Endothelial activation with prothrombotic response in irradiated microvascular recipient veins.

BACKGROUND: Surgical wounds within previously irradiated tissues are common in reconstructive surgery and subject to an increased incidence of postoperative complications due to vascular dysfunction, including thrombosis in both microvascular anastomosis and the microcirculatory bed. However, there is no study that has described gene expression patterns in radiated human blood vessels. This study aims to determine if radiation can induce changes in gene expression that can promote thrombus formation in human microvascular recipient veins. METHODS: Paired biopsies from radiated recipient veins and non-radiated flap veins were simultaneously harvested from 15 patients during free-flap reconstruction, 4-215 weeks from termination of radiation. Radiated and non-radiated veins were compared using a custom-made Taqman(®) low-density array (TLDA) to analyse differential gene expression in a large number of genes involved in inflammation and coagulation. Results were confirmed by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Results from TLDA indicate an acute increase of cytokines and leucocyte adhesion molecules related to activation of transcription factor nuclear factor kappa-B (NF-kB), confined to the first 3 months after radiotherapy treatment. Results were confirmed by RT-PCR and activity localised to the endothelium by immunohistochemistry. RT-PCR analyses of genes associated with coagulation showed sustained expression of plasminogen activator inhibitor-1 (PAI-1) in radiated veins. CONCLUSION: We found an acute inflammatory response with endothelial activation, followed by a sustained PAI-1 gene expression in irradiated microvascular recipient veins that can explain adverse effects years after radiation, such as microvascular occlusion and poor surgical wound healing. We believe that the results contribute to the search for therapeutic adjuncts to cope with the adverse effects of radiation therapy and strongly advocate postoperative, rather than preoperative, radiotherapy whenever possible.

The use of a comprehensive tumour xenograft dataset to validate gene signatures relevant for radiation response.

PURPOSE: To investigate the use of xenograft models in a novel gene signature validation method using gene expression microarrays. MATERIALS AND METHODS: Gene expression profiles of ten human Head and Neck squamous cell carcinomas (HNSCCs) were obtained. Several published prognostic gene expression signatures were evaluated within this set. These consisted of different radiotherapy relevant signatures (i.e. for hypoxia, proliferation and 'stemness'). Signatures were correlated with various endpoints that have been determined in the ten different xenograft models. These include immunohistochemical measures for hypoxia and proliferation, volume doubling time (VDT) and local tumour control after fractionated irradiation or after single dose irradiation under clamp hypoxia. RESULTS: We found several significant correlations between the published gene expression signatures and tumour parameters. Several signatures, like the proliferation and wound signature correlated with BrdU labelling index. Further a 'stemness'-related gene signature showed a strong negative correlation with hypoxic fraction. CONCLUSIONS: Simultaneous assessment of immunohistochemistry, in vivo tumour properties and gene expression profiling in a comprehensive set of xenograft models can be used to validate and potentially infer biological information about prognostic gene signatures.

The proangiogenic factor ephrin-A1 is up-regulated in radioresistant murine tumor by irradiation.

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3'], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors.

Effectiveness of gene expression profiling for response prediction of rectal cancer to preoperative radiotherapy.

BACKGROUND: Our aim was to determine whether the expression levels of specific genes could predict clinical radiosensitivity in human colorectal cancer. METHODS: Radioresistant colorectal cancer cell lines were established by repeated X-ray exposure (total, 100 Gy), and the gene expressions of the parent and radioresistant cell lines were compared in a microarray analysis. To verify the microarray data, we carried out a reverse transcriptase-polymerase chain reaction analysis of identified genes in clinical samples from 30 irradiated rectal cancer patients. RESULTS: A comparison of the intensity data for the parent and three radioresistant cell lines revealed 17 upregulated and 142 downregulated genes in all radioresistant cell lines. Next, we focused on two upregulated genes, PTMA (prothymosin alpha) and EIF5a2 (eukaryotic translation initiation factor 5A), in the radioresistant cell lines. In clinical samples, the expression of PTMA was significantly higher in the minor effect group than in the major effect group (P = 0.004), but there were no significant differences in EIF5a2 expression between the two groups. CONCLUSIONS: We identified radiation-related genes in colorectal cancer and demonstrated that PTMA may play an important role in radiosensitivity. Our findings suggest that PTMA may be a novel marker for predicting the effectiveness of radiotherapy in clinical cases.

Genetic effects of X-ray and carbon ion irradiation in head and neck carcinoma cell lines.

The effects of X-ray and carbon ion irradiation on DNA and genes in head and neck carcinoma cells were examined. Four head and neck cancer cell lines (squamous cell carcinoma, salivary gland cancer, malignant melanoma, normal keratinocyte) were treated with 1, 4, and 7 GyE of carbon ion, or 1, 4, and 8 Gy of X-ray, respectively. DNA and RNA in the treated cells were extracted and purified. PCR-LOH (polymerase chain reaction-loss of heterozygosity) analysis with 6 microsatellite regions on chromosome 17 was performed to determine DNA structural damage, and then microarray analysis was performed to reveal changes in gene expression. PCR-LOH analysis detected high LOH in cells treated by radiation, indicating that most of the damage by X-ray occurred in the target region on one of the homologous chromosomes. However, carbon ion caused homo-deletion, which means deletion of the counterparts in both homologous chromosomes.

Induction of interleukin-6 and oncostatin M by radiation in Kaposi's sarcoma cell lines.

PURPOSE: To define the in vitro radiosensitivity of Kaposi's sarcoma (KS) and to explore the mechanism of its extreme clinical radiosensitivity. METHODS AND MATERIALS: The radiation survivals of the three KS cell cultures (KSY-1, KS-JD, KS6-3E) were determined by clonogenic assay and MTT assay. Supernatants from irradiated cells were collected at different time points for measurement of the interleukin 6 (IL-6), oncostatin M (OSM), and basic fibroblast growth factor (bFGF) by ELISA. Changes in the mRNA expression of these cytokines were examined by reverse transcription polymerase chain reaction and Southern blot hybridization. Fresh KS cells were preincubated with the irradiated supernatant before irradiation, and the change in survivals were assessed. RESULTS: The mean SF-2 (survival fraction after 2 Gy) for KS was 0.43. Preincubation with the irradiated supernatant reduced the SF-2 significantly from 0.43 to 0.33 (p < 0.05). Treatment with irradiated supernatant alone was not cytotoxic to the cells. Radiation induced IL-6 and OSM production by KS cells at the transcription level. A single dose of 2 Gy increased IL-6 and OSM mRNA expression of the KS Y-1 cells. This corresponded to an increase in the IL-6 and OSM levels in the culture medium. There was no significant change in the level of bFGF. Preincubation with recombinant human IL-6 or OSM sensitized KS in a dose dependent manner. CONCLUSION: The low SF-2 value for these KS cell lines correlates with the clinical radiosensitivity of KS. The high radiosensitivity may be due to radiation induction of cytokines such as IL-6 and OSM, which are radiosensitizers for KS cells.

Effect of doxorubicin on wild-type and deoxyadenosine-resistant mouse leukemia L1210 cells.

Wild-type (WT) mouse leukemia L1210 cells express steady-state levels of the mRNA and protein for p53. However, the p53 expressed by the wild-type cells is a mutant form of p53. A deoxyadenosine-resistant L1210 cell line (Y8) derived from the parental WT L1210 cells does not maintain constitutive levels of p53 mRNA or protein. Upon DNA damage, induced by doxorubicin, neither the WT nor the Y8 cells block in G0/G1; the cells block in G2/M. However, treatment of the Y8 cells with doxorubicin results in a much greater fraction of cells becoming apoptotic compared to the WT cells. Doxorubicin treatment resulted in the induction of p53 mRNA in the WT cells, but not the Y8 cells. WAF1, c-myc and Bax mRNAs were also induced by doxorubicin in the WT cells but not in the Y8 cells. The constitutive levels of WAF1 and Gadd45, unexpectedly seen in the p53-deficient Y8 cells, decreased following doxorubicin treatment. The comparison of the effects of DNA damage, as measured by mRNA levels, induced by X-irradiation or doxorubicin were found to vary between the WT and Y8 cells and for the particular mRNA studied. The effect of doxorubicin or X-irradiation on the cell cycle could be overridden in the WT cells by caffeine. Comparisons of DNA damage induced by doxorubicin or X-irradiation show that although the Y8 cells are more sensitive to these damaging agents than the WT cells, the effects on gene expressions are not identical.

Ionizing radiation and TNF-alpha stimulate gene expression of a Thr/Tyr-protein phosphatase HVH1 and inhibitory factor IkappaB alpha in human squamous carcinoma cells.

Exposure of cells to ionizing radiation (IR) or tumor necrosis factor-alpha (TNF-alpha) results in the stimulation of the DNA binding activities of transcription factors, AP-1 and NF-kappaB. HVH1/CL100, a dual specificity protein phosphatase, may attenuate the AP-1 response by dephosphorylating a key upstream element, mitogen-activated protein kinase (MAPK). The members of IkappaB family of proteins regulate the NF-kappaB response. We examined the effects of IR and TNF-alpha on HVH1 and IkappaB alpha gene expression. Our data demonstrate that IR or TNF-alpha treatment of head and neck squamous carcinoma cells (PCI-04A) increased the steady-state levels of HVH1 and IkappaB alpha mRNAs; however, the induction patterns were different. TNF-alpha treatment led to a relatively prolonged stimulation of HVH1 and IkappaB alpha mRNAs lasting at least 7 h, while IR caused a transient stimulation of these mRNAs and the expression returned to basal levels within 6 h post-IR treatment. Treatment of cells with cycloheximide did not prevent the IR orTNF-alpha-inducible expression of HVH1 and IkappaB alpha genes, indicating that these responses were independent of the new protein synthesis. These data imply that protein phosphatase HVH1 and regulatory factor IkappaB alpha may play important roles in cellular response to IR and TNF-alpha. In addition, the kinetics of responsiveness indicates that the mechanisms of IR and TNF-alpha-induced signalling are distinct.

Oncogenic changes in murine lymphoid tumors induced by in utero exposure to ionizing radiation.

We have investigated the oncogenic alterations in murine lymphomas induced by in utero exposure to gamma-radiation. The expression of the myc oncogene increased in 23% of the tumors. Alterations in the expression of the ras oncogenes and in the p53 tumor suppressor gene were not characteristic. The p53 gene was mutated in a low percentage of the tumors (12%). Ras mutations were not detected. Loss of heterozygosity (LOH) at the p53 locus was found in 30% of the tumors, and LOH at the mts tumor suppressor gene was detected in 23% of lymphomas. Multiple oncogenic changes were infrequent in the investigated tumors. There were no essential differences in the frequency of carcinogenic alterations in spontaneous and gamma-radiation-induced lymphomas.

Messenger RNA expression of resistance factors in human tumor cell lines after single exposure to radiation.

Resistance of tumor cells to chemotherapeutic drugs can not only be caused by treatment with antineoplastic agents but also by radiotherapy. The aim of this study was to analyze whether ionizing radiation can influence the mRNA expression of proteins which have been found to be involved in drug resistance of tumor cells. Human tumor cell lines (MCF-7, LXF and Sk-Mel) were treated with single doses of irradiation (5, 10 and 20 Gy). The expression of the resistance related proteins glutathione S-transferase-pi (GST-pi), topoisomerase II alpha (Topo II), thymidylate synthase (TS), O6-methylguanine-DNA-methyltransferase (MGMT), P-glycoprotein (Pgp), glutathione peroxidase (GPX) multidrug resistance-associated protein (MRP) and also of the heat-shock protein 70 (HSP 70) were determined at the mRNA level during the time interval from 1.5 to 72 h post-irradiation and compared with their corresponding controls. We also examined whether a relationship exists between these proteins and the proliferative activity (histone 3, Ki-67, statin) of the cells. We found that exposure of MCF-7, LXF and Sk-Mel cells to ionizing radiation increases the expression of the mRNA of GST-pi. Topo II, TS, HSP 70 and proliferation markers were also altered by exposure to ionizing radiation, but there was no common response of the three cell lines. No significant changes were observed in the expression of MGMT, Pgp, GPX and MRP after radiation treatment. Drug resistance tests revealed that irradiated MCF 7 cells were less sensitive to doxorubicin than non-irradiated control cells. Our results indicate that ionizing irradiation modifies the expression of some proteins involved in drug resistance and the response of MCF 7 cells to doxorubicin and may, therefore, play a role in clinical drug response.

Proteins binding to the leader of the 6.0 kb mRNA of human insulin-like growth factor 2 influence translation.

The leader of the 6.0 kb human insulin-like growth factor 2 (IGF-2) mRNA, leader 3, has been reported to partially repress translation. In the regulation of this phenomenon, RNA-binding proteins may play a role. Using UV-irradiation crosslinking, we found specific binding of four proteins (57, 43, 37 and 36 kDa) to this leader. Binding of these proteins to RNA proved to be highly sensitive to the potassium chloride concentration in the buffer solution, each protein having its own optimum. The 57 kDa protein was indistinguishable by size, binding properties and immunoprecipitation from the polypyrimidine tract binding protein (PTB), first described as a nuclear protein binding to the polypyrimidine tracts (PPTs) in introns. Cross-competition experiments showed that leader 3 has a much higher affinity for this 57 kDa protein than the PPT on which PTB was originally characterized. By competition with different fragments of leader 3, we were able to localize the binding of the 57 kDa protein to a 162 nt RNA fragment (AsnI-PvuII) in the 3'-part of the leader. When placed before a chloramphenicol acetyltransferase (CAT) open reading frame, this RNA fragment stimulated translation in reticulocyte lysate 3-fold, while other fragments of leader 3 repressed translation. The efficient translation directed by the 162 nt AsnI-PvuII fragment fused to CAT could be repressed by adding free AsnI-PvuII RNA fragment, indicating that the high translation efficiency of the AsnI-PvuII-CAT synthetic mRNA was due to the binding of protein and not to the structure of the RNA itself.

Elevated p53 RNA expression correlates with incomplete osteogenic differentiation of radiation-induced murine osteosarcomas.

An important role for the p53 gene in osteogenic sarcomas has been imputed by identification of somatically acquired gene alterations in human osteosarcomas and by the development of osteosarcomas in p53 transgenic mice. To study the involvement of p53 in radiation-induced osteosarcomagenesis, we have investigated gene alterations and expression of p53 in radiation-induced murine osteosarcomas and tumor-derived cell lines. Eighteen of 31 tumors and 8 of 9 cells lines showed alterations in the p53 gene region, or elevated levels of p53 RNA. Expression of the osteoblast marker gene bone gla protein was substantially reduced in tumors which simultaneously showed high steady-state levels of p53 RNA. Our data indicate that p53, in addition to its function in regulating DNA synthesis, may be involved in the control of osteogenic differentiation in osteosarcomagenesis.

In vitro effects of pulsed near-ultraviolet laser exposure on human larynx carcinoma cells.

Effects of pulsed near-ultraviolet laser beam on structural characteristics and macromolecular synthesis of carcinoma HEp2 cells were investigated. Laser irradiation damage induced in these eukaryotic cells could be characterized by two development stages: a) a reversible stage with minor morphological damages (1.5 kJ/m2) and 2) an irreversible one, at higher fluences, characterized by cellular membrane damage, necrobiosis and cells detachment from the substrate (4.5 kJ/m2). A. Studies performed referring to macromolecular syntheses of low laser fluences (1.5 kJ/m2)--irradiated HEp2 cells showed the following aspects: a) syntheses inhibiton phase in the first cycles of cellular replication and b) syntheses stimulation phases in the following cycle with total repair of laser-induced molecular lesions. B. At high laser fluences (3-4.5 kJ/m2), metabolic lesions repair was partially or totally blocked after prolonged culturing at 37 degrees C. Ths paper suggests some mechanisms of laser action on macromolecular synthesis and correlates them with morphological changes induced by laser exposure of carcinoma cells.

Overexpression of P-glycoprotein in mammalian tumor cell lines after fractionated X irradiation in vitro.

We observed that in vitro exposure of mammalian tumor cells to fractionated x irradiation results in the expression of drug resistance. The cause of this resistance was investigated in a series of Chinese hamster ovary cell lines that had survived exposure to multiple lethal doses of radiation. These cell lines had increased levels of P-glycoprotein (Pgp), the multidrug-resistance-associated membrane glycoprotein. Consistent with the classic multidrug resistance phenotype, they exhibited cross-resistance to multiple drugs, as well as sensitivity to reversal of vincristine resistance by verapamil. However, the cell lines showed no change in their sensitivity to x rays. Pgp overexpression occurred in these cells, despite a lack of Pgp gene amplification or of significant alteration in Pgp messenger RNA levels. Although the cause of increased Pgp levels is not yet known, these data suggest a biological basis for the clinical problem of drug resistance that can occur in previously irradiated tumors.

18 S ribosomal RNA is degraded during ribosome maturation in irradiated HeLa cells.

The effects of ionizing radiation (137Cs) on processing of ribosomal RNA (rRNA) were studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. The 45 S rRNA precursor, and its two major daughter species, 28 and 18 S rRNA, were separated by gel electrophoresis and the extent of radiolabel incorporation into each was determined at various times after irradiation. This approach permitted kinetic analysis of processing of the 45 S rRNA which had been predominantly synthesized (radiolabeled) prior to irradiation. Since they both derive from the same 45 S pre-rRNA transcript, 28 and 18 S rRNA are produced with a stoichiometry of 1:1, as observed in control cells in the present studies. However, within 1 h following 10 Gy an altered stoichiometry of 28 S:18 S rRNA was apparent, reaching 1.6:1 by 5-7 h following irradiation. This alteration was also observed following the higher dose of 20 Gy, but not following exposures of 5 Gy or less. The 18 S portion of the 45 S pre-rRNA is transcribed prior to the 28 S portion. Consequently, an increase in the 28 S/18 S ratio can only be due to degradation of the 18 S species during or after processing. This alteration may represent a response to radiation-induced growth arrest, by reducing the number of newly synthesized ribosomes that would otherwise be required for cell propagation.

[Fundamental model assays on chemical damages to ribonucleic acids caused by oxygen radicals in aqueous solution]. / Grundlegende Modellversuche über chemische Schädigungen von Ribonukleinsäuren durch Sauerstoffradikale in wässriger Lösung.

A partial decomposition of RNA molecules in Saccharomyces cerevisiae is caused by photochemically produced superoxide anion radical (O2-.). Hydroxyl radicals (OH.) lead to complete destruction of RNA. Such radicals as originating from peroxidase-hydrogen peroxide reaction cause a partial decomposition of RNA molecules. All RNA species are damaged to the same extent by radicals. Damages due to oxygen radicals can be prevented or reduced by strong radical captors (dimethyl sulfoxide, 1,4-diazabicyclo-(2,2,2)-octane or superoxide dismutase). All these reactions proceed in aqueous solution. Correlations with the use of radicals in radiotherapy are discussed.

Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA.

The positions of intramolecular crosslinks induced by irradiation with ultraviolet light were mapped into potato spindle tuber viroid RNA and HeLa 5S rRNA. Crosslinking in each of these molecules occurred at a single major site, which was located by RNA fingerprinting and secondary analysis (and additional primer extension studies in the case of the viroid). Various lines of evidence suggest that these crosslinks identify a previously undescribed element of local tertiary structure common to these two widely divergent RNA molecules: (i) both crosslinks occur in an identical eight-base context, with the sequence 5' GGGAA 3' on one side and the sequence 5' UAC 3' on the other; (ii) both crosslinks connect bases that are not thought to be involved in conventional hydrogen bonding, within regions usually depicted as single-stranded loops flanked by short helical segments; and (iii) both crosslinks connect a purine and a pyrimidine residue, and both may generate the same G-U dimer. Furthermore, it is likely that the crosslinking site is of functional significance because it is located within the most highly conserved region of the viroid sequence and involves bases that are essentially invariant among eukaryotic 5S rRNA molecules.

Cross-linking of mRNA to proteins by irradiation of intact cells with ultraviolet light.

Ehrlich ascites tumor cells were irradiated with ultraviolet light to cross-link intracellular RNAs to their tightly bound proteins. The efficiency of such cross-linking in vivo was measured by two independent methods, namely by phenol/chloroform extraction and by isopycnic centrifugation. After short irradiation times more than 70% of the ribosomal RNAs and more than 80% of the mRNA were found to be cross-linked to protein. The size of the cross-linked RNAs was essentially unchanged, indicating that the irradiation does not lead to extensive fragmentation of the RNA. Covalent poly(A)-containing mRNA-protein complexes were isolated by oligo(dT)-cellulose chromatography in the presence of sodium dodecylsulphate. The complexed proteins were freed from RNA by extensive nuclease degradation and analysed by polyacrylamide gel electrophoresis. The results show that some specific proteins are associated with poly(A)-containing RNA in vivo.