RESUMEN
Chinese hamster ovary (CHO) K1 and radiosensitive CHO irs-20 cells were synchronized in S phase and labeled for 10 min with 5-[(125)I]-iodo-2'-deoxyuridine ((125)IdU). The cells were washed, incubated in fresh medium for 1 h for incorporation of the intracellular radionucleotides into DNA, and then frozen (-80 degrees C) for accumulation of (125)I decays. At intervals after freezing, when the cells had accumulated the desired number of decays, aliquots of the frozen cells were thawed and plated to determine survival. The survival curves for K1 and irs-20 cells were similar from 100% to 30% survival. At higher (125)I doses (more decays/cell), the survival of K1 cells continued to decline exponentially, but the survival of X-ray-sensitive irs-20 cells remained at approximately 30% even after the cells had accumulated 1265 decays/cell. The results contradict the notion that increased DNA damage inevitably causes increased cell death. To account for these findings, we propose a model that postulates the existence of a second radiation target. According to this model, radiation damage to DNA may be necessary to induce cell death, but DNA damage alone is not sufficient to kill cells. We infer from the survival response of irs-20 cells that damage to a second (non-DNA) structure is involved in cell death, and that this structure directly affects the repair of DNA and cell survival.
Asunto(s)
Células CHO/efectos de la radiación , ADN/efectos de la radiación , Animales , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Cricetinae , Cricetulus , Criopreservación , Daño del ADN , Fragmentación del ADN , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Idoxuridina/toxicidad , Radioisótopos de Yodo/toxicidad , Modelos BiológicosRESUMEN
Minimal residual disease in lymphoma patients is a major problem in the clinical management of their cancer. High-dose chemotherapy followed by autologous bone marrow transplantation has been used to treat the disease. However, residual lymphoma may be reintroduced along with the marrow if it is present in the bone marrow harvest. In this report we describe results of experiments testing the efficacy of 5-[125I]-iodo-2'-deoxyuridine (125IdU) for purging murine RAW117 large cell lymphoma cells (Joshi et al., Oncology 44, 180-185, 1987; Cancer Res. 47, 3551-3557, 1987) from bone marrow in a relevant animal model. Donor BALB/c mice were injected with murine RAW117 cells and euthanized on day 13, and their bone marrow that had been contaminated with tumor cells was harvested and treated in vitro with 125IdU or nonradioactive 127IdU (control). Nine of 10 mice receiving 127IdU-treated bone marrow contaminated with tumor cells died at an average of 17 days after injection. In comparison, 9 of 10 mice injected with 125IdU-treated bone marrow contaminated with tumor cells were still alive after 82 days. In addition, the 125IdU treatment did not diminish the formation of hematopoietic progenitor cell colonies in normal mouse and human peripheral blood stem cells.
Asunto(s)
Células de la Médula Ósea/efectos de la radiación , Purgación de la Médula Ósea/métodos , Idoxuridina/uso terapéutico , Linfoma/radioterapia , Animales , Ciclo Celular , Femenino , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Ratones , Ratones Endogámicos BALB C , Análisis de SupervivenciaRESUMEN
UNLABELLED: Studies were undertaken to determine the relationship between IUdR concentration and the duration of radiolabeled IUdR treatment required to incorporate the equivalent of a D(o) dose in vitro and to estimate the treatment parameters necessary to incorporate a killing dose in vivo. METHODS: W138 (normal human) and HeLa (human cancer) cells were grown axenically or in co-culture. The three cultures were treated for 5 days with 18.5 kBq/ml [125I]IUdR. After treatment, the cells were subcultured and grown for 7 days in medium without [125I]IUdR. In separate experiments, Chinese hamster ovary cells (CHO) were labeled with various ratios of radiolabeled (125I) and nonradiolabeled IUdR and the mole rate of IUdR incorporation in double-stranded DNA was measured. Mitotically selected CHO cells were incubated without treatment until > 98% were in S phase. At this time, the cells were labeled for 15 min with several concentrations of either [123I]IUdR or [125I]IUdR and their colony survival was measured. RESULTS: After incubation with [125I]IUdR, selective eradication of HeLa cells from a co-culture of W138 and HeLa cells was achieved. The incorporation of IUdR into DNA of CHO cells, although the sum of a series of enzymatic steps, has the appearance of and can be analyzed as a Michaelis-Menton type curve. The maximum rate of IUdR incorporation (Vmax) is 4.424 x 10(-18) mol/min and the substrate concentration at 1/2 Vmax (K) is 3.717 x 10(-6) M IUdR. The Do dose rates for [123I]IUdR and [125I]IUdR, respectively, are 18.78 and 1.88 initial decays/cell/hr. CONCLUSION: The D(o) dose for *IUdR can be determined from survival curves versus the mole amount of *IUdR incorporated in DNA. To be effective as an in vivo treatment it will be necessary to manipulate the IUdR delivery time, concentration and volume in a manner that assures that the target cells incorporate a cytocidal dose of *IUdR.
Asunto(s)
Idoxuridina/uso terapéutico , Radioisótopos de Yodo/uso terapéutico , Animales , Supervivencia Celular , Células Cultivadas/efectos de la radiación , Cricetinae , Humanos , Dosis de Radiación , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
To increase tumor incorporation and minimize hepatic degradation of radio-IUdR, compartmental administration routes are being considered as an alternative to intravenous (i.v.) injections. Although there are significant data on the biodistribution and some reports on radiotoxicity of i.v.-administered 125IUdR, similar results for other routes of delivery are not available. We have undertaken a series of experiments intended to examine radiation effects of 125IUdR after intravesical (3 swine; eight 3 mCi doses at 4-day intervals), intracarotid (3 swine; two 10 mCi doses at 2-week intervals), and intra-aortic (5 swine, single dose of 10 mCi) administration in a swine model. Liver, renal functions, and complete blood counts were monitored throughout the duration of the experiment. Pharmacokinetics, systemic distribution of radioactivity and metabolites were measured. The normal tissue 125IUdR uptake and histology were determined after necropsy. No adverse systemic effects were identified. Clinical observations, laboratory data, and necropsy results were within normal range.
Asunto(s)
Idoxuridina/administración & dosificación , Idoxuridina/farmacocinética , Radioisótopos de Yodo/administración & dosificación , Radioisótopos de Yodo/farmacocinética , Administración Intravesical , Animales , Aorta , Arterias Carótidas , Femenino , Inyecciones Intraarteriales , Porcinos , Distribución TisularRESUMEN
Cell progression into mitosis and chromatid aberration frequencies were compared in two Chinese hamster ovary (CHO) cell lines after incorporation of 125IdUrd. Asynchronous, exponentially growing populations of CHO K1 and the DNA repair-deficient, radiation-sensitive CHO irs-20 cells were compared after a 10-min exposure to 14.8 kBq/ml 125IdUrd. Essentially no differences were seen for either end point between the cells of the two cell lines. As the cells in S phase at the time of labeling entered the mitotic cell selection window, the number of mitotic cells of each cell line declined to approximately 60% of the respective unlabeled control. Chromosome analysis of the mitotically selected cells indicated an 125I decay-dependent increase in the number of chromatid aberrations in cells of both cell lines. The appearance of aberrations together with the known rates of production and rejoining of DNA double-strand breaks show that cells are able to progress through G2 phase and into mitosis in the presence of such breaks. The data suggest that DNA damage may be necessary, but is not sufficient to cause a radiation-induced blockade of cell progression through G2 phase.
Asunto(s)
Ciclo Celular/efectos de la radiación , Cromátides/efectos de la radiación , Aberraciones Cromosómicas , Daño del ADN , Idoxuridina/metabolismo , Radioisótopos de Yodo , Animales , Células CHO , Deleción Cromosómica , Cricetinae , Fase G2/efectos de la radiación , Rayos gamma , Idoxuridina/farmacología , Isocromosomas , Cinética , Mitosis , Tolerancia a Radiación , Factores de TiempoRESUMEN
A radionuclide release assay for measuring the in vitro kinetics of cell death has been developed. CHO cells were labelled for 24 h with 3.0 hBq/ml of [125I] iododeoxyuridine (125IUdR) and the fate of the labelled cells and their progeny was monitored at daily intervals by measuring the rate of 125I release. Prelabelling with 125IUdR did not alter the plating efficiency, the doubling time or the selection of mitotic cells. The rate of 125I release from labelled (but otherwise untreated) CHO cells was approximately equal to 4% day. Treatment with a lethal dose of X-rays (30 Gy), heat (46 degrees C, 1 h), cold (-90 degrees C, 1 h) or the antibiotic Geneticin (300 micrograms/ml, continuously) resulted in the release of greater than 99% the 125I activity associated with the cells. Cell death was rapid after heating or freezing, and delayed after treatment with X-rays or Geneticin. The results illustrate the efficacy of the 125I release assay for measuring the kinetics of cell death in mammalian tissue culture cells.
Asunto(s)
Supervivencia Celular/fisiología , Idoxuridina , Radioisótopos de Yodo , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Frío/efectos adversos , Gentamicinas/toxicidad , Calor/efectos adversos , Técnicas In Vitro , CinéticaRESUMEN
The precise cell cycle time of association between labeled DNA (the radiation source) and the non-DNA cell structure whose damage is responsible for radiation-induced division delay was measured. Mitotic cells were selected from a monolayer of Chinese hamster ovary cells for 80 min (nine shakes) to establish the rate of cell progression into mitosis. The cell monolayers were then exposed to 0.1295 MBq/ml 125IUdR for 10 min to label the cells in S phase. After pulse labeling, mitotic cell selection was continued for various times (between 0 and 120 min) before 125I decays were accumulated at 4 degrees C. After 2 h in the cold, the cells were rewarmed and the selection of mitotic cells was continued. (Cooling had a small, transient affect on subsequent cell progression.) As the time between labeling and cooling was increased, the fraction of cells selected in mitosis decreased, indicating that an increasing proportion of 125I-labeled cells had entered a sensitive phase of the cell cycle where 125I decays are particularly effective in producing radiation-induced division delay. It is hypothesized that during this sensitive period (from -25 to +90 min of the S/G2 boundary), the labeled DNA comes into sufficiently close contact with a non-DNA structure to facilitate damage to this structure by overlap irradiation from 125I decays in the DNA.
Asunto(s)
Ciclo Celular , División Celular/efectos de la radiación , ADN , Animales , Idoxuridina/metabolismo , Radioisótopos de Yodo , Factores de TiempoRESUMEN
Chinese hamster ovary cells were labeled with [125I]iododeoxyuridine (125IUdR, 0.1184 MBq/ml for 20 min) and the labeled mitotic cells were collected by selective detachment ("mitotic shake off"). The cells were pooled, plated into replicate flasks, and allowed to progress through the cell cycle. At several times after plating, corresponding to G1, S, late S, and G2 plus M, cells were cooled to stop cell cycle progression and to facilitate accumulation of 125I decays. Evaluation of cell progression into the subsequent mitosis indicated that accumulation of additional 125I decays during G1 or S phase was eight to nine times less effective in inducing progression delay than decays accumulated during G2. The results support our previous hypothesis that DNA damage per se is not responsible for radiation-induced progression delay. Instead, 125I-labeled DNA appears to act as a source of radiation that associates during the G2 phase of the cell cycle with another radiosensitive structure in the cell nucleus, and damage to the latter structure by overlap irradiation is responsible for progression delay (M. H. Schneiderman and K. G. Hofer, Radiat. Res. 84, 462-476 (1980].
Asunto(s)
Ciclo Celular , ADN/efectos de la radiación , Radioisótopos de Yodo , Animales , Línea Celular , Cricetinae , Cricetulus , Idoxuridina , Técnicas In Vitro , Interfase , Marcaje IsotópicoRESUMEN
The progression of Chinese hamster ovary (CHO) G2 cells into mitosis and their survival was measured after X-ray doses up to 4.0 Gy. S-phase cells were prevented from reaching mitosis by labeling with 125IUdR for 10 min prior to irradiation of the exponentially growing monolayer of cells. Mitotic cells, located past the radiation-induced division delay transition point, did not suffer a delay and were selected separately prior to the recovery of the G2 cells. The results show that (1) up to 400 min after radiation only 55% of the G2 cells recovered after about 2.5 Gy; (2) the progression delay of the G2 cells that recovered was 52.5 min/Gy; and (3) the survival curve D0 for these cells, 2.45 Gy, indicated a radioresistant population.
Asunto(s)
Interfase/efectos de la radiación , Animales , Supervivencia Celular/efectos de la radiación , Cricetinae , Cricetulus , Relación Dosis-Respuesta en la Radiación , Femenino , Mitosis/efectos de la radiación , Ovario , Factores de TiempoRESUMEN
G2 cells treated with 150 rad X-radiation were isolated from a monolayer culture of exponentially growing Chinese hamster ovary (CHO) cells by a combination of 125Iododeoxyuridine ([125I]UdR)-induced blockade of S-phase cell progression, treatment and mitotic selection (125I-TMS technique). Once the rate at which cells were selected from a small window in mitosis was established (Schneiderman et al., 1972), the cells were exposed to 10 microCi/ml, carrier-free [125I]UdR for 10 min immediately before treatment with 150 rads X-radiation. After X-irradiation the cells located later in the cell cycle than the X-ray-induced division delay transition point (TPx), at or just prior to prophase, progressed without delay and were selected during the next 50 min (Walters & Petersen, 1968; Schneiderman et al., 1972). The G2- and S-phase cells, located prior to the TPx, sustained a transitory delay and resumed progression into mitosis only after recovery from the radiation insult (Terasima & Tolmach, 1963). However, S-phase cells having incorporated [125I]UdR during the pulse label were prevented from entering mitosis (Schneiderman & Hofer, 1980) and only the X-ray-treated G2 cells resumed progression into mitosis and were selected.