Heavy ion beam irradiation regulates the mRNA expression in megakaryocytopoiesis from human hematopoietic stem/progenitor cells.

Heavy ion beams are a high-LET radiation that has greater biological effect than electron beams or X-rays. However, little is known about the effect of heavy ion beams on the proliferation and differentiation of human hematopoietic stem/progenitor cells (HSPCs). The present study examined the effect of heavy ion beams on gene expression in human HSPCs, especially during early stage of megakaryocytopoiesis. Human CD34+ cells were exposed to monoenergetic carbon-ion beams (290 MeV/nucleon, LET = 50 KeV/m) that were generated by an accelerator (Heavy Ion Medical Accelerator in Chiba). The expression of various genes related to early hematopoiesis, megakaryocytopoiesis/erythropoiesis, cytokine receptors and oxidative stress were analyzed by real-time RT-PCR. Friend leukemia virus integration 1, an early hematopoiesis-related gene, showed significantly higher mRNA expression than the control at 6 hr after irradiation. In contrast, no significant differences were observed in almost all of the other early hematopoiesis-related genes, cytokine receptor-coded genes and megakaryocytopoiesis/erythropoiesis-differentiation pathway-related genes, respectively. An analysis of the response of the genes to oxidative stress revealed the expression of heme oxygenase 1 to show a 1.5-fold and 11.9-fold increase from the day 0 control at 24 hr after 0.5 Gy and 2 Gy irradiation, respectively. Similarly, the NAD(P)H dehydrogenase-quinone 1 expression also showed a 22.0-fold and a 21.8-fold increase at 6 hr in comparison to the initial control. These results showed that the heavy ion beams affect megakaryocytopoiesis/ erythropoiesis differentiation of human HSPCs on the gene expression level.

Heavy-ion-induced mutations in the gpt delta transgenic mouse: effect of p53 gene knockout.

The influence of the loss of p53 gene on heavy-ion-induced mutations was examined by constructing a new line of transgenic mice, p53 knockout (p53(-/-)) gpt delta. In this mouse model, deletions in lambda DNA integrated into the mouse genome are preferentially selected as Spi(-) phages, which can then be subjected to molecular analysis. Mice were exposed to 10 Gy of whole-body carbon-ion irradiation. The carbon ions were accelerated to 135 MeV/u by the RIKEN Ring Cyclotron. The p53 defect markedly enhanced the Spi(-) mutant frequency (MF) in the kidneys of mice exposed to C-ion irradiation: the Spi(-) MF increased 4.4- and 2.8-fold over the background level after irradiation in p53(-/-) and p53(+/+) mice, respectively. There was no significant difference in the background Spi(-) MF between p53(-/-) and p53(+/+) mice. Sequence analysis of the Spi(-) mutants indicated that the enhancement of kidney Spi(-) MF in p53(-/-) mice was primarily due to an increase in complex or rearranged-type deletions. In contrast to the kidney, the p53 defect had no effect on the Spi(-) MF in liver: Spi(-) MF increased 3.0- and 2.7-fold after the irradiation in p53(-/-) and p53(+/+) mice, respectively. Our results suggest that p53 suppresses deletion mutations induced by heavy-ion irradiation in an organ-specific manner.

Characteristics of myeloid differentiation and maturation pathway derived from human hematopoietic stem cells exposed to different linear energy transfer radiation types.

Exposure of hematopoietic stem/progenitor cells (HSPCs) to ionizing radiation causes a marked suppression of mature functional blood cell production in a linear energy transfer (LET)- and/or dose-dependent manner. However, little information about LET effects on the proliferation and differentiation of HSPCs has been reported. With the aim of characterizing the effects of different types of LET radiations on human myeloid hematopoiesis, in vitro hematopoiesis in Human CD34(+) cells exposed to carbon-ion beams or X-rays was compared. Highly purified CD34(+) cells exposed to each form of radiation were plated onto semi-solid culture for a myeloid progenitor assay. The surviving fractions of total myeloid progenitors, colony-forming cells (CFC), exposed to carbon-ion beams were significantly lower than of those exposed to X-rays, indicating that CFCs are more sensitive to carbon-ion beams (D(0) = 0.65) than to X-rays (D(0) = 1.07). Similar sensitivities were observed in granulocyte-macrophage and erythroid progenitors, respectively. However, the sensitivities of mixed-type progenitors to both radiation types were similar. In liquid culture for 14 days, no significant difference in total numbers of mononuclear cells was observed between non-irradiated control culture and cells exposed to 0.5 Gy X-rays, whereas 0.5 Gy carbon-ion beams suppressed cell proliferation to 4.9% of the control, a level similar to that for cells exposed to 1.5 Gy X-rays. Cell surface antigens associated with terminal maturation, such as CD13, CD14, and CD15, on harvest from the culture of X-ray-exposed cells were almost the same as those from the non-irradiated control culture. X-rays increased the CD235a(+) erythroid-related fraction, whereas carbon-ion beams increased the CD34(+)CD38(-) primitive cell fraction and the CD13(+)CD14(+/-)CD15(-) fraction. These results suggest that carbon-ion beams inflict severe damage on the clonal growth of myeloid HSPCs, although the intensity of cell surface antigen expression by mature myeloid cells derived from HSPCs exposed to each type of radiation was similar to that by controls.

Terminal maturation of megakaryocytes and platelet production by hematopoietic stem cells irradiated with heavy-ion beams.

Hematopoietic processes, especially megakaryocytopoiesis and thrombopoiesis, are highly sensitive to high-linear energy transfer (LET) radiations such as heavy-ion beams that have greater biological effects than low-LET radiation. This study examined the terminal maturation of megakaryocytes and platelet production derived from hematopoietic stem cells irradiated with heavy-ion beams. CD34(+) cells derived from human placental/umbilical cord blood were exposed to monoenergetic carbon-ion beams (LET  =  50 keV/µm) and then cultured in a serum-free medium supplemented with thrombopoietin and interleukin-3. There was no significant difference in megakaryocyte-specific markers between nonirradiated control and irradiated cells. Expression of Tie-2, a receptor that acts in early hematopoiesis, showed a significant 1.31-fold increase after 2 Gy irradiation compared to control cells on day 7. There was a significant increase in Tie-2 mRNA expression. In addition, the expression of other mRNAs, such as PECAM1, SELP and CD44, was also significantly increased in cells irradiated with heavy-ion beams. However, the adherent function of platelets derived from the irradiated cells showed no difference from that in the controls. These results clarify that the functions of megakaryocytopoiesis and thrombopoiesis derived from hematopoietic stem/progenitor cells irradiated with heavy-ion beams are similar to those in the unirradiated cells, although heavy-ion beams affect the expression of genes associated with cellular adhesion.