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Biomedical subjects

C E Ng

Publications and source records attributed to C E Ng.

At least 37 records · Page 2Linked to original sources
PubMed1995

Stepdown hyperthermia in human melanoma cells: effects on protracted mild hyperthermia for survival and DNA polymerase inactivation.

Human melanoma cells have been used to evaluate whether stepdown heating (SDH) could increase the effectiveness of long-duration mild hyperthermia (LDMH). The effects of these treatments were also evaluated on cell survival and DNA polymerase inactivation. Short treatments (30 min) at 43 degrees C did not result in much SDH effect for subsequent protracted heating at 40 degrees C. The effect on DNA polymerases was also very small. However, heating at 44 degrees C for 30 min had a large SDH heating effect on subsequent heating at 40 degrees C and 41 degrees C. The SDH effect occurred mainly in the first 5-10 h of subsequent LDMH and, at longer heating times, the rate of cell killing was reduced. The 44 degrees C SDH effect was also observed on DNA polymerase inactivation. Comparing the degree of cell killing and polymerase inactivation showed a good correlation for the various SDH protocols.

Cell Death↗
PubMed1994

31P NMR spectroscopic studies of the effects of cyclophosphamide on perfused RIF-1 tumor cells.

To determine whether direct cellular effects of chemotherapy are responsible for 31P NMR spectral changes observed in treated tumors in vivo, RIF-1 fibrosarcoma cells were examined in vitro before, during, and after treatment with 4-hydroperoxycyclophosphamide (4-HC), an activated form of cyclophosphamide. When RIF-1 cells were treated with 4-HC in a metabolically stable but nonproliferating state, the 31P NMR spectra were identical with those of untreated cells for up to 70 h. When actively proliferating RIF-1 cells were treated with 4-HC, the intensities of the nucleotide triphosphate resonances, which increased linearly during control cell growth, remained constant for 50 h or longer. These studies demonstrate that the bioenergetic improvement observed following treatment of RIF-1 tumors in vivo [S.-J. Li, J.P. Wehrle, S.S. Rajan, R.G. Steen, J.D. Glickson, and J. Hilton, Cancer Res. 48, 4736 (1988)] does not result from direct effects of cyclophosphamide metabolites on RIF-1 cell metabolism, but rather from indirect effects of treatment on tumor or host physiology.

Animals↗
PubMed1994

Measurement of the intracellular concentration of oxygen in a cell perfusion system.

[O2] was measured in the embedding material (alginate) in a typical apparatus for conducting studies of viable cells with NMR, using low frequency EPR. In suspension cultures respiration was independent of [O2] in the perfusing media down to about 1 microM while in alginate beads, the comparable value was 70 microM, indicating that the alginate was a very substantial barrier to the free diffusion of oxygen. With knowledge of [O2] in the various compartments, [O2] in the perfusing medium can be increased and the full power of NMR can be used to provide information on metabolism under various conditions. These results also provide evidence supporting the feasibility and usefulness of EPR techniques using nitroxides to measure [O2] in macroscopic samples such as NMR perfusion tubes. This technique is rapid, apparently nonperturbing, and enables one to differentiate between the concentrations of oxygen in different compartments.

Alginates↗
PubMed1994

Inhibition of potentially lethal and sublethal damage repair by camptothecin and etoposide in human melanoma cell lines.

We investigated the ability of camptothecin (CPT), an inhibitor of topoisomerase (topo) I, and etoposide (VP-16), an inhibitor of topo II, to potentiate X-radiation response and to inhibit the repair of potentially lethal damage (PLDR) and sublethal damage (SLDR) in confluent cultures of a radioresistant (Sk-Mel-3) and a radiosensitive (HT-144) human melanoma cell line. CPT or VP-16 were present both during irradiation and during the subsequent delayed plating period allowed for repair of X-radiation damage. When the direct toxicities of CPT or VP-16 were corrected for, we found that a dose of either drug that killed approximately 15% of the clonogenic cells potentiated the effects of radiation differentially on the cell lines. CPT and VP-16 inhibited the increase in survival brought about by delayed plating of HT-144 but not Sk-Mel-3 cells. In both cell lines, CPT inhibited SLDR but not PLDR. VP-16 also inhibited SLDR in both cell lines, however, in contrast with CPT, it also inhibited PLDR in HT-144 cells. Our results therefore suggest that either topo I and II are both implicated in the repair of X-radiation damage, or that the lesions formed by CPT and VP-16 with DNA are able to impair the processing of X-radiation repair. In addition, we found that in the absence of the topo inhibitors, the two cell lines repaired similar amounts of PLD from an isosurvival level. Sk-Mel-3, however, repaired significantly increased SLD from an isosurvival level (about three-fold, p < 0.05) compared with HT-144.

Camptothecin↗
PubMed1994

Sublethal radiation damage repair and its inhibition by hyperthermia in two human melanoma cell lines of different radiosensitivities.

Two human melanoma tumour cell lines, one radiation resistant and one radiation sensitive, were studied for their capacity for repair of sublethal radiation damage (SLDR) and for hyperthermia inhibition of radiation damage. The most resistant cell line demonstrated a survival curve with a large shoulder and a large SLDR capacity. The sensitive cell line was characterized by an exponential survival curve and had less SLDR capacity than the resistant line. Hyperthermia could inhibit SLDR in both cell lines and complete inhibition was achieved at 43 degrees C for 30 min. Hyperthermia was more effective after than before irradiation. The SLDR inhibition was greater in the resistant cell line, showing that hyperthermia may be an effective way to overcome resistance in melanoma, especially when such resistance stems from an increased SLDR capacity. In addition, relatively mild thermal treatments, easily achievable clinically, were sufficient to inhibit SLDR in melanoma cells.

Cell Line↗
PubMed1994

Comparison of thermoradiosensitization in two human melanoma cell lines and one fibroblast cell line by concurrent mild hyperthermia and low-dose-rate irradiation.

Two human melanoma cell lines, one radioresistant (SK-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD.

Cell Line↗
PubMed1993

Gel-entrapment of perfluorocarbons: a fluorine-19 NMR spectroscopic method for monitoring oxygen concentration in cell perfusion systems.

Oxygenation is a major determinant of the physiological state of cultured cells. 19F NMR can be used to determine the oxygen concentration available to cells immobilized in a gel matrix by measuring the relaxation rate (1/T1) of perfluorocarbons (PFC) incorporated into the gel matrix. In calcium alginate gel beads without cells the relaxation rate (1/T1) of the trifluoromethyl group of perfluorotripropylamine (FTPA) varies linearly with oxygen concentration, with a slope of 1.26 +/- 0.15 x 10(-3) s-1 microM-1 and an intercept of 0.50 +/- 0.04 s-1. During perfusion with medium equilibrated with 95%/5% O2/CO2, changes in PFC T1s indicate that the average oxygen concentration was reduced from 894 +/- 102 microM in the absence of cells to 476 +/- 65 microM and 475 +/- 50 microM in the presence of 0.7 x 10(8) EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 microliters of FTPA/ml of gel had no effect on the energy status of the cells as indicated by 31P NMR spectra. To calculate oxygen gradients within the beads from the average PFC T1 of the sample, a mathematical model was used assuming that oxygen is the limiting nutrient for cell metabolism and that the cellular oxygen consumption rate is independent of oxygen concentration. Data for EMT6/Ro cells were fit using experimentally determined perfusion parameters together with literature values for cell volume and oxygen consumption rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Alginates↗
PubMed1993

Postirradiation exposure to hypotonic saline shows normal damage processing in radiation-sensitive cell lines.

Three pairs of cell lines (one human and two Chinese hamster ovary (CHO) cell lines) each comprising a cell line with a normal radiation response and a radiation-sensitive mutant, were evaluated for recovery of potentially lethal damage (PLDR) and recovery of sublethal damage (SLDR). In all cases, the normal cell lines (GM1522, human; AA8-4 and K1, CHO) exhibited capacity for PLDR and SLDR was also normal in the two CHO lines. For the mutants (GM3395, human AT; V3 and 5-11, CHO) there was no ability for PLDR and SLDR was also absent in the two CHO cell lines. Postirradiation exposure to hypotonic NaCl solutions resulted in fixation of radiation damage in all the cell lines. This form of damage is repaired if left unperturbed after irradiation. This shows that cells have a large capacity for repair of this form of damage which accounts for much greater changes in survival than those observed in conventional PLDR experiments. These data show that the mutant cell lines retained their capacity to repair the damage which was susceptible to postirradiation fixation by anisotonic salt solutions. In addition, initial (i.e. preirradiation) DNA polymerase activities were measured in the four CHO cell lines; they were not correlated to radiation sensitivity.

Animals↗
PubMed1993

Repair of potentially lethal damage: its inhibition by hyperthermia in two human melanoma cell lines with different radio- and heat sensitivities.

The effect of hyperthermia on the inhibition of potentially lethal radiation damage repair (PLDR) was measured in two human melanoma cell lines of differing radiosensitivities. The HT144 cell line is radiation sensitive and has an exponential survival curve, while the SK-MEL-3 line is radiation resistant and has a shouldered survival curve. Both cell lines demonstrated a similar level of PLDR at isosurvival level radiation doses of 2.0 and 10 Gy for the HT144 and SK-MEL-3 lines, respectively. Hyperthermia (40-45 degrees C) could inhibit PLDR in both cell lines. This inhibition was greater in HT144 than in SK-MEL-3 at low temperatures (40 degrees C) and was reversed at higher temperatures (45 degrees C). There was also a sequence dependence of PLDR inhibition which showed a greater inhibition for heat before irradiation at 40 degrees C but the reverse for temperatures of 41 degrees, 43 degrees and 45 degrees C. These data show that hyperthermia can be an effective inhibitor of PLDR in both radiosensitive and resistant cell lines and that the degree of inhibition is not related to radiosensitivity.

Dose-Response Relationship, Radiation↗
PubMed1992

31P NMR spectroscopic study of the effects of gamma-irradiation on RIF-1 tumor cells perfused in vitro.

In order to examine the mechanisms underlying radiation-induced changes in phosphorus metabolite levels observed in RIF-1 tumors in vivo, RIF-1 cells in culture were perfused for up to 70 h following gamma-irradiation with 0-25 Gy and monitored continuously by 31P NMR spectroscopy at 8.5 T. Cells immobilized in the sample volume by incorporation into calcium alginate beads were bioenergetically stable, but did not replicate at the cell density used. Following an initial increase in PCr and NTP, which occurred in both control and irradiated cells, a dramatic decline in high-energy phosphates was detected beginning 24-30 h after irradiation with 15 or 25 Gy. In contrast, unirradiated cells or cells treated with 10 Gy remained metabolically stable for up to 72 h. The metabolic changes induced by irradiation of the cultured cells, which reflected cell death and lysis, were distinctly different from those observed in RIF-1 tumors in vivo during the same postirradiation time interval--an increase in high-energy relative to low-energy phosphates. This suggests that the spectral changes in vivo do not result from direct modification of cellular energy metabolism by radiation injury.

Animals↗
PubMed1987

Characterization of radiation sensitivity of human squamous carcinoma A431 cells.

Studies have been performed to investigate the radiosensitivity of human squamous carcinoma cells. A431 cells were grown in vitro as exponential and fed-plateau monolayer cultures or as multicellular spheroids. Radiobiological studies of various cultures showed that fed-plateau phase cells were more sensitive (D0 = 1.3 Gy) than exponentially growing cells (D0 = 1.5 Gy). After a single dose of 12 Gy or two doses of 6 Gy irradiation, A431 cultures exhibited a large capacity for potentially lethal damage (PLD) repair (PLD repair factor = 17), but a relatively small sublethal damage (SLD) repair. In order to measure the radiation sensitivity of proliferating (P) and quiescent (Q) cells, enriched populations of P- and Q-cells were isolated from A431 spheroids. Flow cytometric analysis with acridine orange (AO) staining demonstrated that there was a shift of the RNA histograms in fed-plateau and spheroid cultures towards lower values, suggesting the presence of a subpopulation of Q-cells. Centrifugal elutriation was used to isolate the Q-cells from dissociated spheroid cells. Coulter cell volume distributions and flow cytometric analysis showed that Q-cells had a small cell volume (approximately 1380 microns3), low RNA content and a G1-like DNA content. Continuous labelling experiments with tritiated thymidine confirmed the non-proliferating nature of the Q-cells. Irradiation of the Q-cells after isolation from spheroids with between 0 to 10 Gy showed that they were more radiosensitive (decreased D0) than the P-cells isolated from these spheroids. The latter were, however, similar in radiosensitivity to exponential G1 cells.

Carcinoma, Squamous Cell↗
PubMed1986

Density distributions of human squamous carcinoma cells: influence of growth conditions, proliferative status and DNA content.

Human squamous carcinoma cells (A431, CaSki) were grown in vitro as multicellular spheroids or as exponential and plateau-phase monolayer cultures. Single-cell suspensions were obtained by disaggregating the spheroids and monolayer cultures with trypsin-EDTA. Their buoyant density distributions were then compared by centrifuging the suspensions for 30 min at about 800gave and 4 degrees C in linear, continuous Percoll gradients. We found differences in buoyant density related to the growth condition (i.e. cells grown as spheroids or plateau-phase cultures were more dense than those from exponential cultures) and to the cell-line (i.e. CaSki cells were more dense than the A431 cells). Cells isolated from the inner layers of the spheroids appeared to be less dense than those from the outer layers. In addition, quiescent cells separated from the A431 spheroids by centrifugal elutriation, a technique based on cell size, were more dispersed in buoyant density than the corresponding proliferating cells recovered from the same spheroids. Flow cytometric analysis with mithramycin was performed to measure the DNA content of the cells. We found that the CaSki cells contained relatively more DNA than the A431 cells whereas, for the same cell line, growth as monolayer cultures or as multicellular spheroids did not influence their relative DNA content. We conclude that the growth conditions, the proliferative status of the cells and possibly their relative DNA content may influence the density distribution of these cells.

Carcinoma, Squamous Cell↗
PubMed1980

Buoyant density of EMT6 fibrosarcoma cells: time course of the density changes after growth in hypoxia.

EMT6 fibrosarcoma cells were grown to the exponential phase in tissue culture and incubated at 37 degrees C under hypoxic conditions. Buoyant density was determined as a function of the time in hypoxia. Hypoxia was produced in two ways. The first involved incubation of the cells in sealed aluminum chambers containing 95% N2, 5% CO2 gas, and < 10 ppm oxygen, resulting in the cells rapidly becoming exposed to the hypoxic environment. After incubation at 37 degrees C, they were centrifuged in linear Ficoll gradients to their isopycnic density. A significant decrease in density was found after 4 h, and prolonged incubation up to 24 h did not result in further change. This density change was reversible on transfer back to aerobic conditions, with the hypoxic cells reverting to their aerobic density after about 10 h reincubation in air. The second method of producing hypoxia involved growing about 8 X 10(6) cells in a medium-filled air-tight container. Hypoxia was produced gradually as the oxygen in the medium was consumed by cellular respiration. Similar results were obtained; that is, hypoxic cells became significantly less dense. However, when the level of hypoxia was varied between 4000 and < 10 ppm at 2-h intervals after the cells had depleted all of the original oxygen, no significant difference in density was found between hypoxic and aerobic cells.

Animals↗
PubMed1978

Comparison of the densities of clonogenic cells from EMT6 fibrosarcoma monolayer cultures, multicell spheroids, and solid tumors in ficoll density gradients.

The densities of viable EMT6 cells grown in vitro as monolayer exponential and plateau-phase cells or as multicell spheroids or in vivo as a solid tumor in BALB/c mice were determined with the use of isopyknic centrifugation in linear Ficoll gradients. Exponential cells banded at a density of 1.069 g/ml, whereas plateau-phase cells appeared at 1.073 g/ml. Cells grown as spheroids were more dense than the monolayer cells and were recovered mainly at 1.081. Solid-tumor cells, separated under the same conditions, banded at 1.080. Narrowing the range of the Ficoll gradient failed to resolve more than one band of cells in the solid-tumor separation. This provides evidence that the density of cells obtained from the spheroids is greater than that of the monolayer cells but agrees well with the density of the solid-tumor cells. Ficoll was demonstrated to be nontoxic to the cells, and plating efficiency assays showed similar cell viabilities between noncentrifuged and centrifuged cells. The plating efficiency of the peak fraction of exponential cells after centrifugation was 68%, and that of plateau-phase cells was 50%. Corresponding figures for the multicell spheroid and solid-tumor cells were 62 and 28%, respectively. The recovery of cells after centrifugation in the Ficoll gradients ranged from 62 to 83%. The effects of cell load and centrifugation time on the density distributions of the EMT6 cells were also investigated.

Animals↗