[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.

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.

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.

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.

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.

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.

Paclitaxel in simultaneous radiochemotherapy of head and neck cancer: preclinical and clinical results.

Several studies have presented evidence that paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) displays a radiation-sensitizing effect. We therefore analyzed the influence of paclitaxel and concomitant radiation on the proliferation kinetics of head and neck tumor cells and normal fibroblasts. Our data clearly support the notion that paclitaxel given with radiation exerts an additive effect on the clonogenic survival of squamous cell carcinoma cells and normal fibroblasts. Since concomitant radiochemotherapy has proven to be beneficial for the treatment of locally advanced head and neck cancer, we are testing the feasibility of simultaneous paclitaxel plus radiotherapy. Paclitaxel is given on a one-time weekly basis. Up to now, 13 evaluable patients have been treated, and the maximum tolerable dose has not been reached at 40 mg/m2. Mucositis is expected to be the dose-limiting toxicity.

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.

Radiation sensitivity of human squamous cell carcinoma cells in vitro is modulated by all-trans and 13-cis-retinoic acid in combination with interferon-alpha.

PURPOSE: Retinoids and interferon-alpha (IFN-alpha) have been shown to exert antiproliferative and radiosensitizing effects. The present study was designed to determine differential effects of retinoids in combination with IFN-alpha on radiation toxicity of 5 human squamous cell carcinoma (SCC) cell lines. METHODS AND MATERIALS: Using clonogenic assays, the effects of all-trans (ATRA), 13-cis-retinoic acid (13cRA), and IFN-alpha on radiation toxicity were analyzed. Basal mRNA expression of the cytoplasmic retinoic acid binding protein, CRABP I, was determined in retinoid-sensitive and -insensitive cell lines by reverse transcriptase/polymerase chain reaction (RT-PCR). RESULTS: Treatment with ATRA, 13cRA, or IFN-alpha resulted in a cell line-specific inhibition of clonogenic survival. A comparison of retinoid-sensitive and insensitive cells revealed that retinoid sensitivity seems to be dependent on the basal expression level of CRABP I. ATRA, 13cRA, and IFN-alpha alone or in combination altered radiation sensitivity by affecting predominantly the alpha-component of the linear-quadratic dose-response curve. Likewise, depending upon the treatment condition the surviving fraction at 2 Gy (SF2) was decreased cell line-specifically. Combined treatment with ATRA or 13cRA and IFN-alpha markedly enhanced radiation cytotoxicity. CONCLUSION: These in vitro data indicate that the combined treatment with retinoids, IFN-alpha, and ionizing radiation could be beneficial for patients presenting with SCC.

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.

The radioprotective potential of the Bowman-Birk protease inhibitor is independent of its secondary structure.

The Bowman-Birk protease inhibitor (BBI), a serine-protease inhibitor, has been reported to exert both anticarcinogenic and radioprotective activity. In this work we examined whether this effect is mediated through inhibition of serine-proteases of the trypsin-chymotrypsin type. Using linearized forms of BBI, evidence will be provided that the secondary structure, obligatory for the protease inhibitory function, is not necessary for the radioprotective effect. Detailed analysis indicated that the radioprotective effect is correlated with the chymotrypsin-inhibitory region of the molecule. Using a synthetic nonapeptide lacking protease inhibitor activity, the radioprotective effect of the total BBI molecule could be mimicked, indicating that the radioprotective effect is independent of the protease inhibitor function.

Cellular basis of radiation-induced fibrosis.

Fibrosis is a common sequela of both cancer treatment by radiotherapy and accidental irradiation and has been described in many tissues including skin, lung, heart and liver. The underlying mechanisms of the radiation-induced fibrosis still remain to be resolved. In the present review we tried to illustrate the basic cellular mechanisms of radiation-induced fibrosis based on the newest findings arising from molecular radiobiology and cell biology. Based on these findings the cellular mechanism of radiation-induced fibrosis can be seen as a multicellular process involving various interacting cell systems in the target organ resulting in the fibrotic phenotype of the fibroblast/fibrocyte cell system.

Bowman-Birk proteinase inhibitor (BBI) modulates radiosensitivity and radiation-induced differentiation of human fibroblasts in culture.

The radiosensitivity and differentiation pattern of cultured normal human fibroblasts was analysed as a function of treatment of the cells with the Bowman-Birk proteinase inhibitor (BBI). Upon irradiation with doses from 0 to 8 Gy normal human fibroblasts are induced to a premature terminal differentiation within 14-21 days of postirradiation incubation. Treatment of the cells with 10 microM BBI for 2 h prior to the irradiation procedure resulted in a significant shift of the radiation survival curve, increased SF2 values 0.63 vs. 0.84 and the cell type composition of the test fibroblast cultures. Upon pretreatment with BBI the radiation-induced premature terminal differentiation of progenitor fibroblasts to postmitotic fibrocytes could significantly be inhibited. Based on this data, it can be postulated that BBI may serve as a radioprotector of normal fibroblasts which are involved in radiation-induced tissue injuries like radiation fibrosis.

Reciprocal translocation frequency in irradiated sensitive and resistant human tumor cells in correlation with clonogenic in vitro cell survival: a possibility of tumor radiosensitivity prediction?

We have investigated the yields of radiation-induced translocations in several human tumor cell lines and in normal diploid human fibroblasts by fluorescence in situ hybridization. The translocation yields were determined with respect to chromosome no. 1 in all cell lines investigated, and moreover in chromosomes nos. 2, 4 and 9 in fibroblasts and one tumor cell line. The chromosomes were "painted' with fluorescent whole chromosome-hybridization probes. The clonogenic survival of the studied cell lines was determined by standard colony-formation assay. We observed a higher frequency of reciprocal translocations in the radiosensitive cells MCF-7 and MDA-MB-436 as compared with the radioresistant cells CaSki and normal skin fibroblasts. Thus, the results suggest a possibility to predict the intrinsic tumor radiosensitivity on the basis of reciprocal translocation yield determined in cells irradiated in vitro. The correlation was observed in spite of the trisomy no. 1 which occurred in all three investigated tumor cell lines. On the other hand, the results obtained with different chromosomes in MCF-7 cells suggest that only chromosomes with relatively low "spontaneous' translocation yields are suitable for this kind of analysis.

Differentiation state of skin fibroblast cultures versus risk of subcutaneous fibrosis after radiotherapy.

BACKGROUND AND PURPOSE: There is increasing evidence for patient-to-patient variation in the response of normal tissue to radiotherapy. Recently, it has been suggested that accumulation of functional fibrocytes may be a key step in the development of radiation-induced fibrosis. Therefore, we have examined a possible relationship between the differentiation state of untreated fibroblasts and the risk of radiation-induced subcutaneous fibrosis in individual patients. MATERIALS AND METHODS: We used skin fibroblast cultures isolated from eight postmastectomy radiotherapy patients whose individual clinical radiosensitivity was assessed by the mean excess risk of fibrosis. Different types of potentially mitotic progenitor fibroblasts (MF) and postmitotic functional fibrocytes (PMF) in the terminal differentiation lineage, MFI --> MFII --> MFIII --> PMF, were scored morphologically in clonal culture. Progression of differentiation was quantified by the ratio L/E of colony-forming late (MFIII and late MFII) and early (MFI and early MFII) progenitors. RESULTS: We observed a correlation between the ratio L/E and the mean risk of fibrosis (rs = 0.743, P = 0.03), indicating an approximately 10-fold increase in L/E with an increasing risk of fibrosis. This was paralleled by a decreasing trend in the absolute numbers of early progenitor types. By contrast, there was no significant correlation between the plating efficiency and the risk of fibrosis. CONCLUSIONS: The data suggest that the risk of fibrosis increases with the progression of the differentiation of untreated progenitor fibroblasts, indicating that the progression of fibroblast differentiation may be a co-factor in the development of radiation-induced fibrosis. If this hypothesis is validated, it provides a rationale for a novel predictive test to identify patients with an increased risk of subcutaneous fibrosis.

Spontaneous and radiation-induced differentiationof fibroblasts.

Clonal heterogeneity in fibroblast cultures from donors of all ages has been associated with differentiation of the fibroblast/fibrocyte system. Thus, a terminal differentiation lineage including a sequence of three potentially mitotic progenitor fibroblasts (MFI-->MFII-->MFIII) in the precursor compartment and three types of postmitotic fibrocytes (PMFIV-->PMFV-->PMFVI) in the functional compartment has been identified previously. In the present study, we show that replenishment of fibrocytes lost from the functional compartment is not expected to change the distribution of differentiation types in a steady state population, provided cell loss occurs at the end of a long sequence of cell divisions only. However, premature terminal differentiation of progenitor fibroblasts to postmitotic fibrocytes can be induced by ionising radiation and other cell stressors. Furthermore, even a low dose of 1Gy causes a change in the distribution of surviving MF progenitor cells towards later differentiation stages within the precursor compartment. The role of autocrine transforming growth factor-beta1 production by fibroblasts in mediating terminal differentiation was investigated. We propose that cell stress and DNA damaging agents may contribute to progression of the differentiation state with age and that individual variation may be related to differences in the rate of induced differentiation.

MK-886, a leukotriene biosynthesis inhibitor, induces antiproliferative effects and apoptosis in HL-60 cells.

MK-886, a specific inhibitor of 5-lipoxygenase inhibited DNA replication in leukemic HL-60 cells in a dose-dependent manner. Addition of exogenous leukotriene B4 reversed this effect, whereas addition of leukotriene B4 failed to modulate a prostaglandin D2-induced inhibition of DNA replication. The reversal of MK-886-induced inhibition was not observed with leukotriene C4. These results suggest that the effect of MK-886 is mediated by inhibition of leukotriene B4 biosynthesis. Moreover, MK-886 not only impaired DNA replication in HL-60 cells but also decreased cell proliferation and induced apoptotic cell death. Our results suggest a crucial role of leukotriene B4 in the regulation of cell proliferation and cell survival in HL-60 cells.

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.

Induction of intracellular calcium oscillations in human skin fibroblast populations by sinusoidal extremely low-frequency magnetic fields (20 Hz, 8 mT) is dependent on the differentiation state of the single cell.

Experiments were performed to analyze whether short-term exposure to a sinusoidal extremely low-frequency electromagnetic field (20 Hz, 8 mT) can alter the dynamics of intracellular calcium in diploid human skin fibroblasts. In heterogeneous fibroblast populations, about 30% of the cells responded with a change in the oscillation activity of intracellular calcium within 40 min. It was demonstrated at the level of the single cell that the responsiveness of fibroblast populations to extremely low-frequency electromagnetic fields depends on the specific differentiation state of the exposed cell. The data obtained clearly indicate that mitotic progenitor fibroblasts respond with an enhancement of the dynamics of calcium, whereas in postmitotic fibrocytes a reduction of the dynamics was observed when the cells were co-stimulated with suboptimal concentrations of platelet-derived growth factor. Thus data from our laboratory on terminal differentiation induced by extremely low-frequency electromagnetic fields may be correlated with changes in the dynamics of Ca2+ reported here.

The presence of wild-type TP53 is necessary for the radioprotective effect of the Bowman-Birk proteinase inhibitor in normal fibroblasts.

In the present study we have demonstrated that the Bowman-Birk proteinase inhibitor (BBI) protected normal fibroblasts from a radiation-induced reduction in cell survival, whereas in transformed fibroblasts no radioprotective effect was observed. It was shown that BBI reduced the radiation-induced protein stabilization and DNA-binding activity of TP53 (formerly known as p53) in normal fibroblasts. In transformed fibroblasts, BBI failed to induce these effects. The analysis of the TP53 gene in transformed fibroblasts revealed a mutation in exon 5. As a consequence of this mutation, the expression of the TP53 downstream gene CDKN1A (p21/WAF1/Cip1) is blocked. Based on experiments using TP53 antisense oligonucleotides, the radioprotective effect of BBI could be correlated with the function of wild-type TP53. Thus BBI can be considered as a selective radioprotective agent for normal human fibroblasts.