The nuclear aryl hydocarbon receptor is involved in regulation of DNA repair and cell survival following treatment with ionizing radiation.

In the present study, we explored the role of the aryl hydrocarbon receptor (AhR) for γ-H2AX associated DNA repair in response to treatment with ionizing radiation. Ionizing radiation was able to stabilize AhR protein and to induce a nuclear translocation in a similar way as described for exposure to aromatic hydrocarbons. A comparable AhR protein stabilization was obtained by treatment with hydroxyl-nonenal-generated by radiation-induced lipid peroxidation. AhR knockdown resulted in significant radio-sensitization of both A549- and HaCaT cells. Under these conditions an increased amount of residual γ-H2AX foci and a delayed decline of γ-H2AX foci was observed. Knockdown of the co-activator ARNT, which is essential for transcriptional activation of AhR target genes, reduced AhR-dependent CYP1A expression in response to irradiation, but was without effect on the amount of residual γ-H2AX foci. Nuclear AhR was found in complex with γ-H2AX, DNA-PK, ATM and Lamin A. AhR and γ-H2AX form together nuclear foci, which disappear during DNA repair. Presence of nuclear AhR protein is associated with ATM activation and chromatin relaxation indicated by acetylation of histone H3. Taken together, we could show, that beyond the function as a transcription factor the nuclear AhR is involved in the regulation of DNA repair. Reduction of nuclear AhR inhibits DNA-double stand repair and radiosensitizes cells. First hints for its molecular mechanism suggest a role during ATM activation and chromatin relaxation, both essential for DNA repair.

Cellular and molecular properties of (90)Y-labeled cetuximab in combination with radiotherapy on human tumor cells in vitro.

PURPOSE: Anti-EGFR antibody cetuximab (C225) is used in combination with radiotherapy of head and neck squamous cell carcinoma (HNSCC) patients. We investigated whether conjugation of cetuximab with trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA) and radiolabeling with (90)Yttrium affect the molecular and cellular function of cetuximab and improve its combined effect with external-beam irradiation (EBI). METHODS: The following cell lines were used: HNSCC UT5, SAS, FaDu, as well as A43, Chinese hamster ovary cells (CHO), and human skin fibroblast HSF7. Binding affinity and kinetics, specificity, retention, and the combination of (90)Y-cetuximab with EBI were evaluated. RESULTS: Control cetuximab and CHX-A″-DTPA-cetuximab blocked the proliferation activity of UT5 cells. In combination with EBI, CHX-A″-DTPA-cetuximab increased the radiosensitivity of UT5 to a similar degree as control cetuximab did. In contrast, in SAS and HSF7 cells neither proliferation nor radiosensitivity was affected by either of the antibodies. Binding [(90)Y]Y-CHX-A″-DTPA-cetuximab ((90)Y-cetuximab) to EGFR in HNSCC cells occurred time dependently with a maximum binding at 24 h. Retention of (90)Y-cetuximab was similar in both HNSCC cell lines; 24 h after treatment, approximately 90% of bound activity remained in the cell layer. Competition assays, using cell membranes in the absence of an internalized fraction of cetuximab, showed that the cetuximab affinity is not lost as a result of conjugation with CHX-A″-DTPA. Cetuximab and CHX-A″-DTPA-cetuximab blocked EGF-induced Y1068 phosphorylation of EGFR. The lack of an effect of cetuximab on EGF-induced Akt and ERK1/2 phosphorylation and the inhibition of irradiation (IR)-induced Akt and ERK1/2 phosphorylation by cetuximab were not affected by DTPA conjugation. (90)Y-cetuximab in combination with EBI resulted in a pronounced inhibition of colony formation of HNSCC cells. CONCLUSIONS: Conjugation of CHX-A″-DTPA to cetuximab does not alter the cellular and biological function of cetuximab. (90)Y-labeling of cetuximab in combination with EBI may improve radiotherapy outcome.

[Molecular signaling pathways. Mechanisms and clinical use]. / Molekulare Signalwege. Mechanismen und klinische Anwendung.

This brief summary on the role of experimental radiation oncology highlights several new research topics and research approaches that offer great potential for the optimization of modern radiation oncology. In addition, many areas of research, such as hypoxia, angiogenesis, the immune system, and metabolism, to name a few, comprise a substantial part of our current knowledge of tumor and radiation biology. Which new insights and therapeutic possibilities via the Human Cancer Genome Project or new processes, such as next generation sequencing may offer, cannot be easily foreseen at present. However, we do know for certain: radiation biology has and will continue to contribute to improvements in radiation oncology.

Membrane receptor signaling and control of DNA repair after exposure to ionizing radiation.

Accumulated evidence indicates that activation of erbB family of receptors, when mutated or over-expressed, mediates chemo- and radiotherapy resistance. In this context signaling pathways down-stream of epidermal growth factor receptor (EGFR), when abnormally activated, invoke cell survival mechanisms, which leads to resistance against radiation. In several reports it has been demonstrated that molecular targeting of EGFR signaling enhances the cytotoxic effects of radiotherapy. The radiosensitizing effects of EGFR antagonists correlate with a suppression of the ability of tumor cells to repair radiation-induced DNA double strand breaks (DNA-DSBs) through non-homologous end-joining repair pathway (NHEJ). The purpose of this review is to highlight the function of EGFR and erbB2 receptors on signaling pathways, i. e. PI3K/Akt activated by ionizing radiation (IR) and involved in repair of DNA-DSB which can explain the radiosensitizing effects of related antagonists. Advances in understanding the mechanism of erbB-signaling in regulating DNA-DSB repair will promote translational approaches to test new strategies for clinically applicable molecular targeting.

[Frequency of terminally differentiated fibroblasts in the synovial membrane of rheumatoid arthritis patients]. / Häufigkeit terminal differenzierter Fibroblasten in der Synovialmembran von Rheumapatienten.

The metabolic activation of synovial fibroblasts (SF) and their expression of matrix degrading enzymes and inflammatory cytokines contributes to the pathology of rheumatoid arthritis (RA). It is remarkable that SF of RA patients do not proliferate at higher rates when compared to SF of other patients, but they are resistant to apotposis inducing signals. The chronic inflammation in RA causes fibrosis of the synovial tissue and fibrosis has been associated with terminal differentiation. Therefore we investigated if there are increased numbers of terminally differentiated fibroblasts in the RA synovium and if there is a correlation between terminal differentiation of SF and increased levels of expression of interleukins and matrix metalloproteinases. We analyzed specimen of four RA patients, two patients with osteoarthritis (OA) and two healthy donors suffering from joint injuries. By use of RT-PCR techniques we examined mRNA expression of two genes in SF which are associated with terminal differentiation, p16INK4a and p21-cip. In addition, we labelled differentiated fibroblasts using the SA-beta-galaktosidase assay and investigated differences in protein expression patterns of factor PIVa and the tropomyosin 1 and 2 molecules. We report that the number of terminally differentiated fibrolasts are not increased in the synovial membrane of RA patients. On the contrary we show that the synovia of the much younger patients has higher levels of terminally differentiated fibroblasts. Consequently, the fibrosis of synovial tissues in RA patients at later stages of disorder is not associated with proliferation and differentiation of the fibroblasts but rather a consequence of chronic inflammation.

Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation.

PURPOSE: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells. MATERIALS AND METHODS: Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized. RESULTS: The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post-mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double-strand break repair, radiation-induced increases in p53 and p21(WAF-1,CIP-1) expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen-induced mitotic arrest of fibroblasts was still possible in the hTERT-immortalized clones. CONCLUSIONS: Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.

Bowman-Birk protease inhibitor activates DNA-dependent protein kinase and reduces formation of radiation-induced dicentric chromosomes.

PURPOSE: To test a stimulatory effect of the radioprotector Bowman Birk protease inhibitor (BBI) upon DNA repair processes. MATERIALS AND METHODS: An effect of BBI upon DNA repair was investigated by quantification of radiation-induced dicentric chromosomes. Sensitivity to ionizing radiation was determined by clonogenic survival assay. Quantification of activity of the DNA-dependent kinase was performed by immunoprecipitation and phosphorylation of a TP53-derived peptide. RESULTS: The formation of radiation-induced dicentric chromosomes was reduced significantly after pretreatment of cells with BBI. By using a cell line with an inducible expression of a mutated TP53, it was shown that the BBI-mediated reduction of dicentric chromosome formation depended on the presence of wild-type TP53. To get further insights into the molecular mode of action of BBI, activity of the DNA-dependent protein kinase (DNA-PK) was quantified. BBI treatment resulted in a stimulation of basal (DNA-PK) activity. In SCID mouse fibroblasts deficient in DNA-PK activity, BBI failed to reduce the amount of radiation-induced dicentric chromosomes and the radioprotective effect was absent. Likewise, cells expressing mt.TP53 did not show radioprotection by BBI. CONCLUSIONS: It was observed that BBI exerts its radioprotective effect by a reduction of incorrect DNA repair, resulting in a reduced amount of dicentric chromosomes. This effect on the fidelity of DNA repair is TP53 dependent and correlated with induction of DNA-PK activity.

Radioprotection of normal tissue to improve radiotherapy: the effect of the Bowman Birk protease inhibitor.

Specific radioprotection of normal tissue represents a promising approach to improve radiotherapy. The ultimate feature of a normal tissue selective radioprotector is that tumor tissue is excluded from protection. Radioprotectors of the current generation, such as Ethyol, are not explicit normal tissue specific. In contrast, the Bowman Birk protease inhibitor, which is known to prevent in vitro and in vivo radiation-induced carcinogenesis, was found to be normal tissue specific. Moreover, the molecular restrictions for this specificity were identified. The radioprotective effect is dependent upon the presence of a functional wt. TP53. Since a high amount of tumors have lost TP53 function during tumor development, the clinical application of BBI to protect normal tissue from radiation damage would effectively improve the therapeutic outcome of radiation therapy. We succeeded to identify stimulation of DNA-repair mechanisms, such as nucleotide excision repair (NER) and nonhomologous end joining (NHEJ), as molecular mode of action. These results are in good agreement with the observations that BBI concomitantly exhibits anticarcinogenic effect and radioprotective effects. Taken together, BBI is recommended as a radioprotector for normal tissue expressing wild type TP53 during treatment of tumors characterized by a mutant TP53.

Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain in human tumour and normal cells in vitro.

PURPOSE: Cell-extracellular matrix (ECM) interactions are thought to mediate drug and radiation resistance. Dependence of cell survival, beta1-integrin expression and cell cycling on the ECM proteins and beta1-integrin ligands fibronectin (FN) and laminin (LA) were examined in malignant and normal cells exposed to the cytotoxic drug Ukrain plus/minus irradiation. MATERIALS AND METHODS: Human A549 lung cancer and MDAMB231 (MDA231) breast cancer cells and normal fibroblasts (HSF1) grown on FN, LA, bovine serum albumin (BSA) or polystyrene were treated with Ukrain (1 microg ml(-1), 24 h) plus/minus irradiation (2-8 Gy) and the effects studied using colony formation assays, flow cytometry (beta1-integrin, DNA analysis) and adhesion assays. RESULTS: FN and LA reduced the cytotoxic effect of single Ukrain treatment compared with polystyrene and BSA. FN and LA also abolished Ukrain-dependent radiosensitization in A549 cells and decreased the radiosensitivity of MDA231 and HSF1 cells. Single Ukrain exposure on polystyrene significantly reduced beta1-integrin expression and promoted G2-phase accumulation of A549 cells. In contrast, Ukrain-treated MDA231 and HSF1 cells showed elevated beta1-integrin expression and no Ukrain-specific cell cycle effect. Under Ukrain-radiation exposure, irradiation, FN or LA abolished Ukrain-mediated reduction of beta1-integrin expression and G2-phase accumulation in A549 cells, whereas in MDA231 cells and fibroblasts beta1-integrin expression and cell cycle distribution were stabilized. Cell adhesion to FN or LA was significantly impaired (A549) or improved (MDA231, HSF1) upon Ukrain treatment. CONCLUSIONS: The data corroborate the findings of other groups that cell adhesion-mediated resistance to either single or combined drug and radiation exposure is tightly correlated to specific ECM proteins. By demonstrating a strong modulatory impact of FN and LA on the radiosensitivity-modifying activity of the drug Ukrain, the set findings are also highly important for the assessment of drug and radiation effects within in vitro cytotoxicity studies. The data give the first mechanistic insights into specific FN- and LA-modulated cellular resistance mechanisms as well as into the important role for beta1-integrins using the unique cytotoxic and radiosensitivity-modifying drug Ukrain.

CRABP I expression and the mediation of the sensitivity of human tumour cells to retinoic acid and irradiation.

PURPOSE: To examine the role cytoplasmic retinoic acid binding protein type 1 (CRABP I) and retinoic acid receptor beta 2 (RAR-beta 2) in mediating radiosensitization of human tumour cells in vitro by retinoic acid. MATERIALS AND METHODS: Human squamous cell carcinoma cell lines of different types were treated with retinoic acid followed by irradiation. Radiation response under drug treatment was detected by colony-formation assay. mRNA and protein expression levels of CRABP I, RAR-beta and cyclin D1 were investigated under different treatment conditions by room temperature polymerase chain reaction and Western blotting. The retinoic acid-sensitive cell line HTB35 was transfected for inducible CRABP I overexpression to test the role of this protein in modulating the sensitivity to retinoic acid and radiation as well as in regulating RAR-beta 2 and cyclin D1 expression. RESULTS: The basal CRABP I level clearly correlated with the clonogenic survival of tumour cells and normal fibroblasts after treatment with retinoic acid and ionizing irradiation (IR). Cells expressing high basal CRABP I were more resistant to combined retinoic acid radiation treatment than cells with low basal expression. Overexpression of CRABP I in retinoic acid-sensitive HTB35 cells induced a retinoic acid-insensitive phenotype resistant to combined treatment with retinoic acid and radiation. This effect was independent of RAR-beta 2 expression. CRABP I overexpression resulted in stimulated cyclin D1 expression indicating the dependency of this cell cycle control protein on retinoic acid metabolism. CONCLUSION: CRABP I plays an important role not only in mediating the retinoid effects, but also in modulating the radiation sensitivity of tumour cells after combined retinoic acid radiation treatment.

Irradiated homozygous TGF-beta1 knockout fibroblasts show enhanced clonogenic survival as compared with TGF-beta1 wild-type fibroblasts.

PURPOSE: To study the role of transforming growth factor beta1 (TGF-beta1) on cellular radiation sensitivity by analysing mouse lung fibroblasts of different TGF-beta1 genotypes. MATERIALS AND METHODS: Heterozygous TGF-beta1 knock-out mice were mated to produce offspring of different TGF-beta1 genotypes as confirmed by PCR-genotyping. Primary lung fibroblast populations were established from new-born animals of specific genotypes (TGF-beta1(+/+), TGF-beta1(+/-), TGF-beta1(-/-)). Production of TGF-beta1 was tested by ELISA. TGF-beta1 receptor-II mRNA expression was analysed by RT-PCR. Colony formation of untreated, TGF-beta1-treated and/or irradiated primary lung fibroblasts was determined under different medium conditions. RESULTS: Plating efficiencies under different medium conditions were independent of TGF-beta1 genotype. Production of TGF-beta1 correlated with the genotype: heterozygous TGF-beta1 knock-out fibroblasts (TGF-beta1(+/-)) produced 60-65% of wild-type (TGF-beta1(+/+) cells). As expected, homozygous TGF-beta1 knock-out fibroblasts (TGF-beta1(-/-)) did not produce TGF-beta1. Radiation exposure significantly enhanced TGF-beta1 production in TGF-beta1(+/+) cells by a factor of 2. No such stimulation was observed in TGF-beta1(+/-) cells. TGF-beta1(+/-) and especially TGF-beta1(-/-) cells were significantly more radioresistant than TGF-beta1(+/+) cells. TGF-beta1 treatment significantly reduced clonogenic survival for both TGF-beta1(+/+) and TGF-beta1(-/-) cells. TGF-beta1 treatment of TGF-beta1(-/-) cells resulted in an enhancement of radiation sensitivity. CONCLUSION: The data are the first direct evidence that TGF-beta1 is a major autocrine regulator of intrinsic radiation sensitivity of mouse lung fibroblasts.

Differential response of tumor cells and normal fibroblasts to fractionated combined treatment with topotecan and ionizing radiation.

PURPOSE: The impact of the topoisomerase-I inhibitor topotecan (Hycamtin) as a single agent or in combination with ionizing radiation on clonogenic cell survival has been evaluated in three tumor cell lines and in normal fibroblasts. Both agents have been applied in a fractionated scheme in order to assess clinically relevant conditions. MATERIALS AND METHODS: Cell inactivation was investigated in human glioblastoma cell lines U118 and U138, lung carcinoma cell line A549 and normal fibroblasts of the skin (HSF6) and lung (CCD32) using the colony formation assay. RESULTS: The glioblastoma cell lines were highly sensitive to the drug, whereas normal fibroblasts were much less sensitive. A significant antagonistic effect of the drug combined with irradiation was found for normal fibroblasts, while glioblastoma cells showed no evidence of interaction, indicating additivity. A549 cells showed a biphasic response to topotecan alone and in combination with irradiation, suggesting induction of resistence to the drug. CONCLUSION: Differential effects of combined topotecan/radiation treatment were detected between cells of normal tissue and tumor cells, being antagonistic in fibroblasts of the skin and lung, but not in tumor tissue, especially in glioblastoma cells.

Ukrain, an alkaloid thiophosphoric acid derivative of Chelidonium majus L. protects human fibroblasts but not human tumour cells in vitro against ionizing radiation.

PURPOSE: Ukrain, an alkaloid thiophosphoric acid derivative of Chelidonium majus L., has demonstrated a promising impact on chemotherapy in a variety of malignancies. The effects of the drug on cell survival, alteration of the cell cycle and induction of apoptosis were examined without and in combination with ionizing radiation (IR). The TP53 status of the cell lines used was also investigated. MATERIALS AND METHODS: Exponentially growing human tumour cell lines MDA-MB-231 (breast), PA-TU-8902 (pancreas), CCL-221 (colorectal), U-138MG (glioblastoma), and human skin and lung fibroblastic cells, HSF1, HSF2 and CCD32-LU were studied by colony assay, flow cytometry (cell-cycle, annexin-V staining for apoptosis) and Western blotting. Ukrain was used in concentrations from 0.1 to 50 microg ml(-1) for 1, 3 and 24 h and radiation as single doses of 1-10Gy. Combined drug-radiation exposure employed 1 microg ml(-1) Ukrain for 24h plus 2-8 Gy. RESULTS: Ukrain cytotoxicity was time- and dose-dependent. The combination of Ukrain plus IR gave enhanced toxicity in CCL-221 and U-138MG cells, but not in MDA-MB-231 and PA-TU-8902 cells. Most strikingly, a radioprotective effect was found in normal human skin and lung fibroblasts. Flow-cytometry analyses supported the differential and cell line-specific cytotoxicity of Ukrain. CCL-221 and U-138MG cells accumulated in G2 after 24-h Ukrain treatment, whereas no alterations were detected in the other tumour cells and normal fibroblasts tested. Western blotting of TP53 demonstrated non-functional overexpression in all tumour cell lines without affecting p21. HSF1 presented wild-type TP53 and a p21 response after IR. Flowcytometric analyses of annexin-V staining showed no induction of apoptosis after Ukrain treatment in comparison with untreated controls. CONCLUSIONS: Differential effects of Ukrain in modulating radiation toxicity of human cancer cell lines and its protective effect in normal human fibroblasts suggest that this alkaloid may have potential properties for clinical radiochemotherapy.

Bowman-Birk proteinase inhibitor-mediated radioprotection against UV irradiation is TP53-dependent and associated with stimulation of nucleotide excision repair.

The Bowman-Birk proteinase inhibitor (BBI) has previously been described as a radioprotective agent against ionising radiation. It was demonstrated that BBI is able to significantly increase the clonogenic cell survival of normal fibroblasts when applied before exposure to ultraviolet B (UVB) radiation. In transformed TP53-mutated cell lines, however, the BBI-mediated radioprotection was absent. At the molecular level, the radioprotective effect of BBI can be correlated with BBI-mediated stabilisation of TP53 protein prior to irradiation. Following UVB irradiation, the BBI-treated cells present an accelerated removal of cyclobutane pyrimidine dimers. Thus, the cell and molecular biological data presented suggest that BBI is able to protect cells with functional TP53 from UVB-induced DNA damage. This protective effect is most likely achieved via the activation of the TP53 signalling cascade resulting in the activation of nucleotide excision repair.

Transcriptional downregulation of ATM by EGF is defective in ataxia-telangiectasia cells expressing mutant protein.

There is evidence that ATM plays a wider role in intracellular signalling in addition to DNA damage recognition and cell cycle control. In this report we show that activation of the EGF receptor is defective in ataxia-telangiectasia (A-T) cells and that sustained stimulation of cells with EGF downregulates ATM protein in control cells but not in A-T cells expressing mutant protein. Concomitant with the downregulation of ATM, DNA-binding activity of the transcription factor Sp1 decreased in controls after EGF treatment but increased from a lower basal level in A-T cells to that in untreated control cells. Mutation in two Sp1 consensus sequences in the ATM promoter reduced markedly the capacity of the promoter to support luciferase activity in a reporter assay. Overexpression of anti-sense ATM cDNA in control cells decreased the basal level of Sp1, which in turn was increased by subsequent treatment of cells with EGF, similar to that observed in A-T cells. On the other hand full-length ATM cDNA increased the basal level of Sp1 binding in A-T cells, and in response to EGF Sp1 binding decreased, confirming that this is an ATM-dependent process. Contrary to that observed in control cells there was no radiation-induced change in ATM protein in EGF-treated A-T cells and likewise no alteration in Sp1 binding activity. The results demonstrate that EGF-induced downregulation of ATM (mutant) protein in A-T cells is defective and this appears to be due to less efficient EGFR activation and abnormal Sp1 regulation.

Changes of fibrosis-related parameters after high- and low-LET irradiation of fibroblasts.

PURPOSE: To investigate the radiation-induced, premature terminal differentiation and collagen production of fibroblasts after heavy ion irradiation. These endpoints are discussed as an underlying cellular mechanism of fibrosis. MATERIALS AND METHODS: Normal human foreskin fibroblasts (AG1522B) were used to determine clonogenic survival, the premature differentiation and synthesis of extracellular matrix (ECM) proteins, e.g. collagen after irradiation with X-rays, 195 and 11.0 MeV u(-1) carbon ions and 9.9 MeV u(-1) nickel ions. Additionally, biopsies from the skin of minipigs were taken. Similar experiments were carried out after irradiation with X-rays and 195 MeV u(-1) carbon ions. RESULTS AND CONCLUSIONS: RBE for clonogenic survival as well as for fibrosis-related parameters for high-energy carbon ions are slightly above unity. Low-energy carbon ions with a higher LET are more efficient than X-rays, whereas the RBE of nickel ions is below unity. The results obtained for the differentiation pattern and protein production of porcine fibroblasts after irradiation with X-rays and high-energy carbon ions are in agreement with those obtained with human fibroblasts. An accumulation of fibrocytes with a concomitant increase in ECM protein production could be seen after in vitro irradiation. There is no indication of a higher RBE for fibrosis-related parameters compared with other endpoints (survival, chromosomal and DNA damage). The dose- and LET-dependence suggest that premature differentiation is a survival strategy after radiation damage.

Characterization of the amino acids essential for the photo- and radioprotective effects of a Bowman-Birk protease inhibitor-derived nonapeptide.

The Bowman-Birk protease inhibitor has been reported to exert photo- and radioprotective activity. This effect was assigned to a cyclic nonapeptide sequence which is known to contain the amino acids responsible for the anti-chymotryptic activity of the BBI. The present study indicated that linearization of the nonapeptide resulted in a significant loss of anti-proteolytic activity, whereas the photo- and radioprotective capacity persisted. Substitution of the amino acids Leu or Ser of the nonapeptide, essential for the anti-proteolytic activity, with different amino acids, indicated that rather the hydrophobic features of the amino acids in this position than charge are critical to retain the photo- and radioprotective effect. These results suggest the existence of a bifunctional peptide sequence with anti-proteolytic and photo-/radioprotective capacity. However, the lack of correlation between the photo-/radioprotective activity and the anti-proteolytic activity within the peptides generated by modification of the linear nonapeptide argues for the existence of two closely colocalized domains within the nonapeptide responsible for photo-/radioprotection and protease inhibition.

Loss of heterozygosity at 11p15 and p53 alterations in malignant gliomas.

PURPOSE: Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with prognostic implications. In the later steps of tumor-progression, deletions on chromosome 11p15 and mutations of the tumor suppressor gene p53 were determined for different malignancies. To elucidate the involvement of 11p15 deletions in the tumorigenesis of malignant gliomas, we analyzed a series of 50 glioblastomas for loss of heterozygosity (LOH). METHODS: Paired tissue and blood samples from 50 patients with glioblastoma multiforme were included. Microsatellite markers located on 11p15.1-11p15.5 were used for LOH analysis. Additionally, mutation analysis of the tumor suppressor gene p53 was performed, which might correlate with favorable survival in glioblastomas. RESULTS: The region 11p15.4-5 was deleted heterozygously in 28% of cases representing 15 cM. Twenty-six glioblastomas did not show allelic loss for any locus. Our data revealed close association of LOH 11p15 with p53 mutations, and survival analysis showed a trend indicating better prognosis in glioblastomas characterized by LOH 11p15. CONCLUSION: In the tumorigenesis of malignant gliomas, p53 mutations and 11p15 deletions seem to indicate a genetic subset of tumors with favorable prognostic value.

Rhenium-188 for inhibition of human aortic smooth muscle cell proliferation.

PURPOSE: To evaluate dose-dependent growth-modulating effects of the beta-gamma emitter Rhenium-188 on cultured human aortic smooth muscle cells (haSMC). METHODS AND MATERIALS: HaSMC were plated in 25 cm(2) flasks. Two days after plating, cells were incubated with the Re-188 (beta E(max) 2.12 MeV, tissue range(max) < 10 mm, T(1/2) 17 h) for five days. The doses administered were 0.2 Gy, 1, 4, 6, 8, 16, and 32 Gy. After five days, the radionuclide was removed. Cell growth, cell cycle distribution, and clonogenic activity were analyzed for the following 25 days. RESULTS: The 0.2 and 1 Gy groups did not show relevant growth-inhibiting effects compared to the control groups. The 4 to 32 Gy groups presented dose-dependent growth inhibition, with a complete growth arrest of the 16 and 32 Gy groups. Clonogenic activity of the smooth muscle cell was strongly inhibited from doses > or =8 Gy. Flow cytometry showed a lasting dose-dependent G2/M phase block. CONCLUSION: Smooth muscle cell (SMC) growth can be controlled effectively with Re-188 for at least 25 days after radiation in vitro. As the first four weeks after arterial angioplasty are crucial concerning neointimal formation, Re-188 may be a valuable radionuclide to inhibit restenosis after arterial angioplasty.

Normal tissue radiobiology: from the laboratory to the clinic.

This manuscript is in four parts, presenting the four talks given in a symposium on normal tissue radiobiology. The first part addresses the general concept of the role of parenchymal cell radiosensitivity vs. other factors, highlighting research over the last decade that has altered our understanding of factors underlying normal tissue response. The other three parts expand on specific themes raised in the first part dealing in particular with (1) modifications of fibroblast response to irradiation in relation to the induction of tissue fibrosis, (2) the use of the linear-quadratic equation to model the potential benefits of using different means (both physical and biologic) of modifying normal tissue response, and (3) the specific role of the growth factor TFG-beta1 in normal tissue response to irradiation. The symposium highlights the complexities of the radiobiology of late normal tissue responses, yet provides evidence and ideas about how the clinical problem of such responses may be modified or alleviated.