Role of gene therapy in radiation oncology.
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Animals↗
Biomedical subjects
D E Hallahan
Publications and source records attributed to D E Hallahan.
Enhancement of replication of genetically engineered herpes simplex viruses by ionizing radiation: a new paradigm for destruction of therapeutically intractable tumors.
Human U-87 malignant glioma xenografts in mice were exposed to ionizing radiation, inoculated with a herpes simplex virus 1 mutant R3616 lacking both copies of the gamma 34.5 gene, or received both virus and radiation. Dual treatment caused a significantly greater reduction in volume or total regression of tumors than either radiation or infection alone. The significantly enhanced oncolytic effects of the combined treatment correlate with two-to five-fold enhanced replication in irradiated tumor cells than in tumors receiving virus only. In addition, in situ hybridization with viral DNA probes showed that infected tumor cells were the dominant landscape of irradiated tumors and much less apparent in the nonirradiated tumors administered this virus.
Animals↗
Adenoviral TNF-alpha gene therapy and radiation damage tumor vasculature in a human malignant glioma xenograft.
We evaluated the antitumor effects of ionizing radiation and tumor necrosis factor-alpha (TNF-alpha) gene therapy in human malignant glioma (D54) xenografts. An adenoviral vector (Ad5) containing DNA sequences of the Egr-1 promoter was linked to a cDNA encoding the TNF-alpha gene (Ad. Egr-TNF). Athymic nude mice bearing D54 xenografts received intratumoral injections of Ad.Egr-TNF or the null vector (Ad.null), with and without fractionated radiation, 5 gray (Gy) per day for 6 days, a total dose of 30 Gy. Administration of Ad.Egr-TNF and 30 Gy resulted in complete tumor regression in 71% of xenografts compared with xenografts treated with radiation alone (7.4%, P = 0.006), Ad.Egr-TNF alone (0%, P = 0.012) or Ad.null with 30 Gy (0%, P = 0.002). Combined treatment with Ad.Egr-TNF and 30 Gy significantly reduced mean fractional tumor volumes compared with radiation alone (P = 0.002), Ad.Egr-TNF alone (P = 0.002) and Ad.null plus 30 Gy (P = 0.018). Histopathologic analyses of glioma xenografts treated with Ad.Egr-TNF and radiation revealed tumor vessel thrombosis by day 4 and necrosis by day 7. Thrombosis was not observed in tumors treated with Ad.Egr-TNF alone and was significantly reduced in all other treatment groups. These studies suggest that in the D54 glioma xenograft model, the antitumor effects of combining radiation and Ad.Egr-TNF are mediated, in part, by the destruction of the tumor microvasculature.
Adenoviridae↗
Virally directed cytosine deaminase/5-fluorocytosine gene therapy enhances radiation response in human cancer xenografts.
Gene therapy combined with radiation therapy to enhance selectively radiation cytotoxicity in malignant cells represents a new approach for cancer treatment. We investigated the efficacy of adenoviral (Ad5)-directed cytosine deaminase/5-fluorocytosine (CD/5-FC) enzyme/prodrug gene therapy to enhance selectively the tumoricidal action of ionizing radiation in human cancer xenografts derived from a human squamous carcinoma cell line (SQ-20B). Tumor xenografts grown in hindlimbs of nude mice were transfected with an adenoviral vector (Ad.CMV.CD) containing the cytosine deaminase (CD) gene under the control of a cytomegalovirus (CMV) promoter. Mice were injected i.p. with 800 mg/kg of 5-FC for 12 days, and tumors were treated with fractionated radiation at a dose of 5 Gy/day to a total dose of 50 Gy. In larger tumors with a mean volume of 1069 mm3, marked tumor regression to 11% of the original tumor volume was observed at day 21 (P = 0.01). The volumetric regression of smaller tumors with a mean volume of 199 mm3, which received the same combined treatment protocol, was significant at day 12 (P = 0.014). However, unlike large tumors, regression of the smaller tumors continued until day 36 (P = 0.01), with 43% cured at day 26. No cures or significant volumetric reduction in size was observed in tumors treated with radiation alone; Ad.CMV.CD with or without radiation; or with Ad.CMV.CD and 5-FC. These results suggest that the CD/5-FC gene therapy approach is an effective radiosensitizing strategy and may lead to substantial improvement in local tumor control that would translate into improved cure rates and better survival.
Adenoviruses, Human↗
Intercellular adhesion molecule 1 knockout abrogates radiation induced pulmonary inflammation.
Increased expression of intercellular adhesion molecule 1 (ICAM-1; CD54) is induced by exposure to ionizing radiation. The lung was used as a model to study the role of ICAM-1 in the pathogenesis of the radiation-induced inflammation-like response. ICAM-1 expression increased in the pulmonary microvascular endothelium and not in the endothelium of larger pulmonary vessels following treatment of mice with thoracic irradiation. To quantify radiation-induced ICAM-1 expression, we utilized fluorescence-activated cell sorting analysis of anti-ICAM-1 antibody labeling of pulmonary microvascular endothelial cells from human cadaver donors (HMVEC-L cells). Fluorochrome conjugates and UV microscopy were used to quantify the fluorescence intensity of ICAM in the irradiated lung. These studies showed a dose- and time-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium. Peak expression occurred at 24 h, while threshold dose was as low as 2 Gy. To determine whether ICAM-1 is required for inflammatory cell infiltration into the irradiated lung, the anti-ICAM-1 blocking antibody was administered by tail vein injection to mice following thoracic irradiation. Inflammatory cells were quantified by immunofluorescence for leukocyte common antigen (CD45). Mice treated with the anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure. To verify the requirement of ICAM-1 in the inflammation-like radiation response, we utilized the ICAM-1 knockout mouse. ICAM-1 was not expressed in the lungs of ICAM-1-deficient mice following treatment with thoracic irradiation. ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation. These studies demonstrate a radiation dose-dependent increase in ICAM-1 expression in the pulmonary microvascular endothelium, and show that ICAM-1 is required for inflammatory cell infiltration into the irradiated lung.
Animals↗
Ionizing radiation mediates expression of cell adhesion molecules in distinct histological patterns within the lung.
Inflammatory cell infiltration of the lung is a predominant histopathological change that occurs during radiation pneumonitis. Emigration of inflammatory cells from the circulation requires the interaction between cell adhesion molecules on the vascular endothelium and molecules on the surface of leukocytes. We studied the immunohistochemical pattern of expression of cell adhesion molecules in lungs from mice treated with thoracic irradiation. After X-irradiation, the endothelial leukocyte adhesion molecule 1 (ELAM-1; E-selectin) was primarily expressed in the pulmonary endothelium of larger vessels and minimally in the microvascular endothelium. Conversely, the intercellular adhesion molecule 1 (ICAM-1; CD54) was expressed in the pulmonary capillary endothelium and minimally in the endothelium of larger vessels. Radiation-mediated E-selectin expression was first observed at 6 h, whereas ICAM-1 expression initially increased at 24 h after irradiation. ICAM-1 and E-selectin expression persisted for several days. P-selectin is constitutively expressed in Weibel-Palade bodies in the endothelium, which moved to the vascular lumen within 30 min after irradiation. P-selectin was not detected in the pulmonary endothelium at 6 h after irradiation. The radiation dose required for increased cell adhesion molecule expression within the pulmonary vascular endothelium was 2 Gy, and expression increased in a dose-dependent manner. These data demonstrate that ICAM-1 and E-selectin expression is increased in the pulmonary endothelium following thoracic irradiation. The pattern of expression of E-selectin, P-selectin, and ICAM-1 is distinct from one another.
Animals↗
Sialyl Lewis X mimetics attenuate E-selectin-mediated adhesion of leukocytes to irradiated human endothelial cells.
Ionizing radiation causes histological changes in normal tissues that resemble those resulting from the inflammatory response. Inflammation is a multistep process requiring expression of adhesion molecules on the surface of endothelial cells which results in leukocyte extravasation. E-selectin is an adhesion molecule that mediates leukocyte "rolling" on the endothelium and is required for the inflammatory response. We quantified E-selectin expression and selectin-dependent adhesion of leukocytes to human endothelial cells after X irradiation to determine whether E-selectin participates in the radiation-mediated inflammation-like response. Immunofluorescence staining of irradiated endothelial cells demonstrated expression of E-selectin on the cell surface similar to that elicited by treatment with interleukin-1 (IL-1). Radiation-mediated expression of E-selectin was dependent on dose and time and occurred at doses as low as 0.5 Gy. Furthermore, the increased adhesion of leukocytes to irradiated endothelial cells was prevented by an E-selectin-blocking antibody. Sialyl Lewis X is one of the molecules on the surface of leukocytes that adheres to E-selectin. The anti-inflammatory agents glycyrrhizin and carminic acid, which are structural analogues of sialyl Lewis X, attenuated adhesion of leukocytes to endothelial cells treated with X rays or IL-1. These data implicate a new class of anti-inflammatory agents in the prevention of adhesions of leukocytes to the irradiated vascular endothelium.
Anti-Inflammatory Agents, Non-Steroidal↗
Tumor necrosis factor alpha (TNF-alpha) gene therapy targeted by ionizing radiation selectively damages tumor vasculature.
Intratumoral injection of an adenoviral vector containing radiation-inducible DNA sequences of the Egr-1 promoter linked to a cDNA encoding tumor necrosis factor (TNF) alpha (Ad.Egr-TNF) enhances the tumoricidal action of ionizing radiation in a human epidermoid carcinoma xenograft (SQ-20B). The dominant histopathological feature in tumor-bearing animals treated with Ad.Egr-TNF and irradiation is extensive intratumoral vascular thrombosis and tumor necrosis. Thrombosis and necrosis are not observed in animals treated with either the viral construct encoding TNF-alpha or radiation and did not occur in irradiated normal tissues adjacent to tumor in animals injected with Ad.Egr-TNF. To determine if the occlusive effects of Ad.Egr-TNF and X-irradiation were specific for tumor vessels, non-tumor-bearing mice were irradiated after receiving i.m. injection of Ad.Egr-TNF at viral titers 20-100 times greater than titers injected intratumorally. No vascular thrombosis was observed in the treated normal tissues. Combined Ad.Egr-TNF and radiation produced occlusion of tumor microvessels without significant normal tissue damage. Taken together, these data suggest that the interaction between radiation inducible TNF-alpha and X-irradiation occurs selectively within the tumor vessels.
Animals↗
Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts.
PURPOSE: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. METHODS AND MATERIALS: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1 and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. RESULTS: X-ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. CONCLUSIONS: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.
Ataxia Telangiectasia↗
Cross-talk between ceramide and PKC activity in the control of apoptosis in WEHI-231.
WEHI-231, a murine B-cell lymphoma, readily undergoes programmed cell death following surface immunoglobulin (Ig) cross-linking [1]. Ceramide has been shown to induce apoptosis in WEHI-231 following its exposure to anti-lg antibodies, dexamethasone, and irradiation [2]. Recently, Haimovitz-Friedman et al. have demonstrated in endothelial cells that PMA not only prevented ceramide mediated apoptosis, but inhibited the generation of ceramide following irradiation [3]. In this paper we use highly specific PKC inhibitors to explore the connection between PKC activity, ceramide signaling and apoptosis. Both chelerythrine chloride and calphostin C triggered rapid apoptosis in WEHI-231 and acted in synergy with exogenous ceramide to induce apoptosis. Detailed studies of chelerythrine's mechanism of action revealed that 30 minutes following addition of 10 microM chelerythrine, sphingomyelin and phosphatidylcholine (PC) mass decreased confirming our previous findings of neutral, but not acidic, sphingomyelinase activation following treatment with PKC inhibitors [4]. The novel observation that inhibition of PKC isoforms present in WEHI-231 leads to a rapid rise in cellular ceramide as a results of sphingomyelin hydrolysis further suggests an antagonistic relationship between PKC activity and ceramide in the signaling events preceding apoptosis.
Alkaloids↗
C-jun and Egr-1 participate in DNA synthesis and cell survival in response to ionizing radiation exposure.
Exposure of mammalian cells to ionizing radiation results in the induction of the immediate early genes, c-jun and Egr-1, which encode transcription factors implicated in cell growth as well as the cellular response to oxidative stress. We studied the role of these immediate early genes in cell cycle kinetics and cell survival following x-irradiation of clones containing inducible dominant negatives to c-jun and Egr-1. The dominant negative constructs to c-jun (delta 9) and Egr-1 (WT/Egr) prevented x-ray induction of transcription through the AP-1 and Egr binding sites, respectively. Twenty percent of confluent, serum-deprived SQ20B human tumor cells, normal fibroblasts, and fibroblasts from patients with ataxia telangiectasia entered S phase within 5 h of irradiation. Clones containing inducible delta 9 and WT/Egr dominant negative constructs demonstrated attenuation of the percentage of cells exiting G1 phase and reduced survival following irradiation. These data indicate that the dominant negatives to the stress-inducible immediate early genes Egr-1 and c-jun prevent the onset of S phase and reduce the survival of human cells exposed to ionizing radiation.
Cell Cycle↗
Genetic radiotherapy overcomes tumor resistance to cytotoxic agents.
We report that radiation enhances gene therapy of a radioresistant tumor by upregulating the induction of a chimeric gene encoding a radiosensitizing protein, tumor necrosis factor alpha (TNF-alpha). We ligated the radiation-inducible CArG elements of the radiation-inducible Egr-1 promoter/enhancer region upstream to the transcriptional start site of the human TNF cDNA (pE425-TNF). This construct was transfected using cationic liposomes into the variant murine fibrosarcoma cell line, P4L. The P4L cell line was both radioresistant (D0 = 188) and resistant to TNF. After a single intratumoral injection of 10 micrograms of pE425-TNF in cationic liposomes and two 20-Gy doses of irradiation, mean tumor volumes were significantly reduced in P4L tumors as compared to those receiving either pE425-TNF in liposomes or radiation alone (P = 0.01). TNf protein in P4L tumors was induced by radiation as high as 29 times control levels and remained detectable for 14 days. Our data indicate that combined gene therapy using liposomes, together with ionizing radiation to locally activate the induction of a radiosensitizing protein, is successful at overcoming resistance to both TNF and radiation.
Animals↗
Patterns of failure following total body irradiation and bone marrow transplantation with or without a radiotherapy boost for advanced neuroblastoma.
PURPOSE: To evaluate the patterns of failure and outcome of patients undergoing high-dose chemotherapy, total body irradiation (TBI), and bone marrow transplantation (BMT) for advanced/relapsed pediatric neuroblastoma, with emphasis on the impact of a radiotherapy boost to primary and metastatic sites. METHODS AND MATERIALS: Between May 1986 and June 1993, 26 patients with advanced neuroblastoma underwent high-dose chemotherapy and TBI followed by BMT at our institution. The majority of patients were over the age of 2 years (73%) and were Stage IV at diagnosis (81%). Multiple metastatic sites were involved including bone (17), bone marrow (15), distant nodes (11), liver (5), lung (4) and brain (1). Twenty patients (77%) received cyclophosphamide (50 mg/kg x 4 days) and TBI as consolidation therapy. TBI was delivered to a total dose of 12 Gy given in 2 Gy twice daily (b.i.d.) fractions over the 3 days preceding bone marrow infusion. A local radiotherapy boost of 8-24 Gy was given to 13 out of 26 patients (50%) to the primary and/or metastatic sites immediately prior to or following induction chemotherapy according to physician judgement. Sites not amenable to a radiotherapy boost included the bone marrow, diffuse/bilateral lung involvement, and multiple bone metastases (> four sites). RESULTS: The actuarial overall survival of the 26 patients was 40.4% at 3 and 5 years, with a progression-free survival at 5 years of 38.5%. Six patients died of transplant-related toxicity (23%). The use of cyclophosphamide as high-dose consolidation chemotherapy was significantly better than other multidrug regimens used in terms of overall survival (p < 0.0001) and progression-free survival (p = 0.0004). The presence of liver involvement prior to BMT was a significant adverse prognostic factor by multivariate analysis. Of the 20 patients surviving the transplant, 10 (50%) underwent a local radiotherapy boost. The patterns of failure were as follows: 3 out of 10 "boost" patients failed overall, none in previous (old) sites of disease only, 1 in new sites only, and 2 in old and new sites; 6 out of 10 "no boost" patients failed overall, 4 in old sites only, none in new sites only, and 2 in old and new sites. There was a trend toward improved 5-year progression-free survival in patients surviving the transplant that received a boost (68% vs. 33%, p = 0.24). A failure analysis was also performed for each of the 59 initially involved sites, of which the majority (64%) were amenable to a radiotherapy boost. Overall, there is a trend toward less failure in sites amenable to a radiotherapy boost that were irradiated (1 out of 10) vs. those not irradiated (6 out of 28). Failure in the liver occurred in three out of four of the patients with liver involvement that did not receive boost radiotherapy, whereas all seven patients with distant nodal involvement were controlled without a boost. Long-term sequelae include learning difficulties (2), cataract formation (1), and hearing loss (2). Sequelae attributable to a radiotherapy boost occurred in only one patient who received whole brain radiotherapy and developed a cataract and learning difficulties. CONCLUSION: We have found an actuarial 5-year survival rate of 40.4% for patients with advanced neuroblastoma treated with BMT, which compares favorably with results of other published series. Disease recurrence following BMT was most common in previous sites of disease. The majority (64%) of these sites were amenable to a radiotherapy boost. An analysis of failure suggests that a low-dose radiotherapy boost improves control of these sites.
Adrenal Gland Neoplasms↗
Spatial and temporal control of gene therapy using ionizing radiation.
Activation of transcription of the Egr-1 gene by X-rays is regulated by the promoter region of this gene. We linked the radiation-inducible promoter region of the Egr-1 gene to the gene encoding the radiosensitizing and tumoricidal cytokine, tumour necrosis factor-alpha (TNF-alpha) and used a replication-deficient adenovirus to deliver the Egr-TNF construct to human tumours growing in nude mice. Combined treatment with Ad5.Egr-TNF and 5,000 cGy (rad) resulted in increased intratumoral TNF-alpha production and increased tumour control compared with treatment with Ad5.Egr-TNF alone or with radiation alone. The increase in tumour control was achieved without an increase in normal tissue damage when compared to tissue injury from radiation alone. Control of gene transcription by ionizing radiation in vivo represents a novel method of spatial and temporal regulation of gene-based medical treatments.
Animals↗
Gene therapy targeted by radiation preferentially radiosensitizes tumor cells.
Transcriptional regulation of the promoter/enhancer region of the Egr-1 gene is activated by ionizing radiation. We linked DNA sequences from the promotor region of Egr-1 to a complementary DNA sequence which encodes human tumor necrosis factor (TNF) alpha, a radiosensitizing cytokine. The Egr-TNF construct was transfected into a human cell line of hematopoietic origin, HL525, which was used in an experimental animal system. HL525 (clone 2) cells containing the Egr-TNF construct which exhibits radiation induction of TNF-alpha were injected into human xenografts of the radioresistant human squamous cell carcinoma cell line SQ-20B. Animals treated with radiation and clone 2 demonstrated an increase in tumor cures compared with animals treated with radiation alone or unirradiated animals given injections of clone 2 alone. No increase in local or systemic toxicity was observed in the combined treatment group. The combination of gene therapy and radiation therapy enhances tumor cures without increasing normal tissue toxicity and is a new paradigm for cancer treatment.
Animals↗
Ketoconazole attenuates radiation-induction of tumor necrosis factor.
PURPOSE: Previous work has demonstrated that inhibitors of phospholipase A2 attenuate ionizing radiation induced arachidonic acid production, protein kinase C activation and prevent subsequent induction of the tumor necrosis factor gene. Because arachidonic acid contributes to radiation-induced tumor necrosis factor expression, we analyzed the effects of agents which alter arachidonate metabolism on the regulation of this gene. METHODS AND MATERIALS: Phospholipase A2 inhibitors quinicrine, bromphenyl bromide, and pentoxyfylline or the inhibitor of lipoxygenase (ketoconazole) or the inhibitor of cyclooxygenase (indomethacin) were added to cell culture 1 h prior to irradiation. RESULTS: Radiation-induced tumor necrosis factor gene expression was attenuated by each of the phospholipase A2 inhibitors (quinicrine, bromphenyl bromide, and pentoxyfylline). Furthermore, ketoconazole attenuated X ray induced tumor necrosis factor gene expression. Conversely, indomethacin enhanced tumor necrosis factor expression following irradiation. CONCLUSION: The finding that radiation-induced tumor necrosis factor gene expression was attenuated by ketoconazole suggests that the lipoxygenase pathway participates in signal transduction preceding tumor necrosis factor induction. Enhancement of tumor necrosis factor expression by indomethacin following irradiation suggests that prostaglandins produced by cyclooxygenase act as negative regulators of tumor necrosis factor expression. Inhibitors of tumor necrosis factor induction ameliorate acute and subacute sequelae of radiotherapy. We propose therefore, that ketoconazole may reduce acute radiation sequelae such as mucositis and esophagitis through a reduction in tumor necrosis factor induction or inhibition of phospholipase A2 in addition to its antifungal activity.
Arachidonic Acid↗
Membrane-derived second messenger regulates x-ray-mediated tumor necrosis factor alpha gene induction.
Cells adapt to adverse environmental conditions through a wide range of responses that are conserved throughout evolution. Physical agents such as ionizing radiation are known to initiate a stress response that is triggered by the recognition of DNA damage. We have identified a signaling pathway involving the activation of phospholipase A2 and protein kinase C in human cells that confers x-ray induction of the tumor necrosis factor alpha gene. Treatment of human cells with ionizing radiation or H2O2 was associated with the production of arachidonic acid. Inhibition of phospholipase A2 abolished radiation-mediated arachidonate production as well as the subsequent activation of protein kinase C and tumor necrosis factor alpha gene expression. These findings demonstrate that ionizing radiation-mediated gene expression in human cells is regulated in part by extranuclear signal transduction. One practical application of phospholipase A2 inhibitors is to ameliorate the adverse effects of radiotherapy associated with tumor necrosis factor alpha production.
Amino Acid Sequence↗
The role of intracellular calcium in the cellular response to ionizing radiation.
Calcium is required as a cofactor by primer recognition proteins involved in DNA synthesis and by protein kinase C (PKC), which is activated by ionizing radiation. Because these processes may be involved in radiation-mediated regulation of the progression of cells through the phases of the cell cycle, we studied the effects of the intracellular Ca2+ chelator, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (AM-BAPTA), on PKC activation, expression of c-jun and Gadd45 and distribution of cells in the phases of the cell cycle after irradiation. AM-BAPTA prevented ionizing-radiation-induced activation of PKC and expression of c-jun in cells of human tumor cell lines. Conversely, calcium chelation had no effect on X-ray-induced expression of the Gadd45 gene. To determine whether changes in the intracellular Ca2+ concentration ([Ca2+]i) occurred during irradiation, we measured [Ca2+]i in single cells using fura-2-based microfluorimetry. There was no increase in [Ca2+]i during or after irradiation of cells of the human tumor cell lines RIT-3, SQ-20B or HL-60 or normal human fibroblast strain IMR-90. The percentage of human tumor cells crossing the G1/S-phase border was reduced by pretreatment with AM-BAPTA. These data indicate that calcium is required for ionizing radiation-induced cell cycle regulation and PKC activation, but that increases in [Ca2+]i do not occur in cells of the cell lines irradiated in this study.
Amino Acid Sequence↗