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Plasma membrane fluidity of intact peripheral blood lymphocytes (PBL) of phenytoin-treated nonepileptic patients and phenytoin-treated CD4 + lymphoid cells H9 and K37 was determined by fluorescence anisotropy measurements. Anisotropy values of the membrane probe 6-(9-anthroyloxy) stearic acid were decreased in all cell types as compared with controls, indicating increased plasma membrane fluidity of phenytoin-treated cells. Specific binding of 125 I-labeled vasoactive intestinal peptide (VIP) to its cellular receptor CD4 on PBL was decreased in PBL of phenytoin-treated patients as compared with untreated, healthy subjects. Adsorption of a different ligand to the CD4 receptor on PBL, the human immunodeficiency virus type 1 (HIV-1), was likewise abolished to PBL of phenytoin-treated patients and phenytoin-treated CD4 + H9 and K37 cells, as assessed by indirect immunofluorescence. Subsequent HIV-1 infection of phenytoin-treated H9 and K37 cells was reduced as measured by indirect immunofluorescence and p24 antigen production. These data indicate that CD4 receptor availability for VIP and HIV-1 was reduced in phenytoin-treated cells. Using the DNA-specific dye Hoechst 33258, we examined cell cycle phase distributions of HIV-1 adsorbing and nonadsorbing H9 cells, as separated by flow cytometry. The majority of HIV-1 adsorbing cells were found to be in the G 2 /M phase, while nonadsorbing cells were mainly in the G 0 G 1 phase, during which plasma membrane fluidity is supposed to be increased. This study indicates that plasma membrane fluidization by phenytoin may serve to disrupt CD4 receptor function and emphasizes the impact of plasma membrane properties on HIV-1 adsorption and infection.

Cell kinetics of human renal cell carcinomas xenotransplanted into nu/nu mice were analysed using the bromodeoxyuridine (BrdUrd) labelling method. Tumours were removed 0.5-14 h after injection of the BrdUrd solution. The tumour cells were stained with fluorescein isothiocyanate conjugated anti-BrdUrd antibodies and propidium iodide (DNA content). From the flow cytometry data the relative movement was calculated. Relative movement data of variable intervals after BrdUrd labelling were subjected to a fit procedure using log-normal distributions for S phase transition (T(s)). The log-normal distributions were modified by inflation factors in order to get extremely asymmetric distributions. The best fits to the experimental data were obtained using wide asymmetric T(s) distributions, indicating that progression through S phase in solid human tumours is considerably heterogeneous. This implies that the potential doubling time (T(pot)) is longer than calculated from a single measured relative movement value obtained a few hours after BrdUrd labelling.

The effects of mitomycin C (MTC) alone and in combination with radiation on R1H rhabdomyosarcomas in WAG/Rij rats were studied. Growth delay was determined from tumor volume growth curves and proportion of tumor and host cells from flow cytometry measurements. Only minor growth delays were obtained with MTC at doses near the toxic level while irradiation applied in three fractions of 10 Gy in three weeks yielded growth delays of 27, 44, and 51 days measured at volumes of 2, 5, and 10 cm3, respectively. The combination of 1 micrograms/kg b.w. MTC and 10 Gy irradiation given in three weekly fractions resulted in 21, 28, and 29 days growth delay for the above tumor volumes. The lower effect of combination as compared to irradiation alone is discussed with the flow cytometry data. These data show that early after start of treatment the proportion of tumor cells remains constant in contrast to radiation alone where the fraction of tumor cells decreases because of influx of granulocytes and macrophages into the tumor. In contrast to the reduced effect on tumors the combination treatment was considerably more toxic for the animals than the single treatments.

The authors investigate if cytometric parameters can be used for discerning malignant and benign tumors as well as for the prognostic classification of malignant tumors. Measures on leukemias, brain and prostate tumors, colorectal carcinomas, and renal carcinomas taken by some study groups of the Institute of Biophysics and Radiobiology are cited and discussed with respect to the above mentioned aspects. The analysis showed that fractions of cells in phases of the cellular cycle as indicators of the proliferation rate are only of little diagnostic value because of the too great zone of dispersion. Especially the differentiation between benign and malignant tumors is not clear enough. In case of renal carcinomas, however, there is a marked correlation between prognosis and aneuploidy measured by cytometry. Thus the DNA index as a quantitative parameter of aneuploidy seems to be also of clinical interest for the determination of malignancy.

Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation (CV) from 2 to 16%; (ii) constant measurement CV or CV's increasing from G1 to G2 phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time. The fractions of cells in the G1, S, and (G2+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G1 fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.

Thirty human renal cell carcinomas were transplanted into NMRI (nu/nu) mice. The take rate from surgical specimens was 100%, and all tumors were established as permanent transplantable tumor lines. The xenotransplants were followed up to around passages 10-50. In addition to histology and volume growth the DNA indices (DI), proportion of tumor cells, and fractions of cells in the phases of the cycle were measured by flow cytometry. All 30 tumors retained their primary histologic structure and cellular morphology throughout all passages. The DI, as ascertained by the DNA content per cell of G1-phase tumor cells divided by the DNA content per cell of normal diploid G1-phase cells, remained the same as those in the original tumors in 21 of 27 xenotransplants. Three original lymph nodes or metastases were not available for flow cytometry. During passage, 4 of 30 tumors changed their DI. In 2 of these cases the tumor doubling time (td), the proportion of tumor cells versus the proportion of host cells, and the fractions of cells in the cell cycle phases changed simultaneously. All other tumor lines were stable in td, DI, proportion of tumor cells, and fractions of cells in the phases during serial transplantation. However, the measure of parameters varied considerably between individual tumors of every passage. Tumor growth rate was generally related to the prognosis of the patients from whom the tumor was derived.

128 tumors from three different human renal-cell carcinomas and from one human transitional bladder cancer were transplanted into NMRI mice treated with cyclosporin A at various dosages. Their acceptance rate, proliferation rate, and morphologic and growth characteristics were studied and compared with those of the same human tumors after transplantation into NMRI nu/nu mice. The acceptance rate was 80% in mice treated with cyclosporin A at 100 mg/kg-1 and 150 mg/kg-1/day. The highest acceptance rate was observed after transplantation into nu/nu (thymus-aplastic) mice (100%). Morphologically, the transplanted tumor was similar in the two animal groups and identical with the primary tumors. Metastases were not seen in either animal model. The NMRI mice treated with cyclosporin A had leukocyte infiltration into the transplanted tumor. The growth rate of the human tumors in normal mice depended on the cyclosporin A dosage, and was highest in the nu/nu mice. The proliferation rates of the transplanted human tumors, as judged by flow cytometry, were rather similar in all groups.

Individual properties of gynecologic specimens can produce artifacts in flow cytometric (FMC) measurements, possibly leading to false interpretations. An identification of such artifacts was undertaken by parallel FCM and microscopic investigations. One hundred fifty unselected cervical smears were measured by FCM using the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) for DNA and sulforhodamine (SR 101) for protein. Microscopic specimens were stained by the Papanicolaou technique, and a detailed cytogram was prepared from each smear. FCM discrimination of fluorochrome-stained superficial and intermediate cells was very difficult. On the other hand, a correlation could be established between the fraction of cells from the deeper epithelial layers in the microscopic cytogram and the mean protein content in the FCM histogram. Furthermore, the role of microorganism could be elucidated. Some microorganisms may produce a reduction of the protein content by cytolytic changes. Other microorganisms adhere to the cell surface, resulting in a misleading increase of the DNA fluorescence. Implications for the problem of false alarms are discussed.