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Nuclear factor kappaB-dependent gene expression profiling of Hodgkin's disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity.

Hinz M, Lemke P, Anagnostopoulos I, Hacker C, Krappmann D, Mathas S, Dörken B, Zenke M, Stein H, Scheidereit C - J. Exp. Med. (2002)

Bottom Line: The NF-kappaB-dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells.Intriguingly, NF-kappaB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation.The gene profile underscores a central role of NF-kappaB in the pathogenesis of HD and potentially of other tumors with constitutive NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.

ABSTRACT
Constitutive nuclear nuclear factor (NF)-kappaB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-kappaB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkin's disease (HD) cell lines to identify NF-kappaB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-kappaB. The NF-kappaB-dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-kappaB target genes. Using chromatin immunoprecipitation we demonstrate that NF-kappaB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-kappaB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-kappaB in the pathogenesis of HD and potentially of other tumors with constitutive NF-kappaB activation.

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Verification of novel NF-κB target genes. (A) NF-κB binding activity. Whole cell extracts of HRS cells and control cells were analyzed by EMSA for NF-κB DNA binding activity. (B) RNA was extracted 24 h after infection from L428 and HDLM2 cells infected with Ad5 control (L428c24, HDLM2c24) or Ad5-IκBΔN (L428i24, HDLM2i24), as well as from various control (Reh, Namalwa) and HRS cell lines as indicated. Northern blotting was performed for the indicated genes. As a control, the stripped blot was reprobed with a GAPDH cDNA probe. (C) Total RNA was extracted as described in B and RT-PCR reactions were performed for the indicated genes. As internal control, RT-PCRs were performed for β-actin.
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fig4: Verification of novel NF-κB target genes. (A) NF-κB binding activity. Whole cell extracts of HRS cells and control cells were analyzed by EMSA for NF-κB DNA binding activity. (B) RNA was extracted 24 h after infection from L428 and HDLM2 cells infected with Ad5 control (L428c24, HDLM2c24) or Ad5-IκBΔN (L428i24, HDLM2i24), as well as from various control (Reh, Namalwa) and HRS cell lines as indicated. Northern blotting was performed for the indicated genes. As a control, the stripped blot was reprobed with a GAPDH cDNA probe. (C) Total RNA was extracted as described in B and RT-PCR reactions were performed for the indicated genes. As internal control, RT-PCRs were performed for β-actin.

Mentions: All novel candidates identified for both L428 and HDLM2 cells and a group of known NF-κB target genes were selected for Northern analysis to validate NF-κB–dependent expression in HRS cells. Because the microchip data were obtained from two independent experiments and two different cell lines, the rate of false positives should be minimal. For some candidates, semi-quantitative RT-PCR was performed. We analyzed mRNA expression in Ad5-IκBΔN– or Ad5 control–infected L428 and HDLM2 cells, and in uninfected HRS and control cell lines. For all samples we confirmed that NF-κB DNA binding activity was constitutive only in HRS cells and was repressed by IκBΔN (Fig. 4 A). Pronounced NF-κB–dependent regulation could be verified for the vast majority of genes (Fig. 4, B and C). Moreover, the observed change of mRNA expression levels upon NF-κB inhibition correlated well with the DNA microarray data (Fig. 2). CX3CL, MIP1-α, CCR7, IL-15Rα, CD83, IEX-1, SMAD7, interferon regulatory factor 1, and NF-κB p100, previously proposed to be regulated by NF-κB in other cell types (29, 35–39), could be confirmed as cellular target genes in HRS cells. Abundant CCR7 and CD83 expression was detected exclusively in HRS cells (Fig. 3 B), supporting recent observations (38, 40). Likewise, the mRNA patterns of CX3CL, IEX-1, IL-15Rα, and p100 indicate strongly elevated expression in HRS cells compared with non-HRS cells (Fig. 4 B).


Nuclear factor kappaB-dependent gene expression profiling of Hodgkin's disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity.

Hinz M, Lemke P, Anagnostopoulos I, Hacker C, Krappmann D, Mathas S, Dörken B, Zenke M, Stein H, Scheidereit C - J. Exp. Med. (2002)

Verification of novel NF-κB target genes. (A) NF-κB binding activity. Whole cell extracts of HRS cells and control cells were analyzed by EMSA for NF-κB DNA binding activity. (B) RNA was extracted 24 h after infection from L428 and HDLM2 cells infected with Ad5 control (L428c24, HDLM2c24) or Ad5-IκBΔN (L428i24, HDLM2i24), as well as from various control (Reh, Namalwa) and HRS cell lines as indicated. Northern blotting was performed for the indicated genes. As a control, the stripped blot was reprobed with a GAPDH cDNA probe. (C) Total RNA was extracted as described in B and RT-PCR reactions were performed for the indicated genes. As internal control, RT-PCRs were performed for β-actin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194004&req=5

fig4: Verification of novel NF-κB target genes. (A) NF-κB binding activity. Whole cell extracts of HRS cells and control cells were analyzed by EMSA for NF-κB DNA binding activity. (B) RNA was extracted 24 h after infection from L428 and HDLM2 cells infected with Ad5 control (L428c24, HDLM2c24) or Ad5-IκBΔN (L428i24, HDLM2i24), as well as from various control (Reh, Namalwa) and HRS cell lines as indicated. Northern blotting was performed for the indicated genes. As a control, the stripped blot was reprobed with a GAPDH cDNA probe. (C) Total RNA was extracted as described in B and RT-PCR reactions were performed for the indicated genes. As internal control, RT-PCRs were performed for β-actin.
Mentions: All novel candidates identified for both L428 and HDLM2 cells and a group of known NF-κB target genes were selected for Northern analysis to validate NF-κB–dependent expression in HRS cells. Because the microchip data were obtained from two independent experiments and two different cell lines, the rate of false positives should be minimal. For some candidates, semi-quantitative RT-PCR was performed. We analyzed mRNA expression in Ad5-IκBΔN– or Ad5 control–infected L428 and HDLM2 cells, and in uninfected HRS and control cell lines. For all samples we confirmed that NF-κB DNA binding activity was constitutive only in HRS cells and was repressed by IκBΔN (Fig. 4 A). Pronounced NF-κB–dependent regulation could be verified for the vast majority of genes (Fig. 4, B and C). Moreover, the observed change of mRNA expression levels upon NF-κB inhibition correlated well with the DNA microarray data (Fig. 2). CX3CL, MIP1-α, CCR7, IL-15Rα, CD83, IEX-1, SMAD7, interferon regulatory factor 1, and NF-κB p100, previously proposed to be regulated by NF-κB in other cell types (29, 35–39), could be confirmed as cellular target genes in HRS cells. Abundant CCR7 and CD83 expression was detected exclusively in HRS cells (Fig. 3 B), supporting recent observations (38, 40). Likewise, the mRNA patterns of CX3CL, IEX-1, IL-15Rα, and p100 indicate strongly elevated expression in HRS cells compared with non-HRS cells (Fig. 4 B).

Bottom Line: The NF-kappaB-dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells.Intriguingly, NF-kappaB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation.The gene profile underscores a central role of NF-kappaB in the pathogenesis of HD and potentially of other tumors with constitutive NF-kappaB activation.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.

ABSTRACT
Constitutive nuclear nuclear factor (NF)-kappaB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-kappaB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkin's disease (HD) cell lines to identify NF-kappaB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-kappaB. The NF-kappaB-dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-kappaB target genes. Using chromatin immunoprecipitation we demonstrate that NF-kappaB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-kappaB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-kappaB in the pathogenesis of HD and potentially of other tumors with constitutive NF-kappaB activation.

Show MeSH
Related in: MedlinePlus