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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 is highly expressed in the majority subtypes of NHL. (A) Immunohistochemistry for ISL-1 expression in normal lymph nodes and multiple subtypes of lymphoma specimens was performed. Representative images of ISL-1 expression level and cellular distribution in different samples are shown (200 ×). Scale bars = 100 μm. (B) Staining scores of ISL-1 in normal lymph nodes, HL and NHL were statistically analyzed by χ2 test. (C to D) The mRNA and protein levels of ISL-1 in NHL cell lines and health human peripheral white blood cells (PBC) were analyzed by RT-PCR (C) and Western blot (D) analysis. Numbers 1–7 represent PBC samples from different donors.
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Figure 1: ISL-1 is highly expressed in the majority subtypes of NHL. (A) Immunohistochemistry for ISL-1 expression in normal lymph nodes and multiple subtypes of lymphoma specimens was performed. Representative images of ISL-1 expression level and cellular distribution in different samples are shown (200 ×). Scale bars = 100 μm. (B) Staining scores of ISL-1 in normal lymph nodes, HL and NHL were statistically analyzed by χ2 test. (C to D) The mRNA and protein levels of ISL-1 in NHL cell lines and health human peripheral white blood cells (PBC) were analyzed by RT-PCR (C) and Western blot (D) analysis. Numbers 1–7 represent PBC samples from different donors.

Mentions: To examine the pathological relevance of ISL-1 in human lymphoma development, we analyzed the expression level and cellular distribution of ISL-1 in collected specimens and tissue microarrays by immunohistochemical staining. These tissue specimens included 23 normal lymph nodes and 211 lymphoma samples. The lymphoma specimens could be classified into two types: 195 NHL (159 B-cell lymphoma, 36 T-cell lymphoma) and 16 Hodgkin lymphoma (HL). As summarized in Table 1, ISL-1 expression level is markedly elevated in 75% of 195 NHL samples. Only 3 cases of normal lymph nodes exhibited moderate ISL-1 immunostaining, none of the 23 normal lymph nodes or 16 HL showed any strong positive staining for ISL-1. Figure 1A shows representative immunohistochemistry images of ISL-1 staining in human normal lymph node, HL and NHL. ISL-1 staining was predominantly detected in the nuclear of a series of NHL lymphoma cells and, to a much lesser extent, in the normal lymph nodes and HL samples. Statistical analysis revealed that there was no significant difference in the expression of ISL-1 between normal lymph nodes and HL samples (p = 0.13), whereas, the positive staining of ISL-1 was significantly correlated with NHLs compared with that in normal lymph nodes (p < 0.001) (Figure 1B).Meanwhile, we found a predominant expression of ISL-1 in a variety of NHL cell lines (Figure 1C,D). These data establish that ISL-1 expression is highly elevated in the majority of NHLs and might be tightly linked to lymphomagenesis.


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 is highly expressed in the majority subtypes of NHL. (A) Immunohistochemistry for ISL-1 expression in normal lymph nodes and multiple subtypes of lymphoma specimens was performed. Representative images of ISL-1 expression level and cellular distribution in different samples are shown (200 ×). Scale bars = 100 μm. (B) Staining scores of ISL-1 in normal lymph nodes, HL and NHL were statistically analyzed by χ2 test. (C to D) The mRNA and protein levels of ISL-1 in NHL cell lines and health human peripheral white blood cells (PBC) were analyzed by RT-PCR (C) and Western blot (D) analysis. Numbers 1–7 represent PBC samples from different donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: ISL-1 is highly expressed in the majority subtypes of NHL. (A) Immunohistochemistry for ISL-1 expression in normal lymph nodes and multiple subtypes of lymphoma specimens was performed. Representative images of ISL-1 expression level and cellular distribution in different samples are shown (200 ×). Scale bars = 100 μm. (B) Staining scores of ISL-1 in normal lymph nodes, HL and NHL were statistically analyzed by χ2 test. (C to D) The mRNA and protein levels of ISL-1 in NHL cell lines and health human peripheral white blood cells (PBC) were analyzed by RT-PCR (C) and Western blot (D) analysis. Numbers 1–7 represent PBC samples from different donors.
Mentions: To examine the pathological relevance of ISL-1 in human lymphoma development, we analyzed the expression level and cellular distribution of ISL-1 in collected specimens and tissue microarrays by immunohistochemical staining. These tissue specimens included 23 normal lymph nodes and 211 lymphoma samples. The lymphoma specimens could be classified into two types: 195 NHL (159 B-cell lymphoma, 36 T-cell lymphoma) and 16 Hodgkin lymphoma (HL). As summarized in Table 1, ISL-1 expression level is markedly elevated in 75% of 195 NHL samples. Only 3 cases of normal lymph nodes exhibited moderate ISL-1 immunostaining, none of the 23 normal lymph nodes or 16 HL showed any strong positive staining for ISL-1. Figure 1A shows representative immunohistochemistry images of ISL-1 staining in human normal lymph node, HL and NHL. ISL-1 staining was predominantly detected in the nuclear of a series of NHL lymphoma cells and, to a much lesser extent, in the normal lymph nodes and HL samples. Statistical analysis revealed that there was no significant difference in the expression of ISL-1 between normal lymph nodes and HL samples (p = 0.13), whereas, the positive staining of ISL-1 was significantly correlated with NHLs compared with that in normal lymph nodes (p < 0.001) (Figure 1B).Meanwhile, we found a predominant expression of ISL-1 in a variety of NHL cell lines (Figure 1C,D). These data establish that ISL-1 expression is highly elevated in the majority of NHLs and might be tightly linked to lymphomagenesis.

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