Amaury Perez

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.

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.

Differences in the composition and in the efficiency of red cell production of normal and CML erythroid progenitor populations are highlighted by response to human c-kit ligand☆

Previous studies have suggested that erythroid progenitors derived from patients with chronic myelogenous leukemia (CML) in chronic phase may have reduced proliferative capacity. Considering recent evidence that mast cell growth factor (MGF) enhances the proliferative capacity of normal erythroid burst-forming units (BFU-E), we examined whether MGF could increase the proliferative potential of CML erythroid progenitors to normal capacity. To evaluate the total proliferative capacity achieved, the BFU-E were divided into four subpopulations (XL = extra large, L = large, M = medium, S = small) and colonies were aspirated to determine the cellularity of BFU-E from each subpopulation. MGF alone or in combination with MoT cell line conditioned medium (MoCM) or granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-3 (IL-3) significantly increased the proliferative capacity of erythropoietin (EPO) dependent CML and normal BFU-E. Although the total number of BFU-E generated were similar, the number of BFU-E with high proliferative potential were considerably less in CML BFU-E populations. BFU-E designated XL (129,000-431,000 cells) were only found in MGF cultures and only normal BFU-E had this proliferative capacity. BFU-E designated L were increased in both normal and CML BFU-E populations but less CML BFU-E had this proliferative capacity (mean number 25% of normal) and CML L BFU-E from 2/3 CML patients comprised fewer cells than normal L BFU-E. Normal BFU-E populations comprised 16-24% high proliferative BFU-E (XL + L) in contrast to 4-5% high proliferative BFU-E (L only) comprising CML BFU-E populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cytosine arabinoside and daunorubicin on survival and cell cycle progression of Chinese hamster ovary cells.

Abstract. Kinetic and cytotoxic effects of cytosine arabinoside (Ara‐C) and daunorubicin (DNR) on exponentially growing Chinese hamster ovary (CHO) cells were measured by flow cytometry and by a colony‐forming assay, respectively. With Ara‐C alone, increasing drug concentrations between 10‐7 M, for up to 27 hr, were associated with increased inhibition of cell progression through the S phase. Even at the very toxic concentration of 10‐4 M, however, cells were able to enter and progress slowly through S. DNR, which appears to enter these cells relatively slowly, was highly toxic even at 2 times 10‐7 M. It decreased the rate of progression through S phase and caused cells to accumulate in G2, except at the highest concentration (2 times 10‐5 M), at which progression was inhibited throughout the cycle. Simultaneous exposure of the cells to Ara‐C and DNR yielded cell cycle distributions similar to those of the former drug alone. When cells were exposed to a non‐lethal dose of Ara‐C and to a dose of DNR which was lethal to a fraction of the cell population (or conversely), either simultaneously or separated by a drug‐free interval, small, but in some cases significant, drug interactions were observed. These effects were not caused by druginduced redistribution of cells within the cell cycle, but may have been related to the effects of the non‐lethal drug on DNA synthesis rate.

Discrepancy between flow cytometric analyses of CALLA using two monoclonal antibodies.

Both the J5 and BA-3 monoclonal antibodies are considered to be specific for epitopes on the common acute lymphoblastic leukemia antigen (CALLA). Flow-cytometric analyses of three cell lines and one normal bone marrow sample using these antibodies as CALLA markers demonstrated that J5-labeled cells were always brighter than those labeled with BA-3, and that the ratio of their fluorescence intensities varied widely in the different systems. Furthermore, one of the lines, RPMI 8226, while positive for J5, appeared to be negative when labeled with BA-3, except for a slight displacement of the fluorescence distribution relative to the control. A possible explanation for the observed results is that the BA-3 binding epitope or epitopes on CALLA may vary in their number and/or accessibility to the antibody. These observations suggest that the use of a single monoclonal antibody to detect a cell surface antigen may be misleading, particularly when a negative result is obtained.