The addition of Valproic acid to concurrent radiation therapy and temozolomide improves patient outcome: a Correlative analysis of RTOG 0525, SEER and a Phase II NCI trial.

PURPOSE/OBJECTIVES: Valproic Acid (VPA) is an antiepileptic agent with HDACi (histone deacetylase inhibitor) activity shown to radiosensitize glioblastoma (GBM) cells. We evaluated the addition of VPA to standard radiation therapy (RT) and temozolomide (TMZ) in an open-label, phase II study (NCI-06-C-0112). The intent of the current study was to compare our patient outcomes with modern era standard of care data (RTOG 0525) and general population data (SEER 2006-2013). MATERIALS/METHODS: 37 patients with newly diagnosed GBM were treated in a phase II NCI trial with daily VPA (25 mg/kg) in addition to concurrent RT and TMZ (2006 - 2013) and 411 patients with newly diagnosed GBM were treated in the standard TMZ dose arm of RTOG 0525 (2006 - 2008). Using the SEER database, adult patients (age > 15) with diagnostic codes 9440-9443 (third edition (IDC-O-3) diagnosed between 2006 - 2013 were identified and 6083 were included in the analysis. Kaplan-Meier method was used to estimate OS and PFS. The effect of patient characteristics and clinical factors on OS and PFS was analyzed using univariate analysis and a Cox regression model. A landmark analysis was performed to correlate recurrence to OS and conditional probabilities of surviving an additional 12 months at diagnosis, 6, 12, 18, 24 and 30 months were calculated for both the trial data and the SEER data. RESULTS: Updated median OS in the NCI cohort was 30.9m (22.2- 65.6m), compared to RTOG 0525 18.9m (16.8-20.3m) (p= 0.007) and the SEER cohort of 11m. Median PFS in the NCI cohort was 11.1m (6.6 - 49.6m) compared to RTOG 0525 with a median PFS of 7.5m (6.9-8.2m) (p = 0.004). Younger age, class V RPA and MGMT status were significant for PFS in both the NCI cohort and the RTOG 0525 cohort, in addition KPS was also significant for OS. In comparison to RTOG 0525, the population in the NCI cohort had a more favorable KPS and RPA, and a higher proportion of patients receiving bevacizumab after protocol therapy however with the exception of RPA (V) (8% vs 18%) (0.026), the effects of these factors on PFS and OS were not significantly different between the two cohorts. CONCLUSION: Previously reported improvements in PFS and OS with the addition of VPA to concurrent RT and TMZ in the NCI phase II study were confirmed by comparison to both a trial population receiving standard of care (RTOG 0525) and a contemporary SEER cohort. These results provide further justification of a phase III trial of VPA/RT/TMZ.

Cyclooxygenase-2 expression in human gliomas: prognostic significance and molecular correlations.

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the growth and progression of a variety of human cancers. Although COX-2 overexpression has been observed in human gliomas, the prognostic or clinical relevance of this overexpression has not been investigated to date. In addition, no study has analyzed the relationship between COX-2 expression and other molecular alterations in gliomas. Consequently, we examined COX-2 expression by immunohistochemistry in tumor specimens from 66 patients with low- and high-grade astrocytomas and correlated the percentage of COX-2 expression with patient survival. We also analyzed the relative importance of COX-2 expression in comparison with other clinicopathological features (age and tumor grade) and other molecular alterations commonly found in gliomas (high MIB-1 level, p53 alteration, loss of retinoblastoma (Rb) protein or p16, and high bcl-2 level). Kaplan-Meier analyses demonstrated that high COX-2 expression (>50% of cells stained positive) correlated with poor survival for the study group as a whole (P < 0.0001) and for those with glioblastoma multiforme in particular (P < 0.03). Cox regression analyses demonstrated that COX-2 expression was the strongest predictor of outcome, independent of all other variables. In addition, high COX-2 expression correlated with increasing histological grade but did not correlate with positive p53 immunostaining, bcl-2 expression, loss of p16 or retinoblastoma protein expression, or high MIB-1 expression. These findings indicate that high COX-2 expression in tumor cells is associated with clinically more aggressive gliomas and is a strong predictor of poor survival.

Radiation-induced transcription factor activation in the rat cerebral cortex.

PURPOSE: To determine the effects of ionising radiation on the DNA-binding activity of the injury-related transcription factors AP-1, Sp-1, p53 and NFkappaB in the rat brain. MATERIALS AND METHODS: Male Sprague-Dawley rats were irradiated with 137Cs gamma-rays at 3.8Gy/min and the cerebral cortex was isolated at intervals up to 24h. Nuclear protein extract of the cerebral cortex was analysed by electrophoretic mobility shift assay for DNA-binding activity of AP-1, Sp-1, p53 and NFkappaB. In addition, total RNA was extracted from the cerebral cortex and subjected to northern analysis. RESULTS: The DNA-binding activity of each of the transcription factors increased in a time- and dose-dependent manner after irradiation. Maximum increase in the activity of AP-1, Sp-1, and p53 DNA binding was seen after exposure to 10Gy and then decreased after higher doses. In contrast, NFkappaB DNA-binding activity continued to increase out to at least 30Gy. The levels of bFGF and p21WAF-1 mRNA increased after irradiation, suggesting an increase in the transactivating activity of AP-1, Sp-1, and p53. CONCLUSIONS: These data indicate that the response of the CNS to irradiation includes the activation of a similar set of transcription factors as previously observed after other types of insults.

Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor.

The antitumor effects of the selective cyclooxygenase (COX)-2 inhibitor SC-236 alone and in combination with radiation were investigated using the human glioma cell line U251 grown in monolayer culture and as tumor xenografts. On the basis of Western and Northern blot analyses, these cells express COX-2 protein and mRNA to levels similar to those in the human colon carcinoma cell line HT29. Treatment of U251 cells in monolayer culture with 50 microM SC-236 resulted in a time-dependent decrease in cell survival as determined by a clonogenic assay. The cell death induced by SC-236 was associated with apoptosis and the detachment of cells from the monolayer. After 2 days of drug treatment, the cells that remained attached were exposed to graded doses of radiation, and the clonogenic assay was performed. Comparison of the survival curves for drug-treated and untreated cultures revealed that SC-236 enhanced radiation-induced cell death. In these combination studies, SC-236 treatment resulted in a dose-enhancement factor of 1.4 at a surviving fraction of 0.1, with the surviving fraction at 2 Gy (SF2) reduced from 0.61 to 0.31. These data indicate that in vitro SC-236 induces U251 apoptotic cell death and enhances the radiosensitivity of the surviving cells. To extend these investigations to an in vivo situation, U251 glioma cells were grown as tumor xenografts in the hind leg of nude mice, and SC-236 was administered in drinking water. SC-236 alone slowed tumor growth rate, and when administered in combination with local irradiation, SC-236 caused a greater than additive increase in tumor growth delay. These in vitro and in vivo results suggest that the selective inhibition of COX-2 combined with radiation has potential as a cancer treatment.

The radioresponse of the central nervous system: a dynamic process.

Radiation continues to be a major treatment modality for tumors located within and close to the central nervous system (CNS). Consequently, alleviating or protecting against radiation-induced CNS injury would be of benefit in cancer treatment. However, the rational development of such interventional strategies will depend on a more complete understand-ing of the mechanisms responsible for the development of this form of normal tissue injury. Whereas the vasculature and the oligodendrocyte lineage have traditionally been considered the primary radiation targets in the CNS, in this review we suggest that other phenotypes as well as critical cellular interactions may also be involved in determining the radio-response of the CNS. Furthermore, based on the assumption that the CNS has a limited repertoire of responses to injury, the reaction of the CNS to other types of insults is used as a framework for modeling the pathogenesis of radiation-induced damage. Evidence is then provided suggesting that, in addition to acute cell death, radiation induces an intrinsic recovery/repair response in the form of specific cytokines and may

Preferential enhancement of tumor radioresponse by a cyclooxygenase-2 inhibitor.

Cyclooxygenase-2 (COX-2), an inducible isoform of cyclooxygenase, is overexpressed in many types of malignant tumors, where it mediates production of prostaglandins (PGs), which in turn may stimulate tumor growth and protect against damage by cytotoxic agents. This study investigated whether SC-'236, a selective inhibitor of COX-2, potentiates antitumor efficacy of radiation without increasing radiation injury to normal tissue. Mice bearing the sarcoma FSA in the hind legs were treated daily for 10 days with SC-'236 (6 mg/kg given in the drinking water) when tumors were 6 mm in diameter. When tumors reached 8 mm in diameter, the mice were given 11- to 50-Gy single-dose local tumor irradiation with or without SC-'236. SC-'236 inhibited tumor growth on its own, and it greatly enhanced the effect of tumor irradiation. The growth delay was increased from 14.8 days after 25-Gy single dose to 28.4 days after the combined treatment (P = 0.01). SC-'236 reduced TCD50 (radiation dose yielding 50% tumor cure) from 39.2 Gy to 20.9 Gy (enhancement factor = 1.87). SC-'236 did not appreciably alter radiation damage to jejunal crypt cells and tissue involved in the development of radiation-induced leg contractures. The SC-'236-induced enhancement of tumor radioresponse was associated with a decrease in PGE2 levels in FSA tumors. The drug had no effect on radiation-induced apoptosis. Neoangiogenesis was inhibited by SC-'236, which could account for some of the increase in tumor radioresponse. Overall, our findings demonstrated that treatment with a selective inhibitor of COX-2 greatly enhanced tumor radioresponse without markedly affecting normal tissue radioresponse. Thus, COX-2 inhibitors have a high potential for increasing the therapeutic ratio of radiotherapy.

Adenovirus-mediated p53 gene therapy for human gliomas.

OBJECTIVE: The rationale and current evidence for using p53 gene replacement as a potential treatment for human gliomas are reviewed. The possible benefits of and obstacles to this approach are delineated. METHODS: One approach to overcoming the poor outcomes associated with conventional glioma therapies involves the replacement of tumor suppressor genes that are fundamental to glioma development. The p53 gene is one of the most frequently mutated genes in human gliomas, and loss of p53 function is critical to the development of glial neoplasms. Consequently, replacement of the p53 gene using viral vectors may be a potential treatment for human gliomas. RESULTS: In vitro studies demonstrate that adenovirus-mediated p53 gene transfer into gliomas with mutant p53 results in massive apoptosis. Similarly, transfer of p53 inhibits tumor growth in vivo. In contrast to mutant p53 gliomas, wild-type p53 glioma cells are resistant to the apoptotic effects of p53 transfer, but this resistance can be overcome by the addition of deoxyribonucleic acid-damaging agents such as ionizing radiation or chemotherapy. The main obstacle to p53 gene therapy involves the limitations associated with current modes of delivery. CONCLUSION: Preclinical data strongly support the use of p53 gene transfer as a potential treatment for human gliomas.

Radiosensitization of human tumor cell lines induced by the adenovirus-mediated expression of an anti-Ras single-chain antibody fragment.

The expression of activated ras genes has been implicated as a contributing factor to the radioresistance of tumor cells. As a strategy for compromising Ras protein activity and potentially enhancing the radiosensitivity of tumor cells, we have investigated the application of the AV1Y28 adenovirus, which expresses a single-chain antibody fragment directed against p21 Ras proteins. The ability of AV1Y28 transduction to modulate radioresponse was investigated using four human tumor cell lines--U251 glioblastoma, MIA PaCa-2 pancreatic carcinoma, and the colon carcinomas SW620 and HT29. Cultures were exposed to sufficient levels of AV1Y28 to transduce more than 90% of the cells; 24 h later, cultures were exposed to ionizing radiation, and clonogenic cell survival was determined. Tumor cell survival was reduced by 40-50% when the tumor cell lines were exposed to AV1Y28 only. In addition, for each tumor cell line, AV1Y28 exposure enhanced the level of radiation-induced cell killing. Dose enhancement factors at a surviving fraction of 0.1 ranged from 1.3 to 1.5. Furthermore, for each of the cell lines, the surviving fraction at 2 Gy was significantly reduced by AV1Y28 exposure. In contrast to the results seen in tumor cells, the radiosensitivity of a normal human fibroblast cell line was not affected by AV1Y28. These data indicate that this anti-Ras adenovirus enhances the radiosensitivity of tumor cells but does not affect the radiosensitivity of normal cells.

Ionizing radiation-induced alterations in the electrophysiological properties of Aplysia sensory neurons.

It is clear that ionizing radiation can alter neuronal function. Recently it has been suggested that radiation can directly influence neurons and/or the neuronal microenvironment. We have developed a simple in vitro model system utilizing the marine mollusc Aplysia californica to test this hypothesis. We show that ionizing radiation at doses of 5, 10 or 15 Gy produces complex effects on the electrophysiological properties of a population of Aplysia nociceptive sensory neurons at 24 and 48 h post irradiation. These results add support to the notion that ionizing radiation can directly influence neurons and/or the neuronal microenvironment. Furthermore, they demonstrate that Aplysia may be used as a useful model system to study radiation-induced neuronal plasticity.

NF kappa B activity and target gene expression in the rat brain after one and two exposures to ionizing radiation.

The central nervous system injury that can result after radiotherapy has been suggested to involve induced gene expression and cytokine production. We have previously shown that irradiation of primary cultures of rat astrocytes results in the activation of NF kappa B. To determine whether such an effect also occurs in vivo, NF kappa B activity was analyzed in the cerebral cortex of the rat brain after whole body irradiation. After a single dose of 15 Gy, NF kappa B activity was increased by 2 h postirradiation, returning to unirradiated levels by 8 hours. The increase was dose-dependent beginning at 2 Gy and continuing to at least 22.5 Gy. NF kappa B activity in the irradiated cortex was not accompanied by I kappa B alpha degradation. When 7.5 Gy was delivered 24 h before the 15 Gy, the increase in NF kappa B activity after 15 Gy was significantly reduced. These results suggest that an initial exposure to radiation induced a refractory period in the brain during which the susceptibility of NF kappa B to activation by subsequent irradiation was significantly reduced. This period of reduced sensitivity to radiation was also apparent for the induction of the NF kappa B-regulated cytokines IL-1 beta, IL-6, and TNF alpha.

X-irradiation-induced loss of O-2A progenitor cells in rat spinal cord is inhibited by implants of cells engineered to secrete glial growth factor 2.

The loss of O-2A progenitor cells has been implicated as a critical event in radiation-induced spinal cord demyelination. To investigate whether glial growth factor 2 (GGF2) affects the number of O-2A cells in the irradiated rat cervical spinal cord, an ex vivo gene therapy approach was applied in which CHO cells engineered to express recombinant human GGF2 were injected into the cisterna magna of adult rats. Spinal cord irradiation reduced the number of O-2A cells in a dose-dependent manner. However, this radiation-induced decrease in O-2A progenitor cells was significantly attenuated by the delivery of GGF2 after irradiation. These data indicate that the cell-mediated delivery of GGF2 can reduce the loss of O-2A progenitors after irradiation.

Increased expression of prohormone convertase-2 in the irradiated rat brain.

Changes in gene expression have been suggested to play a role in radiotherapy-induced central nervous system (CNS) injury. To begin to identify radiation-inducible genes in the CNS, we have applied the differential display of reverse transcription-polymerase chain reaction products to RNA extracted from the brain of adult rats. RNA was isolated from a rat brain 6 h after whole-body exposure to 10 Gy and compared with RNA from unirradiated brain. A cDNA band was consistently observed at about 600 bp in samples from the irradiated rat but not from unirradiated (control) rat. Amplification and sequencing of the cDNA revealed that it corresponded to the prohormone convertase-2 (PC2) gene, which is involved in the processing of inert prohormones and neuropeptides to their bioactive forms. Enhanced PC2 expression was detected after irradiation of neuronal cultures but not in cultures of astrocytes, suggesting that the cell type in the CNS responsible for the PC2 induction after in vivo irradiation is the neuron. These results indicate that radiation induces the expression of a neuronal enzyme that is critical to the activation of a number of prohormones and neuropeptides, which may influence the radioresponse of the CNS.

IkappaBalpha degradation is not a requirement for the X-ray-induced activation of nuclear factor kappaB in normal rat astrocytes and human brain tumour cells.

PURPOSE: To investigate the mechanism of NFkappaB activation by X-rays in normal primary rat astrocytes. MATERIALS AND METHODS: Primary cultures of type I astrocytes generated from the cortex of neonatal rats were exposed to X-rays with and without various kinase inhibitors and a protease inhibitor. The nuclear or cytoplasmic protein extracts were collected at specified times after treatment and analysed for NFkappaB-DNA binding activity and IkappaB protein levels. RESULTS: The NFkappaB-DNA binding activity was induced by X-rays in a dose- and time-dependent manner in the absence of IkappaB protein degradation in astrocytes as well as in the human glioma cell line U-373MG. Whereas a protease inhibitor (calpain inhibitor 1) and a protein kinase C inhibitor (CGP-41251) did not affect X-ray-induced NFkappaB-DNA binding, treatment of astrocytes with the tyrosine kinase inhibitor (erbstatin) completely prevented the increase in NFkappaB activity after irradiation. Erbstatin also reduced the phosphorylation of IkappaBalpha after X-ray exposure. CONCLUSIONS: These results indicate that, in contrast with the more frequently investigated activators of NFkappaB, radiation-induced activation of this transcription factor proceeds in the absence of IkappaBalpha degradation and requires tyrosine phosphorylation.

Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene.

OBJECT: The authors sought to determine whether combining p53 gene transfer with radiation therapy would enhance the therapeutic killing of p53 wild-type glioma cells. It has been shown in several reports that adenovirus-mediated delivery of the p53 gene into p53 mutant gliomas results in dramatic apoptosis, but has little effect on gliomas containing wild-type p53 alleles. Therefore, p53 gene therapy alone may not be a clinically effective treatment for gliomas because most gliomas are composed of both p53 mutant and wild-type cell populations. One potential approach to overcome this problem is to exploit the role p53 plays as an important determinant in the cellular response to ionizing radiation. METHODS: In vitro experiments were performed using the glioma cell line U87MG, which contains wild-type p53. Comparisons were made to the glioma cell line U251MG, which contains a mutant p53 allele. Monolayer cultures were infected with an adenovirus containing wild-type p53 (Ad5CMV-p53), a control vector (dl312), or Dulbecco's modified Eagle's medium (DMEM). Two days later, cultures were irradiated and colony-forming efficiency was determined. Transfection with p53 had only a minor effect on the plating efficiency of nonirradiated U87MG cells, reducing the plating efficiency from 0.23 +/- 0.01 in DMEM to 0.22 +/- 0.04 after addition of Ad5CMV-p53. However, p53 transfection significantly enhanced the radiosensitivity of these cells. The dose enhancement factor at a surviving fraction of 0.10 was 1.5, and the surviving fraction at 2 Gy was reduced from 0.61 in untransfected controls to 0.38 in p53-transfected cells. Transfection of the viral vector control (dl312) had no effect on U87MG radiosensitivity. In comparison, transfection of Ad5CMV-p53 into the p53 mutant cell line U251 MG resulted in a significant decrease in the surviving fraction of these cells compared with controls, and no radiosensitization was detected. To determine whether Ad5CMV-p53-mediated radiosensitization of U87MG cells involved an increase in the propensity of these cells to undergo apoptosis, flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling-stained cells was performed. Whereas the amount of radiation-induced apoptosis in uninfected and dl312-infected control cells was relatively small (2.1 +/- 0.05% and 3.7 +/- 0.5%, respectively), the combination of Ad5CMV-p53 infection and radiation treatment significantly increased the apoptotic frequency (18.6 +/- 1.4%). To determine whether infection with Ad5CMV-p53 resulted in increased expression of functional exogenous p53 protein, Western blot analysis of p53 was performed on U87MG cells that were exposed to 9 Gy of radiation 2 days after exposure to Ad5CMV-p53, dl312, or DMEM. Infection with Ad5CMV-p53 alone increased p53 levels compared with DMEM- or dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in a further increase in p53 that reached a maximum at 2 hours postirradiation. To determine whether exogenous p53 provided by Ad5CMV-p53 had transactivating activity, U87MG cells were treated as described earlier and p21 messenger RNA levels were determined. Infection of U87MG cells with Ad5CMV-p53 only resulted in an increase in p21 compared with DMEM- and dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in an additional time-dependent increase in p21 expression. CONCLUSIONS: These data indicate that adenovirus-mediated delivery of p53 may enhance the radioresponse of brain tumor cells containing wild-type p53 and that this radiosensitization may involve converting from a clonogenic to the more sensitive apoptotic form of cell death. Although the mechanism underlying this enhanced apoptotic susceptibility is unknown, the AdSCMV-p53-infected cells have a higher level of p53 protein, which increases further after irradiation, and this exogenous p53 is transcriptionally active. (ABSTRACT TRUNCATE

Astrocytes protect against X-ray-induced neuronal toxicity in vitro.

The role of neuronal damage in the radiation-induced CNS injury resulting from brain tumor therapy remains poorly understood. To begin to define the radioresponse of neurons, the survival of rat cortical neuron cultures was investigated. Neuronal survival was reduced by approximately 40% 24-48 h after irradiation with 3.5 Gy. The addition of the free radical scavenger NAC after irradiation increased neuronal survival. Neurons were also significantly less sensitive to radiation in co-cultures with astrocytes or in the presence of astrocyte-conditioned medium. Medium conditioned on astrocytes was found to acquire significant free radical scavenging capability. However, this antioxidant property does not appear to be responsible for neuronal radioprotection. The ability of astrocytes to reduce radiation-induced neuronal toxicity appears to be mediated by a soluble protein(s) of mol. wt > 10 kDa.

Failure of a second X-ray dose to activate nuclear factor kappaB in normal rat astrocytes.

Induced gene expression and subsequent cytokine production have been implicated in the normal tissue injury response to radiotherapy. However, studies of radiation-induced gene expression have used single radiation doses rather than the fractionated exposures typical of the clinical situation. To study the effects of multiple radiation doses on gene expression, we investigated nuclear factor kappaB (NFkappaB) DNA binding activity in primary astrocyte cultures after one and two exposures to x-rays. After a single dose of x-rays (3.8-15 gray (Gy)), NFkappaB binding activity in astrocytes increased in a dose-dependent manner, reaching a maximum by 2-4 h and returning to control levels by 8 h after irradiation. In split-dose experiments, when an interval of 24 h was used between two doses of 7.5 Gy, the second 7.5-Gy exposure failed to induce NFkappaB activation. The period of desensitization induced by the first radiation exposure was dose-dependent, persisting approximately 72 h after 7.5 Gy compared with 24 h after 1.5 Gy. No changes in IkappaBalpha protein levels were detected. However, the presence of a transcription inhibitor prevented the desensitizing effect of the initial irradiation. Irradiation also prevented NFkappaB activation in astrocytes by a subsequent exposure to H2O2, but it had no effect on the activation induced by tumor necrosis factor-alpha. These data indicate that an initial x-ray exposure can desensitize astrocytes to the NFkappaB-activating effects of a subsequent radiation exposure. Furthermore, they suggest that this desensitization depends on gene transcription and may have some specificity for NFkappaB activation mediated by reactive oxygen species.

X-ray-mediated reduction in basic fibroblast growth factor expression in primary rat astrocyte cultures.

Injury of the normal central nervous system is a major concern in the radiotherapy of brain tumors, but the pathogenesis of injury remains poorly understood. Modulation of the production of growth factors is associated with ischemia and traumatic injury in the central nervous system. Ionizing radiation has been shown to induce basic fibroblast growth factor in endothelial cells and in cells of a human breast carcinoma cell line. The inducibility of basic fibroblast growth factor after irradiation and its potential role in the recovery response of the central nervous system led us to investigate the effects of radiation on the expression of this growth factor in primary cultures of normal rat type 1 astrocytes. Astrocyte monolayers were exposed to ionizing radiation (1 to 10 Gy). Northern blot analysis revealed that doses of 2 to 10 Gy markedly reduced the expression of basic fibroblast growth factor as early as 1 h after irradiation, and that it remained below levels in unirradiated cells for at least 24 h. The effect was not associated with astrocyte cytotoxicity, and it appears to have some specificity for basic fibroblast growth factor since the levels of mRNA coding for ciliary neurotrophic factor and glial fibrillary acidic protein were not affected.

Microglial cytokine gene induction after irradiation is affected by morphologic differentiation.

Microglia are known to play an important role in the CNS cytokine network, and their response after irradiation may be associated with the development of radiation-induced tissue damage. Radiation effects on this cytokine network have not yet been elucidated. We investigated the effect of gamma-irradiation on microglia stimulated with Zymosan A and lipopolysaccharide (LPS), which alone induce the expression of some cytokines and neurotoxic products by microglial cells. In the resting condition (ramified microglia), radiation had no effect on the mRNA level corresponding to cytokines such as IL1beta or IL-6, although TGF-beta1 mRNA was minimally enhanced by irradiation. However, in the activated microglia (amoeboid microglia) stimulated with Zymosan A, radiation-induced IL-6 mRNA expression was increased about two-fold in comparison with non-irradiation. IL-1beta was slightly induced by 2 Gy irradiation, but was not induced by higher doses. TGF-beta1 mRNA was not enhanced by radiation following Zymosan stimulation. In the LPS-stimulated condition, IL-6 mRNA was induced only by 2 Gy of irradiation, but no change in the expression of other genes was detected. These results suggested that radiation exerted different effects on cytokine gene transcription in microglia depending on their morphological state.

Repair of chromosome and DNA breaks versus cell survival in Chinese hamster cells.

Clonogenic and non-clonogenic parameters of cell survival were compared in irradiated Chinese hamster cells. Clonogenic survival, chromatid break and repair kinetics, as well as DNA damage and repair, were assessed in synchronized cells in different parts of the cell cycle. C2 chromatid damage and repair was examined in metaphase chromosomes of cells irradiated during S and G2 phase, treated with or without inhibitors of DNA repair. Bromodeoxyuridine labelling of S phase cells starting at the time of irradiation made it possible to determine precisely, while scoring metaphase chromosomes, whether cells were irradiated in mid S, late S, or G2 phases of the cycle. The results showed that chromatid breaks induced in S phase are efficiently repaired until the moment cells progress into G2, when repair stops abruptly. Chromatid damage in G2 phase is not repaired. On the other hand, DNA double-strand breaks are repaired in all phases of the cycle, even during G2 phase which has no concurrent chromatid break repair. Finally, there is no consistent correlation between chromatid damage and repair, DNA damage and repair, and cell survival, thus indicating that the interaction of different parameters of radiosensitivity must be better understood for them to be useful predictors of cell survival.