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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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JNK or JAK/STAT signaling inhibitors inhibit NHL cells proliferation through down-regulating ISL-1 expression. (A) The relative proliferation rate of lymphoma cell lines were measured using CCK-8 analysis after treated with JNK signaling pathway inhibitor (SP60012, 10 μM) or JAK/STAT signaling pathway inhibitor (STATTIC, 6 μM). The cell treated with DMSO were used as the control. (B to C) The effect of SP600125 (10 μM) and STATTIC (6 μM) on ISL-1 and c-Myc expression at both mRNA (B) and protein levels (C) were analyzed by real-time RT-PCR and Western blot. The cells treated with DMSO at different time point were used as the corresponding control. (D) The luciferase activity of c-Myc-luc (wide type or M1) was measured in Ly3 cells with or without ISL-1 transcfection after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. (E) The growth inhibition of Ly3 cells with or without ISL-1 transcfection was measured by CCK-8 analysis after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. 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).
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Figure 7: JNK or JAK/STAT signaling inhibitors inhibit NHL cells proliferation through down-regulating ISL-1 expression. (A) The relative proliferation rate of lymphoma cell lines were measured using CCK-8 analysis after treated with JNK signaling pathway inhibitor (SP60012, 10 μM) or JAK/STAT signaling pathway inhibitor (STATTIC, 6 μM). The cell treated with DMSO were used as the control. (B to C) The effect of SP600125 (10 μM) and STATTIC (6 μM) on ISL-1 and c-Myc expression at both mRNA (B) and protein levels (C) were analyzed by real-time RT-PCR and Western blot. The cells treated with DMSO at different time point were used as the corresponding control. (D) The luciferase activity of c-Myc-luc (wide type or M1) was measured in Ly3 cells with or without ISL-1 transcfection after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. (E) The growth inhibition of Ly3 cells with or without ISL-1 transcfection was measured by CCK-8 analysis after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. 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).

Mentions: We have shown that both JNK and JAK/STAT signaling inhibitors can suppress ISL-1 expression. However, it is not clear whether these inhibitors could affect the proliferation of NHL cells by decreasing ISL-1 expression level. CCK-8 analysis was firstly performed to analyze the influence of JNK and JAK/STAT inhibitors on NHL cells proliferation. As shown in Figure 7A, both SP600125 and STATTIC could significantly decrease the proliferation rate of NHL cells.


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)

JNK or JAK/STAT signaling inhibitors inhibit NHL cells proliferation through down-regulating ISL-1 expression. (A) The relative proliferation rate of lymphoma cell lines were measured using CCK-8 analysis after treated with JNK signaling pathway inhibitor (SP60012, 10 μM) or JAK/STAT signaling pathway inhibitor (STATTIC, 6 μM). The cell treated with DMSO were used as the control. (B to C) The effect of SP600125 (10 μM) and STATTIC (6 μM) on ISL-1 and c-Myc expression at both mRNA (B) and protein levels (C) were analyzed by real-time RT-PCR and Western blot. The cells treated with DMSO at different time point were used as the corresponding control. (D) The luciferase activity of c-Myc-luc (wide type or M1) was measured in Ly3 cells with or without ISL-1 transcfection after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. (E) The growth inhibition of Ly3 cells with or without ISL-1 transcfection was measured by CCK-8 analysis after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4125377&req=5

Figure 7: JNK or JAK/STAT signaling inhibitors inhibit NHL cells proliferation through down-regulating ISL-1 expression. (A) The relative proliferation rate of lymphoma cell lines were measured using CCK-8 analysis after treated with JNK signaling pathway inhibitor (SP60012, 10 μM) or JAK/STAT signaling pathway inhibitor (STATTIC, 6 μM). The cell treated with DMSO were used as the control. (B to C) The effect of SP600125 (10 μM) and STATTIC (6 μM) on ISL-1 and c-Myc expression at both mRNA (B) and protein levels (C) were analyzed by real-time RT-PCR and Western blot. The cells treated with DMSO at different time point were used as the corresponding control. (D) The luciferase activity of c-Myc-luc (wide type or M1) was measured in Ly3 cells with or without ISL-1 transcfection after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. (E) The growth inhibition of Ly3 cells with or without ISL-1 transcfection was measured by CCK-8 analysis after treated with 10 μM SP600125 or 6 μM STATTIC for 24 h. 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).
Mentions: We have shown that both JNK and JAK/STAT signaling inhibitors can suppress ISL-1 expression. However, it is not clear whether these inhibitors could affect the proliferation of NHL cells by decreasing ISL-1 expression level. CCK-8 analysis was firstly performed to analyze the influence of JNK and JAK/STAT inhibitors on NHL cells proliferation. As shown in Figure 7A, both SP600125 and STATTIC could significantly decrease the proliferation rate of NHL cells.

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