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ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

Zhang Q, Yang Z, Jia Z, Liu C, Guo C, Lu H, Chen P, Ma K, Wang W, Zhou C - Mol. Cancer (2014)

Bottom Line: Recently, ISL-1 has been found in some types of human cancers.Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples.Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, 38 Xueyuan Road, 100191 Beijing, China. wwp@bjmu.edu.cn.

ABSTRACT

Background: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear.

Methods and results: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL.

Conclusions: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

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ISL-1 promotes NHL cells proliferation and affects cell-cycle phase distributions. (A) The relative proliferation rate of stably transfected cells was determined by CCK-8 assay at indicated time post-seeding. (B) Cell cycle distributions were analyzed by flow cytometry. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01 vs. each control). (Control, ISL-1, Non-silencer and ISL-1 siRNA represent the cells transfected with pcDNA3.1, pcDNA3.1-ISL-1, pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid, respectively)
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Figure 2: ISL-1 promotes NHL cells proliferation and affects cell-cycle phase distributions. (A) The relative proliferation rate of stably transfected cells was determined by CCK-8 assay at indicated time post-seeding. (B) Cell cycle distributions were analyzed by flow cytometry. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01 vs. each control). (Control, ISL-1, Non-silencer and ISL-1 siRNA represent the cells transfected with pcDNA3.1, pcDNA3.1-ISL-1, pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid, respectively)

Mentions: We have previously shown that ISL-1 promoted the proliferation of adult pancreatic islets cells[6]. We wonder whether up-regulated ISL-1 in NHL plays a role in promoting NHL cells proliferation and tumorigenesis. Therefore, Raji, Jurkat and Ly3 were electroporated with pcDNA3.1-ISL1, or pLL3.7-ISL1-siRNA plasmid to establish stable ISL-1 overexpressing or knockdown NHL cell lines. ISL-1 expression level in stably transfected NHL cell lines was measured by Western blot analysis and the degree of ISL-1 expression changes was analyzed by gray scanning using Bio-Rad Quantity One software on the images from 3 independent experiments. The results showed that the expression level of ISL-1 was ameliorated approximately 7 folds in ISL-1 overexpressed Raji cells (Additional file1: Figure S1A) and around 2.7 folds in ISL-1 overexpressed Ly3 and Jurkat cells (Additional file1: Figure S1B,C), while the level of ISL-1 was attenuated to less than 10% in ISL-1 knockdown Ly3 and Jurkat cells (Additional file1: Figure S1B,C), indicating that both overexpression and knockdown cell lines are successfully established. When ISL-1 protein level was up or down-regulated, notable promotion or inhibition of cell growth were observed in corresponding cell lines (Figure 2A). To further determine the role of ISL-1 on proliferation of NHL cells, the cell cycle profiles were analyzed. Compared with the control, Raji, Ly3 and Jurkat cells with ISL-1-overexpression showed a decreased cell population in G1 phase and a remarkably increased cell population in the S and G2/M phases. Conversely, Ly3 and Jurkat cells with ISL-1 knockdown exhibited an increase in the proportion of cells in G1 phase and a decrease in the proportion of cells in S and G2/M phases (Figure 2B). These results indicate that ISL-1 could significantly change the cell cycle dynamics and thus promote NHL cells proliferation.


ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

Zhang Q, Yang Z, Jia Z, Liu C, Guo C, Lu H, Chen P, Ma K, Wang W, Zhou C - Mol. Cancer (2014)

ISL-1 promotes NHL cells proliferation and affects cell-cycle phase distributions. (A) The relative proliferation rate of stably transfected cells was determined by CCK-8 assay at indicated time post-seeding. (B) Cell cycle distributions were analyzed by flow cytometry. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01 vs. each control). (Control, ISL-1, Non-silencer and ISL-1 siRNA represent the cells transfected with pcDNA3.1, pcDNA3.1-ISL-1, pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid, respectively)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: ISL-1 promotes NHL cells proliferation and affects cell-cycle phase distributions. (A) The relative proliferation rate of stably transfected cells was determined by CCK-8 assay at indicated time post-seeding. (B) Cell cycle distributions were analyzed by flow cytometry. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01 vs. each control). (Control, ISL-1, Non-silencer and ISL-1 siRNA represent the cells transfected with pcDNA3.1, pcDNA3.1-ISL-1, pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid, respectively)
Mentions: We have previously shown that ISL-1 promoted the proliferation of adult pancreatic islets cells[6]. We wonder whether up-regulated ISL-1 in NHL plays a role in promoting NHL cells proliferation and tumorigenesis. Therefore, Raji, Jurkat and Ly3 were electroporated with pcDNA3.1-ISL1, or pLL3.7-ISL1-siRNA plasmid to establish stable ISL-1 overexpressing or knockdown NHL cell lines. ISL-1 expression level in stably transfected NHL cell lines was measured by Western blot analysis and the degree of ISL-1 expression changes was analyzed by gray scanning using Bio-Rad Quantity One software on the images from 3 independent experiments. The results showed that the expression level of ISL-1 was ameliorated approximately 7 folds in ISL-1 overexpressed Raji cells (Additional file1: Figure S1A) and around 2.7 folds in ISL-1 overexpressed Ly3 and Jurkat cells (Additional file1: Figure S1B,C), while the level of ISL-1 was attenuated to less than 10% in ISL-1 knockdown Ly3 and Jurkat cells (Additional file1: Figure S1B,C), indicating that both overexpression and knockdown cell lines are successfully established. When ISL-1 protein level was up or down-regulated, notable promotion or inhibition of cell growth were observed in corresponding cell lines (Figure 2A). To further determine the role of ISL-1 on proliferation of NHL cells, the cell cycle profiles were analyzed. Compared with the control, Raji, Ly3 and Jurkat cells with ISL-1-overexpression showed a decreased cell population in G1 phase and a remarkably increased cell population in the S and G2/M phases. Conversely, Ly3 and Jurkat cells with ISL-1 knockdown exhibited an increase in the proportion of cells in G1 phase and a decrease in the proportion of cells in S and G2/M phases (Figure 2B). These results indicate that ISL-1 could significantly change the cell cycle dynamics and thus promote NHL cells proliferation.

Bottom Line: Recently, ISL-1 has been found in some types of human cancers.Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples.Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, 38 Xueyuan Road, 100191 Beijing, China. wwp@bjmu.edu.cn.

ABSTRACT

Background: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear.

Methods and results: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL.

Conclusions: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

Show MeSH
Related in: MedlinePlus