23rd Annual NASA Space Radiation Investigators' Workshop (2012)
8040.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 94720
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. Radiation-induced
carcinogenesis must either involve 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 less than 50 years old 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 organoids in serum-free medium, 2)
acute irradiation, 3) overnight recovery, 4) dissociation to single cells, 5) growth and differentiation assays.
RESULTS
Beginning with the fall 2011 run (NSRL11C), we examined the effects of radiation on a set of cell surface markers,
CD44 and CD24, that has proven useful for distinguishing stem-like cells in both malignant and non-malignant
human breast tissues. We first determined that the dissociation procedure itself was not a selective influence. Next
we determined, using organoids from several individuals, each run in triplicate, that the baseline fraction of cells
with the CD44hi/CD24Lo phenotype was relatively stable for each specimen, but ranged between 0.5 and 5% among
specimens. During NSRL11C, we exposed organoid aliquots from two specimens to 50 and 100 cGy of gamma
rays, 25 and 50 cGy of Si (300 MeV/amu), as well as 10 and 25 cGy doses of Fe (300 MeV/amu and 1 GeV/amu).
The results indicated that, at the low to moderate doses of radiation employed, no significant changes in the fractions
of CD44hi/CD24Lo cells were detectable 24 hours after irradiation, regardless of the ion employed. To assess the
differentiation potential of primary cells derived from control or irradiated organoids, isolated cells were incubated
at clonal densities in adherent cultures for 7 days before immunofluorescent characterization of the resulting
colonies using antibodies against keratins 14 (basal marker) and/or 19 (luminal marker). In preliminary experiments,
when irradiated with 1 Gy of X-rays, the percentages of mixed colonies containing both luminal and basal cells were
significantly higher for one specimen and marginally higher for another specimen, than for corresponding unirradiated controls. However, cells from a third specimen exposed to X-rays, gamma rays, or 1 GeV/amu Fe ions
showed no significant differences in the fraction of mixed colonies formed. Interestingly, total colony forming
efficiency was relatively unchanged for cells from organoids exposed to as much as 1 Gy of gamma rays, suggesting
that when left intact, the local physical environment exerts a protective effect on potential clonogens for the sparsely
ionizing reference radiation. In contrast, exposure to doses as low as 0.1 Gy of 1 GeV/amu Fe ions resulted in
detectable cytotoxicity. Cells and colonies from additional specimens and exposures during NSRL 12A are currently
being evaluated.
CONCLUSIONS
Using a human primary cell culture system in which the local physical environment of epithelial cells was retained,
we assessed two measures of stem/progenitor cell frequency. However, baseline differences among specimens were
greater than differences between irradiated and control samples, and no consistent radiation-associated trends were
noted. 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.