Research Article: Cellular responses and gene expression profile changes due to bleomycin-induced DNA damage in human fibroblasts in space

Date Published: March 1, 2017

Publisher: Public Library of Science

Author(s): Tao Lu, Ye Zhang, Yared Kidane, Alan Feiveson, Louis Stodieck, Fathi Karouia, Govindarajan Ramesh, Larry Rohde, Honglu Wu, Eric Y. Chuang.


Living organisms in space are constantly exposed to radiation, toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage is essential for assessing the radiation risk for astronauts and the mutation rate in microorganisms. In a study conducted on the International Space Station, confluent human fibroblasts in culture were treated with bleomycin for three hours in the true microgravity environment. The degree of DNA damage was quantified by immunofluorescence staining for γ-H2AX, which is manifested in three types of staining patterns. Although similar percentages of these types of patterns were found between flight and ground cells, there was a slight shift in the distribution of foci counts in the flown cells with countable numbers of γ-H2AX foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. We also performed a microarray analysis of gene expressions in response to bleomycin treatment. A qualitative comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. The microarray data was confirmed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly upregulated in both flight and ground cells after bleomycin treatment. Our results suggest that whether microgravity affects DNA damage response in space can be dependent on the cell type and cell growth condition.

Partial Text

Living organisms are exposed to radiation in space that consists of high energy protons and heavy charged particles. For humans, exposure to this environment is expected to cause cancer and other deleterious effects. Current assessment of space radiation risk in astronauts is based on the knowledge gained primarily from human data and animal experiments on the ground under the 1 g gravity condition. If spaceflight factors, microgravity in particular, affect the repair of space radiation-induced damage, then one would expect an additional impact on the mutation rate in living cells [1], and consequently bias current ground-based risk assessment methods. For astronauts, effects of space radiation exposure have been manifested in the increased frequency of chromosome aberrations in the astronauts’ lymphocytes post mission [2–4], early onset of cataracts [5, 6], and light flashes [7, 8].

Whether spaceflight factors, microgravity in particular, affect cellular responses to DNA damages is a critical question to be addressed for human space exploration. In this flight study, we induced DNA damage intentionally on the ISS with bleomycin and investigated early responses by measuring the phosphorylation of histone protein H2AX for quantification of DNA damages, and by analyzing gene expressions using both the microarray and the PCR array methods. By classifying γ-H2AX staining patterns in different types, we found a clear increase in the percentage of cell nuclei with pan-nucleus staining (Type I) as the concentration of bleomycin increased (Fig 2). Comparison of different types of γ-H2AX staining patterns in cells after bleomycin treatment between flight and ground revealed a similar percentages of Type I (~6%) and Type II (~15%) cells, and the percentages were not significantly different (Tables 2). However, detailed counting of the number of foci in the countable γ-H2AX stained cells (Type III) indicated differences in the low quantiles of the distribution in cells after bleomycin treatment (Table 3, Fig 2B). In the present study, the cells were fixed at one time point of 3 hours after bleomycin treatment, due primarily to the limitation of samples allowed in a spaceflight experiment. This is typically the time point optimal for investigations of gene expressions. With acute damage to cells by ionizing radiation, it is known that the γ-H2AX intensity would peak within one hour post irradiation, and decrease as the DNA damage is repaired [38]. In the present study, the cells were treated continuously with bleomycin, and the γ-H2AX signals reflected a mix of accumulated damage over the 3 hr period and the repair of some of the damages [32]. Unlike ionizing radiation, γ-H2AX foci induced by toxic chemicals or UV may not necessarily represent DNA double strand breaks [33]. These cells in G1 phase of the cell cycle do not go through apoptosis.