Expression and distribution of PPP2R5C gene in leukemia

Samples

Seventy-seven newly diagnosed and untreated cases of leukemia, including 24 with acute myeloid leukemia (AML); 14 with chronic phase chronic myeloid leukemia (CML); 18 with T-cell acute lymphocytic leukemia/lymphoma (T-ALL); 12 with B-cell ALL (B-ALL); and nine with B-cell chronic lymphocytic leukemia (B-CLL). Twenty-six cases with leukemia in complete remission (CR) (nine with AML-CR, three with B-ALL-CR and 14 with CML-CR) were selected, along with 20 healthy individuals as controls. The samples were collected with informed consent. All procedures were conducted in accordance with the guidelines of the medical ethics committees of the Health Bureau of Guangdong Province, China. Human leukemia cell lines Hut-78, CCRF, Jurkat, Molt-3, Molt-4, K562, NB4, Raji and Daudi were used in the study. The RNA extraction and cDNA synthesis were performed according to the manufacturer's instructions.

Real-time quantitative RT-PCR (qRT-PCR)

Expression levels of PPP2R5C and the reference gene β2-MG were determined by SYBR Green I real-time PCR. PCR was performed as our previous description [8]. The 2^(-ΔΔCT) method was used to present the data of the genes of interest relative to an internal control gene [8, 9]. The sequences of primers used in qRT-PCR were PPP2R5C-for: 5'-GTAATAAAGCGGGCAGCAGG-3' and PPP2R5C-bac: 5'-CAAAGT CAAAGAGGACGCAACA-3' for PPP2R5C gene amplification, β₂M-for: 5'-CAGCAAGG ACTGGTCTTTCTAT-3' and β₂M-bac: 5'-GCGGCATCTTCAAACCTC-3' for β₂M gene amplification.

Primer design and RT-PCR

Expression level of PPP2R5C in leukemia

PPP2R5C as a potential tumor suppressor plays a crucial role in cell proliferation and differentiation [4]. Based on our recent finding of a novel gene rearrangement from TRAJ7 to PPP2R5C, it could be interesting to analyze the expression features of PPP2R5C in hematological malignancies. In the present study, we analyzed the expression level of the PPP2R5C gene in leukemia samples. In comparison with healthy controls (1.24 ± 1.09), significantly higher expression of PPP2R5C was found in the AML (2.06 ± 0.85) (p = 0.0076), CML (6.78 ± 2.75) (p < 0.0001), T-ALL/NHL (3.73 ± 3.66) (p = 0.0062) and B-CLL (2.21 ± 1.22) (p = 0.0417) groups (Figure 2). A high tendency toward expression of PPP2R5C was detected in the B-ALL group (1.39 ± 1.31); however, the expression was not significantly different from that in the controls (p = 0.7089) (Figure 2). The expression level of PPP2R5C in the CML-CR group (1.75 ± 0.55) decreased significantly in comparison with the CML group (p < 0.0001), but showed no significant difference compared with the healthy group (p = 0.2895). Although the expression level of PPP2R5C gene decreased in the AML-CR (1.53 ± 0.60) and B-ALL groups (0.54 ± 0.27), there was no significant difference compared with the AML (p = 0.1100) and B-ALL groups (p = 0.2142) or with healthy controls. Overexpression of PPP2R5C was found in T-cell lines like Hut-78, CCRF, Jurkat, Molt-3 and Molt-4, and the expression level was 5-9 times higher than that from healthy CD3⁺ T cells (Figure 3A). Interesting, the tendency of the expression level of PPP2R5C in Raji, Daudi, NB4 and K562 cells was accordant to the results from primary leukemia cells (Figure 3B), which showed higher expression level of PPP2R5C in K562 (CML cell line), and lower expression level in B-cell lines (Raji and Daudi).

Mutation, deletion, or abnormal expression of tumor-suppressor genes is frequently identified in leukemia [1, 11, 12]. To the best of our knowledge, no study has been reported that examined the expression of PPP2R5C in leukemia, except for B-CLL. The present results identifying PPP2R5C overexpression in most cases of leukemia might have a similar significance in cell transformation. Definitive results for the association of the expression level of PPP2R5C with disease progression and outcome could be demonstrated by a comparison of the data for de novo CML and CML in complete remission. In contrast, Fält et al. have described that downregulated PPP2R5C is related to progressive B-CLL by using the Affymetrix GeneChip technique, and they have concluded that PPP2R5C could be a marker of progressive disease in B-CLL. They have compared expression of PPP2R5C between stable and progressive B-CLL, but not with a healthy control group [13]. In the present study, we found that expression of PPP2R5C was significantly increased in the B-CLL group, similar to AML, CML, and T-ALL; however, we were unable to collect samples from patients in complete remission and from those with progressive disease to compare the expression level and evaluate the significance of PPP2R5C detection in B-CLL. Further investigation is needed. Unlike most leukemia cases, the expression level of PPP2R5C in the B-ALL group, as well as in B-cell lines did not differ significantly from that in the controls, but it remains unknown whether this was due to the limited number of samples or whether it reflects a true feature of PPP2R5C in B-ALL.

Distribution of PPP2R5C transcript variants in healthy individuals and leukemia

It has been reported that five transcript variants of PPP2R5C might contribute to the specificity of PP2A [4]. However, little is known about the distribution of different variants in different organs, tissues, and cells, as well as in leukemia cells. Based on the structure of the PPP2R5C gene reported from Genbank and previous studies [4], we drew a schematic diagram of the genomic organization of PPP2R5C with five transcript variants (Figure 1), designed four primer pairs to amplify different exons, and tried to identify different variants in the same sample. By using PPP2R5C-1f/PPP2R5C-1b, which covered exons 12-14, two expected PCR products were detected. The small one comprised 277 bp containing exon 12 and 14 segments (corresponding to B56γ2 or B56γ6), and the large one comprised 394 bp containing exon 12, 13, and 14 segments (corresponding to B56γ3 or B56γ5). PPP2R5C-3f/PPP2R5C-3b, PPP2R5C-5f/PPP2R5C-5b, and PPP2R5C-6f/PPP2R5C-6b primer pairs covered exons 10 to 12a, exon III to 2, and exon IV to 2, respectively, and the expected PCR products were 304, 242, and 213 bp (corresponding to transcript B56γ1, B56γ5, and B56γ6 respectively) (Table 2 Figure 4). According the structure of transcript variants of PPP2R5C gene and the size of the amplicons, we confirmed the B56γ1, B56γ5, and B56γ6 variants. However, using the designed primer pairs, we were unable to distinguish between transcripts 1 and 5 or between transcripts 2 and 6 because PCR products of the same size were amplified by using the PPP2R5C-1f/PPP2R5C-1b, and it could not distinguish these variants using different primer pair combinations, except for whole gene sequencing. B56γ3 (variant 1) and B56γ2 (variant 2) are the frequency variants; therefore, it is thought that they might be expressed when positive PCR products are found. More importantly, using the present methods, we confirmed the expression of B56γ5 and B56γ6, which have been newly identified. Therefore, it could be concluded that all variants can be detected in healthy as well as leukemia samples with a similar frequency and distribution of PPP2R5C.

In conclusion, to the best of our knowledge, this is the first description of the expression level of the PPP2R5C gene as well as the distribution of PPP2R5C transcript variants in PBMCs from different types of leukemia. Overexpression of PPP2R5C is a common feature in most types of leukemia; thus, the change in expression pattern might influence the activity of PP2A and could be related to abnormal cell proliferation, differentiation, and transformation. Further research on the downregulation of PPP2R5C in leukemia cells is needed to investigate its biological function.