Effects Of Sam68 On Proliferation And Apoptosis In T-ALL Cells

BackgroundAcute lymphoblastic leukemia(ALL) is a heterogenous hematologic disease characterized by the proliferation of immature lymphoid cells in the bone marrow, peripheral blood, and other organs. Immunophenotypic classification of ALL involves the use of flow cytometry to determine the presence of cell surface antigens on lymphocytes. ALL can be classified broadly into 3 distinct groups based on immunophenotyping, which include precursor-B-cell ALL, mature B-cell ALL, and T-cell ALL(T-ALL). T cell acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors resulting from the malignant transformation of T cell progenitors. The most specific marker is CD3. Cytoplasmic CD3 is expressed earlier in T-cell differentiation and therefore is more helpful, but surface CD3 is more specific. T-ALL tumor cells are arrested at different stages of cell differentiation. In an attempt to place T-ALL in the context of normal T-cell development, T-ALLs are classified into four groups:pro-T (CD7+), pre-T (CD2+ and/or CD5+and/or CD8+), cortical T (CD1a+), and mature T (surface CD3+, CD1a-). Most T-ALL patients have a poorer prognosis than those in B-ALL. The cure rates and survival outcomes for patients with ALL have improved dramatically over the past several decades. With current treatment regimens, the cure rate among chidren with T-ALL is approximately 75%, while 50% in adult T-ALL. Improvements are largely owed to advances in the understanding of the molecular genetics and pathogenesis of the disease, incorporation of risk-adapted therapy, and the advent of new targeted agents. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains poor. Study the molecular mechanisms of T-ALL can benefit the clinical treatment. T-ALL development is a multi-step process in which different genetic alterations cooperate to alter the normal mechanisms that control cell growth, proliferation, survival, and differentiation during thymocyte development. A variety of oncogenes and functionally inactivating mutations occurs in the development. The diversity of genetic lesions involved in the pathogenesis of T-ALL is further complicated by a number of recurrent cytogenetic and molecular alterations that are common between all molecular subtypes and cause deregulation in specific cellular processes, including cell cycle signaling, cell growth and proliferation, chromatin remodeling, T cell differentiation, and self-renewal.Sam68 (Src-associated in mitosis 68 kDa) is one of the most studied members of the STAR family, containing KH domains and SH3 domains. Sam68 is both an RNA-binding protein and adapter protein. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation. Recent evidence has linked aberrant expression of Sam68 to the onset and progression of endocrine tumors, such as prostate, cervical and breast carcinomas. Notably, all the biochemical activities reported for Sam68 seem to be implicated in carcinogenesis. Sam68 expression is higher in a variety of tumors and have a close relationship with tumor cells growth, invasion and metastasis, apoptosis. What’s more, the abnormal expression was related to adverse clinical prognosis in breast cancer, renal cancer and other solid tumors. Sam68 is closely related to T cells, involving the progresses of T cell activation, TCR signaling pathway and the oncogenic transformation of MLL. However, whether Sam68 participates in the pathological progress of T-ALL is not reported yet.ObjectiveThe present study is aimed to detect the expression level of Sam68 in T-ALL patients, normal donors and T-ALL cell lines. Then investigate the biological roles and molecular mechanism of Sam68 in the proliferation and apoptosis of Jurkat and CCRF-CEM cell lines.Methods1. We collected bone marrow samples from 32 patients diagnosed T-ALL in Tianjin Hematonosis Hospital from June 2014 to May 2015.9 healthy honors were collected. Bone marrow mononuclear cells were prepared by Ficoll-Hypaque density gradient centrifugation.The expression level of Sam68 in primary bone marrow cells was assayed by Real time quantitative PCR.2. The expression of Sam68 in T-ALL cell lines Jurkat and CCRF-CEM was detected by Real time quantitative PCR and Western Blot.3. ShRNA expressing plasmids specifically targeting Sam68 were constructed using pLKO-Tet-On vector. These plasmids were:then transfected into Jurkat and CCRF-CEM cells and stable clones were selected by puromycin. Upon the addition of 2μg/ml doxycycline to the growth media, shRNA expression is triggered resulting in Sam68 knock-down. The efficiency of Sam68 knockdown and then recovery was detected by Real time quantitative PCR and Western Blot.4. MTT assay and cell colony formation assay were used to detected cell viability and monoclonal formation ability. Hoechst staining and Annexin-V-FITC/PI Apoptosis Analysis were used to dectect apoptosis. The western blotting were used to determine the change of expression and activities of cell cycle proteins, apoptotic proteins and AKT/mROR signaling pathways.Results1. The mean expression level of Sam68 in T-ALL patients was significantly higher than that in normal control.2. The expression level of Sam68 in T-ALL cell lines Jurkat and CCRF-CEM were significantly higher than that in normal control.3. Interference plasmid were structured. The expression of Sam68 in Jurkat and CCRF-CEM cells were remarkably reduced with doxycycline and restored with the removal of doxycycline.4. MTT and colony formation assays showed that cell proliferation and colony formation efficiencies of Jurkat and CCRF-CEM cells were both significantly suppressed with Sam68 downregulation and in the wake of Sam68 expression restoration, the proliferation of Jurkat and CCRF-CEM cells almost repaired. Cell cycle distribution showed that Sam68 knockdown cells underwent S arrest and after 1 week rescue from doxycycline, S arrest in both cells were obviously recovered. The apoptotic population in Sam68 silencing cells were significantly higher than the control groups and restored according to the recovery of Sam68 expression. Our results showed the upregulation of p21, Bad, cleaved caspase-9, caspase-3, PARP and downregulation of CDK2, Bad and Bcl-xl in Sam68 knockdown Jurkat and CCRF-CEM cells and the changes in the above proteins were reversed with Sam68 recovery. What’s more, AKT/mTOR signaling pathway activity was downregulated during Sam68 decreasing and restored with Sam68 restoration.ConclusionsIn conclusion, we showed that Sam68 was overexpressed in T-ALL primary cell and cell lines. Interfering the expression of Sam68 affected the cell proliferation and apoptosis at least partially through AKT/mTOR signaling. Our data may provide a new insight of understanding the molecular events in T-ALL.