Changes in growth characteristics and macromolecular synthesis on recovery from severe hypoxia

Abstract

Summary Chinese hamster ovary cells subjected to severe hypoxia stop growing. When oxygen was reintroduced growth resumed, but at a slower rate. The longer the hypoxic stress, the slower the recovery growth rate. Six hours of hypoxia caused very little decrease in growtb rate while a 24 h period almost halved the rate.Short hypoxic periods resulted in almost no growth lag, while longer periods caused significant lag. Clonogenic survival was 60% after 12 h of hypoxia and rose slowly during recovery, reaching control levels after 60 h. Following 24 h of hypoxia, survival remained around 60% throughout recovery. The cell cycle distribution after hypoxia was similar to that of aerobic cultures. After 4-6 h of recovery, a subpopulation of cells entered S phase, and reached G2 by 12 h. During this time few G2-M cells divided. With longer recovery, cells much larger than aerobic cells emerged, containing greater than 4C DNA content and enhanced amounts of RNA. When these cells were isolated, they exhibited slightly slower growth kinetics, greatly lengthened lag time and decreased survival when compared to aerobic cells or the smaller cells. Most of the extra DNA and RNA was lost within one cell cycle.We have found (Wilson et al., 1986) (Koch et al., 1973;Tannock, 1972) and to certain drugs (Born & Eichholtz-Wirth,. 1981;Martin & McNally, 1980, Smith et al., 1980. Hypoxic regions have also been demonstrated in poorly oxygenated human tumours (Wendling et al., 1985). While We have found a set of proteins, the oxygen regulated proteins (ORPs), whose synthesis is greatly enhanced by hypoxia (Heacock & Sutherland, 1986;Sutherland et al., 1986; al., 1985). Briefly, live cells were spun down and resuspended in complete medium to a concentration of 106 cells ml-'. The cells were then kept at 4°C for up to 4 h before staining and flow cytometry. To stain the cells, they were first permeablised by mixing 0.2 ml of the cell suspension with 0.2 ml of a solution containing 0.1 5N NaCl, 0.08N HCI and 0.1% Triton X-100. After one minute, 0.9 ml of the AO stain (121tgml-' AO, 0.15N NaCI, 0.125M Na2 HPO4, 0.037M citric acid, pH 6.0) was added. The sample was then filtered to remove clumps and placed in the flow cytometer. The excitation wavelength was 488 nm. Green fluorescence (IGFL, DNA content) was collected through a 530nm long-pass filter and red fluorescence (IRFL, RNA content) through a 640nm long-pass filter after the fluorescence signals were separated by a 560nm dichroic filter. Forward angle light scatter (FALS), an indicator of cell size, was also measured. Twenty thousand cells were collected for each histogram. Centrifugal elutriationCells were separated into populations enriched in different phases of the cell cycle using centrifugal elutriation. Populations of cells larger than that of normal G2-M phase cells were also obtained using this method. The protocol of Keng et al. (1980) was used. Briefly, 0.5-1 x 10' trypsinised CHO cells were loaded into the separation chamber at a flow rate of 35 ml min-' and rotor spe...

Hypoxia-induced angiogenesis and vascular endothelial growth factor secretion in human melanoma

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