Some long-term cell kinetic effects of ionizing radiation on mouse bladder urothelium

The cell kinetics of the mouse bladder urothelium were followed with tritiated thymidine pulse labelling and flow cytometry for one year after irradiation with electrons. No perturbations were registered after 10 Gy. Three to four weeks after 20 Gy an elevation of the labelling index with a subsequent return to normal was seen. Flow cytometry revealed some increase in the proportion of octaploid nuclei at the same time. From about six months after irradiation the normal polyploidization decreased. The urothelium turned into a mainly diploid cell population. The proportion of diploid S phase cells also increased. The data give some support to the model hypothesis of reactive proliferation in a 'flexible' tissue, according to Wheldon et al. (1982).     

Some cell kinetic effects of combined injury with ionizing radiation and cyclophosphamide on mouse bladder urothelium

Cyclophosphamide was given intraperitoneally to groups of eight female mice 48 h after local electron irradiation to the bladder with 0, 10 and 20 Gy respectively. The reactions in the urothelium were monitored by histology, incorporation of tritiated thymidine and flow cytometry. A wave of increased thymidine incorporation combined with an increase in the proportion of diploid S-phase cells was seen in the unirradiated bladders 24 h after the drug treatment, followed by normalization after 1 week. This response was significantly less pronounced in the irradiated animals. In the unirradiated animals a similar wave characterized by an increased proportion of octaploid cells was also seen, but this wave occurred later in the irradiated animals. Severe injury was observed in the rectum of the 20 Gy-irradiated animals. Irradiation prior to drug treatment led to only small effects, but a decreased ability for regenerative DNA synthesis after drug injury seems to persist. This affects both proliferation and the building up of polyploidy.     

Response to ionizing radiation of human bladder transitional cell carcinomas grown in the nude mouse

The sensitivity to ionizing radiation of three human bladder transitional cell carcinomas grown in the nude mouse was investigated at four different radiation levels, 500, 1,000, 2,000 and 4,000 rad. Each tumor line exhibited a response pattern which reflected to a certain degree tumor differentiation and growth rate in the nude mouse. Exposure to 1,000 rad was the minimum amount of radiation required to produce a distinct, although transient, tumor response in all three tumor lines characterized by a growth delay of 3-4 weeks, whereas maximum tumor response was observed at the 4,000 rad radiation level. These studies would permit a better understanding of the behavior of human bladder cancer to ionizing radiation and may further facilitate efforts at identifying effective radiosensitizing agents that may result in maximizing tumor response.     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

The effects of ionizing radiation on cell cycle progression in ataxia telangiectasia

Although ataxia telangiectasia (AT) cells are more sensitive than normal cells to killing by ionizing radiation, their DNA synthesis is more resistant to inhibition by radiation. It was thought that this anomaly in DNA synthesis was likely to perturb cell cycle progression. Flow cytometry and the fraction of labelled mitoses (FLM) were used to investigate effects of irradiation in normal and AT cell lines. The FLM indicated that radiation apparently induced a longer G2 delay in normal cells than in AT cells. However, flow cytometry showed that radiation induced much larger and more prolonged increases in the proportion of G2 cells in AT than in normals. AT populations also showed much larger postirradiation decreases in viable cell numbers. These data suggest that a large proportion of the radiosensitive AT cells are not reversibly blocked in G2 but die there, and never proceed through mitosis. The less radiosensitive normal cells are delayed in G2 and then proceed through mitosis. We suggest that the apparently shorter radiation-induced mitotic delay seen in AT cells by FLM is not real but is an artifact arising from perturbation of steady state conditions by selective elimination of a particular cohort of AT cells. Accumulation of AT cells in G2 is compatible with radiosensitivity of these cells and may arise from a defect in DNA repair or an anomaly in DNA replication.     

Some effects of radiation on the healing of transected mouse ear

Both histological assessments and measurements of epidermal and dermal components are used to describe some effects of radiation on wound healing in mouse ear. A pattern of early wound closure followed by wound reopening was seen after doses of about 20 Gy and above. After 10 Gy X rays wound closure was comparable with that in unirradiated wounds but the nature of the tissue repair was different. The results suggested that the severity of radiation damage to epidermis is relatively unaffected by wounding but that the time course of expression of the radiation damage is appreciably accelerated. The observations are discussed in terms of their clinical relevance and of current radiobiological hypotheses.     

The psychological effects of ionizing radiation

This paper presents a case study of eleven men who were exposed to non-background ionizing radiation as active participants in the United States' atmospheric nuclear tests. Each of the subjects has developed a virtually identical complex of debilitating psychiatric symptoms. The content of these symptoms is almost entirely focused upon the health effects of the radiation to which each of the subjects was exposed. This symptom complex appears to comprise a syndrome. The symptom structure and course of this syndrome suggests three hypotheses: The syndrome appears to be a pathological development of the self diagnostic belief (that one has been physically harmed by radiation) into a set of symptoms that elaborate upon and express this belief. The self diagnostic belief develops as a means of resolving any one of the various medical mysteries that an individual can experience subsequent to exposure to radiation. Development of the syndrome is a consequence of exposure to non-background ionizing radiation. The paper discusses the evidence for these hypotheses and suggests future research directions.     

Limited short term effects on number of nuclei after electron irradiation of mouse bladder urothelium. A morphometric study

By use of morphometry the number of urothelial nuclei per mouse bladder on days 1 and 7 after electron irradiation with 0, 10, 20 and 30 Gy was calculated. The results indicate only limited cell loss.     

Effects of ionizing radiation on cell cycle progression

Irradiation of normal eukaryotic cells results in delayed progression through the G1, S, and G2 phases of the cell cycle. The G1 arrest is regulated by the p53 tumor suppressor gene product. Irradiation results in increased expression of p53, which in turn induces a 21 kDa protein, WAF 1/Cip 1, that inhibits cyclin CDK kinases. S-phase delay is observed after relatively high doses of radiation. This delay has both radiosensitive and radioresistant components, corresponding to inhibition of DNA replicon initiation and DNA chain elongation, respectively. The mechanism for this delay is as yet undefined, but the extent of the delay appears to be under genetic control and is sensitive to the kinase inhibitor staurosporine. A delay in G2 has been demonstrated in virtually all eukaryotic cells examined in response to irradiation. Our studies have focused on the mechanisms responsible for this delay. Cyclin B1 and p34^cdc2 are cell cycle control proteins that together form a kinase complex required for passage through G2 and mitosis [22]. Control of radiation-induced G2 delay is likely therefore to involve modulation of cyclin B1/p34^cdc2 activity. We have shown in HeLa cells that cyclin B1 expression is decreased in a dose-dependent manner following irradiation. This decrease is controlled at both the level of mRNA and protein accumulation. We have also shown that radiation-sensitive rat embryo fibroblast lines (REF) immortalized with v- or c-myc display a minimal G2 delay when compared to radiation resistant cells transformed with v-myc + H-ras. These REF lines respond to irradiation with a decrease in cyclin B mRNA, which parallels the extent of their respective G2 delays. The duration of the G2 delay in radiation-resistant REF can be shortened by treatment with low doses of the kinase inhibitor staurosporine. We have also been able to markedly reduce the radiation-induced G2 delay in HeLa cells using either staurosporine or caffeine. Attenuation of the G2 delay is accompanied by reversal of the radiation-induced inhibition of cyclin B mRNA accumulation. The results of these studies are consistent with the hypothesis that reduced expression of cyclin B in response to radiation is in part responsible for the G2 delay. The duration of the G2 delay may also be influenced by the activation state of the cyclin B/p34_cdc2 complex.Invited paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Damaging and protective cell signalling in the untargeted effects of ionizing radiation

The major adverse consequences of radiation exposures are attributed to DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes. However, the dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells either directly or via media transfer (radiation-induced genomic instability) or in cells that have communicated with irradiated cells (radiation-induced bystander effects). Radiation-induced genomic instability is characterized by a number of delayed responses including chromosomal abnormalities, gene mutations and cell death. Bystander effects include increases or decreases in damage-inducible and stress-related proteins, increases or decreases in reactive oxygen and nitrogen species, cell death or cell proliferation, cell differentiation, radioadaptation, induction of mutations and chromosome aberrations and chromosomal instability. The phenotypic expression of untargeted effects and the potential consequences of these effects in tissues reflect a balance between the type of bystander signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. Thus, in addition to targeted effects of damage induced directly in cells by irradiation, a variety of untargeted effects may also make important short-term and long-term contributions to determining overall outcome after radiation exposures.     

The response of junctional complexes to induced desquamation in mouse bladder urothelium

During desquamation, the cells of mouse urinary bladder epithelium undergo detachment. In this process we examined the disconnection of cell adhesion molecules. Two proteins of cell junctions were studied: ZO1 of tight junctions and desmoplakin of desmosomes. Desquamation was induced by intravesical injection of LPS, constant illumination of mouse for 96 h, application of a combination of stress hormones hydrocortisone and norepinephrine or by removal of calcium with EGTA. All the inducers caused penetration of lanthanum tracer through the tight junctions, indicating paracellular permeability. Dilatation of extracellular spaces between neighboring cells was seen whenever desquamation was induced in bladders containing urine. Desquamation of single cells as well as groups of cells was observed. Contrary to obvious disconnection of cell junctions, as a precondition for desquamation, the distribution of junctional proteins did not change either in urothelial tissue or in desquamated cells. This study demonstrates that all the inducers of desquamation cause first an extensive dysfunction of a blood urine barrier and after that an occasional mechanical disconnection of adhesive junctions which consequently leads to desquamation.     

Membrane effects of ionizing radiation and hyperthermia

Results of numerous studies demonstrate that membranes are important sites of cell damage by both ionizing radiation and hyperthermia. Modification of membrane properties (mainly lipid fluidity) affects the cellular responses to radiation and hyperthermia but former concepts that membrane rigidification sensitizes cells to radiation while membrane fluidization potentiates hyperthermic damage have now been seriously challenged. It seems that the effects of membrane fluidity on cell responses to hyperthermia and radiation are due to an indirect influence on functional membrane proteins. The major role of lipid peroxidation in radiation damage to membranes has also been questioned. The existing evidence makes it unlikely that the interaction between radiation and hyperthermia is determined by the action of both agents on the same membrane components.