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Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G1 fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G2/M and cells with lower G0/G1 DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14–27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.

The effect of the targeted cytotoxic somatostatin (SST) analog AN-162, consisting of doxorubicin (DOX) conjugated to SST carrier RC-121, was investigated on the growth of human colorectal cancer (CRC) cell lines HT-29, HCT-15, and HCT-116 and a DOX-resistant mouse leukemia cell line P388/R84. mRNA for SST-receptors and high affinity binding sites for SST were detected in all CRC cell lines and in P388/R84 cells. In contrast to DOX alone, AN-162 blocked HCT-116 cells and P388/R84 cells in S/G2 phase and increased the number of apoptotic cells. In vivo, AN-162 reduced the volume of CRC xenografts more effectively than its unconjugated components. Our results suggest that AN-162 inhibits growth of experimental CRC more effectively than DOX and increases sensitivity of DOX resistant human leukemia cells.

The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10 muM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (Delta Psi m). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21(Waf1/Cip1) was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p < 0.05) and extended tumor doubling time (p < 0.001). Our observations suggest that GHRH antagonist JMR-132 exerts its antiproliferative effect on experimental colon cancer cells through p21(Waf1/Cip1) mediated S-phase arrest along with apoptosis involving the intrinsic pathway.

The purpose of this study was to analyse the frequency and type of mutations in the coding region of androgen receptor (AR) and to determine the role of polymorphisms in the intron 1 of ERalpha, exon 5 of ERbeta, intron 7 of progesterone, exon 7 of the aromatase (CYP19) and exon 9 of VDR genes in the risk of prostate cancer. PCR-RFLP analysis of all above the genes was on 100 prostate cancer patients and an equal number of matching controls. The study also included PCR-SSCP analyses of exons 2-8 of AR gene. The genotype containing -/- allele of ERalpha gene was statistically significant for the risk of prostate cancer pose (OR, 2.70; 95% CI, 1.08-6.70, P = 0.032) Rr genotype of ERbeta gene also have a higher risk (OR, 1.65; 95% CI, 0.52-5.23) for prostate cancer. The Cys allele of CYP19 gene was also associated with statistically significant increased risk of prostate cancer (OR; 2.28, 95% CI, 1.20-4.35, P = 0.012). tt genotype of codon 352 of VDR gene showed an OR of 0.43 for (95% CI, 0.13-1.39) and an OR for Tt genotype was 0.65 (95% CI, 0.36-1.16). Taken together, the results showed that in North Indian population, ERalpha and CYP19 genes may be playing a role in the risk of prostate cancer.

Coulter volume is far more accurate measure of cell volume than forward angle light scatter. In this report, we have used Coulter volume to determine the mean cell volume and diameter of normal human peripheral blood cells and hematopoietic progenitor cells obtained by apheresis (HPC-A) from patients with hematological malignancies. Fresh peripheral blood samples (treated with Beckman Coulter IMMUNOPrep erythrocyte lysis solution), HPC-A samples (treated with BD Biosciences FACSLysing solution), or processed by Ficoll Hypaque sedimentation method were stained with CD45-FITC and PE-labeled CD34, CD90, CD117, and CD133 antibodies and analyzed for electronic volume and two color fluorescence. The mean electronic volume and diameter of mononuclear cells from fresh peripheral blood samples prepared with IMMUNOPrep were lymphocytes (191 microm(3), 7.16 microm), monocytes (370 microm(3), 9.91 microm), and granulocytes (328 microm(3), 8.56 microm). In mononuclear cells of HPC-A samples prepared by Histopaque-1077 sedimentation, the lymphocytes had volume and diameter of 311 microm(3), 8.4 microm, monocytes were 486 microm(3), 9.76 microm, and granulocytes were 515 microm(3), 9.95 microm. In contrast, HPC-A samples prepared after lysis with FACSLysing solution had mean electronic volume and diameter of lymphocytes (414 microm(3), 9.25 microm), monocytes (797 microm(3), 11.5 microm), and granulocytes (670 microm(3), 10.85 microm). Cell volume of mononuclear cells in the HPC-A samples prepared by Histopaque-1077 sedimentation method was correlated with the expression of stem cell markers CD34, CD90, CD117, and CD133. CD90 positive cells had the smallest mean electronic volume of 299.93 microm(3) when compared with cells with positive expression of CD133 (322 microm(3)), CD117 (349 microm(3)), CD34 (407 microm(3)), and CD45 (453 microm(3)). Correlation of cell volume with stem cell marker expression may allow for the identification of small stem cells, which may not express the conventional markers used for the identification of stem cells in HPC-A samples.

The Hoechst side population (SP) technique is a critical method of identifying stem cells and early progenitors in rodent, nonhuman primate, and human hematopoietic and nonhematopoietic tissues. In this technique, the cell-permeable DNA-binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells and early progenitors subsequently pump this dye out via an ATP-binding cassette membrane pump-dependent mechanism, resulting in a low-fluorescence "tail" (the SP) when the cells are analyzed by flow cytometry. This population contains stem cells and early progenitors. One significant drawback of this method is the requirement of an UV laser to excite the Hoechst 33342. Unfortunately, flow cytometers equipped with UV sources are expensive to own and operate and are not readily available to many laboratories or institutions. In the interests of designing a less expensive flow cytometric system for stem cell analysis, we determined the minimum UV excitation and instrumentation requirements for measuring Hoechst SP. Less than 3 mW of UV laser output was required for adequate resolution of Hoechst SP on two cuvette-based flow cytometers, one of which was a simple, inexpensive benchtop analyzer (the Quanta Analyzer; NPE Systems). Furthermore, Hoechst SP could also be adequately resolved on this epifluorescence-based cytometer platform using two nonlaser UV sources, a mercury arc lamp with a UV bandpass filter and a UV-emitting light-emitting diode. These results suggest that an economical flow cytometric system can be designed that is capable of resolving Hoechst SP, with a cost far lower than most UV laser-equipped commercial systems. An inexpensive system of this type would make Hoechst SP analysis available to a much broader group of stem cell investigators.

To investigate the involvement of the CYP17, SRD5A2, CYP1B1, and CYP2D6 variants with prostate cancer, a case-control study of 100 patients and an equal number of age-matched control men was conducted. There appears to be a nonsignificant increase with risk of prostate cancer for individuals carrying one copy of the CYP17 A2 allele (OR, 1.80; 95% CI, 0.99-3.29, P=0.05). The risk was increased in individuals having two A2 alleles (OR; 2.81, 95% CI, 1.06-7.40, P=0.03). Compared with men having the VV genotype of SRD5A2 gene, there was no significant association between the VL genotype and the risk of prostate cancer (OR; 0.54, 95% CI; 0.29-1.03, P=0.06). There was no difference in the occurrence of the genotype LL between controls and prostate cancer patients (OR; 0.90, 95% CI; 0.43-1.89, P=0.79). There was a nonsignificant increased risk of prostate cancer for individuals carrying the CYP1B1Leu/Val genotype (OR, 1.70, 95% CI, 0.91-3.17, P =0.09), which was increased in those having the Val/Val allele (OR, 3.38; 95% CI, 1.13-10.07, P=0.02). Relative to men homozygous for the wild-type allele in CYP2D6 gene, those heterozygous for the B allele had an odds ratio of 1.78 (95% CI, 0.76-4.17, P=0.18) for patients, and for homozygous individuals, it was 1.95 (0.55-6.93, P=0.30). These observations have suggested that the CYP17 A2/A2, CYP1B1 Val/Val, and CYP2D6 genotypes may be associated with an altered risk of prostate cancer, while the CYP2D6 and SRD5A2 V89L polymorphism have no association with its risk in the North Indian population.

The number of CAG repeats on the androgen receptor (AR) gene is inversely proportional to transcriptional activity. The purpose of this study was to determine if short-term androgen deprivation therapy (RT + HT) can improve outcome in patients with tumors with short CAG repeats (19).Prostate cancer patients were randomized to receive either radiotherapy (RT) alone or (RT + HT) in the RTOG 86-10 study. CAG repeats were measured in 94 tumor specimens (21%; test cohort) of the 456 (parent cohort) analyzable cases. AR flow cytometry measurements were done on 13 patients. The effect on local failure (LF), distant metastases (DM), prostate cancer survival (PSS), and overall survival (OS) was studied.Pretreatment characteristics and assigned treatment arm were not significantly different between the parent and test groups except for a significantly higher risk of death (P = 0.049) in the test group. The median CAG repeat was 19. There were no significant differences in stage, or Gleason score between high (19 or greater) and low CAG (19) patients within each treatment group. Number of CAG repeats alone did not significantly influence LF, DM, PSS, and OS. However, when the CAG repeat outcome was studied in conjunction with androgen deprivation therapy, patients with CAG19 who received H + RT had improved local control as compared with patients who received RT alone (P = 0.026, 5-year rates 4.6% versus 36.4%) and improved local control over patients with CAGor =19 that received H + RT (P = 0.028).Patients with short CAG repeats show a local control benefit with short-term androgen deprivation therapy, but no improvement in survival.

In DNA histograms of some human solid tumors stained with nuclear isolation medium-4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G0/G1 peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.

The study consisted of 100 patients (97 males and 3 females) suffering from bladder cancer and 76 matching controls. The maximum number of patients in this study was in the age group of 61–70 years. The prevalence of genetic polymorphism in the CYP2D6 , GSTM1 , and GSTT1 genes has been investigated to find their association with risk of bladder cancer. While there was no association between the heterozygous (HEM) genotype of the CYP2D6 gene with the risk of bladder cancer [odds ratio (OR)=1.00; 95% confidence interval (CI)=0.46 – 2.16], it was 1.5-fold with poor metabolizers (PM) genotype. When stratified according to different grades of bladder cancer, a significant association was found with an OR=3.54 (95% CI=0.89 – 13.98) in grade II, 3.3 (95% CI=0.12 – 20.6) in grade III, and 1.67 (95% CI=0.15 – 18.45) in grade IV. When stratified in relation to smoking status, significant association of the disease was found in heavy smokers with an OR=2.13 (95% CI=0.71 – 6.43). Subjects with the null genotype for GSTM1 had a slightly significant association with the bladder cancer risk and the risk increased to 2-fold with the GSTT1 null genotype. Smoking status also revealed an impact on the prevalence of bladder cancer in the individuals with GSTM1 and GSTT1 null genotypes. The results indicated that there is a 3-fold increase in risk of developing this cancer in the presence of one copy of the variant CYP2D6 (HEM) allele and null GSTT1 .

Background Flow cytometry is a rapid and reliable method for measuring nuclear DNA content and genome size. Fluorochrome binding characteristics, sample preparation and differences in DNA condensation, and availability of binding sites can cause variations in results obtained. Methods Blood samples from 82 vertebrate species were collected in 10% dimethyl sulfoxide and stained with propidium iodide/hypotonic citrate or 4,6-diamidino-2-phenylindole dihydrochloride for analysis of DNA content and electronic nuclear volume (ENV). Trout red blood cells (TRBCs), human peripheral blood lymphocytes, and human buccal cavity cells were used as internal standards. Results Mean fluorescence channel (MFC) values of TRBC and buccal cavity cells used as internal standards were stable at 15 to 120 min of propidium iodide staining. TRBCs mixed with other cells especially human peripheral blood cells showed an increase in MFC. ENV and MCF values were less variable in different species of birds than in reptiles or mammals. Genome size based on use of buccal cavity cells as the internal standard showed a high degree of correlation with previous reports. Conclusions Proper selection and use of internal standards and sample preparation are essential for reliable determination of DNA content and genome size in vertebrates by flow cytometry. © 2005 Wiley-Liss, Inc.

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE Quanta for measuring EV and DNA content of different normal mice tissues. Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.

In order to investigate the hypothesis that cells found in hypoxic areas of solid tumors are more sensitive to glycolytic inhibitors than cells growing aerobically, we have previously characterized three distinct in vitro models (A, B and C) that simulate this condition. In all of the models it was shown that cells growing under hypoxic conditions are hypersensitive to the glycolytic inhibitor 2-deoxy- d-glucose (2-DG). However, in those studies cytostatic and cytotoxic effects were not distinguished from one another. Since successful treatment of cancer includes not only slowing down but also actually killing tumor cells, studies were undertaken to assess the effects of 2-DG on cell cycle progression and cell death.Using flow cytometry and cell viability assays, it was found that 2-DG caused significantly greater cell cycle inhibition and cell death in all three hypoxic models as compared to aerobically growing control cells. In model A (a chemically induced model of hypoxia in which rhodamine-123 is used to block oxidative phosphorylation), 1200 microg/ml of 2-DG was shown to induce more cell cycle arrest in late S/G(2) and more cell death than in the aerobic cell counterpart treated with 3600 microg/ml 2-DG. In rho(0) cells which are genetically constructed to be unable to perform oxidative phosphorylation (model B), an even greater window of selectivity (more than tenfold) between hypoxic and aerobic cells was found when considering 2-DG's effects on cell cycle arrest and cell death. In the environmental model (model C), where cells were grown under reduced amounts of external oxygen (0.1%), hypersensitivity to the effects of 2-DG with respect to cell cycle arrest and cell death were also observed.Overall, these results indicate that cells growing under anaerobic conditions respond with greater sensitivity to the effects of 2-DG on cell cycle inhibition and cell death than those growing under aerobic conditions. This supports our contention that glycolytic inhibitors added to standard chemotherapeutic protocols should increase treatment efficacy by selectively killing the slow-growing cells, which are found in the hypoxic portions of solid tumors, while sparing most of the normal cells that are also slow-growing but are living under aerobic conditions.

Pyrazoloacridine (NSC 366140, PD115934, PZA) is a new class of acridine anticancer agents under investigation in Phase II clinical trials in patients with advanced cancers. Although poor responses in patients to the treatment with PZA alone have been observed, this class of agents remains of interest because of its distinct mechanism of action from other topoisomerase poisons. Therefore, the combination of PZA with conventional anticancer agents presents an attractive approach to treat drug-resistant human tumors. In the present study, the cytotoxic effects of PZA combined with doxorubicin, topotecan, and etoposide were determined using paired parental and doxorubicin-resistant human colon carcinoma (SW-620 and SW620/AD-300) and breast cancer cell lines (MCF-7 and MCF-7/TH). Cytotoxicity was measured by soft agar clonogenic assays. Dose effect and combination effects were analyzed by the method of Chou and Talalay. The combination of PZA with doxorubicin, topotecan, and etoposide in fixed ratios demonstrated synergistic cytotoxicity on both SW-620 and SW620/AD-300 cell lines. The combination of PZA with doxorubicin also exhibited synergistic cytotoxicity against both MCF-7 and MCF-7/TH cell lines. The mechanism of synergism appeared independent of topoisomerase I and II inhibition, and interference with protein-DNA complexes. Strategies to define optimal drug combinations are proving to be of significant value when considering potential clinical applications of new and established agents.

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.

Cellular resistance to a broad spectrum of natural products used as antitumor drugs is believed to be a major cause for the failure of chemotherapy. Flow cytometry has been used for monitoring the expression of drug resistance markers, determining accumulation of fluorescent drugs, and for screening of drugs that enhance chemosensitivity by blocking efflux and enhancing drug retention. This article reviews recent developments in our understanding of the multiple drug resistance phenotype and the use of flow cytometry for the study of drug efflux and its modulation in human tumor cells.

Background The NPE Analyzer® flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit.

Hormone receptors play a major role in growth and hormonal therapy of breast and prostate tumors. Quantitative results from the ligand binding assays cannot determine heterogeneity in receptor expression nor can they discriminate between expression of the stromal and the tumor cells. Availability of antibodies to hormone receptors has led to the development of immunohistochemistry as a standard method for monitoring of hormone receptor expression under a microscope. However, this method is based on examination of a small number of cells. Laser flow cytometry has been extensively used for monitoring of receptor expression in human liquid tumors. As most of the hormone receptor expression is nuclear, we have developed methods for flow cytometric analysis of receptor expression in nuclei isolated from enzyme treated paraffin sections. The present report based on gated analysis of androgen receptor expression in nuclei isolated from archival formalin fixed/paraffin embedded breast tumors shows that receptor expression in aneuploid sub-populations is greater than that of the diploid cells.

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.

Background In the present paper, we describe the effect of 4', 6-diamidino-2-phenylindole (DAPI) dihydrochloride concentration and pH on the resolution of DNA distribution histograms generated by dual-parametric simultaneous analysis of DNA content and electronic nuclear volume (ENV). Methods Nuclei from tissue culture cell lines and frozen human solid tumors were isolated in nuclear isolation media containing different concentrations of DAPI, at various pH levels, and analyzed on a NASA/American Cancer Society (ACS) flow cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL flow cytometer were used for comparison. Results Nuclei stained with DAPI concentration of 1–3 μg/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetraploid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under these conditions identified subpopulations that otherwise could not be detected by DNA analysis alone. Conclusions Staining at 1–3 μg/ml DAPI, pH 6.0, was optimal for the detection of aneuploid populations, especially the near-diploid and/or near-tetraploid populations in human tumors. Cytometry 43:12–15, 2001. © 2001 Wiley-Liss, Inc.

Background The NASA/American Cancer Society (ACS) flow cytometer can simultaneously measure electronic nuclear volume (ENV) and DNA content of nuclei. The preceding articles in this volume (“NASA/American Cancer Society High-Resolution Flow Cytometer Project-I”) described the schematics, performance, and procedures used for the preparation of nuclei for analysis on this unit. In the present article, we describe the analysis of selected human tumors using the ratio of ENV/DNA content (nuclear packing efficiency [NPE]). Methods Tumor specimens (frozen) were minced with scalpels and stained with 1–10 μg/ml of 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride at pH 6.0–7.2. Trout erythrocytes were used as internal standards. Data on ENV and DNA content were collected in list mode files. Propidium iodide-stained nuclei, analyzed on a Coulter XL cytometer, were used for comparison. Results Simultaneous measurement of ENV and DNA makes it possible to discriminate between hypodiploid or hyperdiploid tumor cells, as well as to differentiate between near-diploid aneuploid and diploid cells on the basis of their increased ENV. The NPE ratio is a valuable parameter for the detection of small quantities of tumor cells, separating overlapping diploid and aneuploid populations for cell cycle analysis and characterizing the level of differentiation in some tumors. Conclusion NPE analysis provides unique measuring capabilities for the study of human solid tumors by flow cytometry. Cytometry 43:16–22, 2001. © 2001 Wiley-Liss, Inc.

Background: Cellular expression of receptors for the hormones estrogen and progesterone in human mammary tumors is of diagnostic and prognostic value. Ligand binding assays have been replaced by immunohistochemical analysis of receptor expression. However, both of these techniques are slow, and in the ligand-binding assay it is difficult to measure heterogeneity of receptor expression in individual cells. Flow cytometry has been used extensively for monitoring the expression of cellular receptors in hematopoietic tumors but has been of limited value in the analysis of mammary tumors, which are difficult to disaggregate into single cells for flow analysis. Hormone receptors have a predominant nuclear localization, and it is relatively easy to isolate nuclei from paraffin-embedded archival tissues for flow cytometric analysis of receptor expression. Methods: Thick sections from formalin-fixed paraffin-embedded archival mammary tumors were digested by different enzyme solutions for the isolation of single nuclei. Different fixatives were used to compare the results on subsequent staining of the nuclei for estrogen receptor (ER) expression. Double staining with propidium iodide and fluorescein isothiocyanate labeled secondary antibodies for ER expression was used for multiparametric analysis of ER and DNA content. Results: Digestion of paraffin sections with low concentration of pepsin and detergents was ideal for isolation of single nuclei. Fixation with paraformaldehyde in the presence of Triton X-100 improved staining of the cells. Isolated nuclei had enhanced immunoreactivity compared with the whole cells, and subpopulations differing in reactivity could be identified in the nuclear fractions. Double staining of nuclei for ER expression and DNA content could allow for multiparametric analysis of these two important parameters. Conclusions: The procedures described can be used for processing of archival paraffin-embedded mammary tumors for monitoring of ER expression and aneuploidy. These two parameters have important diagnostic and prognostic significance in mammary tumors. Laser flow cytometry by providing multiparametric analysis can allow for correlation of these cellular markers with other important cellular and clinical parameters. Cytometry 36:131–139, 1999. © 1999 Wiley-Liss, Inc.

Flow cytometric analysis of estrogen (ER) and progesterone (PgR) receptor expression in archival human breast tumors is relatively difficult. We have used enzyme digestion and microwave antigen retrieval procedures for multiparametric flow cytometric analysis of ER and PgR expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded primary breast tumors. Deparaffinized rehydrated tissue sections treated with pepsin were subjected to microwave irradiation for unmasking of ER and PgR antigenic sites. Biotinylated ER antibody and streptavidin–fluorescein isothiocyanate (FITC) were used for ER labeling and PgR antibody with phycoerythrin labeled goat anti-mouse antibody was used for PgR labeling. Counter staining with propidium iodide-RNase was used for determination of cellular DNA content. Our results show that enzyme digestion and microwave treatment of formalin-fixed, paraffin-embedded breast tumors can be successfully used for the multiparametric analysis of nuclear hormone receptor expression and DNA content by flow cytometry. Cytometry (Comm. Clin. Cytometry) 38:61–69, 1999. © 1999 Wiley-Liss, Inc.

BACKGROUND PSP94 (prostate secretory protein of 94 aa; also called PIP), one of the predominant proteins secreted into the seminal fluid, was proposed as an independent diagnostic/prognostic marker for prostate cancers. It was also shown to inhibit rat prostate cancer growth. In this study, we investigated the effect of purified PSP94 on the growth of androgen-independent human prostate cancer cells (PC3) and its potential mechanism of action. METHODS AND RESULTS PSP94, in a dose- and time-dependent manner, inhibited the growth of PC3 cells. The protein demonstrated a stronger inhibitory effect on the colony-forming ability of PC3 cells in soft agar. A daily injection of PSP94 at 5 μg/kg/body weight resulted in a 50–60% inhibition in the growth of PC3 xenografts in athymic mice. PC3 cell growth inhibition by PSP94 resulted from cell death characteristic of morphological apoptosis, which was confirmed by dual fluorescence microscopy, electron microscopy, and DNA fragmentation assays. Mechanistic studies indicated that PSP94 enhanced the expression of proapoptotic protein Bax without affecting Bcl-2 levels. CONCLUSIONS This study suggests that PSP94 may represent a novel, apoptosis-based, antitumor agent applicable to the treatment of hormone-refractory human prostate cancers. Prostate 38:118–125, 1999. © 1999 Wiley-Liss, Inc.

The discordance between P-glycoprotein (P-gp) expression and functionality [as measured by the efflux of doxorubicin (DOX)] was analyzed in a DOX-sensitive human breast cancer cell line (HTB-123) with high reactivity against four P-gp specific monoclonal antibodies (C219, MRK-16, UIC2, and 4E3). Reverse transcription–polymerase chain reaction (RT–PCR) and Western blotting analyses confirmed the overexpression of MDR1 mRNA and P-gp in this cell line. However, incubation of cells with efflux blockers, verapamil (VPL) or dipyridamole (DPD), did not enhance cellular (DOX) accumulation or cytotoxicity. Upon incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA), HTB-123 cells retained less DOX than control cells and were sensitive to the efflux blockers verapamil or dipyridamole. These observations suggest that 12-O-tetradecanoylphorbol-13-acetate-induced P-gp phosphorylation may be associated with induction of P-gp-mediated drug efflux in the HTB-123 cell line.

Multiple drug resistance (MDR) in tumor cells has been related to the expression of transport proteins which alter cellular drug transport and distribution. Three different genes (mdr, MRP, and LRP) and their products have been implicated in MDR. Several fluorescent dyes have been used to monitor the effect of these transport proteins on drug retention, as well as for screening of drugs which block drug efflux and thus enhance cellular drug retention and cytotoxicity. The present review summarizes current knowledge about MDR phenotypic markers and techniques available for study of MDR by flow cytometry.

Twenty-two fresh surgical specimens of human sarcomas (soft tissue and bone) from 20 patients were analyzed by flow cytometry for the expression of drug resistance-related P-glycoprotein (P-gp) and cellular daunorubicin (DNR) accumulation with or without the presence of DNR efflux blockers. Single-cell suspensions prepared from the tumor specimens were analyzed by dual-color flow cytometry after reaction with MRK-16 (anti-P-gp) and anti-CD45 (pan-leukocyte) antibodies. MRK-16 reactivity of tumor cells was evaluated after exclusion of CD45-positive cells by electronic gates. Parallel samples were incubated with DNR alone or in combination with DNR efflux blockers, verapamil (VPL), or dipyridamole (DPD) for determination of cellular DNR accumulation and the effect of the efflux blockers. Extensive heterogeneity was observed in both P-gp expression and DNR accumulation of the tumor specimens examined. Eight of the 22 tumor specimens had significant numbers of P-gp-positive cells. In three of the eight P-gp-positive tumors, cellular DNR accumulation was significantly increased by co-incubation with the efflux blockers VPL or DPD. These results indicate that both quantitative and functional analysis of P-gp expression may be essential in determining the cellular drug resistance phenotype of tumor cells and its correlation with therapeutic outcome.

Laser flow cytometry is increasingly used for quantitation of cellular fluorescent drug retention, effect of efflux blockers and for expression of drug resistance related cellular surface markers. Several intrinsic and extrinsic factors can affect the results obtained from drug retention functional assays and lead to artifacts. In the present study, we have used a panel of well-characterized parental and drug resistant cell lines, fluorochromes and efflux blockers to identify the possible sources of artefacts in flow cytometric analysis of the multiple drug resistance phenotype.

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 μg/d; and a 18.4% reduction with 50 μg/d ( P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κβ/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes ( P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.

Rapid cellular efflux of certain natural products used in cancer chemotherapy leads to reduced cytotoxicity and resistance. Multiple Drug Resistance (MDR) gene related glycoproteins are believed to act as the drug efflux pump. Several non-cancer therapy drugs (e.g., verapamil, phenothiazines) compete for the p-glycoprotein pump and thus can block efflux of a chemotherapeutic agent and overcome cellular resistance. Anthracyclines, such as adriamycin, are intrinsically fluorescent and thus their cellular retention can be determined by laser flow cytometry. This method has been used to study heterogeneity in cellular retention of anthracyclines and response to efflux blockers in human tumor cells. These studies have led to generation of clinical protocols where laser flow cytometry is used to monitor and overcome heterogeneity in drug retention of tumor cells from patients treated with a combination of adriamycin and efflux blockers. © 1995 Wiley-Liss, Inc.

The expression of drug resistance-associated mdr-1, GST pi, and topoisomerase II genes was analyzed in cell cycle phase enriched populations of doxorubicin-resistant murine leukemic P388/R-84 cells. Flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation and staining with anti-BrdU antibodies was used to confirm the purity of cell cycle phase enriched populations obtained by centrifugal elutriation. Doxorubicin (DOX) and daunorubicin (DNR) accumulation was significantly lower in S-phase cells, and coincubation with verapamil (VPL) or chlorpromazine (CPZ) enhanced DOX and DNR accumulation more in S-phase than in G1- and G2/M-phase cells. While the cellular content of mdr-1 and topoisomerase II mRNAs changed, GST pi mRNA content remained constant during the cell cycle. S-phase cells had about 3-fold higher mdr-1 mRNA content than G1- and G2/M-phase cells. In G1 cells, P-glycoprotein expression, as determined by C219 monoclonal antibody, was 12% less than that of S and G2/M cells. Topoisomerase II mRNA content increased with the progression of cell cycle and peaked in G2/M cells. These observations suggest that cell cycle stage related changes in expression of drug resistance markers may have a major bearing on chemosensitivity of drug-resistant cells.

BACKGROUND Benign prostatic hyperplasia (BPH) affects aging men. Combined therapy with antagonists of growth hormone-releasing hormone (GHRH) and of luteinizing hormone-releasing hormone (LHRH or GnRH) induces prostate shrinkage in rat models. We investigated the mechanisms of action of this combination on cell cycle traverse and expression of prostatic genes. METHODS Effects of GHRH antagonist, JMR-132 (40 µg/day), the LHRH antagonist, cetrorelix (0.625 mg/kg), and their combination were evaluated on testosterone-induced benign prostatic hyperplasia in male Wistar rats. Influence of JMR-132, cetrorelix, and their combinations on cell viability was assessed by MTS assay in BPH-1 human prostate epithelial cells and WPMY-1 normal prostate stromal cells. Cell cycle was analyzed by laser flow cytometry. Real-time PCR arrays were performed. RESULTS The combination of antagonists caused marked shrinkage of rat prostate (29.5%). In vitro, JMR-132 plus cetrorelix (both 5µM) produced synergistic (57.4%) inhibition of growth of BPH-1 cells, but a lesser inhibition (46%) of WPMY-1 cells. Co-treatment of with JMR-132 plus cetrorelix induced a significant increase of BPH-1 cells blocked in S-phase plus cells with lower G0/G1 and G2/M DNA content. Significant changes in expression of >40 gene transcripts related to growth factors, inflammatory cytokines, and signal transduction were identified. CONCLUSIONS GHRH antagonist and LHRH antagonist combination potentiates rat prostate weight reduction and synergistically inhibits of growth of BPH-1 leading to cell cycle arrest in S-phase. These effects were lesser in normal stromal prostate cell line, WPMY-1. Our findings suggest that GHRH antagonists could be useful for BPH therapy, possibly in combination with LHRH antagonists. Prostate 73: 873–883, 2013. © 2012 Wiley Periodicals, Inc.

Eight cell lines were established from the pleural effusion of 4 patients with malignant mesothelioma. The most sensitive (FCCMES-4) and the most resistant (FCCMES-2) mesothelioma cell lines had IC50 of 0.66 and 1.85 μM for doxorubicin in clonogenic assays, respectively. In comparison with murine leukemic P388 cells, mesothelioma cell lines were 7.5- to 21 -fold more resistant to doxorubicin. Co-incubation with verapamil significantly increased doxorubicin retention in one of the cell lines (FCCMES-2) expressing P-glycoprotein in 16.8% of the cells. These results indicate that doxorubicin resistance may be intrinsic in refractory mesothelioma patients and P-glycoprotein-mediated drug efflux may be involved in resistance of some of the mesotheliomas. © 1994 Wiley-Liss, Inc.

We have analyzed five human melanoma cell lines, displaying variable doxorubicin resistance (1- to 6-fold), for drug-induced DNA breaks, topoisomerase II activity and mRNA expression. Enhanced drug efflux was not the reason for doxorubicin resistance of these tumor cells although they overexpressed the transmembrane 170 kDa P-glycoprotein. Doxorubicin-induced DNA lesions (2-fold) and topoisomerase II activity (7-fold) were higher in HM-1 and G361 cells than in the less doxorubicin-sensitive NH and FCCM-9 cells. Topoisomerase II mRNA expression was also 2-fold higher in HM-1 and G361 cells. Doxorubicin-induced DNA breaks and topoisomerase II activity inversely correlated with the degree of doxorubicin sensitivity. Southern blot analysis showed variation in the hybridization pattern of topoisomerase II gene in doxorubicin-resistant cells when compared to sensitive cells. This study portrays the low doxorubicin sensitivity of NH and FCCM-9 cells as “atypical” and emphasizes the importance of DNA damage and topoisomerase II activity in cellular low doxorubicin resistance.

Cellular drug resistance is believed to involve P-glycoprotein-related drug efflux as well as xenobiotic detoxification. In the present study, we analyzed five human melanoma cell lines with 1- to 6-fold doxorubicin resistance for doxorubicin retention and MDR-1 and GST π gene expression. All the cell lines had high doxorubicin retention, and efflux blockers such as trifluoperazine and verapamil did not have a major effect on drug retention or cytotoxicity. Even though all the cell lines carried the MDR-1 and GST π genes, gene amplification was not associated with drug resistance. Both laser flow cytometry and immunoperoxidase staining showed high expression of C-219 reactive P-glycoprotein in some of the resistant cells which was not accompanied by either high drug efflux or sensitivity to doxorubicin efflux blockers.

Doxorubicin (DOX) efflux in drug-resistant cells is blocked by phenothiazines such as trifluoperazine (TFP) and prochlorperazine (PCZ) in vitro. The present phase I study was conducted in 13 patients with advanced, incurable, nonhematologic tumors to determine whether PCZ plasma levels high enough to block DOX efflux could be achieved in vivo. The treatment schedule consisted of prehydration and i.v. administration of 15, 30, 50, and 75 mg/m2 PCZ followed by a standard dose of 60 mg/m2 DOX. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose used. Toxicities attributable to PCZ were sedation, dryness of the mouth, cramps, chills, and restlessness. The maximal tolerated dose (MTD) of PCZ in this schedule was 75 mg/m2. Pharmacokinetic analysis indicated a large interpatient variation in peak plasma PCZ levels that ranged from 95 to 1100 ng/ml. The three plasma half-lives of PCZ were: t1/2 alpha (+/- SE), 20.9 +/- 5.3 min; t1/2 beta, 1.8 +/- 0.3 h; and t1/2 gamma, 21.9 +/- 5.3 h. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) for PCZ were 2254 +/- 886 l/m2, 60.2 +/- 13.5 l m-2 h-1, and 1624 +/- 686 ng ml-1 h, respectively. DOX retention in tumor cells retrieved from patients during the course of therapy indicated the appearance of cells with enhanced DOX retention. The combination of DOX and high-dose i.v. PCZ appeared to be safe, well tolerated, and active in non-small-cell lung carcinoma.

Lung-tumor cells from pleural effusion of four refractory patients and in cell lines established from them were analyzed for anthracycline retention, cytotoxicity, and MDR-1 gene and P-glycoprotein expression. Murine leukemic P388 and doxorubicin-resistant P388/R84 lines were used as controls. The 50% growth-inhibitory concentration (IC50) for doxorubicin among lung-tumor lines varied from 0.16 to 0.31 microM in soft agar. Heterogeneity in doxorubicin or daunorubicin retention and response to the efflux-blocking action of 25 microM prochlorperazine was noted in pleural effusion of FCCL-1, -4, and -8. Among the cell lines established, an efflux-blocking effect in a subpopulation was noticed only in FCCL-1 and -4. Although the MDR-1 gene was present in all cell lines, including P388, its expression was pronounced only in P388/R84 and FCCL-1. In situ hybridization of antisense RNA probe to tumor cells showed high heterogeneity for MDR-1 message in the human lung-tumor cells as compared with the murine cells. Northern and slot blot hybridization confirmed in situ hybridization in lines with high levels of MDR-1 expression. The synthesis of MDR-1 mRNA and P-glycoprotein in tumor lines was correlated. The results suggest that because of extensive tumor-cell heterogeneity in human tumors, monitoring of MDR expression by in situ hybridization, quantitation of P-glycoprotein content by laser flow cytometry (and/or immunohistochemical methods), and drug efflux (by laser flow cytometry) may be the best ways to monitor multidrug resistance in human tumors.

CONTRIBUTORS. PREFACE. 1 Basics of Flow Cytometry (H. Krishnamurthy and L. Scott Cram). 2 Practical Considerations for Flow Cytometric Sorting of Stem Cells (Geoffrey W. Osborne). 3 Stem Cell Analysis and Sorting Using Side Population Analysis (William Telford). 4 Flow Cytometry in the Study of Proliferation and Apoptosis (Michael G. Ormerod and Ronald M. Hamelik). 5 Flow Cytometric Analysis of Drug Transport and Efflux in Stem Cells (Awtar Krishan and Ronald M. Hamelik). 6 Stem Cell Biology and Application (Swapnil Totey, Rajarshi Pal, Murali Krishna Mamidi, Vijayendran Govindasamy, and Satish Totey). 7 Identification and Isolation of Very Small Embryonic-like Stem Cells from Murine and Human Specimens (Ewa K. Zuba-Surma, Dong-Myung Shin, Izabella Klich, Janina Ratajczak, Magda Kucia, and Mariusz Z. Ratajczak). 8 Electronic Volume of Hematopoietic Stem Cells (Siddharth Sharma and Awtar Krishan0. 9 Hematopoietic Stem Cells: Issues in Enumeration (Michael Keeney and D. Robert Sutherland). 10 Embryonic Stem Cells: Development and Characterization (Vijay Bhaskar R. Konala, Villoo Morawala-Patell, and Aparna Khanna). 11 Human Embryonic Stem Cells: Long-Term Culture and Cardiovascular Differentiation (Maneesha Inamdar). 12 Mesenchymal Stromal Cells and Their Clinical Applications (Jyoti Kode and Vivek Tanavde). 13 Circulating Adult Stem Cells of Hematopoietic Origin for Vascular and Neural Regeneration (Lissy K. Krishnan). 14 Flow Cytometric Characterization of Neural Progenitors Derived from Human Pluripotent Stem Cells (Raj R. Rao, Sujoy K. Dhara, Shilpa Iyer, and David W. Machacek). 15 Limbal Stem Cells and Corneal Regeneration (Geeta K. Vemuganti, Murali Mohan Sagar Balla, and Shubha Tiwari). 16 Flow Cytometric Sorting of Spermatogonial Stem Cells (B. S. Srinag, J. M. Kalappurakkal, G.H. Mohan, and H. Krishnamurthy). 17 Breast Cancer Stem Cells (Devaveena Dey and Annapoorni Rangarajan). 18 Tumor Stem Cell Marker Expression in Cells from Body Cavity Fluids (Awtar Krishan, Deepti Sharma, and Ronald M. Hamelik). INDEX.

The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G(1) peak) and shorter processing time by eliminating the fixation and permeabilization steps.

Background: Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers (CD44+/CD24−) in stem cells from breast tumors has been reported. This study was undertaken to monitor expression of these markers in cells from body cavity fluids of female patients suspected to have a malignancy. Methods: Cells from peritoneal and pleural fluids of 100 female patients were examined by diagnostic cytology and analyzed by laser flow cytometry for enhanced ALDH1 expression. Cells from 36 body cavity fluids with ALDH1bright fluorescence were then analyzed for the expression of CD44 and CD24 markers. Results: In samples positive for malignancy, ALDH1bright cells with both SSClow and SSChigh were seen. In 15 body cavity fluids positive for malignancy, the percentage of ALDH1bright cells ranged from 0.26 to 6.34% of the total cells. The percentage of ALDH1bright cells with CD44+/CD24− expression in these samples ranged from 0.02 to 3.66%. ALDH1bright cells with CD44+/CD24− expression were also present in body cavity fluids of patients in whom diagnostic cytology could not detect any malignancy. However, the percentage of ALDH1bright and CD44+/CD24− cells amongst the 21 body cavity fluids with negative cytology was lower than that of samples with malignancy. Conclusions: Expression of ALDH1bright and the CD44+/CD24− phenotype in body cavity fluids in which diagnostic cytology could not find any malignant cells suggests that this phenotype may not be restricted to the putative breast tumor stem cells. It is possible that only subsets of cells with this phenotype are the putative breast tumor stem cells. © 2010 Clinical Cytometry Society