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Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells.

ABSTRACT: Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G 1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G 1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.

SUBMITTER: Shimura T

PROVIDER: S-EPMC4049961 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.

Project description:PURPOSE:The delivery of radiation therapy to cure gastrointestinal (GI) cancers is often limited by normal tissue toxicity of the GI tract. Studies using genetically engineered mice have demonstrated an essential role of the cyclin-dependent kinase inhibitor p21 in protecting against GI acute radiation syndrome (GI-ARS). Here, we examined the impact of the Food and Drug Administration-approved, selective, cyclin-dependent kinase 4/6 inhibitor palbociclib (PD-0332991) on the development of GI-ARS induced by single-dose versus fractionated radiation in mice. METHODS AND MATERIALS:For the single-dose radiation study, C57BL/6J mice were treated with palbociclib or vehicle 28 and 4 hours before subtotal body irradiation (SBI). For the fractionated radiation study, C57BL/6J mice were exposed to fractionated SBI for 5 consecutive days. These mice were treated with palbociclib or vehicle either 28 and 4 hours before the first dose of irradiation or 4 hours before the first, third, and fifth doses of irradiation. RESULTS:Our data indicate that treatment with palbociclib before, but not after, a single fraction of SBI significantly ameliorated GI-ARS, improved the integrity of the GI barrier, and increased the number of surviving crypts in the small intestine. In addition, palbociclib did not protect tumor cell lines from radiation in vitro. In contrast to the results from the single-dose exposure, treatment with palbociclib before 5 daily fractions of SBI did not prevent GI-ARS. Moreover, we unexpectedly observed that GI-ARS was exacerbated in mice treated with palbociclib before and during 5 daily fractions of SBI. CONCLUSIONS:Our results demonstrate that treatment with palbociclib before a single dose of SBI protects mice from GI-ARS. In contrast, treatment with palbociclib before and during 5 daily fractions of SBI exacerbates GI-ARS in mice. These results emphasize the importance of conducting preclinical studies of radioprotectors with single-dose and fractionated radiation therapy.

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Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells.

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