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Objectives: The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time- and labour-consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier method, also enabling estimation of the fraction of cells in S or S+G2+M (SG2M) cell-cycle phases as indicators of cell proliferation. Methods: Cell suspensions from 35 human embryonic gonads at days 37 to 68 post-conception (pc) were immunomagnetically sorted into C-KIT positive (germ) cells and negative (somatic) cells. They were stained for DNA content and analysed by flow cytometry. S and SG2M fractions could be measured for 13 of the female and 20 of the male gonads. The number of cells was estimated using fluorescent reference beads. Results: During the period from 37 to 68 days pc, female germ and somatic cells had a stable S and SG2M fractions indicating steady growth of both subpopulations, whereas they decreased in both male germ and somatic cells. The number of germ and somatic cells estimated by flow cytometry was significantly lower than in stereological estimates, suggesting loss of cells during preparation. Conclusions: Cell proliferation as indicated by S and SG2M fractions could be estimated specifically for primordial germ and somatic cells. Estimation of total number of germ and somatic cells was not feasible.

In the 50 newly diagnosed, unselected, untreated B-CLL patients, the absolute numbers of blood T cells, T-helper cells, and T-suppressor/cytotoxic cells were determined by flow cytometric counting of mononuclear cells labelled with the monoclonal antibodies Leu5 (T cells), Leu3a (T-helper cells), and Leu2a (T-suppressor/cytotoxic cells). These estimations and the serum concentrations of IgG, IgA, and IgM were correlated to clinical stage (International Workshop System) and pretreatment observation time. For all patients together, the mean counts of Leu5 +, Leu3 +, and Leu2 + cells were significantly increased compared with the mean counts in 12 healthy controls (Mann-Whitney). In patients with advanced disease (stage B + C), both T-subset mean cell counts were significantly increased, whereas in patients with early-stage disease (stage A), although some high T-helper cell counts were noted, only the T-suppressor/ cytotoxic mean cell count increase reached significance. Thus a trend was observed of a more frequent T-suppressor/cytotoxic cell predominance in early-stage disease, which is the opposite of the findings in most other prognostic studies. However, there was no significant difference in pre-treatment observation time according to T-helper: T-suppressor cell ratio below vs. above 1.0, irrespective of stage, whereas according to clinical stage, the pretreatment observation time in stage A was highly significantly longer than in stage B + C (logrank test). Thus, no independent prognostic significance of T-subset counts was found as judged by pretreatment observation time. No correlation was found between the occurrence of hypogammaglobulinaemia, T-subset ratios or T-subset counts. In 24 patients, the number of Leu7+ cells (natural killer/ cytotoxic cells) were also counted and found significantly increased compared with normal values.

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m2) and non-lethal (2 g/m2) doses were found to inhibit cell-cycle progression primarily by impairing the G1/S transition. The results were obtained by flow cytometric DNA analysis. The inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). The greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. The time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G1 to S phase (at the onset of the dark period). The main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G1 cells. G1 cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m2, injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [3H]TdR and [3H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [3H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)

Flow cytometric DNA analysis was performed on fine-needle aspirates from frozen tumour biopsies from 421 node-negative, non-adjuvantly-treated breast-cancer patients with a median observation time of 6.75 years. Among premenopausal patients (n = 175), those having at least one DNA "hypoploid" subpopulation defined as DNA index (DI) < 0.96 or 1.44 < or = 1.92 (n = 81) were characterized by early recurrences (log-rank p = 0.05), Wilcoxon p = 0.007), poor overall survival (OS) (p < 0.001) and poor survival after recurrence (p < 0.001). In the postmenopausal group (n = 246), there were no significant difference among 7 different DI classes regarding either recurrence-free survival (RFS) or OS. S-phase fraction (SPF), divided into quartiles, predicted OS in premenopausal patients only (p = 0.02). Conventional multivariate Cox analysis of OS in the premenopausal group revealed hypoploidy to be the only independent prognostic factor involving a relative risk (RR) of 22.8. Age < or = 40 years was of marginal significance, whereas SPF, histological grade (WHO), oestrogen and progesterone receptor (PgR) content, tumour size and number of lymph nodes removed were excluded from the model. Application of the conventional Cox model to the premenopausal group regarding RFS was found inappropriate due to lack of proportionality of the hazards of hypoploidy due to lack of proportionately of the hazards of hypoploidy, SPF and histological grade. However, introduction of time-dependent co-variates using 2 years as cut-off level showed hypoploidy with a RR of 3.52 and age < or = 40 years with a RR of 3.28 to be independent prognostic factors. In the postmenopausal group, the conventional Cox model identified the number of lymph nodes removed to be the only independent prognostic factor regarding RFS as well as OS, whereas SPF < 9% (lowest quartile) was of marginal significance in RFS analysis. Hypoploidy was correlated to high SPF, low PgR content and low differentiation, indicating that hypoploid tumours proliferate rapidly and hormone-independently. These patients may therefore benefit from adjuvant chemotherapy administered while tumour burden and risk of drug resistance are still low.

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Gαi as determined by a receptor-mediated inhibition of forskolin-induced cAMP production and an induction of the serum response element-driven transcriptional activity in a pertussis toxin-sensitive manner. Gαs and Gαq were not activated constitutively as determined by the lack of cAMP production, the lack of inositol phosphate turnover, and the lack of activities of the transcription factors: cAMP response element-binding protein and nuclear factor-κB. Immunohistochemistry and confocal microscopy of FLAG- and green fluorescent protein-tagged EBI2 revealed cell-surface expression. A putative N-terminal truncated version of EBI2, Δ4-EBI2, showed similar expression and signaling through Gαi as full-length EBI2. By using a 32P-labeled EBI2 probe we found a very high expression in lymphoid tissue (spleen and lymph node) and peripheral blood mononuclear cells and a high expression in lung tissue. Real-time PCR of EBV-infected cells showed high expression of EBI2 during latent and lytic infection, in contrast to the EBV-encoded 7TM receptor BILF1, which was induced during lytic infection. EBI2 clustered with the orphan GPR18 by alignment analysis as well as by close proximity in the chromosomal region 13q32.3. Based on the constitutive signaling and cellular expression pattern of EBI2, it is suggested that it may function in conjunction with BILF1 in the reprogramming of the cell during EBV infection.

High hyperdiploid acute lymphoblastic leukemia in children is related to a good outcome. Because these patients may be stratified to a low-intensity treatment, we have investigated the sensitivity of flow cytometry (FCM), G-band karyotyping (GBK), and high-resolution comparative genomic hybridization (HR-CGH) in detecting high hyperdiploid leukemic clones. Twenty-six girls and 34 boys with acute lymphoblastic leukemia diagnosed in 1998 to 1999 were analyzed by FCM, GBK, and HR-CGH. The correlations between DNA indices obtained by FCM, GBK, and HR-CGH were significant (rs=0.61 to 0.77; P

SummaryIn a number of cancer types high tumor tissue levels of plasminogen activator inhibitor type 1 (PAI-1) protein are strongly associated with shorter cancer patient survival. This association has been intriguing since PAI-1 is known to inhibit urokinase plasminogen activator (uPA) that converts plasminogen to plasmin, which is actively involved in tumor progression and invasion. In order to further explore the biological role of PAI-1 in cancer, we have prepared fibroblasts from PAI-1 gene deficient mice and from their wild type littermates. From these fibroblasts fibrosarcoma cell lines were established and characterized. Both types of fibroblasts underwent spontaneous transformation as indicated by aneuploidy, immortalization, clonogenicity in soft agar and tumor formationin vivo. While both PAI-1 deficient and PAI-1 expressing cell lines showed similar proliferation ratesin vitro, cells devoid of PAI-1 were significantly more sensitive to apoptotic stimuli. When inoculated subcutaneously into nude mice PAI-1 expressing cells rapidly established tumors, while PAI-1 deficient cells had a significantly longer lag-phase before they started to grow (p

The cytogenetic quality of boars used for breeding determines the litter outcome and thus has large economical consequences. Traditionally, quality controls based on the examination of simple karyograms are time consuming and sometimes give uncertain results. As an alternative, the use of high-resolution DNA flow cytometry on DAPI-stained sperm cell nuclei (CV≤1.3%) was investigated as a screening method for the cytogenetic quality of boars. By analyzing a series of 25 animals judged normal by their fertility statistics and a series of seven animals with known reciprocal translocations, a model for identifying sperm cells from cytogenetically aberrant animals was proposed. This model was applied to a series of 50 uncharacterized animals. The model successfully identified a mosaic or chimaeric carrier of an aberrant X chromosome. However, implementation of this technique for screening purposes would necessitate essential improvements in standardization and measurement precision.

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor–associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

Different opinions about flow cytometric estimates of DNA aneuploidy and/or S‐phase fraction (SPF) as supplementary prognostic markers in colorectal cancer are to some degree associated with methodology. Using univariate DNA analysis, we have previously investigated the DNA ploidy in colorectal cancer, its heterogeneity within and between tumors and its relation to survival. To improve detection of DNA aneuploid subpopulations and particularly estimation of their SPF's we investigated a method for bivariate DNA/cytokeratin analysis on fine‐needle aspirates of 728 frozen biopsies from 157 colorectal tumors. Unfixed aspirates were stained with propidium iodide and FITC‐conjugated anti‐cytokeratin antibody in a saponin‐buffer. A significant association between SPF and debris was observed. There were no substantial difference in DNA ploidy patterns between univariate and bivariate measurements (concordance was 92–95%). No new DNA aneuploid subpopulations were detected in cytokeratin‐gated compared to ungated or univariate histograms. Debris‐adjusted SPF's of cytokeratin‐gated histograms were significantly higher than of ungated histograms, also for subpopulations with DI>1.4 (p < 0.0001). There was no significant association between SPF and survival.

Background Whole-genome amplification of minute samples of DNA for the use in comparative genomic hybridization (CGH) analysis has found widespread use, but the method has not been well validated. Methods Four protocols for degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and fluorescence labeling were applied to test DNA from normal and K-562 cells. The DNA products were used for CGH analysis. Results The DOP-PCR–amplified DNA from each protocol produced hybridizations with different qualities. These could be seen primarily as differences in background staining and signal-to-noise ratios, but also as characteristic deviations of normal/normal hybridizations. One DOP-PCR-protocol was further investigated. We observed concordance between CGH results using unamplified and DOP-PCR–amplified DNA. An example of an analysis of an invasive carcinoma of the breast supports the practical value of this approach. Conclusions DOP-PCR–amplified DNA is applicable for high- resolution CGH, the results being similar to those of CGH using unamplified DNA. Cytometry 44:317–325, 2001. © 2001 Wiley-Liss, Inc.

Interaction between CD40 and the CD40 ligand (CD40L) is critical for the survival and proliferation of B cells during immunopoiesis. However, the role of CD40L in the pathogenesis of malignant lymphomas is ambiguous. Primary mantle cell lymphoma (MCL) cells were cultured in the presence of recombinant human CD40L trimer (huCD40LT), and a significant time- and dose-dependent induction of DNA synthesis was observed in thymidine incorporation assays (n = 7,P

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.

The aim of this study was to elucidate some of the possible mechanisms of action of the vitamin D analogue calcipotriol in vivo. Calcipotriol is finding increasing use in the treatment of psoriasis, but the primary target cell in vivo has not yet been identified. We treated psoriatic patients and healthy volunteers with calcipotriol and placebo ointment for 4 and 7 days, and obtained epidermal cell suspensions from treated areas. Epidermal cells were cocultured with autologous T cells, isolated from peripheral blood, in the absence or the presence of a classical antigen or a superantigen. In both psoriatic and normal skin, calcipotriol treatment did not alter the capacity of epidermal antigen-presenting cells to stimulate the proliferation of autologous T cells, either in the absence or in the presence of exogenous antigen. Epidermal cell suspensions were analysed further by staining for infiltrating leucocytes (CD45+) and Langerhans cells (CD1a+). Flow cytometric analyses showed that calcipotriol did not alter the number of CD45+ cells or Langerhans cells in psoriatic skin. These results indicate that calcipotriol does not alter either the number or the function of epidermal antigen-presenting cells in psoriatic epidermis. In contrast, we found that calcipotriol significantly inhibited the proliferation of epidermal cells isolated from psoriatic skin after in vivo treatment, as determined by propidium iodide staining and flow cytometry. More specifically, we stained for CD29+ keratinocytes and found an even more significant reduction in proliferative capacity. This cell type contains the population of hyperproliferative keratinocytes in psoriatic epidermis. In conclusion, calcipotriol seems to act via an inhibitory effect on hyperproliferative basal keratinocytes of psoriatic epidermis, rather than via an effect on infiltrating leucocytes, including antigen-presenting cells.

The Brown Norwegian rat transplanted with promyelocytic leukaemic cells (BNML) has been used as a model for human acute myeloid leukaemia. We have previously shown that both the blood supply to the bone marrow and the metabolic rate decrease in relation to the leukaemic development in these rats. Here we have investigated how the development and progression of this leukaemia affect oxygenation, pH and proliferation of normal and leukaemic cells in vivo. Bone marrow pH was measured by a needle electrode. Nitroimidazol-theophylline (NITP) was used to identify hypoxic cells, and we applied bromodeoxyuridine (BrdUrd) to identify DNA replicating cells. The leukaemia progressed slowly until day 27 after which a rapid deterioration could be observed leading to severe changes over the following 5 d. In whole blood there was evidence of progressing metabolic acidosis. In bone marrow the fraction of leukaemic cells increased to > 90% and the pH dropped to about 6.5. The fraction of NITP+ cells increased to > 80% in bone marrow and to about 40% in blood. The fraction of BrdUrd+ cells was unchanged in blood, but decreased in bone marrow both for normal cells (from about 20% to 5%), and for leukaemic cells (from about 45% to 25%), evidently as a result of the severely changed micro- environment. In this study we have demonstrated in vivo the development of an acidic and hypoxic bone marrow hampering normal haemopoiesis during leukaemic growth. Our data support the notion of BNML as a valuable tool for studying leukaemogenesis.

Of 560 consecutive, newly diagnosed untreated patients with B CLL submitted for chromosome study, G-banded karyotypes could be obtained in 480 cases (86%). Of these, 345 (72%) had normal karyotypes and 135 (28%) had clonal chromosome abnormalities: trisomy 12 (+12) was found in 40 cases, 20 as +12 alone (+12 single ), 20 as +12 with additional abnormalities (+12 complex ). Other frequent findings included abnormalities of 14q, chromosome 17, 13q and 6q. The immunophenotype was typical for CLL in 358 patients (CD5 + , Slg weak , mainly FMC7 − ) and atypical for CLL in 122 patients (25%) (CD5 − , or Slg strong or FMC7 + ). Chromosome abnormalities were found significantly more often in patients with atypical (48%) than in patients with typical CLL phenotype (22%) ( P complex , 14q +, del6q, and abnormalities of chromosome 17 were significantly more frequent in patients with atypical CLL phenotype, whereas +12 single was found equally often in patients with typical and atypical CLL phenotype. The cytomorphology of most of the +12 patients was that of classical CLL irrespective of phenotype. In univariate survival analysis the following cytogenetic findings were significantly correlated to a poor prognosis: chromosome 17 abnormalities, 14q +, an abnormal karyotype, +12 complex , more than one cytogenetic event, and the relative number of abnormal mitoses. In multivariate survival analysis chromosome 17 abnormalities were the only cytogenetic findings with independent prognostic value irrespective of immunophenotype. We conclude that in patients with typical CLL immunopenotype, chromosome abnormalities are somewhat less frequent at the time of diagnosis than hitherto believed. +12 single is compatible with classical CLL, and has no prognostic influence whereas chromosome 17 abnormalities signify a poor prognosis. In patients with an atypical CLL immunophenotype, chromosome abnormalities including +12 complex , 14q +, del 6q and chromosome 17 are found in about 50% of the patients, and in particular chromosome 17 abnormalities suggest a poor prognosis.

Flow cytometric DNA analysis was performed on unfixed frozen samples from 56 breast cancer patients. From each patient, two samples were analyzed. The only difference in the handling of the paired samples was the mechanical disaggregation of one sample by fine-needle aspiration compared to an automated mechanical disaggregation method (Medimachine) of the other sample. With the two methods for tissue disaggregation, the same resolution of the DNA histograms was obtained, indicated by median coefficients of variation (CV) of 1.5% for the DNA diploid G1 peaks. Also, the frequencies of DNA diploid and aneuploid cases as well as the fractions of DNA aneuploid cells were comparable. This indicates that the two methods did not differ in ability to detect DNA aneuploid tumor clones. Automated mechanical disaggregation resulted in DNA histograms with significantly less debris and with lower S-phase fractions. In practice, the procedure of automated mechanical disaggregation was rapid, easy, and safer because of minimal handling of the unfixed tissue compared to the fine-needle aspiration. © 1996 Wiley-Liss, Inc.

High-resolution flow cytometric (FCM) DNA analysis was performed on 148 unfixed, frozen tissue samples from four groups of early breast cancers: invasive carcinomas (ICs) with predominance of carcinoma in situ (DCIS) (group I), small clinical cancers < or = 15 mm (group II), node-negative, clinical cancers (group III) and small screening-detected cancers < or = 15 mm (group IV). The median tumour size was 12 mm. The aim of the study was to support, with a larger sample, our recent findings with respect to DNA ploidy pattern in the selected group of ICs with predominance of DCIS (group I). Similar results to this group were found for both the small clinical cancers and the node-negative cancers, with respect to frequency of DNA aneuploidy (79% and 90%), DNA index (DI) distribution, intratumoral DNA heterogeneity and S-phase fraction. A high frequency of DNA hyperdiploid clones was found, in particular related to highly differentiated tumours. A significant difference was found compared with the screening-detected cancers, which were characterised by a much lower frequency of DNA aneuploid samples (49%) and may represent a biologically specific group of low-malignant, slowly growing tumours. Associations were shown between histological grade and DI subclasses, and between lymph node status and DNA diploidy/aneuploidy, whereas DI was not correlated with tumour size. The DNA ploidy findings in this series of early cancers are concordant to our own results from preinvasive lesions as well as those reported from series of more advanced cancers.

In a population of 110 primary breast cancers with medullary features, registered in the Danish Breast Cancer Cooperative Group (DBCG) from 1977-82, we have determined ploidy and S-phase fraction (SF) by flow cytometry (FCM) on paraffin embedded tumour tissue. The distribution of DNA ploidy is not different from the distribution described for breast cancers in general. No difference is found between the subgroups of medullary and non-medullary cancer when using a new simplified histopathological definition of medullary carcinoma of the breast, recently proposed by us. When using the definition proposed by Ridolfi et al. in 1977, we find significantly more tumours with aneuploidy and high SF in the groups of typical medullary carcinoma (TMC) and atypical medullary carcinoma (AMC) than in the small group of non-medullary carcinoma (NMC), which seems a paradox, as patients with NMC have the worst prognosis. However, the number of patients in the NMC group is very small, and the percentage of aneuploid tumours is very low. In 84 protocolled patients we found no statistically prognostic importance of ploidy or SF, either in the whole group assessed or when stratifying for the histopathological subgroups. However, a prognostic influence of SF can be traced for the non-medullary cancers, according to the new definition, but not for the medullary cancers of the breast. The result emphasizes the impression of MC as being biologically different from other histological types of breast cancer.

To investigate whether growth factors derived from T cells in psoriatic lesions are able to stimulate keratinocyte growth, T-cell lines were initiated from lesional psoriasis skin and cloned by limiting dilution. Eight clones with good proliferative capacity out of 40 clones from one patient were stimulated. After 24 h, the conditioned medium was harvested and the growth modulatory effect of the conditioned medium on keratinocytes was assessed. Seven of the eight T-cell clones stimulated keratinocyte growth to an extent ranging from 22% +/- 19 to 64% +/- 9 (mean +/- SD of three experiments) of maximal inducible keratinocyte growth, and one T-cell clone had no effect (-5% +/- 2) on keratinocyte growth. Keratinocyte growth was also induced by T-cell clones obtained from two other patients. Several cytokines were tested in this system to determine which T-cell growth factor may induce the keratinocyte growth. None of the cytokines interferon-g, transforming growth factor-beta, interleukin (IL)-2, IL-3, IL-4, IL-6, IL-8, or granulocyte-macrophage colony stimulating factor alone was found to possibly be responsible for the T-cell-induced keratinocyte growth. Thus the nature of the T-cell keratinocyte growth-promoting stimulus remains to be elucidated.

RNA synthesis can be analysed in nuclei or cells labelled with 5-bromouridine (BrUrd) and stained using cross-reacting anti-bromodeoxyuridine ( BrdUrd) antibody. Flow cytometric dual parameter analysis of BrUrd incorporation and DNA content in nuclear suspensions of human blood lymphocytes showed that RNA synthesis increased within the first 24 hours of phytohemagglutinin (PHA) stimulation, reaching a maximum at 48 hours, when cells had entered the cell cycle. Using a new method for flow cytometric dual parameter analysis of BrUrd incorporation and a cell surface antigen, spontaneous RNA synthesis in HL-60 and K-562 cells was measured simultaneous with CD13 expression.

Nuclear RNA synthesis can be analysed by flow cytometry of cells labelled with 5-bromouridine (BrUrd) and stained with anti-bromodeoxyuridine (BrdUrd) antibody and FITC-conjugated secondary antibody. A panel of 5 different commercially available anti-BrdUrd antibodies was tested on cells of a HL-60 human leukemia cell line, stained as a methanol-fixed nuclear suspension. The BrUrd-induced fluorescence signals were highest with the antibody ABDM (Partec), moderate but reproducible with B-44 (Becton Dickinson), variable or low with BR-3 and IU-4 (Caltag), and not detectable with Bu20a (DAKO). Treatment of BrUrd-labelled nuclei with ribonuclease before staining with antibodies indicated that ABDM and B-44 antibodies specifically recognized BrUrd-substituted RNA, whereas BR-3 and IU-4 antibodies also bound to BrUrd-unlabelled RNA. Combined analysis of BrUrd and DNA contents demonstrated the variation of RNA synthesis during the cell cycle. The BrUrd incorporation was high in the S and G2 phase, variable in G1, and negligible in mitosis. Similar results were obtained using other cell types.

In this study we show the expression of the newly identified carbohydrate ligand, sialosyl-Le(X) on Langerhans cells. The receptor for sialosyl-Le(X) is the endothelial leukocyte adhesion molecule-1 (ELAM-1) present on activated endothelial cells. Using flow cytometry, Langerhans cells were selected due to positivity for an antibody against CD1a and low orthogonal light scatter. The CD1a antigen stained by the OKT6 antibody is considered a maturational marker of Langerhans cells in agreement with the specific labeling of dendritic cells in the epithelium only. Double immunostaining (OKT6/anti-sialosyl-Le(X)) using flow cytometry and immunohistochemistry demonstrated that almost all OKT6-positive cells in normal stratified epithelium expressed sialosyl-Le(X). Conversely, by immunohistochemistry of oral epithelium with acute inflammation, additional dendritic cells negative for OKT6 were found to express sialosyl-Le(X). In addition, sialosyl-Le(X)-positive but not OKT6-positive dendritic cells were found in the submucosa. These findings indicate that the carbohydrate antigen sialosyl-Le(X) is expressed earlier than the CD1a antigen in the maturation of the Langerhans cell lineage. Future studies should aim at investigating the importance of adhesion between sialosyl Le(X) and ELAM-1 in epithelial recruitment of Langerhans cells.

Blood mononuclear cells from 540 newly diagnosed, unselected patients with B-cell chronic lymphocytic leukemia (CLL) were examined by immunofluorescence flow cytometry for a panel of surface membrane markers, including IgM and IgD, the monoclonal antibodies anti-CD3, -5, -20, -21, -22, -FMC7, and, for the final 125 patients, anti-CD23. There were 503 CD5+ and 37 CD5- cases. In the CD5+ cases, the cells typically expressed IgM, IgD, CD20, CD21, CD22, and CD23. In univariate analysis, age, clinical stage, IgM-fluorescence intensity, CD23, and FMC7 had significant prognostic importance, with high IgM-fluorescence intensity, high FMC7, and low CD23 expression being associated with a short survival. There was no significant difference in survival between 351 cases expressing IgMD and 55 cases expressing IgM without IgD, or between kappa and lambda light chain monoclonal cases. CD20, CD21, and CD22 had no prognostic importance. In Cox multiple regression analyses, age, CD23, IgM-fluorescence intensity, and clinical stage (International Workshop System) had independent prognostic importance. Thus, besides clinical variables, CD23 and IgM intensity might be useful prognostic markers in the management of CD5+, B-cell CLL. The survival of CD5- patients was on the borderline of being significantly shorter than that of CD5+ patients. The majority of the CD5- cases were FMC7+, CD23-, had strong IgM fluorescence, and had splenomegaly.

Washless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)-stimulated normal human blood lymphocytes for the double staining of (1) Ki-67 antigen and DNA and (2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd-labeled cells. With respect to the discrimination between antigen-positive and -negative subpopulations, there was no statistically significant differences between the results from direct (Ki-67) or indirect, (Ki-67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells.

The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.

Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is one of four inhibitors of the matrix metalloproteinases, which are capable of degrading most components of the extracellular matrix. However, in recent years, TIMP-1 has been recognised as a multifunctional protein, playing a complex role in cancer. In this regard, several studies have demonstrated an antiapoptotic effect of TIMP-1 in a number of different cell types. Since chemotherapy works by inducing apoptosis in cancer cells, we raised the hypothesis that TIMP-1 promotes resistance against chemotherapeutic drugs. In order to investigate this hypothesis, we have established TIMP-1 gene-deficient and TIMP-1 wild-type fibrosarcoma cells from mouse lung tissue. We have characterised these cells with regard to TIMP-1 genotype, TIMP-1 expression, malignant transformation and sensitivity to chemotherapy-induced apoptosis. We show that TIMP-1 gene deficiency increases the response to chemotherapy considerably, confirming that TIMP-1 protects the cells from apoptosis. This is to our knowledge the first study investigating TIMP-1 and chemotherapy-induced apoptosis employing a powerful model system comprising TIMP-1 gene-deficient cells and their genetically identical wild-type controls. For future studies, this cell system can be used to uncover the mechanisms and signalling pathways involved in the TIMP-1-mediated inhibition of apoptosis as well as to investigate the possibility of using TIMP-1 inhibitors to optimise the effect of conventional chemotherapy.

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor-mediated inhibition of forskolin-induced cAMP production and an induction of the serum response element-driven transcriptional activity in a pertussis toxin-sensitive manner. Galpha(s) and Galpha(q) were not activated constitutively as determined by the lack of cAMP production, the lack of inositol phosphate turnover, and the lack of activities of the transcription factors: cAMP response element-binding protein and nuclear factor-kappaB. Immunohistochemistry and confocal microscopy of FLAG- and green fluorescent protein-tagged EBI2 revealed cell-surface expression. A putative N-terminal truncated version of EBI2, delta4-EBI2, showed similar expression and signaling through Galpha(i) as full-length EBI2. By using a 32P-labeled EBI2 probe we found a very high expression in lymphoid tissue (spleen and lymph node) and peripheral blood mononuclear cells and a high expression in lung tissue. Real-time PCR of EBV-infected cells showed high expression of EBI2 during latent and lytic infection, in contrast to the EBV-encoded 7TM receptor BILF1, which was induced during lytic infection. EBI2 clustered with the orphan GPR18 by alignment analysis as well as by close proximity in the chromosomal region 13q32.3. Based on the constitutive signaling and cellular expression pattern of EBI2, it is suggested that it may function in conjunction with BILF1 in the reprogramming of the cell during EBV infection.

Udgivelsesdato: 2004-Jan Several studies have shown that oral nickel exposure can elicit systemic contact dermatitis (SCD) in nickel-sensitive individuals. The current study describes some of the immunological mechanisms underlying such nickel-allergic reactions elicited by oral exposure to nickel. Following oral exposure to graded concentrations of nickel or placebo, blood samples were taken from nickel-sensitive individuals and from non-nickel-sensitive controls. T-cell subtypes (CD3+, CD4+, CD8+ and CD45RO+), expression of skin-homing receptor, cutaneous lymphocyte-associated antigen (CLA) and cytokine profiles [interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, interferon-gamma and tumour necrosis factor-alpha] were investigated. A definite dose-response reaction pattern to oral nickel exposure was observed among nickel-sensitive individuals. Nickel-sensitive individuals whose dermatitis flared after oral challenge with nickel showed significant decreases in fractions of CD3+ CD45RO+ CLA+ and CD8+ CD45RO+ CLA+ blood lymphocytes, suggesting migration of CD8+ 'memory' CLA+ T lymphocytes from the blood to peripheral tissues. Only those nickel-sensitive individuals who clinically reacted to oral challenge with nickel (4 mg) had elevated levels of IL-5 in the serum, indicating an activation of type 2 T lymphocytes in the peripheral blood. In conclusion, the study indicates that CD8+ CD45RO+ CLA+ T lymphocytes and T lymphocytes with a type 2 cytokine profile are involved in SCD elicited by nickel.

Interpretation of data from comparative genomic hybridization (CGH) analysis of testicular neoplasms located within normal parenchyma is complicated, because the results may be influenced by a heterogeneity of subpopulations with different chromosomal aberrations and ploidy. In this study, therefore, early stages of testicular germ cell neoplasia were cytogenetically analyzed after flow sorting of nuclei according to their DNA ploidy. DNA from subpopulations with different ploidy was globally amplified by means of degenerate oligonucleotide primed polymerase chain reaction, labeled with FITC-dCTP and -dUTP by nick translation, and analyzed with high resolution CGH. A characteristic pattern of chromosomal abnormalities associated with testicular germ cell cancer (gains in 1q, 7, 8, 12, 14, 21, X; losses from 4, 5, 9, 11, 13, 18, Y) was observed in the tri- to hexaploid but not in the hyperdiploid or in pure tetraploid subpopulations. Our data suggest that subpopulations with a triploid to hexaploid DNA content purified from testes with germ cell neoplasia harbor a mixture of overt tumor and carcinoma-in-situ cells (CIS) and DNA content of CIS cells being in the triploid to hypotetraploid range, supporting the current theory of polyploidization as one of the first events of malignant transformation.

Publisher Summary The biological significance of RNA can be examined by different cytometric approaches, which can be divided into “static” approaches, describing the momentary distribution of the RNA content in the cell population, and “dynamic” approaches, describing the distribution of the content of RNA precursors that have been incorporated during a preset time period. This chapter focuses on the dynamic approach, with emphasis on the flow cytometric analysis limited to applications with mammalian cells. The RNA content of a cell can be estimated by direct staining with fluorochromes such as pyronin Y and acridine orange, the latter binding metachromatically to single-stranded RNA and DNA. For studies of RNA synthesis in experimental animals, in vivo labeling with 5'-bromouridine (BrUrd) (or [ 3 H]uridine) is required. The BrUrd-substituted RNA may be detected by permeabilizing the cells and staining with certain anti-BrdUrd antibodies. Owing to their relatively short lifetime in comparison to other types of RNA molecules, specific RNA transcripts may provide estimates of the cellular expression of the corresponding genes at the RNA level.

Publisher Summary This chapter discusses the detection of proliferating cell nuclear antigen (PCNA). The expression of the PCNA molecule is associated with cell proliferation, being relatively low in quiescent cells and higher in cycling cells, especially during S phase. The possibility of utilizing PCNA expression as an empirical marker of cell proliferation, assessed by quantitative immunoftuorescence cytometry without dependency on labeling of cells with DNA precursors, has motivated numerous investigations due to its potential value in toxicological, pathological, and oncological applications. According to the choice of anti-PCNA antibody and the procedure for permeabilization, fixation, and immunofluorescent staining, it is possible in cell suspensions or tissue sections to identify the subpopulation of S-phase cells or of cycling cells. The level of expression of PCNA can be quantitatively analyzed by flow cytometry; however, image cytometry must be applied for a quantitative analysis of the subcellular distribution of PCNA.

Flow cytometric (FCM) DNA analysis of 41 clinical cases of ductal carcinoma in situ (DCIS) of the breast was performed on fine-needle aspirates of unfixed, frozen tissue. Based on analysis of a single tissue sample, abnormal DNA content was found in 35 (85%) of the cases, and 15 (37%) disclosed heterogeneity. If five or more samples were analyzed, then heterogeneity was found in eight of ten cases. No conclusive correlation could be found between DNA index (DI) and histopathology. Our results on DCIS demonstrate a very high concordance with invasive breast cancer with respect to the frequency of DNA nondiploid cases, distribution of DI, occurrence of multiple clones, and S-phase fraction. This indicates that major genetic alterations and DNA heterogeneity are early events in carcinogenesis that are already established at the preinvasive stage. © 1995 Wiley-Liss, Inc.

CD1+ antigen-presenting cells in involved epidermis of patients with cutaneous T-cell lymphoma exhibit and enhanced functional capacity to activate autologous CD4+ T cells compared with CD1+ antigen-presenting cells from uninvolved and normal epidermis. Class II major histocompatibility complex molecules are involved in antigen presentation, and their expression on CD1+ Langerhans cells is known to vary. The expression of all three class II (HLA-DR, -DQ, -DP) molecules was therefore determined on CD1+ epidermal cells from both involved and uninvolved epidermis, using flow cytometry. The involved CD1+ epidermal cells exhibited a 1.5-1.6-fold, statistically significant increase in fluorescence intensity after staining of the class II molecules (HLA-DR, -DQ, -DP) compared with CD1+ epidermal cells from uninvolved epidermis. The autologous CD4+ T cells, activation was almost completely blocked by anti-HLA-DR, and partly by anti-HLA-DQ and anti-HLA-DP. In contrast, an antibody against class I, and an irrelevant control antibody, had no blocking effect. In a pokeweed mitogen assay it was demonstrated that autologous CD4+ T cells, activated by involved epidermal cells, demonstrated suppressor activity rather than helper activity. The suppressor activity was dependent on the presence of HLA-DR-positive epidermal cells. Thus, in cutaneous T-cell lymphoma, class II molecules on the individual CD1+ antigen-presenting cell are upregulated in clinically involved compared with uninvolved epidermis, and these molecules are crucially involved in activation of CD4+ T cells.

Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 105 ethanolfixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodine. Labelled mitoses could be discriminated from unlabelled mitoses, and from labelled and unlabelled G2 cells, by their intermediate log FITC fluorescence intensity. In addition, mitoses and G2 nuclei differed in forward and orthogonal light scattering, but had equal intensity of propidium iodide fluorescence. This method for discrimination of labelled mitoses was also tested on cultured normal adult human keratinocytes labelled with iododeoxyuridine (IdUrd). In keratinocytes, where the cell structure was preserved after pepsin/HCl, IdUrd labelled mitotic cells were similarly discriminated in the log FITC/propidium iodide fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses. © 1994 Wiley-Liss, Inc.

Antigen-dependent activation of T cells occurs through the T-cell antigen-receptor complex (TCR/CD3). Antigen-independent T-cell activation may occur through the surface molecules CDw60, CD2, and CD28. We wished to determine whether these antigen-independent T-cell-activation pathways could be involved in proliferation of leukemic T cells from patients with cutaneous T-cell lymphoma (CTCL). Whereas CDw60 was only expressed on 28% +/- 7% (mean +/- SEM) of blood T cells obtained from healthy control subjects (n = 4), CDw60 was expressed on 94% +/- 3% of blood T cells obtained from patients with CTCL (n = 4). Dual color immunofluorescence microscopy of the T-cell infiltrate in involved skin of these patients demonstrated that almost 100% of the T cells expressed CDw60. Not only did T cells in the patients with CTCL express CDw60, but triggering of the T cells with anti-CDw60 resulted in enhanced proliferation relative to anti-TCR/CD3 and mitogenic lectins. Other antigen-independent pathways also appeared highly active in the T cells from patients with CTCL because enhanced proliferation relative to anti-TCR/CD3 or mitogenic lectins was found when anti-CD2 or anti-CD28 plus phorbol ester was used as stimulant. Despite the brisk proliferation induced by anti-CDw60, anti-CD2, or anti-CD28, T cells from the patients did not produce detectable amounts of gamma-interferon. The inability to produce gamma-interferon correlates with our finding of absent (n = 3) or weak (n = 1) intercellular adhesion molecule-1 expression in the lesional keratinocytes in these patients. In conclusion, T cells of patients with CTCL demonstrate elevated expression of a T-cell-independent signaling molecule CDw60 and respond to antigen-independent activating signals.

In 42 tumour samples of human neuroblastoma, histological classification by differentiation (Shimada) was significantly correlated with strong positivity for neuron-specific enolase (NSE) and inversely correlated with rosette formation. Most ganglioneuroblastomas were positive for S-100 protein and reacted strongly with NSE antibody. Histological signs of high proliferative activity included intermediate or high mitosis-karyorrhexis index, necrosis and lack of calcification, which were significantly correlated with each other. Flow cytometric DNA analysis demonstrated that 88% of the tumour samples had DNA aneuploid stem lines. High S phase fraction (greater than or equal to 0.20) was significantly correlated with necrosis and lack of calcification. Univariate analysis of prognosis for 26 patients whose tumour samples were obtained before adjuvant treatment showed that five factors were significantly related to a better outcome: early stage of the disease (stages I, II, IV-S), S phase fraction less than 0.20, favourable Shimada histology, positivity for S-100 protein, and strong positivity for NSE. In multivariate analysis, only S phase fraction or stage of disease remained significantly associated with prognosis. DNA index did not correlate with prognosis in this study.

In vitro tetraploidy (IVT) in skin fibroblasts cultures measured by flow cytometry was compared with histological type and degree of dysplasia in 22 patients with adenomas of the colon and rectum. Furthermore, IVT was compared with stage and differentiation in 36 patients with carcinoma of the colon and rectum. In vitro tetraploidy in skin fibroblasts was correlated to type as well as dysplasia in adenomas and differentiation in carcinomas but was not correlated to Dukes' stage in carcinomas. Skin fibroblast genetic instability, expressed as increased IVT (IVT+), has been reported to reflect a genetic predisposition to colorectal cancer in the hereditary nonpolyposis colorectal cancer syndrome. Because IVT+, which appears to be associated with the progression of adenomas to carcinomas, also is found in many of the non-syndrome colorectal cancers, we suggest that development of colorectal cancer is considerably influenced by the constitutive genetic instability of the autosomal dominant colorectal cancer syndromes.

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B-lymphoblastoid cell lines, LICR-LON-HMy-2 (HMy-2) and WI-L2-729-HF2 (729-HF2), to generate hybridomas synthesizing antibodies reacting with tumor-associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been further analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showed no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.

Publisher Summary This chapter presents a new methodological approach to the double staining of a nuclear antigen and DNA. In the first step of this procedure, the cells are lysed with detergent into a suspension of pure nuclei. This is done to optimize the exposure of DNA and the specific nuclear antigen to the staining reagents without denaturing the antigen and to minimize the amount of extranuclear material that might contribute nonspecifically to the measured nuclear fluorescence. If a fluorescein isothiocyanate (FITC)-conjugated specific antibody is available, the method is simplified to two steps. If not, an antibody sandwich is built up in the single nucleus by the addition of a specific monoclonal nonconjugated antibody and a secondary, FITC conjugated antibody, in a second and a third step, sequentially. For the special purpose of staining bromodeoxyuridine (BrdUrd) with an antibody recognizing BrdUrd in single-stranded DNA, two additional steps are inserted in the procedure before the application of specific antibody to denature the DNA by HCl and subsequently to restore the pH.

Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.

The effect of 10(-6) M tamoxifen on MCF-7 cells adapted to growth in 0.5% foetal calf serum has been studied. The growth inhibitory effect of this tamoxifen concentration was abolished by simultaneous addition of 10(-8) M oestradiol, indicating that tamoxifen may exert its effect via binding to the oestrogen receptor. Flow cytometric cell cycle analysis of tamoxifen-treated cultures showed an increase in the proportion of cells in the G1 phase of the cell cycle. By exposing the cells to BUdR before flow cytometry the growth fraction was determined and found to be dramatically decreased in tamoxifen-treated cultures. Cells were not only arrested in the G1 phase but also in the G2 phase of the cell cycle. A few colonies of MCF-7 cells were resistant to 10 days treatment with 10(-6) M tamoxifen.

Seven patients with mycosis fungoides early plaque stage with nondiagnostic histology had single-cell DNA content measured by flow cytometry for estimation of clonal ploidy. A total of 63 skin specimens were examined by histology and DNA measurements concurrently during the course of the disease. In addition, six patients had blood samples studied. All seven patients demonstrated aneuploid DNA histograms when the specimens were obtained from skin lesions. In 36 specimens the aneuploid peaks were hyperdiploid. By sequential studies one patient demonstrated two different aneuploid cell clones, one located in the hyperdiploid region and one located in the hypotetraploid region. All patients developed mycosis fungoides which were histologically confirmed, and the time from first aneuploid DNA histogram until diagnostic histology varied from 5 to 21 months (median, 12 months). In six of the patients a normal diploid DNA histogram was found of peripheral blood lymphocytes. The finding of aneuploidy in patients with early mycosis fungoides who still have a nondiagnostic histology emphasizes the value of flow cytometry as a complementary diagnostic aid which facilitates an early diagnosis.