Jerrold Fried

Method for the quantitative evaluation of data from flow microfluorometry

Abstract A new method is proposed for estimating the distribution of cells among the phases of the mitotic cycle using data from a flow microfluorometer. The method can be applied to asynchronous or synchronous cell populations, and is not limited to systems with very low coefficients of variation of fluorescence intensity. It is based on a mathematical model of the cell population having the following properties: (i) the population is separated into compartments, one consisting of G1 cells, another of G2 and M cells, and several of S phase cells which have synthesized different specified fractions of their DNA; (ii) the fluorescence intensity of cells in each compartment is normally distributed with the mean of the G2 + M compartment having a channel location twice that of G1; the S phase compartments have means at intermediate values. Coefficients of variation are the same for all compartments. The sizes of the compartments are determined by a least squares fitting procedure utilizing the observed data. The method and some of its limitations are illustrated by selected examples of simulated data from asynchronous and partially synchronized cell populations.

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. 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 CVs 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.

Multi-user system for analysis of data from flow cytometry.

A new program is described for the analysis of DNA histograms from flow cytometry. The fundamental model representing the cell population is similar to one described previously. It assumes the population is grouped into compartments, each consisting of cells having approximately the same DNA content. After staining the cells with an appropriate fluorochrome, the fluorescence distribution of cells within each compartment is assumed to be Gaussian. In the present algorithm, the parameters of the model can either be computed directly by the program from the data, or can be specified as input by the user. When synchronous cell populations lacking distinct G1 and G2/M phases are analyzed, the parameter values must first be obtained using an appropriate control. Percentages of cells in the various compartments are computed using a gradient search method described by Bevington.

Proliferative kinetics of central nervous system (CNS) leukemia

The proliferative kinetics of the leukemic cells in the cerebrospinal fluid (CSF) were studied in three adults with CNS leukemia. Two (G.H. and A.K.) had lymphoblastic leukemia and developed CNS leukemia after 18 and 8 months while receiving an intensive treatment regimen. One (J.L.) had acute myeloblastic leukemia and developed CNS disease after 5 years treatment with arabinosylcytosine and 6‐thioguanine. All were in marrow remission at the time of study. G.H. had had no therapy for CNS disease, A.K. had had intrathecal methotrexate 2 months previously, and J.L. had recently completed radiotherapy (1900 R) to the head. An Ommaya reservoir was placed in a lateral ventricle and 3H‐thymidine was injected into the reservoir every 12 hours for 10 days; samples of cells were obtained by lumbar puncture periodically for autoradiography. In all patients the flash 3H‐thymidine labeling index (LI) of the leukemic cells was < 2% (determined in vitro) and the mitotic index < 0.1%. After 10 days of 3H‐thymidine injections in vivo, the LIs of the leukemic cells were 55%, 36%, and 21% in G.H., A.K., and J.L., respectively. These findings indicate that leukemic cells may proliferate very slowly in the CNS, and stress the difficulty of eradicating CNS leukemia with chemotherapeutic agents which are only active against proliferating cells.

Quantitative Analysis of Flow Microfluorometric Data from Asynchronous and Drug-Treated Cell Populations*

Abstract A new method for the mathematical analysis of data from flow microfluorometry is applied to data from cell populations growing exponentially or exposed to hydroxyurea or cyclic AMP. The analysis is not completely automatic, but requires interaction with the investigator. An algorithm for carrying out the analysis is described and illustrated with an example. Possible methods for simplifying the procedure are discussed. Studies with hydroxyurea-treated cells indicated that many cells were arrested in early S phase as well as in G 1 . Some of the drug treated cells, especially those exposed for 3 to 4 days, had abnormally low fluorescence intensities. Cells treated with cyclic AMP for 72 hr accumulated mainly in the G 1 and G 2 phases, but some remained in S. Relatively pure fractions of G 1 cells (but not G 2 ) were obtained by cyclic AMP treatment followed by sucrose gradient fractionation.

Enhancement of the radiation response of cultured tumor cells by chloroquine

The treatment of tumor cells in culture with chloroquine after irradiation significantly reduces their survival, but no enhancement is seen when the cells are treated with the drug before irradiation. This suggests that the potentiation of the radiation response by chloroquine results from the impairment of the post‐irradiation recovery processes. Incorporation studies with melanotic melanoma cells in culture derived from human malignant melanoma demonstrate significantly higher uptake of the compound than in amelanotic melanoma, HeLa, or nonpigmented human diploid cells in culture. The significance of these findings is discussed in relation to the potential use of the drug as a radiosensitizer in selected human tumors.

Cell-mediated toxicity of interleukin-2-activated lymphocytes against autologous and allogeneic human myeloma cells

We studied the sensitivity of human myeloma (plasma cell leukemia) toward autologous and allogeneic lymphokine-activated killer (LAK) cells. Fresh plasma cell leukemia (PCL)-derived peripheral blood mononuclear cells (PBMC) and PBMC from 3 normal donors were cultured in the presence of recombinant interleukin-2 (rIL2; 1,000 U/ml) for subsequent use as cytotoxic effectors against fresh and continuously cultured myeloma cells. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, rIL2-induced PCL-PBMC lysed 48 +/- 19% (mean +/- 1 SD) of autologous myeloma targets, as compared to 89 +/- 5, 95 +/- 15, and 100 +/- 9% lysis of standard LAK-sensitive Daudi cells and allogeneic myeloma cell lines SKO-007, and RPMI-8226, respectively. Normal PBMC-derived rIL2-induced (LAK) cells exhibited a slightly lower cytotoxic reactivity against allogeneic targets (61 +/- 9, 60 +/- 6, and 81 +/- 8% cytolysis of SKO-007, RPMI-8226, and Daudi cells, respectively, at a 50:1 E:T ratio). Cytotoxicity against myeloma (PCL) of autologous PCL-derived killer cells could be significantly (at least 2-fold) enhanced when rIL-2-induced effector cells were preincubated for 18 h in the presence of recombinant Interferon-alpha rIFN-alpha; 1,000 U/ml). In summary, our results indicate the potential antitumor efficacy of rIL2- and rIL2 + rIFN-alpha-activated killer cells in human myeloma (PCL).

Establishment of a Near-Tetraploid B-Cell Lymphoma Line with Duplication of the 8;14 Translocation

The neoplastic cells of a patient with diffuse histiocytic lymphoma were grown in three systems: Soft agar, liquid culture, and by heterotransplantation into the nude mouse. In each case, the growing cells were shown to bear the same immunoglobulin heavy and light chain type as the original neoplastic cells (IgM-lambda). In both the liquid culture and nude mouse heterotransplant tumors, karyotypic analysis revealed the presence of pseudodiploid and near-tetraploid metaphases bearing one and two copies of an 8;14 translocation, respectively. From the liquid culture separate clones of pseudodiploid and near-tetraploid cells were isolated in soft agar, and separate cell lines SK-DHL2A and SK-DHL2B were established. Studies comparing the proliferative characteristics of the two lines showed no difference in cell population doubling time or clonigenicity in soft agar. Hence, the presence of two copies of the translocated material did not confer a proliferative advantage on cells containing it. Theses lines may be valuable in future studies of cell proliferation and oncogenesis in the human immune system.

Radiosensitivity of confluent density-inhibited cells.

Dose∕survival curves were obtained following x-irradiation of plateau- and log-phase 3T3 and HeLa cells. The 3T3 line, whose proliferative behavior resembles that of normal tissue in certain respects, showed striking differences in radiation response according to the phase of growth. The neoplastic HeLa cell line exhibited much smaller differences. These findings may have potential radiotherapeutic significance.

Quantitative analysis of cell cycle progression of synchronous cells by flow cytometry

Abstract Chinese hamster ovary (CHO) cells were synchronized by the mitotic selection procedure and their subsequent progression through the cell cycle was monitored by flow cytometry. It is shown that our previously proposed method for DNA histogram analysis is applicable even to highly synchronous cell populations lacking significant G1 and G2/M components, provided an appropriate control is used as a reference standard. We have also obtained estimates of cell cycle parameters from the DNA histograms in this system, including phase durations, relative cellular DNA synthesis rates as a function of position in S, and synchrony indices. In particular, the rate of DNA synthesis appears to be very low in early S phase in this cell line, giving rise to an unexpected early S phase peak in the histogram of asynchronous, exponentially growing cells.