Henness, S., Davey, M. W., Harvie, R. M., Banyer, J., Wasinger, V., Corthals, G. and Davey, R. A. Changes in Gene Expression Associated with Stable Drug and Radiation Resistance in Small Cell Lung Cancer Cells are Similar to those Caused by a Single X-Ray Dose. Radiat. Res. 161, 495–503 (2004).

Small cell lung cancer (SCLC) initially responds well to chemotherapy and fractionated radiotherapy, but resistance to these treatments eventually develops in the vast majority of cases. To understand how resistance develops in the H69 SCLC cell line, we compared the changes in gene expression associated with 37.5 Gy fractionated X-ray treatment that produced the stable radiation- and drug-resistant H69/R38 cell subline to the changes associated with a single 4- or 8-Gy X-ray treatment. Gene expression was determined by suppression subtractive hybridization combined with Northern blot analysis and two-dimensional (2D) protein electrophoresis. Stable radiation and drug resistance was associated with coordinate changes in the expression of genes of the cytoskeleton, protein synthesis, cell cycle, redox/stress and metabolic pathways. The pattern of these changes was remarkably similar to the changes seen 24 h after a single X-ray treatment of the H69 cells but differed from the changes in expression associated with a single X-ray treatment of the resistant H69/ R38 cells. Stable radiation and drug resistance may be caused by the constitutive expression of those genes transiently expressed by sensitive cells in response to a single X-ray dose. The repeated treatments received during fractionated irradiation may promote the change from a transient to a constitutive pattern of gene expression.

Franken, N. A. P., van Bree, C. and Haveman, J. Differential Response to Radiation of TP53-Inactivated Cells by Overexpression of Dominant-Negative Mutant TP53 or HPVE6. Radiat. Res. 161, 504–510 (2004).

The inactivation of TP53 by transfection of a dominant- negative mutated TP53 (MP53.13 cells) was compared with inactivation of TP53 by transfection with the HPV E6 gene (RC10.1 cells) with respect to PLD repair, G₁-phase arrest, and induction of color junctions. Functional G₁ arrest was demonstrated in parental (RKO) cells with wild-type TP53, while in RC10.1 cells the G₁ arrest was eliminated. In MP53.13 cells an intermediate G₁ arrest was found. Functionality of endogenous TP53 was confirmed in RKO and MP53.13 cells by accumulation of TP53 protein and its downstream target CDKN1A (p21). Radiation survival of MP53.13 cells was higher than that of RKO cells, and PLD repair was found in RKO cells and MP53.13 cells but not in RC10.1 cells. Both with and without irradiation, the number of color junctions was 50 to 80% higher in MP53.13 cells than in RKO and RC10.1 cells. In the MP53.13 cells, the genetic instability appears to lead to more aberrations and to radioresistance. In spite of the presence of an excess of mutated TP53, wild- type TP53 functions appear to be affected only partly or not at all.

van Bree, C., Franken, N. A. P., Rodermond, H. M., Stalpers, L. J. A. and Haveman, J. Repair of Potentially Lethal Damage does not Depend on Functional TP53 in Human Glioblastoma Cells. Radiat. Res. 161, 511–516 (2004).

The functionality of G₁-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after γ irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G₁ arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G₁ arrest.

Karlsson, K. H. and Stenerlöw, B. Focus Formation of DNA Repair Proteins in Normal and Repair-Deficient Cells Irradiated with High-LET Ions. Radiat. Res. 161, 517–527 (2004).

To investigate the repair of clustered lesions within the DNA/chromatin, the focus formation and persistence of foci of the phosphorylated histone protein H2AX and the repair protein MRE11 were studied in normal cells and in cells lacking DNA-PKcs (M059J) or ATM (GM2052D) after irradiation with high-LET nitrogen ions or low-LET photons. There was a rapid formation of MRE11 and γ-H2AX foci, and 0.5 h after high-LET irradiation, the number of foci in normal cells correlated well with the number of particle hits per cell nucleus. After 8 h of repair, there were significantly more γ-H2AX foci than MRE11 foci remaining in the normal cells, independent of radiation quality. The difficulty in repairing clustered breaks was detected as slower rejoining of DSBs (measured by DNA fragmentation analysis), as quantification of the amount of γ-H2AX over time, and as a larger fraction of repair foci remaining after 24 h in cells irradiated with high- LET ions. These data indicate that clustered lesions are repaired by a pathway involving the same proteins that repair sparsely distributed breaks. Further, for both low- and high- LET radiation, no reduction of the initial number of γ-H2AX and MRE11 foci was detected in M059J cells up to 21 h after irradiation, which was in accordance with a complete absence of DSB rejoining in these cells. In the GM2052D cells there was also a higher level of foci remaining after 21 h; however, this was not accompanied by unrejoined DSBs, indicating that these foci not only represent DSBs but also may be a sign of persistent problems even when breaks are rejoined.

Nakajima, T., Yukawa, O., Azuma, C., Ohyama, H., Wang, B., Kojima, S., Hayata, I. and Hama-Inaba, H. Involvement of Protein Kinase C-Related Anti-apoptosis Signaling in Radiation-Induced Apoptosis in Murine Thymic Lymphoma (3SBH5) Cells. Radiat. Res. 161, 528–534 (2004).

Protein kinase C (PKC; also known as PRKC) is known to be an important participant in radiation-induced apoptosis. However, its role is not fully clarified. Using 3SBH5 cells, which are radiation-sensitive thymic lymphoma cells, the involvement and functions of PKC were assessed in radiation- induced apoptosis. PMA (phorbol 12-myristate 13-acetate), a PKC activator, inhibited the radiation-induced apoptosis in 3SBH5 cells. On the other hand, chelerythrine, a PKC inhibitor, potentiated apoptosis. In addition, Gö6976, a classical PKC (cPKC) inhibitor, which specifically inhibits PKC (α and βI), also promoted apoptosis. Interestingly, post-treatment (20 min after irradiation) with Gö6976 had no effect on the radiation-induced apoptosis. These results suggest that cPKC is activated early after irradiation for anti-apoptosis signaling and contributes to the balance between cell survival and death. Indeed, an increase of cPKC activity involving PKC (α, βI and βII) was observed in the cytosolic fraction 3 min after irradiation with 0.5 Gy. However, no translocation of cPKC was observed in the cells after irradiation. Our findings indicate that activation of cPKC (α or β) soon after irradiation is critical to the understanding of the regulation of radiation-induced apoptosis in radiation-sensitive cells.

Morgan, M. A., Poynter, J. N., Maybaum, J. and Lawrence, T. S. The Role of Bcl-X_S in Radiation Sensitivity. Radiat. Res. 161, 535–539 (2004).

Bcl-X_S is a pro-apoptosis member of the Bcl2 family that has been shown to induce cell death and enhance chemosensitivity. We have investigated the effect of Bcl-X_S overexpression on radiation sensitivity. Using a tetracycline-repressible system, we found that removal of tetracycline for 16 h induced Bcl-X_S and reduced the surviving fraction of NIH 3T3 cells to 25%. However, radiation sensitivity was not significantly affected by Bcl-X_S expression; the mean inactivation doses for Bcl-X_S repressed and Bcl-X_S induced cells were 2.7 ± 0.3 and 2.3 ± 0.1 Gy, respectively. We conclude that Bcl-X_S induces cell death without affecting radiation sensitivity. These results suggest that mitochondrial pathways to apoptosis may not have a significant role in survival after irradiation.

Johannes, C., Horstmann, M., Durante, M., Chudoba, I. and Obe, G. Chromosome Intrachanges and Interchanges Detected by Multicolor Banding in Lymphocytes: Searching for Clastogen Signatures in the Human Genome. Radiat. Res. 161 540–548 (2004).

Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.

Van der Meeren, A., Mouthon, M-A., Vandamme, M., Squiban, C. and Aigueperse, J. Combinations of Cytokines Promote Survival of Mice and Limit Acute Radiation Damage in Concert with Amelioration of Vascular Damage. Radiat. Res. 161, 549–559 (2004).

Recovery from hematopoietic aplasia is a predominant factor in the survival of total-body-irradiated mice within 30 days after exposure. However, other radiation-induced pathophysiological events have been shown to play a role, among which an inflammatory reaction must be considered. In the present study, we evaluated the therapeutic potential of a hematopoietic growth factor (thrombopoietin, Tpo) and pleiotropic cytokines (Il4 or Il11), used alone or in combination, on the survival of mice, hematopoietic reconstitution, inflammatory reaction and vascular changes. All treatments including Tpo induced a higher level of survival than did treatment with a placebo, with combinations being the most efficient. The increased survival could not be explained solely by an improved hematopoietic recovery. Treatments with Tpo also reduced the level of the chemokine KC in plasma and the level of expression of mRNA for inflammatory and coagulation proteins in the lungs of irradiated mice. In addition, radiation- induced vascular hyperpermeability was reduced with the use of Tpo. In summary, our results show that Tpo may improve survival by limiting vascular leakage, which in turn could limit inflammatory reactions and the ensuing tissue damage.

Williams, J. P., Hernady, E., Johnston, C. J., Reed, C. M., Fenton, B., Okunieff, P. and Finkelstein, J. N. Effect of Administration of Lovastatin on the Development of Late Pulmonary Effects after Whole-Lung Irradiation in a Murine Model. Radiat. Res. 161, 560–567 (2004).

Our group's work on late radiation effects has been governed by the hypothesis that the effects observed in normal tissues are a consequence of multicellular interactions through a network of mediators. Further, we believe that inflammation is a necessary component of this process. We therefore investigated whether the recruitment of mononuclear cells, observed during the pneumonitic period in the irradiated normal lung, is dependent on the expression of chemokines, notably Mcp1. Since statins have been shown to reduce chemokine expression and inflammatory cell recruitment, we specifically examined whether statins could be used to reduce monocyte recruitment. Mice received 15 Gy whole-lung irradiation; treated groups were administered lovastatin three times weekly starting either immediately or 8 weeks postirradiation. At subsequent intervals, animals were killed humanely, and cellular, mRNA and protein analyses were undertaken. Statin-treated animals demonstrated a statistically significant reduction in both macrophage and lymphocyte populations in the lung compared to radiation alone as well as improved rates of survival and decreased collagen content. In addition, ELISA measurements showed that radiation-induced increases in Mcp1 protein were reduced by statin treatment. Additional experiments are needed to assess whether statins offer a potential treatment for the amelioration of late effects in breast and lung cancer patients undergoing radiation therapy.

Hahn, F. F., Romanov, S. A., Guilmette, R. A., Nifatov, A. P., Diel, J. H. and Zaytseva, Y. Plutonium Microdistribution in the Lungs of Mayak Workers. Radiat. Res. 161, 568– 581 (2004).

The degree of nonuniform distribution of plutonium in the human lung has not been determined; thus current dosimetric models do not account for nonuniform irradiation. A better scientific basis is needed for assessing the risk of developing radiation-induced disease from inhaled α-particle-emitting radionuclides. We measured the distribution of plutonium activity in the lung by autoradiography and related the activity to specific compartments of the lung. The study materials were lung specimens from deceased workers employed by the Mayak Production Association. The approach to analyzing these lung samples used contemporary stereological sampling and analysis techniques together with quantitative α-particle autoradiography. For the first time, plutonium distribution has been quantified in the human lung. The distribution of long-term retained plutonium is nonuniform, and a significant portion of plutonium was retained in pulmonary scars. In addition, a large fraction of plutonium was present in the parenchyma, where it was retained much longer than was estimated previously. The sequestration of plutonium particles in scars would greatly reduce the radiation exposure of the critical target cells and tissues for lung cancer. Thus the prolonged retention of plutonium in lung scars may not increase the dose or risk for lung cancer.

Shukla, L. I., Pazdro, R., Huang, J., DeVreugd, C., Becker, D. and Sevilla, M. D. The Formation of DNA Sugar Radicals from Photoexcitation of Guanine Cation Radicals. Radiat. Res. 161, 582–590 (2004).

In this investigation of radical formation and reaction in γ- irradiated DNA and model compounds, we report the conversion of the guanine cation radical (one-electron oxidized guanine, G^· ) to the C1′ sugar radical and another sugar radical at the C3′ or C4′ position (designated C3′^·/C4′^·) by visible and UV photolysis. Electron spin resonance (ESR) spectroscopic investigations were performed on salmon testes DNA as well as 5′-dGMP, 3′-dGMP, 2′-deoxyguanosine and other nucleosides/nucleotides as model systems. DNA samples (25– 150 mg/ml D₂O) were prepared with Tl³ or Fe(CN)³⁻₆ as electron scavengers. Upon γ irradiation of such samples at 77 K, the electron-gain path in the DNA is strongly suppressed and predominantly G^· is found; after UV or visible photolysis, the fraction of the C1′ sugar radical increases with a concomitant reduction in the fraction of G^· . In model systems, 3′- dGMP ^·; and 5′-dGMP ^· were produced by attack of Cl^·−₂ on the parent nucleotide in 7 M LiCl glass. Subsequent visible photolysis of the 3′-dGMP ^· (77 K) results predominantly in formation of C1′^· whereas photolysis of 5′-dGMP ^· results predominantly in formation of C3′^·/C4′^·. We propose that sugar radical formation is a result of delocalization of the hole in the electronically excited base cation radical into the sugar ring, followed by deprotonation at specific sites on the sugar.

Wilson, W. E., Miller, J. H., Lynch, D. J., Lewis, R. R. and Batdorf, M. Analysis of Low-Energy Electron Track Structure in Liquid Water. Radiat. Res. 161, 591–596 (2004).

An implementation is presented of interaction cross sections for non-relativistic electron track structure simulations. The model, incorporating liquid-phase cross sections for inelastic interactions and improved algorithms for elastic scattering, is applied to Monte Carlo simulation of the track structure of low-energy electrons. Benchmark distributions and mean values are presented for several measures of penetration distances that characterize the general physical extent of the track structure. The results indicate that, except for the last approximately 500 eV of energy loss, electron tracks have a quasi-linear character; this suggests that a major part of an electron track may be reasonably described by a lineal-energy-like characterization.

Ropenga, A., Chapel, A., Vandamme, M., and Griffiths, N. M. Use of Reference Gene Expression in Rat Distal Colon after Radiation Exposure: A Caveat. Radiat. Res. 161, 597– 602 (2004).

Research on the effects of ionizing radiation exposure includes transcriptome studies using real-time reverse transcription polymerase chain reaction (RT-PCR). These studies require the use of a reference gene that normalizes for cDNA quantity and corrects for transcription between different samples. In this study, several criteria are reviewed that allow the choice of a reference gene. With the example of five genes selected from the widely used standard housekeeping genes, Gapd (glyceraldehyde-3-phosphate dehydrogenase), Hprt (hypoxanthine-guanine phosphoribosyl transferase), cyclophilin A, AcRP0 (acidic ribosomal protein P0) and 18S, we show that the use of a reference gene without a preliminary study is hazardous. We have shown in rat colon after a hemi-body irradiation that expression of a gene of interest, the serotonin receptor type 1F (5-HT_1F), was either increased or unchanged, with the result depending on the reference gene used. This work has led us to propose the use of two reference genes, a ribosomal gene, 18S, and another gene with a level of expression closer to that of the gene of interest. The methodology reported here may be applied to other studies of gene expression levels to evaluate the effects of experimental treatment on the expression of potential reference genes.

Finch, S. C. Myelodysplasia and Radiation. Radiat. Res. 161, 603–606 (2004).

Reasons are given why myelodysplasia should be considered in any assessment in humans of radiation exposure as an etiological factor for the development of leukemia.

Lambert, B., Cybulla, M., Weiner, S. M., Van De Wiele, C., Ham, H., Dierckx, R. A. and Otte, A. Renal Toxicity after Radionuclide Therapy. Radiat. Res. 161, 607–611 (2004).

During the past 10 years, a variety of radiolabeled monoclonal antibodies, antibody fragments, and low-molecular- weight oncophilic peptides have been used to deliver radioactivity to target cells for therapeutic purposes. The high and persistent localization of several of these radiolabeled molecules in the kidneys raised concern about potential renal radiation toxicity compromising therapeutic effectiveness. In particular, radiolabeled peptides, such as yttrium-90-labeled synthetic somatostatin analogues, have initiated a discussion on the safety profiles of the various somatostatin derivatives in recent clinical trials. In general, the toxicity risk seems to depend on the characteristics of the oncophilic molecule, such as the molecular weight, electric charges and clearance pathways as well as the chemical and physical characteristics of the applied radionuclide. Encouraging results for the prevention of radiation-induced renal damage by radiolabeled peptides have been obtained by co-infusion of positively charged amino acids. The available literature on nephrotoxicity after radiolabeled peptide therapy is reviewed, and therapeutic options that have become available as a result of greater insights into putative pathogenic mechanisms are discussed.

Scholz, M. and Kraft, G. The Physical and Radiobiological Basis of the Local Effect Model: A Response to the Commentary by R. Katz. Radiat. Res. 161, 612–620 (2004).

The physical and biological basis of our model to calculate the biological effects of charged particles, termed the local effect model (LEM), has recently been questioned in a commentary by R. Katz. Major objections were related to the definition of the target size and the use of the term cross section. Here we show that the objections raised against our approach are unjustified and are largely based on serious misunderstandings of the conceptual basis of the local effect model. Furthermore, we show that the approach developed by Katz and coworkers itself suffers from exactly those deficiencies for which Katz criticizes our model. The essential conceptual differences between the two models are discussed by means of some illustrative examples, based on a comparison with experimental data. For these examples, the predictions of the local effect model are fully consistent with the experimental data. In contrast, e.g. for very heavy ions, there are significant discrepancies observed for the Katz approach. These discrepancies can be attributed to the inadequate definition of the target size in this model. Experimental data are thus clearly in favor of the definition of the target as used in the local effect model. Agreement with experimental data is achieved for protons within the Katz approach but at the cost of questionable approximations in combination with the violation of the fundamental physical principle of energy conservation.