Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Breast Cancer Res. 2010;12(1):R11. doi: 10.1186/bcr2477. Epub 2010 Feb 10.
Most human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated beta-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth.
Pre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or gamma-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated beta-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses.
Cultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated beta-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism.
Our studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential.
从组织学上正常的乳腺组织中培养的大多数人乳腺上皮细胞（HMEC）在经过 5 到 20 次细胞倍增后进入称为停滞的衰老状态。这些衰老细胞体积增大，含有衰老相关的β-半乳糖苷酶活性，并表达细胞周期蛋白依赖性激酶抑制剂 p16INK4A（CDKN2A;p16）。然而，在无血清培养基中生长的 HMEC 自发地以低频率产生能够长期生长并且易发生基因组不稳定性的变异（v）HMEC。我们研究了电离辐射（增加女性乳腺癌的风险）是否会影响 vHMEC 生长的速度。
在预停滞 HMEC 培养物中，用稀疏（X 或γ射线）或密集（1 GeV/amu 56Fe）电离辐射照射 5 至 200 cGy。在辐射暴露后 4 至 6 周，当出现 vHMEC 斑片时，在亚培养的辐照或未辐照群体中检测增殖（溴脱氧尿苷掺入）、衰老（衰老相关的β-半乳糖苷酶活性）和 p16 表达。还监测了随后传代中的长期生长潜力和 p16 启动子甲基化。基于代理的建模，结合了一组简单的规则和基本假设，用于模拟 vHMEC 的生长并评估机制假设。
与源自未辐照细胞的培养物相比，源自辐照细胞的培养物含有显著更多缺乏衰老相关β-半乳糖苷酶或 p16 表达的 vHMEC。正如预期的那样，源自未辐照和辐照细胞的后停滞 vHMEC 培养物的 p16 基因甲基化程度均高于前停滞 HMEC 培养物。然而，vHMEC 样品中单个 CpG 位点的甲基化程度与传代数或处理无关。与未辐照对照相比，稀疏或密集电离辐射的暴露导致 vHMEC 数量相似的增加。基于代理的建模表明，正常 HMEC 的辐射诱导过早衰老极有可能通过减轻空间限制来加速 vHMEC 的生长。随后使用定义明确的 vHMEC 和衰老细胞共培养进行的实验支持了这一机制。
我们的研究表明，电离辐射可以促进具有潜在恶性前体的表观遗传改变细胞的生长。
