E.A. Blakely1, M.P. McNamara1, P.Y. Chang1,
K.A. Bjornstad1, D. Sudar1, and A.C. Thompson2
1Life Sciences Division, Lawrence Berkeley National Laboratory,
Berkeley, California 94720; 2Advanced Light Source Division,
Lawrence Berkeley National Laboratory, Berkeley, California 94720
INTRODUCTION
The human lens is one of the most radiosensitive organs of the body. Cataract, the opacification of the lens, is a late-appearing response to radiation damage. There are few data available on the late radiation effects of exposure in space flight to charged particle beams, the most prevalent of which are protons. Basic research in this area is needed to integrate the responses of both critical and other representative tissues into our understanding and estimation of radiation risk for space travel. Radiation damage to the lens is not life threatening but, if severe, can affect vision unless surgically corrected with synthetic lens replacement. The lens, however, may be a sensitive detector of radiation effects for other cells of ectodermal origin in the body for which there are not currently clear endpoints of low dose radiation effects.
Basic fibroblast growth factor (FGF-2) has an essential role in the proliferation, migration and differentiation of human lens cells. We are investigating a role for proton radiation-induced changes in FGF-2 gene expression in mechanism(s) underlying lens cell injury (Chang et al., Radiat. Res. 154:477-484, 2000). Cell Adhesion Molecules (CAMs) are important to the regulation of cell motility, division, differentiation and apoptosis. CAMs link cells to the extracellular matrix (ECM) and mediate mechanical and chemical signals from them. These signals regulate kinases, growth factor receptors, and ion channels, and control the organization of the cytoskeleton. Radiation effects on CAMs have been reported in other tissues. We have investigated the dose-dependent expression of several radiation-responsive endpoints using our in vitro model of differentiating human lens epithelial cells. Our hypothesis is that low-dose and low-dose rates of low-LET ionizing radiations trigger signal transduction pathways that deregulate cell cycle, apoptosis and differentiation of irradiated, as well as neighboring cells, by aberrant expression of the cell-cycle regulator p21Cip1(CDKN1A). The dose-range studied currently extends down to 10 cGy.
CURRENT STATUS OF RESEARCH
Methods
Normal human lens epithelial (HLE) cells cultured in vitro on ECM derived from bovine corneal endothelial cells demonstrate morphological and biochemical markers for differentiation into lens fiber cells (Blakely et al., Investigative Ophthalmology & Visual Sciences, 41(12):3898-3907, 2000). HLE at various stages of differentiation have been irradiated with single acute doses of either Xrays (150 kVp, 320 kVp, or 12.5 keV from a synchrotron source), 55 MeV protons or 32 MeV/amu helium ions. After 6 hrs. of post-incubation at 37C, cultures were fixed and prepared for immunofluorescence to study the incidence of TUNEL-positive apoptotic bodies and the prevalence and cellular distribution of b1-integrin protein and p21Cip1(CDKN1A).
Results
· Radiation-Induced Apoptosis
HLE cells are resistant to radiation-induced apoptosis, while differentiating fiber cells show a radiation-dose-dependent induction, but with LET-dependent differences in the dose response 6 hrs. after exposure. Measurements of TUNEL-positive differentiating cells after single graded X-ray doses from 0.5 Gy to 12 Gy show a dose-dependent increase that appears linear up to 2 Gy, and then levels off at higher doses. Proton studies also indicate a dose-dependent increase in TUNEL-positive differentiating lens cells after 4 Gy, 8 Gy or 12 Gy. However, after exposure to 4 Gy, 8 Gy, or 12 Gy helium ions, there is no increase in the percentage of TUNEL-positive cells. These data suggest that there are LET-dependent differences in the dose-dependent appearance and/or time course of TUNEL-positive cells.
· p21Cip1(CDKN1A) Expression
Lens epithelial cells at low density demonstrate moderate nuclear expression of p21 in only a few sparse cells, while very high, nuclear expression of p21 was evident in more than 90% of the cells after a single 4 Gy dose of Xrays to the entire cell population on the petri dish. X-ray experiments at low doses down to 10 cGy and 20 cGy also show evidence of the enhanced expression of p21 and cytoskeletal changes at the same time point post-irradiation. Unirradiated, three week confluent, differentiating cultures show uniform moderate, nuclear, p21 expression in all cells, while after 4 Gy Xrays, p21 expression was enhanced in most nuclei. These data suggest a role for p21 in the early signaling for normal lens fiber cell differentiation, and implicate X-ray-induced modulation of p21 expression in both the epithelial and differentiating cells.
· b1-Integrin Expression
The constitutive expression of adhesion molecules is dependent on the differentiation status of the lens cells. The cellular distribution and level of expression of b1-integrin changes in the transition from lens epithelial to fiber cell. The b1-integrin moves from a focal adhesion point to a more diffuse distribution with increased expression. Proton-irradiation induces changes in the immunoreactivity of adhesion molecules in the lens. Epithelial cells show a dose-dependent increase in b1-integrin expression with changes in intracellular localization, but a decrease in immunoreactivity in the differentiating fiber cells at after exposure to protons. The decrease in immunoreactivity is also seen after 20 cGy of Xrays.
CONCLUSION
We hypothesize that proton irradiation promotes lens cell dysfunction by initiating premature differentiation via events that include modulation of FGF-2 regulation, inhibition of cell cycle control, altered expression of cell adhesion molecules, and altered apoptotic/enucleation program machinery in human lens cells. This misregulation of cell proliferation, cell adhesion and apoptosis depends upon the differentiation status of the cell at the time of irradiation. Radiation-induced cataracts are typically characterized as posterior subcapsular opacifications, arising at the point where lens fiber cells lose their attachment to the capsule, which is primarily basement membrane type ECM. Our work indicates that irradiated epithelial cells, resistant to apoptosis by upregulation of both FGF-2 and anti-apoptotic gene expression, survive the radiation exposure and show premature and defective differentiation and expression of b1-integrin.
FUTURE PLANS
Studies of the time course of radiation-induced changes in CAMs and p21 at lower radiation doses are in progress. We propose to delineate the specific molecular role(s) of FGF-2 trafficking and signaling in the response of human lens epithelial and fiber cells to proton or X-ray irradiation. The definition of these events will allow us to develop strategies to intervene with countermeasures.
This work was supported by NASA Grant #T-965W.