Radiosensitivity of four human tumor xenografts. Influence of hypoxia and cell-cell contact.

Contact effect (CE) and hypoxia have been studied in human tumor cell lines transplanted in athymic nude mice. Four cell lines--one melanoma (Bell) and three colorectal adenocarcinomas (HT29, HRT18, and HCT8)--were studied. Cell survival was determined with an in vivo to in vitro colony-forming assay. Survival curves were obtained under three different conditions: (1) tumor cells irradiated in air-breathing mice, (2) tumor cells irradiated in animals asphyxiated for 10 min, and (3) tumor cells plated and irradiated either immediately or 5 hr later. For all cell lines, radiosensitivity appeared to be lower when cells were irradiated in vivo than when they were irradiated in vitro. Only in the case of the HCT8 tumor did the relative in vivo radioresistance seem to be linked to hypoxia; in the other cell lines, hypoxia alone could not account for the lower in vivo radiosensitivity. Our results suggest that a CE plays an important role in the response of human tumor xenografts to irradiation.

Mean inactivation dose: a useful concept for intercomparison of human cell survival curves.

The mean inactivation dose (D) is calculated for published in vitro survival curves obtained from cell lines of both normal and neoplastic human tissues. Cells belonging to different histological categories (melanomas, carcinomas, etc.) are shown to be characterized by distinct values of D which are related to the clinical radiosensitivity of tumors from these categories. Compared to other ways of representing in vitro radiosensitivity, e.g., by the multitarget parameters D0 and n, the parameter D has several specific advantages: (i) D is representative for the whole cell population rather than for a fraction of it; (ii) it minimizes the fluctuations of the survival curves of a given cell line investigated by different authors; (iii) there is low variability of D within each histological category; (iv) significant differences in radiosensitivity between the categories emerge when using D. D appears to be a useful concept for specifying intrinsic radiosensitivity of human cell lines.

Quantification of NGF-dependent neuronal differentiation of PC-12 cells by means of neurofilament-L mRNA expression and neuronal outgrowth.

We demonstrate that the degree of neuronal development of PC-12 cell differentiation can be quantified by the expression of neurofilament-L (NF-L) mRNA, when an optimal concentration of NGF (50 ng/ml) is used. During the first 7 days of NGF treatment, the relative amount of NF-L mRNA was found to increase continuously and to correlate with the outgrowth of neurites in a statistically significant way. Thus, mRNA expression is, under these conditions, a suitable means for reliably monitoring the differentiation of PC-12 cells as early as after 3 days of NGF treatment. The results obtained with 5 ng/ml NGF differ from those with 50 ng/ml: during the first 3 days of NGF treatment, neuronal outgrowth was less than with 50 ng/ml, although the NF-L mRNA levels did not depend significantly on NGF concentration. Beyond day 3, NF-L mRNA levels did not increase further at 5 ng/ml as opposed to 50 ng/ml NGF. These differences point to different signal transduction processes involved in neuronal differentiation at high and low NGF concentration. Expression of NF-L protein in response to NGF treatment was also demonstrated. In summary, our results stress that stable and sustained differentiation of PC-12 cells can only be achieved with 50 ng/ml NGF.

The DNA content of some mammalian cells measured by flow cytometry and its influence on radiation sensitivity.

The DNA content of nine mammalian cell lines was determined by flow cytometry. Using radiobiological data from this and other laboratories a correlation between DNA mass and 1/D0 for X-rays, alpha-particles, and heavy ions could be established when the quantities were plotted on a log-log scale. The slopes of the regression lines amounted to 0.65 (X-rays), 0.64 (alpha-particles) and 0.74 (heavy ions). A similar correlation was found between DNA content and mean inactivation dose. The rather uniform slopes close to 2/3 suggest that radiosensitivity may depend on the surface area of the sensitive target, (cell nucleus) indicating a possible non-uniform distribution of radiosensitive sites within the nucleus.

Induction of sister chromatid exchanges in fibroblasts from normal donors and from patients with xeroderma pigmentosum after combined treatment with ultraviolet radiation and modulated low frequency electric currents.

Dermal fibroblasts derived from normal donors as well as from patients with xeroderma pigmentosum (XP) were exposed in vitro to ultraviolet radiation of 254 nm wavelength and to weak electric 4, 000 Hz currents modulated in amplitude with 50 Hz. Treatment with this so-called interferential current (IFC) increased the rate of sister chromatid exchanges (SCE) in all fibroblast types up to roughly 50% above the spontaneous level. When IFC was applied in combination with UV-radiation, it lowered UV-induced increase of the SCE-rate. A reduction of UV-induced SCEs was seen when XP-cells were exposed to IFC prior to UV-treatment, compared to the reverse order of treatment. The order of exposure has only been analysed in 5 XP patients, not in normal donors. The relationships between different combinations of exposure parameters (IFC-amperage, UV-dose, cell pathology, order of treatment) were examined by analysis of variance (ANOVA).

Interferential current is effective in palmar psoriaris: an open prospective trial.

Interferential current (IFC) has been shown to improve psoriasis in a small case series. So far no formal clinical trial had been conducted. As IFC is associated with slight prickling sensations a blinded study design was not feasible. Therefore, an open type prospective study was conducted with the assumption of 18% spontaneous remission rate. A response rate of 50% or less was judged as indicating no effect (hypothetical control), while 80% or more was considered as success (alternative hypothesis). In this "quasi-controlled" study 12 patients with therapy resistant palmar psoriasis received local treatment with IFC during a 12 week period. Treatment was performed at low current density in two daily sessions, each of 6 minutes duration. Erythema, scaling, induration, fissures and pustules were recorded in separate scores every 4 weeks. Response of a patient was judged positive when the total score of these criteria was reduced at least by two points at the end of treatment. After 12 weeks of treatment, 11 of 12 patients were cured or showed marked remission with the median overall score reduced by 4 points. An interim analysis was performed in order to decide whether the results had already reached significance (a < 0.05). The analysis revealed a statistically significant response rate of 90% (95% confidence interval 62-99%). These results are highly encouraging and should focus attention on this new therapy modality, which, in contrast to other treatments is not associated with side effects and discomfort.

Analgesic and disease modifying effects of interferential current in psoriatic arthritis.

Interferential current (IFC) was suggested to improve the skin manifestations of psoriasis vulgaris, possibly by enhancing the intracellular concentration of cyclic AMP. We assessed the efficacy of IFC on psoriatic arthritis (PsA). Nine consecutive patients were analyzed at baseline and after 16 weeks of IFC therapy. Bipolar IFC was applied twice daily to hands, feet plus all affected joints. IFC improved SF-36 assessed body pain, but not other SF-36 subscales. Morning stiffness, tender joint counts, and physician assessed disease activity improved. In contrast, visual analogue scale assessed pain, CRP and ESR measurements were unchanged. MRI of the most affected hand or foot documented a tendency towards worsened tendinitis, soft tissue swelling, and new joint space narrowing and erosions. Bone scintigraphy showed a trend towards deterioration. New joints became inflamed within treated sites. Thus IFC has analgesic effects in PsA, but does not have a satisfactory disease modifying effect.

Neuronal outgrowth of PC-12 cells after combined treatment with nerve growth factor and a magnetic field: influence of the induced electric field strength.

In view of possible therapeutic applications of magnetic fields, the effect of an enhancement of neuronal outgrowth at higher figures of flux density and induced field strength was investigated. On the average sinusoidal magnetic field treatment at 100 microTrms/50 Hz did not change nerve growth factor (NGF) induced neurite outgrowth to a statistically significant extent. These results suggest that further increasing the induced field strength by using either higher flux densities and/or more sophisticated wave forms might be necessary to cause the neuronal response of PC-12 cells, as seen in other experiments.

Kinetics of regrowth of V79 spheroids after gamma- and pi--irradiation.

Spheroids of V79 cells were irradiated with 180 MeV/c pi--mesons in the peak region and with gamma-rays. Doses of 900 and 1500 rad were applied, respectively, yielding 14% surviving cells. Regrowth of the spheroids was followed by measuring volume changes, cell multiplication as well as viability and proliferation of cycling and resting spheroid cells. While volume growth after irradiation was equal for both radiations, the time profiles of cell density, viability and mean DNA content per cell were found to be different. Consistently, a more prompt and pronounced response to pion irradiation could be inferred and was discussed in terms of LET dependent proliferation and elimination of inactivated cells and their subsequent substitution by viable progeny cells. Recruitment of resting cells was observed with both radiations.

The action of ionizing radiation on DNA in the presence of quinacrine. II. DNA strand breakage.

Strand breakage of DNA irradiated in solution and in the dry state in the presence of quinacrine was investigated by sedimentation analysis. Determination of single strand breaks in solution combined with binding studies permits to conclude that bound quinacrine protects DNA more effectively than the free compound. In the dry state quinacrine is without detectable effect on both single and double strand break formation, neither under aerobic nor anaerobic conditions.

Energy requirements for damaging DNA molecules. III. The mechanisms of inactivation of bacteriophage chiX 174 DNA by vacuum ultra-violet radiation.

Strand break formation and biological inactivation of infectious DNA of bacteriophage phi chi 174 exposed to vacuum-ultra-violet radiation of 4-9 to 21-2 eV quantum energy is investigated. At 21-2 eV as many as 84 per cent of the DNA molecules are inactivated by breaks whereas breaks do not contribute to inactivation at 4-9 eV. The quantum yield of break formation increases from 1-7 X 10(-5) (4-9 EV) to 0-55 (21-2 eV) and shows a dependence on energy similar to that of electron emission (due to ionization) above 8 eV. The mechanisms leading to break formation and inactivation are discussed taking the absorption spectrum of DNA in the vacuum-ultra-violet region into consideration.

[Recovery and cycle progression in multicell spheroids after fractionated gamma-irradiation and combined hyperthermic treatment (author's transl)]. / Erholungs- und Proliferationseffekte in multizellulären Sphäroiden bei fraktionierter gamma-Bestrahlung und kombinierter Hyperthermiebehandlung.

Spheroids of V79 cells were subjected to fractionated irradiation with two doses of gamma-radiation. In addition, a two hours hyperthermic treatment at 42 degrees C immediately following the first dose was applied. Cycling and resting cells of this in-vitro tumour model were then assayed for survival as function of the fractionation interval. In parallel, changes in cycle progression between the doses were measured by means of cytofluorometry. As main proliferative effects induced by this combined radiation and heat treatment transient S-phase blocking of cycling and recruitment of resting cells were observed. The split-dose survival curve displayed considerable synergistic action of heat and radiation and a six hours delay of Elkind-recovery in both cycling and resting cells.

Action of a 50 Hz magnetic field on proliferation of cells in culture.

Proliferation of SV40-3T3 mouse fibroblasts and human HL-60 promyelocytes was studied after treatment with a sinusoidal 2 mT 50 Hz magnetic field. A single exposure of 60 minutes caused quasicyclic changes in the number of SV40-3T3 cultures as function of time after treatment, which was interpreted to be due to the induction of chronobiological mechanisms by the field. Moreover, small variations in cell cycle distribution were measured during postexposure incubation for both cell lines. To discriminate between the effect of the magnetic vector and the induced electric field, HL-60 cell exposure was also performed on organ culture dishes. These dishes consist of two coaxially centered, isolated compartments in which different electric field levels are induced in the medium during treatment. Cell growth was affected in the outer compartment only where the induced electric field ranged from 8 to 12 mVpeak/meter at 2 mT, but it was not affected in the inner compartment (field range 0-4 mVpeak/meter). This suggests that the effects on cell growth are due to the induced electric field and are expressed only above a threshold of between 4 and 8 mVpeak/meter.

Increased radioresistance of cells in cultured multicell spheroids. I. Dependence on cellular interaction.

Monolayers of six different cell lines were investigated with respect to ionic coupling using micro-electrode techniques. In parallel, survival after Co-gamma-irradiation of monolayer- and spheroid cultures of these lines was compared. It was found that spheroids of coupled cell lines were more radioresistant than monolayers ("contact effect"). however, cell coupling did not enhance the survival of monolayers over single cells. This suggests that the contact effect is a "tissue phenomenon" requiring cellular interaction but is expressed only under conditions of three-dimensional growth.

Increased radioresistance of cells in cultured multicell spheroids. II. Kinetic and cytogenetic studies.

Using the Chinese hamster cell line B14 FAF28, several specific properties of the contact effect (CE) of radiation action in spheroids were investigated. CE was found to "protect" the spheroid cells against several types of radiation-induced cytogenetic misfunctions such as blockage in S and G2+M-phase, mutagenesis, and chromosome damage. However, repair of DNA strand-breaks was the same in monolayers and spheroids. Furthermore, CE is a property of the single cell and does not depend on the proliferative status (cycling or non-cycling) of the cells. We conclude that CE is the result of a physiological modification of the cells occurring during growth in the three-dimensional spheroid matrix and requiring metabolic cooperation and cyclic AMP for its induction.

Response of cytosolic calcium, cyclic AMP, and cyclic GMP in dimethylsulfoxide-differentiated HL-60 cells to modulated low frequency electric currents.

The action of interferential current (IFC), an amplitude-modulated 4000 kHz current used in therapeutic applications, upon intracellular calcium, adenosine 3':5'-cyclic monophosphate (cAMP), and guanosine 3':5'-cyclic monophosphate (cGMP) was investigated. Human promyelocytes (HL-60) were differentiated to granulocytes by dimethylsulfoxide (DMSO) treatment and exposed for 5 min at 25, 250, and 2500 microA/cm2 current density. No significant changes in cytosolic free calcium were detected as a function of modulation frequency of the IFC. However, intracellular cAMP reacted in a complex way to modulation frequency, resulting in stimulations and depressions within the range of frequencies studied (0-125 Hz). The "windows" of modulation frequency, where statistically significant increases or decreases in cAMP were noted, coincided with those published earlier for mouse fibroblasts. Cellular cGMP content was always lowered by IFC treatment. Furthermore, no significant influence of IFC current density upon the three second messengers was noted. These results, which also include data relating to treatment with sinusoidal 50 Hz current, contribute to a more detailed understanding of the primary biophysical mechanisms of signal transduction by time-varying electric fields.