Validation of a single point flow cytometric assay for determining P-glycoprotein activity in multidrug resistant cell lines

Summary. P-glycoprotein, a transmembrane protein which acts as an energy dependent efflux pump, has been implicated as one mechanism of multidrug resistance (MDR) in human tumours. Commonly employed assays measure P-glycoprotein immunohistochemically or mdrl messenger RNA. In this study we compared a single point flow cytometric assay for determining activity of P-glycoprotein with cellular expression of P-glycoprotein determined by Western blot. Five cell lines, with varying levels of multiple drug resistance, were incubated with daunorubicin (DNR) in the presence (treated) and absence (control) of cyclosporine or verapamil, agents known to inhibit the activity of P-glycoprotein. The treated cell lines, along with non-treated controls were examined for intracellular concentrations of DNR measured by fluorescence intensity using a flow cytometer. The ratio of fluorescence intensity expressed in the treated/control was used as an index of functional activity of P-glycoprotein. Functional activity of the P-glycoprotein as determined by flow cytometry correlates highly with cellular content of P-glycoprotein measured by western blot (correlation coefficients of r= 0.90–0.98 for the various cell line combinations). This method represents a rapid single point flow cytometric assay which may be suitable for screening clinical samples for P-glycoprotein activity.     

Differential activity of P-glycoprotein in normal blood lymphocyte subsets

To better understand the phenomenon of P-glycoprotein (P-170) expression we investigated lymphocyte subpopulations for P-170 function in healthy volunteers. Studies were based on three-colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P-170. Marked Rh123 efflux was detected in CD8⁺ T lymphocytes with CD8⁺/CD45RA⁺ T cells (naive cells) showing significantly higher P-170 activity as compared with CD8⁺/CD45RA^? cells (P < 0.04). Vice versa, CD8⁺/CD45RO⁺ T cells (memory cells) demonstrated less P-170 activity than CD8⁺/CD45RO^? cells (P < 0.04). P-170 function was less prominent in CD4⁺ T cells, however, Rh123 efflux was higher in the CD4⁺/CD45RA⁺ and CD4⁺/CD45RO^? subpopulations (P < 0.025) corresponding to the CD8⁺ results. Dye efflux differed significantly between activated and non-activated CD8⁺ and CD4⁺ as well as CD8⁺/CD11b⁺ and CD8⁺/CD11b^? T lymphocytes. Since CD16⁺ natural killer cells (NK) expressed the highest level of P-170, the NK cytotoxicity against ⁵¹Cr-labelled K562 target cells was assayed in the presence or absence of P-170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R-verapamil and dexnigaldipine-HCP in a dose-dependent manner. The differential expression of P-170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR-modulators suggest a physiological role of P-170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.     

流式细胞术分析冻存前后脐血CD34+细胞的分布

目的探讨低温冻存对脐血CD34+细胞的影响.方法采用流式细胞仪分析冻存前后脐血CD34+细胞百分率、CD45+细胞和CD34+细胞的荧光强度变化及死细胞群的分布情况.结果冻存后CD34+细胞占CD45+细胞的百分率[(0.84±0.39)%]明显高于冷冻前[(0.51±0.24)%](P<0.01),冻存前后CD34+细胞绝对数无明显变化[(9.372±6.072) ×106/L和(9.246±6.132)×106/L](P>0.05),冻存前后CD34+细胞百分率呈正直线相关(r=0.564, P<0.01).冻存后CD45+细胞荧光强度减弱(P<0.01),CD34+细胞荧光强度无明显变化(P>0.05);中性粒细胞比例下降,淋巴细胞和单核细胞比例增高.死细胞组分中以中性粒细胞为主,占81.52%;活细胞组分中以淋巴细胞为主,占59.44%.结论冻存后CD34+细胞占CD45+细胞的百分率增高,但低温冻存对CD34+细胞绝对数量影响不大.死细胞主要为较成熟的粒细胞,冻存后CD34+细胞的分析需排除死细胞的干扰.     

Flow cytometric DNA histograms and type of growth

Summary DNA histograms of human tumors should be interpreted together with the type of growth. In order to prove this connection the Ehrlich ascites tumors of 144 mice were investigated by absolute cell counting and flow cytometry. The exponential, transition and steady-state phases of tumor development were defined as types of growth based on the logistic function (Verhulst-Pearl). Flow-cytometric histograms (ethidium-bromide, olivomycin) were evaluated by two methods presupposing lognormal distribution and resulting in the same trend of changes concerning the percentage of cells in the three cell-cycle phases. In exponential growth the histograms show a high S-phase compartment and a decrease of the G2M peak. The increased percentage of S-phase cells is caused by a recruitment from G0-1 and G0-2 after transplantation. The decrease of the G2M peak is due to the exponential growth, a recruitment from G0-2 and a relative shortening of G2M duration. A high G2M peak and a low S-phase portion occur in steady state. Besides a decrease of DNA-synthezising cells the S compartment decreases as a result of a relative S-phase shortening. For the increase of the G2M peak, transition into steady state, prolongation of G2M duration, recruitment of G0-1 and the occurrence of G0-2 cells are responsible. The same histogram may reflect different biological behavior patterns depending on the type of growth.     

Flow cytometry study of cell cycle, apoptosis and drug resistance in acute leukemia

The response to therapy of leukemic cells is largely determined by their capacity of proliferation and apoptosis in presence of the administered drugs. We describe here the main markers used in flow cytometry (FCM) and involved in the assessment of cell cycle parameters: single labeling by Propidium Iodide (PI) and double labeling anti-Bromodeoxyuridine (BrdUrd)/PI which, both in vitro and in vivo, gives cell percentages in the different cell cycle phases. The markers of cell cycle progression can be divided into proliferation markers such as PCNA (proliferating cell nuclear antigen) or Ki-67 and cell cycle progression markers. The latter, which are the core of the cell cycle machinery, are molecules recently characterized (Cyclins, CDKs (cell dependent kinases), CDIs (cyclin-dependent kinase inhibitors)) and their cell expression can be analyzed using FCM. FCM is also one of the best means to detect and quantitate appoptotic cells. Several techniques are described: Nuclear labeling using Hoechst 33342: mitochondrial labeling using DiOC6(3): detection of DNA fragmentation using 1) labeling of fixed and permeabilized cells with a DNA marker or 2) labeling of the free 3 DNA ends using incorporation of labeled deoxynucleotides; detection of antigens in apoptotic cells (Bcl-2, Fas, phospholipids...). At last, we analyzed flow cytometry methods to study the cell resistance to Ara-C and anthracyclins. In combination with cell kinetic studies and detection of apoptotic cells, they should increase the efficiancy of the acute leukemia treatment.     

Absence of correlation between cytotoxicity and drug transport by P-glycoprotein in clinical leukemic cells.

Development of resistance to cytotoxic agents is a common problem in the treatment of acute leukemia. In cell lines having multidrug resistance (MDR) phenotype, a decrease in the intracellular accumulation of drugs has been closely related to the overexpression of P-glycoprotein/mdr1 genes. We analyzed the relationship between the cytotoxicity of adriamycin (ADR) in vitro, intracellular accumulation of ADR, and the expression of P-glycoprotein on fresh leukemic cells from 19 patients at their initial presentation and from 9 relapsed patients. Pretreatment patients showed significantly higher ratio of complete remission than relapsed patients, and mean value of IC50 for adriamycin in initial presentation was higher than at relapse. But we found no significant relationship between in vitro cytotoxicity and drug transport. In addition, only 2 of the 5 relapsed patients examined by monoclonal antibody C219 expressed the P-glycoprotein. These results suggest that the acquisition of clinical drug resistance may involve various mechanisms other than the reduction of drug accumulation with P-glycoprotein expression.     

流式细胞术分析活化血小板及其在急性肺血栓栓塞症中的临床意义

目的应用全血流式细胞术(FCM)结合血小板活化特异性单抗检测急性肺血栓栓塞症(PTE)患者血小板活化状态,探讨血小板活化在急性PTE中的作用及临床意义.方法以凝血因子I受体(FIB-R)、P-选择素(CD62P)作为分子标志物,利用FCM荧光标记法检测36例急性PTE患者和20例健康对照者微量全血FIB-R、CD62P血小板表面阳性表达的百分率,并比较急性PTE患者治疗前后FIB-R、CD62P在血小板表面阳性表达的变化.结果急性PTE患者血小板表面活性标志蛋白FIB-R、CD62P阳性率分别为(18.30±12.23)%、(12.07±6.54)%,均显著高于对照组[(1.81±0.88)%、(2.18±1.50)%,(P＜0.01)].溶栓和抗凝治疗后各项指标均较治疗前明显下降(均P＜0.01).结论急性PTE患者体内存在着明显的血小板活性增强,全血FCM能准确地反映体内血小板的活化水平.     

耐多药结核病患者外周血中多药耐药相关跨膜转运蛋白的表达

目的 评价耐多药结核病(MDR-TB)患者外周血中单核细胞P糖蛋白和多药耐药相关蛋白(MRP)表达水平的变化.方法选择2004年9月至2007年12月在武汉市结核病防治所住院治疗的MDR-TB患者89例为耐多药组,其中男52例,女37例;年龄17~62岁,平均(45±6)岁;平均病史3.5年;均符合MDR-TB的诊断标准.同期在武汉市结核病防治所住院的初治结核病患者55例为结核病组,其中男34例,女21例;年龄18~60岁,平均(48±8)岁;平均病史1.2年;痰涂片均为阳性.对照组为武汉市结核病防治所无肺结核史的工作人员31例,其中男19例,女12例;年龄25~62岁,平均(44±5)岁.抽取外周血,采用实时定量PCR法,分别检测单核细胞P糖蛋白和MRP的mRNA水平.多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验.结果 耐多药组P糖蛋白mRNA表达的吸光度值(1.34±0.32)与结核病组(1.12±0.23)和对照组(1.05±0.16)比较,差异无统计学意义(F=0.536,P>0.05).耐多药组MRP的mRNA表达(3.45±0.43)明显高于结核病组(1.23±0.34)和对照组(1.04±0.12),3组比较,差异有统计学意义(F=24.241,P<0.05),耐多药组分别与其他2组比较,差异均有统计学意义(P<0.01).结论 MRP高表达与MDR-TB患者的多耐药存在一定的相关性.

Abstract：

Objective To evaluate the expression of P-glycoprotein (P-pg) and multidrug resistance-associated protein (MRP) mRNA levels in peripheral blood mononuclear cells from multidrug resistant tuberculosis (MDR-TB) patients. Methods The subjects of this study included 3 groups: a non-TB control group, a TB control group and a MDR-TB group. The 31 subjects in the non-TB control group were staff from Wuhan Tuberculosis Prevention and Treatment Institute. The 55 cases in the TB control group were in-patients during September 2004 to December 2007 who were diagnosed as having pulmonary tuberculosis. The 89 cases in the MDR-TB group were in-patients during the same period, but who were diagnosed as having MDR-TB. Peripheral mononuclear cells were isolated and mRNA levels of P-pg and MRP were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK q was used for comparison between 2 groups.Results There was no significant difference in the relative P-pg mRNA levels among the MDR-TB group (1.34±0.32), the non-TB control group (1.05±0.16) and the TB control group (1.12±0.23), F=0.536, P>0.05. The relative MRP mRNA level (3.45±0.43) was the highest in the MDR-TB group (3.45±0.43), as compared to the TB control group (1.23±0.34) and the non-TB control group (1.04±0.12), F=24.241, P<0.05. Conclusion Higher expression of MRP in peripheral mononuclear cells might be related to multidrug resistance in MDR-TB patients.     

Flow cytometric analysis of T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia: Correlation with clinical stage

Summary Several authors have studied the T-lymphocyte subpopulations in B-cell chronic lymphocytic leukemia (B-CLL), but previous studies were performed after preceding enrichment procedures, which are known to cause selective losses of certain subpopulations. To correct for this deficiency we used flow cytometric analysis, which enabled us to measure subpopulations directly on total blood samples. We studied T-lymphocyte subsets with OKT monoclonal antibodies in 45 patients with B-CLL. Serum levels of IgG, IgA and IgM were assayed simultaneously and findings were correlated with clinical stage (Rai classification). The absolute number of CD 4-positive cells decreased in more advanced Rai stages, while the absolute number of CD 8-positive cells increased, resulting in a progressive reduction in CD 4/8 ratio. Results from patients in stages with equal prognosis (Rai I and II, Rai III and IV) were similar and when these results were grouped the observed differences were highly significant and clearly correlated with all prognostic groups.     

Flowcytometric analysis of multidrug-resistance-associated antigen (P-Glycoprotein) and DNA ploidy in human colon cancer

Summary In many cell systems, resistance to cytotoxic drugs is acquired by the amplification and/or overexpression of the multidrug resistance (mdr) gene, which codes for the glycoprotein, p170 (P-glycoprotein). Moreover, in a variety of malignant tumours there is increasing evidence of the relationship between the DNA ploidy pattern of patients and their prognosis. In this study we aimed to evaluate these two potential indicators of constitutive drug resistance in human colorectal tumours. We employed a method to quantify simultaneously, on a per cell basis,mdr gene expression (using the C219 monoclonal antibody for P-glycoprotein) and nuclear DNA content with high-resolution bivariate flow cytometry. The study was performed on a human coloncarcinoma-derived cell line (LoVo) and its doxorubicinresistant variant (LoVo/Dx) and on tumour samples and adjacent normal mucosa from 35 untreated patients with colon cancer. The P-glycoprotein was found in both LoVo and LoVo/Dx cells with levels slightly lower in the parental than in the resistant subline (P, NS). A multidrug-resistant specific probe for mRNA expression and Western blot assay confirmed the specificity of p170 expression. All of the colon cancer with unimodal diploid DNA distribution and all the normal colonic mucosa samples showed P-glycoprotein expression, without a statistically significant difference in median values between tumours and normal samples. Tumours with bimodal DNA distribution showed median values of P-glycoprotein expression of their hyperdiploid cell clones significantly higher than those of their diploid clones and of the tumours with unimodal DNA distribution (P<0.005). Our results show the feasibility of bivariate flow-cytometric analysis of P-glycoprotein expression and DNA content on clinical material and support the hypothesis that the MDR phenotype and DNA ploidy together may influence the biological behaviour of colon cancer in vivo.Abbreviations MDR multidrug resistance phenotype - mdr multidrug resistance gene - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline Research supported in part by C.N.R. target projects Oncology and Biotechnology and Bioinstrumentation, by A.I.R.C. and by I.R.C.C.S. San Matteo     

Flow cytometric analysis of tissue factor (TF) expression on stimulated monocytes — comparison to procoagulant activity of mononuclear blood cells

Summary Whereas tissue factor (TF), a 47 kDa transmembrane glycoprotein, is constitutively present in certain tissues such as epithelial tissue, brain, and placenta, it is normally not expressed by cells within the vasculature. However, inflammatory mediators including bacterial lipopolysaccharide (LPS) can stimulate the expression of cell surface procoagulant activity (PCA) on monocytes. In our present study the kinetics (over 24 h) of molecular TF expression on LPS-stimulated monocytes analyzed by flow cytometry corresponds closely to functional PCA of human mononuclear blood cells (MBC). Both PCA and TF expression on monocytes were rapid events reaching their maximum after about 6 h of stimulation. At this time approximately 70–80% of monocytes had also achieved maximum anti-TF MAb receptor density. For certain analytical applications, monitoring of molecular TF expression on monocytes by flow cytometry using anti-TF MAb is favorable because there is no influence by PCA inhibitors.     

Flow cytometric DNA analysis of malignant lymphomas with primary bone manifestation

Summary Malignant lymphomas with primary skeletal manifestation have received controversial evaluation with regard to histological classification and histogenesis. Recent histological and immunohistological studies on the rare bone lymphomas conducted by our team, have shown that they do not differ from primary nodal lymphomas with regard to the spectrum of histological subtypes. The present flow cytometric DNA analysis of paraffin-embedded material from 17 lymphomas documented in the Bone Tumor Registry of Westfalia yielded the following distribution pattern of DNA ploidy: among 12 non-Hodgkin's lymphomas (NHL) (according to the Kiel classification) there was only 1 case of low grade malignancy; this centroblastic-centrocytic lymphoma showed a unimodal diploid DNA histogram. Of 11 highly malignant NHL, 6 were DNA hyperdiploid. Among the 5 cases of Hodgkin's lymphoma, 4 were DNA diploid, (1 nodular sclerosing, 3 mixed types) and one DNA tetraploid (lymphocytic depletion type). Comparison with data from the literature reveals that even with regard to DNA ploidy, malignant lymphomas primarily manifesting in bone do not differ from those of exclusively nodal manifestation.     

Flow cytometric analysis of intracellular CD6 8 molecule expression in normal and malignant haemopoiesis

Summary. CD68 molecules are heavily glycosylated lysosomal membrane constituents of unknown function with strong expression in monocytes and macrophages. Using flow cytometry, we quantified expression levels of CD68 molecules in normal and malignant haemopoietic cells. CD68 molecules are intensely expressed in the cytoplasm and weakly on the surface of mature CD14⁺ monocytes. CD68 expression seems to start very early during granulo-monopoietic differentiation. Virtually all myeloperoxidase (MPO)⁺ bone marrow cells coexpress CD68 and similar proportions of CD34⁺ progenitor cells weakly express CD68 or MPO molecules. During further differentiation, CD68 expression is strongly up-regulated in early MP0⁺ precursor cells which lack lactoferrin (LF) and CD14 molecules. Compared to these, more mature MPO⁺LF⁺ bone marrow and peripheral blood granulocytes express considerable lower levels of CD68. In-line with this broad expression, all investigated acute myeloid leukaemia (AML) cases, classified as FAB M1-M5, were CD68 positive, and compared to normal CD34⁺ bone marrow cells, CD34⁺ AML blast cells expressed increased levels. CD68 expression is, however, not restricted to cells of myeloid origin, because a subset (40 – 15%, n = 6) of CD19⁺ peripheral blood B-lympho-cytes and 50% of B-ALL are also weakly positive. In contrast, normal CD3+ lymphocytes lack (<3%. n = 6) CD68 and only low proportions (6 – 3%, n= 6) of CD56⁺ NK cells are CD68⁺. Also, all investigated T-ALL cases (n = 6) lacked CD68.     

Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic red blood cells

Reactive oxygen species (ROS) contribute to the pathogenesis of several hereditary disorders of red blood cells (RBCs), including thalassaemia. We report here on a modified flow cytometric method for measuring ROS in normal and thalassaemic RBCs. RBCs were incubated with 0.4 mM 2',7'-dichlorofluorescin diacetate (DCFH-DA), then washed and further incubated either with or without 2 mM H2O2. Flow cytometric analysis showed that RBC fluorescence increased with time; it increased faster and reached higher intensity (by 10-30-fold) in H2O2-stimulated RBCs as compared to unstimulated RBCs. In both cases, the antioxidant N-acetyl-l-cysteine reduced fluorescence, confirming previous reports that DCFH fluorescence is mediated by ROS. While the fluorescence of unstimulated RBCs increased with time, probably because of exposure to atmospheric oxygen, in H2O2-stimulated RBCs fluorescence decreased after 30 min. The latter effect is most likely related to H2O2 decomposition by catalase as both sodium azide, an antimetabolite that inhibits catalase and low temperature increased the fluorescence of stimulated RBCs. Washing had a similar effect, suggesting that maintenance of the oxidised DCF requires a constant supply of ROS. We next studied RBCs of beta-thalassaemic patients. The results demonstrated a significantly higher ROS generation by stimulated and unstimulated thalassaemic RBCs compared to their normal counterparts. These results suggest that flow cytometry can be useful for measuring the ROS status of RBCs in various diseases and for studying chemical agents as antioxidants.     

P-glycoprotein in blood CD4 cells of HIV-1-infected patients treated with protease inhibitors

Objective

Many factors are involved in the virological failure of antiretroviral treatments such as low pharmacological plasma levels of drugs, poor adherence to therapy and emergence of viral resistance. P‐glycoprotein (P‐gp) has been demonstrated to play a role in multidrug resistance in the therapy of solid tumours, haematological malignancies and Plasmodium falciparum infection. HIV‐1 protease inhibitors (PIs) have been described to be substrates of P‐gp. In vitro and in vivo studies performed in mice have demonstrated that P‐gp may affect the oral bioavailability and intracellular accumulation of PIs. P‐gps have been detected on peripheral CD4 blood cells in HIV‐1‐infected, but antiretroviral‐naive patients.

Method

We quantified P‐gp expression and performed functional tests of P‐gp activity in the CD4 cells in HIV‐1‐infected patients, with and without virological failure, treated with PIs, and in healthy patients (control group).

Result

Out of the 18 HIV‐infected patients studied, P‐gp expression and function were found in the CD4 cells of six patients (four of 10 without, and two of eight with virological failure). Out of the 43 healthy patients studied, P‐gp expression and function were found in the CD4 cells of 11 patients (26%). We found P‐gp in peripheral CD4 cells of patients treated with PIs, with and without virological failure, within the same frequency than in antiretroviral naive patients or than in non HIV‐infected patients.

Conclusions

P‐gp expression in peripheral CD4 blood cells does not seem to be enhanced by PI treatment and does not seem to be linked particularly to virological failures. These facts do not preclude of the role of P‐gp on PI absorption or efficacy in other compartments of the body such as gut, lymph nodes or brain in HIV‐1 PI‐treated patients.

     

Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34⁺CD33⁻ cells expressed P-gp strongly, CD34⁺CD33⁺ cells moderately, and CD34⁻CD33⁺ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34⁻CD33⁺ but not CD34⁺CD33⁻ at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.     

Flow cytometric analysis of T and Tn epitopes on chronic lymphocytic leukemia cells

Immunophenotyping of peripheral blood lymphocytes from six patients with B-cell chronic lymphocytic leukemia (B-CLL) and five normal volunteers was done and their T and Tn epitopes analyzed using specific monoclonal antibodies and flow cytometry. Lymphocytes from all patients showed strong Tn expression as compared to normal control lymphocytes. By contrast, T antigen was not expressed. The Tn expression may be a useful diagnostic and prognostic marker for B-CLL. © 1996 Wiley-Liss, Inc.     

造血干细胞多药耐药P-糖蛋白的表达及多药耐药逆转剂对其功能的影响

了解多药耐药Ｐ－糖蛋白在造血干／祖细胞的表达,探讨多药耐药逆转剂在临床应用过程中可能对造血干细胞的损害。方法：采用ＭＲＫ１６单克隆抗体,利用流式细胞仪测定健康人骨髓ＣＤ３４阳性细胞表达Ｐ－ｇｐ的表达;将多药耐药逆转剂ＭＳ－２０９作用于造血细胞,检测ＭＳ－２０９对ＣＤ３４阳性细胞摄取染料Ｒｈｏｄａｍｉｎ－１２３的影响。     

Cytotoxicity of adriamycin,idarubicin, and vincristine in acute myeloid leukemia: Chemosensitization by verapamil in relation to P-glycoprotein expression

Summary A 4-day colorimetric tetrazolium dye (MTT) assay was used to assess the cytotoxicity of adriamycin (ADM), vincristine (VCR), and idarubicin (IDA) in blasts isolated from 37 patients with newly diagnosed and pretreated acute myeloid leukemia (AML). The effect of verapamil (VRP) as a chemosensitizer was studied in relation to the expression of the membrane efflux pump P-glycoprotein (PGP) as determined by a semiquantitative flow-cytometric procedure. A slight positive correlation was found between the fraction of cells expressing PGP and the ID₅₀ values for ADM and VCR, but not between cellular PGP content and sensitivity to IDA. The overall data showed no significant sensitization effect of VRP. However, in specimens with more than 10% cells expressing PGP, 2M VRP sensitized cells to ADM and VCR significantly. The median of sensitization ratios (SRs), i.e., the ratios of cytotoxic drug ID₅₀ in the absence/presence of VRP, were 1.89 and 2.0, respectively. No sensitizing effect of VRP on the cytotoxicity of IDA was observed. Related to the clinical status, the median fraction of PGP-positive blasts was elevated fourfold in pretreated patients (n=16) in comparison to patients with de novo AML (n=19). No differences in ID₅₀ values were observed between newly diagnosed and pretreated patients. However, SRs for ADM and VCR were higher in samples of pretreated patients compared with de novo AML. PGP-mediated cellular drug resistance may thus be circumvented in leukemic blasts by application of chemosensitizers or, potentially, alternative anthracyclines.     

Flow cytometric analysis of defensins in blood and marrow neutrophils

Polymorphonuclear neutrophils (PMN) are vital in host defense against microbial infections. This study provides a flow cytometric method for the quantitative analysis of microbicidal peptides (defensins) in cells of PMN lineage. Rabbit neutrophil peptides, NP-2 and NP-5, were measured in all PMN and in subpopulations of PMN expressing 1-selectin. PMN lineage counts were made on Wright's-stained blood smears and marrow cytospins. Immunoreactivity for NP-2, and NP-5 was detected by using the alkaline phosphatase anti-alkaline phosphatase technique. The results show that marrow PMN express higher levels of NP-2 and NP-5 than blood PMN, p < 0.001 and that these levels are associated with elevated numbers of myeloid precursors. In both blood and marrow, NP-2 occurs in two PMN subpopulations and the mean fluorescence intensity of NP-2 is consistently higher than that of NP-5. Increased levels of defensins are observed in circulating PMN depicting the most 1-selectin p < 0.05. Immunocytochemical results indicate that PMN defensins reside in cytoplasmic granules and are not constitutively expressed on the cell surface. Furthermore, defensins are not detected in monocytes, eosinophils, lymphocytes and erythrocytes. The flow cytometric method described here provides a novel means of quantitating host natural defenses, allows the characterization of PMN subpopulations and has clinical applications.