Constitutively overexpressed 21 kDa protein in Hodgkin lymphoma and aggressive non-Hodgkin lymphomas identified as cytochrome B5b (CYB5B).
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Murphy
Derek
D
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.
Parker
Jeremy
J
Zhou
Minglong
M
Fadlelmola
Faisal M
FM
Steidl
Christian
C
Karsan
Aly
A
Gascoyne
Randy D
RD
Chen
Hong
H
Banerjee
Diponkar
D
D000595:Amino Acid Sequence; D000912:Antibodies, Neoplasm / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D055028:Comparative Genomic Hybridization; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D015972:Gene Expression Regulation, Neoplastic; D006689:Hodgkin Disease / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D008198:Lymph Nodes / Q000378:metabolism; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014066:Palatine Tonsil / Q000378:metabolism; D040081:Protein Array Analysis; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions; D013601:T-Lymphocytes / Q000378:metabolism; D046888:Tissue Array Analysis
D000912:Antibodies, Neoplasm; D015786:Cytochromes b5