Differential expression of protein kinase C epsilon protein in lung cancer cell lines by ionising radiation.

The effect of ionising radiation on the regulation of gene and protein expression is complex. This study focuses on the translational regulational of the epsilon isoform of protein kinase C by ionising radiation. We found that protein kinase C epsilon is rapidly increased in the human lung adenocarcinoma cell line A549 following irradiation. Western blots showed increased accumulation of this protein at doses as low as 75 cGy after 15 min post irradiation. Maximal induction (11-fold over unirradiated cells) of PKC epsilon occurred at 150 cGy within 1 h after treatment by X-rays in A549 cells. The increased levels of PKC epsilon protein after X-rays does not require de novo protein or RNA synthesis, suggesting that this increase is post-translationally controlled. In contrast to A549 cells PKC epsilon levels in the large cell lung carcinoma cell line NCI H661 were not induced by radiation. In the small cell lung carcinoma cell line NCI N417, PKC epsilon was also not induced but a higher molecular weight PKC epsilon protein, suggestive of phosphorylation, appeared at 2 h after irradiation. The variation in induction or phosphorylation of PKC epsilon by ionising radiation in the cell lines tested in this study suggested that no clear correlation existed between intrinsic radiation sensitivity and PKC epsilon induction. To determine whether PKC epsilon does play a role in cell survival to irradiation, we used the protein kinase inhibitor staurosporin to decrease PKC activity and found that staurosporin sensitised cells to killing by ionising radiation. Pulsed field gel electrophoresis, however, indicated that DNA double-strand break repair was not decreased, suggesting that PKC epsilon is modifying the fidelity of rejoining and not the overall magnitude of repair. The regulation of PKC by ionising radiation will be discussed with respect to the biological consequences of gene induction by DNA damage agents.     

Enhanced induction of tissue-type plasminogen activator in normal human cells compared to cancer-prone cells following ionizing radiation.

Normal human fibroblast (i.e., GM2936B, GM2907A, and IMR-90) and cancer-prone human fibroblast (i.e., Fanconi's anemia, Bloom's syndrome, and Ataxia telangiectasia) cells demonstrated the induction of intracellular and extracellular levels of tissue-type plasminogen activator (t-PA) at 6 and 12 hr, respectively, following ionizing radiation. Induced t-PA enzymatic activities following ionizing radiation were blocked by actinomycin D treatments. t-PA enzymatic activities were induced over 14-fold in Ataxia telangiectasia cells, over 9-fold in Bloom's syndrome cells, and over 6-fold in Fanconi's anemia cells, as compared to normal human fibroblasts. Similarly, the induction of t-PA mRNA levels in cancer-prone cells were between 5- to 10-fold higher than those observed in normal cells following equitoxic doses of ionizing radiation. Temporal induction of t-PA mRNA levels for normal and cancer-prone human cells were consistent with quantifiable enzymatic activities. The elevated induction of an intracellular protease (i.e., t-PA) in cancer-prone human cells is reminiscent of an "SOS"-like response observed in yeast and bacteria.     

Influence of Ionizing Radiation on Ovarian Carcinoma SKOV-3 Xenografts in Nude Mice under Hypoxic Conditions

Purpose: We aimed to detect the expression of HIF-1α, VEGF, HPSE-1 and CD31 in SKOV3 xenografts innude mice treated with different doses of ionizing radiation, trying to explore the possible mechanism of hypoxiaand radioresistance. Methods: Nude mice bearing SKOV3 xenografts were randomly divided into 4 groups:Group A (control group, no ionizing radiation), Group B (treated with low dose of ionizing radiation: 50cGy),Group C (treated with high dose of ionizing radiation: 300cGy), Group D ( combined ionizing radiation, treatedwith ionizing radiation from low dose to high dose : 50cGy first and 300cGy after 6h interval). The mRNAlevels of HIF-1 and VEGF in each group were detected by real time polymerase chain reaction, while HPSE-1expression was measured by ELISA. The microvessel density (MVD) and hypoxic cells were determined throughimmunohistochemical (IHC) staining of CD31 and HIF-1a. Results: Significant differences of HIF-1α mRNA levelcould be found among the 4 groups (F=74.164, P<0.001): Group C>Group A>Group D> Group B. The mRNAlevel of VEGF in Group C was significantly higher than in the other three groups (t=-5.267, P=0.000), while nosignificant difference was observed among Group A, B and D (t=1.528, 1.588; P=0.205, 0.222). In addition, theMVD was shown to be the highest in Group C (t=6.253, P=0.000), whereas the HPSE-1 level in Group A waslower than in Group B (t=14.066, P=0.000) and higher than in Group C (t=-21.919, P=0.000), and similar withGroup D (t=-2.066, P=0.058). Through IHC staining of HIF-1a, the expression of hypoxic cells in Group A was(++), Group B was (+), Group C was (+++) and Group D was (+). Conclusion: Ionizing radiation with lowerdosesmight improve tumor hypoxia through inhibiting the expression of HIF-1 and HPSE-1, whereas higherdosesworsen tumor hypoxic conditions by up-regulating HIF-1α, HPSE-1, VEGF and CD31 levels. A protocolof low-dose ionizing radiation followed by a high-dose irradiation might at least partly improve tumor hypoxiaand enhance radiosensitivity.     

Acquired radioresistance of hematopoietic progenitors (granulocyte/monocyte colony-forming units) during chronic radiation leukemogenesis.

Protracted exposure of dogs to low daily doses of whole-body gamma-radiation (7.5 cGy/day for duration of life) elicits a high incidence of myeloid leukemia or related myeloproliferative disorders. Under such exposure, vital hematopoietic progenitors [granulocyte/monocyte colony-forming units in agar (CFU-GM)] acquire increased radioresistance along with renewed proliferative capacity at an early phase of evolving myeloid leukemia. To further characterize the expression of acquired radioresistance by CFU-GM, we evaluated the effects of various exposure rates, cumulative radiation doses, and times of exposure and postexposure in several groups of long-lived dogs under two conditions of irradiation: (a) continuous, duration-of-life exposures at dose rates of 0.3-7.5 cGy/day; and (b) discontinuous, fraction-of-life exposures at dose rates of 3.8-26.3 cGy/day, with cumulative doses of 450-3458 cGy and postexposure times of 14-4702 days. Results indicated that (a) under protracted continuous irradiation, the degree of radioresistance expressed by CFU-GM in vitro increased markedly in a biphasic pattern with rising daily rates of exposure; (b) under discontinuous, fraction-of-life exposure regimens, elevated levels of radioresistance were expressed and stably maintained by CFU-GM only following large radiation doses accumulated at high dose rates; and (c) with extended postexposure times, the magnitude of expressed radioresistance appeared to wane. These results continue to support the hypothesis that the acquisition of radioresistance and associated repair functions by vital lineage-committed progenitors, under the strong selective and mutagenic pressure of chronic irradiation, is tied temporally and causally to leukemogenic transformation elicited by radiation exposure.     

Expression levels of the DNA repair enzyme HAP1 do not correlate with the radiosensitivities of human or HAP1-transfected rat cell lines.

Apurinic/apyrimidinic (AP) sites in DNA are potentially lethal and mutagenic. They can arise spontaneously or following DNA damage from reactive oxygen species or alkylating agents, and they constitute a significant product of DNA damage following cellular exposure to ionizing radiation. The major AP endonuclease responsible for initiating the repair of these and other DNA lesions in human cells is HAP1, which also possesses a redox function. We have determined the cellular levels of this enzyme in 11 human tumour and fibroblast cell lines in relation to clonogenic survival following ionizing radiation. Cellular HAP1 levels and surviving fraction at 2 Gy (SF2) varied five- and tenfold respectively. However, no correlation was found between these two parameters following exposure to gamma-irradiation at low (1.1 cGy per min) or high (108 cGy per min) dose rates. To examine this further, wild-type and mutant versions of HAP1 were overexpressed, using an inducible HAP1 cDNA expression vector system, in the rat C6 glioma cell line which has low endogenous AP endonuclease activity. Induction of wild-type HAP1 expression caused a > fivefold increase in the capacity of cellular extracts to cleave an oligonucleotide substrate containing a single abasic site, but increased expression did not confer increased resistance to gamma-irradiation at high- or low-dose rates, or to the methylating agent methyl methanesulphonate (MMS). Expression in C6 cell lines of mutant forms of HAP1 deleted for either the redox activator or DNA repair functions displayed no apparent titrational or dominant negative effects. These studies suggest that the levels of endogenous AP endonuclease activities in the various cell lines examined are not limiting for efficient repair in cells following exposure to ionizing radiation or MMS. This contrasts with the correlation we have found between HAP1 levels and radiosensitivity in cervix carcinomas (Herring et al (1998) Br J Cancer 78: 1128-1133), indicating that HAP1 levels in this case assume a critical survival role and hence that established cell lines might not be a suitable model for such studies.     

电离辐射对人外周血线粒体编码基因mRNA表达的影响

目的： 观察γ射线对人外周血线粒体编码基因mRNA表达水平的影响,探讨线粒体编码基因用于辐射生物剂量估算的可行性。方法：用不同剂量水平 (0~5 Gy)的60Co γ射线照射3名正常人离体外周血样本,照射6 h后,提取总RNA,应用实时荧光定量PCR技术检测ND1,ND6,细胞色素C氧化酶亚基 (cytochrome C oxidase subunit,COX)I、II、III,三磷酸腺苷酶6 (adenosine triphosphatase,ATPase6),三磷酸腺苷酶8(adenosine triphosphatase,ATPase8) mRNA表达水平的变化,采用Origin 7.5拟合照射剂量与基因表达水平的剂量-效应关系曲线。结果：γ射线照射人外周血后,线粒体编码基因COXI及 ATPase6的相对表达水平在0~5 Gy剂量范围内表达先增加,3 Gy剂量点之后显现下降趋势,对这两个基因在0~3 Gy剂量范围内分别进行拟合,得到COXI 剂量-效应关系方程为Y=0.625 9+0.256 1D (r2=0.916 3,P<0.01); ATPase6基因表达剂量-效应关系方程为Y=0.218 9+ 0.806 0 D (r2=0.912 6,P<0.01),其他5个基因的相对表达水平与照射剂量间不存在明显的剂量-效应关系 (P>0.05)。结论：γ射线对人外周血线粒体编码基因COXI 及 ATPase6的mRNA表达水平具有明显的影响,这两个基因的表达水平与γ射线的照射剂量存在线性关系,有望用于辐射生物剂量的估算。     

Radiation-initiated, immortal Syrian hamster embryo fibroblasts release elevated levels of H2O2 and show divergent MnSOD and catalase activities

To characterize molecular events associated with the neoplastic conversion of primary cells by ionizing radiation, we studied the activities and mRNA expression of manganese superoxide dismutase (MnSOD) and catalase (CAT) in Syrian hamster embryo fibroblasts during the early stages of immortalization after treatment with gamma-rays. The irradiated cells showed divergent MnSOD and CAT responses relative to unirradiated controls. At passage 6, MnSOD activity was increased about 50-fold, although the concentration of MnSOD mRNA increased only 1.6-fold. By contrast, CAT activity diminished 2-fold despite an increase of 1.6-fold in the concentration of CAT mRNA. This divergence between the MnSOD and CAT activities was maintained upon culturing and, at passage 12, MnSOD was 35-fold increased and CAT 3.7-fold decreased, relative to unirradiated cells. The amount of H2O2 released into the culture medium by the radiation-initiated cells was 6-fold greater than in control media. Because H2O2 is a causative agent in the induction of malignant transformation in vitro, our results suggest that the elevated production of H2O2 caused by the imbalance between the activities of MnSOD and CAT may participate in the immortalization and subsequent malignant conversion of Syrian hamster embryo fibroblasts by ionizing radiation.     

Low dose fractionated radiation enhances the radiosensitization effect of paclitaxel in colorectal tumor cells with mutant p53

BACKGROUND: The current study was undertaken to investigate the influence of wild-type or mutant p53 status on the radiosensitizing effect of paclitaxel in colorectal tumor cell lines. METHODS: HCT-116 (contains wild-type p53) and HT-29 (contains mutant p53) established from moderately differentiated colorectal carcinomas were used in this study. Colony-forming assay was performed after exposure to either different radiation doses (0.5-6 gray [Gy]) or paclitaxel (1-10 nM) or in combination. Induction of p53 and p21(waf1/cip1) by these treatments were determined by immunocytochemistry and Western blot analysis. RESULTS: Radiation caused an increase in nuclear p53 and p21(waf1/cip1) proteins in HCT-116 cells, indicating that p53 functionally induced p21(waf1/cip1). However, induction of nuclear p53 and p21(waf1/cip1) protein was not evident in HT-29 cells, suggesting that p53 was not functional in these cells. Survival data showed that the HCT-116 cells (survival fraction of exponentially growing cells that were irradiated at the clinically relevant dose of 2 Gy [SF(2)] = 0.383; dose required to reduce the fraction of cells to 37% [D(0)] = 223 centigray [cGy]) were significantly sensitive to ionizing radiation (P < 0.008) when compared with the HT-29 cells (SF(2) = 0.614; D(0) = 351 cGy). Paclitaxel caused a higher degree of clonogenic inhibition in HCT-116 (D(0) = 0.7 nM) than HT-29 (D(0) = 1.11 nM) cells (P < 0.06). When paclitaxel and radiation were combined, an enhanced radiosensitizing effect (P < 0.05) was observed in HCT-116 cells (SF(2) = 0.138; D(0) = 103 cGy), whereas in HT-29 cells no significant radiosensitization of paclitaxel was observed (SF(2) = 0.608; D(0) = 306 cGy). However, pretreatment with paclitaxel followed by multifractionated low dose radiation (0.5- or 1-Gy fractions for a total dose of 2 Gy) significantly enhanced the radiosensitizing effect in both HCT-116 and HT-29 cells. CONCLUSIONS: The results of the current study suggested that multifractionated radiation given at very low doses after exposure of cells to paclitaxel conferred a potent radiation sensitizing effect irrespective of p53 status.     

Extremely low-dose ionizing radiation causes activation of mitogen-activated protein kinase pathway and enhances proliferation of normal human diploid cells.

We demonstrated here that X-ray irradiation at very low doses of between 2 and 5 cGy stimulated proliferation of normal human diploid cells and human tumor cells. Higher doses of irradiation at >1 Gy accumulated p53 protein and induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Phosphorylation of ERK1/2 decreased with dose down to 50 cGy, however, doses of between 5 cGy and 2 cGy phosphorylated ERK1/2 as efficiently as higher doses of X-rays, whereas the p53 protein level was not changed by doses <50 cGy. We found that mitogen-activated protein /ERK kinase (MEK) 1 was phosphorylated with both 2 cGy and 6 Gy of X-rays, and that activated ERK1/2 augmented phosphorylation of Elk-1 protein. The specific epidermal growth factor receptor tyrosine kinase inhibitor, AG1478, decreased phosphorylation of the ERK1/2 proteins induced by 2 cGy or 6 Gy of X-rays, and similar suppressive effect was observed with MEK inhibitor, PD98059. Suppression of ERK1/2 phosphorylation with these inhibitors alleviated enhanced proliferation of normal human cells by low-dose irradiation. Furthermore, overexpression of ERK2 in NCI-H1299 human lung carcinoma cells potentiated enhanced proliferation, whereas down-regulation of ERK2 using the antisense ERK2 gene abrogated the stimulative effect of low-dose irradiation. These results indicate that a limited range of low-dose ionizing radiation differentially activates ERK1/2 kinases via activation of epidermal growth factor receptor and MEK, which causes enhanced proliferation of cells receiving very low doses of ionizing radiation.     

Dose-rate dependence of heat radiosensitization

The dose rate dependence of heat radiosensitization was studied using rat astrocytoma cells in culture and a clinically relevant protocol of heat dose and heat radiation sequence. Cells were treated with a minimally toxic heat dose of 43°C for 30 minutes, after which they were irradiated with varying doses of radiation at dose rates ranging from 0.567 to 300 cGy/min. This heat dose substantially reduced the extrapolation number ($\text{n}\text{?}$), but had little effect on D_o of the radiation survival curve at dose rates of 50 cGy/min or greater. At dose rates less than 10 cGy/min, 43°C for 30 min had little a on $\text{n}\text{?}$ and only for the lowest dose rate studied (0.567 cGy/min) was there a significant reduction in D_o (60%). The thermal enhancement ratio did not vary inversely with radiation dose rate over the dose rate range studied but, instead, was maximal at the two dose rate extremes (0.567 and 300 cGy/min). These data demonstrate that a clinically relevant heat dose enhances very low dose rate, as well as high dose rate, ionizing radiation, but suggest that little benefit is to be gained from using dose rates intermediate between conventional radiotherapeutic high dose rates or dose rates representative of interstitial implants.     

Transition of Ki-67 index of uterine cervical tumors during radiation therapy. Immunohistochemical study

Histopathologic and Ki-67-staining features of cancer cells were investigated in biopsy specimens before and during radiation therapy in 29 patients with cervical squamous cell carcinoma. No morphologic changes were observed up to doses of 540 cGy. A few intact cancer cells remained up to doses of 2700 cGy. Moderate changes in the cancer cells were noticed in patients who received 900 cGy or more, i.e., multinuclei, swollen nuclei and cytoplasms, and prominent large nucleoli. At doses of 1800 cGy or greater, many cancer nests had severely damaged cancer cells with features such as cytolysis, karyolysis, karyorrhexis, pyknosis, and bizarre giant cells. There was no mitosis in the cells of patients who received doses greater than 1800 cGy. The Ki-67-positive cancer cells showed diffuse nuclear-stainings and dot-stainings before radiation therapy. Radiation doses more than 900 cGy changed the staining pattern of the Ki-67 antibody; large irregular spot-stainings and ring-stainings were observed predominantly. The Ki-67 index initially increased with the radiation dose; the mean Ki-67 indices before radiation therapy and at radiation doses of 180 cGy, 540 cGy, and 900 cGy were 41%, 50%, 63%, and 68%, respectively. The indices decreased when the dose was increased further, and they were 39% and 20% at doses of 1800 cGy and 2700 cGy, respectively. Possible explanations, including recruitment of quiescent cells, for the change in Ki-67 staining are discussed.     

Transient increases of apoptosis and Bax expression occurring during radiotherapy in patients with invasive cervical carcinoma.

BACKGROUND: Apoptosis plays a crucial role in radiation therapy (RT) in various carcinomas. This study was designed to investigate the relation between apoptosis and RT in invasive squamous cell carcinoma (ISCC) of the uterine cervix METHODS: Thirty-five specimens were obtained from 7 patients with ISCC before and during a fractionated RT. The occurrence of apoptosis was examined by end labeling of DNA gel fractionation and in situ 3' end labeling of DNA. The expression of Bax and Bcl-2 proteins was investigated by immunohistochemical staining. RESULTS: Autoradiographic analysis revealed that high molecular weight DNA was predominant in the untreated ISCC specimens. However, a ladder-like pattern, characteristic of the apoptotic breakdown of DNA, was identified at doses of 900 cGy and 1980 cGy. At doses >1980 cGy, DNA laddering disappeared without any extensive smearing. Quantitative analysis of low molecular weight fragments of DNA revealed significant increases at doses of 900 cGy and 1980 cGy compared with those before RT and at doses of >1980 cGy. Labeling of DNA in situ indicated that cells undergoing apoptosis increased dramatically at a dose of 900 cGy. However, apoptotic cells decreased at a dose of 3960 cGy. In addition, a large fraction of tumor cells was immunonegative for Bcl-2 before and during RT. By contrast, immunoreactive Bax was observed intensely in many neoplastic cells at doses of 900 cGy and 1980 cGy. CONCLUSIONS: The current investigation indicates that low doses of RT result in apoptotic cell death in ISCC in association with the increased expression of Bax but not with increased Bcl-2 expression.     

Ionizing radiation-induced adenovirus infection is mediated by Dynamin 2

Specific viral targeting into intrahepatic tumors remains critical for adenovirus gene therapy in liver cancer. We previously showed that ionizing radiation increases adenovirus uptake and transgene expression in cells and colon cancer xenografts. Here, we tested whether radiation induces viral uptake through virus-cell membrane interaction. We found that radiation (8 Gy) induced adenoviral gene transfer in rat hepatocytes (WB) and human colon carcinoma cells (LoVo). This induction (24.4- and 6.5-fold, respectively) and viral uptake were significantly diminished by preincubation with antibody for Dynamin 2 but not for Coxsackie adenovirus receptor or for integrin alpha(v). Radiation-induced Dynamin 2 expression was detected by immunohistochemical staining and by increased mRNA levels for Dynamin 2 in WB (1.5-fold) and LoVo (2.2-fold) cells. Specific small interference RNA (siRNA) transfection significantly inhibited Dynamin 2 expression in various tumor cell lines (LoVo, D54, and MCF-7) and abolished the radiation induction of Dynamin 2. Likewise, radiation-induced viral gene transfer in these cells (6.5-, 5.5-, and 9.0-fold, respectively) was significantly reduced in siRNA-transfected cells (2.7-, 3.7-, and 5.0-fold, respectively). Moreover, viral uptake in LoVo tumor xenografts was significantly increased in s.c. tumors (10.9-fold) when adenovirus was given i.v. at 24 hours after tumor irradiation, coincident with an elevated Dynamin 2 expression in irradiated tumors. These data suggest that ionizing radiation induces adenovirus gene transfer in cells and tumor xenografts by regulating viral uptake, potentially through interaction with cellular Dynamin 2 and thus should provide insight into improving adenovirus targeting in tumors.     

Modulation of expression of virus-like elements following exposure of mice to high- and low-LET radiations

Modulation of virus expression has been reported following exposure to a variety of cellular stresses, including UV radiation and heat-shock. The experiments reported here were designed to examine expression of endogenous VL30 (virus-like 30 S) elements following exposure of whole mice to ionizing radiations. Whole mice were exposed to doses of neutrons (50 cGy) or gamma-rays (300 cGy) shown to be equally efficient in cancer production in the whole animal, and tissues were harvested at 10 and 60 min following completion of the exposure. RNA extracted from these tissues and from tissues of untreated controls was examined for VL30 RNA accumulation by dilution dot blot and Northern blot analyses. These studies revealed that neutrons repressed VL30 RNA accumulation evident within 10 min following exposure in brain, gut, thymus and spleen but not in liver, in which VL30 RNA was unaffected by radiation exposure. During this same time interval, gamma-rays induced VL30 expression in gut and brain and to a lesser extent in liver. These experiments suggest the presence of a differential molecular response following whole-body exposure to high- versus low-LET radiations. In addition, this work demonstrates that ionizing radiations may affect expression of murine endogenous viral sequences.