Opioid receptors expression in the skin of haemodialysis patients suffering from uraemic pruritus.

BACKGROUND: Uraemic pruritus is a common symptom in patients with chronic kidney disease undergoing haemodialysis (HD) treatment. Etiopathogenesis of uraemic pruritus is complex and not fully explained, but there are reports indicating a possible role of peripheral opioid system in its pathomechanism. OBJECTIVES: This study was undertaken to analyse the expression of mu- and kappa-opioid receptors in the skin of HD patients with and without uraemic pruritus. METHODS: Forty patients suffering from chronic kidney disease, treated with haemodialysis (21 pruritic and 19 non-pruritic), were included. The expression of mu- and kappa-opioid receptors in their skin was evaluated with immunohistochemistry. RESULTS: The significant (P < 0.02) decrease in kappa-opioid receptor expression was shown in the skin of pruritic patients in comparison with the skin of those without itch (1.07 ± 0.62 and 1.66 ± 0.85, respectively). Moreover, there was a significant negative correlation between intensity of pruritus and expression of kappa-opioid receptors (r = -0.63, P = 0.002). There was no difference in the expression of mu-opioid receptor expression in the skin of pruritic and non-pruritic HD patients. CONCLUSIONS: This study indicates that changes in peripheral opioid system may play an important role in the uraemic itch pathogenesis and that future studies are necessary to fully understand this mechanism.

Postpartum two- and three-dimensional ultrasound evaluation of anal sphincter complex in women with obstetric anal sphincter injury.

OBJECTIVE: To compare the sensitivity and specificity of two- (2D) and three- (3D) dimensional transperineal ultrasound (TPUS) and 3D endovaginal ultrasound (EVUS) with the gold standard 3D endoanal ultrasound (EAUS) in detecting residual defects after primary repair of obstetric anal sphincter injuries (OASIS). METHODS: External (EAS) and internal (IAS) anal sphincters were evaluated by the four ultrasound modalities in women with repaired OASIS. 2D-TPUS was evaluated in real-time, whereas 3D-TPUS, 3D-EVUS and 3D-EAUS volumes were evaluated offline by six blinded readers. The presence/absence of any tear in EAS or IAS was recorded and defects were scored according to the Starck system. Sensitivity, specificity and predictive values were calculated, using 3D-EAUS as reference standard. Inter- and intraobserver analyses were performed for all 3D imaging modalities. Association between patients' symptoms (Wexner score) and ultrasound findings (Starck score) was calculated. RESULTS: Images from 55 patients were analyzed. Compared with findings on 3D-EAUS, the agreement for EAS evaluation was poor for 3D-EVUS (κ = 0.01), fair for 2D-TPUS (κ = 0.30) and good for 3D-TPUS (κ = 0.73). The agreement for IAS evaluation was moderate for both 3D-EVUS (κ = 0.41) and 2D-TPUS (κ = 0.52) and good for 3D-TPUS (κ = 0.66). Good intraobserver (3D-EAUS, κ = 0.73; 3D-TPUS, κ = 0.78) and interobserver (3D-EAUS, κ = 0.68; 3D-TPUS, κ = 0.60) agreement was reported. Significant association between Starck and Wexner scores was found only for 3D-EAUS (Spearman's rho = 0.277, P = 0.04). CONCLUSIONS: 2D-TPUS and 3D-EVUS are not accurate modalities for the assessment of anal sphincters after repair of OASIS. 3D-TPUS shows good agreement with the gold standard 3D-EAUS and a high sensitivity in detecting residual defects. It, thus, has potential as a screening tool after primary repair of OASIS. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Perineal body anatomy seen by three-dimensional endovaginal ultrasound of asymptomatic nulliparae.

AIM: The perineal body (PB) plays an important role in supporting the pelvic floor and the posterior vaginal wall, but its attachments and relationships are still debated. This study aimed to assess the normal anatomy of the PB using high-resolution three-dimensional endovaginal ultrasound (3D-EVUS) in asymptomatic nulliparae. METHOD: To validate the identification of perineal structures, 3D-EVUS was initially performed on nulliparous cadavers. Fresh frozen pelves were prepared and echogenic structures thought to be the PB, the external anal sphincter, the superficial and deep transverse perineii, pubovaginalis, puboperinealis, puboanalis, puborectalis and iliococcygeus muscles were tagged with biopsy needles, and marked with indigo carmine dye for localization during dissection. In the second part of the study, consecutive asymptomatic nulliparae were prospectively imaged with the same ultrasound modality. Interrater reproducibility was assessed off-line from stored 3D US volumes using a standardized technique. RESULTS: Five fresh frozen pelves and 44 asymptomatic nulliparae were assessed with 3D-EVUS. The PB was seen as an ovoid structure of mixed echogenicity between the rectum and vagina. It appeared to be divided into a superficial level, in contact with the external anal sphincter, the bulbospongiousus and the superficial transverse perineii muscle and a deep level, in contact with puboperinealis and puboanalis muscles. Interobserver repeatability was excellent for the measurements of PB height [intraclass correlation coefficient (ICC) 0.927], PB depth (ICC 0.969) and PB width (ICC 0.932). CONCLUSION: The PB is divided into two levels with different anatomical relationships with the pelvic floor muscles. 3D-EVUS yields reproducible assessment of this complex structure.

Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates.

It is unclear how polyglutamine expansion is associated with the pathogenesis of Huntington disease (HD). Here, we provide evidence that polyglutamine expansion leads to the formation of large intracellular aggregates in vitro and in vivo. In vitro these huntingtin-containing aggregates disrupt normal cellular architecture and increase in frequency with polyglutamine length. Huntingtin truncated at nucleotide 1955, close to the caspase-3 cleavage site, forms perinuclear aggregates more readily than full-length huntingtin and increases the susceptibility of cells to death following apoptotic stimuli. Further truncation of huntingtin to nucleotide 436 results in both intranuclear and perinuclear aggregates. For a given protein size, increasing polyglutamine length is associated with increased cellular toxicity. Asymptomatic transgenic mice expressing full-length huntingtin with 138 polyglutamines form exclusively perinuclear aggregates in neurons. These data support the hypothesis that proteolytic cleavage of mutant huntingtin leads to the development of aggregates which compromise cell viability, and that their localization is influenced by protein length.

Changes in the anaerobic threshold in an annual cycle of sport training of young soccer players.

The aim of the study was to assess changes in the anaerobic threshold of young soccer players in an annual training cycle. A group of highly trained 15-18 year old players of KKS Lech Poznan were tested. The tests included an annual training macrocycle, and its individual stages resulted from the time structure of the sports training. In order to assess the level of exercise capacities of the players, a field exercise test of increasing intensity was carried out on a soccer pitch. The test made it possible to determine the 4 millimolar lactate threshold (T LA 4 mmol · l(-1)) on the basis of the lactate concentration in blood [LA], to establish the threshold running speed and the threshold heart rate [HR]. The threshold running speed at the level of the 4 millimolar lactate threshold was established using the two-point form of the equation of a straight line. The obtained indicators of the threshold running speed allowed for precise establishment of effort intensity used in individual training in developing aerobic endurance. In order to test the significance of differences in mean values between four dates of tests, a non-parametric Friedman ANOVA test was used. The significance of differences between consecutive dates of tests was determined using a post-hoc Friedman ANOVA test. The tests showed significant differences in values of selected indicators determined at the anaerobic threshold in various stages of an annual training cycle of young soccer players. The most beneficial changes in terms of the threshold running speed were noted on the fourth date of tests, when the participants had the highest values of 4.01 m · s(-1) for older juniors, and 3.80 m · s(-1) for younger juniors. This may be indicative of effective application of an individualized programme of training loads and of good preparation of teams for competition in terms of players' aerobic endurance.

Pelvic floor disorders: role of new ultrasonographic techniques.

The paper presents the role of various ultrasound modalities in the diagnostics of female pelvic floor disorders (PFD). It describes the use of two/three/four-dimensional transperineal ultrasound and endocavitary transducers, which, up to now, have been used for proctological examinations and prostate cancer brachytherapy. Ultrasonography is the most widely available imaging modality. As a result of technical progress, novel transducers and more sophisticated software have recently been introduced to the market providing more information about the anatomy of pelvic organs. Some features of these transducers, such as higher frequency and multiplanar imaging, enable better visualisation of pelvic floor organs. In-depth knowledge of the technical and physical properties of modern ultrasonography, as well as its advantages and limitations, could provide an integrated approach to imaging of PFD. Technical modalities, the wide availability of ultrasonographic techniques, and an understanding of the imaging possible with modern ultrasonography could improve our understanding of PFD and allow better assessment in pre- and post-surgical management.

Structure-based rescue of common tumor-derived p53 mutants.

The p53 tumor suppressor protein induces cell-cycle arrest or cell death in response to DNA-damaging agents, such as radiation and many of the chemotherapeutics used in cancer therapy. The function of p53 is dependent on its ability to bind DNA in a sequence-specific manner, but in one-half of all human tumors, its sequence-specific DNA binding domain is compromised by single-amino acid substitutions. The nature of these substitutions, which target residues that directly contact DNA or that stabilize the structure of the DNA binding domain, has raised concerns as to whether the function of p53 mutants could ever be rescued. Nevertheless, pharmaceuticals that restore function to p53 mutants could specifically suppress proliferation of cancer cells in patients. To determine whether tumor-derived p53 mutants are irreversibly inactivated, we introduced basic residues in their DNA binding domains, aiming to establish novel contacts between p53 and the DNA phosphate backbone. In three of the seven most common p53 mutants, replacement of Thr284 with Arg significantly enhanced DNA binding affinity, without affecting DNA binding specificity, and rescued their transactivation and tumor suppressor functions. Thus, many tumor-derived p53 mutants retain their sequence-specific DNA binding determinants and can be activated to suppress tumor growth.

State of the art: an integrated approach to pelvic floor ultrasonography.

Surgical management of pelvic floor disorders depends on a comprehensive understanding of the structural integrity and function of the pelvic floor. For visualizing this region, ultrasonography has emerged as a procedure that is relatively easy to perform, cost-effective and widely available. In this review, pelvic floor ultrasonography, including two-dimensional (2D), three-dimensional (3D) and 4D imaging as well as transvaginal, endoanal and transperineal techniques, is discussed from a global and multicompartmental perspective, rather than using a compartmentalized approach. The role of the different sonographic modalities in the major disorders of the pelvic floor-urinary and fecal incontinence, pelvic organ prolapse and obstructed defecation syndrome-is evaluated critically.

Central nervous system relapse in high-risk stage 4 neuroblastoma: The HR-NBL1/SIOPEN trial experience.

BACKGROUND: There is rising concern on the impact of new strategies, such as high-dose chemotherapy (HDC) and immunotherapy, on the pattern of relapse in high-risk neuroblastoma (HR-NBL). Our aim is to evaluate the incidence and identify risk factors for first recurrence in the central nervous system (CNS) in HR-NBL. PATIENTS AND METHODS: Data from patients with stage 4V HR-NBL included from February 2002 to June 2015 in the prospective HR-NBL trial of the European International Society of Pediatric Oncology Neuroblastoma Group were analysed. Characteristics at diagnosis, treatment and the pattern of first relapse were studied. CNS imaging at relapse was centrally reviewed. RESULTS: The 1977 included patients had a median age of 3 years (1 day-20 years); 1163 were boys. Among the 1161 first relapses, 53 were in the CNS, with an overall incidence of 2.7%, representing 6.2% of all metastatic relapses. One- and three-year post-relapse overall survival was 25 ± 6% and 8 ± 4%, respectively. Higher risk of CNS recurrence was associated with female sex (hazard ratio [HR] = 2.0 [95% confidence interval {CI}: 1.1-3.5]; P = 0.016), MYCN-amplification (HR = 2.4 [95% CI: 1.2-4.4]; P = 0.008), liver (HR = 2.5 [95% CI: 1.2-5.1]; P = 0.01) or >1 metastatic compartment involvement (HR = 7.1 [95% CI: 1.0-48.4]; P = 0.047) at diagnosis. Neither HDC nor immunotherapy was associated with higher risk of CNS recurrence. Stable incidence of CNS relapse was reported over time. CONCLUSIONS: The risk of CNS recurrence is linked to both patient and disease characteristics, with neither impact of HDC nor immunotherapy. These findings support the current treatment strategy and do not justify a CNS prophylactic treatment.

Prognostic values of shear wave elastography in adolescent boys with varicocele.

INTRODUCTION: Shear wave elastography is an ultrasound technique for non-invasive quantification of tissue stiffness. It was used in assessing testis elasticity in some scrotal abnormalities, such as undescended pediatric testes or adult varicocele testes. In this study, its usefulness in adolescent patients with varicocele was examined. OBJECTIVE: The aim of this study was to quantify elasticity of testes with the use of elastography and comparison of the results with typical threshold values used in varicocele management in adolescent patients with varicoceles. STUDY DESIGN: In 30 patients with clinically diagnosed left varicoceles, quantitative 2D shear wave imaging of varicocele testes and contralateral ones were performed. RESULTS: The relationships between the grade of varicocele and elastography were calculated. The stiffness was 2.5 ± 0.49 kPa in testes with grade I of varicocele, 2.59 ± 0.81 in grade II and 2.80 ± 0.72 kPa in grade III. In contralateral testes, it was respectively grade I 2.39 ± 0.49 kPa, grade II 2.41 ± 0.61 kPa, and grade III 2.42 ± 0.85 kPa. The statistical significance was close to importance in grade III (P = 0.153). There was a statistically significant difference between elastography results in patients with volume difference over 20%. In testes with varicocele, it was 2.77 ± 0.75 kPa and in contralateral testes, 2.37 ± 0.65 kPa (P < 0.05). In patients with testis volume difference between 0 and 20%, elastography results were comparable, and it was 2.45 ± 0.57 kPa in testes with varicocele and 2.44 ± 0.61 kPa in contralateral testes (Table). DISCUSSION: Ultrasound is currently the most widely used imaging technique for the assessment of varicocele, but its role in the diagnostic algorithm is still controversial. Therefore, many attempts are made to determine the manifestations of testicular damage that precede morphological deterioration, which could increase the importance of imaging techniques in treatment planning. CONCLUSION: The changes of tissue elasticity due to varicocele seem to confirm the need of surgery in patients with testis volume difference more than 20% and in grade III of varicocele (Table). In case of validation of diffuse testis changes, they could indicate the need for surgery also in other stages.

CFU-megakaryocytic progenitors expanded ex vivo from cord blood maintain their in vitro homing potential and express matrix metalloproteinases.

BACKGROUND: In patients transplanted with cord blood (CB), prolonged thrombocytopenia is a major complication. However, this could be alleviated by supplementing the CB graft with ex vivo-expanded megakaryocytic progenitors (CFU-Meg), provided that the homing properties of these cells are not affected negatively by expansion. METHODS AND RESULTS: We assessed the in vitro homing potential of CFU-Meg progenitors expanded from CB and showed that the combination of thrombopoietin (TPO) with interleukin-3 (IL-3) used for expansion not only results in optimal proliferation of CFU-Meg but also protects these cells from apoptosis. Moreover, we found that ex vivo-expanded CFU-Meg maintained expression of the CXCR4 receptor throughout a 9-day culture and were chemoattracted towards a stromal cell-derived factor-1 (SDF-1) gradient. They also expressed matrix metalloproteinase-9 (MMP-9) and membrane-type (MT) 1-MMP, and transmigrated across the reconstituted basement membrane Matrigel. Finally, we observed that SDF-1 up-regulated the expression of both MMP-9 and MT1-MMP in CB CD34(+) cells and ex vivo-expanded CFU-Meg. DISCUSSION: We suggest that CB-expanded CFU-Meg, in particular those from day 3 of expansion, when their proliferation and in vitro homing potential are maximal, could be employed to supplement CB grafts and speed up platelet recovery in transplant recipients.

Prediction of outcome of fetal congenital heart disease using a cardiovascular profile score.

OBJECTIVES: Congestive heart failure in fetuses with congenital heart defects (CHD) is associated with high perinatal mortality. The clinical condition can be characterized by five ultrasound markers that comprise the 10-point cardiovascular profile (CVP) score. Our aim was to assess the value of the CVP score in evaluating the condition and in maintaining surveillance of fetuses with CHD. METHODS: We evaluated retrospectively 131 singleton pregnancies with a diagnosis of fetal CHD, which had been assessed by serial echocardiographic examinations, during which the CVP score was obtained. Fetal and neonatal outcomes, including perinatal mortality and Apgar scores, were assessed. RESULTS: Fetuses with a final CVP score or= 8 (87.5% vs. 15.2% mortality; P < 0.0001, chi square = 24.5). Significance was maintained after controlling for birth weight, lag time between the final examination and delivery and the dichotomized 5-min Apgar score (odds ratio, 22.3; P = 0.024). For low Apgar score and mortality, the CVP score had low sensitivity (0.25 and 0.27, respectively) but high specificity (0.98 and 0.99, respectively). The presence of hydrops and severe cardiomegaly were statistically significantly associated with mortality (P < 0.05). CONCLUSIONS: Fetuses with CHD and a CVP score below 8 are at risk of perinatal death. The CVP score may be used to assess the severity of fetal CHD and to plan perinatal management.

Evaluation of Single-Chip, Real-Time Tomographic Data Processing on FPGA SoC Devices.

A novel approach to tomographic data processing has been developed and evaluated using the Jagiellonian positron emission tomography scanner as an example. We propose a system in which there is no need for powerful, local to the scanner processing facility, capable to reconstruct images on the fly. Instead, we introduce a field programmable gate array system-on-chip platform connected directly to data streams coming from the scanner, which can perform event building, filtering, coincidence search, and region-of-response reconstruction by the programmable logic and visualization by the integrated processors. The platform significantly reduces data volume converting raw data to a list-mode representation, while generating visualization on the fly.

Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.

Normal and malignant cells shed from their surface membranes as well as secrete from the endosomal membrane compartment circular membrane fragments called microvesicles (MV). MV that are released from viable cells are usually smaller in size compared to the apoptotic bodies derived from damaged cells and unlike them do not contain fragmented DNA. Growing experimental evidence indicates that MV are an underappreciated component of the cell environment and play an important pleiotropic role in many biological processes. Generally, MV are enriched in various bioactive molecules and may (i) directly stimulate cells as a kind of 'signaling complex', (ii) transfer membrane receptors, proteins, mRNA and organelles (e.g., mitochondria) between cells and finally (iii) deliver infectious agents into cells (e.g., human immuno deficiency virus, prions). In this review, we discuss the pleiotropic effects of MV that are important for communication between cells, as well as the role of MV in carcinogenesis, coagulation, immune responses and modulation of susceptibility/infectability of cells to retroviruses or prions.

Beneficial effects of enrichment of chicken meat with n-3 polyunsaturated fatty acids, vitamin E and selenium on health parameters: a study on male rats.

Consumption of chicken meat enriched with bioactive compounds such as n-3 polyunsaturated fatty acids (PUFAn-3), vitamin E (vE) and selenium (Se) can help prevent many diseases and can be used to deliver those substances to humans. This might be of importance as chicken meat consumption is increasing worldwide. The effects of enriching chicken meat with PUFAn-3, vE and Se through dietary interventions were studied in rats. Four groups of Ross 308 female broilers from day 22 to day 35 of age were fed control diet (L) that contained lard and 80 mg vE and 0.3 mg Se/kg, or diets that contained rape seeds and fish oil with the same level of Se and vE as in the control diet, the same level of Se as in the control and 150 mg vE/kg, or 150 mg of vE and 0.7 mg Se/kg. Broiler carcasses were boiled, deboned, lyophilized and pooled by group. Boiled edible components of chicken carcass (BECC) were included (240 g/kg) in the diets fed to four groups of ten 10-week-old Wistar male rats for 8 weeks. Inclusion of BECCs modulated dietary fatty acid profile in the rat diets. Feeding these diets did not influence parameters related to growth or relative weights of internal organs in the rats. Feeding BECCs with lower PUFAn-6/n-3 decreased the n-6/n-3 ratio in the rat brain and liver, and increased the proportion of docosahexaenoic acid in the brain lipids. Liver cholesterol level was similar among the experimental groups, whereas the concentration of vE in the liver of rats fed BECC with increased vE levels was higher than that in the rats fed BECC with the basal vE level. Haematological and biochemical parameters in blood were within the normal range for rats, but a few rats showed a tendency towards increased levels because of the higher vE and Se level. The health-promoting effect of feeding rats PUFAn-3 enriched BECC was more pronounced when an increased dietary level of vE was used, but the increased level of Se did not provide the rats with additional benefits. Thus, the findings indicate that BECC enriched with PUFAn-3 and vE by a dietary intervention is a functional food with great potential of implementation.

Calculation of the time resolution of the J-PET tomograph using kernel density estimation.

In this paper we estimate the time resolution of the J-PET scanner built from plastic scintillators. We incorporate the method of signal processing using the Tikhonov regularization framework and the kernel density estimation method. We obtain simple, closed-form analytical formulae for time resolution. The proposed method is validated using signals registered by means of the single detection unit of the J-PET tomograph built from a 30 cm long plastic scintillator strip. It is shown that the experimental and theoretical results obtained for the J-PET scanner equipped with vacuum tube photomultipliers are consistent.

Diagnostic and prognostic value of RNA-proteolipid in sera of patients with malignant disorders following therapy: first clinical evaluation of a novel tumor marker.

The circulating level of a novel RNA-proteolipid complex associated with malignant diseases was critically evaluated as a tumor marker in clinical oncology. The complex, isolated from the sera of cancer patients, exhibited unvarying chemical composition regardless of the cell type and clinical staging. Clearance from blood was rapid with a half-life of approximately 2 days. Tumor mass could be correlated with the circulating level. After effective treatment the level fell and rose again 10 months prior to the conventional clinical diagnosis of relapse.

A comparison of the abilities of nitrobenzylthioinosine, dilazep, and dipyridamole to protect human hematopoietic cells from 7-deazaadenosine (tubercidin).

Nitrobenzylthioinosine, dilazep, and dipyridamole are potent inhibitors of equilibrative transport of nucleosides that may have pharmacological applications in modulating the therapeutic index of nucleoside antimetabolites used in cancer chemotherapy. We have compared the relative abilities of these inhibitors to reduce the toxicity of in vitro exposures to tubercidin against clonogenic progenitor cells of normal human bone marrow (CFU-GEMM, BFU-E, CFU-GM) and of two leukemic human cell lines (HL-60/C1, CCRF-CEM) that differ in their expression of transporter subtypes. Short (1-h) exposures to 1 microM tubercidin alone inhibited colony formation (a) of normal human hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) by 100%, and (b) of HL-60/C1 and CCRF-CEM cells by > 90%. Pretreatment (30 min) with nitrobenzylthioinosine, dilazep, or dipyridamole followed by simultaneous treatment (1 h) with these transport inhibitors during tubercidin exposures reduced toxicity against hematopoietic progenitors and cell lines. Greater reductions of toxicity were consistently seen with bone marrow progenitors and CCRF-CEM cells than with HL-60/C1 cells. For CFU-GEMM, BFU-E, and CFU-GM cells, reductions in tubercidin toxicity of 50-100% were achieved at these concentrations: > or = 0.1 microM (nitrobenzylthioinosine); > or = 0.1 microM (dilazep); and > or = 3.0 microM (dipyridamole). Pretreatment (30 min) followed by simultaneous treatment (1 h) with any of the transport inhibitors (> or = 0.1 microM) and 0.1 microM [3H]-tubercidin blocked the uptake of radioactivity completely in CCRF-CEM cells and only partially in HL-60/C1 cells. These effects, which were consistent with the nucleoside transport phenotypes of CCRF-CEM cells (inhibitor-sensitive) and HL-60/C1 cells (inhibitor-sensitive and inhibitor-resistant), suggested that protection was due to the inhibition of tubercidin uptake via equilibrative nucleoside transport system(s). Light-density mononuclear cells from human bone marrow, of which the clonogenic progenitors represented only a minor (< 0.01%) subpopulation, possessed far fewer nitrobenzylthioinosine-binding sites (2 x 10(4) sites/cell, Kd = 0.7 nM) than either HL-60/C1 cells (1.7 x 10(5) sites/cell, Kd = 0.9 nM) or CCRF-CEM cells (3.3 x 10(5) sites/cell, Kd = 0.5 nM). Initial rates of uptake of 1 microM [3H]adenosine (0-6 s, 20 degrees C) by human bone marrow mononuclear cells were reduced partially by 0.1 microM inhibitor (nitrobenzylthioinosine > dipyridamole > dilazep) and completely by 10 microM inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of duration of exposure to verapamil on vincristine activity against multidrug-resistant human leukemic cell lines.

Verapamil sensitizes multidrug-resistant cell lines to various heterocyclic anticancer drugs by inhibition of energy-dependent release of drug, presumably by interaction with membrane glycoproteins involved in drug efflux. This work assessed verapamil sensitization of human multidrug-resistant lymphocytic and myeloid leukemic cell lines (CEM/VLB100, HL-60/AR) to vincristine during exposures of short duration (4 h). When cells were transferred to drug-free medium immediately after simultaneous 4-h exposures to vincristine and verapamil, the antiproliferative activity of vincristine was not altered in CEM/VLB100 cells and was only moderately increased in HL-60/AR cells. In contrast, when cells were transferred to verapamil-containing medium, vincristine activity was greatly increased against both CEM/VLB100 and HL-60/AR cells. Verapamil enhanced accumulation and inhibited release of [3H]vincristine by CEM/VLB100 and HL-60/AR cells, indicating that the sensitization was due to an increase in cell-associated vincristine after transfer of cells to vincristine-free medium. Slot blot analysis of cellular RNA with the pMDR1 probe revealed high levels of expression of the mdr1 gene in CEM/VLB100 cells but no detectable expression in HL-60/AR cells. Consistent with this finding, polypeptides (Mr 170,000 to 180,000) that were recognized by a monoclonal antibody (C219) against P-glycoprotein were greatly overexpressed in CEM/VLB100 cells, but were expressed at low levels, if at all, in HL-60/AR cells. These results demonstrate the importance of duration of exposure to verapamil in reversing multidrug resistance, not only in cells that overexpress P-glycoprotein but also in cells, such as HL-60/AR, that express little, if any, P-glycoprotein.