Measurement of proliferation activities in human tumor models: a comparison of flow cytometric methods

Proliferating cells in tumors may be of considerable relevance in cancer therapy. Not only do such cells dictate the rate of tumor progression, but evidence exists that they may also play an important role in the diagnosis and prognosis of tumor regrowth. Consequently, the identification of this subset of cells in the overall neoplastic cell population is of considerable importance. The aim of the present investigations was to compare four flow cytometric methodologies commonly used to study cell proliferation. These included nuclear antigen Ki67 detection, acridine orange (AO) and bromodeoxyuridine (BrdUrd) staining, and percent S-phase determinations. Three human tumor cell lines (HEp3, A549, H226) were examined in various stages of growth. Further, a direct comparison was made of the proliferation activities of HEp3 cells grown in culture or as xenografts in nude mice. The results showed that of the techniques investigated, detection of the nuclear antigen Ki67 may be most useful for marking proliferating tumor cells and determining tumor growth fractions.     

Heterogeneity in early and advanced gastric carcinomas by flow cytometric DNA analysis

We investigated 230 systematically sampled fresh specimens from 12 early and 26 advanced gastric cancer patients by DNA flow cytometry for heterogeneity in DNA content. Fifty-eight percent of the 12 early gastric cancers were uniformly diploid and 42% were uniformly aneuploid. Fifty-four percent of advanced cancers were uniformly diploid in superficial layers and 42% were uniformly diploid in deep layers, whereas 46% were uniformly aneuploid in superficial layers, and 50% were uniformly aneuploid and 8% were heterogeneously aneuploid and diploid in deep layers. Both diploid and aneuploid samples were obtained from 15% for advanced cancers, but ploidy heterogeneity did not occur in early cancers. Heterogeneity for DNA index (more than one aneuploid DNA index) occurred in 46% of whole thickness of advanced cancers, in 19% of superficial layers of advanced cancers, and in 8% of early cancers. We concluded that DNA ploidy determination using superficial layer specimens may be reliable in early gastric cancer but must be interpreted with care in advanced cancer.     

Intracellular calcium and pH alterations induced by tri-n-butyltin chloride in isolated rainbow trout hepatocytes: a flow cytometric analysis

The effects of tri-n-butyltin chloride (TBT) on ionic homeostasis on isolated trout hepatocytes were investigated by flow cytometry (FCM), using the Ca(2+)-sensitive and pH-sensitive fluorescent probes Indo-1 and SNARF-1, respectively. Cell viability was monitored concurrently. Treatment of hepatocytes with 1 and 5 microM TBT caused a rapid and sustained elevation of cytosolic free Ca2+ concentration [Ca2+]i and an important cytoplasmic acidification. These changes were dependent upon TBT concentration and were maintained over 60 min, the maximum exposure period investigated. At 0.5 microM TBT, there was a slight but not significant increase in [Ca2+]i and a significant reduction in intracellular pH (pHi) only after 60 min of exposure. A rise in [Ca2+]i and cytoplasmic acidification were observed before loss of viability was detectable. Experiments carried out in Ca(2+)-free medium suggest that TBT mainly mobilizes Ca2+ from intracellular stores in trout hepatocytes. The cytoplasmic acidification following TBT exposure seems to be caused by the combination of intracellular Ca2+ mobilization and by direct action of TBT. The present results suggest that ionic homeostasis perturbations could be early events in the mechanism of cell injury by TBT.     

Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms

Genetic damages are frequently found in both tumor and normal cells at carcinogen exposed areas in the patients with upper aerodigestive tract cancer. These phenomena are explained by the multistage process and/or field cancerization theories. The c-erbB-2 proto-oncogene has been amplified in many human tumors including breast, stomach, kidney and lung cancers. To study the possible evidence of multistage process and/or field cancerization in the development of gastric adenocarcinoma, the amplification statuses of c-erbB-2 proto-oncogene using the Southern hybridization technique were evaluated at the 45 gastric adenocarcinoma specimen sets consisting of tumor tissue, adjacent normal tissue (within 2 cm of the primary tumor), metastatic tissue and normal stomach tissue (at least 5 cm away from primary tumor). As a result, c-erbB-2 proto-oncogene at 2 specimen sets (4.4%) was amplified 2- to 4-fold to normal control status. In these 2 cases, c-erbB-2 proto-oncogene at histologically normal tissue adjacent to tumor tissue was amplified. And, the metastatic tissue of 1 case also exhibited c-erbB-2 proto-oncogene amplification of which the degree was less than that of tumor tissue. From these results, we were able to suspect that c-erbB-2 proto-oncogene amplification in the normal tissue adjacent to tumor tissue could be a biomarker of premalignant changes in a small proportion of gastric adenocarcinoma patients. And, this result might suggest the possible role of multistage process and/or field cancerization in the development of gastric adenocarcinoma.     

Flow cytometric determination of metallothionein levels in human peripheral blood lymphocytes: utility in environmental exposure assessment

Metallothioneins (MT) are ubiquitous, low-molecular-weight proteins that exhibit high binding affinities for heavy metal ions. The expression of these cysteine-rich proteins is induced in response to various types of chemical and physical stresses and therefore can be used to assess human exposure to cytotoxic environmental agents. In the current study, MT levels of human peripheral blood lymphocytes were determined using an MT-specific antibody and flow cytometry. Treatment of human whole blood ex vivo with CdCl2 was found to induce a concentration- and time-dependent increase in lymphocyte MT levels at concentrations as low as 0.3 microM and within a 12-h period. Interestingly, differences were observed in the magnitude of cadmium-induced MT levels in the lymphocytes of six human test subjects. Two members of the study population exhibited CdCl2-induced cellular MT levels that were up to twofold greater than the lymphocytes of other human subjects. While the lymphocytes of most test subjects exhibited a symmetric (unimodal) distribution of cadmium-induced MT-specific fluorescence, the cells of two individuals displayed a heterogeneous (nonuniform) distribution of MT levels. Dual-parameter flow cytometric analysis using phenotype-specific antibodies indicated that variations in the responsiveness of subpopulations of lymphocytes to CdCl2 were responsible for the heterogeneous distribution of MT-specific cellular fluorescence. T-helper (CD4-positive) and T-suppressor/cytotoxic (CD8-positive) lymphocytes expressed higher cellular levels of MT than other lymphocyte subpopulations (i.e., B lymphocytes, natural killer cells). Our results suggest that MT protein levels of peripheral blood lymphocytes, as determined by this flow cytometric method, may be used to assess human exposure to toxic metals and to characterize various quantitative/qualitative aspects of the response of individuals to cadmium and possibly to other types of environmental stresses.     

Prognostic value of S-phase fraction in lymph-node-negative breast cancer by image and flow cytometric analysis

A two-year multicentre prospective study was performed from 1992 to 1995 in order to evaluate the real value of various kinds of coral blocks as bone substitute in maxillofacial surgery. This study was supported by the French National Agency for Research Valorization (GBM/TEP procedure). Ten Maxillofacial Surgery Units were included. During this time, 28 coral blocks (23 patients) of two different shapes were used as malar implants for correction of congenital or acquired zygomatic hypoplasia. The mean follow-up was 1.8 year (min: 1.5; max: 2). The tolerance was perfect for 89% of cases. The radiologic opacity never decreased more than 30% and the volume augmentation was always stable at the end of the follow-up period. Three implants were removed because of septic complications. Rigid fixation between the implant and the zygomatic bone appears to be the most important factor of success. On the other hand, the surgical approach (endo- or exo-buccal) does not seem to influence the success rate. The aesthetic improvement was always evaluated as satisfactory and stable by the patients and the surgeons. The authors discuss the real value of the various kinds of biomaterials and especially coral, comparing their personal data with those of the literature. Coral blocks clearly constitute a safe and reliable bone substitute, but further investigations are required to determine its long-term behavior.     

Increased apoptosis of adult rat lymphocytes after single neonatal vitamin A treatment (hormonal imprinting). A flow cytometric analysis

Newborn rats were treated with a single dose of vitamin A (retinol) and apoptosis of peripheral lymphocytes was studied by flow cytometry in adult age. Vitamin A treatment (hormonal imprinting) caused a moderate, however significant elevation in the number of apoptotic lymphocytes after three months. Dexamethasone or Concanavalin-A alone did not influence apoptosis significantly. However, in the neonatally retinol treated rats dexamathasone significantly elevated the quantity of apoptotic lymphocytes related to the control or Concanavalin-A treated control cells. The results call attention to the prolonged effect of hormonal imprinting in a new index and to the possible dangerous effects in human, neonatally treated with vitamin A.     

Regulation of alphaIIb beta3 function in human B lymphocytes

We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.     

CD48 expression on leukocytes in infectious diseases: flow cytometric analysis of surface antigen

BACKGROUND: The function of CD48, one of the pan leukocyte cell surface antigens, is not yet well understood. CD48 was recently shown to enhance the CD40-mediated activating signal to B lymphocytes. As CD48 is one of the activation antigens of monocytes, neutrophils and lymphocytes, a change of its expression on the cells could be expected in infectious diseases. METHODS AND RESULTS: Leukocytes from 27 healthy controls and 97 patients with various infectious diseases were stained with anti CD48 antibody and analyzed by flow cytometry. On monocytes and neutrophils, the CD48 expression was increased in all of the patients with varicella, measles, rubella, infectious mononucleosis, streptococcus tonsillitis, sepsis and appendicitis. On lymphocytes, a significant increase of CD48 was also detected in the patients with the same diseases, except those with sepsis or appendicitis. The normalization of increased CD48 expression was confirmed on monocytes at the convalescent phase. CONCLUSION: These data suggest that CD48 expression on leukocytes reflects the disease activity of infectious diseases, especially of viral infections.     

Prognostic significance of DNA flow cytometric analysis in patients with nasopharyngeal carcinoma

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a prevalent malignant tumor among Southern Chinese. Previously, the authors described the prognostic significance of a serum antibody assay to a recombinant Epstein-Barr virus Bam HI-Z replication activator protein (ZEBRA) in NPC patients with long term follow-up. In this study, the authors further reported the use of DNA flow cytometry (DNA-FCM) as an additional technique for determining the prognosis of NPC patients in the same series. METHODS: One hundred and forty-three archival biopsies from 110 NPC patients were deparaffinized and subjected to DNA-FCM analysis. DNA ploidy state and various proliferative indices (PI) of the tumors were correlated with patient survival and frequency of recurrence. RESULTS: Among the biopsies analyzed, 119 were histologically positive NPC and 24 were negative. Fifty-one tumor biopsies that fulfilled the guideline criteria of the DNA Cytometry Consensus Conference were correlated with the clinical manifestations of the patients. Among them, 43 tumors (84%) were DNA diploid and 8 (16%) were aneuploid. Two PI, S-phase fraction (SPF) and proliferation fraction (PF), appear to be potentially useful prognostic indicators. For example, PF in patients who developed locoregional recurrence (15.1%) and distant recurrence (16.4%) after radiation therapy both were significantly higher than PF in patients who were in complete remission (8.2%) (P = 0.0005 and P = 0.004, respectively). Significant differences in SPF between patients with distant recurrence (10.6%) and those in remission (5.7%) also was found (P = 0.005). Using Kaplan-Meier analysis, patients with high PF, high SPF, and aneuploid tumors had significantly poorer 12-year survival rates (35%, 26%, and 28%, respectively) than those patients with low PF, low SPF, and diploid tumors (77%, 67%, and 59%, respectively) (P < 0.0009, P < 0.004, and P < 0.01, respectively). CONCLUSIONS: Determination of tumor PI and DNA ploidy state by DNA-FCM at diagnosis of NPC can be potentially useful in selecting a poor prognostic subgroup of NPC patients. These parameters may enable oncologists to plan for more stringent treatment strategies such as hyperfractionated and accelerated radiation therapy or concomitant chemoradiotherapy for these patients.     

Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by a flow cytometric assay

BACKGROUND: Chemoresistance in some hematologic malignancies has been associated with overexpression of P-glycoprotein, which is encoded by the MDR1 gene (also known as PGY1). However, inconsistencies in data on frequency and clinical relevance of multidrug resistance in B-cell chronic lymphocytic leukemia (B-CLL) may reflect a need for improved techniques to detect this overexpression. PURPOSE: Our purpose was to measure P-glycoprotein activity in peripheral blood cells of B-CLL patients and to analyze possible clinical correlations (disease duration, prior treatment, Rai disease stage, lymphocyte counts, and disease progression). METHODS: P-glycoprotein activity was assayed in peripheral blood cells of 42 consecutive B-CLL patients (22 treated and 20 untreated). We used dual fluorescence in a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123, which is transported from the cell by the P-glyprotein pump. Leukemia cells were costained with monoclonal antibody Leu12/CD19, and rhodamine 123 efflux was measured. Expression of MDR1 and MDR3 (also known as PGY3) messenger RNA (mRNA) was quantitatively evaluated by polymerase chain reaction (PCR) in 26 cases. RESULTS: Marked rhodamine 123 efflux was observed in 34 (81%) of the 42 cases and was abolished in the presence of multidrug resistance inhibitors. Rhodamine 123 efflux was not associated with Rai stage, lymphocyte counts, duration of disease, or disease progression. Although rhodamine 123-negative cases were about equally distributed among untreated and previously treated patients, the percentage of cells with rhodamine 123 efflux was significantly lower for untreated patients than for those treated with chemotherapy regimens including at least one multidrug resistance-associated drug. MDR1 mRNA was detected in 25 of 26 cases and MDR3 mRNA in all 26. MDR1 mRNA expression was significantly correlated with rhodamine 123 efflux, whereas MDR3 mRNA expression was not significantly correlated; MDR1 and MDR3 mRNA expression was not significantly associated with Rai stage, prior treatment, or disease progresssion. CONCLUSIONS: These findings suggest that P-glycoprotein overexpression in B-CLL is intrinsic rather than acquired and that P-glycoprotein activity is enhanced after exposure to multidrug resistance-associated drugs. This enhanced activity does not seem to be associated with more aggressive disease. Our results also indicate that an assay of P-glycoprotein function combined with PCR is suitable for clinical multidrug resistance screening. IMPLICATIONS: Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.     

Determination and derivatization of protein thiols by n-octyldithionitrobenzoic acid

n-Octyl-5-dithio-2-nitrobenzoic acid (ODNB), the reaction product of n-octane-1-thiol and Ellman's reagent, can be isolated in crystalline form. Like Ellman's reagent, ODNB can be used for the titration of SH groups in proteins, but with considerable advantages: Due to the absence of one negative charge and the presence of a lipophilic hydrocarbon chain, titrations of protein thiols are significantly faster. Normally, endpoints are reached after 5-30 min, while the corresponding reaction with Ellman's reagent may take 1-2 h. Another advantage of ODNB is that it finds access to thiol groups which normally remain undetected. With myosin S1, for example, ODNB reacts with a thiol group which is not reactive with Ellman's reagent. Although ODNB has detergent properties, reaction of this thiol group as a consequence of denaturation seems unlikely, because ODNB was used below its critical micelle concentration and all other thiols of myosin S1 remained buried. The n-octylthiol derivatives of proteins formed by reaction with ODNB can be used for transient protection of thiols, for example, during purification procedures, or for a reversible blocking of SH groups involved in biochemical reactions.     

Coexpression of CD69 and HLADR activation markers on synovial fluid T lymphocytes of patients affected by rheumatoid arthritis: a three-colour cytometric analysis

Haploid and diploid strains of Aspergillus nidulans have been repeatedly treated with the strong mutagen 6-N-hydroxylaminopurine (HAP) which causes only base substitutions. An enormous amount of variability may be rapidly accumulated in haploid or diploid strains of A. nidulans. In particular, in the diploids the analysis of the results shows that after 12 cycles of treatment the conidia differ from each other for about ten recessive lethals and therefore probably for several hundreds of mutations. The viability of the heterozygous multiply mutant diploids is not appreciably different from that of untreated controls. In the diploid strains the accumulated variability was very high. The treatment of a haploid strain during vegetative growth also caused a strong accumulation of mutations, even though deleterious, because they can be maintained in the heterokaryotic condition.     

Preparation of fine needle aspiration biopsy samples for flow cytometric analysis

In order to improve the quality of flow cytometric (FCM) DNA histograms, a new preparatory method was tested on samples obtained by fine needle aspiration biopsy (FNAB) of breast tumors. Twenty-four samples were obtained in vivo (group 1), and 20 were obtained from surgically resected specimens (group 2). Tumors from both groups were aspirated twice each. The first sample was injected directly into 70% ethanol, whereas the second sample was pretreated with a mixture of Tween-20 and citric acid solution (Tween-20 CA) before ethanol fixation. The coefficient of variation (CV) of G0-G1 peaks of Tween 20 CA-pretreated samples varied from 1.85 to 5.10 (mean, 3.3) in group 1 and from 1.87 to 3.72 (mean, 2.77) in group 2. The CV of G0-G1 peaks of ethanol-preserved samples ranged from 2.28 to 7.22 (mean, 5.23) in group 1 and from 1.78 to 4.04 (mean, 3.48) in group 2. The CV values of histograms obtained by the new protocol were significantly lower (group 1, P < .05; group 2, P < .01).     

DNA flow cytometric analysis in patients with operable non-small cell lung carcinoma

Fresh surgical specimens of tumors from 60 patients with previously untreated non-small cell lung carcinoma (NSCLC) who underwent radical surgery between January 1991 and October 1992 were investigated by means of flow-cytometry. The nuclear DNA measurement was carried out using a Facscan (Becton, Dickinson, USA). Analysis of the DNA content was performed in all 60 patients whilst cell cycle analysis was possible in 41 cases (68.3%). Forty-two of the 60 cases (70%) were aneuploid and 18 (30%) were diploid. The overall mean value of DNA index was 1.5. Diploid NSCLC were compared with aneuploid tumors: no significant differences in age distribution, sex ratio, histology and staging were found between the two groups (P > 0.05). An S-phase proportion of more than 10% was found in 30 out of 41 patients (73.2%). Early cancer deaths were reported in four patients (6.6%): the aneuploidy rate was very close in these patients (75%) and in the remaining surviving patients (69.6%). An S-phase proportion of more than 10% was found in 100% of early cancer deaths and in 70.2% of the remaining cases; such a difference seems of some importance although it was not statistically significant (P = 0.071). In conclusion, flow-cytometry studies seem to be a useful tool in the understanding of the biological behavior of patients with NSCLC. In the present prospective report there were no significant correlations between DNA measurements and clinical outcome, however, these results suggest that a high S-phase proportion should be seen as a possible prognostic indicator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular mechanisms of erythrophagocytosis: flow cytometric quantitation of in vitro erythrocyte phagocytosis by macrophages

A vaccine that uses a live, attenuated human immunodeficiency virus (HIV) may offer the best hope of a vaccine against acquired immunodeficiency syndrome (AIDS). A recent improvement should increase the safety of the live-virus approach: a "suicide gene" inserted into the viral RNA, which causes infected cells to die when treated with ganciclovir. We envision using this strategy not only to prevent AIDS, but also to treat it.     

Investigations on the pharmacokinetics of alpha-lipoic acid in healthy volunteers

Recent medical publications postulate a connection between the Chronic Fatigue Syndrome (CFS) and disturbed regulation of the circulation, manifesting itself during orthostatic stress testing. Four studies were published on the circulatory response on prolonged head up tilt testing. Numerous CFS patients displayed postural tachycardia or syncope during the test. However, many CFS patients examined had had orthostatic symptoms prior to the examination. It is not certain that cardiovascular dysregulation is present in CFS patients without orthostatic symptoms. It is also not clear whether such a dysregulation would be the effect of physical inactivity or a manifestation of a subtle form of autonomic neuropathy.     

Use of flow cytometric CD34 analysis to quantify hematopoietic progenitor cells

This review summarizes our experiments on flow cytometric analysis of CD34 positive mononuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only 1%-9% of the CD34+ MNC. Due to the variable numbers of CD34+ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA.(ABSTRACT TRUNCATED AT 250 WORDS)