H. P. Rodemann

TGF-beta1-mediated alterations of rat lung fibroblast differentiation resulting in the radiation-induced fibrotic phenotype

Purpose : To study the influence of TGF-beta1 and TGF-beta-neutralizing antibodies on the clonogenic activity and terminal differentiation of rat lung fibroblasts following radiation exposure. Material and methods : Early passage rat lung fibroblasts were used in this study. Colony formation assays were applied to determine the radiation sensitivity as well as radiation-induced alterations in differentiation pattern. Based on a TGF-beta1-specific ELISA system, the amount of TGF-beta1 in the culture medium of sham-irradiated and irradiated cultures was determined. Results : Applying immediate (ip) and delayed plating (dp) procedures for cells irradiated in the subconfluent state, distinct differences in the radiation dose–response curves could be observed (SF2 ip 0.20 +/- 0.025 versus SF2 dp 0.51 +/- 0.0077). Upon irradiation with a single dose of 4 Gy the level of TGF-beta1 found in the culture medium increased by about 60%. Radiation-induced terminal differentiation of progenitor fibroblasts (MF) to postmitotic fibrocytes (PMF) was expressed by the change of the ratio of PMF:MF (0.8 at 0 Gy versus 3.0 at 4 Gy). Neutralizing antibodies directed against TGF-beta inhibited both the radiation-induced reduction in clonogenic activity of rat lung fibroblasts as well as the radiation-induced terminal differentiation of MF progenitor fibroblasts to PMF postmitotic fibrocytes. Conclusion : The results indicate the important role of TGF-beta1 in triggering radiation-induced inhibition of clonogenic activity as well as terminal differentiation of rat lung fibroblasts. The data presented support the hypothesis that terminal differentiation is an essential cellular process in the development of radiation-induced fibrosis in the lung.PURPOSE To study the influence of TGF-beta1 and TGF-beta-neutralizing antibodies on the clonogenic activity and terminal differentiation of rat lung fibroblasts following radiation exposure. MATERIAL AND METHODS Early passage rat lung fibroblasts were used in this study. Colony formation assays were applied to determine the radiation sensitivity as well as radiation-induced alterations in differentiation pattern. Based on a TGF-beta1-specific ELISA system, the amount of TGF-beta1 in the culture medium of sham-irradiated and irradiated cultures was determined. RESULTS Applying immediate (ip) and delayed plating (dp) procedures for cells irradiated in the subconfluent state, distinct differences in the radiation dose-response curves could be observed (SF2ip 0.20+/-0.025 versus SF2dp 0.51+/-0.0077). Upon irradiation with a single dose of 4Gy the level of TGF-beta1 found in the culture medium increased by about 60%. Radiation-induced terminal differentiation of progenitor fibroblasts (MF) to postmitotic fibrocytes (PMF) was expressed by the change of the ratio of PMF:MF (0.8 at 0 Gy versus 3.0 at 4 Gy). Neutralizing antibodies directed against TGF-beta inhibited both the radiation-induced reduction in clonogenic activity of rat lung fibroblasts as well as the radiation-induced terminal differentiation of MF progenitor fibroblasts to PMF postmitotic fibrocytes. CONCLUSION The results indicate the important role of TGF-beta1 in triggering radiation-induced inhibition of clonogenic activity as well as terminal differentiation of rat lung fibroblasts. The data presented support the hypothesis that terminal differentiation is an essential cellular process in the development of radiation-induced fibrosis in the lung.

Analysis of mdm2 and p53 gene alterations in glioblastomas and its correlation with clinical factors.

Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with probably prognostic implications. To elucidate the involvement of murine-double-minute (mdm)2 gene amplifications and mutations of the tumor suppressor gene p53 in the tumorigenesis of malignant gliomas we analyzed a series of 75 glioblastomas. The p53 mutations occur in one-third of glioblastomas, mdm2 amplifications were found in 13% of cases. Our analysis revealed a hot spot in the p53 gene locus in codon 156, the same point mutation was detected in 4 tumor samples. None of the mdm2 amplified tumors had p53 mutations, supporting the hypothesis, that mdm2 amplifications are alternative mechanisms for p53 inactivation. Patients with p53 mutated tumors were significantly younger characterized by a mean age of 44 years. Additionally association with longer overall survival could be detected for this subgroup of patients. In our study, survival estimation revealed a significant correlation of mdm2 gene amplification with shorter survival time, and support the hypothesis, that mdm2 oncogene activation appears to occur late in tumor progression and may be characteristic as negative prognostic marker.

BCL-2 Family Proteins Modulate Radiosensitivity in Human Malignant Glioma Cells

Radiotherapy is the standard treatment for glioblastoma. Here, we assessed the radiosensitivity of 12 human malignant glioma cell lines in vitro and correlated these data with irradiation-induced cell cycle changes, chemosensitivity profiles and BCL-2 family protein expression. Irradiation at 3 Gy failed to cause major cell cycle perturbations. Radioresistance was associated with collateral sensitivity to the topoisomerase II inhibitors, teniposide and doxorubicin. High levels of BCL-XL and low levels of BAX were independently linked to radioresistance. Ectopic expression of a BAX transgene induced radiosensitization in the LN-18 cell line. Thus, BCL-2 family protein expression modulates radiosensitivity in human glioma cells and targeted alterations in BCL-2 family protein expression are a promising strategy to improve the therapeutic efficacy of radiotherapy for gliomas.

Irradiated homozygous TGF- β 1 knockout fibroblasts show enhanced clonogenic survival as compared with TGF- β 1 wild-type fibroblasts

Purpose : To study the role of transforming growth factor β 1 (TGF- β 1) on cellular radiation sensitivity by analysing mouse lung fibroblasts of different TGF- β 1 genotypes. Materials and methods : Heterozygous TGF- β 1 knock-out mice were mated to produce offspring of different TGF- β 1 genotypes as confirmed by PCR-genotyping. Primary lung fibroblast populations were established from new-born animals of specific genotypes (TGF- β 1 +/+, TGF- β 1 +/-, TGF- β 1 -/-) . Production of TGF- β 1 was tested by ELISA. TGF- β 1 receptor-II mRNA expression was analysed by RT-PCR. Colony formation of untreated, TGF- β 1-treated and/or irradiated primary lung fibroblasts was determined under different medium conditions. Results : Plating efficiencies under different medium conditions were independent of TGF- β 1 genotype. Production of TGF- β 1 correlated with the genotype: heterozygous TGF- β 1 knock-out fibroblasts (TGF- β 1 +/-) produced 60-65% of wild-type (TGF- β 1 +/+ cells). As expected, homozygous TGF- β 1 knock-out fibroblasts (TGF- β 1 -/-) did not produce TGF- β 1. Radiation exposure significantly enhanced TGF- β 1 production in TGF- β 1 +/+ cells by a factor of 2. No such stimulation was observed in TGF- β 1 +/- cells. TGF- β 1 +/- and especially TGF- β 1 -/- cells were significantly more radioresistant than TGF- β 1 +/+ cells. TGF- β 1 treatment significantly reduced clonogenic survival for both TGF- β 1 +/+ and TGF- β 1 -/- cells. TGF- β 1 treatment of TGF- β 1 -/- cells resulted in an enhancement of radiation sensitivity. Conclusion : The data are the first direct evidence that TGF- β 1 is a major autocrine regulator of intrinsic radiation sensitivity of mouse lung fibroblasts.

The radioprotective effect of BBI is associated with the activation of DNA repair-relevant genes

PURPOSE To investigate the molecular mechanisms of the radioprotective effect of the Bowman-Birk proteinase inhibitor (BBI) in normal human skin fibroblasts (HSF). MATERIAL AND METHODS The effect of BBI pre-treatment on p53 protein level and on mRNA levels of downstream genes (ERCC3, Gadd45 and p53) was investigated. RESULTS As indicated by time-course experiments based on clonogenic assays, a 6 h pre-incubation with BBI before irradiation of HSF with a single dose of 6 Gy resulted in maximum radioprotection. In non-irradiated cells, pre-incubation with BBI resulted in an increased level of p53 protein. Concomitantly, enhanced mRNA levels of the ERCC3 and the Gadd45 genes were observed. As a consequence, BBI-treated cells showed accelerated DNA repair compared with untreated cells when irradiated. CONCLUSIONS The radioprotective effect of the Bowman-Birk proteinase inhibitor was accompanied by elevated mRNA expression of repair-relevant genes prior to irradiation. Activation of the DNA-repair machinery induced by pre-treatment with BBI is one possible mechanism of the radioprotective effect of BBI.

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.

Bowman-Birk protease inhibitor reduces the radiation-induced activation of the EGF receptor and induces tyrosine phosphatase activity

PURPOSE To investigate the molecular action of the radioprotective Bowman Birk protease inhibitor (BBI) on radiation-induced tyrosine kinase activity. MATERIALS AND METHODS Radiation-induced activation of tyrosine kinases and phosphatases was measured in normal human skin fibroblasts by in vitro kinase assays after pre-incubation with BBI. RESULTS Pre-incubation with BBI resulted in a time-dependent block of the radiation-induced activation of tyrosine kinases. Whilst radiation-induced pp60c-Src activity was not modulated due to BBI pre-treatment, activation of epidermal growth factor receptor (EGFR) was inhibited. Additionally, pre-incubation with BBI resulted in enhanced tyrosine specific phosphatase (PTP) activity. CONCLUSIONS BBI might exert its radioprotective activity by stabilizing specific tyrosine-phosphatases that interfere with EGFR activation in response to radiation exposure.

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.

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.

Präklinische und klinische Aspekte zur Therapie des Zervixkarzinoms mit 13cis-Retinsäure, Interferonalpha und Bestrahlung

ZusammenfassungTrotz der weit verbreiteten Karzinomvorsorge stellt das Zervixkarzinom in den USA und den westlichen Industrienationen immer noch den vierthäufigsten Tumor bei Frauen dar. Die Anzahl von Neuerkrankungen beträgt ca. 14 000 neue Fälle pro Jahr. In unterentwickelten Ländern ist das Zervixkarzinom weit verbreitet und oft der häufigste Tumor[18].Neben dem primär chirurgischen Vorgehen stellt die Strahlentherapie die wesentliche kurative Behandlungsmodalität dar, wobei die 5-Jahres-Überlebensraten nach Therapie bei 90% für das Stadium I und 7% für das Stadium IV liegen (FIGO Annual Report). Es erscheint daher sinnvoll, nach therapeutischen Konzeptionen zu suchen, um durch neue Kombinationen der Bestrahlung mit einer System-Therapie die gegenwärtigen Ergebnisse weiter zu verbessern.SummaryAlthough carcinoma screening is now widely available, cervical carcinoma is still the fourth most common tumour among women in the USA and in the western industrialized nations. There are about 14,000 new cases every year. In underdeveloped countries carcinoma of the cervix is widespread and often the most frequent tumour[18]. Besides primarily surgical treatment radiotherapy is the main curative treatment modality, the 5-year survival rates after therapy being around 90% for stage I disease and 7% for stage IV (FIGO Annual Report). It therefore seems desirable to look for therapeutic concepts, with the aim of improving the present results further by means of new combinations of irradiation with a systemic therapy.