Biomedical subjects

E Wang

Publications and source records attributed to E Wang.

At least 91 records · Page 5Linked to original sources

PubMed2001

[The influence of labyrinthectomy on the expression of NMDA R1 mRNA].

OBJECTIVE: To study the effect of labyrinthectomy on the expression of NMDAR1 (N-methyl-D-aspartate receptor subunit R1). METHOD: The in situ hybridization was used to observe the expression of NMDA R1mRNA post-unilateral labyrinthectomy and investigate its effect on vestibular compensation. RESULT: 20 h post-unilateral labyrinthectomy, the NMDA R1mRNA positive neurons in the medial vestibular nucleus increased significantly compared with the unlesioned side(P < 0.01). 40 h-6 d, the positive cells decreased gradually. 6 d post lesion, the positive neuron counts of the lesioned side had no significant difference compared with those of the un-lesioned side (P > 0.05). CONCLUSION: The changing trend of the expression of NMDA R1mRNA in the medial vestibular nucleus is related to the recovery of the vestibular static symptoms after unilateral labyrinthectomy. It is suggested that the modulation of the efficacy of the NMDA R1 play an important role in the compensation of the static symptoms.

Animals↗

PubMed2000

Ion channel behavior of supported bilayer lipid membranes on a glassy carbon electrode.

A new kind of solid substrate, a glassy carbon (GC) electrode, was selected to support lipid layer membranes. On the surface of the GC electrode, we made layers of didodecyldimethylammonium bromide (a synthetic lipid). From electrochemical impedance experiments, we demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We studied the ion channel behavior of the supported bilayer lipid membrane. In the presence of perchlorate anions as the stimulus and ruthenium(II) complex cations as the marker ions, the lipid membrane channel was open and exhibited distinct channel current. The channel was in a closed state in the absence of perchlorate anions.

Carbon↗

PubMed2000

Studies on the interaction between TWEAK and the death receptor WSL-1/TRAMP (DR3).

WSL-1/TRAMP (DR3) is a member of the tumour necrosis factor (TNF) receptor superfamily which exhibits effects on NF-kappaB activation and apoptosis. TWEAK, a novel TNF-related molecule, has been proposed as the ligand for this receptor. Utilising both human and murine TWEAK ligand, it is shown that TWEAK and WSL-1/TRAMP do not interact in an in vitro binding assay and that TWEAK binds strongly to cells that do not express WSL-1/TRAMP on the cell surface. Biological activity of TWEAK is also observed in these cells. Finally, cells isolated from WSL-1/TRAMP knockout mice are shown to retain their ability to interact with TWEAK. These results suggest that WSL-1/TRAMP is not the major receptor for TWEAK

Animals↗

PubMed2000

Sulfoxide-containing aromatic nitrogen mustards as hypoxia-directed bioreductive cytotoxins.

A series of diaryl and alkylaryl sulfoxide-containing nitrogen mustards were synthesized and evaluated for their hypoxia-selective cytotoxicity against V-79 cells in vitro as well as for their metabolism profiles with the rat S-9 fractions. In general, the diaryl sulfoxides (4, 5, and 7-9) showed much greater hypoxia selectivity (11-27-fold) than the alkylaryl sulfoxides (approximately 3-fold) (1 and 3). The fused diphenyl sulfoxides (10 and 11), on the other hand, showed very low hypoxia selectivity (1.3-3-fold). Compound 10 was highly cytotoxic under both aerobic and anaerobic conditions, while 11 showed low cytotoxicity under both conditions. The bioreduction of 8 by the rat S-9 fraction under anaerobic conditions was inhibited by menadione and enhanced by benzaldehyde, acetaldehyde, or 2-hydroxypyrimidine suggesting the involvement of aldehyde oxidase in the reduction of the sulfoxides. Bioreductive metabolism studies of selected model sulfoxides suggested that diaryl sulfoxides are better substrates for aldehyde oxidase than alkylaryl sulfoxides.

Aerobiosis↗

PubMed2000

Cholesterol interaction with the daunorubicin binding site of P-glycoprotein.

The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent. Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition. Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter. In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM. Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Modulation of cytokines and chemokines, limited pulmonary vascular bed permeability, and prevention of septicemia and death with ceftriaxone and interleukin-10 in pneumococcal pneumonia.

Interleukin (IL)-10 is a biologically active anti-inflammatory and immunomodulatory cytokine. The respective effects or combined effect of ceftriaxone (Ctri) and IL-10 on host response was studied in a mouse model of lethal pneumococcal pneumonia. A once daily intraperitoneal (ip) injection of IL-10 (1 microg/mouse) for 2 days did not affect inflammation but accelerated bacterial dissemination to the bloodstream. Of mice treated with 1 ip 20 mg/kg Ctri injection, 40% developed septicemia, and only 52% survived. However, the addition of IL-10 to Ctri enhanced bacterial clearance, prevented septicemia, and yielded a 95% survival rate (P<.001). This approach also significantly (P<.05) decreased IL-1beta, IL-6, macrophage inflammatory protein-2, and myeloperoxidase levels in lungs and the production of nitric oxide in bronchoalveolar lavage fluid. Furthermore, Ctri plus IL-10 significantly (P<.05) reduced pulmonary vascular leakage and the appearance of red blood cells in alveoli. These data indicate a beneficial role for IL-10 as an adjunctive therapy to antibiotics against pneumococcal pneumonia.

Animals↗

PubMed2000

Rapid up-regulation of peptide elongation factor EF-1alpha protein levels is an immediate early event during oxidative stress-induced apoptosis.

Hydrogen peroxide (H(2)O(2)) induces apoptosis in cultured cells, in a dose-dependent manner. Treatment with H(2)O(2) causes decreased mitochondrial respiration, along with DNA degradation and the formation of an oligonucleosomal ladder, all hallmarks of apoptotic cell death. In this report, we investigate alterations in expression of a peptide elongation factor, EF-1alpha, during oxidative challenge. EF-1alpha protein levels undergo rapid increase upon treatment with H(2)O(2); however, whereas sublethal doses of H(2)O(2) stimulate only transient increases of EF-1alpha protein levels, lethal doses produce sustained elevation of EF-1alpha levels. Furthermore, pretreatment of H9c2(2-1) cells with transcriptional inhibitors fails to abolish the oxidant-induced increase in EF-1alpha, and Northern blotting analysis reveals that EF-1alpha mRNA levels remain steady throughout the H(2)O(2) treatment period, suggesting that the up-regulation of EF-1alpha is mediated posttranscriptionally. Transient transfection with an antisense EF-1alpha cDNA protects against hydrogen peroxide-mediated cytotoxicity in proportion to the degree of repression of EF-1alpha protein levels, suggesting that up-regulation of EF-1alpha plays a role in expediting the execution of the apoptotic program in response to oxidative stress.

Actins↗

PubMed2000

Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPK-dependent pathway.

We have established that the serum- and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5' deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation of sgk gene promoter activity. Gel shift analysis identified four major DNA-protein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction of sgk promoter activity or protein production. Using an in vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.

Animals↗

PubMed2000

Status of activation of circulating vaccine-elicited CD8+ T cells.

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209-217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-gamma expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-gamma expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.

Antigen Presentation↗

PubMed2000

Molecular classification of cutaneous malignant melanoma by gene expression profiling.

The most common human cancers are malignant neoplasms of the skin. Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease. Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm. Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities. Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas. Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.

Adult↗

PubMed2000

Optical detection of polycations via polymer film-modified microtiter plates: response mechanism and bioanalytical applications.

Microtiter plate wells modified with thin (approximately 20 microm) polymeric films capable of optically sensing macromolecular protamine and other polycationic species are described. The plates are prepared by coating the bottom of each well of a conventional 96-well polypropylene plate with an adherent polymer film (a mixture of poly(vinyl chloride) and polyurethane) containing a lipophilic 2',7'-dichlorofluorescein derivative. Surprisingly, optical response toward polycations is shown to result from the extraction of the fluorescein derivative from the polymer film into a lyophobic colloidal phase at the sample/film interface. This new phase is likely composed of a micellular-type ion pair complex between the analyte polycation from aqueous sample phase and the deprotonated form of the fluorescein derivative. Accumulation of the deprotonated fluorescein species in this interfacial region induces an absorbance change measured at 540 nm. Optimized plates can be used to sense protamine concentrations in the range of 0-100 microg/mL in 10 min with little or no response to physiological levels of common cationic species (Na+, K+, Ca2+, etc.). The modified plates are shown to be useful as simple optical detectors for measuring heparin levels in plasma via titrations with protamine and for monitoring protease activities (trypsin and plasmin) that cleave polycationic peptides/proteins such as protamine into smaller peptide fragments that are not detected by the sensing films. Assays for "clot busting" plasminogen activators (streptokinase, urokinase, and tissue plasminogen activator) are also demonstrated using this relatively simple microtiter plate-based polycation detection system.

Cations↗

PubMed2000

Threshold levels of gene expression of the melanoma antigen gp100 correlate with tumor cell recognition by cytotoxic T lymphocytes.

The level of expression of melanoma antigens (MA) may modulate the host immunologic response. Thus, the accurate measurement of MA expression may allow proper patient selection for antigen-specific therapies and yield important information for the evaluation of clinical results. In this study, we measured the absolute levels of MA messenger ribonucleic acid (mRNA) in tumor cell lines utilizing real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). mRNA levels of MART-1, gp100, tyrosinase, TRP-1 and TRP-2 melanoma differentiation antigens and MAGE-1, MAGE-3 and ESO-1 cancer testis (CT) antigens were compared in 24 early-passage (<5 passages in culture) and 12 archival melanoma cell lines. MA mRNA expression was extremely variable among cell lines, occasionally reaching levels comparable to ribosomal RNA (rRNA). gp100 and MART-1 mRNA levels correlated with protein expression measurement obtained by FACS analysis. More significantly, a threshold of gp100 mRNA expression required for T-cell stimulation and target-cell killing was identified. This threshold level corresponded to approximately 500 mRNA copies per 10(8) copies of rRNA. Our results suggest that the measurements of MA mRNA levels may yield useful information relevant to the interpretation of clinical outcome during antigen-specific treatments.

Antigens, Neoplasm↗

PubMed2000

A high-performance liquid chromatographic method to measure sphingosine 1-phosphate and related compounds from sphingosine kinase assays and other biological samples.

Sphingosine 1-phosphate is an intermediate of sphingosine catabolism as well as a potent signaling compound. Conditions were established for the extraction and analysis of sphingosine 1-phosphate and other sphingoid base 1-phosphates from in vitro sphingosine kinase assays and other biological samples. The sphingoid base 1-phosphates were extracted in high yield (85%) using small C-18 reverse-phase columns (LiChroprep RP-18). After the extracts were treated with 0.1 N KOH to remove glycerolipids, the sphingoid base 1-phosphates were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-18 columns with a mobile phase of methanol:10 mM potassium phosphate (pH 7.2):1 M tetrabutylammonium dihydrogen phosphate (in water) (83:16:1, v/v/v). The o-phthalaldehyde derivative of sphingosine 1-phosphate was reasonably stable (t(1/2) > or = 18 h) when EDTA was present and could be detected in picomole amounts. The HPLC retention time of the sphingoid base 1-phosphates could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as sphingosine 1-phosphate and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base 1-phosphates in biological samples. The extraction procedure and HPLC method facilitated assays of sphingosine kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base 1-phosphates in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism.

Blood Platelets↗

PubMed2000

Morphological symmetry breaking during epitaxial growth at grazing incidence

It is shown that, in submonolayer growth at off-normal incidence, even much less than 1% of transfer from the condensation energy of the deposited atoms into adatom motion is sufficient to induce a net adatom current from the illuminated edge of a two-dimensional island to the other edges, thereby breaking the island symmetry. Such a symmetry breaking phenomenon is most pronounced for deposition at grazing incidence. Comparison between our theoretical predictions and existing experimental results confirms the general validity of the model.

Journal Article↗

PubMed2000

Expansion of tumor-T cell pairs from fine needle aspirates of melanoma metastases.

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.

Antigens, Neoplasm↗

PubMed2000

Hospitalization for community-acquired, rotavirus-associated diarrhea: a prospective, longitudinal, population-based study during the seasonal outbreak. The Greater Toronto Area/Peel Region PRESI Study Group. Pediatric Rotavirus Epidemiology Study for Immunization.

OBJECTIVES: To determine the age-specific hospitalization rate for rotavirus-associated diarrhea in Canadian children during the seasonal outbreak, and to characterize children and their households, for assessment of the need for a rotavirus vaccine. DESIGN: Prospective multisite cohort study. SETTINGS AND PARTICIPANTS: Children with an admission diagnosis of diarrhea admitted to 18 hospitals serving 132 study census tracts of a major urban region, from November 1, 1997, through June 30, 1998. Prospective centralized testing of stools was performed; research nurses administered a follow-up questionnaire to parents. MAIN OUTCOME MEASURE: Age-specific diarrhea and rotavirus-associated hospitalization rates. RESULTS: Of 224160 children younger than 5 years, the diarrhea hospitalization rate was 4.8 in 1000 (n = 1086) during the seasonal epidemic. Based on testing of 65% of the hospitalized children, the rotavirus-associated diarrhea hospitalization rate was 1.3 in 1000; the cumulative incidence to 5 years of age was 1 in 160. Rotavirus-associated diarrhea was reported in 37% of the 1001 hospitalized children undergoing testing inside and outside of the census tracts; in children aged 6 to 35 months, this rose to more than 70% during April and May. Ages of children with rotavirus-associated diarrhea were 0 to 2 months (2%), 3 to 5 months (5%), 6 to 23 months (60%), 24 to 35 months (15%), and 36 months or older (19%). Of children aged 0 to 5 and 6 to 11 months, 4 (19%) of 21 and 6 (10%) of 59, respectively, had been born prematurely; 20 (24%) of 83 younger than 1 year were breastfed at the time of illness. Of children younger than 36 months, 77% were cared for in their homes; 13%, in family day care homes; and 8%, in child care centers. The mean (+/- SD) duration of rotavirus hospitalization based on hospital records and parental questioning was 2.4 +/- 1.7 and 3.1 +/- 1.6 days, respectively; it was significantly longer (P < or = .001) in children with an underlying medical condition. One child required intensive care unit hospitalization. Diarrhea occurred concurrently in 74% of household contacts younger than 3 years; 38%, aged 3 to 18 years; and 29%, older than 18 years. Seventy-six percent of parents were married. Household incomes in Canadian dollars in the 81% reporting were less than $20000 in 20%, $20000 to $60000 in 44%, and greater than $60000 in 36%. Ethnicity was reported as 53% white, 15% black, 10% Asian, 12% East Indian, and 11% other. CONCLUSIONS: Based on testing of 65% of children with diarrhea, rotavirus resulted in hospitalization in a minimum of 1 in 160 children by 5 years of age during the seasonal outbreak. Had 100% of young children with diarrhea undergone testing, the extrapolated cumulative incidence of rotavirus-associated diarrhea by 5 years of age may have been 1 in 106.

Acute Disease↗

PubMed2000

Rotavirus-associated diarrhea in outpatient settings and child care centers. The Greater Toronto Area/Peel Region PRESI Study Group. Pediatric Rotavirus Epidemiology Study for Immunization.

OBJECTIVES: To determine the prevalence of rotavirus infection in outpatient and child care center (CCC) settings during the seasonal rotavirus outbreak and to describe associated health care utilization. DESIGN: Prospective, multisite cohort study in various ambulatory settings. SETTINGS AND PARTICIPANTS: Participants were children with diarrhea (1) presenting to hospital emergency departments (EDs) and receiving intravenous (IV; n = 8) or oral (n = 1) hydration, (2) seen in pediatric practices (n=4), or (3) attending CCCs (n = 19) between November 1, 1997, and June 30, 1998. Prospective centralized testing of stool samples for rotavirus was performed using enzyme-linked immunosorbent assay and electron microscopy. Study nurses administered follow-up parent questionnaires for rotavirus-positive children. MAIN OUTCOME MEASURE: Prevalence of rotavirus-associated diarrhea. RESULTS: During the 8-month study, rotavirus was identified in 92 children with diarrhea: ED-IV, 20 (44%) of 45; ED-oral, 9 (47%) of 19; pediatric practices, 30 (20%) of 147; and CCCs, 33 (18%) of 186. Of 226 children with diarrhea in pediatric practices, all 5 who progressed to ED-IV hydration or hospitalization were tested, and 3 (60%) were rotavirus positive. Of 211 children in CCCs with diarrhea, 84% who required no health care visits were tested, and of these 10% were positive; of 56 who went on to require a health care visit and 8 who required ED-IV hydration or hospitalization, all were tested, and 27% and 75%, respectively, were rotavirus positive. Among 16 children with rotavirus followed up with ED-IV hydration, 4 (25%) returned and were hospitalized. Maximal health care intervention among 29 children with rotavirus enrolled in pediatric practices included 22 (76%) seeing the pediatrician only, 5 (17%) seeking further care in the ED, 1 (3%) receiving further ED-IV hydration, and 1 (3%) being hospitalized briefly. Maximal health care intervention for 33 children with rotavirus enrolled in CCCs included 13 (39%) who did not visit a physician, 11 (33%) who did, 3 (9%) who sought care in the ED, 1 (3%) who received ED-IV hydration, and 5 (15%) who were hospitalized. In CCCs, rates of diarrhea per 100 child-months of observation were as follows: ages 0 to 23 months, 6.6 episodes; ages 24 to 35 months, 1.9 episodes; and 3 years and older, 0.07 episodes; rates of rotavirus-associated diarrhea were as follows: ages 0 to 23 months, 1.1 episodes (28 of 2547); ages 24 to 35 months, 0.23 episodes (5 of 2185); and 3 years and older, 0 episodes (0 of 4124). CONCLUSION: Across a variety of outpatient and CCC settings, rotavirus is an important cause of diarrhea and a major cause of health care utilization.

Child↗

PubMed2000

Lysosomal accumulation of drugs in drug-sensitive MES-SA but not multidrug-resistant MES-SA/Dx5 uterine sarcoma cells.

Sequestration of drugs in intracellular vesicles has been associated with multidrug-resistance (MDR), but it is not clear why vesicular drug accumulation, which depends upon intracellular pH gradients, should be associated with MDR. Using a human uterine sarcoma cell line (MES-SA) and a doxorubicin (DOX)-resistant variant cell line (Dx-5), which expresses p-glycoprotein (PGP), we have addressed the relationship between multidrug resistance, vesicular acidification, and vesicular drug accumulation. Consistent with a pH-dependent mechanism of vesicular drug accumulation, studies of living cells vitally labeled with multiple probes indicate that DOX and daunorubicin (DNR) predominately accumulate in lysosomes, whose lumenal pH was measured at < 4.5, but are not detected in endosomes, whose pH was measured at 5.9. However, vesicular DOX accumulation is more pronounced in the drug-sensitive MES-SA cells and minimal in Dx5 cells even when cellular levels of DOX are increased by verapamil treatment. While lysosomal accumulation of DOX correlated well with pharmacologically induced differences in lysosome pH in MES-SA cells, lysosomal accumulation was minimal in Dx5 cells regardless of lysosomal pH. We found no differences in the pH of either endosomes or lysosomes between MES-SA and Dx5 cells, suggesting that, in contrast to other MDR cell systems, the drug-resistant Dx5 cells are refractory to pH-dependent vesicular drug accumulation. These studies demonstrate that altered endomembrane pH regulation is not a necessary consequence of cell transformation, and that vesicular sequestration of drugs is not a necessary characteristic of MDR.

Animals↗