Laboratory of Molecular Oncology, Quilmes National University, Buenos Aires, Argentina.
Mol Med Rep. 2010 Mar-Apr;3(2):253-9. doi: 10.3892/mmr_00000248.
Extraocular dissemination is the main cause of death in patients with retinoblastoma (RB) in developing countries, and there are few molecular markers that are useful for the evaluation of minimal disseminated disease. The GD2 ganglioside is known to be expressed by RB cells that metastasize in bone marrow, and the activity of the enzyme responsible for its synthesis, GD2 synthase, can be detected in neuroblastoma, which shares many phenotypic features with RB. The purpose of the present study was to optimize the detection of GD2 synthase expression by reverse transcription-polymerase chain reaction (RT-PCR) followed by nested-PCR in human RB cell lines and patient samples. The optimization strategy was carried out using the RB cell lines Y79 and WERI-Rb1 and specific primers designed for the human sequence of GD2 synthase mRNA. We detected GD2 synthase expression with at least 200 and 40 pg of total RNA extracted from cultured RB cells using a first round of RT-PCR amplification or a second round of nested-PCR, respectively. We also confirmed the expression of GD2 synthase by RT-PCR and immunohistochemical detection of the ganglioside in human RB tumors xenotransplanted in nude mice. Using tumor bank specimens from eight RB patients, we were able to demonstrate the presence of GD2 synthase mRNA in blood and cerebrospinal fluid samples in cases of extraocular dissemination of the tumor. The sequence was not detected in samples derived from children with low-risk disease or healthy adult volunteers. Hence, GD2 synthase mRNA detection through an optimized nested RT-PCR assay is a promising tool for the assessment of minimal disseminated disease in enucleated patients.
眼外播散是发展中国家视网膜母细胞瘤 (RB) 患者死亡的主要原因，目前用于评估微量播散疾病的分子标志物很少。GD2 神经节苷脂已知在骨髓中转移的 RB 细胞中表达，而负责其合成的酶 GD2 合酶的活性可在神经母细胞瘤中检测到，神经母细胞瘤与 RB 具有许多表型特征。本研究的目的是通过逆转录-聚合酶链反应 (RT-PCR) 优化检测人 RB 细胞系和患者样本中 GD2 合酶表达的优化策略使用 RB 细胞系 Y79 和 WERI-Rb1 以及针对人 GD2 合酶 mRNA 序列设计的特异性引物进行。我们使用第一轮 RT-PCR 扩增或第二轮嵌套-PCR 分别从培养的 RB 细胞中提取至少 200 和 40 pg 总 RNA 来检测 GD2 合酶表达。我们还通过 RT-PCR 和在裸鼠中移植的人 RB 肿瘤的免疫组织化学检测证实了 GD2 合酶的表达。使用来自 8 名 RB 患者的肿瘤库标本，我们能够证明在肿瘤眼外播散的情况下，血液和脑脊液样本中存在 GD2 合酶 mRNA。在来自低危疾病患儿或健康成年志愿者的样本中未检测到该序列。因此，通过优化的嵌套 RT-PCR 检测 GD2 合酶 mRNA 是评估眼窝切除患者微量播散疾病的有前途的工具。
