Expression and distribution of PPP2R5C gene in leukemia
- Haitao Zheng†1,
- Yu Chen†1, 2,
- Shaohua Chen1,
- Yuzhe Niu1, 2,
- Lijian Yang1,
- Bo Li1,
- Yuhong Lu1,
- Suxia Geng3,
- Xin Du3 and
- Yangqiu Li1, 2Email author
© Zheng et al; licensee BioMed Central Ltd. 2011
Received: 22 March 2011
Accepted: 6 May 2011
Published: 6 May 2011
Recently, we clarified at the molecular level novel chromosomal translocation t(14;14)(q11;q32) in a case of Sézary syndrome, which caused a rearrangement from TRAJ7 to the PPP2R5C gene. PPP2R5C is one of the regulatory B subunits of protein phosphatase 2A (PP2A). It plays a crucial role in cell proliferation, differentiation, and transformation. To characterize the expression and distribution of five different transcript variants of the PPP2R5C gene in leukemia, we analyzed the expression level of PPP2R5C in peripheral blood mononuclear cells from 77 patients with de novo leukemia, 26 patients with leukemia in complete remission (CR), and 20 healthy individuals by real-time PCR and identified the different variants of PPP2R5C by RT-PCR.
Significantly higher expression of PPP2R5C was found in AML, CML, T-ALL, and B-CLL groups in comparison with healthy controls. High expression of PPP2R5C was detected in the B-ALL group; however, no significant difference was found compared with the healthy group. The expression level of PPP2R5C in the CML-CR group decreased significantly compared with that in the de novo CML group and was not significantly different from the level in the healthy group. By using different primer pairs that covered different exons, five transcript variants of PPP2R5C could be identified. All variants could be detected in healthy samples as well as in all the leukemia samples, and similar frequencies and distributions of PPP2R5C were indicated.
Overexpression of PPP2R5C in T-cell malignancy as well as in myeloid leukemia cells might relate to its proliferation and differentiation. Investigation of the effect of target inhibition of this gene might be beneficial to further characterization of molecular mechanisms and targeted therapy in leukemia.
Molecular genetic aberrations could provide the basis for assays that can predict prognosis of individual patients as well as potential molecular targets for novel therapies [1–3]. The process of malignant transformation in leukemia is complex, and many factors such as abnormal gene expression and mutation, chromosomal aberrations, deregulation of various cellular signaling pathways, and deregulation of epigenetic regulation are involved in the development of leukemia . Therefore, new data regarding molecular genetic aberrations in different types of leukemia are needed for further characterization.
Based on our recent finding of a novel chromosomal translocation t(14;14)(q11;q32) that was involved in a gene rearrangement from TRAJ7 to PPP2R5C in a patient with Sézary syndrome (data unpublished), in the present study, we further characterized the expression and distribution of five different transcript variants of the PPP2R5C gene in leukemia.
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 . 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
Information on primers used in RT-PCR for PPP2R5C segments amplification
5'-TGAAAGAACGGGAAGAAGCAT - 3'
1407 bp (12 exon)
5'-TGATTGGTATGGCACAGGAAG - 3'
1801 bp (14 exon)
5'-CAGTGACAACGCAGCGAAGAT - 3'
1216 bp (10 exon)
1520 bp (12a exon)
230 bp (III exon)
472 bp (2 exon)
5'-AGCCTTGTTGCTGTCCCGTCT - 3'
210 bp (IV exon)
5'-GTCAAAGAGGACGCAACACTG - 3'
423 bp (2 exon)
Amplified PCR products using different primer pairs
12 + 13 + 14 exons
12 + 14 exons (277 bp)
12 + 13 + 14 exons
12 + 14 exons (277 bp)
10 + 11 + 12 + 12a exons
III + 2 exons
IV + 2 exons
Results and Discussion
Expression level of PPP2R5C in leukemia
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 . 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
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.
This work was supported by Grants from National Natural Science Foundation of China (no. 30871091), the Fundamental Research Funds for the Central Universities (No. 21610604) and Science and Technology Innovation Key Project of Guangdong Higher Education Institutes (kjcxzd1013).
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