Cellular growth and metabolic adaptations to nutrient stress environments in tumor microregions

Sutherland, Robert M.; Freyer, James P.; Mueller‐Klieser, Wolfgang; Wilson, R. E.; Heacock, Craig S.; Sciandra, James J.; Sordat, Bernard

doi:10.1016/0360-3016(86)90070-2

Cited by 49 publications

(25 citation statements)

References 8 publications

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“…Factors such as nutrient supply, oxygen tension, pH and other microenvironmental conditions may affect the cellular sensitivity to paclitaxel (Carlsson et al, 1983;Mueller-Klieser, 1987). The three-dimensional arrangement of multicellular spheroids provides a cellular micromilieu that includes different environmental conditions in different parts of the spheroid, and it has been shown that spheroids from both normal and malignant tissues maintain several biochemical and morphological characteristics similar to those present in the corresponding tissue in vivo (Sutherland et al, 1986;Sutherland, 1988). Thus, testing paclitaxel on spheroids may provide additional information concerning paclitaxel effects in vivo.…”

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confidence: 99%

Proliferation, migration and invasion of human glioma cells exposed to paclitaxel (Taxol) in vitro

Terzis

Thorsen²,

Heese³

et al. 1997

Br J Cancer

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Summary Paclitaxel (Taxol), an anti-cancer drug derived from Taxus species, was tested for its anti-migrational, anti-invasive and antiproliferative effect on two human glioma cell lines (GaMg and D-54Mg) grown as multicellular tumour spheroids. In addition, the direct effect of paclitaxel on glioma cells was studied using flow cytometry and scanning confocal microscopy. Both cell lines showed a dose-dependent growth and migratory response to paclitaxel. The GaMg cells were found to be 5-10 times more sensitive to paclitaxel than D-54Mg cells. Paclitaxel also proved to be remarkably effective in preventing invasion in a co-culture system in which tumour spheroids were confronted with fetal rat brain cell aggregates. Control experiments with Cremophor EL (the solvent of paclitaxel for clinical use) in this study showed no effect on tumour cell migration, cell proliferation or cell invasion. Scanning confocal microscopy of both cell lines showed an extensive random organization of the microtubules in the cytoplasm. After paclitaxel exposure, the GaMg and the D-54Mg cells exhibited a fragmentation of the nuclear material, indicating a possible induction of apoptosis. In line with this, flow cytometric DNA histograms showed an accumulation of cells in the G/M phase of the cell cycle after 24 h of paclitaxel exposure. After 48 h, a deterioration of the DNA histograms was observed indicating nuclear fragmentation.

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confidence: 99%

Proliferation, migration and invasion of human glioma cells exposed to paclitaxel (Taxol) in vitro

Terzis

Thorsen²,

Heese³

et al. 1997

Br J Cancer

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“…The advantage of cell line spheroids is that they are relatively easy to obtain and to maintain in culture. Treatment-related changes of the growth kinetic of spheroids and the outgrowth of tumour cells present established and reproducible endpoints (25,32,33). Using spheroids of two biologically different human glioblastoma cell lines, we have shown in this study that irradiation can enhance the cytotoxic effect of TZM, but the exerted effect is less than additive in terms of spheroid growth and migrational behaviour.…”

Section: Discussionmentioning

confidence: 59%

“…After 10 days in culture, spheroids with diameter of 250 μm were selected for the experiments. These spheroids do not present central necrosis and have very few hypoxic cells (24,25).…”

Section: Methodsmentioning

confidence: 99%

The inhibition of proliferation and migration of glioma spheroids exposed to temozolomide is less than additive if combined with irradiation

Fehlauer

Muench²,

Richter³

et al. 2007

Oncol Rep

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Abstract. The aim of this study was to investigate the effect of temozolomide (TZM) in combination with X-rays on proliferation and migration in human glioma spheroids. Multicellular spheroids were derived from GaMg and U87 cell lines. Spheroids were treated with various concentrations of TZM (5 μmol, 0.025 mmol, 0.05 mmol) and irradiation (RT). Proliferation and migration assays were performed. For GaMg spheroids, the proliferation inhibition was 30% (RT), 71%, 79%, 85% (for various TZM concentrations) and 78%, 83%, 90% following RT+TZM. For U87 spheroids, the inhibition of proliferation was 52% (RT), 62%, 78%, 88% (TZM), and 73%, 87%, 92% (RT+TZM). Inhibition of migration for GaMg was 30% (RT), 37%, 63%, 78% (TZM), and 56%, 75%, 84% (RT+TZM). For U87, migration inhibition was 29% (RT), 48%, 52%, 67% (TZM), and 62%, 67%, 73% (RT+TZM). Radiotherapy enhancement ratio (RER) of GaMg/U87 spheroid proliferation was 1.4/1.7 (5 μmol TZM), 1.3/1.8 (0.025 mmol TZM), and 1.4/1.4 (0.05 mmol TZM). RER for migration of GaMg/U87 was 2.2/1.9 (5 μmol TZM), 1.7/1.8 (0.025 mmol TZM), and 1.5/1.4 (0.05 mmol TZM). In terms of inhibition of proliferation and migration, irradiation can lead to an enhancement of the TZM effect in human glioma spheroids, which is less than additive. IntroductionGliomas are the most common primary human brain tumours and prognosis is poor, in particular with high-grade tumours such as glioblastoma multiforme (GBM). Intensive surgery followed by radiotherapy with or without adjuvant chemotherapy is considered as standard therapy for primary brain tumours (1). Treatment of brain tumours with adjuvant chemotherapy, mainly 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), has only resulted in minor improvements (2-4). Phase I-II clinical studies with temozolomide (TMZ) have demonstrated that this drug crosses the blood-brain barrier and has activity against malignant glioma (5-8).TMZ in the treatment of primary and recurrent malignant brain tumours is still a topic of clinical trials due to the promising pre-clinical and clinical reports. Recently, Stupp et al found a clinically meaningful increase, by a factor of 2.5, in the survival rate at two years, from 10% with radiotherapy alone to 27% with radiotherapy plus TZM in a randomized phase III trial. The addition of temozolomide to irradiation was associated with improvements in median progression-free survival (6.9 vs. 5.0 month) and overall survival (14.6 vs. 21.1 month) (9).Since radiotherapy in combination with TMZ might be assumed as gold standard in the treatment of brain malignancies in future (10), it is of interest to investigate further what the pre-clinical effects are on various human glioma cell lines. In a study with the human glioma cell line U373 treated as monolayer cultures, an additive effect was reported for TMZ and X-rays (11). In glioma cells lines U251 and D384, combinations of TMZ and X-rays showed additional as well as supra-additional cytotoxic effects (12).We have chosen spheroid...

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“…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-'.…”

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confidence: 95%

“…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;. Concurrently with the increased rate of synthesis of the ORPs during hypoxia, we have seen increased resistance to adriamycin .…”

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confidence: 96%

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

Wilson

Keng²,

Sutherland³

1990

Br J Cancer

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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...

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Cellular growth and metabolic adaptations to nutrient stress environments in tumor microregions

Cited by 49 publications

References 8 publications

Proliferation, migration and invasion of human glioma cells exposed to paclitaxel (Taxol) in vitro

Proliferation, migration and invasion of human glioma cells exposed to paclitaxel (Taxol) in vitro

The inhibition of proliferation and migration of glioma spheroids exposed to temozolomide is less than additive if combined with irradiation

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

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