Alternative expression of TCRζ related genes in patients with chronic myeloid leukemia

Abstract

A previous study has demonstrated a significant decrease in the TCRζ gene expression level in chronic myeloid leukemia (CML); thus, we further investigated the expression of TCRζ-regulating factors, the distribution of the TCRζ 3' untranslated region (3'-UTR) splice variants, and the expression level and correlation of the alternative splicing factor/splicing factor 2 (ASF/SF-2), FcεRIγ and ZAP-70 genes. TCRζ 3'-UTR splice variants were identified in peripheral blood mononuclear cells (PBMCs) from 14 healthy individuals, 40 patients with CML and 22 patients with CML in complete remission (CML-CR) by RT-PCR. The expression level of the TCRζ, FcεRIγ, ASF/SF-2 and ZAP-70 genes was analyzed by real-time quantitative PCR. While the expression of TCRζ gene in the CML group was significantly lower than that in the healthy individual and CML-CR groups, a significantly higher expression of the FceRIγ and ASF/SF-2 genes was found in the CML group. Two types of splicing forms were detected in all of the healthy individual CML-CR cases: wild type (WT) TCRζ 3'-UTR and alternatively splieced (AS) TCRζ 3'-UTR which have been alternatively splieced in the WT TCRζ 3'-UTR . However, 35% of the CML cases contained only the wild type TCRζ 3'-UTR isoform. Based on the TCRζ 3'-UTR isoform expression characteristic, we divided the patients with CML into two subgroups: the WT+AS- CML group, containing patients that express only the wild type TCRζ 3'-UTR, and the WT+AS+ CML group, which contained patients that expressed two TCRζ 3'-UTR isoforms. A significantly different ASF/SF-2 and FcεRIγ gene expression pattern was found between the WT+AS- and WT+AS+CML groups. We concluded that defective TCRζ expression may be characterized in the WT+AS-and WT+AS+CML subgroups by the different gene expression pattern. The overexpression of ASF/SF2, which alternatively splices the TCRζ 3'-UTR, is thought to participate in feedback regulation. The characteristics of TCRζ 3'-UTR alternative splicing may be a novel immunological marker for the evaluation of the CML immune status.

Figure 1

TCRζ 3'-UTR PCR amplification in healthy individual and CML samples. Two splice forms, wild type TCRζ 3'-UTR (906 bp) and alternatively spliced TCRζ 3'-UTR (344 bp), could be detected in healthy individuals (lanes 1 and 2), patients with CML-CP (lane 3 and 4) and patients with CML-CR (lane 5). Thirty-five percent of the CML cases contained only the wild type TCRζ 3'-UTR isoform (lane 3). Lane 6 is an 100 bp DNA ladder.

Figure 2

Melting curve and agarose gel analysis of ASF/SF-2 gene expression. A: Amplification and Melting curve of the ASF/SF-2 gene by SYBR Green I real-time PCR. B: The ASF/SF-2 PCR products were analysis by agarose gel electrophoresis. M: 100 bp DNA ladder, lanes 1–3: samples from the healthy control, CML-CP and CML-CR samples, respectively.

Figure 3

A: A schematic view of ASF/SF-2 gene bearing the isoform 1 (bottom) and 2 (top). B: Sequence of the ASF/SF-2 transcript isoform 2 PCR product. The box indicates the 200 bp sequence that was cut in transcript isoform 2, and the gray indicates the PCR primer.

Figure 4

The expression level of the ASF/SF-2, TCRζ, ZAP-70and FcεRIγ genes in the CML, CML-CR and healthy control groups (HI). A: ASF/SF-2; B: FcεRIγ; C: TCRζ; and D: ZAP-70.

Figure 5

Differential expression pattern of the ASF/SF-2, TCRζ, ZAP-70 and FcεRIγ genes in the WT⁺AS^- and WT⁺AS⁺CML groups. A: ASF/SF-2; B: FcεRIγ; C: TCRζ; and D: ZAP-70.

Figure 6

Correlation analysis of the TCRζ and ASF/SF-2 genes. A: Healthy controls; B: CML-CR group; C: WT⁺AS⁺ CML group; and D: WT⁺AS^- CML group.

Figure 7

Correlation analysis of the TCRζ and FcεRIγ genes. A: Healthy controls; B: CML-CR group; C: WT⁺AS⁺ CML group; and D: WT⁺AS^- CML group.

Figure 8

Correlation analysis of the TCRζ and ZAP-70 genes. A: Healthy controls; B: CML-CR group; C: WT⁺AS⁺ CML group; and D: WT⁺AS^- CML group.