The role of heterogeneity of tumour cell populations in biological activity and sensitivity to chemotherapeutics.

The biological activity of a rat ascites leukaemia subline isolated by isopycnic centrifugation was compared to that of the original tumour both in vivo and in vitro. The subline had different marker chromosomes, a longer cell cycle time and decreased sensitivity to vinblastine as compared to the original tumour.     

The relationship between the RBE of alpha particles and the radiosensitivity of different mutations of Chinese hamster cells

The RBE of α-particles in different mutations of Chinese hamster cells was determined with the aim of identifying differences in the sensitivity to x-ray and α-particle-induced DNA damage. Two parental lines of Chinese hamster cells and four radiosensitive mutants were irradiated with different single doses of x-rays and α-particles and clonogenic cell survival was determined. Radiosensitivity to x-rays varied by a factor of 5 between the cell strains whereas sensitivity to α-particle irradiation was almost identical among all strains. The RBE is only determined by the sensitivity of the cells towards x-rays. Since cells with different defects of repair or cell cycle control have different radiosensitivities, we conclude that the effects of x-ray irradiation and the RBE are mostly determined by the activity of repair processes. Received: 20 August 2000 / Accepted: 7 May 2001     

Photocontrol of the Germination of Onoclea Spores: II. Analysis of Germination Processes by Means of Anaerobiosis

The oxygen requirements during the three phases of photoinduced germination of Onoclea sensibilis L. spores were analyzed by temporarily applying nitrogen atmosphere. The dark preinduction phase, during which the spores imbibe water and establish sensitivity to irradiation, involves an oxidative process which can be reversibly inhibited and stimulated by nitrogen and air, respectively. The induction phase of germination is characterized by a pure photochemical reaction, independent of temperature and oxygen. The postinduction phase, when the photoproduct triggers dark processes eventually leading to the protrusion of the rhizoidal or protonematal cells, involves an oxidative process which occurs within the first 10 hours of this phase. This oxidative process differs in kinetic characteristics from that in the preinduction phase. The oxidative process is inhibited by nitrogen treatment, but following nitrogen inhibition the ability of the spores to germinate can be reinstated by a long period of air intervening between the nitrogen treatment and a second irradiation. This suggests that enzymes or reactants which are needed in the postinduction process decay under anaerobic conditions and are resynthesized when the spores are transferred to air. Spores take up acetocarmine stain towards the latter part of the postinduction phase. Stain uptake is apparently succeeded very closely by cell division, and some time later by protrusion of the germling cells.     

Cell cycle phase sensitivity to cis-dichloro-bis (isopropylamine) trans-dihydroxy platinum (IV) (CHIP)

Cis-dichloro-bis (isopropylamine) trans-dihydroxy platinum (IV) (CHIP) is a second generation platinum coordination complex now in Phase II clinical trials. In vitro studies with Chinese Hamster Ovary cell cultures show that CHIP is a phase-sensitive drug, being most cytotoxic to cells in early G1 phase and least toxic to late S and G2 phase cells. The dose-modifying factor between the drug sensitivity of cells treated in G1 and in late S phase is 1.6. These findings and their clinical significance are discussed with respect to the phase sensitivity of other cytotoxic agents.     

Effect of low temperature on radiation-induced genetic damage in Drosophila melanogaster: response of motile sperm and late spermatids.

The response of fully mature motile sperm and late spermatids when challenged with X-radiation at 0 degrees C has been studied in sex-linked recessive lethals, II-III translocations and dominant lethality experiments. At 0 degrees C a significant increase in both mutagenic and clastogenic damage was detected compared to that obtained at 24 degrees C. Furthermore, the results of experiments performed with different postirradiation temperatures demonstrate that the low temperature during irradiation was the sole factor responsible for the observed increase. In the recessive lethal and translocation tests the response of late spermatids was higher than that shown by motile spermatozoa. As a whole, the results, which are rather similar to data reported on the effect of irradiation in oxygen of the same cell stages, suggest that the low temperature acted as a dose-modifying factor.     

Cell Cycle Phase Sensitivity to Cis-Dichloro-Bis (Isopropylamine) Trans-Dihydroxy Platinum (Iv) (Chip)

Abstract. Cis-dichloro-bis (isopropylamine) trans-dihydroxy platinum (IV) (CHIP) is a second generation platinum coordination complex now in Phase II clinical trials. In vitro studies with Chinese Hamster Ovary cell cultures show that CHIP is a phase-sensitive drug, being most cytotoxic to cells in early G₁ phase and least toxic to late S and G₁ phase cells. the dose-modifying factor between the drug sensitivity of cells treated in G₁ and in late S phase is 1.6. These findings and their clinical significance are discussed with respect to the phase sensitivity of other cytotoxic agents.     

Radical mediators and mitogen-activated protein kinase signaling in oxygen-dependent radiosensitivity of human tumor cell lines

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2',7'-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N(G)-monomethyl-L-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK-independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.     

Effect of High Oxygen Tension on Potassium Retentivity and Colony Formation of Bakers' Yeast

The ability of yeast cells to retain potassium and to form colonies was studied after exposure to pressures ranging from 2 to 143 atmospheres of oxygen. The investigations allow comparison of these responses with those found after x-ray exposure. Exposure to 2 to 8 atmospheres of oxygen for 2, 20, and 40 hours showed decreased potassium leakage as measured by an elution technique. Further experiments using 0.5 to 22 hour exposures to 10 to 143 atmospheres of oxygen showed decreased potassium leakage when glucose was present in the test media, but increased leakage (as did x-ray effects) in the absence of substrate. There was increased potassium leakage into the suspending media (distilled water) during oxygen exposure but this usually did not affect the leakage rates measured subsequently. Marked inability to form colonies was observed after 20 hour exposures to 100 atmospheres of oxygen, with a much smaller response at lower pressures. Increased oxygen concentrations, not pressure, evidently caused these effects, since comparable pressures of nitrogen produced almost no change. The ratio of potassium leakage to survival sensitivity was found to be approximately unity when comparing exposures causing 50 per cent damage. This is quite different from that seen with x-ray or ultraviolet irradiation.     

Analysis of Potential Radiosensitizing Materials for X-Ray-Induced Photodynamic Therapy

For a development of deep tumor treatment in photodynamic therapy, a feasibility of novel radiosensitizers induced by x-ray was investigated. The sensitizers are designed to generate reactive oxygen species (ROS) inside or outside the cell, possibly leading to damage exclusively on tumor cells and reservation of normal cells along the x-ray path. Taking note of the similarity in energy transfer mechanism in photocatalysts, scintillators, and particulate semiconductors, we chose TiO₂, ZnS:Ag, CeF₃, and quantum dots (CdTe and CdSe) in particulate form, which contain heavy atoms for efficient absorption of x-rays. A parameter study for x-ray operating conditions showed that in a typical scenario, photons with 20 to 170 keV energy are attenuated by 90% through the region of particle dispersed aqueous solution at varying concentration between 0.01 and 100 wt%. The amount of ROS generation under the exposure of polychromatic x-ray was measured using dihydroethidium reagent which detects an integrated amount of several species. Proportional increase in ROS generation to x-ray dose was observed for varying concentrations of TiO₂, ZnS:Ag, CeF₃, and CdSe quantum dot dispersions. Then, HeLa cells were mixed with aqueous solutions dispersed with sensitizing materials at a concentration of 3.0 mg/ml and were exposed to x-ray. Their survival fraction obtained by a cell proliferation reagent WST-1 immediately after the irradiation showed insignificant effects of sensitizing materials except at large doses. To enhance the sensitization effect, bio-conjugated CdSe quantum dots were internalized in the cytoplasm up to a concentration of 1.0 ng/ml. The cells were irradiated by x-ray up to 5 Gy, and their survival fraction was measured by the colony forming ability 9 days after irradiation. Survival fraction of the cells treated with quantum dots were less than those without quantum dots for all doses, suggesting that the colony forming ability is impaired by the internalized quantum dots.     

Gene expression and tumor cell escape from host effector mechanisms in murine large cell lymphoma

Using in vivo selection methods, we obtained metastatic sublines of the murine RAW117 large cell lymphoma that form multiple liver metastases. The highly metastatic subline RAW117-H10 has a low number of gp70 molecules expressed at the cell surface and low cytostatic sensitivity to activated syngeneic macrophages. This subline was infected with endogenous RNA tumor virus isolated from a high virus-expressing RAW117-P subline of low metastatic potential. After superinfection the H10 subline gradually increased its expression of cell surface gp70 and showed enhanced sensitivity to macrophage-mediated cytostasis, suggesting that gp70 might be involved in host macrophage-mediated surveillance. Culture of RAW117-P and H10 cells in media conditioned by activated macrophages indicated that parental cells are severely growth inhibited in a dose dependent fashion while H10 cells showed almost no effect. Examination of differentially expressed genes in the highly metastatic RAW117-H10 cells by analysis of RNA blots indicated that a mitochondrial gene was expressed at a level that was approximately 10 times higher in H10 cells than in parental cells. This gene was identified as ND5, which codes for a subunit of NADH dehydrogenase (complex I of the mitochondrial electron transport chain); this complex is the target for an activated macrophage-released cytostatic factor. Among other possibilities, the results are consistent with the suggestion that highly metastatic RAW117 cells may escape macrophage surveillance by decreasing the synthesis of specific cell-surface receptors for cytostatic molecules and increasing the synthesis of specific cellular targets for such molecules.     

Differentiated cells from BALB/c mice differ in their radiosensitivity

Various isolated cells of an inbred mouse strain (BALB/c) differed widely in their sensitivity to gamma irradiation: fibroblasts are five times more resistant than peripheral lymphocytes. Among lymphocytes, T cells are more resistant than B cells. Cell lines derived from the primary cells conserved their radiosensitivity. Cytofluorometric measurements show that the differential reaction of a cell to gamma irradiation can be detected already 2–3 h after the irradiation event. Radiation-sensitive cells are delayed for a longer time in S phase and G2 phase of the cell cycle than radiation-resistant cells. No difference in the capacity of the cells to perform single-strand break repair, double-strand break repair or unscheduled DNA synthesis could yet be detected.     

Effect of hyperbaric stress on yeast morphology: study by automated image analysis

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO₂.     

Radiosensitization of mammalian cells by diamide.

The effect of diamide on the radiosensitivity of T-cells was investigated under oxic and anoxic conditions. The compound was found to sensitize the cells under both conditions. Under oxic conditions, exposure for 10 min before and during irradiation to 0.1, 0.5 and 1.0 mM diamide produced dose-modifying factors of 0.81, 0.60 and 0.55, respectively. Under anoxic conditions, exposure for 10 min before and during irradiation to 0.5 mM produced a dose-modifying factor of 0.34. When the cells in oxic conditions were exposed for just 20 min before irradiation, the sensitizing effect was smaller, but some sensitization effect was still apparent after a 120 min interval between diamide treatment and irradiation. Diamide also sensitized the cells after irradiation, but this effect was less than when it was present during irradiation. The presence of whole rat-blood in the incubation medium prevented sensitization. No sensitization could be detected in the whole animal. It is proposed that sensitization is due to lack of capacity for repair of radicals by hydrogen transfer and biochemical repair processes.     

Effects of the differentiating agents sodium butyrate and N-methylformamide on the oxygen enhancement ratio of human colon tumor cells

We have previously shown that chronic adaptation of human tumor cells to the differentiation-inducing agents N-methylformamide (NMF) and sodium butyrate (NAB) increases the sensitivity of oxic cells to graded single doses of X rays. These studies were carried out to define the sensitivity of hypoxic cells after adaptation. Clone A colon tumor cells were grown for three passages in medium containing 170 mM NMF or 2 mM NAB and irradiated in suspension culture, after gassing with either oxygen (60 min) or ultrapure nitrogen (90 min), and complete survival curves were generated. Using the linear-quadratic equation to describe the data, it was found that NMF and NAB produced increased X-ray killing of hypoxic cells. At the 10% level of survival, the dose-modifying factors were about 1.20 and 1.25 for NMF- and NAB-adapted hypoxic cells, respectively, as compared to hypoxic control cells. However, since both oxic and hypoxic cells exhibited increased sensitivity after NMF and NAB adaptation, there was no major change in the oxygen enhancement ratio.     

Alterations in DNA repair efficiency are involved in the radioresistance of esophageal adenocarcinoma

To study radioresistance in esophageal adenocarcinoma, we generated an isogenic cell line model by exposing OE33 esophageal adenocarcinoma cells to clinically relevant fractionated doses of radiation (cumulative dose 50 Gy). A clonogenic assay confirmed enhanced survival of the radioresistant OE33 subline (OE33 R). To our knowledge, we are the first to generate an isogenic model of radioresistance in esophageal adenocarcinoma. This model system was characterized in terms of growth, cell cycle distribution and checkpoint operation, apoptosis, reactive oxygen species generation and scavenging, and DNA damage. While similar properties were found for both the parental OE33 (OE33 P) cells and radioresistant OE33 R cells, OE33 R cells demonstrated greater repair of radiation-induced DNA damage. Our results suggest that the radioresistance of OE33 R cells is due at least in part to increased DNA repair.     

Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells

There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase. In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting. This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels. We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses. For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells. Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase. For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells. None of these age fractions showed a dependence of OER on survival level. Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells. These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations. For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.     

Cyclic x-ray responses in Mammalian cells in vitro

Various radiation responses in mammalian cells depend on the position of the cell within its generation cycle (that is, its age) at the time of irradiation. Studies have most often been made by irradiating synchronized populations of cells in vitro. Results in different cell lines are not easy to compare, but an attempt has been made here to point out similarities and differences with regard to cell killing and division delay. In general, survival data obtained so far show that, in cells with a short G(1), cells are most sensitive in mitosis and in G(2), less sensitive in G(1), and least sensitive during the latter part of the S period. In cells with a long G(1), in addition to the above, there is usually a resistant phase early in G(1) followed by a sensitive stage near its end. (The latter may be as sensitive as mitosis.) Exceptions to the above, especially in some L cell sublines, have been noted, and a possible explanation is given. In Chinese hamster cells, maximum survival after irradiation occurs during S, but it does not coincide with the time of the maximum rate of DNA synthesis or with the time of the maximum number of cells in DNA synthesis, and changes in survival also occur in cells inhibited from synthesizing DNA. Rather, survival depends on the position the cell has reached in the cycle at that time, which involves not only DNA synthesis but other processes as well. Survival is not completely correlated with DNA synthesis, since halting DNA synthesis just before or just after irradiation only slightly affects survival at its maximum. Division delay exhibits a pattern of response which is similar in most cell lines. Delay is considerable for cells irradiated in mitosis, is small for cells in G(1), increases to a maximum for cells during S, and declines for cells in G(2). L cells or human kidney cells may have a longer delay for cells irradiated in G(2) than for those irradiated in S. The results can be explained in terms of a two-component model of division delay. One component results from the prolongation of the S period due to the reduced rate of DNA synthesis, and the other, a block in G(2), is independent of DNA synthesis. The proportion of the two components may vary in different cell lines.     

Analysis of cell flow and cell loss following X-irradiation using sequential investigation of the total number of cells in the various parts of the cell cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. The generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G2 blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. The experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Suramin sensitizing cells to ionizing radiation by inactivating DNA-dependent protein kinase

Here we report that suramin sensitizes LM217, MDA-MB-468, T98G and A431 cells to ionizing radiation. Suramin sensitized cells to X radiation in a dose-dependent fashion, and longer exposure to suramin before X irradiation resulted in more efficient sensitization. The dose-modifying factors calculated from the survival curves were 1.18 in LM217 cells and 1.37 in MDA-MB-468 cells. Suramin did not sensitize Scid cells that had no DNA-dependent protein kinase activity. Suramin inhibited DNA-dependent protein kinase activity in vitro and in vivo. The concentration of suramin resulting in 50% inhibition in vitro was 1.7 microM in LM217 cells and 2.4 microM in MDA-MB-468 cells. Exposure of LM217 and MDA-MB-468 cells to suramin did not affect the level of Ku70 (G22P1) or Ku80 (XRCC5), but it increased the level of DNA-PKcs(PRKDC). Suramin did not sensitize LM217 or MDA-MB-468 cells to UV radiation. Suramin's effects were not caused by accumulation of cells in a specific phase of the cell cycle. These results suggest that suramin sensitizes cells to ionizing radiation by inhibiting DNA-dependent protein kinase activity.