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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 enhances xenografted lymphoma development in vivo. (A to D) NOD-SCID (nonobese diabetic/severe combined immunodeficient) mice were injected s.c. with different NHL cells that were stably transfected with pcDNA3.1 (Control), or pcDNA3.1-ISL-1 (ISL-1) construct (A,C), pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid (B,D). The tumor size was monitored at indicated days post-injection. Statistical analysis was carried out with 2-way ANOVA. (E) The mice were killed after the last measurement of tumor volume, whole-cell lysate of 2 tumor samples of each group were prepared and subjected to Western blot analysis for ISL-1 level detection. GAPDH served as an internal control.
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Figure 3: ISL-1 enhances xenografted lymphoma development in vivo. (A to D) NOD-SCID (nonobese diabetic/severe combined immunodeficient) mice were injected s.c. with different NHL cells that were stably transfected with pcDNA3.1 (Control), or pcDNA3.1-ISL-1 (ISL-1) construct (A,C), pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid (B,D). The tumor size was monitored at indicated days post-injection. Statistical analysis was carried out with 2-way ANOVA. (E) The mice were killed after the last measurement of tumor volume, whole-cell lysate of 2 tumor samples of each group were prepared and subjected to Western blot analysis for ISL-1 level detection. GAPDH served as an internal control.

Mentions: To further confirm whether ISL-1 could promote tumor growth in vivo, we used the SCID mice xenograft model to study the impact of ISL-1 on NHL genesis and development. We found that the initiation and the growth of tumor were significantly earlier and faster with ISL-1 overexpressing cells than those with the control cells (Figure 3A,C). Conversely, the tumor growth was obviously impaired with ISL-1 knockdown cells (Figure 3B,D). After the last measurement, the tumors were isolated and weighed. The ISL-1-overexpressing cells produced significantly larger and heavier tumors than the control cells, in contrast, the ISL-1-knockdown cells produced smaller and lighter tumors compared with the control cells (Additional file2: Figure S2). We further compared the expression of ISL-1 in the tumor tissues isolated from the mice. As shown in Figure 3E, the protein level of ISL-1 in the tumors was positively correlated with the tumor volumes in each group. Therefore, our animal experiments confirm that ISL-1 potentiates NHL growth in vivo.


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 enhances xenografted lymphoma development in vivo. (A to D) NOD-SCID (nonobese diabetic/severe combined immunodeficient) mice were injected s.c. with different NHL cells that were stably transfected with pcDNA3.1 (Control), or pcDNA3.1-ISL-1 (ISL-1) construct (A,C), pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid (B,D). The tumor size was monitored at indicated days post-injection. Statistical analysis was carried out with 2-way ANOVA. (E) The mice were killed after the last measurement of tumor volume, whole-cell lysate of 2 tumor samples of each group were prepared and subjected to Western blot analysis for ISL-1 level detection. GAPDH served as an internal control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125377&req=5

Figure 3: ISL-1 enhances xenografted lymphoma development in vivo. (A to D) NOD-SCID (nonobese diabetic/severe combined immunodeficient) mice were injected s.c. with different NHL cells that were stably transfected with pcDNA3.1 (Control), or pcDNA3.1-ISL-1 (ISL-1) construct (A,C), pLL3.7-Non-silencer or pLL3.7-ISL1-siRNA plasmid (B,D). The tumor size was monitored at indicated days post-injection. Statistical analysis was carried out with 2-way ANOVA. (E) The mice were killed after the last measurement of tumor volume, whole-cell lysate of 2 tumor samples of each group were prepared and subjected to Western blot analysis for ISL-1 level detection. GAPDH served as an internal control.
Mentions: To further confirm whether ISL-1 could promote tumor growth in vivo, we used the SCID mice xenograft model to study the impact of ISL-1 on NHL genesis and development. We found that the initiation and the growth of tumor were significantly earlier and faster with ISL-1 overexpressing cells than those with the control cells (Figure 3A,C). Conversely, the tumor growth was obviously impaired with ISL-1 knockdown cells (Figure 3B,D). After the last measurement, the tumors were isolated and weighed. The ISL-1-overexpressing cells produced significantly larger and heavier tumors than the control cells, in contrast, the ISL-1-knockdown cells produced smaller and lighter tumors compared with the control cells (Additional file2: Figure S2). We further compared the expression of ISL-1 in the tumor tissues isolated from the mice. As shown in Figure 3E, the protein level of ISL-1 in the tumors was positively correlated with the tumor volumes in each group. Therefore, our animal experiments confirm that ISL-1 potentiates NHL growth in vivo.

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