A unique feature of Hodgkin disease (HD) is the small number of malignant Hodgkin and Reed-Sternberg (HRS) cells in diseased tissue. Growth of HD-involved lymph nodes is mostly the result of an infiltration of benign T cells. Because of their scarcity, HRS cells are not easily isolated from the vast number of surrounding T cells, and this explains why functional data from single HRS cells are difficult to obtain.
HRS cells, which originate from germinal center B cells, frequently contain crippling somatic mutations within rearranged immunoglobulin heavy chain genes.1Because such crippling mutations trigger apoptosis in germinal center B cells, their detection in HRS tumor cells has supported the view that the lack of surface immunoglobulin–mediated protection from apoptosis must be compensated through expression of surrogate survival factors. Since receptor activator of nuclear factor κB (RANK) promotes survival of dendritic cells, detection of RANK in HRS cells could help explain their ability to escape apoptosis.
In a recent report, Fiumara et al examined the expression of RANK in 4 HD-derived cell lines,2 assuming that these cell lines retained the molecular signature of the founder HRS tumor cells from which the lines originated. RANK expression was clearly demonstrated in 2 HD cell lines (HDLM-2 and L-428 cells). The other 2 HD cell lines (HD-MYZ and KM-H2) did not express significant amounts of RANK. Among all HD cell lines tested, RANK ligand (RANKL)–mediated activation of nuclear factor κB (NFκB) was most pronounced in HD-MYZ cells, and addition of exogenous RANKL to these cells strongly induced interleukin-8 mRNA. This result is not easily explained because HD-MYZ cells fail to synthesize detectable amounts of RANK, the receptor that transduces RANKL-mediated signals. Equally puzzling is a lack of NFκB activation in RANKL-stimulated L428 cells, which, contrary to HD-MYZ cells, express high levels of RANK. Finally, the high-level expression of RANKL in all 4 HD cell lines remains largely unexplained.
Fiumara et al further examined whether exogenous RANKL would affect the proliferation of HD-MYZ, HDLM-2, L428, and KM-H2 cells, thereby testing a possible role for RANK in regulating cell growth of HD tumor lines. The authors found that proliferation rates remained completely unaffected by the addition of RANKL. This result casts further doubt on the significance of RANK expression in HD cell lines.
The problems associated with the use of tumor cell lines as a model for HD are demonstrated by the expression of the interleukin-3 receptor (IL-3R). IL-3R rescues cells from apoptosis3 and, like RANK, is expressed in some but not all HD cell lines. As shown in Table1, whether HD cell lines express IL-3R depends, to a large extent, on the types of cell lines examined. More uncertainty about the possible role of IL-3R in HD arises from the fact that none of the IL-3R–expressing HD cell lines show IL-3 dependency.
The study by Fiumara et al is interesting in that it addresses the important issue of tumor-cell survival in HD. A possible role for RANK in promoting survival of HRS tumor cells, however, remains to be determined.
Receptor activator of nuclear factor–κB ligand activates mitogen-activated protein kinases signaling pathways in Hodgkin and Reed-Sternberg cells
Dr Bosshart raises several interesting issues related to our recent report of the functional expression of receptor activator of nuclear factor–κB (RANK) in Hodgkin disease (HD).1-1Contrary to Bosshart's statement, all HD cell lines that we examined expressed RANK protein and mRNA.1-1 The relatively low level of RANK protein expression in the HD-MYZ and the KM-H2 cell lines that was shown in our original report is related to the amount of protein that was loaded on gel. When a higher amount of protein (40 μg) is loaded, RANK protein expression becomes more evident in all of these cell lines (Figure 1-1A). Thus, because all HD cell lines expressed RANK, it is not surprising that all of them responded to stimulation with RANK ligand (RANKL) by either activating nuclear factor–κB (NF-κB), up-regulating cytokine and chemokine mRNA expression, or inducing cytokine secretion.1-1
![Fig. 1-1. RANK expression and activation of MAPK pathways in the HD-LM2 cell line. / (A) RANK expression in 4 HD cell lines as determined by Western blot (anti-RANK antibody used was from Santa Cruz, CA; SC9072). For the activation studies, HD-LM2 cell line cells (2 x 106/mL) were treated with 10 nM RANKL for 0 to 60 minutes. (B) Fifty micrograms of the protein extracts was probed with phospho-ERK (p44/42) antibodies (Santa Cruz, CA) (upper panel). Thirty micrograms of the same protein extracts was probed with ERK1 antibodies (lower panel). (C) Fifty micrograms of the protein extracts was resolved on the gels and probed with phospho-p38 MAPK antibodies (New England BioLabs, Beverly, MA) (upper panel). The same blot was stripped and reprobed with antibodies against the p38 MAPK (lower panel). (D) One hundred micrograms of whole cell protein was reacted with JNK1 antibodies (Santa Cruz, CA) and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). The c-Jun kinase assay was performed by a modified method as described,1-4 using washed beads as a source of enzyme and glutathione S-transferase-Jun (1-79) as substrate (2 μg/sample) in the presence of 10 μCi (.37 MBq) [32P] adenosine 5′-triphosphate (ATP) per sample. The kinase reaction was carried out by incubating the above mixture at 30°C in kinase assay buffer for 15 minutes. The reaction was stopped by boiling beads in sodium dodecyl sulfate (SDS) sample buffer. Finally, protein was resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The radioactive bands of the dried gel were visualized and quantitated by phosphorImager. Forty micrograms of these same protein extracts was probed with JNK1 antibodies (lower panel). (E) HD-LM2 cells were treated with increasing concentrations of RANKL (0-10 nM) for 20 minutes. One hundred micrograms of whole cell protein was incubated with JNK1 antibodies and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). Forty micrograms of the same protein extracts was probed with JNK1 antibodies (lower panel).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/9/10.1182_blood-2002-01-0122/6/m_h80922588001.jpeg?Expires=1735850443&Signature=zyH39GF2OkjFZfEK2-5WyoPO7NIstPw0GQ0oHnyMPSNdTqMLD9L-sxyXeIMEfsOULIGbkYjCj7GwmbUGEqYmbqxs3VrlttA~22Y5Yad53Ihdjh1ufLmZoSdXeIFasykNy7aGyzc2SGCmYE~e6Iq5cusqiX-LTm3lRiZAS-ObqG4fIeGksLEdV7jJGm24bsYitfXp90AToKE7F75JXbQyTu2fOaLK-Hg-9ymx5BTyX6TdRDICXhZmMH~hK7KgebGB5UjIZxYBLKFnhuE5d91LNYEC4t9JiMWD5NT2GSAFb9rRdt4wgy48XxRSOAqGGymbovpp1VwmUE6onDDbj2njfg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
RANK expression and activation of MAPK pathways in the HD-LM2 cell line.
(A) RANK expression in 4 HD cell lines as determined by Western blot (anti-RANK antibody used was from Santa Cruz, CA; SC9072). For the activation studies, HD-LM2 cell line cells (2 x 106/mL) were treated with 10 nM RANKL for 0 to 60 minutes. (B) Fifty micrograms of the protein extracts was probed with phospho-ERK (p44/42) antibodies (Santa Cruz, CA) (upper panel). Thirty micrograms of the same protein extracts was probed with ERK1 antibodies (lower panel). (C) Fifty micrograms of the protein extracts was resolved on the gels and probed with phospho-p38 MAPK antibodies (New England BioLabs, Beverly, MA) (upper panel). The same blot was stripped and reprobed with antibodies against the p38 MAPK (lower panel). (D) One hundred micrograms of whole cell protein was reacted with JNK1 antibodies (Santa Cruz, CA) and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). The c-Jun kinase assay was performed by a modified method as described,1-4 using washed beads as a source of enzyme and glutathione S-transferase-Jun (1-79) as substrate (2 μg/sample) in the presence of 10 μCi (.37 MBq) [32P] adenosine 5′-triphosphate (ATP) per sample. The kinase reaction was carried out by incubating the above mixture at 30°C in kinase assay buffer for 15 minutes. The reaction was stopped by boiling beads in sodium dodecyl sulfate (SDS) sample buffer. Finally, protein was resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The radioactive bands of the dried gel were visualized and quantitated by phosphorImager. Forty micrograms of these same protein extracts was probed with JNK1 antibodies (lower panel). (E) HD-LM2 cells were treated with increasing concentrations of RANKL (0-10 nM) for 20 minutes. One hundred micrograms of whole cell protein was incubated with JNK1 antibodies and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). Forty micrograms of the same protein extracts was probed with JNK1 antibodies (lower panel).
RANK expression and activation of MAPK pathways in the HD-LM2 cell line.
(A) RANK expression in 4 HD cell lines as determined by Western blot (anti-RANK antibody used was from Santa Cruz, CA; SC9072). For the activation studies, HD-LM2 cell line cells (2 x 106/mL) were treated with 10 nM RANKL for 0 to 60 minutes. (B) Fifty micrograms of the protein extracts was probed with phospho-ERK (p44/42) antibodies (Santa Cruz, CA) (upper panel). Thirty micrograms of the same protein extracts was probed with ERK1 antibodies (lower panel). (C) Fifty micrograms of the protein extracts was resolved on the gels and probed with phospho-p38 MAPK antibodies (New England BioLabs, Beverly, MA) (upper panel). The same blot was stripped and reprobed with antibodies against the p38 MAPK (lower panel). (D) One hundred micrograms of whole cell protein was reacted with JNK1 antibodies (Santa Cruz, CA) and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). The c-Jun kinase assay was performed by a modified method as described,1-4 using washed beads as a source of enzyme and glutathione S-transferase-Jun (1-79) as substrate (2 μg/sample) in the presence of 10 μCi (.37 MBq) [32P] adenosine 5′-triphosphate (ATP) per sample. The kinase reaction was carried out by incubating the above mixture at 30°C in kinase assay buffer for 15 minutes. The reaction was stopped by boiling beads in sodium dodecyl sulfate (SDS) sample buffer. Finally, protein was resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The radioactive bands of the dried gel were visualized and quantitated by phosphorImager. Forty micrograms of these same protein extracts was probed with JNK1 antibodies (lower panel). (E) HD-LM2 cells were treated with increasing concentrations of RANKL (0-10 nM) for 20 minutes. One hundred micrograms of whole cell protein was incubated with JNK1 antibodies and then immunoprecipitated with protein A/G sepharose. The beads were washed and subjected to kinase assay (upper panel). Forty micrograms of the same protein extracts was probed with JNK1 antibodies (lower panel).
Because these cell lines constitutively express high levels of NF-κB,1-2 1-3 it is not surprising that RANK activation did not result in further increase in NF-κB activation above the high baseline level in all Hodgkin cell lines. However, RANK may also induce some of its biologic functions through other signaling pathways. In fact, RANK can activate all 3 major mitogen-activated protein kinases (MAPK) pathways: ERK, p38, and JNK (Figures 1B-E). When the HD-LM2 cells were treated with RANKL for different times, MAPKK was strongly activated, causing ERK phosphorylation within 10 minutes and peaking at 60 minutes (Figure 1-1B, upper panel). This activation was not due to increased protein expression because ERK total protein level did not change with RANK activation (Figure 1-1B, lower panel). Similarly, RANKL phosphorylated the p38 MAPK within 5 minutes of RANKL treatment, reached maximum at 10 minutes, and declined after 20 minutes (Figure1-1C, upper panel). During the same time points, total p38 level did not change, indicating that the increased phosphorylated state was not due to an increase in protein level (Figure 1-1C, lower panel). Finally, RANKL activated jun kinase (JNK), causing phosphorylation of c-jun in a time- and dose-dependent manner. JNK was activated within 5 minutes, reaching maximum activation between 15 and 30 minutes (Figure 1-1D). The activation of JNK was also dose-dependent, reaching 2.2-fold at 0.01 nM RANKL and 21-fold at 10 nM RANKL (Figure 1-1E).
In our original study, we reported that RANKL failed to stimulate the growth of HD cell lines.1-1 However, because these cell lines have a high doubling time, it is difficult to show a significant increase in the proliferative rate above the baseline level. Studying the effect of RANKL on primary Hodgkin and Reed-Sternberg cells may yield more valuable information. However, the ability of RANKL to activate survival pathways such as NF-κB and MAPK suggest that RANK may indeed be involved in providing survival signals to the malignant cells.
