The effect of prolonged hypoxia on growth and viability of Chinese hamster cells

Summary Cultured cells maintained in very low oxygen levels alter their structure, metabolism and genetic expression. Culture conditions for cells were modified to minimise variation of nutrients and to allow normal survival levels after 24 h of hypoxic exposure. Under these hypoxic conditions, glucose consumption and lactate production rates were similar to aerobic rates until about 12 h after which the hypoxic rates increased. DNA and protein synthesis rates are continuously inhibited to about 48% or 55% of the respective aerobic rates. During this period of decreased protein synthesis, a set of proteins termed oxygen regulated proteins (ORPs), exhibits enhanced relative synthesis. The molecular weights of the five major ORPs are approximately 260, 150, 100, 80 and 33 kDa. While increased relative synthesis of oxygen regulated proteins is partly due to increased levels of mRNA which encode these proteins, the mechanism of enhanced synthesis of ORPs may be more complex.Hypoxia and related pathophysiologic environmental and cellular metabolic changes can be determining factors for the effectiveness of treatment of tumours using radiation therapy (Hall, 1978), hyperthermia (Gerweck et al., 1979) or chemotherapy (Teicher et al., 1981). Cells are resistant to irradiation virtually immediately upon introduction of severe hypoxia and are sensitised quickly after oxygen addition (Michael et al., 1973). Slower responses to hypoxia include increased hyperthermia and adriamycin resistance (Li & Shrieve, 1982;Smith et al., 1980).The most notable metabolic change during hypoxia is the increased rate of glucose consumption. Enhanced synthesis of selected proteins after hypoxia has been reported in plants (Guttman et al., 1980) and mammalian cell lines (Sciandra et al., 1984;Heacock & Sutherland, 1986). These stress proteins have been termed oxygen regulated proteins (ORPs) to denote proteins whose synthesis is enhanced during hypoxia and repressed after addition of oxygen. ORPs may be very similar to proteins synthesised after prolonged glucose deprivation (Sciandra et al., 1984). To understand the molecular basis of resistance to various treatments and rationally plan therapeutic treatments, the basic biochemical events occurring during hypoxia must be studied. (Chen, 1977). Additionally, rodent cell lines were free of human cell contamination by lactate dehydrogenase isozyme determination (Dietz & Lubrano, 1967).Experimental cultures were initiated 20-24 hours before each experiment, except where noted, such that approximately 106 cells existed per 100 mm diameter glass Petri dish. When dishes of differing size were used, the number of cells and volume of medium was changed proportionally to the measured surface area. Loosely attached and floating cells were removed by pipetting off the growth medium and BME rinse. Attached cells were removed by 5-O min incubation at 37'C with 5 ml of 0.1% trypsin (Worthington). Trypsin was inactivated by addition of 5 ml of growth medium before determination of cell number and cell vol...

Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Enhanced synthesis of stress proteins caused by hypoxia and relation to altered cell growth and metabolism

Heacock

Sutherland²

1990

“…The adenylate energy charge of tumour cells subjected to microenvironmental stress is controlled by the balance between the rates of energy generation and energy consumption (Calderwood et al, 1985;Rotin et al, 1986;Gerweck et al, 1992). Exposure of cells to hypoxia leads to continuously decreasing rates of DNA and protein synthesis and cessation of cell proliferation (Born et al, 1976;Shrieve et al, 1983;Pettersen et al, 1986;Heacock and Sutherland, 1990). Cell lines might differ in the capacity to regulate and turn off these energy-requiring processes (Gerweck et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The ATP formation is reduced markedly in tumour cells in a hypoxic microenvironment, mainly because of inhibition of respiration but also because of inhibition of glycolysis by low PHe (Rotin et al, 1986;Gerweck et al, 1993). ATP utilization is subsequently reduced, leading to decreased DNA and protein synthesis and cessation of cell proliferation (Born et al, 1976;Heacock and Sutherland, 1990;Casciari et al, 1992). The rate of energy deprivation during exposure to hypoxia at normal or low PHe might thus differ between tumours because of differences in the capacity of the tumour cells to generate energy and/or to reduce the energy consumption.…”

mentioning

confidence: 99%

Energy metabolism in human melanoma cells under hypoxic and acidic conditions in vitro

Skøyum

Eide²,

Berg³

et al. 1997

Summary The response to treatment and the malignant progression of tumours are influenced by the ability of the tumour cells to withstand severe energy deprivation during prolonged exposure to hypoxia at normal or low extracellular pH (pHe). The objective of the present work was to demonstrate intertumour heterogeneity under conditions of microenvironment-induced energy deprivation and to investigate whether the heterogeneity can be attributed to differences in the capacity of the tumour cells to generate energy in an oxygen-deficient microenvironment. Cultures of four human melanoma cell lines (BEX-c, COX-c, SAX-c, WIX-c) were exposed to hypoxia in vitro at PHe 7.4, 7.0 or 6.6 for times up to 31 h by using the steel-chamber method. High-performance liquid chromatography was used to assess adenylate energy charge as a function of exposure time. Cellular rates of glucose uptake and lactate release were determined by using standard enzymatic test kits. The adenylate energy charge decreased with time under hypoxia in all cell lines. The decrease was most pronounced shortly after the treatment had been initiated and then tapered off. BEX-c and SAX-c showed a significantly higher adenylate energy charge under hypoxic conditions than did COX-c and WIX-c whether the PHe was 7.4, 7.0 or 6.6, showing that tumours can differ in the ability to avoid energy deprivation during microenvironmental stress. There was no correlation between the adenylate energy charge and the rates of glucose uptake and lactate release. Intertumour heterogeneity in the ability to withstand energy deprivation in an oxygen-deficient microenvironment cannot therefore be attributed mainly to differences in the capacity of the tumour cells to generate energy by anaerobic metabolism. The data presented here suggest that the heterogeneity is rather caused by differences in the capacity of the tumour cells to reduce the rate of energy consumption when exposed to hypoxia.

“…Since a retarding effect of hypoxia on cycle progression has been observed both in vitro (Born et al, 1976) and in vivo , the tumour size related increase in T, may be due to increased tumour hypoxia. When tested functionally however, the time course of repopulation was found identical in small and large AT478 tumours .…”

Section: Can T Of Explants Be Measured In Vitro?mentioning

confidence: 99%

Cell kinetic analysis of murine squamous cell carcinomas: a comparison of single versus double labelling using flow cytometry and immunohistochemistry

Schultz-Hector¹,

Begg²,

Hofland³

et al. 1993

Summary The study was originally set up to measure accurate cell kinetic parameters in two murine squamous cell carcinomas (scc) for comparison with radiobiological data on proliferation during radiotherapy. The tumours, AT84 and AT478, were both moderately well differentiated aneuploid scc. In the course of the study, several Tumour cell kinetic studies are not only essential in investigating the many factors regulating proliferation but have also been shown to be useful for predicting the response to therapy (Silvestrini et al., 1984;Tubiana et al., 1989;Begg et al., 1992;Begg, 1993). Most cell kinetic studies have been carried out using either radio-labelled thymidine or a thymidine analog, which are incorporated into cells undergoing DNA synthesis. Such labelling of S-phase cells can be carried out in vivo or in vitro. In patients, administration of radiolabelled DNA precursors is precluded because of radiation hazards. Consequently, clinical studies have employed labelling of biopsy material in vitro (Silvestrini et al., 1984;Tubiana et al., 1989; The original goal of these studies was to measure accurately the cell kinetic parameters in two murine tumours for correlations with radiobiological characteristics of tumour proliferation during radiotherapy . The radiobiological data were available for tumours at two sizes, necessitating kinetic measurements at both these sizes. It was planned to use both FCM and IHC methods to ensure an accurate and correct description of the cell kinetics. The study was then also used to attempt to answer the questions posed above in these two well described animal tumour models. It thus provides comparisons of FCM and IHC methods, in vitro with in vivo labelling, single vs double labelling, the magnitude of intratumoural variations in kinetic parameters, in addition to the influence of tumour size. Materials and methodsTumours and tumour growth rate AT84 and 478 are both fairly well differentiated murine squamous cell carcinoma lines, derived from spontaneous tumours of the oral and vaginal mucosa respectively. An early, more slowly growing (AT478/4) and a late, much faster (AT478/25) generation of one tumour line were compared.