The Effect of X-Irradiation On Cell Loss In Five Solid Murine Tumours, As Determined By the 125Iudr Method

The rate of cell loss in irradiated RIF-1, EMT6, KHJJ, B16 and KHT tumours was studied using the ¹²⁵IUdR loss technique. Administration of ¹²⁵IUdR preceded localized tumour irradiation by 2 days. Loss of tumour radioactivity was measured for 6–8 days after irradiation. the blood flow to some tumours was occluded during, and for 30 min following, injection of the label to measure the amount of radioactivity entering the tumour as a result of reutilization of label from the gut epithelia and influx of labelled host cells. Irradiation did not significantly alter the amount of radioactivity entering these clamped tumours during the 8–10 days after injection of ¹²⁵IUdR. This permitted comparison of irradiated and control groups based on the loss of radioactivity from the non-occluded tumours. Irradiation of RIF-1, EMT6, KHJJ or B16 tumours with doses of 600, 1400, 2400 or 4400 rads produced no significant increase in the rate of loss of tumour radioactivity. This suggested that, in the population of labelled cells, cell lysis following irradiation proceeded slowly. In contrast, KHT tumours showed a significant increase in loss rate following each radiation dose, although the increase was dose-independent. In all tumour systems, the constant rate of cell loss after radiation appeared to coincide with published reports of tumour growth responses after irradiation. the present data suggest that the manner of expression of radiation-induced cell killing results from the cellular proliferative status, i.e. whether a cell is cycling or non-cycling.     

Radiation carcinogenesis in experimental animals and its implications for radiation protection

Cancer induction is generally considered to be the most important somatic effect of low doses of ionizing radiation. It is therefore of great concern to assess the quantitative cancer risk of exposure to radiations of different quality and to obtain information on the dose-response relationships for carcinogenesis. Tissues in the human with a high sensitivity for cancer induction include the bone marrow, the lung, the thyroid and the breast in women. If the revised dosimetry estimates for the Japanese survivors of the atomic bomb explosions are correct, there is no useful data base left to derive r.b.e. values for human carcinogenesis. As a consequence, it will be necessary to rely on results obtained in biological systems, including experimental animals, for these estimates. With respect to radiation protection, the following aspects of experimental studies on radiation carcinogenesis are of relevance: Assessment of the nature of dose-response relationships. Determination of the relative biological effectiveness of radiations of different quality. Effects of fractionation or protraction of the dose on tumour development. For the analysis of tumour data in animals, specific approaches have to be applied which correct for competing risks. These methods include actuarial estimates, non-parametric models and analytical models. The dose-response curves for radiation-induced cancers in different tissues vary in shape. This is exemplified by studies on myeloid leukaemia in mice and mammary neoplasms in different rat strains. The results on radiation carcinogenesis in animal models clearly indicate that the highest r.b.e. values are observed for neutrons with energies between 0.5 and 1 MeV. On the basis of such results it might be concluded that the maximum quality factor of 10 for neutrons should be increased. Based on current evidence, an increase by a factor of 2 to 3 seems more realistic than a tenfold rise. The diversity of dose-response relationships point to different mechanisms involved in the induction of different tumours in various species and even in different strains of the same species.     

Cell kinetics and radiation biology

The cell cycle, the growth fraction and cell loss influence the response of cells to radiation in many ways. The variation in radiosensitivity around the cell cycle, and the extent of radiation-induced delay in cell cycle progression have both been clearly demonstrated in vitro. This translates into a variable time of expression of radiation injury in different normal tissues, ranging from a few days in intestine to weeks, months or even years in slowly proliferating tissues like lung, kidney, bladder and spinal cord. The radiosensitivity of tumours, to single doses, is dominated by hypoxic cells which arise from the imbalance between tumour cell production and the proliferation and branching of the blood vessels needed to bring oxygen and other nutrients to each cell. The response to fractionated radiation schedules is also influenced by the cell kinetic parameters of the cells comprising each tissue or tumour. This is described in terms of repair, redistribution, reoxygenation and repopulation. Slowly cycling cells show much more curved underlying cell survival curves, leading to more dramatic changes with fractionation, dose rate or l.e.t. Rapidly cycling cells redistribute around the cell cycle when the cells in sensitive phases have been killed, and experience less mitotic delay than slowly proliferating cells. Reoxygenation seems more effective in tumours with rapidly cycling cells and high natural cell loss rates. Compensatory repopulation within a treatment schedule may spare skin and mucosa but does not spare slowly proliferating tissues. Furthermore, tumour cell proliferation during fractionated radiotherapy may be an important factor limiting the overall success of treatment.     

Differential effects of genes of the <Emphasis Type="Italic">Rb1</Emphasis> signalling pathway on osteosarcoma incidence and latency in alpha-particle irradiated mice

Osteosarcoma is the most frequent secondary malignancy following radiotherapy of patients with bilateral retinoblastoma. This suggests that the Rb1 tumour suppressor gene might confer genetic susceptibility towards radiation-induced osteosarcoma. To define the contribution of the Rb1 pathway in the multistep process of radiation carcinogenesis, we evaluated somatic allelic changes affecting the Rb1 gene itself as well as its upstream regulator p16 in murine osteosarcoma induced by (227)Th incorporation. To distinguish between the contribution of germline predisposition and the effect of a 2-hit allelic loss, two mouse models harbouring heterozygote germline Rb1 and p16 defects were tested for the incidence and latency of osteosarcoma following irradiation. We could show that all tumours arising in BALB/c×CBA/CA hybrid mice (wild-type for Rb1 and for p16) carried a somatic allelic loss of either the Rb1 gene (76.5%) or the p16 gene (59%). In none of the tumours, we found concordant retention of heterozygosity at both loci. Heterozygote knock-out mice for Rb1 exhibit a significant increase in the incidence of osteosarcoma following (227)Th incorporation (11/24 [corrected] in Rb1+/- vs. 2/18 in Rb1+/+, p=4×10(-5)), without affecting tumour latency. In contrast, heterozygote knock-out mice for p16 had no significant change in tumour incidence, but a pronounced reduction of latency (LT(50%) =355 days in p16+/- vs. 445 days in p16+/+, p=8×10(-3)). These data suggest that Rb1 germline defects influence early steps of radiation osteosarcomagenesis, whereas alterations in p16 mainly affect later stages of tumour promotion and growth.     

Radiation combines with immune checkpoint blockade to enhance T cell priming in a murine model of poorly immunogenic pancreatic cancer

Radiation has been a pillar of cancer therapy for decades. The effects of radiation on the anti-tumour immune response are variable across studies and have not been explicitly defined in poorly immunogenic tumour types. Here, we employed combination checkpoint blockade immunotherapy with stereotactic body radiation therapy and examined the effect on tumour growth and immune infiltrates in subcutaneous and orthotopic mouse models of pancreatic cancer. Although immune checkpoint blockade and radiation were ineffective alone, their combination produced a modest growth delay in both irradiated and non-irradiated tumours that corresponded with significant increases in CD8+ T cells, CD4+ T cells and tumour-specific T cells as identified by IFNγ ELISpot. We conclude that radiation enhances priming of tumour-specific T cells in poorly immunogenic tumours and that the frequency of these T cells can be further increased by combination with immune checkpoint blockade.     

Optimization of tumour radiotherapy: Part V--Radiosensitization by 2-deoxy-D-glucose and DNA ligand Hoechst-33342 in a murine tumour

Radiosensitizing effects of combination of a minor groove DNA ligand, Hoechst-33342, with the glucose analogue and inhibitor of glycolysis, 2-deoxy-D-glucose (2-DG) have been investigated in Ehrlich ascites tumour (EAT) bearing mice following focal irradiation of the tumour with 60Co gamma-rays. Treatment-induced tumour growth delay and tumour free animal survival were evaluated as parameters of radiation response. Focal irradiation of the tumour with a single fraction of 10 Gy induced a moderate delay in tumour growth but did not lead to complete regression in any of the tumours. Intravenous administration of H-342 1 hr before irradiation enhanced radiation-induced growth delay in a dose dependent manner. Complete regression of the tumour was observed only at a dose of 10 mg/kg body wt, leading to a cure (tumour free survival for more than 100 days) rate of 55%. Administration of 2-DG (2 g/kg body wt; i.v.), immediately before irradiation significantly enhanced radiation-induced growth delay and resulted in a cure rate of 45%. In combination with this dose of 2-DG (2 g/kg body wt), H-342 at a lower dose (5 mg/kg body wt) significantly enhanced the cure rate to 66%. H-342 or 2-DG given alone or in combination at the doses investigated here did not show any significant effects on the unirradiated tumour.     

The Evolution of Tumour Composition During Fractionated Radiotherapy: Implications for Outcome

Current protocols for delivering radiotherapy are based primarily on tumour stage and nodal and metastases status, even though it is well known that tumours and their microenvironments are highly heterogeneous. It is well established that the local oxygen tension plays an important role in radiation-induced cell death, with hypoxic tumour regions responding poorly to irradiation. Therefore, to improve radiation response, it is important to understand more fully the spatiotemporal distribution of oxygen within a growing tumour before and during fractionated radiation. To this end, we have extended a spatially resolved mathematical model of tumour growth, first proposed by Greenspan (Stud Appl Math 51:317–340, 1972), to investigate the effects of oxygen heterogeneity on radiation-induced cell death. In more detail, cell death due to radiation at each location in the tumour, as determined by the well-known linear-quadratic model, is assumed also to depend on the local oxygen concentration. The oxygen concentration is governed by a reaction-diffusion equation that is coupled to an integro-differential equation that determines the size of the assumed spherically symmetric tumour. We combine numerical and analytical techniques to investigate radiation response of tumours with different intratumoral oxygen distribution profiles. Model simulations reveal a rapid transient increase in hypoxia upon regrowth of the tumour spheroid post-irradiation. We investigate the response to different radiation fractionation schedules and identify a tumour-specific relationship between inter-fraction time and dose per fraction to achieve cure. The rich dynamics exhibited by the model suggest that spatial heterogeneity may be important for predicting tumour response to radiotherapy for clinical applications.     

Tumour size can have an impact on the outcomes of epidemiological studies on second cancers after radiotherapy

Obtaining a correct dose–response relationship for radiation-induced cancer after radiotherapy presents a major challenge for epidemiological studies. The purpose of this paper is to gain a better understanding of the associated uncertainties. To accomplish this goal, some aspects of an epidemiological study on breast cancer following radiotherapy of Hodgkin’s disease were simulated with Monte Carlo methods. It is demonstrated that although the doses to the breast volume are calculated by one treatment plan, the locations and sizes of the induced secondary breast tumours can be simulated and, based on these simulated locations and sizes, the absorbed doses at the site of tumour incidence can also be simulated. For the simulations of point dose at tumour site, linear and non-linear mechanistic models which predict risk of cancer induction as a function of dose were applied randomly to the treatment plan. These simulations provided for each second tumour and each simulated tumour size the predicted dose. The predicted-dose–response-characteristic from the analysis of the simulated epidemiological study was analysed. If a linear dose–response relationship for cancer induction was applied to calculate the theoretical doses at the simulated tumour sites, all Monte-Carlo realizations of the epidemiological study yielded strong evidence for a resulting linear risk to predicted-dose–response. However, if a non-linear dose–response of cancer induction was applied to calculate the theoretical doses, the Monte Carlo simulated epidemiological study resulted in a non-linear risk to predicted-dose–response relationship only if the tumour size was small (<?1.5 cm). If the diagnosed breast tumours exceeded an average diameter of 1.5 cm, an applied non-linear theoretical-dose–response relationship for second cancer falsely resulted in strong evidence for a linear predicted-dose relationship from the epidemiological study realizations. For a typical distribution of breast cancer sizes, the model selection probability for a resulting predicted-dose linear model was 61% although a non-linear theoretical-dose–response relationship for cancer induction had been applied. The results of this study, therefore, provide evidence that the shapes of epidemiologically obtained dose–response relationships for cancer induction can be biased by the finite size of the diagnosed second tumour, even though the epidemiological study was done correctly.     

Most common skin tumours in correlation with solar ultraviolet radiation in the area of West Herzegovina

Incidence rate of skin tumours, both, non-melanoma and melanoma, is increasing nowadays. Various etiological factors are of relevance for the occurrence of the diseases. The solar radiation, as well, long-term exposure to ultraviolet (UV) radiation, have the greatest impact on development of these skin tumours. Non-melanoma skin tumours, Basal Cell Carcinoma (BCC) and Squamous Cell Carcinoma (SCC), are the most common skin tumours in humans, and usually develop on the chronically photo-exposed areas. As for the Malignant Melanoma (MM), one of the most aggressive skin tumours, the exposure to solar radiation also plays an important role. This study investigates the correlation between the skin tumours and UV radiation in the area of West Herzegovina, on the sample of 1676 patients. It presents the occurrence of skin tumours in the period from 1997 to 2003. The study investigates the incidence and the risk factors separately for every skin tumour which can be etiologically related to the occurrence of skin tumours and UV radiation: occupation, exposure to UV radiation, skin type, and family history on malignan tumours within the patient's family. The exact incidence rate of non-melanoma and melanoma skin tumours in Bosnia and Herzegovina is still unknown, for the reason that the united National Cancer Register does not exist yet.     

Changes in G1-phase populations in human glioblastoma and neuroblastoma cell lines influence p66/Be neutron-induced micronucleus yield

Some photon resistant tumours are sensitive to neutrons but no predictive methods exist which could identify such tumours. In a recent study addressing this clinically important issue, we demonstrated that relative biologic effectiveness (RBE) values for p(66)/Be neutrons estimated from micronucleus (MN) data correlate positively with RBE values obtained from conventional clonogenic survival data. However, not all photon-resistant cell lines showed high RBE values when the MN endpoint was used. Now, we examine how the functional status of the p53 tumour suppressor gene and radiation-induced changes in cell cycle phase populations may contribute to this discrepancy. No significant association was established between p53 status and MN yield for both photon and neutron irradiation. The data demonstrated that neutron-, but not photon-, induced MN yield is dependent on the intrinsic ability of cells to activate a G1-phase arrest. In cell lines of comparable photon sensitivity, those showing more extensive depletion of the G1 population express significantly more micronuclei per unit dose of neutrons. These results suggest that differences in cell cycle kinetics, and not the p53 status, may constitute an important factor in damage induction by high linear energy transfer (LET) irradiation and need to be considered when radiation toxicity in clinical radiobiology or radiation protection is assessed using damage endpoints.     

X-ray-induced telomeric instability in Atm-deficient mouse cells

The gene responsible for ataxia telangiectasia (AT) encodes ATM protein, which plays a major role in the network of a signal transduction initiated by double strand DNA breaks. To determine how radiation-induced genomic instability is modulated by the dysfunction of ATM protein, we examined radiation-induced delayed chromosomal instability in individual cell lines established from wild-type Atm(+/+), heterozygote Atm(+/-), and knock-out Atm(-/-) mouse embryos. The results indicate that Atm(-/-) mouse cells are highly susceptible to the delayed induction of telomeric instability and end-to-end chromosome fusions by radiation in addition to the elevated spontaneous telomeric instability detected by telomere fluorescence in situ hybridization (FISH). The telomeric instability was characterized by abnormal telomere FISH signals, including loss of the signals and the extra-chromosomal signals that were associated and/or not associated with chromosome ends, suggesting that Atm deficiency makes telomeres vulnerable to breakage. Thus, the present study shows that Atm protein plays an essential role in maintaining telomere integrity and prevents chromosomes from end-to-end fusions, indicating that telomeres are a target for the induction of genomic instability by radiation.     

The effect of alterations in haematocrit on tumour sensitivity to X-rays

Hypoxic cells in human tumours probably contribute to the failure of radiotherapy in some sites. Changes in the oxygen carrying capacity of the blood, such as in anaemia, have been shown to influence tumour response. The effect of acute and chronic changes in haematocrit on the radiosensitivity of three mouse tumours (EMT6, KHT and RIF-1) were studied. Alterations in haematocrit were achieved by bleeding followed by retransfusion. When radiation was preceded immediately by an acute reduction in haematocrit (anaemia), radiosensitivity was markedly reduced in each tumour. An acute rise in haematocrit (polycythaemia) increased or decreased X-ray sensitivity depending on its severity. The optimum haematocrit for maximum sensitivity was always found to be at a level 5-10 per cent above normal. When the time between induction of anaemia and irradiation was increased, simulating a progressively longer duration of anaemia, marked changes in radiosensitivity of all the tumours were observed. A short duration of anaemia resulted in a resistant tumour with each cell line, but the resistance was gradually lost as the anaemia was prolonged, even though no recovery in haematocrit occurred. The rate of recovery to normal radiosensitivity varied from 24 to 72 hours in the different tumours. Therefore, only haematocrit changes which occurred within 1-3 days of a dose of radiation affect the radiosensitivity of these tumours.     

Incidence of radiation-induced skin tumours in mice and variations with dose rate

Mice were exposed to weakly penetrating beta-particles from an external source, using 12 different surface doses ranging from 5.4 to 260 Gy and given at four different dose rates from 200 to 1.7 cGy/min. As in previous investigations, both epidermal and dermal tumours occurred with the latter predominating. The lowest surface dose to produce a statistically significant increase in skin tumours was 21.7 Gy, no effect being detected with doses of 5.4-16.3 Gy. The dose-response curves rose steeply when obvious increases occurred. Consideration of these findings and the fact that radiation-induced skin tumours can have an exceptionally long latent period leads to the suggestion that there is some relatively radioresistant factor which normally restrains potential radiation-induced cancer cells in the skin from becoming tumours until the skin is subjected to high local doses. Tumour-induction was unaffected by reducing the highest dose rate by a factor of 10 and the dose-response curves were almost identical. Further reductions of dose rate, encompassing a further factor of 10, in general resulted in fewer tumours.     

CELL LOSS FROM SEVERAL TYPES OF SOLID MURTNE TUMOUR: COMPARISON OF 125I-IODODEOXYURIDINE AND TRITIATED THYMIDINE METHODS

The method of measuring tumour cell loss rates in situ following radioactivity loss after a single injection of ¹²⁵I-iododeoxyurudine (¹²⁵I-UdR) was tested for its accuracy in five different types of murine tumour. To achieve this the method was compared with two others: (1) using ¹²⁵I-UdR, but excising tumours before the radioactivity determinations, with or without extracting DNA; (2) using tritiated thymidine and autoradiography. A third method was used on three of the tumours, in which ¹²⁵I-UdR-labelled tumours were grown in unlabelled hosts, followed by whole body counting of the tumour-bearing mice. In two of the tumours an increase was observed in total tumour radioactivity with time after ¹²⁵I-UdR injection. This prevented the estimation of cell loss parameters in these tumours. Approximately half the increase was due to reutilization of ¹²⁵I-UdR supplied from tissues within the mouse; approximately a third to an influx of labelled inflammatory cells (probably in response to infection accompanying ulceration of overlying skin); and the remainder to an increase in non-DNA radioactivity. In these tumours cell loss rates could be obtained from the whole body counting technique in which influxes of labelled cells and reutilizable radioactivity were eliminated. A comparison of either ¹²⁵I-UdR technique with the ³H-TdR technique showed good agreement of the cell loss factors for the low cell loss tumours. However, for tumours with high cell loss factors the ¹²⁵I-UdR technique gave lower values for cell loss. This implied that reutilization of ¹²⁵I-UdR within the tumour (i.e. from internal, not external sources) occurred in the high cell loss tumours. It is concluded that equating radioactivity loss with cell loss after an injection of ¹²⁵I-UdR is reasonable for some tumours, but will result in significant underestimates in others. For high cell loss tumours the ³H-TdR technique will give the     

Mutation induction by inhaled radon progeny modeled at the tissue level

The observable responses of living systems to ionizing radiation depend on the level of biological organization studied. Understanding the relationships between the responses characteristic of the different levels of organization is of crucial importance. The main objective of the present study is to investigate how some cellular effects of radiation manifest at the tissue level by modeling mutation induction due to chronic exposure to inhaled radon progeny. For this purpose, a mathematical model of the bronchial epithelium was elaborated to quantify cell nucleus hits and cell doses. Mutagenesis was modeled considering endogenous as well as radiation-induced DNA damages and cell cycle shortening due to cell inactivation. The model parameters describing the cellular effects of radiation are obtained from experimental data. Cell nucleus hits, cell doses, and mutation induction were computed for the activity hot spots of the large bronchi at different exposures. Results demonstrate that the mutagenic effect of densely ionizing radiation is dominated by cell cycle shortening due to cell inactivation and not by DNA damages. This suggests that radiation burdens of non-progenitor cells play a significant role in mutagenesis in case of protracted exposures to densely ionizing radiation. Mutation rate as a function of dose rate exhibits a convex shape below a threshold. This threshold indicates the exhaustion of the tissue regeneration capacity of local progenitor cells. It is suggested that progenitor cell hyperplasia occurs beyond the threshold dose rate, giving a possible explanation of the inverse dose-rate effect observed in the epidemiology of lung cancer among uranium miners.     

Tumour bed effect: hypoxic fraction of tumours growing in preirradiated beds

The reduction in tumour growth rate seen when tumours are implanted into preirradiated sites, the tumour bed effect (TBE), is believed to be due to radiation damage to vascular stroma, leading to defective angiogenesis in the tumour. The present work examined whether or not the functional inadequacy of irradiated stroma was accompanied by an increased hypoxic fraction in tumours growing in irradiated beds. Mouse flank skin was given 0 or 20 Gy X-rays and RIF-1 fibrosarcoma cells were implanted i.d. into the centre of the treatment field one week later. Tumours of 200 mm3 were irradiated under clamped or unclamped conditions and the hypoxic fraction measured from the displacement of the corresponding survival curves, assayed in vitro. Results indicated a small increase in the hypoxic fraction. Averaging values from three independent experiments, the percentage of hypoxic cells increased from 2.5 per cent for cells in tumours growing in unirradiated beds to 4.6 per cent for those from tumours in beds given 20 Gy. Thus an irradiated vascular bed is still to some extent able to maintain the proportion of oxic: hypoxic tumour cells found in tumours growing in unirradiated beds, despite manifest changes in tumour necrosis and growth rate.     

Tumour escape mechanisms and their therapeutic implications in combination tumour therapy

Most tumours arise from a single normal cell through a sequential evolutionary process of mutation and selection. Tumours are initiated by escaping non‐immune surveillance, which includes defective DNA repair, epigenetic gene alternation, resistance to apoptosis and loss of intercellular contact inhibition. Tumour cells harbour mutations in a number of critical genes that provide selective advantages at various stages during the evolution of the tumour. The tumour cells that circumvent the tumour suppressor mechanisms of the non‐immune surveillance process are edited by the immune system, resulting in the selection of a resistant tumour variant. The selection of the tumour cell is further shaped by its interactions with cells and other factors in its microenvironment. Tumour evolution is thought to adhere to Darwinian principles by escaping both non‐immune (intrinsic) and immune (extrinsic) responses against self‐altered tumour cells. At end‐stage, tumours have escaped both non‐immune and immune surveillance with increased threshold of apoptosis. Combination therapy has been proposed, by exploring the non‐immune and immune suppressive nature of the tumour, and has been found to have a therapeutic efficiency on tumour regression as compared with monotherapies. The combination of immunotherapy and other different modalities, especially vaccines, with conventional anticancer therapies with optimized dosage and scheduling can offer synergistic antitumour effects. Here, we focus on the mechanism of tumour evolution and its implication in combination therapy.     

Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness

Cancer stem cells have been described in various cancers including squamous tumours of the skin by their ability to reform secondary tumours upon transplantation into immunodeficient mice. Here, we used transplantation of limiting dilution of different populations of FACS‐isolated tumour cells from four distinct mouse models of squamous skin tumours to investigate the frequency of tumour propagating cells (TPCs) at different stages of tumour progression. We found that benign papillomas, despite growing rapidly in vivo and being clonogenic in vitro, reformed secondary tumours upon transplantation at very low frequency and only when tumour cells were co‐transplanted together with tumour‐associated fibroblasts or endothelial cells. In two models of skin squamous cell carcinoma (SCC), TPCs increased with tumour invasiveness. Interestingly, the frequency of TPCs increased in CD34^HI but not in CD34^LO SCC cells with serial transplantations, while the two populations initially gave rise to secondary tumours with the same frequency. Our results illustrate the progressive increase of squamous skin TPCs with tumour progression and invasiveness and reveal that serial transplantation may be required to define the long‐term renewal potential of TPCs.     

Radiation-induced apoptosis and its relationship to loss of clonogenic survival

Ionizing radiation can be an effective inducer of apoptosis and studies of many aspects of the pathways and mechanisms involved in this apoptosis induction have been published. This review stresses two aspects: the relationship between apoptosis and loss of clonogenic ability in irradiated cells and the time course for the appearance of apoptosis after radiation exposure. Although it was initially assumed that apoptosis occurred relatively quickly (within hours) after irradiation, evidence is presented and discussed here showing that apoptosis can occur at long times after irradiation (out to 20 days) in some cell types. This late, or delayed, apoptosis occurs after the cells have divided once or several times. The impact of delayed apoptosis on loss of clonogenicity after irradiation remains unclear. It seems likely that in some cell types, e.g., fibroblasts, the occurrence of late apoptosis is minimal and may have little impact on long term cell survival of the population, but in at least one instance, with a cell line of hematopoietic origin, it appears that late apoptosis can account for all the loss of clonogenicity in irradiated cells. The role of p53 in radiation-induced apoptosis is also discussed, with data presented showing that both p53-dependent and independent pathways for radiation-induced apoptosis exist, depending on the cell type.     

Expression of DeltaNLef1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours.

To examine the consequences of repressing beta-catenin/Lef1 signalling in mouse epidermis, we expressed a DeltaNLef1 transgene, which lacks the beta-catenin binding site, under the control of the keratin 14 promoter. No skin abnormalities were detected before the first postnatal hair cycle. However, from 6 weeks of age, mice underwent progressive hair loss which correlated with the development of dermal cysts. The cysts were derived from the base of the hair follicles and expressed morphological and molecular markers of interfollicular epidermis. Adult mice developed spontaneous skin tumours, most of which exhibited sebaceous differentiation, which could be indicative of an origin in the upper part of the hair follicle. The transgene continued to be expressed in the tumours and beta-catenin signalling was still inhibited, as evidenced by absence of cyclin D1 expression. However, patched mRNA expression was upregulated, suggesting that the sonic hedgehog pathway might play a role in tumour formation. Based on our results and previous data on the consequences of activating beta-catenin/Lef1 signalling in postnatal keratinocytes, we conclude that the level of beta-catenin signalling determines whether keratinocytes differentiate into hair or interfollicular epidermis, and that perturbation of the pathway by overexpression of DeltaNLef1 can lead to skin tumour formation.