Modulation of fibrinolysis by ionizing radiation

Summary Radiation-induced damage to the central nervous system is believed to be targeted to glial or endothelial cells or both, although the pathophysiology of this process is still poorly understood. A series of experiments were, therefore, conducted, including irradiation to primary rat astrocytes (in vitro) and rat spinal cords (in vivo). The levels of plasminogen activators (uPA and tPA) and their inhibitors (PNI and PAI-1) were determined by fibrin zymography, ELISA, amidolytic activity assay, complex formation, and Western blot analysis. Fibrin zymography revealed the presence of M_r 48,000 (uPA) and M_r 68,000 (tPA) lytic bands that were increased in irradiated samples. Three- to four-fold higher levels of tPA and 8- to 10-fold higher levels of uPA were detected in irradiated samples. Western blot analysis confirmed the presence of a 51-kDa band (PAI-1) in irradiated samples. PAI-1 is undetectable in nonirradiated spinal cord. Serum-free medium and cell and spinal cord extracts of nonirradiated samples showed a 43-kDa band (PNI), the intensity of which is decreased in irradiated samples. Four- to five-fold decreased levels of PNI were detected in irradiated serum-free media and cell extracts, but no levels of PNI were detected in irradiated spinal cord extracts. This study provides additional information regarding the proposed roles of plasminogen activators and their inhibitors in the development of CNS damage after irradiation.     

BRCA1 promotes induction of ssDNA by ionizing radiation

The BRCA1 tumor suppressor contributes to the repair of DNA double-strand breaks (DSB) through homologous recombination, but the mechanism is unknown. The rapid accumulation of BRCA1 into nuclear foci in response to induction of DNA breaks suggests that BRCA1 may function in an early step in the repair pathway. We examined the role of BRCA1 in one such early step, the resection of DSBs to generate ssDNA. The appearance of ssDNA in response to ionizing radiation is similar to that of BRCA1 foci formation, suggesting that the two processes are related. Furthermore, BRCA1 colocalizes to ssDNA sites induced by ionizing radiation. Overexpression of BRCA1 causes an increase in cells exhibiting ssDNA induced by ionizing radiation. Mutant BRCA1 that lacks the COOH-terminal BRCT domain also promotes ssDNA but fails to form nuclear foci. Knockdown of BRCA1 expression reduces ssDNA and Rad51 foci formation in response to ionizing radiation. These results indicate that BRCA1 promotes induction of ssDNA in response to ionizing radiation and accumulates at sites of ssDNA.     

Cyclin B expression in HeLa cells during the G2 block induced by ionizing radiation.

After exposure to ionizing radiation, eukaryotic cells undergo a division delay which is reflected by increased time spent in the G2 portion of the cell cycle. Recent information identifies increased levels of mitotic cyclins as key biochemical events initiating mitosis. In HeLa cells cyclin B mRNA and protein levels have been shown to increase in G2 and to decrease after division is completed. Cyclin B protein binds to cdc2, resulting in histone kinase activity which is necessary for the initiation of mitosis. Accordingly, we chose to investigate how cyclin B mRNA and protein levels were perturbed by irradiation in order to gain further understanding of the mechanisms by which ionizing radiation leads to a division delay. Our experiments revealed at least two effects on cyclin B regulation which might contribute to the division delay: (a) when HeLa cells were irradiated in S phase, there was a delay in the accumulation of cyclin B mRNA; (b) when cells were radiated in G2 phase, at a time when mRNA levels were increasing, a division delay was induced which coincided with a markedly lowered level of cyclin B protein despite high levels of the mRNA.     

Response of malignant B lymphocytes to ionizing radiation: gene expression and genotype

The human malignant B-lymphocyte cell lines Reh and U698 show arrest in G2 phase after ionizing radiation (IR), but only Reh cells arrest in G1 phase and die by apoptosis. We have used cDNA microarrays to measure changes in gene expression at 2, 4 and 6 hr after irradiation of Reh and U698 cells with 0.5 and 4 Gy in order to begin exploring the molecular mechanisms underlying the phenotypic changes. We also investigated whether gene expression changes could be caused by possible aberrations of genes, as measured by comparative genomic hybridization. Reh cells showed upregulation of CDKN1A that likely mediated the G1 arrest. In contrast, U698 cells have impaired function of TP53 protein and no activation of CDKN1A, suppressing the arrest in G1. The G2 arrest in both cell lines was likely due to repression of PLK1 and/or CCNF. IR-induced apoptosis in Reh cells was probably mediated by TP53 and CDKN1A, whereas a high expression level of MCL1, caused by gene amplification, and activation of the NFKB pathway may have suppressed the apoptotic response in U698 cells. Genes suggested to be involved in apoptosis were activated long before this phenotype was detectable and showed the same temporal expression profiles as genes involved in cell cycle arrest. Our results suggest that differences in functionality and/or copy number of several genes involved in IR-regulated pathways contributed to the phenotypic differences between Reh and U698 cells after IR, and that multiple molecular factors control the radiation response of malignant B lymphocytes.     

Targeting angiogenic processes by combination rofecoxib and ionizing radiation

Tumor growth and angiogenesis are interdependent. Cyclooxygenase (COX) catalyzes the synthesis of prostaglandins from arachidonic acid. Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit COX-mediated synthesis of prostaglandins. COX-1 is constitutively expressed in a wide range of tissues, whereas COX-2 is cytokine inducible. Enhanced COX-2 expression has been attributed a key role in the development of inflammation and related processes observed in pathologically altered disease states. Two specific COX-2 inhibitors, namely rofecoxib (Vioxx) and celecoxib (Celebrex), both oral agents and U.S. Food and Drug Administration approved, have been shown preclinically and clinically to have efficacy comparable to that of NSAIDs for relief of pain and inflammation in osteoarthritis, with decreased risk of gastrointestinal damage. Little is known about how angiogenesis is affected by the combination of rofecoxib and radiation. We have evaluated the combination of rofecoxib, at various concentrations, and radiation on cytokine-induced angiogenesis in vitro. We have found that rofecoxib inhibited endothelial cell proliferation, migration, and tube formation (differentiation) at clinically relevant doses. In combination with radiation, inhibition of endothelial cell function further increased twofold. The combination of rofecoxib and radiation suggests a complementary strategy with clinical ramifications to target angiogenesis-dependent malignancies.     

Modification of multidrug resistance of tumor cells by ionizing radiation

Purpose The effect of ionizing radiation on multidrug resistance (MDR) of human larynx cancer HEp-2 cells has been investigated. We studied the dependence of the radiation effect on radiation dose, time after irradiation and cell density. Methods MDR was determined from an increase in cell sensitivity to daunorubicin, taxol and vincristine by the inhibitors of multidrug resistance cyclosporin A and avermectin B₁, and from the suppression by cyclosporin A of the transport of rhodamine 123 out of the cells. The cells were irradiated with X-ray beams (dose rate 1.12 Gy min⁻¹) at room temperature. Results It was shown that, at 8 and 16 h after irradiation with doses up to 4 Gy, the multidrug resistance of cells increases, and at 24 h it decreases to the control level. The effect was maximal by 16 h after irradiation with a dose of 1 Gy. Both, the contribution of active transport to the rate of rhodamine 123 efflux from cells and their resistance to vincristine, increased. The effect of irradiation on multidrug resistance of HEp-2 cells depended on the density of cells on the substrate, being maximal at a density of 80,000–100,000 cm⁻². Conclusion The irradiation-induced changes in the MDR of tumor cells should be taken into account when combining radiotherapy with chemotherapy. It was assumed that the dependence of multidrug resistance of HEp-2 cells on radiation dose and cell density is determined by changes in the amount of reactive oxygen species in the cells.     

Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.     

电离辐射引起血管损伤的研究

放射治疗是目前恶性肿瘤治疗的重要方法之一,但照射可能引起正常组织器官的放射性炎症、纤维化和坏死等限制了放射治疗的有效使用,并在一定程度上影响了疗效,而引起这些放疗并发症的确切机制目前还不十分明确.研究表明,照射后早期血管的辐射反应或损伤与照射后正常组织器官的损伤有密切的关系,血管内皮细胞（vascular endothelial cell,VEC）和血管平滑肌细胞（vascular smooth muscle cell, VSMC）是血管中重要的辐射敏感的靶细胞,VEC和VSMC的辐射损伤是引起血管及周围组织器官损伤的重要原因.从1）照射后VEC与白细胞黏附的变化,也就是炎症及免疫反应的早期改变的变化;2）射线对VEC表达黏附分子的影响,阐述了辐射对选择素家族、免疫球蛋白超家族及整合素的影响;3）射线对VSMC增值的影响,三方面阐述电离辐射在引起血管损伤中的作用.最后根据本文给临床治疗提出展望.     

Urinary bladder lesions induced by persistent chronic low-dose ionizing radiation

The incidence of urinary bladder cancer in the Ukraine increased from 26.2 to 43.3 per 100 000 population between 1986 and 2001 after the Chernobyl accident. The present study was conducted to evaluate the development of radiation-dependent lesions in the urinary bladders of people living in cesium 137 (¹³⁷Cs) radio-contaminated areas of the Ukraine. Bladder urothelial biopsies from 159 male and 5 female patients were subjected to histological examination and immunohistochemical study of p38 mitogen-acti-vated protein kinase (MAPK), as well as the p50 and p65 subunits of nuclear factor kappa B (NF- k B). A pattern of chronic proliferative atypical cystitis accompanied with large areas of sclerosis of connective tissue in the lamina propria was commonly observed in all cases. Interestingly, these lesions were associated with a dramatic increase in the incidences of dysplasia/carcinoma in situ , and, moreover, small urothelial carcinomas were incidentally detected. We defined the overall condition as "Chernobyl cystitis.'Greatly elevated levels of p38, p65 and p50 expression in the urothelium were evident and the patients showed increased ¹³⁷Cs in urine. The data support conclusions from our previous studies of a critical role for increased oxidative stress in generation of urinary bladder urothelial lesions in individuals chronically exposed to low-dose ¹³⁷Cs radiation. Alterations in the p38 MARK cascade and accumulation of NF- k B subunits could be crucial early molecular events in the pathogenesis of Chernobyl cystitis. (Cancer Sci 2003; 94: 328–333)     

Inhibition of constitutive NF-kappa B activity by I kappa B alpha M suppresses tumorigenesis

We have demonstrated that nuclear factor-kappa B (NF-kappa B) is constitutively activated in human pancreatic adenocarcinoma and human pancreatic cancer cell lines but not in normal pancreatic tissues or in immortalized, nontumorigenic pancreatic epithelial cells, suggesting that NF-kappa B plays a critical role in the development of pancreatic adenocarcinoma. To elucidate the role of constitutive NF-kappa B activity in human pancreatic cancer cells, we generated pancreatic tumor cell lines that express a phosphorylation defective I kappa B alpha (S32, 36A) (I kappa B alpha M) that blocks NF-kappa B activity. In this study, we showed that inhibiting constitutive NF-kappa B activity by expressing I kappa B alpha M suppressed the tumorigenicity of a nonmetastatic human pancreatic cancer cell line, PANC-1, in an orthotopic nude mouse model. Immunohistochemical analysis showed that PANC-1-derived tumors expressed vascular endothelial growth factor (VEGF) and induced angiogenesis. Inhibiting NF-kappa B signaling by expressing I kappa B alpha M significantly reduced expression of Bcl-x(L) and Bcl-2. The cytokine-induced expression of VEGF and Interleukin-8 in PANC-1 cells is also decreased. Taken together, these results suggest that the inhibition of NF-kappa B signaling can suppress tumorigenesis of pancreatic cancer cells and that the NF-kappa B signaling pathway is a potential target for anticancer agents.