pathway of uncovering enzyme, a crucial player in the process of lysosomal biogenesis.

Information technology research has played a significant role in the genomics revolution over the past decade, from aiding with large-scale sequence assembly to automating gene identification to efficiently searching databases by sequence similarity. The tremendous amount of information gathered from genomics will be dwarfed in the next decade by the knowledge to be gained from comprehensive, systematic studies of the properties and behaviors of all proteins and other biomolecules. High-resolution imaging of molecules and cells will be critical for understanding complex systems such as the nervous system, whether it be for the localization of specific neuron types within a region of the central nervous system, the branching pattern of dendritic trees, or the localization of molecules at the subcellular level. Furthermore, knowing how these distribution patterns and subcellular locations change as a function of time is critical to understanding how cells respond to stress, injury, aging, and disease. We are developing sophisticated information technologies for collecting and interpreting the enormous volume of biological image data. A major outcome of the research will be a unique, fully operational, distributed digital library of biomolecular image data accessible to researchers around the world. Such searchable databases will make it possible to optimally understand and interpret the data, leading to a more complete and integrated understanding of cellular structure, function and regulation.

Hypericin, a polycyclic aromatic dianthroquinone, is a natural pigment derived from the plant Hypericum perforatum (St John's Wort). The compound has been synthesized and shown to inhibit the growth of malignant glioma cell lines in vitro via inhibition of protein kinase C. Oral hypericin has entered clinical trials in adults with recurrent malignant glioma. Purpose: The present study was performed to characterize the plasma pharmacokinetics (PK) and cerebrospinal fluid (CSF) penetration of hypericin in nonhuman primates. Methods: Hypericin was administered as an intravenous bolus dose of 2 mg/kg (n=3) or 5 mg/kg (n=1). Plasma and CSF (ventricular or lumbar) were sampled prior to administration and at frequent intervals for up to 50 h after administration of the drug. Hypericin concentrations in plasma and CSF were determined using a specific reverse-phase HPLC assay. Results: Mean peak plasma concentration of hypericin following the 2 mg/kg dose was 142±45 µM. Elimination of hypericin from plasma was biexponential, with an average alpha half-life of 2.8±0.3 h and average terminal half-life of 26±14 h. Conclusions: The 2 mg/kg dose in the nonhuman primate was sufficient to maintain plasma concentrations above 10 µM (the in vitro concentration required for growth inhibition of human glioma cell lines) for up to 12 h. No hypericin was detected in the CSF of any animal (lower limit of detection 0.1 µM); the CSF penetration is therefore less than 1%. A severe dose-limiting photosensitivity skin rash was seen at the 5 mg/kg dose level.

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35 S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of

PURPOSE To determine the maximum-tolerated dose (MTD) of all-trans-retinoic acid (ATRA) administered on an intermittent oral schedule with interferon-alpha2a (IFN-alpha2a) in children with refractory cancer, and whether the marked reduction in plasma ATRA concentrations observed with chronic daily oral dosing could be circumvented with an intermittent dosing schedule. PATIENTS AND METHODS Thirty-three children with refractory cancer (stratified by age, < or = 12 and > 12 years) were treated with ATRA 3 consecutive days per week and IFN-alpha2a 3 x 10(6) U/m2 5 consecutive days per week, both repeated weekly. The starting dose of ATRA was 60 mg/m2/d divided into three doses, with planned escalations to 90 and 120 mg/m2/d. Because severe headaches have been noted to occur on the initial day of ATRA administration, only two of three doses of ATRA were administered on day 1 of each week. RESULTS Pseudotumor cerebri or dose-limiting headache was observed in two of five patients older than 12 years treated at the 120-mg/m2/d dose level and in one of six < or = 12 years at the 90-mg/m2/d level. Other non-dose-limiting toxicities of ATRA included reversible elevations in hepatic transaminases and triglycerides, dry skin, cheilitis, and nausea/vomiting. One child with recurrent neuroblastoma had an objective response of 6 months' duration, and one with recurrent Wilms' tumor had histologic maturation of multiple tumors. This intermittent schedule allowed for exposure to relatively high plasma concentrations of ATRA on a repetitive basis. Following 30-mg/m2 doses, the ATRA area under the concentration-time curve (AUC) decreased from 96 +/- 14 micromol/L/min on day 1 to 26 +/- 24 micromol/L/min by day 3 of drug administration, but on day 1 of the fourth consecutive week of therapy, the AUC averaged 110 +/- 16 micromol/L/min. The recommended pediatric phase II dose of ATRA administered on this schedule is 90 mg/m2/d. CONCLUSION An intermittent schedule of ATRA administration appears to circumvent the low plasma drug exposure that is a result of the sustained upregulation of metabolism when this drug is administered on a chronic daily schedule. Based on the results of this trial, a phase II trial of ATRA/IFN-alpha2a in neuroblastoma and Wilms' tumor using this schedule is in progress.

Location proteomics is concerned with the systematic analysis of the subcellular location of proteins. In order to perform comprehensive analysis of all protein location patterns, automated methods are needed. With the goal of extending automated subcellular location pattern analysis methods to high resolution images of tissues, 3D confocal microscope images of polarized CaCo 2 cells immunostained for various proteins were collected. A three-color staining protocol was developed that permits parallel imaging of proteins of interest as well as DNA and the actin cytoskeleton. The collection is composed of 11 to 21 images for each of the 9 proteins that depict major subcellular patterns. A classifier was trained to recognize the subcellular location pattern of segmented cells with an accuracy of 89.2%. Using the Prior Updating method allowed improvement of this accuracy to 99.6%. This study demonstrates the benefit of using a graphical model approach for improving the pattern classification in tissue images.

While basic principles of microtubule organization are well understood, much remains to be learned about the extent and significance of variation in that organization among cell types and conditions. Large numbers of images of microtubule distributions for many cell types can be readily obtained by high throughput fluorescence microscopy but direct estimation of the parameters underlying the organization is problematic because it is difficult to resolve individual microtubules present at the microtubule-organizing center or at regions of high crossover. Previously, we developed an indirect, generative model-based approach that can estimate such spatial distribution parameters as the number and mean length of microtubules. In order to validate this approach, we have applied it to 3D images of NIH 3T3 cells expressing fluorescently-tagged tubulin in the presence and absence of the microtubule depolymerizing drug nocodazole. We describe here the first application of our inverse modeling approach to live cell images and demonstrate that it yields estimates consistent with expectations.

The thiopurines have a wide array of effects on purine metabolism, but the primary mechanism of cytotoxicity for both 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) appears to be incorporation of drug into DNA following conversion to the thioguanylate form. In murine leukemic cell lines exposed to a range of thiopurine concentrations in vitro, cell survival curves have displayed a phenomenon termed paradoxical cytotoxicity, defined as a decrease in cytotoxicity with increasing drug concentration. The paradoxical cytotoxicity of thiopurines has usually been attributed to concentration-dependent perturbations in the cell cycle. The present study assessed whether the paradoxical cytotoxicity of 6-MP occurred in cultured human leukemic cells, and investigated the biochemical and cell-cycle alterations occurring in these lines at thiopurine concentrations associated with the reverse of cytotoxicity. Paradoxical cytotoxicity was observed in the two human leukemic cell lines examined, but only when 6-MP concentrations exceeded 100 microM. The extent of incorporation of 6-MP metabolites into DNA as thiol-versus non-thiol-containing metabolites was analyzed by performing parallel experiments with 14C- and 35S-radiolabeled drug. With 5 microM 6-MP, approximately 50% of drug was incorporated into DNA as a thionucleotide; however, with increasing drug concentrations, the degree of thionucleotide incorporation remained unchanged or decreased, and the amount incorporated as the desulfurated metabolite (presumably adenylate or guanylate) increased. With 500 microM 6-MP, less than 10% of the drug was incorporated as the thionucleotide. Perturbations in cell cycle reflected the relative amounts of thiol- and non-thiol-containing nucleotide formed at various concentrations of 6-MP. These results suggest that thiopurines may be vulnerable to a unique mechanism of detoxification, in which a human cell can metabolize a cytotoxic drug to a comparatively potent "self-rescue" agent.

Purpose To assess the role of the recombinant bacterial enzyme, glucarpidase (carboxypeptidase-G2), leucovorin, and thymidine in the management and outcome of patients with high-dose methotrexate (HDMTX) –induced nephrotoxicity. Methods Patients with HDMTX-induced nephrotoxicity received one to three doses of intravenous (IV) glucarpidase and leucovorin rescue. The initial cohort (n = 35) also received thymidine by continuous IV infusion. Subsequently, thymidine was restricted to patients with prolonged exposure (> 96 hours) to methotrexate (MTX) or with substantial MTX toxicity at study entry. Plasma MTX, leucovorin, and 5-methyltetrahydrofolate (5-mTHF) concentrations were measured pre- and postglucarpidase. Toxicities were monitored, and logistic regression analysis was used to assess the relationship of baseline characteristics to the development of severe toxicity and death. Results Glucarpidase was administered at a median of 96 hours (receiving thymidine, n = 44) and 66 hours (not receiving thymidine, n = 56) after the start of the MTX infusion. Plasma MTX concentrations decreased within 15 minutes of glucarpidase by 98.7%. Plasma 5-mTHF concentrations also decreased more than 98% after administration of glucarpidase. Of 12 deaths, six were directly attributed to irreversible MTX toxicity. Presence of grade 4 toxicity before administration of glucarpidase, inadequate initial increase in leucovorin dosing, and administration of glucarpidase more than 96 hours after the start of the MTX infusion were associated with development of grade 4 and 5 toxicity. Conclusion Early intervention with the combination of leucovorin and glucarpidase is highly effective in patients who develop HDMTX-induced renal dysfunction. Severe toxicity and mortality occurred in patients in whom glucarpidase rescue was delayed and occurred despite thymidine administration.

The usefulness and efficacy of cisplatin, a chemotherapeutic drug, are limited by its toxicity to normal tissues and organs, including the kidneys. The uptake of cisplatin in renal tubular cells is high, leading to cisplatin accumulation and tubular cell injury and death, culminating in acute renal failure. While extensive investigations have been focused on the signaling pathways of cisplatin nephrotoxicity, much less is known about the mechanism of cisplatin uptake by renal cells and tissues. In this regard, evidence has been shown for the involvement of organic cation transporters (OCT), specifically OCT2. The copper transporter Ctr1 is highly expressed in the renal tubular cells; however, its role in cisplatin nephrotoxicity is not known. In this study, we demonstrate that Ctr1 is mainly expressed in both proximal and distal tubular cells in mouse kidneys. We further show that Ctr1 is mainly localized on the basolateral side of these cells, a proposed site for cisplatin uptake. Importantly, downregulation of Ctr1 by small interfering RNA or copper pretreatment results in decreased cisplatin uptake. Consistently, downregulation of Ctr1 suppresses cisplatin toxicity, including cell death by both apoptosis and necrosis. Cimetidine, a pharmacological inhibitor of OCT2, can also partially attenuate cisplatin uptake. Notably, cimetidine can further reduce cisplatin uptake and cisplatin toxicity in Ctr1-downregulated cells. The results have demonstrated the first evidence for a role of Ctr1 in cisplatin uptake and nephrotoxicity.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.

In order to better understand the cellular delivery of iron from serum transferrin (Tf), we compared iron release from receptor-bound and free Tf. While free Tf did not release all iron until below pH 4.6, receptor-bound Tf released significantly more iron at mildly acidic pH, with essentially all iron released between pH 5.6 and 6.0. Since Tf is acidified to a minimum pH of 5.4 in K562 cells, this result accounts for the nearly complete extraction of iron from Tf by these cells. Comparison of fluorescence from Tf conjugated with lissamine rhodamine sulfonyl chloride (LRSC-Tf) free in solution and bound to receptor provides further evidence that the Tf receptor modulates low pH-mediated conformational changes in Tf. As pH was decreased from neutrality, the fluorescence of free LRSC-Tf began to increase below pH 6.2; the fluorescence of LRSC-Tf bound to human receptors did not increase until below pH 5.6. Binding to the Tf receptor, while facilitating iron release from Tf, appears to partially inhibit a conformational change that causes the increase in LRSC-Tf fluorescence at low pH. The fluorescence of human LRSC-Tf bound to murine receptors increases at a higher pH, 6.0, indicating that there are differences in conformational stabilization of Tf by receptors of different species. The results suggest that the Tf receptor, in addition to providing a means by which cells may internalize Tf, functions to increase the release of iron from Tf in the endosome.

Transferrin (Tf) acidification has been shown to be limited to pH 6 in murine Balb/c 3T3 fibroblasts, human A549 epidermoid carcinoma cells, and Chinese hamster ovary cells and is followed by alkalinization during recycling. In contrast, Tf acidification in the human erythroleukemic cell line K562 proceeds to below pH 5.5, and alkalinization of internal Tf during recycling is not observed. To explore the regulation of endosomal pH in K562 cells, we determined whether the existence of an early endosome of pH 6 could be demonstrated in K562 cells. The kinetics of Tf internalization, acidification, and recycling were determined at temperatures which block recycling of Tf in 3T3 cells. As in 3T3, Tf recycling in K562 was inhibited at 24 degrees C and below. At these temperatures, Tf internalization and acidification were delayed relative to 37 degrees C, yet the minimum pH achieved was below 5.5. Temperatures at or below 19 degrees C resulted in a complete block in recycling (at least over 40 min), which was rapidly reversible by incubation at 37 degrees C. Ouabain (a specific inhibitor of the Na+,K(+)-ATPase) had no effect on K562 Tf acidification, indicating that K562 endosomal pH is probably not regulated by the Na+,K(+)-ATPase. The results suggest that differentiation of the early endocytic pathway in erythroid cells involves changes such that the pH of Tf-containing compartments is not limited to 6 by the Na+,K(+)-ATPase.

Previous studies have shown that catecholamine secretion patterns have been imperfect predictors of clinical behavior of neuroblastomas. Recently, studies of nuclear DNA content in neuroblastoma have shown that an aneuploid DNA content predicts favorable clinical behavior. To determine if a correlation exists between these tumor biologic indicators, the authors analyzed both in a series of 39 patients with neuroblastoma. Flow cytometric DNA analysis performed on paraffin blocks determined that 23 patients had tumors with aneuploid DNA content (aneuploid tumors) and 16 patients showed no demonstrable anomalies of tumor DNA content (nonaneuploid tumors). Comparison of catecholamine levels in urine and tumor homogenates with DNA content data indicate that nonaneuploid neuroblastomas include a significant number (P less than 0.02) of biochemically primitive tumors which secrete high levels of 3,4 dihydroxyphenylalanine (DOPA), dopamine and homovanillic acid (HVA). This suggests a dopamine-norepinephrine pathway block, which supports previous reports of deficiency of dopamine beta-hydroxylase activity in some neuroblastomas. The study shows that in contrast to aneuploid tumors, nonaneuploid neuroblastomas secrete higher levels of early pathway catecholamine metabolites and are more likely to present in higher (unfavorable) clinical stages of disease.

The kinetics of exposure of endocytosed material to two lysosomal enzymes were determined for a number of cultured cell lines using fluorogenic substrates. Hydrolysis of endocytosed substrates for cathepsin B and acid phosphatase was observed to begin within 3-10 min of substrate addition and to proceed linearly for up to 60 min thereafter. Hydrolysis of the cathepsin B substrate was not affected by inhibition of protein synthesis with cycloheximide, indicating that the enzymes present in early endosomes are not exclusively newly synthesized. As had been observed previously for a cathepsin B substrate (Roederer, M., Bowser, R., and Murphy, R. F., J. Cell. Physiol., 131:200-209, 1987), hydrolysis of the acid phosphatase substrate was not blocked at temperatures below 20 degrees C. The results suggest that the endosome is the primary site of initial exposure of endocytosed material to hydrolytic enzymes.

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.

The effects of bafilomycin, nocodazole, and reduced temperature on recycling and the lysosomal pathway have been investigated in various cultured cell lines and have been shown to vary dependent on the cell type examined. However, the way in which these treatments affect recycling and transport to lysosomes within the same cell line has not been analyzed. In the current study, we used fluorophore-labeled transferrin and dextran as typical markers for the recycling and the lysosomal pathways, respectively, to explore the morphology and the intravesicular pH of endocytic compartments in HeLa cells. The V-ATPase inhibitor bafilomycin selectively inhibited the transport of marker destined for lysosomal degradation in early endosomes, whereas the transport of transferrin to the perinuclear recycling compartment (PNRC) still occurred. The kinetics of transferrin acidification was found to be biphasic, indicative of fast and slow recycling pathways via early endosomes (pH 6.0) and PNRC (pH 5.6), respectively. Furthermore, the disruption of microtubules by nocodazole blocked the transport of transferrin to the PNRC in early endosomes and of lysosome-directed marker into endosomal carrier vesicles. In contrast, incubation at 20 degrees C affected the lysosomal pathway by causing retention of internalized dextran in late endosomes and a delay in transferrin recycling. Taken together, these data clearly demonstrate, for the first time, that the transferrin recycling pathway and transport of endocytosed material to lysosomes are differentially affected by bafilomycin, nocodazole, and low temperature in HeLa cells. Consequently, these treatments can be applied to investigate whether internalized macromolecules such as viruses follow a recycling or degradative pathway.

Knowing the subcellular location of a protein is critical to a full understanding of its function, and automated, objective methods for assigning locations are needed as part of the characterization process for the thousands of proteins expressed in each cell type. Fluorescence microscopy is the most common method used for determining subcellular location, and we have previously described automated systems that can recognize all major subcellular structures in 2D fluorescence microscope images. Here we show that 2D pattern recognition accuracy is dependent on the choice of the vertical position of the 2D slice through the cell and that classification of protein location patterns in 3D images results in higher accuracy than in 2D. In particular, automated analysis of 3D images provides excellent distinction between two Golgi proteins whose patterns are indistinguishable by visual examination.

The goal of methods for ligand acidification by nonadherent cells is to obtain nearly continuous measurements of the pH to which a receptor-bound ligand is exposed after endocytosis. The protocols presented below are based on the method developed by Sipe and Murphy [1] for adherent (BALB/c3T3) cells and modified by Sipe et al. [2] for nonadherent (K562) cells. Reviews of acidification of endocytic compartments [3, 4] and flow cytometric methods for analysis of endocytosis [5, 6] may be consulted for additional background. The protocols below describe analysis of transferrin (Tf) acidification, but they may be easily modified for use with a different ligand.

The use of flow cytometry for measuring the pH of endocytic vesicles is the focus of this chapter. The protocols described below are based on the dual fluorescence method first described by Murphy et al. and are adaptations from previous work in the laboratory and previous reviews. Methods for measuring the pH of endocytic vesicle are described for both adherent and non-adherent cells. In addition, the use of two different endocytic markers, dextran and transferrin (Tf), are described. Transferrin is an iron-biding protein which binds to the Tf receptor on the cell surface. Upon reaching the acidic interior of the endosome, iron is release, while Tf remains bound to its receptor and recycle to the cell surface where it may bind more iron. Therefore, Tf is a convenient marker of early endosomes (2–5 min labeling) and recycle endosomes (10–12) min labeling). Dextran is non-specific marker which enters the cell via fluids-phase endocytosis. Pulse-labeling cells for a period of time (10–60 min) followed by chasing for a long time allows dextran to be used as a probe for measuring the pH of late endocytic compartment such as late endosomes and lysosomes. The pH of earlier compartment may be measured with dextran if shorter pulse and chase times are use. However, it is sometimes difficult to obtain enough signal during such a short pulse.

Background: Bevacizumab and dexamethasone are commonly administered to patients with brain tumors, and may alter the central nervous system penetration of concurrently administered chemotherapeutic agents by affecting vascular permeability and blood brain barrier (BBB) composition. We compared plasma UF and CSF PKs of intravenously (IV) administered carboplatin given alone and in combination with dexamethasone or bevacizumab in a non-tumor bearing NHP model, that has been predictive of human PKs of anti-cancer drugs. Methods: Carboplatin (8.75 mg/kg IV over 1 hour) combined with bevacizumab (12.5 mg/kg IV over 1 hour immediately prior to carboplatin) or dexamethasone (0.4 mg/kg IV bolus given 24 hours and immediately prior to carboplatin), was administered on separate occasions to each of three adult rhesus monkeys. Each monkey received carboplatin alone as control. Serial paired plasma and CSF samples were obtained over 24 hours. Plasma UFs were prepared immediately. Platinum was quantified using a validated atomic absorption spectroscopy assay with a lower limit of quantitation of 0.03 μM. Area under concentration x time curves (AUC) were derived using the linear trapezoidal method. CSF penetration (ratio of CSF AUC0-∞: plasma UF AUC0-∞) and CSF penetration relative to control (ratio of the CSF penetration of carboplatin with bevacizumab or dexamethasone to the CSF penetration of carboplatin alone) were calculated. Results: Plasma UF and CSF PK parameters for carboplatin (median and range) Plasma ultrafiltrate Cerebrospinal fluid CSF penetration Cmax (μM) AUC 0-∞ (μM*h) T ½ (h) Cmax (μM) AUC 0-∞ (μM*h) T ½ (h) (%) Relative to control (%) Carboplatin 78.3 (76.7–102.1) 182.0 (161.7–259.2) 5.2 (4.4–6.2) 0.71 (0.65–1.21) 8.26 (7.48–13.98) 9.6 (5.5–10.1) 4.6 (3.2–7.7) - Carboplatin+ Bevacizumab 88.2 (74.7–117.2) 197.5 (149.4–274.0) 5.6 (5.1–5.9) 0.62 (0.58–0.76) 6.82 (6.23–9.13) 9.4 (8.9–11.6) 4.2 (2.5–4.6) 78.1 (60.2–90.1) Carboplatin + Dexamethasone 104.0 (68.6–104.1) 202.5 (126.1–226.2) 6.5 (5.6–9.4) 0.60 (0.49–0.88) 6.38 (5.15–9.43) 7.9 (7.5–10.5) 4.1 (2.8–4.7) 88.3 (60.6–88.5) Cmax = maximal concentration, AUC = area under the concentration x time curve, T1/2 = terminal half life Conclusions: Plasma UF and CSF PK of carboplatin in NHP were similar to previous reports and showed limited inter-subject variability. Bevacizumab and dexamethasone consistently decreased the CSF penetration of carboplatin (median decrease 22% and 12% respectively). In patients with brain tumors and disrupted BBB, these differences may be larger and could be clinically relevant. Citation Format: Srivandana Akshintala, Cynthia M. Lester-McCully, Robert F. Murphy, Brigitte C. Widemann, Katherine E. Warren. Effect of bevacizumab and dexamethasone on plasma ultrafiltrate (UF) and cerebrospinal fluid (CSF) pharmacokinetics (PKs) of carboplatin in a non-human primate (NHP) model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3374. doi:10.1158/1538-7445.AM2013-3374

PURPOSE Intralumbar methotrexate is one of the primary therapeutic modalities for the prevention and treatment of meningeal leukemia. However, methotrexate distribution to the ventricles is limited and highly variable following intralumbar dosing, and cytotoxic concentrations of methotrexate are not always achieved or sustained in the ventricular CSF. We used a nonhuman primate model to determine the effect of body position on the caudal distribution of an intralumbar dose of methotrexate. METHODS Methotrexate (1.0 mg) was administered by intralumbar injection to four animals, which were then immediately placed either in an upright sitting position or in a prone position for 1 hour, then upright. Each animal served as its own control and was studied in each position on at least one occasion. RESULTS The mean peak ventricular methotrexate concentration was 0.12 mumol/L (range, 0.091 to 0.20) in animals that were immediately placed upright, compared with 2.81 mumol/L (range, 0.21 to 8.9) in animals that remained prone for 1 hour. The mean area under the concentration-versus-time curves (AUC) was 0.51 mumol/L.h (range, 0.26 to 1.1) in the upright animals and 12.0 mumol/L.h (range, 0.9 to 35.4) in the prone animals. CONCLUSION Maintaining a prone position for 1 hour after an intralumbar dose increased the peak methotrexate concentration and drug exposure in ventricular CSF. CSF drug distribution following intralumbar therapy can be influenced by body position after the injection.

Nat. Methods 9, 697–710 (2012); published online 28 June 2012; corrected after print 20 July 2012; corrected after print 29 August 2012. In the version of this article initially published, the disclaimer was omitted. The error has been corrected in the HTML and PDF versions of the article.

5025 Background: We have found clinical activity of carboplatin (C) with the PARP inhibitor olaparib (O) in BRCA1/2mut or BRCA-like breast and ovarian cancers. Current clinical trials are testing the hypothesis that PARP inhibition will sensitize tumors to platinum agents. We have examined sequence specificity of C and O combinations in cell lines, including two BRCA1mut (HCC1937, UWB1.289) and two BRCA-WT lines (OVCAR8, HeyA8), on the development of DNA damage and cell injury. Methods: Cell injury was measured with XTT assays. DNA damage was examined using Comet assay with tail quantification and γH2AX foci by immunofluorescence. Platinum DNA-adduct formation was measured using atomic absorption spectrometry and normalized to DNA input. Results: Exposure to O prior to C for 24 hours (O>C) reduced the efficiency of double stranded DNA damage compared to C alone or O+C (p C vs. C or O+C) measured by γH2AX foci/nucleus in BRCA1mut cell lines, UWB1.289: 11.0 (O>C), 40.9 (C), 46.4 (O+C); HCC1937: 1...

Intracellular hydrolases degrade all types of biological polymers. Acting in concert, they are capable of degrading protein, polysaccharide, lipid, DNA, and RNA. In addition, sulfate and phosphate groups can be specifically removed from these polymers. Cytochemistry, immunolocalization, subcellular fractionation, and analysis of the kinetics of hydrolysis of endocytosed substrates have all been used to localize and identify the intracellular compartments in which these enzymes are contained (for reviews see Kornfeld, 1987; Glaumann and Ballard, 1987; Storrie, 1988; Holtzman, 1989; Kornfeld and Mellman, 1989). Most acid hydrolases are targeted to lysosomes by the addition of mannose 6-phosphate (M6P), which is recognized by mannose 6-phosphate receptors (MPR) that are thought to cycle between the Golgi apparatus and endosomes or lysosomes (for reviews see von Figura and Hasilik, 1986; Kornfeld and Mellman, 1989). The pH optima of many of these enzymes are less than 5 (for reviews see Barrett and McDonald, 1980; McDonald and Barrett, 1986) consistent with their acting in lysosomes, whose pH is in this vicinity (Ohkuma and Poole, 1978). However, some enzymes have a higher pH optimum, and activity has been detected in early endosomal compartments (Storrie et al., 1984; Diment and Stahl, 1985; Roederer et al., 1987) that have a pH near 6 (Murphy et al., 1984; Roederer and Murphy, 1986; Kielian et al., 1986; Sipe and Murphy, 1987). Therefore, it appears that some of these enzymes may be present (perhaps in varying amounts) in many, if not all, compartments of the endocytic pathway, and that pH may play an important role in regulating their activity (Murphy, 1988; Yamashiro and Maxfield, 1988). In this chapter we discuss the endocytic pathway in various cell types, the regulation of pH within its compartments, and the enzymatic components of the pathway. In addition, we discuss the processing of known physiological ligands and implications for drug targeting.

Background: Satraplatin is an orally bioavailable platinum analog with preclinical activity (IC50 range in tumor cell lines 0.04 - 16 µM) in cisplatin sensitive and resistant models and clinical activity in adults with refractory cancers including prostate cancer. The cerebrospinal fluid (CSF) penetration of cisplatin and carboplatin in non-human primates (NHP) is limited (3.6% and 3.8%, respectively). We evaluated the plasma and CSF pharmacokinetics (PK) of satraplatin, which is more lipophilic than other platinum analogs, after an intravenous (IV) dose in a NHP model. Methods: Satraplatin 120 mg/m2 was administered as 1 h IV infusion in DMSO (5%) and normal saline to 5 NHP. Serial blood and CSF samples were obtained over 48 hours. Plasma was separated immediately and ultrafiltrate (UF) was prepared by centrifugation through a Microcon 10K MWCO filter. CSF was obtained from an indwelling Ommaya reservoir (n=4) or from a temporary lumbar catheter (n=1). Platinum was quantified in the plasma UF and CSF using a validated atomic absorption spectroscopy assay with lower limit of quantification of 0.03 µM in UF and CSF. Pharmacokinetic parameters were estimated using non-compartmental analyses. CSF penetration was calculated from the AUCCSF:AUCPlasma. Results: Satraplatin was well tolerated by all NHP. Pharmacokinetic parameters (median and range) for satraplatin plasma UF were Cmax 8.3 (5.7-10.6) µM, AUC0-48h 28.3 (22.6-33.2) µM·h, AUC0-inf 34.2 (24.7-38.2) µM·h, clearance 3.6 (2.4-4.3) L/h, and t1/2 18.8 (13.4-25) h. Satraplatin was detected in the CSF of all NHP. For the 4 NHP with Ommaya reservoirs PK parameters were: tmax 1.5 (1.3-2.1) h from the start of infusion, Cmax 0.07 (0.02-0.12) µM, AUC0-48h 1.2 (0.49-2.43) µM·h, ratio AUC0-48h CSF:AUC0-48h Plasma was 4.3 (2.2-7.4) %. For the NHP with lumbar catheter and limited CSF samples the tmax was 1.1 h, and Cmax was 0.62 µM. Conclusions: Satraplatin penetration into CSF is similar to that of carboplatin and cisplatin, despite its greater lipophilicity. The development of a phase I trial of satraplatin for refractory childhood solid tumors including brain tumors is in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3599.

Background Orally administered all-trans-retinoic acid (all-trans-RA) can induce remission in a high proportion of patients with acute promyelocytic leukemia. Purpose To further define the drug's pharmacokinetics, a study of intravenous all-trans-RA was performed in rhesus monkeys. Methods A total of nine monkeys received intravenous bolus injections of all-trans-RA. Three different doses (20, 50, and 100 mg/m2) were each tested in three monkeys. Blood samples for determination of all-trans-RA concentration were obtained prior to drug administration and at 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 360, and 480 minutes after drug administration. Results Plasma disappearance of all-trans-RA was characterized by three distinct phases: a brief, initial exponential decline, followed by a relative plateau in the disappearance curve (the duration of which was dose dependent), and finally a terminal exponential decay. This profile is consistent with a capacity-limited (saturable) elimination process. The first-order (terminal) half-life for all-trans-RA averaged 19 minutes, and the mean clearances were 77, 52, and 59 mL/min for the 20-, 50-, and 100-mg/m2 dose groups, respectively. The mean +/- SD Michaelis constant (Km) for the capacity-limited process was 3.2 +/- 1.9 microM. Conclusions Peak plasma concentrations following oral administration of 45 mg/m2 all-trans-RA in humans approach the Km for the capacity-limited process; thus, the dose-dependent pharmacokinetics of all-trans-RA described here may occur within the clinically used dosage range.

The goal of the protocols described here is to measure the affinity of a ligand for cell surface receptor(s) and to determine the number of receptors per cell, following the classic method of Scatchard [1]. The preferred method for analysis of ligand binding is to measure ligand bound to cells at equilibrium without removing unbound ligand, since removing unbound ligand allows ligand dissociation to begin and may result in underestimation of the equilibrium value. As first described by Bohn [2] and Steinkamp and Kraemer [3], the small diameters of the sample stream and excitation beam used in most flow cytometers provide excellent discrimination between cell-associated and free ligand. Further discussion of the basis of this discrimination and analysis of the effects of ligand affinity, number of receptor per cell, and sample stream diameter on expected signal (fluorescence from bound ligand) to noise (fluorescence from free ligand) may be found elsewhere [4]. When the equilibrium method is not suitable, the rapid dilution method is frequently an acceptable substitute (see second Procedure).

The mechanisms by which the recycling and degradation of endocytosed material are regulated have been the subject of intense study since the initial description of lysosomes (for reviews see Steinman et al 1983; von Figura & Hasilik 1986). While a number of questions remain, significant evidence points to the important role played by low in controlling and facilitating processing of endocytosed ligands. The dissociation of many ligand-receptor complexes is induced by acidification below 7, and many lysosomal hydrolases have low optima (Mellman et al 1986). Within the last five years, it has become clear that, at least in many non-erythroid cells, the to which material destined for lysosomes is exposed decreases continuously with time after internalization (Murphy et al 1984; Roederer & Murphy 1986; Kielian et al 1986; Roederer et al 1987; Yamashiro & Maxfield 1987).

The nuclear cycle kinetics of Physarum polycephalum plasmodia were examined using flow cytofluorometry. The dyes Hoechst 33342 and propidium iodide were used to stain the DNA of isolated nuclei. In asynchronously growing microplasmodia. S phase consists of 13--15% of the nuclear division cycle time. Nuclei isolated from individual macroplasmodia, which have previously been demonstrated to divide in synchrony, were shown to be less synchronized during late S phase than during mitosis. The results obtained demonstrate the feasibility of flow cytometric measurement of the properties of nuclei isolated from a single cell.

Fourteen children (aged 3 to 14 years) with average-risk acute lymphoblastic leukemia were studied after an oral dose of 6-mercaptopurine (6-MP) (75 mg/m2) administered alone and, on the next day, concurrently with oral methotrexate (20 mg/m2). When 6-MP was administered alone, both the peak plasma concentration (15 to 150 ng X ml-1) and the AUC (36 to 340 ng X ml-1 X hr) were highly variable. Concurrent methotrexate resulted in a 31% increase in the AUC (P less than 0.01) and a 26% increase in peak plasma levels (P less than 0.05) of 6-MP. The AUC of methotrexate correlated with the degree of increase in 6-MP plasma concentrations. These findings are consistent with previous in vitro studies demonstrating that methotrexate is an inhibitor of xanthine oxidase, the enzyme that catabolizes 6-MP to the inactive metabolite thiouric acid. Although the increases in 6-MP AUC and peak plasma concentrations resulting from concurrent methotrexate administration were statistically significant, this interaction is probably not clinically significant at standard low oral doses of methotrexate in light of the wide interpatient variability in these pharmacokinetic parameters of 6-MP.

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.

Flow cytometric DNA analysis of isolated nuclei was performed on 14 lesions occurring in ten children with adrenocortical tumors. Unimodal DNA content distributions were obtained from seven tumors occurring in patients without metastases 2 to 18 years after diagnosis. Abnormal DNA contents were detected in all four primary lesions, which subsequently metastasized, and in the tumor of one patient who was followed for less than 2 years. Paraffin and frozen preparations were virtually identical, as were the analyses of the primary, recurrent, and metastatic disease occurring in one patient. These observations suggest that DNA content abnormalities detected by flow cytometry correlate with metastases, and may provide an objective measure of the biologic potential of these tumors.

The existence of three distinct classes of endocytic vesicles that are part of the pathway of fluid-phase endocytosis has been demonstrated by flow cytometry. Amounts of fluorescent and scattered light were measured on a particle-by-particle basis for unfractionated whole cell lysates from cells incubated with fluorescein isothiocyanate-dextran. After 20 min two different fluorescent populations were observed, and after a 180-min incubation a third highly fluorescent population was found. Since the fluorescein isothiocyanate-dextran per fluid-phase vesicle should be a function solely of the external fluorescein isothiocyanate-dextran concentration, the existence of endocytic compartments with widely different amounts of fluorescence could result from a wide range of sizes of initial endocytic vesicles. However, the kinetics of appearance of the intermediate and highly fluorescent vesicles suggest that these compartments become labeled through fusion with the smaller primary endocytic vesicles.

To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification.

The effects of five lysosomotropic amines on the growth of Swiss 3T3 fibroblasts were measured and compared with effects on intravesicular pH. Tributylamine and benzylamine, amines that affect intravesicular pH without causing vacuolation, were found to inhibit cell growth to a similar extent as vacuologenic amines previously tested. Excellent correlation between the half-maximal concentrations for the growth and pH effects were found for tributylamine, benzylamine, chloroquine, and ammonium chloride. The results suggest that growth inhibition by these amines is a direct result of their effects on pH and not due to other effects (such as vacuolation). In contrast, a 100-fold difference in the half-maximal concentrations was found for methylamine, suggesting that methylamine inhibits growth by a mechanism unrelated to pH.

Retrospective quantitative DNA analysis was done on 147 samples from 89 patients with neuroblastoma and ganglioneuroma using flow cytometry. In the neuroblastoma patients, nuclear DNA content was found to be a stable tumor marker irrespective of site (primary versus metastatic) and despite changes with time in tumor progression, maturation, or therapy. The occurrence of DNA aneuploidy, which was detected in 60% of the neuroblastoma patients, paralleled other favorable indicators and was highly associated with survival (P less than 0.001). Of clinical stage, age, primary site, sex, and DNA content, only stage and DNA content correlated with survival. Those patients with favorable stage and DNA aneuploidy had higher survival rates. Further, favorable stage and the presence of DNA aneuploidy were independent prognostic indicators. Abnormal DNA content was also detected in samples from ganglioneuromas in which significant numbers of ganglion cell nuclei were recovered. These results indicate a striking difference between neuroblastoma and adult tumors in which DNA aneuploidy is generally a poor prognostic sign and provide a molecular link between ganglioneuromas and their malignant counterparts.

Chromatin from Friend leukemia cells labeled with [14C]thymidine for 24 hr followed by [3H]thymidine for 10 min is converted into nucleosomes by staphylococcal nuclease at only half the rate that total chromatin is converted. Polyacrylamide gel electrophoresis of nucleosomal DNA from cells labeled for 24 hr with [14C]thymidine followed by 10 min with [3H]thymidine demonstrates that the internucleosomal spacer of newly replicated chromatin is approximately 20 base pairs shorter than that of total chromatin. The implications of this difference for models of chromatin structure are discussed.