Two newly characterized germinal center B-cell-associated genes, GCET1 and GCET2, have differential expression in normal and neoplastic B cells

Abstract

A group of genes are highly expressed in normal germinal center (GC) B cells and GC B-cell-derived malignancies based on cDNA microarray analysis. Two new genes, GCET1 (germinal center B-cell expressed transcript 1) and GCET2, were cloned from selected expressed sequence tags (IMAGE clone 1334260 and 814622, respectively). GCET1 is located on chromosome 14q32 and has four splicing isoforms, of which the longest one is 1787 bp and encodes a 435-amino acid protein. GCET2 is located on 3q13.13, and the cloned fragment is 3270 bp, which encodes a protein of 178 amino acids. Blast search showed that GCET1 has a highly conserved serine proteinase inhibitor (SERPIN) domain and is located on a chromosomal locus containing seven other SERPIN family members. GCET2 is a likely homologue of the mouse gene M17, a GC-expressed transcript. Analysis of the GCET2 protein sequence indicated that it may be involved in signal transduction in the cytoplasm. Northern blot and real-time polymerase chain reaction analyses confirmed that GCET1 is highly restricted to normal GC B cells and GCB-cell-derived cell lines. Although GCET2 is also a useful marker for normal and neoplastic GC B cells, it has a wider range of expression including immature B and T cells. Real-time polymerase chain reaction assay showed that both GCET1 and GCET2 are preferentially expressed in follicular lymphoma and diffuse large B-cell lymphoma with GC B-cell differentiation, confirming previous microarray gene expression analysis, but neither one is entirely specific. Multiple markers are necessary to differentiate the GCB from the activated B-cell type of diffuse large B-cell lymphoma with a high degree of accuracy.

Figure 1.

Microarray gene expression profiles of GCET1 and GCET2. A: Both GCET1 and GCET2 are highly expressed in normal GC B cells (a) and FL (f); down-regulated in activated and resting peripheral B cells (b, g), resting and activated T cells (c, e), and CLL (h). B: They have high expression levels in GCB-DLBCL but low expression in the ABC group.

Figure 2.

A: 5′ RACE for GCET1 reveals two major bands on agarose gel electrophoresis. B: Distribution of GCET1-splicing isoforms and other known SERPIN family members on chromosome 14q32. C: Northern blot of GCET1 shows a transcript around 1.8 kb in DHL16. D: Alignment of GCET1-splicing isoforms. GCET1A is 1787 bp long encoding a protein of 435 amino acids. GCET1B is 300 bp shorter at the 5′ end encoding a protein of 335 amino acids. GCET1C is 190 bp shorter at the 5′ end encoding a protein of 337 amino acids. GCET1D has 393 bp missing at the 5′ end compared with GCET1A and encodes a protein of 286 amino acids, but it has an extra exon as shown. E: Nucleotide and protein sequence data of GCET1A. Bolded sequence is the conserved SERPIN motif and the underlined nucleotides represent the possible element for polyadenylation.

Figure 3.

A: Locations of GCET2 and adjacent genes on chromosome 3q13.13. B: Northern blot hybridization of GCET2. C: Nucleotide and protein sequence data of GCET2. Bolded nucleotides are the possible elements for polyadenylation.

Figure 4.

A: Sequence alignment between human GCET2 and AAH24174. The homology is 34% (46 of 135) (does not include positively charged amino acid residues). B: Homology between GCET2 and mouse M17 is 57% (89 of 157) (does not include positively charged amino acid residues). Bolded amino acid sequences are the possible conserved tyrosine phosphorylation sites.

Figure 5.

A: Top, Standard amplification plot for reference gene, β actin (ACTB); bottom, ACTB standard curve. The serial dilutions of the standard are expressed in nature log values in the y axis. B: Top, Standard amplification plot for GCET1; bottom, GCET1 standard curve. C: Top, Standard amplification plot for GCET2; bottom, GCET2 standard curve. Standard amplification plots and curves for B2M and GAPDH were similar (not shown).

Figure 6.

Expression profiling of GCET1 and GCET2. A: GCET1 is highly expressed in microdissected normal GCs, significantly lower in MZB, and very low in MB. In normal tonsil and spleen, it has a moderate expression level. B: GCET1 has a high expression in FL and GCB-DLBCL samples, and its expression is significantly lower in CLL and ABC-DLBCL samples. C: Compared with MB and MZB, normal GCs also have a high expression of GCET2. Normal tonsil and spleen express a moderate level of GCET2. GCET2 also has moderately high expression in thymus and Nalm6. D: In FL, CLL, and DLBCL samples, the expression of GCET2 is very similar to that of GCET1 except for one CLL sample. All of the real-time PCR data are expressed as a ratio to DHL16, which is arbitrarily set at 1.

Figure 7.

Comparison of real-time PCR with microarray data for GCET1 and GCET2. All of the real-time PCR data were multiplied by 16 and converted into log2 values, with the value of DHL16 standard set at 4 to obtain a better spread of the colors. These values were converted into pseudocolors with green representing low expression and red representing high expression according to the predefined scheme shown. R, Real-time PCR data; M, microarray data.