Differences in Growth Factor Sensitivity between Primary and Transformed Murine Cell Cultures Revealed by BrdU/Hoechst Flow Cytometry

Abstract

Spontaneous cell transformation is a common feature of all murine cell cultures grown in vitro for extended periods of time. During early passages, these cultures show either progressively reduced growth or complete cessation of growth; after such a ‘crisis’ they display increasing growth rates and unlimited lifespan. Here we use a novel bromodeoxyuridine/Hoechst flow-cytometric technique to examine the growth response of diploid and transformed cells of the murine species Micromys minutus under a variety of growth conditions. After spontaneous transformation, growth factor exposure results in increased G0/Gl cell recruitment and higher growth rates than shown by the nontransformed diploid cell fraction. Despite clonal heterogeneity, this difference is seen at all fetal calf serum (FCS) concentrations, although it is most pronounced with low serum. Epidermal growth factor and insulin are shown to act synergistically and promote growth equal to exposures of transformed cultures to 10% FCS. The observed differences in growth factor response between diploid and aneuploid cells could explain the reported lack of a classical growth crisis in growth factor-supplemented media during the spontaneous in vitro transformation of primary murine cell cultures.

Influence of Iron on Proliferation and Cell Cycle Kinetics on Cultured Malignant and Nonmalignant Cells

“…The width and the presence of more than one peak ( fig. 3a,b) in each cell cycle phase suggest the presence of more than one cell population with dif ferent contents of DNA [9], The increased number of cells in the G1 and the early S phase suggests that iron blocked the cell cycle of transformed cells before or immediately after the beginning of DNA synthesis ( fig. 3a,b.…”

“…2a,b). We suppose this was caused by an in creased dependence of transformed cells on growth factor stimulation [8,9], In serum-free conditions, extremely low concentrations of iron (0.5 \xM ferric nitriloacetate) were sufficient to supply HL-60 and K562 cells with iron, necessary for normal growth. The suppressive effect of iron in serum-free medium was not observed at such low concentrations as ( fig.…”

Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst‐propidium iodide stain

“…Many such investigations have used human peripheral blood lymphocytes (PBLs), normally in GO phase of the cell cycle, which were stimulated into cycle by the addition of a mitogen such as phytohaemagglutinin (PHA) (14,(16)(17)(18)(21)(22)(23)(24)(25)(36)(37)(38)(39)(40). Another approach has been to render fibroblasts quiescent by the removal of serum and then to stimulate them by the re-addition of serum (5,6,19,21,35,37,41). Melanoma cells (42), mouse CTLL-cells (17), and rat keratinocytes (34) have also been studied by this method.…”

“…If the resolution of the cytogram and the concentration of BrdUrd is sufficient, three rounds of replication can be observed. The entry and exit kinetics of the cells into successive S, GYM, and G1 phases can be measured and the effect of various growth factors (6,14,16,19,20,39), cytotoxic agents (14,17,18,22,33,35,41,42) or endogenous cell cycle disturbances (22,25,38,39) can be observed. Figure 2 shows typical data from initially synchronous GO/Gl phase PBLs stimulated with PHA.…”

“…The continuous BrdUrd labelling technique applying the BrdUrd/Hoechst quenching effect has been used to examine the cell cycle kinetics of quiescent cells which have been stimulated into cycle (6,14,16,19,21,(23)(24)(25)31,32,36,37). The method can also be applied to asynchronously dividing cells (10,11,13,29,30); this has the advantage that it does not require artificial cell synchronisation with possible interference in the pro- ever, to date, this has been used less extensively, possibly due to the more complex nature of the data generated.…”