Design: Expression of GH and for Growth Hormonebinding-protein (GH-bp) in eutopic and ectopic endometrium during the menstrual cycle of women with endometriosis, and in normal endometrium of women not affected by endometriosis, were studied by immunohistochemistry.Materials and Methods: We evaluated tissue specimens of eutopic and ectopic endometrium obtained from 10 patients with endometriosis who underwent operative laparoscopy in different phases of the menstrual cycle and from 15 normal women matched for menstrual phase, as controls.The indirect avidin-biotin complex immunoperoxidase assay was performed on dewaxed and rehydrated sections of 10% formalin fixed, paraffin embedded tissue.Anti GH and anti GH-bp monoclonal primary antibodies with a dilution 1:50 phosphate-buffer solution were used.Results: Immunohistochemical analysis for GH showed in controls and in eutopic endometrium of patients with endometriosis a strong immunostaining in the glandular cells during secretory phase only.In ectopic endometrium of endometriosis lesions a positive staining for GH was found in 50% of cases in the ovarian cysts and in the peritoneal adhesions there was no staining in the epithelial endometrial cells.Immunohistochemical analysis for GH-bp showed in controls and in eutopic endometrium of patients with endometriosis a strong immunostaining in the stromal cells during secretory phase and a weak staining in the proliferative phase.No staining was observed in the epithelial cells of ovarian cyts.Conclusions: In human endometrium there is an active role of GH produced by epithelial cells which is probably active on the stroma that expresses the specific receptor for this hormone during the secretory phase.Furthermore in endometriosis there is an alteration of mechanisms regulating cell proliferation and differentiation.The progressive modifications of the pattern of expression of GH and of GH-bp in eutopic endometrium and in ectopic endometrium of different endometriotic lesions, suggests that the the different lesions present different cellular populations with different cell growth characteristics.

Although cyclosporine (CsA*) is a widely used immunosuppressive agent, the side effects of CsA therapy constitute a major drawback in immunosuppression. Mainly, these effects are those associated with suppression of the immune system and organ toxicities such as renal dysfunction. Use of drugs that may be clinically safe and synergize with CsA may be a useful approach to minimize CsA dosage and alleviate CsA side effects. Pentoxifylline (PTX), a phosphodiesterase inhibitor initially characterized as a hemorheologic drug, was shown to inhibit tumor necrosis factor (TNF) production and has several in vivo benefits that may be attributed to PTX inhibition of TNF production (1-5). In this article, we addressed the possibility that PTX may potentiate CsA-mediated suppression of immunological functions. The results showed that PTX in a selective manner potentiates cell-mediated lymphocytotoxicity (CML) inhibition by CsA among several other in vitro functions. The median-effect analysis developed by Chou (6) was performed and demonstrated synergism and dose reductions in PTX and CsA combination treatment in CML inhibition. Peripheral blood mononuclear cells were separated by Ficoll-Hypaque density gradient, washed, and cultured in complete RPMI 1640 supplemented with 10% fetal bovine serum (ultralow endotoxin Hyclone, Inc., Logan, UT) or 10% human AB serum in case of mixed lymphocyte reaction (MLR). PTX (Sigma) was diluted in phosphate-buffered saline while CsA (Sandoz, Geneva, Switzerland) as a white powder was solubilized initially in ethanol and further diluted in the culture medium. Cells were seeded at 2×105 cell/well in 96-microwell plates and cultured in triplicate with 5 μg/ml phytohemagglutinin (PHA) or 10 ng/ml staphylococci enterotoxin A (SEA) for 3 days in the presence or absence of various concentrations of drugs. MLR was performed in triplicate by the coculture of 105 irradiated allogenic cells with 105 responder cells in the presence or absence of drugs for 6 days. Cells were pulsed with 1 μCi of 3[H]thymidine (Amersham, Bucking-hamshire, England) for an additional 18 hours and harvested to quantitate incorporated radioactivity. CML was generated by 4-hour incubation of effector cells, which were drug- or medium-treated MLR-generated responders, with 5×10451Cr-labeled Con A-blast target cells. Specific 51Cr release was quantitated as follows: 100 × (experimental - spontaneous)/(maximum - spontaneous). TNF activity (cytotoxin) was quantitated by the actinomycin-D-treated WEHI-13VAR cells as described in detail elsewhere (7); titers were taken at ED50 that gave 50% toxicity. TNF-α sandwich-type ELISA (monoclonal and polyclonal rabbit anti-TNF-α antibodies were obtained from Endogen, Boston, MA) was also performed. Samples, described in text, were analyzed by flow cytometry using FACScan (Becton Dickinson, BD, San Jose, CA) using fluorescein isothiocyanate-conjugated antibodies for interleukin (IL)-2R (CD25) and LFA-1α (CD11a) and phycoerythrin-conjugated antibodies for ICAM-1 (CD54). Mean channel fluorescence was obtained using LYSIS II software. The median-effect analysis was performed and assisted by computer software (6,8). The model analyzed dose-response parameters and quantitated synergism and dose-reduction indices. Details of the analysis are given in the text. PTX inhibited the proliferative responses of peripheral blood mononuclear cells due to the mitogen (PHA), antigen (SEA), and alloantigen (MLR) in dose-dependent manners; the ED50s were 400 μM, 700 μM, respectively. However, these concentrations exceed in many folds the physiological concentrations that can be attained in vivo (1,9). This is also true for the concentrations, though lower, required to inhibit TNF production in PHA, SEA, and MLR responses (ED50s were 50 μM, 35 μM, and 150 μM, respectively). Therefore, we addressed the possibility that the combination of CsA and PTX might result in synergistic inhibition of immunological functions. We chose therapeutically attained low concentrations of CsA (12.5 nM-100 nM) that would give 50% or less inhibition of immunological functions to facilitate comparison between the effect of CsA when alone and the effect of CsA in combination with PTX (also at therapeutic levels: 250 nM-2 μM). The combination of PTX (which alone was ineffective) and CsA results in isoeffective (addictive), rather than synergistic, inhibitions (Table 1). This is also the case with inhibition of TNF activity (Table 1). The adhesion molecules LFA-1α and ICAM-1 were also tested by flow cytometry in antigen and alloantigen cultures; there were no synergistic inhibitions (data not shown). However, PTX seems to selectively potentiate CsA-mediated suppression of CML (Table 1). Thus, the inclusion of therapeutic, yet ineffective, in vitro doses of PTX on CML may result in potentiation of CML inhibition by CsA. However, proper assessment of synergism and quantitation of dose reductions requires that the effects of drugs at their effective doses, alone or in combination, be carefully analyzed, such as by the use of the median-effect model. For reliable analysis (10), the drugs were combined in an equipotent combination (approximately in a ratio of 1:10,000) which reflects the relative difference in the activities of the drugs. Figure 1 shows the median-effect plots for the drugs alone and in combination. The plots were constructed to generate the median-effect parameters, median-effect dose (Dm), and slope (m), needed for the analysis. The linear correlation coefficients (r) indicate excellent correlation linearity (r>0.93) and applicability of the model. The slopes that were near 0.5-0.6 indicate semiflat dose-response curves for the effects of the drugs on CML. Table 2 shows detailed analysis of doses of each drug alone (Dx) and combination doses (Dc) that were calculated by the median-effect equation for the entire range of fractions affected (i.e. CML inhibitions). Dose-reduction indices (DRIs) indicated the degree of the fold decrease of the drug dose when in combination to give the same effect when the drug was alone. CsA dose reduction was almost constant (DRI = 5-6) over the entire range of fractions affected; approximately 80% decrease was achieved over the dose alone that gave the same effect. Interestingly, the PTX dose in combination required to inhibit CML at a given fraction was dramatically reduced and PTX reduction indices correlated with increasing doses of CsA, PTX, and higher CML inhibitions (r=0.87, 0.84, and 0.93, respectively, at P<0.01) (Table 2). Combination indices over the entire CML-inhibited fractions confirmed the synergistic effect. For example, at 0.5 CML fraction inhibited, combination index (CI) was calculated using the CI equation as follows: Equation which is less than 1 and thus is synergistic. The DRI for CsA and PTX at 0.5 fraction (i.e., 50% CML inhibition) was calculated using the formula:Equation with DRI defined as Dx/Dc. The results show that PTX potentiates CsA-mediated suppression of CML and this action is probably selective among other immune functions. This observation was confirmed by the use of the median-effect analysis for CML (Table 2). Median-effect analysis was also performed for MLR and showed no synergistic inhibitions with combination treatment (data not shown). The potency of PTX action in CML increased in combination treatment, as suggested by the increased dose-reduction indices of PTX (Table 2). This may suggest that most of the potentiation is on PTX-mediated pathways. The mechanism underlying this observation is not understood. The possible selective potentiation in CML among other functions suggested that unique pathways in the action of CML are the target of PTX. However, in vitro studies on CML lytic processes, such as granule exocytosis, perforin action, and DNA fragmentation, may be advisable to test whether they are subject to PTX regulation. PTX at therapeutic levels does not seem to directly influence cell-mediated immune functions and PTX inhibits CML at doses rather larger than those required to inhibit antigen and alloantigen responses. Thus, the direct benefits of PTX as a single drug in bone marrow transplantation are controversial (2,3,9). The in vivo mechanism of PTX in modulating immunological reactions is not known, but several lines of evidence suggest it is more potent in vivo than in vitro (1,2,11). It is more likely that PTX influences CML in vivo in synergism with CsA than alone. This may explain the reduction of organ toxicities seen in graft-versus-host disease patients in some clinical trials (1,2,5). Thus, the discrepancy of usefulness of PTX in clinical trials (1,2,5,9,12) may be explained in part by lack of controlling CsA synergism, which requires consideration of the two drug schedules. Also, in vivo discontinuous treatment with PTX yielded inhibition not only of TNF-α, but also of other proinflammatory cytokines (IL-1β, IL-6, and IL-8) (11), which may explain why oral but not continuous intravenous administration of PTX is beneficial. Although it is still to be confirmed in vivo, the results may be useful in two ways: (1) CsA dose reduction with combined treatment of PTX may reduce long-term CsA side effects, such as nephrotoxicity; and (2) prophylactic therapy for graft-versus-host disease and possibly acute rejection episodes may be achieved with the combination therapy. Careful assessment of PTX in conjunction with CsA therapy may be needed, such as in animal models or in randomized clinical trials. Acknowledgments. The authors thank Dr. Peter Ernst for his helpful comments. We also thank Ali El-Sofi for his technical assistance.Figure 1: Median-effect plot for CsA (•), PTX (▪), and their combination (▴) in inhibiting CML. Different doses (8 or 9 0.5 log data points) of CsA alone, PTX alone, or in combination (1 μM:10 mM ratio, i.e., 1:10,000) were tested for inhibition of CML. Dosage of drugs is expressed as log μM and log mM in case of CsA and PTX, respectively. Combination dosage is expressed as log sum of combined doses. At each dose, the fraction of CML inhibited (fa) is divided by the fraction of CML uninhibited (fu) and expressed as log (fa/fu). Data points were fitted by linear regression. Linear correlation coefficients (r), slope (m), and the median-effect dose, i.e., the dose that gives 50% inhibition in the median-effect plots (Dm), were generated from each plot. For CsA, r=0.99, m=0.64, and Dm=77 nM. For PTX, r=0.94, m=0.52, and Dm=2.5 mM. For combination, r=0.93, m=0.64, and Dm=0.15 (13.6 nM CsA/136 μM PTX). Drug doses were not toxic to cells as assessed by trypan blue exclusion dye. Data are mean of two independent experiments (of four performed) with approximate similar parameters.Footnotes Abbreviations: CI, combination index; CML, cell-mediated lymphocytotoxicity; CsA, cyclosporine; DIR, dose reduction index; IL, interleukin; MLR, mixed lymphocyte reaction; PHA, phytohemagglutinin; PTX, pentoxifylline; SEA, staphylococci enterotoxin A; TNF, tumor necrosis factor. REFERENCES 1. Bianco JA, Almgren J, Kern DL, et al. Evidence that oral pentoxifylline reverses acute renal dysfunction in bone marrow transplant recipients receiving amphotericin B and cyclosporine. Transplantation 1991; 51:925. Cited Here | View Full Text | PubMed | CrossRef 2. Bianco JA, Appelbaum FR, Nemunaitis J, et al. Phase I-II trial of pentoxifylline for the prevention of transplant-related toxicities following bone marrow transplantation. Blood 1991;78:1205. Cited Here | PubMed | CrossRef 3. Strieter RM, Remick DG, Ward PA, Spengler RN, Lynch JP, Larrick J. Cellular and molecular regulation of tumor necrosis factor production by pentoxifylline. Biochem Biophys Res Commun 1988;155:1230. Cited Here | PubMed | CrossRef 4. Tilg H, Eibl B, Pichl M, et al. Immune response modulation by pentoxifylline. Transplantation 1993;56:196. Cited Here | View Full Text | PubMed | CrossRef 5. Vincenti FG, Vasconcelos M, Birnbaum JL, et al. Pentoxifylline reduces the first-dose reactions following OKT3. Transplant Proc 1990;25:57. Cited Here | PubMed 6. Chou TC. The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Chou TC, Rideout DC, eds. Synergism and antagonism in chemotherapy. New York: Academic Press, 1991:61. Cited Here 7. Khabar KSA, Siddiqui S, Armstrong JA. WEHI-13VAR: a stable and sensitive cell line for tumor necrosis factor activity. Immunol Lett 1995;46:107. Cited Here | PubMed | CrossRef 8. Chou J, Chou T.C. Dose effect analysis with microcomputers: quantitation of ED50, LD50, synergism, antagonism, low dose receptor binding and enzyme kinetics [IBM-PC series]. Cambridge, UK: Elsevier, 1988. Cited Here 9. Beelen DW, Sayer HG, Franke M, et al. Constant intravenous pentoxifylline infusions in allogenic marrow transplant recipients: results of a dose escalation study. Bone Marrow Transplant 1993;12:363. Cited Here | PubMed 10. Khabar KSA, Armstrong, JA, Ho M. Analysis and examination of cytokine interactions by the median-effect model: an example with antiviral action of tumor necrosis factor and interferon-γ. J Interferon Res 1992;12:161. Cited Here | PubMed | CrossRef 11. Neuner P, Klosner G, Schauer E, et al. Pentoxifylline in vivo down-regulates the release of IL-1β, IL-6, IL-8 and tumor necrosis factor-α by human peripheral blood mononuclear cells. Immunology 1994;83:262. Cited Here | PubMed 12. Stockschlader M, Kalhs P, Peters S, et al. Intravenous pentoxifylline failed to prevent transplant-related toxicities in allogenic bone marrow transplant recipients. Bone Marrow Transplant 1993;12:357. Cited Here | PubMed © Williams & Wilkins 1996. All Rights Reserved.

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.

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.

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.

In the present study, both post-irradiation DNA synthesis and G1 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 cyle 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 anomally in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle.

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.

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.

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.

Both radiation-induced lethality and transformation frequency have been observed to plateu 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 (T(t)). Anomalies (discontinuities) in dose-response relationships and effects of dose fractionation previously noted are qualitatively explained in terms of this model.

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.

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.

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.

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.

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.

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.

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.

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.