Centre for Translational and Applied Genomics, British Columbia Cancer Agency, Provincial Health Services Authority Laboratories, 600 West 10th Avenue, Vancouver, British Columbia, V5Z 4E6, Canada.
Mol Cancer. 2010 Jan 26;9:14. doi: 10.1186/1476-4598-9-14.
We have previously reported a novel constitutively overexpressed 21 kDa protein in Hodgkin Lymphoma (HL) and aggressive Non-Hodgkin Lymphomas (NHL). The objective of the current study was to 1) identify this protein using two independent methods, 2) study the expression of the protein and its encoding mRNA in reactive lymph nodes, normal lymphocytes and CD34+ bone marrow precursor cells, 3) analyse patterns of expression of the protein in tissue microarrays assembled from a large number of diagnostic clinical biopsies from patients with HL, and 4) determine the copy number variation and mutation status of the encoding gene in HL cell lines.
Peptide sequencing by LC-MS/MS and protein identification by protein array screening identified a single protein, CYB5B. No mutations were detected in the CYB5B gene in HL cell lines. Quantitative PCR showed CYB5B gene expression was increased in HL and NHL cell lines. Array CGH using a submegabase resolution tiling array revealed gains in the CYB5B locus in HL cell lines KMH2 and L428. Membrane expression was seen in Reed-Sternberg cells in clinical biopsies from patients with HL but not in reactive lymph nodes. Bone marrow CD34+ precursor cells were CYB5B negative on the cell surface. RT-PCR assays of RNA extracted from T and B cell enriched fractions obtained from normal peripheral blood mononuclear cells, reactive lymph nodes, tonsils and normal bone marrow samples showed no evidence of increased mRNA levels of CYB5B in comparison to housekeeping gene GAPDH.
The 21 kDa protein overexpressed in HL and aggressive NHL is identical to CYB5B. CYB5B gene expression is increased in a subset of HL and NHL cell lines tested. This is associated with CYB5B gene amplification in HL cell lines KMH2 and L428. CYB5B may be a potential target for antibody-based therapy of HL and aggressive NHL as although cytoplasmic expression is present in reactive lymphocytes, it is not expressed on the cell surface of non-neoplastic lymphocytes or bone marrow precursor cells.
我们之前报道过霍奇金淋巴瘤（HL）和侵袭性非霍奇金淋巴瘤（NHL）中一种新型组成性过表达的 21kDa 蛋白。本研究的目的是：1）使用两种独立的方法鉴定这种蛋白；2）研究该蛋白及其编码 mRNA 在反应性淋巴结、正常淋巴细胞和 CD34+骨髓前体细胞中的表达情况；3）分析该蛋白在大量来自 HL 患者的诊断性临床活检组织微阵列中的表达模式；4）确定 HL 细胞系中编码基因的拷贝数变异和突变状态。
通过 LC-MS/MS 进行肽测序和通过蛋白质阵列筛选进行蛋白质鉴定，鉴定出一种单一的蛋白质，即 CYB5B。在 HL 细胞系中未检测到 CYB5B 基因突变。定量 PCR 显示 CYB5B 基因在 HL 和 NHL 细胞系中表达增加。使用亚兆碱基分辨率平铺阵列的 array CGH 显示 HL 细胞系 KMH2 和 L428 中 CYB5B 基因座的增益。在来自 HL 患者的临床活检中观察到 Reed-Sternberg 细胞的膜表达，但在反应性淋巴结中未见。骨髓 CD34+前体细胞在细胞表面呈 CYB5B 阴性。从正常外周血单核细胞、反应性淋巴结、扁桃体和正常骨髓样本中获得的 T 和 B 细胞富集部分提取的 RNA 的 RT-PCR 检测显示，与管家基因 GAPDH 相比，CYB5B 的 mRNA 水平没有增加的证据。
在 HL 和侵袭性 NHL 中过表达的 21kDa 蛋白与 CYB5B 相同。在测试的 HL 和 NHL 细胞系亚组中，CYB5B 基因表达增加。这与 HL 细胞系 KMH2 和 L428 中的 CYB5B 基因扩增有关。CYB5B 可能成为 HL 和侵袭性 NHL 抗体治疗的潜在靶点，因为尽管在反应性淋巴细胞中存在细胞质表达，但它不在非肿瘤性淋巴细胞或骨髓前体细胞的细胞表面表达。
