Impact of Inter-Individual Variance in the Expression of a Radiation-Responsive Gene Panel Used for Triage.

In previous studies we determined a gene expression signature in baboons for predicting the severity of hematological acute radiation syndrome. We subsequently validated a set of eight of these genes in leukemia patients undergoing total-body irradiation. In the current study, we addressed the effect of intra-individual variability on the basal level of expression of those eight radiation-responsive genes identified previously, by examining baseline levels in 200 unexposed healthy human donors (122 males and 88 females with an average age of 46 years) using real-time PCR. In addition to the eight candidate genes ( DAGLA, WNT3, CD177, PLA2G16, WLS, POU2AF1, STAT4 and PRF1), we examined two more genes ( FDXR and DDB2) widely used in ex vivo whole blood experiments. Although significant sex- (seven genes) and age-dependent (two genes) differences in expression were found, the fold changes ranged only between 1.1-1.6. These were well within the twofold differences in gene expression generally considered to represent control values. Age and sex contributed less than 20-30% to the complete inter-individual variance, which is calculated as the fold change between the lowest (reference) and the highest Ct value minimum-maximum fold change (min-max FC). Min-max FCs ranging between 10-17 were observed for most genes; however, for three genes, min-max FCs of complete inter-individual variance were found to be 37.1 ( WNT3), 51.4 ( WLS) and 1,627.8 ( CD177). In addition, to determine whether discrimination between healthy and diseased baboons might be altered by replacing the published gene expression data of the 18 healthy baboons with that of the 200 healthy humans, we employed logistic regression analysis and calculated the area under the receiver operating characteristic (ROC) curve. The additional inter-individual variance of the human data set had either no impact or marginal impact on the ROC area, since up to 32-fold change gene expression differences between healthy and diseased baboons were observed.

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors.

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.

Radiation-chemotherapy interactions.

The design of radiobiological experiments to study radiation-chemotherapy interactions in tumors and normal tissues is critically reviewed. It is suggested that such experiments should not be designed in such a way as to optimize the treatment strategy for the respective animal model, but to study the influence of one modality on the effectiveness of the other modality with regard to one specific endpoint at a time and its dependence on dose and timing, preferentially in multifraction experiments.

Cell repopulation and overall treatment time.

Prolongation of the overall treatment time in radiotherapy, especially of squamous cell carcinomas of the head and neck, decreases the chances of cure. This is most likely because of the proliferation of surviving clonogenic tumor cells between dose fractions. Between the 3rd and 7th week of conventional radiotherapy of head and neck cancers, on average 0.5 to 0.7 Gy are lost per day by repopulation. As this represents an average value, repopulation may be even more efficient in subgroups of faster tumors. There is little information on repopulation rates during the first 2 weeks of radiotherapy. Prolongation of the overall treatment time in radiotherapy helps to avoid severe side effects from acutely responding tissues, especially oral mucosa. This sparing effect of increasing overall treatment time is mainly caused by the regeneration of mucosal stem cells and transit cells. This repopulation is slow in the first 2 weeks and accelerates dramatically thereafter. The nature of the trigger for acceleration is not known but seems to be related to a critical threshold of acute tissue hypoplasia and the development of the inflammatory reaction of the connective tissue. It is not known whether accelerated tumor repopulation is stimulated by the same or a similar mechanism as the normal epithelium. During accelerated repopulation the oral mucosa is able to compensate a considerably higher proportion of the daily dose fraction than the tumor. These factors have to be taken into account when treatment strategies are designed to cope with the problem of accelerated repopulation.

Chronic damage after radiation therapy: challenge to radiation biology.

After local irradiation of the heart or the recto-sigmoid of rats, chronic radiation damage develops after several months, leading to well-defined clinical syndromes of fatal pericarditis and myocardial necrosis or fatal ileus. In both organs, the LD-50 is below 20 Gy, and a pronounced split dose recovery has been measured with fractionated irradiation. In both organs, the primary radiation damage appears to be to the capillaries, which then leads to secondary parenchymal atrophy. Damage to structured vessels and fibrosis was only seen in areas of necrosis, which in the gut might be precipitated by secondary trauma to the atrophic mucosa.

Radiation induced heart disease in hypertensive rats.

Spontaneously hypertensive Wistar rats were given single doses of X rays to their heart. Irradiation decreased the blood pressure before any myocardial radiation damage was apparent. Male rats, which were more hypertensive than female rats, had a shorter survival time after local heart irradiation than female rats. Antihypertensive treatment with hydralazine did not increase the survival time. It is considered that myocardial hypertrophy is the cause of the increased susceptibility of spontaneously hypertensive rats to local heart irradiation.

Experimental studies on the pathogenesis of the chronic radiation ulcer of the large bowel in rats.

Following local irradiation of a 24 mm segment of the large bowel with 23 Gy, 90% of Wistar rats developed a chronic radiation ulcer leading to progressive large bowel obstruction within 8 weeks. The incidence and latency of the chronic radiation damage was markedly altered by local treatments after irradiation, especially those which modified the amount and texture of the feces. The results of these studies suggest that the primary radiation damage to the large bowel is to the microvasculature of the mucosal and submucosal stroma leading to progressive mucosal atrophy which thus becomes very vulnerable. The chronic radiation ulcer and the hypertrophic, cystic mucosa (which is the result of hyperregeneration of subclinical ulcers) are secondary to the interaction of the primary radiation damage to the vascular-connective tissue of the intestinal wall with mechanical and infectious damage to the chronically atrophic mucosa.

The influence of the number of fractions and of overall treatment time on local control and late complication rate in squamous cell carcinoma of the larynx.

Three hundred and ten patients with T3/T4, N0, M0 squamous cell carcinoma were irradiated with 200 kV X rays with total doses ranging from 4,900 to 6,200 rad, given in 21 to 35 fractions in 32-63 days. After a minimum follow-up period of 3 years, the local control rate was 50%; 21 severe late complications were observed among the patients. The dependence of local control rate and of late complication rate on the dose per fraction and on overall treatment time was analyzed by various statistical methods. Whereas the late complication rate depended significantly on dose per fraction, local tumor control depended strongly on overall treatment time.

Effect of X-irradiation on the stomach of the rat.

A model for localized 300 kV X-irradiation of the rat stomach was developed. After irradiation with single doses, three distinct gastric disorders were observed which occurred at different latency times. Acute death 2-3 weeks after irradiation was caused by an erosive and ulcerative gastritis and occurred in all animals given 28.5 Gy without diet, in 17% of the animals given 28.5 Gy plus diet, and in 13% of the animals given 23 Gy. Subacute to chronic fatal disorders 4 weeks to 7 months after irradiation were seen as stomach dilatation and gastroparesis, associated with the replacement of the normal gastric mucosa by a hyperkeratinized multilayered squamous epithelium. These disorders occurred in 40-100% of the animals after doses between 16 Gy and 28.5 Gy (+diet). An ED 50 value of 19.2 Gy (16.5-21.2 Gy, 95% confidence interval) was calculated for this gastroparesis. Late gastric obstruction exceeding 7 months after irradiation was seen in the rats because of profound changes in the gastric wall in 13-18% of the animals after doses between 23 Gy and 14 Gy. In animals surviving these three periods, an atrophic mucosa and intestinal metaplasia developed. From functional and morphohistological studies, it can be concluded that there are differences in the pathogenesis of the fatal radiation damage for each of these periods after irradiation.

Physiologic consequences of local heart irradiation in rats.

Noninvasive methods have been used to study the long-term cardiovascular and pulmonary functional changes at rest and after exercise in adult rats following local heart irradiation with single x-ray doses of 15, 17.5 or 20 Gy, and in non-irradiated control animals. Rats that had undergone a chronic exercise program were compared with untrained cohorts. The earliest dysfunction detected was an increased respiratory rate (f) at 10 weeks after irradiation in the highest dose group. In contrast, both telemetric heart-rate (HR) and rhythm and indirect systolic blood pressure measurements performed at rest only revealed changes starting at 43 weeks after irradiation with 20 Gy, up to which point the rats showed no clinical signs of heart failure. However, the number of minutes required for the recovery of the HR to pre-exercise levels following the implementation of a standardized exercise challenge was elevated in untrained rats compared with their trained cohorts at 18 weeks after irradiation with 20 Gy. Increases in recovery times were required in the two lowest dose groups, starting at 26 weeks after irradiation. It was concluded that the reserve capacity of the cardiopulmonary system masks functional decrements at rest for many months following local heart irradiation, necessitating the use of techniques which reveal reductions in reserve capacities. Further, the influence of local irradiation to the heart and lungs deserves closer scrutiny due to mutual interactions.

The influence of autologous tumor fibroblasts on the radiosensitivity of squamous cell carcinoma megacolonies.

PURPOSE: To study the influence of tumor fibroblasts on radiosensitivity and stem cell fraction of tumor cells in squamous cell carcinoma megacolonies by determining colony cure and clonogen survival. METHODS AND MATERIALS: Murine squamous cell carcinoma cells (AT478c) grown as flat but multilayered megacolonies were co-cultured with pre-irradiated tumor fibroblasts derived from the same carcinoma, and irradiated with 1, 2, 4, or 8 fractions. Recurrent clones and their growth pattern in situ were recorded. From megacolony cure data and clonogen survival data, the clonogen number and the parameters of cellular radiosensitivity were calculated. RESULTS: The curability of the co-cultured megacolonies, as determined by TCD50 values, was significantly increased compared to the megacolonies without fibroblasts (p < 0.01). Both the megacolony cure and clonogen survival data suggested a decrease of the clonogen fraction in the co-cultured megacolonies. CONCLUSIONS: The presence of tumor fibroblasts increases megacolony radiosensitivity. This is due to a decrease in the fraction of clonogens in the tumor megacolony, apparently caused by a downregulation of the stem cell fraction of the tumor cells.

Radiation-induced heart disease in rats.

After local irradiation of the rat heart with X ray doses of over 10 Gy (single dose), animals developed symptoms of radiation-induced heart disease, which at higher doses would lead to fatal cardiac failure. The LD 50 at 1 year was between 15 Gy and 20 Gy. The pericardium and epicardium responded to irradiation with exudative pericarditis after 4 months. Focal myocardial damage was secondary to progressive capillary damage.

Tumour stem cells: the biological concept and its application in cancer treatment.

Experiments in different transplantable mouse tumours suggest that a proportion ranging from 0.1% to 100% of all tumour cells in these different tumours meet the functional definition criteria of tumour stem cells, i.e. regrowth of the tumour proceeded by clonal expansion from a single cell with unlimited proliferative potential. It is concluded that the proliferative organization of many or most human tumours may resemble that of the tissue of origin with a small proportion of stem cells and the majority of transit cells, both proliferating or resting. Cell loss and accelerated repopulation are due to regulated changes in the symmetry constraint of stem cell divisions.

Volume effects in radiobiology as applied to radiotherapy.

PURPOSE: To explore the radiobiological evidence for a dependence of normal tissue complication probability on irradiated normal tissue volume. MATERIALS AND METHODS: Data from experimental studies on the volume effect in different organs, using different criteria of structural or functional organ damage and in different animals, were evaluated to investigate the volume effects for structural radiation damage as opposed to functional radiation damage, and the importance of organ anatomy and dose distribution within the organ on the development of chronic radiation damage in the respective organ. RESULTS: There is little or no volume effect for structural radiation damage, however, some very pronounced volume effects have been reported for functional damage. Volume, as such, is not the relevant criterion, since critical, radiosensitive structures are not homogeneously distributed within organs. CONCLUSION: Volume effects in patients and experimental animals are more related to organ anatomy and organ physiology than to cellular radiobiology.

Rapid repopulation in radiotherapy: a debate on mechanism. Accelerated repopulation in tumours and normal tissues.

Accelerated repopulation is a well established response pattern of normal epithelial to fractionated irradiation. It is delayed until the tissue recognises functional injury. It is well regulated to maintain a steady state and continues until integrity of the tissue is restored. We assume that some of these features of the parental normal tissue are preserved and still operate in squamous cell carcinoma, although probably in a less well controlled and organised manner.

Experimental irradiation of the rat ureter: the effects of field size and the presence of contrast medium on incidence and latency of hydronephrosis.

Following X-irradiation of a 1.5 cm length of rat ureter, hydronephrosis developed after doses down to 10 Gy. The estimated ED50 was 11.8 Gy. In the dose range 37.4 Gy to 17.5 Gy there was a significant increase in latency with decreasing dose, but at lower doses the latency did not increase further. Reducing the length of ureter irradiated to 0.5 cm or 0.8 cm caused a decrease in incidence of hydronephrosis and longer latency periods. The ED50 for rats irradiated to 0.5 cm of ureter was 29.6 Gy. The possibility that secondary radiation produced as a result of interaction between X-radiation and iodinated contrast medium might affect the radiation induction of hydronephrosis was investigated. No difference was found between groups of rats irradiated with or without injection of contrast media.

Chronic radiation damage in the rectum of the rat after protracted fractionated irradiation.

In this experiment, a protracted fractionation regime (18 fractions/170 days, dose/fraction: 6,7,8 Gy) caused late rectal obstruction in rats independently of any clinically detectable acute damage. Rectal obstruction was due to an ulcer which developed secondary to an atrophic mucosa and capillary damage. In agreement with our earlier protracted fractionation schedules, the results were not fitted well by the linear-quadratic model. However, there are indications of relatively low alpha/beta values. Hydronephroses and hydroureters were the only competing risks to rectal obstruction and they occurred independently of it.

The effect of actinomycin D on split-dose recovery and repopulation in jejunal crypt cells in vivo.

The effect of actinomycin D in combination with radiation on the survival of mouse jejunal crypt cells was measured using the microcolony technique. When administered 30 min before irradiation the drug was seen to enhance the cell killing of single doses of radiation. No significant changes in the rate or amount of repair in split-dose irradiation were found. Protracted split-dose intervals revealed cell repopulation commenced within 12 h after the first radiation dose. When given 6 h post-conditioning dose actinomycin D did not inhibit cell repopulation.