Abstract
purpose. Uveal melanomas are notoriously radioresistant and thus necessitate treatment with extremely high radiation doses that often cause ocular complications. The p53 tumor suppressor pathway is a major mediator of the cellular response to radiation-induced DNA damage, suggesting that this pathway may be defective in uveal melanoma. The current study was conducted to analyze the functional integrity of the p53 pathway in primary uveal melanoma cells.
methods. The p53 gene was sequenced in three primary uveal melanoma cells lines. Cultured primary uveal melanoma cells (MM28, MM50, Mel202, Mel270, and Mel290), MCF7 breast carcinoma cells, normal uveal melanocytes (UM47), and normal human diploid fibroblasts (NHDFs) were irradiated at 250 kVp and 12 mA at a dose rate of 1.08 Gy/min for a total dose of up to 20 Gy. Cell viability was analyzed with trypan blue exclusion. Western blot analysis was used to analyze the expression of p53, p53-phospho-Ser15, p21, Bax, PUMA, and Bcl-xL.
results. No p53 gene mutations were found in MM28, MM50, or Mel270 cells. Upstream signaling to p53 was intact, with normal induction of p53 and phosphorylation of p53-Ser15, in all five cell lines. Radiation-induced downstream activation of p21 was defective in MM28 and MM50 cells, and activation of Bax was defective in MM50 and Mel290 cells. MM28, MM50, and Mel202 cells failed to deamidate Bcl-xL in response to radiation-induced DNA damage. Overall, four of the five uveal melanoma cell lines exhibited at least one downstream defect in the p53 pathway.
conclusions. Expression of p53 and upstream signaling to p53 in response to radiation-induced DNA damage appear to be intact in most uveal melanomas. In contrast, functional defects in the p53 pathway downstream of p53 activation appear to be common. Further elucidation of p53 pathway abnormalities in uveal melanoma may allow therapeutic interventions to increase the radiosensitivity of the tumors.
Primary uveal melanomas are highly radioresistant.
1 Nevertheless, these cancers are most commonly treated with episcleral plaque brachytherapy or charged-particle radiotherapy at very high doses (85–100 Gy).
2 This high-dose radiotherapy usually results in good local control but frequently leads to vision-threatening radiation complications, such as cataract, retinopathy, papillopathy, and neovascular glaucoma.
3 4 A better understanding of the molecular basis for radioresistance in uveal melanoma could lead to novel therapeutic interventions to render tumors more radiosensitive.
Radiation therapy generates DNA damage, which leads to cell cycle arrest and/or apoptosis, depending on the cellular context.
5 The p53 tumor suppressor plays an important role in the cellular response to radiation-induced DNA damage.
6 7 Cellular p53 levels are very low in normal cells, due to an inhibitory interaction with HDM2 that targets p53 for degradation.
8 After radiation-induced DNA damage, p53 undergoes posttranslational modifications, such as phosphorylation at Ser15, that stabilize the p53 protein by inhibiting its binding to HDM2.
9 Consequently, p53 accumulates rapidly and activates downstream target genes encoding p21 (a cell cycle inhibitor), Bax (a proapoptotic protein), and other proteins.
8
Over half of human cancers contain p53 mutations, and most others contain genetic lesions in the p53 pathway that render p53 ineffective as a tumor suppressor.
10 Untreated uveal melanomas rarely contain p53 mutations,
11 12 13 which suggests that they may contain functional defects that interfere with the p53 pathway. Such defects could contribute to the radioresistant phenotype of uveal melanoma. The Bcl2 family of apoptotic
arbitrators is a critical downstream target of p53 signaling.
14 Activated p53 signals directly to Bcl2 family proteins by activating Bax and other proapoptotic family members. Disruption of this signaling from p53 to the Bcl2 family proteins can block the proapoptotic p53 stimulus and is encountered commonly in cancer. Deamidation of Bcl-x
L at asparagines 52 and 66 in the unstructured loop of the protein has recently been shown to be a hallmark of an intact p53-Bcl2 response to DNA damage.
15 This deamidation conveys susceptibility to DNA damage-induced apoptosis by abrogating the ability of Bcl-x
L to block proapoptotic BH3 domain-only proteins. There is growing evidence that disruption of this deamidation may be a common mechanism by which tumor cells acquire resistance to DNA damage-induced apoptosis.
16
In this study, we evaluated the functional status of the p53 pathway in response to ionizing radiation in primary uveal melanoma cell lines by analyzing key molecular events upstream and downstream of p53.