22nd Annual NASA Space Radiation Investigators' Workshop (2011)
7031.pdf
Epigenetic effects of radiation on epithelial cell self-renewal
P. Yaswen, G. Kaur, S. Gauny, B. Parvin, and A. Kronenberg
Life Sciences Division, Lawrence Berkeley National Lab, Berkeley, California, USA
INTRODUCTION: A current weakness in cancer risk assessment models is the lack of consideration of heritable
“epigenetic” factors that influence the susceptibility of different cell populations to malignant transformation. A
fundamental property of cancer cells, the capacity for extensive self-renewal, is only found in small subsets of cells
found in normal somatic tissues – those with “stem” or “progenitor” qualities. In more differentiated cells, this
property is repressed through epigenetic changes in function that are independent of changes in the primary DNA
sequence. Early events in radiation-induced carcinogenesis must either cause the expansion of pre-existing cells
with extensive self-renewal potential or the acquisition of extensive self-renewal potential by cells that have
repressed it. We are developing a tissue specific risk model using human breast cells to determine the effects of
ionization density and dose on the frequency of altered differentiation / self-renewal.
METHODS: Surgically discarded reduction mammoplasty specimens from several anonymous donors were
obtained from the Cooperative Human Tissue Network. After mechanical dissociation and collagenase digestion to
remove stromal components, enriched pools of epithelial tissue fragments called “organoids” were suspended in
cryogenic medium and immediately frozen for future use. To avoid selection bias associated with expansion in
adherent cultures, irradiation experiments were performed using thawed organoids in suspension. A general scheme
was developed that involved the following steps: 1) overnight incubation of thawed organoids in serum-free
medium, 2) acute irradiation, 3) overnight recovery, 4) dissociation to single cells, 5) growth and differentiation
assays. X-ray exposures were performed using a 160 kVp Faxitron X-ray generator with 0.5 mm of Cu and 0.5 mm
of Al filtration. The effects of adding Matrigel (a commercial laminin-rich extracellular matrix preparation) prior to
or after irradiation, and/or lowering the temperature during recovery, on cell survival and growth were measured.
Several growth and differentiation assays were evaluated. These included: a) the Hoechst dye-exclusion assay, b) a
two-dimensional colony-forming assay, and c) a three-dimensional assay to quantify polarized “acinus” formation.
Side population cells identified using the Hoechst dye-exclusion assay are those cells that do not retain the DNAbinding dye, Hoechst 33342, usually because they express high levels of the ATP-binding Cassette Transporter
ABCG2 that actively pumps out the Hoechst dye. This assay is one of the oldest and most trusted techniques for
identifying and sorting stem and early progenitor cells in a variety of tissues and species. To optimize conditions for
two-dimensional colony-forming assays, a variety of media formulations were tested with or without mitomycin Ctreated NIH3T3 “feeder cells” present to condition the media with growth and attachment factors. Finally, various
markers, including keratins 8, 14, 18, and 19, DLL1, DNER, MUC1, and α-6 integrin, were evaluated for their
potential to distinguish colonies derived from multi-potential versus lineage-restricted cells by immunofluorescent
staining.
RESULTS: 1) The Hoechst dye-exclusion assay showed reproducible dose-dependent increases in the percentage of
“side-population” cells present in organoids irradiated with 0-5 Gy of X-rays, with some evidence of toxicity at the
highest dose of 7 Gy. 2) Two-dimensional colony-forming ability was markedly improved in the presence of feeder
cells. Interestingly, in the presence of feeder cells, but not in their absence, the frequency of colonies derived from
irradiated organoids was significantly greater than that from unirradiated controls. 3) Keratins 14 and 19 were highly
expressed in a mutually exclusive manner in most cultured primary human mammary epithelial cells, and provided a
clear means of distinguishing colonies established from multi-potential versus lineage-restricted cells. 4) X-ray
irradiation with 1 Gy resulted in reproducible decreases in basal (Keratin 14) and luminal (Keratin 19) lineage
restricted colonies and increases in the proportions of mixed colonies comprised of both cell types, as well as in
individual cells in which both keratins were co-expressed.
CONCLUSIONS: We have developed a method to use primary human breast cells, in a culture system in which
positional information is retained, to determine the effects of radiation on differentiation and self-renewal. Using this
method, we have detected X-ray-induced alterations in two measures of stem/progenitor cell frequency in
preliminary experiments. Confirmation of these results and further assessment of the effects of ionization density
and dose are now in progress.
Supported by NASA grant NNA10DE03I carried out at Lawrence Berkeley National Laboratory under Contract No.
DE-AC02-05CH11231.