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Constitutive expression of NF-κB is a characteristic feature of mycosis fungoides: Implications for apoptosis resistance and pathogenesis
Constitutive Expression of NF-B Is a Characteristic Feature of Mycosis Fungoides: Implications for Apoptosis Resistance and Pathogenesis KEITH F. IZBAN, MD, MELEK ERGIN, MD, JIAN-ZHONG QIN, PHD, ROBERT L. MARTINEZ, BA, HT (ASCP), ROBERT J. POOLEY, JR, MD, SHAHNAZ SAEED, MD, AND SERHAN ALKAN, MD The NF-B family of transcription factors is an important regulator of genes expressed during inflammatory responses, immunoglobulin (Ig) class switching, cellular differentiation, and apoptosis. Recently, members of the NF-B family, including p65(Rel A), have been implicated in promoting survival of various hematopoeitic neoplasms, including T cell malignancies such as adult T cell leukemialymphoma. We investigated the expression of active NF-B p65(Rel A) in cases of mycosis fungoides (MF) and the effect of chemical inhibitors of NF-B on apoptosis in cutaneous T cell lymphoma (CTCL) cell lines. Paraffin-embedded tissues from 23 cutaneous lesions and a single lymph node biopsy from patients diagnosed with MF were evaluated for p65(Rel A) expression by using a monoclonal mouse antibody that detects the activated form of p65(Rel A). Apoptosis after treatment with the NF-B inhibitors gliotoxin, MG132, BAY 11-7082, and BAY 11-7085 was quantitatively measured in the CTCL cell lines HuT-78 and HH by propidium iodide (PI)/cell cycle analysis for detection of a hypodiploid (sub-G0) population and by determination of increased Annexin V/7-amino-actinomycin D (7AAD) expression. Nuclear extracts from CTCL cells before and after chemical inhibition were analyzed for NF-B nuclear DNA-binding activity by electrophoretic mobility shift assay (EMSA) with quantitative densitometry. Nuclear expression of p65(Rel A) before and after treatment with the various inhibitory compounds was measured by
immunofluorescence staining in each CTCL cell line. Neoplastic T lymphocytes from 22 of 24 cases of MF showed strong nuclear and cytoplasmic expression of active p65(Rel A). Compared with untreated control cells, a marked increase in apoptosis, a significant decrease in NF-B DNA-binding activity, and a marked decrease in nuclear p65(Rel A) expression were seen in cells from both CTCL cell lines after chemical NF-B inhibition. These data show that the active form of NF-B p65(Rel A) is commonly expressed in neoplastic T lymphocytes in patients with MF. In CTCL cell lines, the significant decrease in nuclear NF-B expression and the marked increase in spontaneous apoptosis caused by chemical NF-B inhibition suggest a critical role for NF-B in the pathogenesis and tumor cell maintenance of CTCLs. HUM PATHOL 31:1482-1490. Copyright © 2000 by W.B. Saunders Company Key words: cutaneous T cell lymphoma, mycosis fungoides, NFB, apoptosis. Abbreviations: MF, mycosis fungoides; CTCL, cutaneous T cell lymphoma; 7-AAD, 7-amino-actinomycin D; EMSA, electrophoretic mobility shift assay; HI-FBS, heat-inactivated fetal bovine serum; TBE, tris-borate-EDTA; PI, propidium iodide; CLL, chronic lymphocytic leukemia; HRS, Hodgkin/Reed-Sternberg; TNF-␣, tumor necrosis factor-␣; IL, interleukin; Ig, immunoglobulin; EDTA, ethylene diaminetetra-acetic acid; NIH, National Institute of Health.
Over a decade ago, the first Rel/NF-B protein was originally described as a constitutive nuclear factor in B lymphocytes and an inducible factor in pre-B cells that is required for expression of the immunoglobulin light chain gene.1,2 Today, however, the Rel/NF-B protein factors are known as a family of ubiquitously expressed transcription factors found in the cytoplasm of most cells. Several different Rel/NF-B protein subunits have been characterized, including p65/Rel A, Rel B, c-Rel, p50/p105, and p52/p100.3,4 These proteins are structurally related through a 300 –amino acid N-terminal domain called the Rel homology domain, which contains sequences essential for dimerization, DNA binding, nuclear transport, and inhibitor (IB) binding. Nearly all protein subunits of the Rel/NF-B
family are able to form homodimers and heterodimers, which bind to DNA target sites (B sites) to influence gene expression. The classical and most common dimeric form of the Rel/NF-B proteins is a p50p65(Rel A) heterodimer specifically called NF-B. Other identified Rel-related subunits, as mentioned previously, also may be part of activated NF-B; it is further possible that different forms of NF-B regulate unique sets of target genes. The Rel/NF-B family of transcription factors modulates a diverse group of genes that play a major role in regulating the expression of proteins involved in immune, inflammatory, and acute phase responses.4-6 In its latent form, NF-B is sequestered in the cytoplasm, bound by the IB family of inhibitory proteins, which include IB␣, IB, IB␥, and IB.6-9 After cellular stimulation by a variety of agents, including tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), endotoxin, ␥-radiation, and phorbol ester, NF-B is activated by signal-induced, specific phosphorylation, ubiquitination, and subsequent rapid degradation of IB by proteasomes.9-11 As a result of this process, NF-B is released from IB and translocated to the nucleus, where it plays an important role in the tran-
From the Department of Pathology and Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL. Accepted for publication September 5, 2000. Address correspondence and reprint requests to Serhan Alkan, MD, Department of Pathology, Loyola University Medical Center, EMS Building, Suite 2230, 2160 S First Ave, Maywood, IL 60153. Copyright © 2000 by W.B. Saunders Company 0046-8177/00/3112-0006$10.00/0 doi:10.1053/hupa.2000.20370
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scriptional activation of the B site in the promoter region of a large number of inducible target genes. Genes regulated by NF-B include a variety of adhesion molecules, major histocompatibility proteins, and several cytokines, including IL-2, IL-6, TNF-␣, transforming growth factor-, and interferon-.9,10,12,13 With notable exception of B lymphocytes, which show constitutive NF-B activity, most cells show only transient NF-B activity because NF-B also induces the transcription of IB␣, among other genes.5,14 As a result, activation normally results in a negative feedback mechanism in which newly synthesized IB binds NFB, leading to resequestration of NF-B in the cytoplasm.14 Among the Rel/NF-B family of transcription factors, the viral oncoprotein v-Rel was the first to be identified, with subsequent investigators showing in vitro and in vivo transforming properties and resistance to apoptosis.15-17 Subsequently, the constitutive activity of the Rel/NF-B protein factors has been correlated with proliferation, growth progression, and resistance to apoptosis in several neoplasms.18-20 These include several neoplastic cell lines of lymphoid origin such as B cell lymphoma WEHI231 cells,21,22 multiple myeloma,23 Hodgkin/Reed-Sternberg (HRS) cells,24-26 and T cell lymphoma HuT-78 cells.27,28 In the current study, we show for the first time constitutive expression of active NF-B p65(Rel A) in neoplastic T lymphocytes in most mucosis fungoides (MF) patient samples. Analysis of the cutaneous T-cell lymphoma (CTCL) cell lines HuT-78 and HH also showed strong constitutively active nuclear NF-B p65(Rel A) as demonstrated by immunohistochemistry and electrophoretic mobility shift assay (EMSA). Chemical inhibition of NF-B by the proteasome inhibitors MG13211,29,30 and gliotoxin30-32 and the IB␣ phosphorylation inhibitors BAY 11-708230,33 and BAY 11-708530,33 resulted in a marked increase in apoptosis, and an equally impressive decrease in nuclear NF-B expression in both cell lines. These results indicate a critical role for NF-B in cell survival and apoptosis resistance in CTCL.
vated fetal bovine serum (HI-FBS) (Sigma Chemical Co., St Louis, MO), 2 mmol/L L-glutamine (GIBCO-BRL), 25 mmol/L Hepes (Sigma), and antibiotic-antimycotic solution (Sigma). All cell lines were maintained at 37°C in a humidified incubator at 5% CO2.
NF-B Inhibitors To evaluate the possible apoptotic effects of the NF-B inhibitors, 1 ⫻ 106/mL cells from each cell line were cultured in 24-well tissue culture plates (Falcon, Lincoln Park, NJ) and incubated with either 10 mol/L z-Leu-Leu-Leu-aldehyde (MG132, Biomol Research Laboratories, Inc., Plymouth Meeting, PA), 10 mol/L gliotoxin (Calbiochem, La Jolla, CA), 10 mol/L (E)3-[(4-t-Butylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7085, Calbiochem), or 10 mol/L (E)3-[(4methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082, Calbiochem) for 72-hour periods. All reactions were performed at least in duplicate.
Immunohistochemical Determination of NF-B p65(Rel A) After fixation in a 1:1 mixture of acetone and methanol for 10 minutes, cytospins from both CTCL cell lines were stained with a monoclonal mouse anti-human p65 antibody (clone 12H11, 1:75 titer, Boehringer Mannheim Corporation, Indianapolis, IN), which recognizes only the unbound, active form of p65(Rel A) dissociated from IB␣35 and a secondary polyclonal goat anti-mouse immunoglobulin G (IgG)/IgM antibody conjugated to fluorescein isothiocyanate (Biosource International, Camarillo, CA). Analysis of paraffin-embedded patient material for expression of active p65(Rel A) was performed by using a Ventana NEXES automated stainer (Ventana Medical Systems, Tucson, AZ) and streptavidin/horseradish peroxidase detection kit (Ventana) without antigen retrieval. The chromogen used was 3,3⬘-diaminobenzidine tetrahydrochloride. Cases were considered to be positive for p65(Rel A) expression if 50% or more of neoplastic cells showed nuclear positivity with or without cytoplasmic staining. Negative (normal skin), positive (nodular sclerosis Hodgkin’s disease) and isotypic controls were included in each staining run. In addition, the staining pattern of lymphocytes from 4 reactive lymph nodes was also analyzed.
Detection of Apoptosis MATERIALS AND METHODS Patient Samples and Cell Culture Formalin-fixed, paraffin-embedded tissue sections from 24 cases of MF were selected from the surgical pathology archives of Loyola University Medical Center for immunohistochemical determination of active p65 (Rel A). For each specimen, the diagnosis of MF was made retrospectively and confirmed by using established histologic criteria.34 Of the 23 cutaneous specimens analyzed, most fit criteria consistent with plaque or patch lesions of MF. A single lymph node involved by MF was also included in the study. All specimens analyzed were initial diagnostic specimens without previous therapy. CTCL cell lines HuT-78 and HH were obtained from the American Type Culture Collection (ATCC; Rockville, MD). Cell lines were cultured in RPMI 1640 (GIBCO-BRL, Grand Island, NY) supplemented with 20% (vol/vol) heat-inacti-
For propidium iodide staining, treated and untreated cells (1 ⫻ 106 cells/mL) were washed in cold FACS buffer (5 mL phosphate-buffered saline [PBS] without Mg2⫹ or Ca2⫹, 2% sodium azide/deionized water, and 5% [vol/vol] HI-FBS [Sigma]) and resuspended in a 1:6 mixture of 100% HI-FBS and absolute ethanol. After 30 minutes incubation on ice, the cells were washed once again in FACS buffer. The cell pellet was resuspended and incubated at 37°C with 10 g/mL RNAse (Boehringer Mannheim) for 15 minutes. After brief cooling at room temperature, 100 g/mL propidium iodide (PI) was added to each cell suspension followed by overnight incubation at 4°C in the dark. PI fluorescence of individual nuclei from each sample (10,000 events per sample) was analyzed on an Epics XL-MCL flow cytometer (Coulter, Miami Lakes, FL) and expressed on a logarithmic scale as a measure of DNA content using Coulter System II software. Apoptosis was quantitatively measured by PI/cell cycle analysis for the detection of a hypodiploid (sub-G0) population within the cell suspensions.
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The Annexin V/7-AAD staining method was performed per manufacturer’s protocol by using Annexin V-PE (PharMingen, San Diego, CA) and Via-Probe 7-amino-actinomycin D (7-AAD) (PharMingen). Treated and untreated cells (0.5 ⫻ 106 cell/mL) were washed twice in cold PBS and resuspended in 1⫻ binding buffer (10 mmol/L Hepes/NaOH [pH 7.4], 140 mmol/L NaCL, and 2.5 mmol/L CaCl2). Resuspended cells were then incubated for 20 minutes at 20°C to 25°C in the dark with 2 L Annexin V-PE and 5 L 7-AAD. Samples (10,000 events per sample) were then quantitated on a Coulter Epics XL-MCL flow cytometer, recorded in LIST mode, and registered on logarithmic scales. 7-AAD emission was detected in the FL-3 channel (⬎650 nm). Analysis was performed by using Coulter System II software.
Determination of Active NF-B by the Electrophoretic Mobility Shift Assay (EMSA) Nuclear protein was extracted using the method of Gerber et al.36 Protein content was measured by the Bradford method. A consensus double-stranded NF-B probe, 5⬘-AGT TGA GGG GAC TTT CCC AGG C-3⬘ (Integrated DNA Technologies, Inc., Coralville, IA) was end-labeled by using ␥-32Padenosine-5-triphosphate. Five micrograms nuclear extract from investigated cells were then incubated in binding buffer consisting of 10 mmol/L HEPES (pH 7.9), 60 mmol/L KCL, 4% Ficoll, 1 mmol/L 1,4-dithiothreitol, 1 mmol/L ethylene diaminetetra-acetic acid (EDTA) (pH 8.0), and 1 g/mL poly dI:dC (Pharmacia Biotech Inc., Piscataway, NJ) in 20 L volume. Afterward, the end-labeled probe was added (200,000 cpm). Samples were then incubated for 30 minutes on ice followed by loading on 4% nondenaturing polyacrylamide gel (0.25 ⫻ Tris-borate-EDTA [TBE]). Electrophoresis was run for 3 hours in 0.25 TBE buffer at 100 V. Protein complexes were identified by autoradiography. Quantitative densitometry was performed for each band corresponding to NF-B (p50 to p65) using National Institute of Health (NIH) Image 1.62 software (NIH, Bethesda, MD). Densitometric ratios were calculated by dividing the densitometry reading of individual bands from chemically treated cells by the densitometry reading of untreated control cells.
RESULTS Neoplastic Cells From Cases of CTCL Demonstrate Strong Immunopositivity for p65(Rel A)
FIGURE 1. Neoplastic T lymphocytes from cases of Mycosis fungoides show strong immunopositivity for active p65 (Rel A). Positive nuclear and cytoplasmic staining for active p65(Rel A) was found in neoplastic lymphocytes in most MF cases as shown in these individual cutaneous cases (A,B,C, original magnification ⫻1,000; p65(Rel A)/DAB). In comparison, no evidence of positive staining was seen in isotypic controls (D, original magnification ⫻1,000, isotypic IgG). As shown here, keratinocytes in cutaneous tissues involved by MF also showed p65(Rel A) expression; however, keratinocytes and scattered lymphocytes (arrows) found in normal skin samples showed no evidence of p65(Rel A) immunopositivity (E, original magnification ⫻500, p65[Rel A]/DAB). As seen in cutaneous cases, neoplastic lymphocytes in the single lymph node biopsy also
Although constitutive nuclear expression of NF-B was previously demonstrated in the CTCL cell line HuT-78,27,28 investigation for such expression in neoplastic T lymphocytes from patient samples of CTCL was not previously performed. To compare and contrast NF-B expression between cells from CTCL cell lines and patient samples, we examined neoplastic T showed strong positive staining (F, original magnification ⫻1,000, p65[Rel A]/DAB). In all positive MF cases, the pattern of nuclear and cytoplasmic expression of p65(Rel A) was similar to that identified in HRS cells (arrows) from Hodgkin’s disease positive controls (G, original magnification ⫻1,000, p65[Rel A]/DAB). Also for comparison, lymphocytes in reactive lymph node specimens showed concentrated p65(Rel A) expression in follicular centers with a lesser degree of staining found in lymphocytes occupying the interfollicular, mantle, and marginal zones (H, original magnification ⫻100, p65[Rel A]/DAB).
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lymphocytes from the initial diagnostic specimens of 24 cases of MF for the expression of active NF-B p65(Rel A). Immunohistochemical staining of paraffin-embedded MF specimens showed strong nuclear and cytoplasmic expression of active p65(Rel A) in neoplastic T lymphocytes from 21 of 23 (91%) cutaneous cases. In most of these cases, more than 80% of neoplastic lymphocytes showed positive p65(Rel A) expression regardless of histologic pattern (range, 65% to 90% positive staining). In these cutaneous cases, p65(Rel A) expression was characterized by strong nuclear expression with or without cytoplasmic positivity (Fig 1A-C) compared with negative isotypic controls (Fig 1D). Interestingly in most of these cases, keratinocytes also showed immunopositivity for p65(Rel A). This finding may be attributable to the stimulation of NF-B expression in keratinocytes in response to various stimulatory cytokines produced by neoplastic MF cells. In comparison, keratinocytes and scattered lymphoid cells found in normal skin samples were negative for p65(Rel A) expression (Fig 1E). As seen in cutaneous cases, neoplastic MF cells in the single lymph node biopsy also showed strong positive staining (Fig 1F). For each staining run, cases of nodular sclerosis Hodgkin’s disease24-26 were used as positive controls. Typically, HRS cells from these cases showed strong nuclear and cytoplasmic expression of p65(Rel A) (Fig 1G). Interestingly, lymphocytes in reactive lymph node specimens showed concentrated p65(Rel A) expression in follicular centers with a lesser degree of staining found in lymphocytes occupying the interfollicular, mantle, and marginal zones (Fig 1H).
CTCL Cell Lines Demonstrate Markedly Increased Apoptosis and Significantly Decreased Nuclear NF-B Expression After Chemical NF-B Inhibition Having established the presence of active NF-B in CTCL cells from both patient and cell line specimens, we sought to determine the effect of chemical NF-B inhibition on CTCL cell lines. To measure the potential apoptotic effect of NF-B inhibition on CTCL cell lines, determination of apoptotic activity was measured after 72-hour incubation with each of the following NF-B inhibitors: MG132, gliotoxin, BAY 11-7082, and BAY 11-7085. Regardless of the cell line examined (HuT-78 or HH), the methodology of apoptosis measurement (PI v Annexin/7-AAD), or the inhibitory compound used, each compound induced a marked increase in apoptosis compared with baseline controls (Figs 2, 3). In each cell line, the mean increase in apoptosis from the baseline was at least 40% and often was much higher in most cases. To determine whether the apoptotic effect generated by the above compounds was related to inhibition of nuclear NF-B, expression of active NF-B was determined by EMSA, using nuclear extracts prepared from HuT-78 and HH after 24-hour incubation with MG132, gliotoxin, BAY 11-7082, or BAY 11-7085. In both cell lines, treatment with each of the chemical NF-B inhibitors resulted in significantly decreased NF-B DNA binding activity compared with untreated controls (Fig 4). Quantitation of NF-B (p50-p65) by densitometry showed markedly decreased DNA binding activity (8 to 9-fold decrease) in both cell lines after 24-hour incuba-
FIGURE 2. Flow cytometric detection of apoptosis by PI staining for determination of a hypodiploid (sub-G0) population in CTCL cell lines. HuT-78 and HH cells were treated with 10-mol/L concentrations of the various NF-B inhibitors. DNA content in the cells was determined 72 hours after treatment. In contrast to untreated control cells, marked increases in apoptosis were seen in both cell lines regardless of the compound used. The percentage of apoptotic cells 72 hours after treatment is shown in the upper right corner of each box. These data are representative of at least 2 separate experiments.
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FIGURE 3. Apoptosis determination by Annexin/7-AAD staining and flow cytometry in CTCL cells. In both HuT-78 (A) and HH cells (B), chemical inhibition of NF-B by 10-mol/L concentrations of each compound resulted in a marked increase in apoptosis compared with untreated controls. Annexin/7-AAD staining was determined at 72 hours after treatment, with the percentage of apoptosis shown in the upper right corner of each box. These data are representative of at least 2 separate experiments.
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FIGURE 4. Nuclear DNA binding activity of NF-B in CTCL cell lines after 24-hour chemical NF-B inhibition. As shown by electrophoretic mobility shift assay, marked decreases in active nuclear NF-B expression were obtained in each cell line after 24-hour treatment with each of the 4 chemical inhibitors used. The NF-B probe identified p50-p50 homodimers as well as classical NF-B (p50-p65 heterodimer). The individual numbers below each set of bands represent the calculated densitometry ratios for NF-B (p50 to p65) as determined in the Materials and Methods section.
tion with gliotoxin or BAY 11-7082 (Fig 4). For the remaining compounds, DNA binding activity was reduced by approximately 40% or greater with the exception of a 20% decrease in HuT-78 cells after incubation with BAY 11-7085. In addition to EMSA, expression of nuclear NF-B p65(Rel A) in CTCL cell lines was also determined by immunofluorescence staining both before and after incubation with each of the inhibitory compounds. Both HuT-78 (Fig 5A) and HH (Fig 5B) control cells showed bright nuclear expression of p65(Rel A). HuT-78 cells showed a marked reduction in nuclear p65(Rel A) expression after incubation with MG132 (Fig 5C), gliotoxin (Fig 5E), BAY 11-7082 (Fig 5G), or BAY 11-7085 (Fig 5I). Similarly, HH cells also showed a significant decrease in nuclear p65(Rel A) staining after incubation with each of the above-mentioned inhibitory compounds (Figs 5D, 5F, 5H, and 5J, respectively). DISCUSSION We examined the expression of NF-B in CTCLs, including 2 human T cell lymphoma cell lines, HuT-78 and HH, and patient samples of MF. Previous investigations have shown constitutively active NF-B p50p65(Rel A) in the HuT-78 cell line27,28; however, primary patient sample material was not analyzed in these studies. In support of these past investigations, we found strong nuclear and cytoplasmic expression of active p65(Rel A) expression by immunofluorescence staining and EMSA in unstimulated T cells from each CTCL cell line (HuT-78 and HH). Furthermore, for the first time, we showed active p65(Rel A) in neoplastic T lymphocytes from patient samples of MF, including 21 of 23 (91%) cutaneous cases and a single lymph node
with involvement. All specimens analyzed were initial diagnostic specimens, without previous therapy. Much like their cell line counterparts, neoplastic T lymphocytes from patient cases also showed strong nuclear and cytoplasmic expression of p65(Rel A). The correlation of constitutive p65(Rel A) expression between cultured and patient CTCL cell samples is important and implies similarities in their NF-B biologic pathways. With this in mind, however, constitutive expression of p65(Rel A) is not unique to CTCL. Among hematopoietic neoplasms alone, constitutive expression of p65(Rel A) has also been found in chronic lymphocytic leukemia,37 multiple myeloma,23 Hodgkin’s disease,24-26 and adult T-cell leukemia/lymphoma.38 In addition to p50 and p65(Rel A), investigations to identify additional members of the Rel/NF-B family in HuT-78 showed high nuclear levels of p52, and 2 aberrantly processed forms of the p100 protein, p84 and p85.39 From this investigation, Zhang and colleagues39 revealed a rearrangement of the nfkb2 gene responsible for the production of these aberrant proteins via loss of an ankyrin repeat domain in the carboxy-terminus of p100. In addition, both p84 and p85 were found in the nucleus in an unprocessed form, suggesting that these proteins may escape the cytoplasmic sequestration typical of normal p100 because of loss of the C-terminus. As a result, these proteins were found to bind B sites specifically and alter the composition of NF-B complexes in HuT-78 cells.39 Two more recent studies, however, showed a relationship between TNF-␣ production and the nuclear localization of p50, p52, p65(Rel A), and p85 in HuT-78 cells.27,28 In these investigations, antibodies against TNF-␣ inhibited proliferation and downregulated the constitutive activation of NF-B in HuT-78 cells, sug-
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FIGURE 5. Nuclear expression of p65(Rel A) in CTCL cell lines after chemical NF-B inhibition. Immunofluorescence staining of untreated control cells from HuT-78 (A, original magnification ⫻1,000, p65[Rel A]/ polyclonal anti-mouse FITC) and HH (B, original magnification ⫻1,000, p65 [Rel A]/polyclonal anti-mouse FITC) showed bright nuclear expression of p65(Rel A). After 18-hour incubation with MG132 (C, original magnification ⫻1,000, p65[Rel A]/polyclonal anti-mouse FITC), gliotoxin (E, original magnification ⫻1,000, p65[Rel A]/ polyclonal anti-mouse FITC), BAY 117082 (G, original magnification ⫻1,000, p65[Rel A]/polyclonal anti-mouse FITC), or BAY 11-7085 (I, original magnification ⫻1,000, p65[Rel A]/polyclonal anti-mouse FITC) HuT-78 cells showed markedly decreased p65 (Rel A) expression. Likewise, HH cells also showed significantly decreased nuclear expression of p65(Rel A) after 18-hour incubation with MG132 (D, original magnification ⫻1,000, p65 [Rel A]/polyclonal anti-mouse FITC), gliotoxin (F, original magnification ⫻1,000, p65[Rel A]/polyclonal antimouse FITC), BAY 11-7082 (H, original magnification ⫻1,000, p65[Rel A]/ polyclonal anti-mouse FITC), or BAY 11-7085 (J, original magnification ⫻1,000, p65[Rel A]/polyclonal antimouse FITC).
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gesting an autocrine role for TNF-␣. These findings suggest that the loss of the C-terminal ankyrin domain in p84 and p85 is alone insufficient to cause nuclear translocation. Importantly, the authors of these studies also showed high levels of constitutively expressed TNF-␣, suggesting both the absence of inhibitory feedback mechanisms for TNF gene expression and a vital interrelationship between TNF-␣ and NF-B necessary for survival of HuT-78 cells. Contrary to these findings, however, Dobbeling et al40 reported significantly reduced cell growth in the CTCL cell line SeAx after treating the cells with the same concentration of TNF-␣ (50 pg/mL) reported to be secreted by HuT-78 cells.40 These findings suggest that the autocrine TNF-␣ growth-promoting effect seen in HuT-78 may be a unique characteristic of these cells, rather than a general feature necessary for growth and survival of all CTCL cells. Survival and apoptosis resistance in CTCL cell lines are dependent on cellular mechanisms that promote constitutive activation of NF-B. From the results of our analysis, the ubiquitin-proteasome pathway was shown to play an important role in apoptosis resistance in CTCL cells, such that the proteasome inhibitors MG132 and gliotoxin were capable of producing both downregulation of NF-B expression and a marked apoptotic effect in HuT-78 and HH cells. Proteolytic processing of the p105 protein subunit for generation of the p50p65(Rel A) heterodimer and degradation of IB␣, which is necessary for nuclear translocation of activated NF-B, are 2 important functions of the ubiquitin-proteasome pathway.4,11,41 The proteasome inhibitors used in this study block the 20s proteasome complex in HeLa cells, with MG132 exerting an additional inhibitory effect on the 26s proteasome.11,29-32,41 As a result, these compounds potently inhibit both processes required for NF-B activation. Recently, we and others have shown the possible therapeutic implications of constitutive NF-B downregulation through proteasome inhibition in other hematopoietic neoplasms.29,38,42 From our investigation of Hodgkin’s disease cell lines, we showed both downregulation of NF-B expression and marked apoptosis of Hodgkin/Reed-Sternberg (HRS) cells after treatment with MG132 and gliotoxin.42 Similar to our findings in cultured HRS cells and recently in CTCL cells, Chandra et al29 showed a relationship between suppression of active NF-B and MG132-induced apoptosis in glucocorticoid-resistant chronic lymphocytic leukemia (CLL) cells.29 In addition, Delic and colleagues37 observed similar findings in therapy-resistant CLL cells using the proteasome inhibitor lactacystin. As a whole, these investigations consistently showed inhibition of constitutively expressed NF-B by proteasome inhibition. In addition to the proteasome inhibitors, we investigated the effects of 2 synthetic compounds (BAY 117082 and BAY 11-7085) that block the activation of NF-B at the level of IB phosphorylation/degradation. By preventing nuclear translocation of NF-B, these compounds have been shown to inhibit TNF-␣–
induced expression of several NF-B target genes, including ICAM-1, VCAM-1, and E-selectin.33 Although the specific mechanism of NF-B inhibition is currently undefined, each compound has been shown to stabilize IB␣ by selectively and irreversibly inhibiting phosphorylation of IB␣.33 Thus, in contrast to the proteasome inhibitors that block degradation of phosphorylated IB␣, these compounds inhibit inducible phosphorylation of IB␣. As seen with the proteasome inhibitors, treatment with either BAY 11-7082 or BAY 11-7085 resulted in significant downregulation of NF-B expression and a marked apoptotic effect in HuT-78 and HH cells, once again showing the significance of NF-B for survival of CTCL cells. In conclusion, we show that constitutive expression of NF-B not only is a feature found in CTCL cell lines, but is also a prominent characteristic of neoplastic T lymphocytes from patient samples of MF. Through chemical inhibition of constitutive nuclear NF-B expression, we were able to induce a marked apoptotic effect in HuT-78 and HH, both via inhibition of the ubiquitin-proteasome pathway by MG132 and gliotoxin, and irreversible blockade of degradation of phosphorylated IB␣ by BAY 11-7082 and BAY 11-7085. Overall, these findings indicate that constitutive nuclear NF-B activity is required for cell survival and resistance to apoptosis in CTCL cells. The exact mechanisms by which NF-B provides a survival advantage to CTCL cells, however, remains undetermined. Additional studies of the NF-B and related biologic pathways will likely provide valuable insights into the pathogenesis, progression, and treatment of CTCL as well as other hematopoietic and nonhematopoietic neoplasms.
REFERENCES 1. Sen R, Baltimore D: Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46:705-716, 1986 2. Sen R, Baltimore D: Inducibility of immunoglobulin enhancer binding protein NF-B by a translational mechanism. Cell 47:921-928, 1986 3. Baeuerle PA: The inducible transcriptional activator NF-B: Regulation by distinct protein subunits. Biochim Biophys Acta 1072: 63-80, 1991 4. Grilli M, Chiu J, Lenardo MJ: NF-B and Rel: Participants in a multiform transcriptional regulatory system. Int Rev Cytol 143:1-62, 1993 5. Baeuerle PA, Henkel T: Function and activation of NF-B in the immune system. Annu Rev Immunol 12:141-179, 1994 6. Baldwin AS: The NF-B and IB proteins: New discoveries and insights. Annu Rev Immunol 14:649-681, 1995 7. May MJ, Ghosh S: Rel/NF-B and IB: An overview. Semin Cancer Biol 8:63-73, 1997 8. Whiteside ST, Israel A: IB proteins: Structure, function, and regulation. Semin Cancer Biol 8:75-82, 1997 9. Ghosh S, May MJ, Kopp EB: NF-B and Rel proteins: Evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16:225-260, 1998 10. May MJ, Ghosh S: Signal transduction through NF-B. Immunol Today 19:80-88, 1998 11. Palombella VJ, Rando OJ, Goldberg AL, et al: The ubiquitinproteasome pathway is required for processing the NF-B1 precursor protein and the activation of NF-B. Cell 78:773-785, 1994
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12. Baeuerle PA, Baltimore D: NF-B: Ten years later. Cell 87: 13-20, 1996 13. O’Neill LAJ, Kaltschmidt C: NF-B: A crucial transcription factor for glial and neuronal cell function. Trends Neurol Sci 20:252258, 1997 14. Siebenlist U, Franzoso G, Brown K: Structure, regulation, and function of NF-B. Annu Rev Cell Biol 10:405-455, 1994 15. Neiman PE, Thomas SJ, Loring G: Induction of apoptosis during normal and neoplastic B-cell development in the bursa of Fabricius. Proc Natl Acad Sci U S A 88:5857-5861, 1991 16. White DW, Roy A, Gilmore TD: The v-Rel oncoprotein blocks apoptosis and proteolysis of I kappa B-alpha in transformed chicken spleen cells. Oncogene 10:857-868, 1995 17. Gilmore TD: Multiple mutations contribute to the oncogenicity of the retroviral oncoprotein v-Rel. Oncogene 18:6925-6937, 1999 18. Gilmore TD, Koedood M, Piffat KA, et al: NF-B/IB protein and cancer. Oncogene 13:1367-1378, 1996 19. Luque I, Gelinas C: Rel/NF-B and IB factors in oncogenesis. Semin Cancer Biol 8:103-111, 1997 20. Rayet B, Gelinas C: Aberrant rel/nfkb gene and activity in human cancer. Oncogene 18:6938-6947, 1999 21. Wu M, Lee H, Bellas RE, et al: Inhibition of NF-B/Rel induces apoptosis in murine B cells. EMBO J 15:4682-4690, 1996 22. Doerre S, Corley RB: Constitutive nuclear translocation of NF-B in B cells in the absence of IB degradation. J Immunol 163:269-277, 1999 23. Feinman R, Koury J, Thames M, et al: Role of NF-B in the rescue of multiple myeloma cells from glucocorticoid-induced apoptosis by Bcl-2. Blood 93:3044-3052, 1999 24. Bargou RC, Leng C, Krappmann D, et al: High level nuclear NF-B and Oct-2 is a common feature of cultured Hodgkin/ReedSternberg cells. Blood 87:4340-4347, 1996 25. Bargou RC, Emmerich F, Krappmann D, et al: Constitutive nuclear factor NF-B-Rel A activation is required for proliferation and survival of Hodgkin’s disease tumor cells. J Clin Invest 100:29612969, 1997 26. Krappmann D, Emmerich F, Kordes U, et al: Molecular mechanisms of constitutive NF-B/Rel activation in Hodgkin/ReedSternberg cells. Oncogene 18:943-953, 1999 27. O’Connell MA, Cleere R, Long A, et al: Cellular proliferation and activation of NFB are induced by autocrine production of tumor necrosis factor ␣ in the human T lymphoma line HuT 78. J Biol Chem 270:7399-7404, 1995 28. Giri DK, Aggarwal BB: Constitutive activation of NF-B causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells. J Biol Chem 273:14008-14014, 1998
29. Chandra J, Niemer I, Gilbreath J, et al: Proteasome inhibitors induce apoptosis in glucocorticoid-resistant chronic lymphocytic leukemia lymphocytes. Blood 92:4220-4229, 1998 30. Epinat JC, Gilmore TD: Diverse agents act at multiple levels to inhibit the Rel/NF-B signal transduction pathway. Oncogene 18:6896-6909, 1999 31. Pahl HL, Krauss B, Schulze-Osthoff K, et al: The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-B. J Exp Med 183:1829-1840, 1996 32. Kroll M, Arenzana-Seisdedos F, Bachelerie F, et al: The secondary fungal metabolite gliotoxin targets proteolytic activities of the proteasome. Chem Biol 6:689-698, 1999 33. Pierce JW, Schoenleber R, Jesmok G, et al: Novel inhibitors of cytokine-induced IB␣ phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem 272:21096-21103, 1999 34. Sharpiro PE, Pinto F: The histologic spectrum of mycosis fungoides/Sezary syndrome (cutaneous T-cell lymphoma): A review of 222 biopsies, including newly described patterns and the earliest pathologic changes. Am J Surg Pathol 18:645-667, 1994 35. Zabel U, Henkel T, Silva MS, Baeuerle PA: Nuclear uptake control of NF-kappa B by MAD-3, an I kappa B protein present in the nucleus. EMBO J 12:201-211, 1993 36. Gerber HP, Georgiev O, Harshman K, et al: In vitro transcription complementation assay with mini extracts of transiently transfected COS-1 cells. Nucl Acids Res 20:5855, 1992 37. Delic J, Masdehors P, Omura S, et al: The proteasome inhibitor lactacystin induces apoptosis and sensitizes chemo- and radioresistant human chronic lymphocytic leukaemia lymphocytes to TNF␣-initiated apoptosis. Br J Cancer 77:1103-1107, 1998 38. Mori N, Fujii M, Ikeda S, et al: Constitutive activation of NF-B in primary adult T-cell leukemia cells. Blood 93:2360-2368, 1999 39. Zhang J, Chang CC, Lombardi L, Dalla-Favera R: Rearranged NFKB2 gene in the HUT78 T-lymphoma cell line codes for a constitutively nuclear factor lacking transcriptional repressor functions. Oncogene 9:1931-1937, 1994 40. Dobbeling U, Dummer R, Laine E, et al: Interleukin-15 is an autocrine/paracrine viability factor for cutaneous T-cell lymphoma cells. Blood 92:252-258, 1998 41. Beg AA, Baldwin AS Jr: The IB proteins: Multifunctional regulators of rel/NF-B transcription factors. Gene Dev 7:2064-2070, 1997 42. Izban KF, Wrone-Smith T, Alkan S: Inhibition of NF-B induced apoptosis in Hodgkin’s disease cell lines is independent of the cell death protease CPP32/Caspase 3. Mod Pathol, 2000 (in press)
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immunofluorescence staining in each CTCL cell line. Neoplastic T lymphocytes from 22 of 24 cases of MF showed strong nuclear and cytoplasmic expression of active p65(Rel A). Compared with untreated control cells, a marked increase in apoptosis, a significant decrease in NF-B DNA-binding activity, and a marked decrease in nuclear p65(Rel A) expression were seen in cells from both CTCL cell lines after chemical NF-B inhibition. These data show that the active form of NF-B p65(Rel A) is commonly expressed in neoplastic T lymphocytes in patients with MF. In CTCL cell lines, the significant decrease in nuclear NF-B expression and the marked increase in spontaneous apoptosis caused by chemical NF-B inhibition suggest a critical role for NF-B in the pathogenesis and tumor cell maintenance of CTCLs. HUM PATHOL 31:1482-1490. Copyright © 2000 by W.B. Saunders Company Key words: cutaneous T cell lymphoma, mycosis fungoides, NFB, apoptosis. Abbreviations: MF, mycosis fungoides; CTCL, cutaneous T cell lymphoma; 7-AAD, 7-amino-actinomycin D; EMSA, electrophoretic mobility shift assay; HI-FBS, heat-inactivated fetal bovine serum; TBE, tris-borate-EDTA; PI, propidium iodide; CLL, chronic lymphocytic leukemia; HRS, Hodgkin/Reed-Sternberg; TNF-␣, tumor necrosis factor-␣; IL, interleukin; Ig, immunoglobulin; EDTA, ethylene diaminetetra-acetic acid; NIH, National Institute of Health.
Over a decade ago, the first Rel/NF-B protein was originally described as a constitutive nuclear factor in B lymphocytes and an inducible factor in pre-B cells that is required for expression of the immunoglobulin light chain gene.1,2 Today, however, the Rel/NF-B protein factors are known as a family of ubiquitously expressed transcription factors found in the cytoplasm of most cells. Several different Rel/NF-B protein subunits have been characterized, including p65/Rel A, Rel B, c-Rel, p50/p105, and p52/p100.3,4 These proteins are structurally related through a 300 –amino acid N-terminal domain called the Rel homology domain, which contains sequences essential for dimerization, DNA binding, nuclear transport, and inhibitor (IB) binding. Nearly all protein subunits of the Rel/NF-B
family are able to form homodimers and heterodimers, which bind to DNA target sites (B sites) to influence gene expression. The classical and most common dimeric form of the Rel/NF-B proteins is a p50p65(Rel A) heterodimer specifically called NF-B. Other identified Rel-related subunits, as mentioned previously, also may be part of activated NF-B; it is further possible that different forms of NF-B regulate unique sets of target genes. The Rel/NF-B family of transcription factors modulates a diverse group of genes that play a major role in regulating the expression of proteins involved in immune, inflammatory, and acute phase responses.4-6 In its latent form, NF-B is sequestered in the cytoplasm, bound by the IB family of inhibitory proteins, which include IB␣, IB, IB␥, and IB.6-9 After cellular stimulation by a variety of agents, including tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), endotoxin, ␥-radiation, and phorbol ester, NF-B is activated by signal-induced, specific phosphorylation, ubiquitination, and subsequent rapid degradation of IB by proteasomes.9-11 As a result of this process, NF-B is released from IB and translocated to the nucleus, where it plays an important role in the tran-
From the Department of Pathology and Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL. Accepted for publication September 5, 2000. Address correspondence and reprint requests to Serhan Alkan, MD, Department of Pathology, Loyola University Medical Center, EMS Building, Suite 2230, 2160 S First Ave, Maywood, IL 60153. Copyright © 2000 by W.B. Saunders Company 0046-8177/00/3112-0006$10.00/0 doi:10.1053/hupa.2000.20370
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scriptional activation of the B site in the promoter region of a large number of inducible target genes. Genes regulated by NF-B include a variety of adhesion molecules, major histocompatibility proteins, and several cytokines, including IL-2, IL-6, TNF-␣, transforming growth factor-, and interferon-.9,10,12,13 With notable exception of B lymphocytes, which show constitutive NF-B activity, most cells show only transient NF-B activity because NF-B also induces the transcription of IB␣, among other genes.5,14 As a result, activation normally results in a negative feedback mechanism in which newly synthesized IB binds NFB, leading to resequestration of NF-B in the cytoplasm.14 Among the Rel/NF-B family of transcription factors, the viral oncoprotein v-Rel was the first to be identified, with subsequent investigators showing in vitro and in vivo transforming properties and resistance to apoptosis.15-17 Subsequently, the constitutive activity of the Rel/NF-B protein factors has been correlated with proliferation, growth progression, and resistance to apoptosis in several neoplasms.18-20 These include several neoplastic cell lines of lymphoid origin such as B cell lymphoma WEHI231 cells,21,22 multiple myeloma,23 Hodgkin/Reed-Sternberg (HRS) cells,24-26 and T cell lymphoma HuT-78 cells.27,28 In the current study, we show for the first time constitutive expression of active NF-B p65(Rel A) in neoplastic T lymphocytes in most mucosis fungoides (MF) patient samples. Analysis of the cutaneous T-cell lymphoma (CTCL) cell lines HuT-78 and HH also showed strong constitutively active nuclear NF-B p65(Rel A) as demonstrated by immunohistochemistry and electrophoretic mobility shift assay (EMSA). Chemical inhibition of NF-B by the proteasome inhibitors MG13211,29,30 and gliotoxin30-32 and the IB␣ phosphorylation inhibitors BAY 11-708230,33 and BAY 11-708530,33 resulted in a marked increase in apoptosis, and an equally impressive decrease in nuclear NF-B expression in both cell lines. These results indicate a critical role for NF-B in cell survival and apoptosis resistance in CTCL.
vated fetal bovine serum (HI-FBS) (Sigma Chemical Co., St Louis, MO), 2 mmol/L L-glutamine (GIBCO-BRL), 25 mmol/L Hepes (Sigma), and antibiotic-antimycotic solution (Sigma). All cell lines were maintained at 37°C in a humidified incubator at 5% CO2.
NF-B Inhibitors To evaluate the possible apoptotic effects of the NF-B inhibitors, 1 ⫻ 106/mL cells from each cell line were cultured in 24-well tissue culture plates (Falcon, Lincoln Park, NJ) and incubated with either 10 mol/L z-Leu-Leu-Leu-aldehyde (MG132, Biomol Research Laboratories, Inc., Plymouth Meeting, PA), 10 mol/L gliotoxin (Calbiochem, La Jolla, CA), 10 mol/L (E)3-[(4-t-Butylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7085, Calbiochem), or 10 mol/L (E)3-[(4methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082, Calbiochem) for 72-hour periods. All reactions were performed at least in duplicate.
Immunohistochemical Determination of NF-B p65(Rel A) After fixation in a 1:1 mixture of acetone and methanol for 10 minutes, cytospins from both CTCL cell lines were stained with a monoclonal mouse anti-human p65 antibody (clone 12H11, 1:75 titer, Boehringer Mannheim Corporation, Indianapolis, IN), which recognizes only the unbound, active form of p65(Rel A) dissociated from IB␣35 and a secondary polyclonal goat anti-mouse immunoglobulin G (IgG)/IgM antibody conjugated to fluorescein isothiocyanate (Biosource International, Camarillo, CA). Analysis of paraffin-embedded patient material for expression of active p65(Rel A) was performed by using a Ventana NEXES automated stainer (Ventana Medical Systems, Tucson, AZ) and streptavidin/horseradish peroxidase detection kit (Ventana) without antigen retrieval. The chromogen used was 3,3⬘-diaminobenzidine tetrahydrochloride. Cases were considered to be positive for p65(Rel A) expression if 50% or more of neoplastic cells showed nuclear positivity with or without cytoplasmic staining. Negative (normal skin), positive (nodular sclerosis Hodgkin’s disease) and isotypic controls were included in each staining run. In addition, the staining pattern of lymphocytes from 4 reactive lymph nodes was also analyzed.
Detection of Apoptosis MATERIALS AND METHODS Patient Samples and Cell Culture Formalin-fixed, paraffin-embedded tissue sections from 24 cases of MF were selected from the surgical pathology archives of Loyola University Medical Center for immunohistochemical determination of active p65 (Rel A). For each specimen, the diagnosis of MF was made retrospectively and confirmed by using established histologic criteria.34 Of the 23 cutaneous specimens analyzed, most fit criteria consistent with plaque or patch lesions of MF. A single lymph node involved by MF was also included in the study. All specimens analyzed were initial diagnostic specimens without previous therapy. CTCL cell lines HuT-78 and HH were obtained from the American Type Culture Collection (ATCC; Rockville, MD). Cell lines were cultured in RPMI 1640 (GIBCO-BRL, Grand Island, NY) supplemented with 20% (vol/vol) heat-inacti-
For propidium iodide staining, treated and untreated cells (1 ⫻ 106 cells/mL) were washed in cold FACS buffer (5 mL phosphate-buffered saline [PBS] without Mg2⫹ or Ca2⫹, 2% sodium azide/deionized water, and 5% [vol/vol] HI-FBS [Sigma]) and resuspended in a 1:6 mixture of 100% HI-FBS and absolute ethanol. After 30 minutes incubation on ice, the cells were washed once again in FACS buffer. The cell pellet was resuspended and incubated at 37°C with 10 g/mL RNAse (Boehringer Mannheim) for 15 minutes. After brief cooling at room temperature, 100 g/mL propidium iodide (PI) was added to each cell suspension followed by overnight incubation at 4°C in the dark. PI fluorescence of individual nuclei from each sample (10,000 events per sample) was analyzed on an Epics XL-MCL flow cytometer (Coulter, Miami Lakes, FL) and expressed on a logarithmic scale as a measure of DNA content using Coulter System II software. Apoptosis was quantitatively measured by PI/cell cycle analysis for the detection of a hypodiploid (sub-G0) population within the cell suspensions.
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The Annexin V/7-AAD staining method was performed per manufacturer’s protocol by using Annexin V-PE (PharMingen, San Diego, CA) and Via-Probe 7-amino-actinomycin D (7-AAD) (PharMingen). Treated and untreated cells (0.5 ⫻ 106 cell/mL) were washed twice in cold PBS and resuspended in 1⫻ binding buffer (10 mmol/L Hepes/NaOH [pH 7.4], 140 mmol/L NaCL, and 2.5 mmol/L CaCl2). Resuspended cells were then incubated for 20 minutes at 20°C to 25°C in the dark with 2 L Annexin V-PE and 5 L 7-AAD. Samples (10,000 events per sample) were then quantitated on a Coulter Epics XL-MCL flow cytometer, recorded in LIST mode, and registered on logarithmic scales. 7-AAD emission was detected in the FL-3 channel (⬎650 nm). Analysis was performed by using Coulter System II software.
Determination of Active NF-B by the Electrophoretic Mobility Shift Assay (EMSA) Nuclear protein was extracted using the method of Gerber et al.36 Protein content was measured by the Bradford method. A consensus double-stranded NF-B probe, 5⬘-AGT TGA GGG GAC TTT CCC AGG C-3⬘ (Integrated DNA Technologies, Inc., Coralville, IA) was end-labeled by using ␥-32Padenosine-5-triphosphate. Five micrograms nuclear extract from investigated cells were then incubated in binding buffer consisting of 10 mmol/L HEPES (pH 7.9), 60 mmol/L KCL, 4% Ficoll, 1 mmol/L 1,4-dithiothreitol, 1 mmol/L ethylene diaminetetra-acetic acid (EDTA) (pH 8.0), and 1 g/mL poly dI:dC (Pharmacia Biotech Inc., Piscataway, NJ) in 20 L volume. Afterward, the end-labeled probe was added (200,000 cpm). Samples were then incubated for 30 minutes on ice followed by loading on 4% nondenaturing polyacrylamide gel (0.25 ⫻ Tris-borate-EDTA [TBE]). Electrophoresis was run for 3 hours in 0.25 TBE buffer at 100 V. Protein complexes were identified by autoradiography. Quantitative densitometry was performed for each band corresponding to NF-B (p50 to p65) using National Institute of Health (NIH) Image 1.62 software (NIH, Bethesda, MD). Densitometric ratios were calculated by dividing the densitometry reading of individual bands from chemically treated cells by the densitometry reading of untreated control cells.
RESULTS Neoplastic Cells From Cases of CTCL Demonstrate Strong Immunopositivity for p65(Rel A)
FIGURE 1. Neoplastic T lymphocytes from cases of Mycosis fungoides show strong immunopositivity for active p65 (Rel A). Positive nuclear and cytoplasmic staining for active p65(Rel A) was found in neoplastic lymphocytes in most MF cases as shown in these individual cutaneous cases (A,B,C, original magnification ⫻1,000; p65(Rel A)/DAB). In comparison, no evidence of positive staining was seen in isotypic controls (D, original magnification ⫻1,000, isotypic IgG). As shown here, keratinocytes in cutaneous tissues involved by MF also showed p65(Rel A) expression; however, keratinocytes and scattered lymphocytes (arrows) found in normal skin samples showed no evidence of p65(Rel A) immunopositivity (E, original magnification ⫻500, p65[Rel A]/DAB). As seen in cutaneous cases, neoplastic lymphocytes in the single lymph node biopsy also
Although constitutive nuclear expression of NF-B was previously demonstrated in the CTCL cell line HuT-78,27,28 investigation for such expression in neoplastic T lymphocytes from patient samples of CTCL was not previously performed. To compare and contrast NF-B expression between cells from CTCL cell lines and patient samples, we examined neoplastic T showed strong positive staining (F, original magnification ⫻1,000, p65[Rel A]/DAB). In all positive MF cases, the pattern of nuclear and cytoplasmic expression of p65(Rel A) was similar to that identified in HRS cells (arrows) from Hodgkin’s disease positive controls (G, original magnification ⫻1,000, p65[Rel A]/DAB). Also for comparison, lymphocytes in reactive lymph node specimens showed concentrated p65(Rel A) expression in follicular centers with a lesser degree of staining found in lymphocytes occupying the interfollicular, mantle, and marginal zones (H, original magnification ⫻100, p65[Rel A]/DAB).
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lymphocytes from the initial diagnostic specimens of 24 cases of MF for the expression of active NF-B p65(Rel A). Immunohistochemical staining of paraffin-embedded MF specimens showed strong nuclear and cytoplasmic expression of active p65(Rel A) in neoplastic T lymphocytes from 21 of 23 (91%) cutaneous cases. In most of these cases, more than 80% of neoplastic lymphocytes showed positive p65(Rel A) expression regardless of histologic pattern (range, 65% to 90% positive staining). In these cutaneous cases, p65(Rel A) expression was characterized by strong nuclear expression with or without cytoplasmic positivity (Fig 1A-C) compared with negative isotypic controls (Fig 1D). Interestingly in most of these cases, keratinocytes also showed immunopositivity for p65(Rel A). This finding may be attributable to the stimulation of NF-B expression in keratinocytes in response to various stimulatory cytokines produced by neoplastic MF cells. In comparison, keratinocytes and scattered lymphoid cells found in normal skin samples were negative for p65(Rel A) expression (Fig 1E). As seen in cutaneous cases, neoplastic MF cells in the single lymph node biopsy also showed strong positive staining (Fig 1F). For each staining run, cases of nodular sclerosis Hodgkin’s disease24-26 were used as positive controls. Typically, HRS cells from these cases showed strong nuclear and cytoplasmic expression of p65(Rel A) (Fig 1G). Interestingly, lymphocytes in reactive lymph node specimens showed concentrated p65(Rel A) expression in follicular centers with a lesser degree of staining found in lymphocytes occupying the interfollicular, mantle, and marginal zones (Fig 1H).
CTCL Cell Lines Demonstrate Markedly Increased Apoptosis and Significantly Decreased Nuclear NF-B Expression After Chemical NF-B Inhibition Having established the presence of active NF-B in CTCL cells from both patient and cell line specimens, we sought to determine the effect of chemical NF-B inhibition on CTCL cell lines. To measure the potential apoptotic effect of NF-B inhibition on CTCL cell lines, determination of apoptotic activity was measured after 72-hour incubation with each of the following NF-B inhibitors: MG132, gliotoxin, BAY 11-7082, and BAY 11-7085. Regardless of the cell line examined (HuT-78 or HH), the methodology of apoptosis measurement (PI v Annexin/7-AAD), or the inhibitory compound used, each compound induced a marked increase in apoptosis compared with baseline controls (Figs 2, 3). In each cell line, the mean increase in apoptosis from the baseline was at least 40% and often was much higher in most cases. To determine whether the apoptotic effect generated by the above compounds was related to inhibition of nuclear NF-B, expression of active NF-B was determined by EMSA, using nuclear extracts prepared from HuT-78 and HH after 24-hour incubation with MG132, gliotoxin, BAY 11-7082, or BAY 11-7085. In both cell lines, treatment with each of the chemical NF-B inhibitors resulted in significantly decreased NF-B DNA binding activity compared with untreated controls (Fig 4). Quantitation of NF-B (p50-p65) by densitometry showed markedly decreased DNA binding activity (8 to 9-fold decrease) in both cell lines after 24-hour incuba-
FIGURE 2. Flow cytometric detection of apoptosis by PI staining for determination of a hypodiploid (sub-G0) population in CTCL cell lines. HuT-78 and HH cells were treated with 10-mol/L concentrations of the various NF-B inhibitors. DNA content in the cells was determined 72 hours after treatment. In contrast to untreated control cells, marked increases in apoptosis were seen in both cell lines regardless of the compound used. The percentage of apoptotic cells 72 hours after treatment is shown in the upper right corner of each box. These data are representative of at least 2 separate experiments.
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FIGURE 3. Apoptosis determination by Annexin/7-AAD staining and flow cytometry in CTCL cells. In both HuT-78 (A) and HH cells (B), chemical inhibition of NF-B by 10-mol/L concentrations of each compound resulted in a marked increase in apoptosis compared with untreated controls. Annexin/7-AAD staining was determined at 72 hours after treatment, with the percentage of apoptosis shown in the upper right corner of each box. These data are representative of at least 2 separate experiments.
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FIGURE 4. Nuclear DNA binding activity of NF-B in CTCL cell lines after 24-hour chemical NF-B inhibition. As shown by electrophoretic mobility shift assay, marked decreases in active nuclear NF-B expression were obtained in each cell line after 24-hour treatment with each of the 4 chemical inhibitors used. The NF-B probe identified p50-p50 homodimers as well as classical NF-B (p50-p65 heterodimer). The individual numbers below each set of bands represent the calculated densitometry ratios for NF-B (p50 to p65) as determined in the Materials and Methods section.
tion with gliotoxin or BAY 11-7082 (Fig 4). For the remaining compounds, DNA binding activity was reduced by approximately 40% or greater with the exception of a 20% decrease in HuT-78 cells after incubation with BAY 11-7085. In addition to EMSA, expression of nuclear NF-B p65(Rel A) in CTCL cell lines was also determined by immunofluorescence staining both before and after incubation with each of the inhibitory compounds. Both HuT-78 (Fig 5A) and HH (Fig 5B) control cells showed bright nuclear expression of p65(Rel A). HuT-78 cells showed a marked reduction in nuclear p65(Rel A) expression after incubation with MG132 (Fig 5C), gliotoxin (Fig 5E), BAY 11-7082 (Fig 5G), or BAY 11-7085 (Fig 5I). Similarly, HH cells also showed a significant decrease in nuclear p65(Rel A) staining after incubation with each of the above-mentioned inhibitory compounds (Figs 5D, 5F, 5H, and 5J, respectively). DISCUSSION We examined the expression of NF-B in CTCLs, including 2 human T cell lymphoma cell lines, HuT-78 and HH, and patient samples of MF. Previous investigations have shown constitutively active NF-B p50p65(Rel A) in the HuT-78 cell line27,28; however, primary patient sample material was not analyzed in these studies. In support of these past investigations, we found strong nuclear and cytoplasmic expression of active p65(Rel A) expression by immunofluorescence staining and EMSA in unstimulated T cells from each CTCL cell line (HuT-78 and HH). Furthermore, for the first time, we showed active p65(Rel A) in neoplastic T lymphocytes from patient samples of MF, including 21 of 23 (91%) cutaneous cases and a single lymph node
with involvement. All specimens analyzed were initial diagnostic specimens, without previous therapy. Much like their cell line counterparts, neoplastic T lymphocytes from patient cases also showed strong nuclear and cytoplasmic expression of p65(Rel A). The correlation of constitutive p65(Rel A) expression between cultured and patient CTCL cell samples is important and implies similarities in their NF-B biologic pathways. With this in mind, however, constitutive expression of p65(Rel A) is not unique to CTCL. Among hematopoietic neoplasms alone, constitutive expression of p65(Rel A) has also been found in chronic lymphocytic leukemia,37 multiple myeloma,23 Hodgkin’s disease,24-26 and adult T-cell leukemia/lymphoma.38 In addition to p50 and p65(Rel A), investigations to identify additional members of the Rel/NF-B family in HuT-78 showed high nuclear levels of p52, and 2 aberrantly processed forms of the p100 protein, p84 and p85.39 From this investigation, Zhang and colleagues39 revealed a rearrangement of the nfkb2 gene responsible for the production of these aberrant proteins via loss of an ankyrin repeat domain in the carboxy-terminus of p100. In addition, both p84 and p85 were found in the nucleus in an unprocessed form, suggesting that these proteins may escape the cytoplasmic sequestration typical of normal p100 because of loss of the C-terminus. As a result, these proteins were found to bind B sites specifically and alter the composition of NF-B complexes in HuT-78 cells.39 Two more recent studies, however, showed a relationship between TNF-␣ production and the nuclear localization of p50, p52, p65(Rel A), and p85 in HuT-78 cells.27,28 In these investigations, antibodies against TNF-␣ inhibited proliferation and downregulated the constitutive activation of NF-B in HuT-78 cells, sug-
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FIGURE 5. Nuclear expression of p65(Rel A) in CTCL cell lines after chemical NF-B inhibition. Immunofluorescence staining of untreated control cells from HuT-78 (A, original magnification ⫻1,000, p65[Rel A]/ polyclonal anti-mouse FITC) and HH (B, original magnification ⫻1,000, p65 [Rel A]/polyclonal anti-mouse FITC) showed bright nuclear expression of p65(Rel A). After 18-hour incubation with MG132 (C, original magnification ⫻1,000, p65[Rel A]/polyclonal anti-mouse FITC), gliotoxin (E, original magnification ⫻1,000, p65[Rel A]/ polyclonal anti-mouse FITC), BAY 117082 (G, original magnification ⫻1,000, p65[Rel A]/polyclonal anti-mouse FITC), or BAY 11-7085 (I, original magnification ⫻1,000, p65[Rel A]/polyclonal anti-mouse FITC) HuT-78 cells showed markedly decreased p65 (Rel A) expression. Likewise, HH cells also showed significantly decreased nuclear expression of p65(Rel A) after 18-hour incubation with MG132 (D, original magnification ⫻1,000, p65 [Rel A]/polyclonal anti-mouse FITC), gliotoxin (F, original magnification ⫻1,000, p65[Rel A]/polyclonal antimouse FITC), BAY 11-7082 (H, original magnification ⫻1,000, p65[Rel A]/ polyclonal anti-mouse FITC), or BAY 11-7085 (J, original magnification ⫻1,000, p65[Rel A]/polyclonal antimouse FITC).
EXPRESSION OF NF-B IN MYCOSIS FUNGOIDES (Izban et al)
gesting an autocrine role for TNF-␣. These findings suggest that the loss of the C-terminal ankyrin domain in p84 and p85 is alone insufficient to cause nuclear translocation. Importantly, the authors of these studies also showed high levels of constitutively expressed TNF-␣, suggesting both the absence of inhibitory feedback mechanisms for TNF gene expression and a vital interrelationship between TNF-␣ and NF-B necessary for survival of HuT-78 cells. Contrary to these findings, however, Dobbeling et al40 reported significantly reduced cell growth in the CTCL cell line SeAx after treating the cells with the same concentration of TNF-␣ (50 pg/mL) reported to be secreted by HuT-78 cells.40 These findings suggest that the autocrine TNF-␣ growth-promoting effect seen in HuT-78 may be a unique characteristic of these cells, rather than a general feature necessary for growth and survival of all CTCL cells. Survival and apoptosis resistance in CTCL cell lines are dependent on cellular mechanisms that promote constitutive activation of NF-B. From the results of our analysis, the ubiquitin-proteasome pathway was shown to play an important role in apoptosis resistance in CTCL cells, such that the proteasome inhibitors MG132 and gliotoxin were capable of producing both downregulation of NF-B expression and a marked apoptotic effect in HuT-78 and HH cells. Proteolytic processing of the p105 protein subunit for generation of the p50p65(Rel A) heterodimer and degradation of IB␣, which is necessary for nuclear translocation of activated NF-B, are 2 important functions of the ubiquitin-proteasome pathway.4,11,41 The proteasome inhibitors used in this study block the 20s proteasome complex in HeLa cells, with MG132 exerting an additional inhibitory effect on the 26s proteasome.11,29-32,41 As a result, these compounds potently inhibit both processes required for NF-B activation. Recently, we and others have shown the possible therapeutic implications of constitutive NF-B downregulation through proteasome inhibition in other hematopoietic neoplasms.29,38,42 From our investigation of Hodgkin’s disease cell lines, we showed both downregulation of NF-B expression and marked apoptosis of Hodgkin/Reed-Sternberg (HRS) cells after treatment with MG132 and gliotoxin.42 Similar to our findings in cultured HRS cells and recently in CTCL cells, Chandra et al29 showed a relationship between suppression of active NF-B and MG132-induced apoptosis in glucocorticoid-resistant chronic lymphocytic leukemia (CLL) cells.29 In addition, Delic and colleagues37 observed similar findings in therapy-resistant CLL cells using the proteasome inhibitor lactacystin. As a whole, these investigations consistently showed inhibition of constitutively expressed NF-B by proteasome inhibition. In addition to the proteasome inhibitors, we investigated the effects of 2 synthetic compounds (BAY 117082 and BAY 11-7085) that block the activation of NF-B at the level of IB phosphorylation/degradation. By preventing nuclear translocation of NF-B, these compounds have been shown to inhibit TNF-␣–
induced expression of several NF-B target genes, including ICAM-1, VCAM-1, and E-selectin.33 Although the specific mechanism of NF-B inhibition is currently undefined, each compound has been shown to stabilize IB␣ by selectively and irreversibly inhibiting phosphorylation of IB␣.33 Thus, in contrast to the proteasome inhibitors that block degradation of phosphorylated IB␣, these compounds inhibit inducible phosphorylation of IB␣. As seen with the proteasome inhibitors, treatment with either BAY 11-7082 or BAY 11-7085 resulted in significant downregulation of NF-B expression and a marked apoptotic effect in HuT-78 and HH cells, once again showing the significance of NF-B for survival of CTCL cells. In conclusion, we show that constitutive expression of NF-B not only is a feature found in CTCL cell lines, but is also a prominent characteristic of neoplastic T lymphocytes from patient samples of MF. Through chemical inhibition of constitutive nuclear NF-B expression, we were able to induce a marked apoptotic effect in HuT-78 and HH, both via inhibition of the ubiquitin-proteasome pathway by MG132 and gliotoxin, and irreversible blockade of degradation of phosphorylated IB␣ by BAY 11-7082 and BAY 11-7085. Overall, these findings indicate that constitutive nuclear NF-B activity is required for cell survival and resistance to apoptosis in CTCL cells. The exact mechanisms by which NF-B provides a survival advantage to CTCL cells, however, remains undetermined. Additional studies of the NF-B and related biologic pathways will likely provide valuable insights into the pathogenesis, progression, and treatment of CTCL as well as other hematopoietic and nonhematopoietic neoplasms.
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