Hematopoietic stem and progenitor cells (HSPC) are responsible for the constant renewal of all mature blood cells (lymphocytes, PBLs included). In view of the increasing application of heavy ions in radiotherapy [1, 2] and as HSPC may be located in the radiation field during radiotreatment, their radiobiological features and those of their descendents have to be investigated. Since the effect of radiation on cells results mainly from the damage induced in the DNA, the aim of the study was to compare the DNA repair capacity and fidelity of human HSPC and PBLs. Because several types of DNA damage exist, which induce different mechanisms of DNA repair, a test system for HSPC was established, to enable the observation of distinct repair pathways after the induction of targeted DNA double strand breaks (DSBs), with focus on correct versus incorrect repair of those breaks (according to [3]). Additionally, after exposure to photons and densely ionizing nitrogen ions early radiation-induced damage in chromosomes and the rejoining was studied (PCC method).

The increasing application of heavy ions in radiotherapy and the growing interest in protracted space travels are a strong motivation to expand the fundamental research in radiation biology. With respect to long term effects in different cell systems, in the majority of studies the occurrence of chromosomal aberrations is assessed, but only in lymphocytes. However, the investigation using hematopoietic stem and progenitor cells (HSPC) which are responsible for the permanent renewal of all blood cells, seem to be more appropriate. For this reason we are interested, if the irradiation of HSPC with X-rays or carbon ions has different effects on the occurrence of chromosomal aberrations and the clonal transmission to subsequent generations. As observed in lymphocytes, both radiation qualities induce distinct types of damage in affected cells [1]. Thus the complexity of DNA damages varies highly, whereas carbon ions should produce more complex chromosomal aberrations than X-rays.

In multicellular organisms stem cells are involved in tissue regeneration, which is regulated by apoptosis (programmed cell death). An early event of apoptosis is the externalisation of membrane phospholipids to the extracellular environment, on which Annexin-V is able to bind with a high affinity. The later stages of apoptosis are characterized by the loss of plasma membrane integrity, DNA fragmentation, and chromatin condensation (overview in [1]). DNA fragmentation can be measured using the TUNEL assay (terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling), in which labeled nucleotides are incorporated into the 3’OH recessed termini of radiation induced DNA breaks. The occurrence of radiation induced apoptosis is in particular in proliferating cells an important factor in assuring the correct transmission of genetic information, because damaged cells are eliminated from the population [2]. To investigate the occurrence of apoptosis hematopoietic stem and progenitor cells (HSPCs) were collected by apheresis of healthy, G-CSF treated donors. Following CD34+ enrichment by immunomagnetic separation, HSPCs were exposed to either x-rays (16mA, 250kV) or carbon ions (100MeV/u, 29keV/μm). The Annexin-V and TUNEL assays were used to measure radiation induced apoptosis by microscopic or flow cytometric analyses (Annexin-V-FLUOS and In Situ Cell Death Detection Kit, Fluorescein, Roche). In first experiments on the protective role of cytokines [3], the cells were cultured in serum-free expansion medium, supplemented or not with different cytokines (StemCell Technologies Inc.). Without cytokines we observed apoptosis in unirradiated HSPCs, with an increasing frequency with progressing culture time (figure 1).

Since 2006 a clinical phase I/II study is carried out treating prostate cancer patients with a combination of Cion irradiation and IMRT (C-ion treatment at GSI, IMRT in Heidelberg). In parallel to this therapy, a study investigating chromosome aberrations in blood lymphocytes of prostate cancer patients is performed, as described in [1]. Chromosome aberrations in blood lymphocytes are widely used in biological dosimetry as they represent a sensitive marker for ionizing radiation. Since they are unavoidably exposed to radiation during tumor treatment, this study makes it possible to investigate in vivo the cytogenetic effects of C-ion radiation and to compare with conventional tumor irradiation. Patients are irradiated with C-ion boost (6x3GyE) and IMRT (30x2Gy) or solely with IMRT (38x2Gy). Blood samples are drawn from each patient before, during, at the end of and one year after therapy. Lymphocytes are cultured according to standard techniques [2]. Chromosome spreads are prepared and slides are stained using Fluorescence-Plus-Giemsa staining (FPG) and multiplex fluorescence in situ hybridization (mFISH), respectively. To investigate the individual radiosensitivity, a blood sample from each patient before therapy is irradiated in vitro (3Gy X-rays). The results are compared to those of healthy donors (fig. 1). Slight differences in the aberration yield were observed, but no significantly increased radiosensitivity of one patient was found, nor an enhanced sensitivity of all patients compared to healthy donors.

The increasing application of heavy ions in radiotherapy is a strong motivation to expand the fundamental research in radiation biology, especially with respect to long term effects in different cell systems. The classical cyt ogenetic assay to estimate the radiation effect relies only on the measurement of chromosome aberrations in lymphocytes, but a large part of the damaged lymphocytes is eliminated from the population via apoptosis [1]. On the other hand, the occurrence of clonal aberrations has been reported f or patients, who had received a chemotherapy years ago [2,3]. Therefore, regarding long term effects after heavy ion therapy, it is important to assess the potential t ransmission of chromosomal aberrations by hematopoietic stem cells (HSCs) to lymphocytes. HSCs are responsible for the constant renewal of blood. On the other hand th ey have also the ability to differentiate to a variety of s pecialized blood cells. As both processes have an impact o n a potential transmission of chromosomal aberrations we focused in first experiments on the differentiation of hematopoietic stem cells after irradiation with Carbon i s in comparison to conventional photon irradiation.

Since the start of C-ion therapy at GSI, Darmstadt, in December 1997 about 340 patients have been treated. Most patients had chordoma or low grade chondrosarcoma of the skull base. Based on the promising clinical results, namely high local control rates and mild side effects [1], the application has been recently extended to intermediate risk prostate cancer. A clinical phase I/II trial combing photon intensity-modulated radiotherapy (IMRT) and C-ion boost has been started in 2006. In parallel to this clinical trial the yields and the types of radiation-induced chromosome aberrations will be investigated in peripheral blood lymphocytes of prostate cancer patients.