AKT inhibition is associated with chemosensitisation in the pancreatic cancer cell line MIA-PaCa-2.

Activation of the serine/threonine kinase AKT is common in pancreatic cancer; inhibition of which sensitises cells to the apoptotic effect of chemotherapy. Of the various downstream targets of AKT, we examined activation of the NF-kappaB transcription factor and subsequent transcriptional regulation of BCL-2 gene family in pancreatic cancer cells. Inhibition of either phosphatidylinositol-3 kinase or AKT led to a decreased protein level of the antiapoptotic gene BCL-2 and an increased protein level of the proapoptotic gene BAX. Furthermore, inhibition of AKT decreased the function of NF-kappaB, which is capable of transcriptional regulation of the BCL-2 gene. Inhibiting this pathway had little effect on the basal level of apoptosis in pancreatic cancer cells, but increased the apoptotic effect of chemotherapy. The antiapoptotic effect of AKT activation in pancreatic cancer cells may involve transcriptional induction of a profile of BCL-2 proteins that confer resistance to apoptosis; alteration of this balance allows sensitisation to the apoptotic effect of chemotherapy.

BCL2 expression correlates with metastatic potential in pancreatic cancer cell lines.

BACKGROUND: Programmed cell death (termed apoptosis) regulates normal tissue homeostasis. Loss of local paracrine signals and intercellular adhesion molecules are potent inducers of apoptosis and thereby eliminate normal cells that may have escaped beyond the confines of the local organ environment. Dysregulation in the expression of the BCL2 gene family, the prototypic regulators of apoptosis, is a common occurrence in cancer and imparts resistance to standard triggers of apoptosis. Therefore, the authors sought to examine whether abnormal BCL2 gene family expression correlated with resistance to apoptosis and increased metastatic potential in pancreatic carcinoma. METHODS: The authors examined BCL2 expression and apoptotic sensitivity in three panels of human pancreatic cancer cell lines that possess varying metastatic potential. Stable transfectants were generated that overexpress BCL2. These transfectants were then analyzed for differences in metastasis formation in athymic mice. RESULTS: Among the isogenic panels of pancreatic cancer cell lines, BCL2 expression levels correlated with metastatic potential. Highly metastatic variants of each family of cell lines were more resistant to induction of apoptosis. Finally, using the BCL2 transfectant in a xenograft model, elevated BCL2 expression led to a higher incidence of metastases. CONCLUSIONS: The authors conclude that increased BCL2 expression correlates with apoptotic resistance and metastatic potential; dysregulation of BCL2 expression may be involved in the metastatic progression of pancreatic carcinoma.

Chemosensitization of pancreatic cancer by inhibition of the 26S proteasome.

BACKGROUND: Pancreatic cancer is extremely resistant to the induction of apoptosis by chemotherapies; agents that regulate sensitivity to apoptosis may lead to chemosensitization of pancreatic cancer. MATERIALS AND METHODS: MIA-PaCa-2 human pancreatic cancer cells were treated in vitro with the 26S-proteasome inhibitor PS-341. Levels of the apoptosis-regulating proteins (BCL-2, BAK, and BAX) were determined by Western blotting. The effect of PS-341 on BCL-2 gene transcription was examined using a BCL-2 promoter/luciferase reporter construct. The chemosensitizing effect of PS-341 was determined by measurement of the cytotoxic effect of gemcitabine in the presence of PS-341 (10-1000 nM) using the MTT assay. A corresponding in vivo experiment using tumor xenografts in athymic mice was also performed. RESULTS: PS-341 decreased BCL-2, without effect on BAX or BAK. The downregulation of BCL-2 by PS-341 appears to be transcriptionally mediated. PS-341 induced apoptosis at high does (1000 nM) and increased the cytotoxicity of gemcitabine at low doses (10-100 nM). Xenograft growth was inhibited 59% by gemcitabine; the addition of PS-341 increased growth inhibition to 75%. CONCLUSIONS: Inhibition of the 26S proteasome disrupts the cellular content of key regulators of cell cycle progression and apoptotic control leading to increased sensitivity to standard chemotherapeutic agents, such as gemcitabine, in pancreatic cancer. Combination therapy may lead to better response rates.

Accumulation of P-selectin in the lumen of irradiated blood vessels.

Ionizing radiation induces the inflammatory response in part through leukocyte binding to cell adhesion molecules that are expressed on the vascular endothelium. We studied the effects of X radiation on the pattern of immunohistochemical staining of CD62P (P-selectin). P-selectin was localized within cytoplasmic granules in the untreated vascular endothelium. Immunohistochemical staining of P-selectin was observed at the luminal surface of vascular endothelium within 1 h of irradiation. Increased P-selectin staining at the blood-tissue interface occurred primarily in pulmonary and intestinal blood vessels. To determine whether localization of P-selectin at the vascular lumen occurs through exocytosis of endothelial cell stores in addition to platelet aggregation, we removed the vascular endothelium from the circulation and irradiated endothelial cells in vitro. In this system, we studied the mechanisms by which ionizing radiation induced translocation of P-selectin by using immunofluorescence of human umbilical vein endothelial cells (HUVEC) and confocal microscopy. Prior to irradiation, P-selectin is localized in cytoplasmic reservoirs of HUVEC. After irradiation of HUVEC, P-selectin was translocated to the cell membrane, where it remained tethered. The lowest dose at which we could expect translocation of P-selectin to the cell membrane was 2 Gy. To determine whether P-selectin in Weibel-Palade bodies requires microtubule-dependent membrane transport, we added two microtubule-depolymerizing agents, Colcemid and nocodazole. Microtubule-depolymerizing agents prevented radiation-induced trans- location of P-selectin to the cell membrane. Thus P-selectin accumulates in irradiated blood vessels through both platelet aggregation and microtubule-dependent exocytosis of storage reservoirs within the vascular endothelium.

Nuclear factor kappaB dominant negative genetic constructs inhibit X-ray induction of cell adhesion molecules in the vascular endothelium.

X-ray-induced expression of inflammatory mediators has been proposed to contribute to radiation injury in normal tissues. Radiation-inducible inflammatory mediators include the cell adhesion molecule (CAM) E-selectin and the intercellular adhesion molecule (ICAM)-1. Nuclear factor (NF)kappaB is activated by X-rays and may participate in the transcriptional regulation of each of these inflammatory mediators. To determine whether NFkappaB inhibition abrogates X-ray induction of inflammatory mediators, we used two experimental approaches including NFkappaB inhibitory drugs and a dominant negative genetic construct. Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells were treated with the NFkappaB inhibitors ALLN, PDTC, NAC, and MG132. After irradiation, E-selectin or ICAM-1 was measured by fluorescence-activated cell-sorting analysis. E-selectin and ICAM-1 expression was measured by use of immunofluorescence and fluorescence-activated cell-sorting analysis. E-selectin expression increased 7-fold, and ICAM-1 expression increased 4-fold after irradiation. All of the inhibitors attenuated E-selectin expression after irradiation. ALLN and MG132 attenuated radiation-induced ICAM expression. However, PDTC and NAC induced increased expression of ICAM-1 in HUVECs. Inhibition of X-ray induction of ICAM by these agents could not be demonstrated. In separate experiments, the NFkappaB dominant negative genetic construct was cotransfected with the promoter-reporter constructs by means of Lipofectin reagent. The ICAM promoter-reporter construct consists of the 1.2-kb segment of the human ICAM promoter upstream of the transcriptional start site linked to the luciferase reporter gene (pGL.FL-Luc). The E-selectin promoter-reporter construct consists of 525 bp upstream of the transcriptional start site of the human E-selectin promoter linked to the human growth hormone reporter gene (pE525-GH). Endothelial cells transfected with the ICAM-1 promoter-reporter construct showed a 3-fold induction after irradiation. Likewise, cells transfected with pE525-GH showed a 7-fold induction after irradiation. When cotransfected with the CAM reporter-promoter constructs, the NFkappaB dominant negative genetic construct abolished X-ray-induced transcriptional activation of the E-selectin and ICAM-1 promoters. NFkappaB inhibition is, therefore, a means of abrogating radiation-induced expression of CAMs.

X-ray-induced P-selectin localization to the lumen of tumor blood vessels.

P-selectin is a cell adhesion molecule that is sequestered in Weibel-Palade storage reservoirs within the vascular endothelium and alpha granules in platelets. P-selectin is rapidly translocated to the vascular lumen after tissue injury to initiate the adhesion and activation of platelets and leukocytes. We studied the histological pattern of P-selectin expression in irradiated tumor blood vessels. We observed that P-selectin was localized within the endothelium of tumor vessels prior to treatment. At 1-6 h following irradiation, P-selectin was mobilized to the lumen of blood vessels. To determine whether radiation-induced vascular lumen localization of P-selectin was tumor type specific or species specific, we studied tumors in rats, C3H mice, C57BL6 mice, and nude mice. P-selectin localization to the vascular lumen was present in all tumors and all species studied. Irradiated intracranial gliomas showed P-selectin localization to the vascular lumen within 1 h, whereas blood vessels in normal brain showed no P-selectin staining in the endothelium and no localization to the irradiated vascular lumen. Radiation-induced P-selectin localization to the vascular lumen increased in time-dependent manner, until 24 h after irradiation. P-selectin in platelets may account for the time-dependent increase in staining within the vascular lumen after irradiation. We therefore used immunohistochemistry for platelet antigen GP-IIIa to differentiate between endothelial and platelet localization of P-selectin. We found that GP-IIIa staining was not present at 1 h after irradiation, but it increased at 6 and 24 h. P-selectin localization to the vascular lumen at 6-24 h was, in part, associated with platelet aggregation. These findings indicate that radiation-induced P-selectin staining in the vascular lumen of neoplasms is associated with aggregation of platelets. Radiation-induced localization of P-selectin to the vascular lumen is specific to the microvasculature of malignant gliomas and is not present in the blood vessels of the irradiated normal brain.

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.

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.

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.

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.

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.

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.

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.

Inhibition of protein kinases sensitizes human tumor cells to ionizing radiation.

Protein kinase C (PKC) is activated rapidly and transiently following ionizing radiation exposure and is postulated to activate downstream nuclear signal transducers. Inhibition of this enzyme attenuates radiation-mediated expression of the c-jun and Egr-1/zif-268 genes which are associated with cellular proliferation. To investigate further the role of PKC in the radiation response of human tumor cell lines, two human squamous cell carcinoma cell lines, SQ-20B and JSQ-3, were exposed to graded doses of X rays in the presence of staurosporine, sangivamycin, or H7, all PKC inhibitors. The protein kinase inhibitors staurosporine and sangivamycin produced dose-dependent cytotoxicity in cells of the SQ-20B and JSQ-3 cell lines while H7 did not. Nontoxic concentrations of sangivamycin (10 nM) and staurosporine (1 nM), added to cell cultures from 1 to 7 h before X irradiation, enhanced cell killing by radiation in both cell lines. Maximal sensitization of killing occurred when inhibitors were added 1 h prior to irradiation. The enhanced radiation-induced cell killing was not due to any measurable alteration in the induction or rejoining of DNA single- or double-strand breaks as determined by alkaline and neutral filter elution assays. These data suggest that protein kinase activity is important for cell survival following radiation exposure, although the specific role of PKC in radiation responses is unknown.

The isoquinoline sulfonamide H7 attenuates radiation-mediated protein kinase C activation and delays the onset of x-ray-induced G2 arrest.

Protein kinase C activation by ionizing radiation in human tumor cell lines participates in the transcriptional activation of genes which may be associated with the phenotypic response of cells to x-rays. We gamma-irradiated cell line RIT-3 (radiation-induced human sarcoma) and quantified the phosphorylating capacity of protein kinase C. Protein kinase C activity increased rapidly and transiently in these cells. The selective protein kinase C inhibitor H7 attenuated radiation-mediated protein kinase C activation when added to cells prior to irradiation. To determine whether protein kinase C activation is associated with radiation-induced G2 arrest, we analyzed the cell cycle distribution of cells following gamma-irradiation. Following irradiation, RIT-3 cells rapidly progressed through G1 and S and subsequently underwent a dose dependent G2 arrest. At concentrations which are selective for protein kinase C inhibition, H7 delayed the onset of radiation-induced G2 arrest. However, there was no difference in the duration of G2 arrest following the addition of inhibitor as compared to cells irradiated without inhibitor. We propose that protein kinase C activation by ionizing radiation is associated with radiation-mediated cell cycle regulation.

Mechanisms of X-ray-mediated protooncogene c-jun expression in radiation-induced human sarcoma cell lines.

c-jun is a protooncogene associated with neoplastic transformation and is transcriptionally induced by ionizing radiation. To examine the possible mechanisms of radiation-induced c-jun transcription, we analyzed RNA from human tumor cell lines RIT-3 and STSAR-5 following x-irradiation in the presence of protein kinase inhibitors, or the absence of serum and calcium. Protooncogene c-jun expression increased several fold following irradiation of these radiation-induced human sarcoma cell lines. The expression of c-jun was not altered following irradiation in conditioned medium containing serum as compared to that of cells in serum free medium. Depletion of PKC by prolonged TPA treatment resulted in inhibition of c-jun expression. In addition, nonspecific protein kinase inhibitors, staurosporin and H7 attenuated c-jun expression, whereas the analogue of ATP (sangivamycin) did not. Furthermore, the selective inhibitor of cAMP dependent protein kinase HA 1004 did not alter radiation-mediated c-jun induction. These data indicate that ionizing radiation exposure results in c-jun induction which is dependent upon the activation of PKC. Protein kinase C activation and the subsequent expression of the protooncogene c-jun by ionizing radiation may further define the molecular mechanisms of radiation-induced neoplastic transformation.

Tumor necrosis factor gene expression is mediated by protein kinase C following activation by ionizing radiation.

Tumor necrosis factor (TNF) production following X-irradiation has been implicated in the biological response to ionizing radiation. Protein kinase C (PKC) is suggested to participate in TNF transcriptional induction and X-ray-mediated gene expression. We therefore studied radiation-mediated TNF expression in HL-60 cells with diminished PKC activity produced by either pretreatment with protein kinase inhibitors or prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Both treatments resulted in attenuation of radiation-mediated TNF induction. Consistent with these results, we found no detectable induction of TNF expression following X-irradiation in the HL-60 variant deficient in PKC-mediated signal transduction. The rapid activation of PKC following gamma-irradiation was established using an in vitro assay measuring phosphorylation of a PKC specific substrate. A 4.5-fold increase in PKC activity occurred 15 to 30 s following irradiation, which declined to baseline at 60 s. Two-dimensional gel electrophoresis of phosphoproteins extracted from irradiated cells demonstrated in vivo phosphorylation of the PKC specific substrate Mr 80,000 protein at 45 s following X-irradiation. These findings indicate that signal transduction via the PKC pathway is required for the induction of TNF gene expression by ionizing radiation.

Protein kinase C mediates x-ray inducibility of nuclear signal transducers EGR1 and JUN.

The cellular response to ionizing radiation includes growth arrest and DNA repair followed by proliferation. Induction of immediate early response genes may participate in signal transduction preceding these phenotypic responses. We analyzed mRNA expression for different classes of immediate early genes (JUN, EGR1, and FOS) after cellular x-irradiation. Increased expression of the EGR1 and JUN genes was observed within 0.5-3 hr following x-ray exposure. Preincubation with cycloheximide was associated with superinduction of JUN and EGR1 in x-irradiated cells. Inhibition of protein kinase C activity by prolonged stimulation with phorbol 12-myristate 13-acetate or the protein kinase inhibitor H7 prior to irradiation attenuated the increase in EGR1 and JUN transcripts. FOS expression was not coregulated with that of EGR1 following x-irradiation, suggesting a distinct regulatory pathway of this gene as compared with its regulation following serum and phorbol ester. These data implicate the EGR1 and JUN proteins as signal transducers during the cellular response to radiation injury and suggest that this effect is mediated in part by a protein kinase C-dependent pathway.

Two prostate carcinoma cell lines demonstrate abnormalities in tumor suppressor genes.

Two prostate carcinoma cell lines, DU-145 and PC-3, were examined for abnormalities in the retinoblastoma (Rb) and the p53 putative tumor suppressor genes. We found an abnormal Rb gene product in DU-145 using Western blot analysis. Polymerase chain reaction amplification followed by direct DNA sequencing demonstrated a base substitution mutation that generates a stop codon in exon 21. On Northern, Southern, and Western blot analysis, the p53 gene and its product appear to be normal in DU-145. PC-3, however, failed to demonstrate expression of either the p53 transcript on Northern blot analysis or the p53 protein on Western blot analysis, while the Rb gene products appeared to be normal on both Northern and Western blot analysis. This work extends the correlation between abnormal expression of putative tumor suppressor genes and human malignancies.