High fertilization prediction by flow cytometric analysis of the CD46 antigen on the inner acrosomal membrane of spermatozoa.

The study was set up to determine the relationship between the human sperm acrosome reaction and fertilization in couples undergoing routine in-vitro fertilization (IVF) treatment. Prospective data analysis was carried out on all IVF patients during a 6 month period. Exceptions were those patients having insufficient sperm concentration to allow both acrosome reaction determination and insemination. The main outcome measures were the prediction of fertilization in IVF patients using flow cytometric analysis of the spontaneous and ionophore-induced acrosome reaction [giving the acrosomal response to ionophore challenge (ARIC) score] in the male partner's spermatozoa versus standard analytical methods of sperm motion parameters and morphology. Stepwise logistic regression indicated only two independent factors predictive of fertilization: ARIC score (chi 2 = 109.6, P < 0.0001) and post-Percoll % motility (chi 2 = 8.8, P < 0.003). Of patients with an ARIC score of > 10, 92% had > 30% of oocytes fertilized; 100% of patients with an ARIC score of < 10 had < 30% fertilization of oocytes. The sensitivity and specificity of the assay system were 1.00 and 0.82 respectively. The results would indicate that the ARIC test as measured by flow cytometric analysis of CD46 binding is a sensitive and specific assay for use in the prediction of fertilization in IVF patients, thus enabling direct channelling of those patients with ARIC scores of < 10 into the more invasive micro-assisted fertilization schemes.

Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody.

The study was designed in order to investigate the action of progesterone on the spontaneous and ionophore-induced human spermatozoa acrosome reaction in vitro. The principle of the assay system is flow cytometric analysis of CD46 antibody binding to the inner acrosomal membrane. The technique is a simple and objective method of analysis, allowing fluorescent analysis of a large segment (5000 spermatozoa) of the spermatozoa population under investigation, with concomitant isolation of the live fraction of the spermatozoa population. Four concentrations of progesterone (1, 25, 50, and 100 microg/ml) were examined for their effects on spermatozoa capacitated for 4 and 24 h. In addition, motility parameters were examined by the CellSoft 2000 automated semen analyser system. Analysis of variance revealed that progesterone had no effect on either the spontaneous acrosome reaction or the ionophore-induced acrosome reaction at both 4 h and 24 h of spermatozoa capacitation times. Further, no effects on sperm motility parameters or on spermatozoa viability could be attributed to progesterone. We therefore conclude that progesterone has no objectively measurable effects on either the sperm acrosome reaction or sperm motility parameters, as measured in normal sperm populations.

Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: an indication of cell cycle defects.

In the present study, both post-irradiation DNA synthesis and G2 phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by 3H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G2 phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G2 phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G2 accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cycle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G2 phase accumulation developed by Lavin et al. (1992) in characterizing AT heterozygote-like cell cycle anomaly in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle.

Reduced induction of P53 protein by gamma-irradiation in ataxia telangiectasia cells without constitutional mutations in exons 5, 6, 7, and 8 of the p53 gene.

Ataxia telangiectasia (AT) is an autosomal recessive disease of childhood with several phenotypic characteristics. One of the hallmarks of this syndrome is its hypersensitivity to ionizing radiation, which is believed to be due to defects in DNA repair/processing. In addition to radio-resistant DNA synthesis, both fibroblasts and lymphoblastoid cell lines derived from these patients have been shown to have an impaired G1 arrest and prolonged G2 accumulation of cells indicating a defect in the regulation of cell cycle after irradiation. Since the (tumor suppressor) p53 protein has been reported to participate in the regulation of G1 arrest after irradiation, the possibility of p53 gene mutation and deregulating cell cycle in AT needed to be examined. We used the PCR amplification and DNA sequencing methods to detect mutations in the hypermutable exons (5-8) of germline p53 in fibroblast cells from 3 AT homozygotes. No mutation was found in any of these exons. In order to determine the role of the p53 protein in G1 arrest, its levels were measured before and after gamma-irradiation by flow cytometry in both AT and normal cells. Radiation-induced p53 protein levels in the AT cells varied from 6 to 60% compared to the normal cells, indicating a reduced induction of the protein in AT. These results suggest that mutation in the AT gene affects the p53 induction by irradiation and may, thus, alter the cell cycle regulation in the AT patients.

Pentoxifylline potentiates ionophore (A23187) mediated acrosome reaction in human sperm: flow cytometric analysis using CD46 antibody.

The study was designed to investigate the effects of pentoxifylline on the acrosome reaction of human spermatozoa in vitro, and to determine whether the reaction is differently modulated after sperm selection by multiple tube swim-up and Percoll buoyant density centrifugation. The acrosome reaction was induced in vitro by using calcium ionophore (A23187) and was detected by measuring the fluorescence of FITC-conjugated goat anti-mouse immunoglobulin bound to CD46 antibody (which binds to the CD46 receptor site on the inner acrosomal membrane) by flow cytometry. Spermatozoa separated on Percoll displayed significantly lower spontaneous acrosome reactions (P = 0.002) than did those separated by the swim-up technique. Pentoxifylline did not, by itself, induce acrosome reaction, but after induction with ionophore, it significantly increased the reaction (P = 0.003) and this increase was seen to be greater when Percoll separation was used as compared to the swim-up technique (P = 0.0002). We therefore conclude that Percoll selection of motile spermatozoa together with pentoxifylline treatment may be of value in assisted reproductive techniques, as an increased ARIC score arose after both treatments, and that flow cytometry allows a precise and rapid quantification of the acrosome reaction.

Mechanism of pentoxifylline mediated down-regulation of killer lineage cell functions.

The authors reported recently that endotoxaemia mediated elevated levels of tumour necrosis factor (TNF-alpha) and interleukin-1alpha (IL-1alpha) were involved in the pathophysiology of acute heat stroke patients. Pentoxifylline (PTX) is known to modulate neutrophil functions. In the present study the effects of PTX on lipopolysaccharide (LPS) and cytokine induced T-cell and macrophage (PhiM) activation, and on natural killer (NK) cell and lymphokine activated killer (LAK) cell mediated cytotoxicity were examined. Finally, the effect of PTX on the expression of adhesion molecules (LFA-1, Mac-1 and ICAM-1), and cytokine (IL-1alpha, IL-2, TNF-alpha, IL-6 and IFN-gamma) production and their surface receptor expression in response to LPS activation was investigated. PTX free cultures served as a control. Results revealed that PTX can down-regulate all the above-mentioned immunological parameters in a dosedependent manner. These findings might have far reaching clinical implications.

Chronic gamma-irradiation results in increased cell killing and chromosomal aberration with specific breakpoints in fibroblast cell strains derived from non-Hodgkin's lymphoma patients.

Cultured skin fibroblast cells from 6 patients with non-Hodgkin's lymphoma (NHL) and 2 clinically normal subjects were compared for cell survival and chromosomal aberration after chronic gamma-irradiation. Fibroblasts from an ataxia telangiectasia (AT) homozygote and an AT heterozygote were used as positive controls. Following irradiation, fibroblasts from all 6 NHL patients showed an increase in both cell death and chromosomal aberration (breaks and rearrangements) compared to the normal subjects. The difference in the frequency of chromosomal aberration between the normals and the NHL patients remained virtually unchanged over a period of 24-72 h post irradiation incubation of the cells. Cell cycle analysis by flow cytometry carried out in 1 normal and 1 NHL fibroblast cell strain showed that more cells representing the NHL patient were in G2/M phase compared to the normal at various times of cytogenetic analysis. While the AT homozygote appeared to be the most radiosensitive, the AT heterozygote showed a slightly higher incidence of cell death and chromosomal aberration than the normals. The cellular and chromosomal radiosensitivity of fibroblast cell lines from the NHL patients differed slightly from that of the AT heterozygote but clearly occupied an intermediate position between the AT homozygote and the normal subjects. Cells from 3 of the NHL patients showed radiation-induced specific chromosomal breaks involving chromosomes 1, 2, 6, 8, 10 and 11 which correspond to known fragile sites. Such breakpoints associated with increased radiosensitivity may be indicative of predisposition to malignancy in the patients studied.

Observations and an interpretation of dose-response relationships for cellular transformation in terms of induced (T) repair.

Both radiation-induced lethality and transformation frequency have been observed to plateau or diminish abruptly at relatively low dose levels and then increase with increasing doses, but at a reduced incremental rate. Discontinuities in dose-response relationships are postulated to correspond to the induction of a repair system ('T' repair) not functional at lower doses, i.e. below the induction threshold dose (Tt). Anomalies (discontinuities) in dose-response relationships and effects of dose fractionation previously noted are qualitatively explained in terms of this model.

Reactivation of neutron killed mammalian cells by gamma irradiation: the observations, possible mechanism and implication.

We have observed that combinations of neutron plus gamma ray exposure can significantly increase the colony forming ability of monkey and human cell cultures over the neutron dose alone. The "reactivation" of neutron killed mammalian cells by gamma rays is analogous to observations made in lower eukaryotic organisms and fits the pattern termed "T repair" previously postulated for yeast and protozoans.

Responses of two mammalian cell lines to low gamma-ray doses.

We have observed that low gamma-ray doses regularly improve the survival (plating efficiency) above that of unirradiated control cultures, which we postulate could arise through inducible DNA repair, and would have profound effects on the interpretation of cell culture experiments.

CHEF electrophoresis, a sensitive technique for the determination of DNA double-strand breaks.

It has been demonstrated that clamped homogeneous electrical field (CHEF) electrophoresis is a suitable method for the determination of DNA double-strand breaks in Chinese hamster ovary (CHO) cells. It allows the separation of DNA molecules up to 10 Mbp. The fraction of DNA fragments of this size is correlated with the number of radiation induced double-strand breaks. The resolution limit of the technique is equivalent to the effect of about 1 Gy (gamma-rays). Double-strand break repair was monitored after irradiation with Co-60 gamma rays and the repair time constant determined to t1/2 = 30-35 min. In combination with the detection of DNA by fluorescence, CHEF electrophoresis provides an easy and sensitive method for the determination of double-strand break repair which does not require the radioactive labelling of cells.

Enrichment of progenitor cells from human bone marrow by flow cytometry.

Mononuclear cells, harvested from fresh human bone marrow specimens by density gradient separation, were suspended in phosphate buffered saline and analyzed by flow cytometry in terms of the forward and right angle scattering of the incident light. The rectilinear distribution, obtained by plotting the intensity of light scattered in the forward and right angle directions, contained three regions of interest in which the percentage of cells (Mean +/- standard deviation) with respect to the total was as follows: Region 1: 17.6 +/- 9.9; region 2: 5.3 +/- 1.4; region 3: 71.7 +/- 9.4. Cells from each region were sorted by flow cytometry and plated in semi-solid agar containing cell conditioned medium supportive of myeloid colony formation. Cells from region 2 contained the majority of progenitor cells that gave rise to such colonies at a plating efficiency that rose in proportion to the extent by which the region 2 cells in samples was increased through sorting. This increase in plating efficiency was 6 to 43 fold. Thus, region 2 of the cytometric distribution of cells from normal, unstained human bone marrows was a good source of myeloid progenitor cells.

The use of flow cytometry to measure glucocorticoid-induced killing of lymphoid cell lines.

A flow cytometer method was developed to measure glucocorticoid-induced death in sensitive lymphoid cells. The murine lymphoma cell lines, R1.1, S49.1 and WEHI 7.1, and the human T-lymphocyte cell line, MOLT-4, were exposed to 10(-8) to 10(-6) mol/L dexamethasone or methylprednisolone. The cytogram for unstained, unfixed cells, produced by plotting the axial light loss versus the right-angle scatter using a He-Ne laser as the light source, showed two clearly separated peaks corresponding to live and dead cells. The ratio of live to dead cells seen in the cytogram correlated with that obtained by trypan blue staining. The flow cytometry method offers a number of advantages: 300 to 500 cells/s can be counted, yielding speed and good counting statistics; unstained, unfixed cells can be used; and the live and dead cells can be sorted for plating or biochemical analysis. S49.1 and R1.1 cells were sensitive to methylprednisolone and dexamethasone in the 10(-6) to 10(-8) mol/L concentration range, while MOLT-4 and WEHI 7.1 cells were less sensitive. After a 48-h exposure to 10(-8) mol/L dexamethasone, S49.1 and R1.1 cell cultures had 30% and 38% dead cells, respectively, while WEHI 7.1 and MOLT-4 cell cultures had less than 5% dead.

Selective cytotoxicity of calmidazolium and trifluoperazine for cycling versus noncycling C3H10T1/2 cells in vitro.

Effects of two anticalmodulin drugs, trifluoperazine and calmidazolium, on normal and transformed C3H10T1/2 cells were examined in vitro. As indicated by reduction of plating efficiencies in the presence of these drugs, the intrinsic sensitivities of normal and transformed cells were similar and showed no consistent differences. Comparison of cell killing kinetics in cycling and noncycling cell populations revealed that both drugs were preferentially cytotoxic for cycling cells. This differential cytotoxicity for cycling versus noncycling cells could provide a basis for exploitation of anticalmodulin drugs in cancer chemotherapy.

Comparison of heat and/or radiation sensitivity and membrane composition of seven X-ray-transformed C3H 10T1/2 cell lines and normal C3H 10T1/2 cells.

C3H 10T1/2 mouse embryo cells were transformed by X-irradiation, and seven transformed clones were isolated and propagated as cell lines. Some of these cell lines produced tumors in syngeneic mice and grew in agarose while the normal C3H 10T1/2 cell line did not possess these characteristics. Exponentially growing cell cultures with comparable cell-cycle distributions as measured by flow cytometry were tested for heat and X-ray sensitivity. The heat and X-ray sensitivity varied randomly compared to the normal cell line. One cell line was more heat resistant and one more heat sensitive than the normal cell line, and the others had sensitivities comparable to the normal cell line. Measurements on some of the biochemical parameters of the particulate fraction of cells after sonication and 24,000 X g centrifugation showed that altered thermal sensitivity was not correlated with protein, cholesterol, or phospholipid content of this fraction.

The use of flow cytometry to measure X-ray survival in cultured T lymphocytes.

Light-scattering signals produced in a flow cytometer containing unstained, irradiated T-lymphocytes (MOLT-4 cell line) were analysed by plotting the axial light loss versus right-angle scatter. The resulting three-dimensional scattergram separated into two regions, corresponding to live and dead cells, as confirmed by trypan blue staining. The method is simple, rapid, allows large numbers of cells to be measured, avoids staining artifacts and is suitable for measuring radiation-induced killing down to 0.5 to 0.1 Gy.

Inhibition of DMSO-induced differentiation by hyperthermia in a murine erythroleukemia cell system.

Friend erythroleukemia cells were induced by dimethyl sulfoxide (DMSO) into erythroid differentiation, as characterized by the production of hemoglobin. Induction increased with DMSO concentrations up to 1.5% v/v, at which point about 90% of the cell population produced hemoglobin as measured by a benzidine-staining technique. Heat treatment at 39.0-40.5 degrees C during a 7-day-incubation period, for differentiation in the presence of DMSO, resulted in the inhibition of hemoglobin induction. Also, acute heat treatments at 41.5-46.0 degrees C before or after the addition of DMSO resulted in the inhibition of DMSO induction. This effect was greatest when DMSO was present during heating. The results support the conclusion that hyperthermia inhibits the differentiation process which is induced by DMSO treatment.

Effects of oxygen and misonidazole on cell transformation and cell killing in C3H 10T1/2 cells by X rays in vitro.

The effects of oxygen (air) and misonidazole on the transformation and killing of 10T1/2 cells by X rays were examined. The oxygen effect for the cell transformation end point was very similar to that for cell killing. Misonidazole enhanced both cell killing and cell transformation to a similar extent. The enhancement of both end points by misonidazole occurred only in the absence of oxygen during irradiation and was of lesser magnitude than that observed for oxygen. These results demonstrate that the radiation chemical processes leading to cell killing and cell transformation, respectively, are affected similarly by these two enhancers of radiation action.