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The alpha-folate receptor (alpha-FR) is a folate transporter with restricted expression levels in normal tissues. It is over-expressed in several cancers, particularly epithelial carcinomas, including nonmucinous ovarian carcinoma. It offers a novel therapeutic target for selective imaging and cytotoxic agents. Measurement of the receptor could be a valuable tool in selecting patients more likely to respond to new drugs that target the alpha-FR, and monitoring them while on treatment. While tumor samples are often unavailable, a number of patients who relapse develop ascites, which are often rich in tumor cells. We have therefore developed a triple antibody flow cytometric method to assess alpha-FR expression on tumor cells from ascites. An antibody to BerEP4, an epithelial cell marker expressed on >90% ovarian cancers, labeled with fluorescein, and an alpha-FR antibody labeled with antimouse-phycoerythrin have been used to label tumor cells, with a CD45-phycoerythrin-cyanine5 antibody used to exclude white blood cells from the analysis. The method was optimized using human carcinoma cell lines (JEG-3, IGROV-1, and KB cells). Calibrated beads were used to quantify the number of antibodies bound per cell. The triple antibody protocol successfully measured alpha-FR expression levels in cell lines spiked with blood. Tumor cells were obtained from ascites in 25 patients with relapsed ovarian cancer. In each case sufficient cells were harvested to identify an epithelial cell population to estimate the number of binding sites/cell. All the samples contained a single population of BerEP4, alpha-FR positive cells between 5x10(3) and 5x10(5) antibody binding sites/cell. The method can be used to determine the number of anti-alpha-FR antibodies bound per epithelial cell in ascites from patients with ovarian carcinoma. The results obtained were reproducible and the method could be applied to specimens that had been stored at -80 degrees C., (Copyright (c) 2007 International Society for Analytical Cytology.)

Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway is a mechanism of cytotoxic drug resistance in ovarian cancer, and inhibitors of this pathway can sensitize to cytotoxic drugs. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) depletes some proteins involved in PI3K/AKT signaling, e.g., ERBB2, epidermal growth factor receptor (EGFR), and phosphorylated AKT (p-AKT). 17-AAG and paclitaxel were combined (at a fixed 1:1 ratio of their IC(50)) in four ovarian cancer cell lines that differ in expression of p-AKT, EGFR, and ERBB2. The EGFR-overexpressing A431 and KB epidermoid cell lines were also included. Combination indices (CI) were calculated using the median-effect equation and interpreted in the context of 17-AAG-mediated inhibition of PI3K signaling. Synergy was observed in IGROV-1- and ERBB2-overexpressing SKOV-3 ovarian cancer cells that express a high level of constitutively activated p-AKT [CI at fraction unaffected (fu)(0.5) = 0.50 and 0.53, respectively]. Slight synergy was observed in A431 cells (moderate p-AKT/overexpressed EGFR; CI at fu(0.5) = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu(50) = 3.0, 3.5, and 2.0, respectively). The observed effects correlated with changes in the rate of apoptosis induction. 17-AAG induced a decrease in HSP90 client proteins (e.g., C-RAF, ERBB2, and p-AKT) or in downstream markers of their activity (e.g., phosphorylated extracellular signal-regulated kinase or p-AKT) in SKOV-3, IGROV-1, and CH1 cells at IC(50) concentrations. A non-growth-inhibitory concentration (6 nmol/L) reduced the phosphorylation of AKT (but not extracellular signal-regulated kinase) and sensitized SKOV-3 cells to paclitaxel. In conclusion, 17-AAG may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by ERBB2 and/or p-AKT.

This study compares the sensitivities and specificities of three techniques for the detection of circulating epithelial cells in the blood of patients with breast cancer. The number of circulating epithelial cells present in the blood of 40 patients with metastatic breast cancer and 20 healthy volunteers was determined by: immunomagnetic separation (IMS) and laser scanning cytometry (LSC), cell filtration and LSC and a multimarker real-time RT-PCR assay. Numbers of cytokeratin-positive cells identified and expression of three PCR markers were significantly higher in the blood of patients with breast cancer than in healthy volunteers. Using the upper 95% confidence interval of cells detected in controls to determine positive patient samples: 30% of patients with metastatic breast cancer were positive following cell filtration, 48% following IMS, and 60, 45 and 35% using real-time RT-PCR for cytokeratin 19, mammaglobin and prolactin-inducible peptide. Samples were significantly more likely to be positive for at least one PCR marker than by cell filtration (83 vs 30%, P<0.001) or IMS (83 vs 48%, P<0.001). The use of a multimarker real-time RT-PCR assay was therefore found to be the most sensitive technique for the detection of circulating epithelial cells in the blood of patients with breast cancer.

Background: Changes in mitochondrial structure and size are observed in response to alterations in cell physiology. Flow cytometry provides a useful tool to study these changes in intact cells. We have used flow cytometry and digital fluorescence microscopy to analyze the variations in mitochondrial size in relation to specific phases of the cell cycle., Methods: Supravital staining of rat fibroblasts was done with Hoechst 33342 and rhodamine 123, and cells were analyzed in a dual-laser flow cytometer. Synchronized cells at various stages of the cell cycle were analyzed for changes in mitochondrial size. These cells were also examined by electron microscopy, digital fluorescence microscopy and computerized image analysis to compare the lengths of the mitochondria., Results: By using fluorescence pulse width analysis, we observed two populations of mitochondria in intact cells. The percentage of cells with small and large mitochondria at specific stages of the cell cycle indicated that mitochondrial size increases during the cell cycle; early G1 phase cells had the smallest mitochondria and the mitotic phase cells had the largest mitochondria. These results were confirmed by microscopic analysis of cells., Conclusions: Flow cytometry can distinguish the relative mitochondrial size in intact cells, and in combination with digital microscopy it can be used to study mitochondrial variation during the cell cycle., (2004 Wiley-Liss, Inc.)

Cells are incubated continuously in bromodeoxyuridine (BrdUrd), which is incorporated into cells synthesizing DNA. At intervals, cells are harvested and nuclei are prepared and stained with a bis-benzimidazole, Hoechst 33258, and propidium iodide. In the flow cytometer, the dyes are excited by UV and blue and red fluorescences recorded. BrdUrd quenches the blue fluorescence of the Hoechst dye. The degree of quenching records the progress of the cell through S phase(s); the red (PI) fluorescence yields the cell cycle phases. By this means, the progress of cells around the cell cycle can be followed and the effects of cytotoxic drugs, radiation, and other treatments observed.

Background: A variety of markers, including Ki67, estrogen receptors (ER), and progesterone receptors (PgR), are frequently measured in fine needle aspirates from human breast carcinomas. Previously, we demonstrated the use of laser scanning cytometry (LSC) for the measurement of Ki67, ER, and PgR in a human breast carcinoma cell line, MCF7 (21). In the present study, we investigated the expression of Ki67 in breast tumour fine needle aspirates (FNAs) using LSC and compared the results to the data obtained using conventional immunocytochemistry (ICC)., Methods: A total of 11 sets of cytospins of FNAs taken from breast tumours at various stages of tamoxifen treatment were used. For LSC, the cytospins were stained for Ki67 with fluorescein using immunofluorescence; the nuclei were counterstained with propidium iodide. A parallel set of cytospins was stained using horseradish peroxidase and diaminobenzidine and scored manually by conventional light microscopy., Results: Values for Ki67 obtained using the LSC were generally lower than ICC scores. The changes in Ki67 measured by the LSC were almost all parallel to those obtained by manual scoring of immunocytochemical stains., Conclusions: It should be possible to use LSC for the routine measurement of Ki67 marker in FNAs from human breast carcinomas., (Copyright 2003 Wiley-Liss, Inc.)

Background: Epithelial cells may be detected in the circulation of the majority of patients with metastatic breast cancer. Quantification of such presumptive cancer cells might allow for the monitoring of patients with early or late stage disease as an early index of relapse. Additionally, biomarker analysis may allow a more rational approach to therapeutics. We have developed a new method for the detection and characterisation of these cells., Methods: Blood was filtered through polycarbonate membranes containing cylindrical pores, 8 microm in diameter. All the red cells and a large majority of the white blood cells passed through the filter while the larger epithelial cells were trapped. Cells on the membrane were fixed in ethanol, stained with propidium iodide and anti-pan-cytokeratin-FITC (to identify epithelial cells). The filters were then examined by laser scanning cytometry (LSC), which allowed enumeration and localisation of cells., Results: With normal blood spiked with cells from breast carcinoma cell lines, 99.9% of the leukocytes passed through the membrane, while close to 100% of the epithelial cells were trapped, with a detection limit of less than one epithelial cell/ml of blood. All of 20 samples from patients with widespread metastatic disease contained cytokeratin-positive cells with the morphological characteristics of carcinoma cells, the number of cells ranging from 0.2 to 5.7/ml of blood., Conclusions: Cell filtration-LSC is a viable technique for detecting and studying breast carcinoma cells in peripheral blood., (Copyright 2003 Wiley-Liss, Inc.)

Methods for using flow cytometry to investigate the relationship between the induction of apoptosis and the cell cycle are discussed. Methods for following cell cycle progression are also briefly reviewed. The methods are illustrated using a specific example of the effect of withdrawing an essential growth factor from a cell line.

Apoptotic cells were originally recognized by their characteristic morphology. Since then, a series of biochemical changes have been described. However, it has yet to be established whether any of these changes unequivocally identify an apoptotic cell. In any study of apoptosis, it is important that the presence, or absence, of apoptotic cells is confirmed by morphological examination.

Flow cytometry has been extensively used to follow the apoptotic cascade and to enumerate apoptotic cells, both in cell cultures and, to a lesser extent, in tissue biopsies. An overview of the apoptotic cascade and how flow cytometric measurements can be used to observe the different elements of this process is presented.

Background: A variety of markers, including Ki67, estrogen receptors (ER), and progesterone receptors (PgR), are frequently measured in fine needle aspirates (FNA) from human breast carcinomas. We used a human breast carcinoma cell line, MCF7, as a model system to investigate the use of laser scanning cytometry (LSC) for the measurement of these markers. Additionally, we measured the number of apoptotic cells., Methods: Cells were treated with drugs to vary the expression of markers and the number of apoptotic cells. They were then fixed on microscope slides. For LSC, the cells were stained for the different markers with fluorescein using immunofluorescence and for apoptotic cells using the TUNEL assay. The nuclei were counterstained with propidium iodide. A parallel set of slides was stained using horseradish peroxidase and diaminobenzidine and scored manually by conventional light microscopy., Results: The results from the LSC closely paralleled those obtained by manual scoring of immunohistochemical stains., Conclusions: It should be possible to use LSC for the routine measurement of nuclear markers in FNAs from human breast carcinomas., (Copyright 2000 Wiley-Liss, Inc.)

Three lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC50 values of 508, 250 and 156 nM respectively. CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1-100 nM for several mouse and human cell types). CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 (W1L2:R865; > 200-fold), which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4-48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action.

Aim: To quantify the changes in biological molecular markers during primary medical treatment in patients with operable breast cancer and to assess their possible relationship with response to treatment., Methods: The treatment group consisted of 31 patients with operable breast carcinomas, median age 57 years (range 41-67), treated with four 3-weekly cycles of chemotherapy with Mitoxantrone, methotrexate (+/- mitomycin C), and tamoxifen before surgery. Fine needle aspiration (FNA) was used to obtain samples from patients prior to and at 10 or 21 days post-treatment. The following molecular markers were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Bcl-2, and Ki67 measured by immunocytochemistry, and ploidy and S-phase fraction (SPF) by flow cytometry. To evaluate the reproducibility of the technique, repeat FNA was performed in a separate non-treatment control group of 20 patients and the same molecular markers assessed, two weeks after the first sample with no intervening treatment., Results: The non-treatment control group showed a high reproducibility for the measurement of molecular markers from repeat FNA. In the treatment group there was a non-significant reduction in SPF and a significant reduction (p = 0.005) in Ki67. Patients who responded to neoadjuvant therapy were more likely to have a reduction in these two markers than those who failed to respond. Similarly, a reduction in ER scores was observed between the first and second samples (p = 0.04). For PgR, the change between the first and second samples was not significant although there was a significant difference between responders and non-responders (p = 0.03). All nine patients with an increase in PgR were responders. No significant changes in p53 or Bcl-2 were observed during treatment., Conclusion: Molecular markers can be adequately measured from FNA samples prior to and during neoadjuvant therapy. Changes in cellular proliferation and hormone receptors have been shown that may be related to tumour response. These relationships should be assessed in a larger cohort of patients.

CB30865 (p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+ ++)-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide) is a quinazoline-based pyridine-containing compound that emerged from a programme aimed at the development of thymidylate synthase (TS) inhibitors as anticancer agents. Its structure is based on the antifolate ICI 198583, but with a pyridine ring replacing the glutamate. Despite its structure, CB30865 does not act in vitro via inhibition of TS or, apparently, other known folate-dependent pathways, and extensive mechanistic studies suggest that it acts via a novel locus with respect to conventional antitumour agents. However, CB30865 is highly potent against a variety of human tumour cell lines (e.g., 50%-inhibitory concentration [IC50] values in the 1-10 nM range). Thus, the cell cycle effects of CB30865 were investigated. DNA histogram analysis of W1L2 human lymphoblastoid, L1210 murine leukaemia, and CH1 human ovarian cells (propidium iodide staining) has demonstrated that CB30865 does not cause a phase-specific arrest at concentrations that have been shown to inhibit colony formation. This is unexpected for an anticancer agent. In W1L2 cells, using bromodeoxyuridine (BrdUrd) labelling and bivariate Hoechst/ propidium iodide staining, it was revealed that 0.003-0.15 microM CB30865 (1-50 x 72 h IC50) caused cells to arrest in all phases of the cell cycle simultaneously after 20-24 h exposure. This effect was also observed in CH1 and L1210 cells, though the arrest was at slightly different times. Thus, using this technique, it has been demonstrated that CB30865 induces an unusual and delayed cell cycle arrest, which provides further evidence for a novel locus of action for this compound.

Numerous recent studies have indicated the central role of apoptosis as a determinant of the growth abnormalities occurring with malignancy and of the effectiveness of a wide range of therapeutic manoeuvres in cancer treatment. However, there has been a relative paucity of studies measuring apoptosis in human solid tumours, because of the low incidence of apoptotic cells, the difficulty of identifying cells undergoing apoptosis, and the ethical and practical restrictions on obtaining repeat biopsies from patients during therapy. Fine-needle aspirates (FNAs) may be obtained from breast carcinomas as a minimally invasive technique allowing repeat sampling. We describe an approach in which the in situ end labelling (TUNEL) assay is applied to cells in FNAs prior to their analysis by flow cytometry, which allows many thousands of cells to be analysed automatically by objective criteria. Cells that were discriminated as apoptotic on flow cytometric analysis were sorted onto microscope slides and found to show nuclear morphology typical of apoptotic cells. A statistically significant relationship was found between the flow cytometric analysis and the conventional application of TUNEL on histological sections (P = 0.03). Repeat analyses of FNAs from 12 carcinomas showed a median 2.05% apoptotic cells and an overall coefficient of variation of 34.9%. Of the total variability in 12 tumours, 80% was attributed to variation between tumours, 12% between batches, and 8% was random. Thus, this technique should be able to detect the major differences in the percentage of apoptotic cells that occur between different tumours (range 0.3-11.3% by flow cytometry) and between different phases of treatment, and should provide a useful tool for further research on this process in solid tumours.

Aim: To determine the effects of tamoxifen on the levels of hormone receptors and proliferation markers in the early phase of treatment and the relationship of the changes with tumor response in patients with primary breast cancer., Methods: Twenty-one women with primary, operable breast carcinomas were treated with tamoxifen 20 mg daily. Fine needle aspiration (FNA) was used to obtain samples prior to the start and at 14 days and 8-weeks post-treatment. From these samples estrogen receptor (ER), progesterone receptor (PgR), and Ki67 levels were determined using immunocytochemistry and ploidy and S-phase fraction (SPF) using flow cytometry. Tumor response was measured clinically according to UICC criteria., Results: There were 12 responders (2 CR, 10 PR) and 9 non-responders (2 NC, 7 PD). Responders were more likely to be ER+ (p = 0.002), PgR+ (p = 0.006), and low SPF (p = 0.06). At 14 days post-tamoxifen, the median decrease in Ki67 (% cells staining) for responders was -4.8 and for non-responders -0.15 (p = 0.005). This decrease was seen predominantly in ER+ tumours. The difference in SPF was not significant. A decrease in ER was seen in 3/15 patients all of whom were responders. A rise in PgR was seen in 7/17 patients and all but one were responders. Similar changes for ER and PgR were seen at 8-weeks post-tamoxifen, although the reductions in Ki67 and SPF at that time point were not related to response., Conclusion: We have observed a decrease in Ki67 and ER and a rise in PgR after 14 days of treatment with tamoxifen that was related to subsequent response. This is the first study in which an early decrease in a proliferation marker has been shown to relate to subsequent clinical response.

Guidelines are given to assist the standardisation of DNA flow cytometry in clinical pathology. They have been agreed by a group of twelve scientists from nine European countries.

A staining artefact associated with the terminal deoxynucleotidyl assay for apoptotic cells is described. the Artefact is caused by particulate matter in the reconstituted dried milk used in the washing buffer. We recommend filtering the washing buffer before use.

This study was undertaken to evaluate our ability to detect multiple molecular markers of prognosis and response to treatment in fine needle aspirates (FNA) from patients with primary breast carcinomas. 147 patients with operable primary breast carcinomas who had been recruited to a randomized trial of primary medical therapy (PMT) versus adjuvant chemoendocrine therapy were analysed. FNAs were taken prior to therapy and from this multiple slides were produced using cytospin cytocentrifugation and stored at -80 degrees C for subsequent immunocytochemical analysis (ICA). ICA was performed for oestrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, and Bcl-2. Part of the aspirate was snap frozen and used for flow cytometric analysis of ploidy and S-phase fraction (SPF). In a subgroup of 50 patients who had surgery prior to systemic therapy, as well as FNAs, sections were also taken from paraffin-embedded blocks and stained by ICA for ER, PgR and p53 for validation. In these patients ER was additionally measured by enzyme immunoassay (EIA) from frozen tissue taken at surgery. ER, PgR, p53, Bcl-2, and Ki67 were successfully detected by ICA while ploidy and SPF were successfully measured by flow cytometry from FNA material. The percentage positive values obtained were reasonable and as follows: 74% for ER, 70% for PgR, 36% for p53, 80% for Bcl-2,68% of tumours were aneuploid and 32% diploid. Significant relationships between these measurements were observed in accordance with expectations. The concordance for ER, PgR, and p53 from FNA when compared to ICA of matching histological sections was 91.5%, 75.5%, and 75% respectively. For ER the concordance between measurement by ICA of cytological and histological samples and by EIA of frozen tissue was 82.5% and 84% respectively. These results indicate that multiple molecular markers can be adequately tested on cytological preparations from primary breast tumours. These markers can be used to determine prognosis and predict response to PMT.

Our aim was to determine whether biological molecular markers can predict response to neoadjuvant chemoendocrine therapy in patients with early breast cancer. Ninety patients (median age 56 years; range, 28-69 years) with primary operable breast carcinoma were studied. They were treated with four 3-weekly cycles of chemotherapy with mitozantrone, methotrexate (+/- mitomycin C), and tamoxifen prior to surgery. Fine-needle aspiration was used to obtain samples from patients prior to therapy, and the following parameters were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, Bcl-2, and c-erbB-2 measured by immunocytochemistry, and ploidy and S-phase fraction (SPF) by flow cytometry. The tumors of 78% of the subjects responded (complete response, 9%; partial response, 69%) and 22% did not (no change, 20%; progressive disease, 2%). Response rates according to disease stage and patient age were as follows: T1, 74%; T2, 79%; T3/T4, 78%; age 50, 79% (P = not significant). Response rates for other parameters were as follows: ER-positive, 82%, and -negative, 70%; PgR-positive, 86%, and -negative, 71%; p53-positive, 74%, and -negative, 81%; Bcl-2-positive, 85%, and -negative 61%; c-erbB-2-positive, 57%, and -negative, 93%; Ki67 high, 77%, and low, 81%; SPF high, 77%, and low, 77%; aneuploid, 71%; and diploid, 85%. Only the difference for c-erbB-2 was statistically significant (P = 0.007). A trend for higher response rates to neoadjuvant chemoendocrine therapy for tumors that were positive for ER, PgR, and Bcl-2 was observed but did not reach statistical significance. Tumors negative for c-erbB-2 had a higher response rate, which was statistically significant. In contrast, Ki67, ploidy, SPF, and p53 failed to predict for response.

A flow cytometric technique utilizing the continuous incorporation of bromodeoxyuridine (BrdU) into asynchronous cells to measure radiation-induced cell cycle delay is described. Following the incorporation of the BrdU label the cells are stained with ethidium bromide and the bis-benzimidazole Hoechst 33258. These fluorochromes have differential staining patterns. Hoechst 33258 fluoresces blue and is quenched by BrdU incorporated into cellular DNA during S phase. Ethidium bromide fluoresces red and is not quenched by BrdU. Therefore in cells that are cycling and synthesizing DNA new G1 and G2 compartments are created and this can be used to measure cell cycle delays following ionizing radiation to asynchronous cells. We have used this technique to evaluate two cell lines: a normal diploid human embryo fibroblast cell line MRC 5, which has inducible p53 and shows delays at both G1 and G2 checkpoints, and the human cervix carcinoma cell line HX 156. This cell line has been infected with human papilloma virus (HPV) 16, and therefore has inactivated p53 function and is blocked only at the G2 checkpoint. Using this method, cell cycle-dependent effects relating to the G2 block can be observed. The radiation-induced G2 block differs from that induced by drugs or heating in that cells are blocked in G2 irrespective of the phase of the cell cycle they are treated in. This method allows these different types of G2 block to be quantified.

We have studied the effects of the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-platin) on three human ovarian carcinoma cell lines - one sensitive to the drug (CH1), one with acquired resistance (CH1cisR) and one with intrinsic resistance (SKOV-3). Previous work has shown that the 50% inhibitory concentrations (IC50 values) after a 2-h exposure to the drug are: CH1, 2.5 microM; CH1cisR, 7.5 microM; and SKOV-3, 33 microM. Despite the variation in sensitivity, the amount of Pt bound to DNA and the rate of removal of Pt was similar for the three lines. There were significant differences in the rates of formation of DNA cross-links but these were not large enough to account for the high resistance of the SKOV-3 line. We have reported that in the L1210 murine leukaemia cell line there are two mechanisms of cisplatin-induced cell death - one of which involves apoptosis. In this paper, we report on an investigation into whether sensitivity to apoptosis played a role in the resistance of these ovarian lines toward cisplatin. After a 2-h incubation with the drug, cells from the three lines showed evidence of death through apoptosis. The cells detached from the culture dish in a time- and dose-dependent fashion. These cells morphologically were quite distinctive from the attached cells and showed changes in their chromatin structure indicative of apoptosis. Their DNA had not been degraded into oligonucleosomal fragments (200 bp and multiples thereof) but had been cut into larger fragments (30 kilobase pairs, kbp) of a size associated with chromatin domains (chromatin loops). At equitoxic doses of drug, the quantity of cells undergoing apoptosis was similar for the three cell lines. The most prominent effect on cell-cycle kinetics was a slowdown in S-phase transit during which the cells underwent apoptosis. Cells that successfully completed the S phase subsequently suffered a temporary G2 block. We propose that the sensitivity of these cell lines to cisplatin was governed by their ability to handle damage caused by platination of the DNA and that the major mechanism of cisplatin-induced cell death in all three cell lines was the induction of apoptosis.

The quality of a DNA histogram as recorded in a flow cytometer can often be improved by gating on forward and orthogonal light scatter. This may be helpful when measuring DNA histograms from formalin-fixed, paraffin-embedded material. We report that nuclei from many human tumours scatter more light orthogonally than those from normal stroma. Gating on light scatter enabled the DNA histogram from the tumour to be recorded with reduced contamination from normal cells. In studies of human mammary carcinomas, we have found that this method has the potential to improve the estimation of S phase and the measurement of DNA index (DI) when the DI is close to 1 (near diploid).

The discrimination of apoptotic and normal rat thymocytes by forward-angle light scatter in the UV depended on the angle over which the scattered light was collected. To achieve good separation, the light needed to be collected over a narrow angle. It was also observed that apoptotic thymocytes gave less forward light scatter than normal cells, whereas, under identical conditions, apoptotic cells from a murine cell line gave more light scatter than their normal counterparts. We suggest that the forward light scatter of apoptotic cells is affected by several factors, including changes in cell size and in the internal structure of the cell.

The increasing use of neoadjuvant chemotherapy and endocrine therapy in the management of breast cancer has lead us to evaluate and optimise the standard technique of cytocentrifugation of a single fine needle aspirate (FNA) taken from a breast tumour in-vivo, to determine a range of both immunocytochemical and flow cytometric factors which are predictive of response to primary medical therapy. Some of these factors are also of prognostic significance in early stage disease. An analysis of the cellularity and immunocytochemical staining characteristics of FNAs obtained from a series of 206 patients with palpable breast cancers indicate that in a sample of 46 cases it is possible to measure oestrogen receptor, progesterone receptor and c-erbB-2 providing over 400 cells per slide are obtained, with material obtained in a single FNA prepared by cytocentrifugation, using standard immunocytochemical methods. The staining results obtained were comparable to those obtained using frozen or paraffin embedded tissue sections taken from the same tumour. In addition an estimate of the proliferation indices could be made by flow cytometric analysis of the residual cell suspension fluid with measurement of DNA index and S-phase fraction in 131/164 (80%) and 110/164 (67%) of cases respectively. Providing all FNAs obtained for cytocentrifugation were taken at first presentation rather than immediately following a standard FNA, then it was possible to obtain adequately cellular (> 400 cells/slide) samples in 96 out of 126 (75%) of the last cohort of breast aspirates. These effects may be independent of T stage but not histological type as patients with lobular tumours only produced cellular aspirates in 1/7 (14%) of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of EGF receptors has been studied on luminal and basal cells of human breast in vitro. Primary cultures of normal adult human breast epithelium were prepared as single cell suspensions containing a mixture of luminal and basal cells. The cells were simultaneously immunolabelled with antibodies recognising EMA (luminal epithelial cells), CALLA/CD10 (basal cells) and the epidermal growth factor receptor (EGFR). Flow cytometric analysis of these triple labelled cells detected low levels of EGFR on both cell types, with proportionally more EGFR on basal cells compared with luminal cells. Separated populations of basal and luminal cells were prepared from single cell suspensions by flow sorting or by immunomagnetic methods and cultured with and without EGF. Increased proliferation was detected in both cell types in the presence of EGF. To determine the localisation of the EGF receptor, purified cell populations were immunolabelled with anti-EGFR antibody and an FITC-labelled second antibody for fluorescence light microscopy and colloidal gold-labelled antibody for scanning electron microscopy (SEM). Low levels of EGFR were detected by indirect immunofluorescence on both cell types with higher levels on basal cells compared with luminal cells. The detailed subcellular distribution of the receptor was examined by SEM, with gold-labelling of EGFR detected using a field emission scanning electron microscope with a YAG crystal backscattered electron detector. Both luminal and basal cells expressed EGFR over the upper surface of individual cells when these were growing in isolation, but when cells formed part of a confluent island, levels of EGFR on the upper surface of cells were obviously reduced. Observations made by SEM on cells at the edges of such confluent islands showed that cultured basal cells expressed much higher levels of EGFR on their basal, as compared with their upper surfaces.

Dexamethasone and etoposide both induce apoptosis in immature rat thymocytes. We investigated the dependence of apoptosis on the phase of the cell cycle after incubation with these drugs. Cell cycle progression was followed by a combination of pulse labelling with 5-bromo-2'-deoxyuridine (BrdU), labelling fixed cells with an anti-BrdU antibody and flow cytometry. Dexamethasone had little effect on the cell cycle progression of proliferating thymocytes, while etoposide caused cell cycle arrest. Normal and apoptotic thymocytes were separated by centrifugation on discontinuous Percoll gradients into four fractions (F1-F4). It was found that both dexamethasone and etoposide induced apoptosis in cells in G0/G1 and G2/M of the cell cycle, whereas only etoposide induced apoptosis of cells in S phase. These results demonstrated that dexamethasone induced apoptosis in quiescent cells while only etoposide could induce apoptosis in cells from the proliferative compartment. Following treatment of thymocytes with etoposide, some of the proliferating thymocytes (F1) were converted to cells with intermediate size and density (F3). We have recently identified these cells as a population of preapoptotic thymocytes, at an early stage of apoptosis. These cells then further progressed to fully apoptotic cells (F4). These data support the hypothesis that normal thymocytes (F1) became apoptotic (F4) via an intermediate population (F3).

After treatment of the human ovarian carcinoma cell line, CH1, with cisplatin, cells detached from the culture dish in a time- and dose-dependent fashion. These cells showed morphological changes indicative of apoptosis. Their DNA had not been degraded into oligonucleosomal fragments, but the DNA had been cut into larger fragments (30 kbp) of a size associated with chromatin loops. We conclude that cisplatin killed these ovarian cells by inducing apoptosis. However, in these cells, apoptosis was not accompanied by internucleosomal degradation of DNA. Our data are consistent with the hypothesis that the introduction of a double-strand break at a specific site in the chromatin loops is an early event in apoptosis. This degradation is accompanied by morphologically observable changes in chromatin structure. Internucleosomal degradation, when it occurs, is a late event.

Etoposide, a DNA topoisomerase II inhibitor, caused a concentration-dependent induction of apoptosis in immature thymocytes. Using a flow cytometric method to separate and quantify normal and apoptotic cells, etoposide-induced apoptosis was inhibited by cycloheximide and actinomycin D but not by zinc. Etoposide induced a marked cleavage of DNA into nucleosomal length fragments or multiples thereof, which was completely inhibited if the thymocytes were also incubated in the presence of zinc. Etoposide, alone, induced the classical ultrastructural features of apoptosis, but in the presence of zinc, the morphological pattern was markedly different and dominated by discrete clumps of condensed chromatin abutting the nuclear membrane. These latter changes resemble those described as the earliest changes in apoptosis. These results support the hypothesis that, in the induction of apoptosis, critical alterations in nuclear chromatin occur prior to endonuclease cleavage of DNA into nucleosomal fragments.

The concentration of phospholipid metabolites was determined in chemical extracts from two types of rat mammary tumours and compared with proliferation data (S-phase fraction). One of the tumours was an oestrogen-sensitive transplanted tumour. In this tumour the concentration of phosphocholine (PC) and glycerophosphorylcholine (GPC) correlated strongly with the S-phase fraction but not with the number of cells actively synthesizing DNA. Oestrogen ablation resulted in tumour regression. Regressing tumours contained less PC and more GPC than those actively growing. The other tumour was induced in rats by intravenous administration of N-methyl N-nitrosourea. Phosphoethanolamine (PE), PC and GPC levels were not associated with the S-phase fraction in this tumour. Oestrogen ablation resulted in tumour regression. There was no significant difference between the regressing and growing tumours in PE, PC or GPC content.