- Email: [email protected]
Distinct Effects of CD30 and Fas Signaling in Cutaneous Anaplastic Lymphomas: A Possible Mechanism for Disease Progression
Distinct Effects of CD30 and Fas Signaling in Cutaneous Anaplastic Lymphomas: A Possible Mechanism for Disease Progression Edi Levi, Zhenxi Wang, Tina Petrogiannis-Haliotis, Walther M. Pfeifer, Werner Kempf, Reed Drews, and Marshall E. Kadin Departments of Pathology and Medicine, Beth Israel-Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, U.S.A.
Lymphomatoid papulosis is part of a spectrum of CD30+ cutaneous lymphoproliferative disorders characterized by spontaneous tumor regression. The mechanism(s) of regression is unknown. In a recent study, a selective increase in CD30 ligand expression in regressing lesions of lymphomatoid papulosis and cutaneous CD30+ anaplastic large cell lymphoma was shown, suggesting that activation of the CD30 signaling pathway may be responsible for tumor regression, whereas no difference in Fas/Fas ligand expression was found between regressing and nonregressing lesions. Therefore we tested the effects of CD30 and Fas activation on three CD30+ cutaneous lymphoma cell lines (Mac-1, Mac-2 A, JK) derived from nonregressing tumors of two patients who had progressed from lymphomatoid papulosis to systemic anaplastic large cell lymphoma. To evaluate the effects of CD30 signaling, the cell lines were incubated with a CD30
P
rimary cutaneous CD30+ lymphoproliferative disorders (LPD) are recognized as a distinct biologic entity in the European Organization for Research and Treatment of Cancer classi®cation of cutaneous lymphomas and the forthcoming World Health Organization classi®cation of lymphomas (Willemze et al, 1997; Jaffe et al, 1999). CD30+ cutaneous LPD are associated with frequent spontaneous regression (Cabanillas et al, 1995; Paulli et al, 1995). Although cutaneous CD30+ LPD generally have a good prognosis, certain cases progress to aggressive systemic lymphomas and become resistant to chemotherapy (Willemze and Beljaards, 1993; Paulli et al, 1995; Willemze et al, 1997; Bekkenk et al, 2000). CD30 antigen expression is a favorable prognostic factor in cutaneous T cell lymphomas (Beljaards et al, 1993; Willemze et al, 1993; Paulli et al, 1995; Bekkenk et al, 2000). With the exception of Manuscript received May 5, 2000; revised July 28, 2000; accepted for publication August 22, 2000. Reprint requests to: Dr. Marshall E. Kadin, Director of Hematopathology, Department of Pathology, YA 309, Beth Israel-Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. Email: [email protected] Abbreviations: ALCL, anaplastic large cell lymphoma; LPD, lymphoproliferative disorders; LyP, lymphomatoid papulosis; MAPK, mitogenactivated protein kinase; NF-kB, nuclear factor kB; TNF, tumor necrosis factor. 0022-202X/00/$15.00
agonistic antibody, HeFi-1. Proliferative responses, mitogen-activated protein kinase, and nuclear factor kB activities were determined with and without CD30 activation. Mac-1 and Mac-2 A showed increased proliferative responses to incubation with CD30 activating antibody, HeFi-1. Inhibition of the mitogen-activated protein kinase activity caused growth inhibition of the Mac-1, Mac-2 A, and JK cell lines. Activation of the Fas pathway induced apoptosis in all three cell lines. Taken together, these ®ndings suggest that resistance to CD30-mediated growth inhibition provides a possible mechanism for escape of cutaneous anaplastic large cell lymphoma from tumor regression. Mitogenactivated protein kinase inhibitors are potential therapeutic agents for the treatment of advanced cutaneous anaplastic large cell lymphoma. Key words: CD30/ cutaneous anaplastic large cell lymphoma/MAPK/NF-kB. J Invest Dermatol 115:1034±1040, 2000
mycosis fungoides, CD30-negative T cell lymphomas of the skin tend to have a more aggressive course and a poorer prognosis than CD30+ cutaneous LPD. Members of the tumor necrosis factor (TNF) receptor superfamily, such as Fas (CD95), TNF receptors types I and II, CD27, CD30, CD40, DR3, DR4, and DR5 play an important role in immune regulation. These members of the TNF receptor family contain a ``death domain'' that triggers programmed cell death when activated by the appropriate ligand (Ashkenazi and Dixit, 1998). Some members of the TNF receptor and ligand superfamily are implicated in the pathogenesis of immune disorders and lymphomas (Gruss and Dower, 1995). An important function of the TNF members is regulation of growth and elimination of peripheral T cells during their expansion in response to antigenic stimuli. T cell apoptosis occurs in two major forms: antigen driven and lymphokine withdrawal induced. Antigen-driven death is induced by the activation of the T cell antigen receptor, and is regulated by Fas and TNFRI (Lenardo et al, 1999). The speci®city of T cell elimination by this pathway is partly due to a requirement for simultaneous activation of T cell antigen receptor and the TNF or Fas receptor (Lenardo et al, 1999). The second mechanism of T cell elimination is cytokinewithdrawal-induced cell death. In this pathway, T cells are deprived of the cytokine support that is associated with the antigen-induced activation state (Telford et al, 1997; Lenardo et al, 1999). This mechanism of apoptosis is prevented by the presence of
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 1034
VOL. 115, NO. 6 DECEMBER 2000
cytokines such as interleukin 2 (IL-2), IL-4, and IL-7 (Lenardo et al, 1999). Telford et al (1997) have shown in a mouse model that antigen-withdrawal-induced apoptosis is independent of Fas and TNFRI but is regulated mainly by CD30 antigen. CD30 is normally detectable on a small number of immunoblasts located in the perifollicular region around germinal centers (Falini et al, 1995). In addition, CD30 is expressed by Reed-Sternberg cells in virtually all cases of classical Hodgkin's lymphoma and by tumor cells in anaplastic large cell lymphoma (ALCL) (Falini et al, 1995). CD30 ligand (CD30L) is expressed on neutrophils, histiocytes, eosinophils, and a small subset of activated T cells (Smith et al, 1993). Accumulating evidence suggests that CD30 acts as a negative regulator of T cells. CD30 knock out mice show thymic hyperplasia and a defect in negative selection of T cells responding to self-antigens in the thymus (Amakawa et al, 1996). In a murine model, CD30 antigen is protective against autoimmune type I diabetes mediated by autoreactive CD8+ cells (Kurts et al, 1999), and Chiarle et al (1999) demonstrated enhanced cell death of T cells that express CD30 in response to antigen stimulation and CD30 costimulation. As CD30+ cutaneous LPD represents clonal expansion of an abnormal T cell clone, the normal fate of these T cells should be elimination by one of the mechanisms mentioned above. The early lesions do regress, but the mechanism of spontaneous regression seen in these lesions is unknown. We believe that Fas and CD30 are two signaling pathways that can mediate regression. The tumor cells in CD30+ cutaneous LPD have been shown to express Fas (Paulli et al, 1998; Mori et al, 1999). CD30L expression shows a correlation with regression of the lesions suggesting a role for CD30 activation in the usual regression of cutaneous CD30+ lymphomas (Mori et al, 1999). Until now, there has not been a functional study of the responses of tumor cells from cutaneous CD30+ LPD to the activation of TNF receptor superfamily members including Fas and CD30, due to the lack of tumor cell lines. We developed three cell lines from cutaneous tumors and circulating tumor cells of two patients who had lymphomatoid papulosis (LyP) and then progressed to fatal systemic lymphomas during the course of their disease (Davis et al, 1992; Kadin et al, 1994; Schiemann et al, 1999). We investigated the responses of these cell lines to Fas and CD30 activation in vitro. We also investigated the intracellular signaling mechanisms associated with CD30 activation, including nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK), in order to understand the mechanism of disease progression and to develop novel therapies targeting these pathways in advanced disease originating from cutaneous CD30+ LPD. MATERIALS AND METHODS Cell lines JK, Mac-1, and Mac-2 A are cutaneous ALCL cell lines (Kadin et al, 1994). The JK cell line was developed from an advanced skin tumor of a patient who progressed over a 10 y period from LyP to ALCL (Schiemann et al, 1999); the clonal relationship of LyP and ALCL lesions has been demonstrated (Chott et al, 1996). JK cells have the same phenotype as the original tumor (CD30+, CD3+). Mac-1 and Mac-2 A are clonally related cell lines derived from a CD30+ cutaneous T cell ALCL that had progressed from LyP over a 17 y period. Details of the cell lines have been reported elsewhere (Davis et al, 1992; Kadin et al, 1994). Immunohistochemistry Immunohistochemical staining for CD30L was performed on 4 mm sections of formalin-®xed paraf®n-embedded material as previously described (Mori et al, 1999). Sections were stained with the M80 antibody against CD30L kindly provided by Immunex, Seattle, WA. Antibodies and inhibitors HeFi-1 (NCI, Frederick, MD) is a mouse monoclonal IgG1 antibody raised against human CD30 (Hecht et al, 1985). HeFi-1 has an agonistic effect on the CD30 pathway mimicking the effects of CD30L. Mouse IgG1 (Sigma, St. Louis, MO) is used as an isotypespeci®c control. SN50 is a synthetic peptide that is cell permeable and inhibits NF-kB by competing with the nuclear localization sequence of NF-kB (p50) (Lin et al, 1995). SN50M is a synthetic peptide mutated at the nuclear localization sequence site so that it can be used as a negative control
CD30 SIGNALING IN CUTANEOUS ALCL
1035
Figure 1. Immunohistochemical staining of CD30L. Immunohistochemistry with antibody M80 reveals staining of Reed-Sternberg-like cells and some smaller cells in LyP lesions of patients from whom Mac-1 and Mac-2 A cells (a and b) and JK cells (c and d) were derived. for SN50. U0126 is a selective inhibitor of MEK1 and MEK2 (Favata et al, 1998). All three chemicals were purchased from Calbiochem (San Diego, CA). Anti-human Fas antibody with agonistic properties was obtained from R&D Systems, Minneapolis, MN (clone DX2.1). 3H-Thymidine incorporation assay Tumor cells were grown in round-bottomed 96 well plates in triplicate. Following incubation with soluble antibodies and 10% fetal bovine serum in RPMI, the wells were pulsed with 1 mCi 3H-thymidine during the ®nal 12 h. Tumor cells were harvested with the aid of a cell harvester (PhD Systems, Cambridge, MA) and radioactivity was measured with a scintillation counter (Wallac 1409; Wallac, Gaithersburg, MD).
Electrophoretic mobility shift experiments Nuclear extracts from tumor cell lines were obtained before and after stimulation with HeFi-1 antibody for 1 h. The extracts were incubated with 32P end-labeled NF-kB binding site oligoprobes (5¢-AGCTTGGGGTATTTCCAGCCG-3¢), mutant oligoprobes (5¢-AGCTTGGCATAGGTCCAGCCG-3¢), and excess cold oligoprobes, and were run on a nondenaturing 6% polyacrylamide gel. Gels were dried and exposed to ®lm (NF-kB/Rel T cell activation Gelshift kit, Geneka, Montreal, Canada). Nuclear extracts were prepared as follows: 10 3 106 cells were incubated with HeFi-1 for 1 h, and then washed in phosphate-buffered saline and lyzed in 400 ml cold buffer A [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl ¯uoride (PMSF), pH 7.9]. Samples were kept on ice for 15 min, and then vortexed for 10 s. Nuclei were pelleted by microcentrifugation for 10 s. Pellets were resuspended in 40 ml buffer B (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediamine tetraacetic acid, 0.5 mM DTT, 0.2 mM PMSF) and incubated on ice for 20 min. Nuclei were pelleted by spinning for 3 min at 4°C. Supernatants were aliquoted and stored at ±80°C. MAPK kinase assay MAPK activity was determined by the MAPK kinase assay kit according to the manufacturer's recommended protocols (New England Biolabs, Beverly, MA). Brie¯y, whole cell lysates were immunoprecipitated with an anti-MAPK antibody. The immunoprecipitated proteins were used in a nonradioactive in vitro kinase assay. A Gst-Elk1 fusion peptide was used as a substrate for the detection of kinase activity in the immunoprecipitates. Following the kinase assay, phosphorylation of the Gst-Elk1 substrate was measured by Western blots using an antiphopho-Elk1 antibody for detection.
RESULTS Immunohistochemical staining for CD30L Immunohistochemical staining con®rmed the presence of CD30L in tissue sections from the LyP mainly in the cytoplasm of 10%-20% of large atypical cells, including Reed-Sternberg-like cells, and some smaller cells (Fig 1). Staining of CD30L appeared to be associated with degenerative changes in some cells of the case from which Mac-1 and Mac-2 A cell lines were derived.
1036
LEVI ET AL
Figure 2. Proliferative responses to CD30 activation. Proliferative activity was measured by a 3H-thymidine incorporation assay. Cutaneous CD30+ ALCL cell lines JK, Mac-1, and Mac-2 A were incubated in 10% fetal bovine serum and RPMI in the presence of CD30 activating antibody HeFi-1 (1 mg per ml) for 48 h and pulsed with 3H-thymidine (1 mCi) during the last 12 h of incubation. Control cells were incubated with isotypespeci®c mouse IgG. The counts are shown as percentages compared to controls (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 4. Signaling pathways triggered by CD30 activation. This cartoon depicts intracellular signaling by CD30 activation through TRAFs, NF-kB, and MAPK. The sites of action of the inhibitors used in this study are shown. GFR, growth factor receptor.
death was con®rmed (not shown). As expected, the Karpas 299 cell line, which is known to be resistant to Fas-mediated apoptosis, did not show inhibition of cell growth upon Fas activation (not shown).
Figure 3. Fas-mediated growth inhibition of the cell lines. Effects of Fas activation on the cell lines was determined by 3H-thymidine incorporation assays following incubation with a Fas agonist antibody for 24 h. The counts are shown as percentages compared to controls (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
Effects of CD30 activation on cell proliferation In order to analyze the functional status of the CD30/CD30L signaling pathway in the cell lines representing advanced cutaneous ALCL, we used a monoclonal antibody, HeFi-1 (Hecht et al, 1985), that recognizes the CD30L binding domain of CD30 antigen and leads to activation of the CD30 signaling cascade. We and others have previously shown that this antibody mimics the action of CD30L and is an effective growth inhibitor of tumor cells of systemic CD30+ ALCL, both in vitro and in vivo (Tian et al, 1995; Pfeifer et al, 1999). Upon activation of the CD30 signaling pathway by HeFi-1, Mac-1 and Mac-2 A cell lines showed proliferative responses whereas the JK cell line did not change its proliferative activity, as measured in a 3H-thymidine incorporation assay (Fig 2). We also tested the effects of CD30 activation of the Karpas 299 cell line, a systemic ALCL cell line with the translocation t(2; 5)(p23; q35), and observed a 50% growth inhibition as previously reported (Pfeifer et al, 1999) (not shown). Effects of Fas activation on cell growth In order to assess their sensitivity to Fas-mediated apoptosis in vitro, the cell lines were incubated with a Fas agonistic antibody (clone DX2.1, R&D systems) for 24 h, and then pulsed with 3H-thymidine for 12 h. All three cell lines demonstrated signi®cant growth inhibition in response to Fas activation whereas control cells incubated with an isotype-speci®c mouse IgG were unaffected (Fig 3). Cell viability was independently assessed by trypan blue vital staining and cell
Role of NF-kB in CD30 signaling Following the assessment of the responses of the cell lines to CD30 and Fas activation, we wanted to analyze the downstream effectors of the CD30 signaling pathway. The immediate transducers of CD30 signaling are the TNF-associated factors (TRAFs). TRAF-1, TRAF-2, TRAF-3, and TRAF-5 are known to bind the intracytoplasmic domain of CD30 (Arch et al, 1998). TRAF-2 is known to activate NF-kB and the c-jun N-terminal kinase (JNK) pathways (Song et al, 1997). In addition, in some cell lines MAPK is activated by CD30 signaling (Wendtner et al, 1995). As NF-kB and MAPK pathways are important for numerous cell functions including cell survival, proliferation, and cytokine secretion, we thought these two pathways would be most important to explain the proliferative responses observed by CD30 activation (Hunter, 1995; Barnes and Karin, 1997; Sonenshein, 1997). As the JNK pathway is usually associated with stress responses and apoptosis, we did not pursue that pathway (Ip and Davis, 1998). The downstream signaling pathways associated with CD30 signaling and the sites of action of the inhibitors used in the study are illustrated in Fig 4. We measured the nuclear NF-kB activity of cell lines Mac-1 and Mac-2 A before and after CD30 activation. Nuclear extracts were obtained from cells that were incubated with antibody HeFi-1. The nuclear extracts were incubated with 32P-labeled oligoprobes representing NF-kB binding sites and run on a polyacrylamide gel. Gelshift experiments showed minimal constitutive NF-kB activity in Mac-1 and Mac-2 A cell lines. In contrast, following incubation with HeFi-1 for 30 min, both cell lines showed strong NF-kB activation (Fig 5). The speci®city of the binding was tested by addition of excess cold probe and excess cold mutant probe. We then investigated the NF-kB pathway by use of a speci®c inhibitor of NF-kB, SN50 (Lin et al, 1995). Mac-1 and Mac-2 A cell lines demonstrated inhibition of cell growth within 24 h when incubated with the NF-kB inhibitor SN50 at 50 mg per ml concentration in the presence of HeFi-1 (1 mg per ml). The JK cell line showed no response to the NF-kB inhibitor (Fig 6). As a negative control for SN50, we incubated the cells with SN50M, which is a synthetic peptide that differs from SN50 in just two amino acids that correspond to the nuclear localization sequence.
VOL. 115, NO. 6 DECEMBER 2000
CD30 SIGNALING IN CUTANEOUS ALCL
1037
Figure 6. Effects of NF-kB inhibition during CD30 activation. The NF-kB inhibitor SN50 was added 30 min before the addition of HeFi-1. 3H-Thymidine was added in the last 12 h of the 24 h incubation. The results are shown as percentages of the control values (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
Figure 5. Activation of NF-kB by CD30 activation. Nuclear extracts obtained from Mac-1 and Mac-2 A cell lines were incubated with 32Plabeled oligoprobes (H), (H) + excess cold oligoprobes (C), and (H) + excess cold mutant oligoprobes (M), with or without HeFi-1 for 1 h. The arrows show the speci®c bands, which are retarded due to the binding of the probes to nuclear proteins, and the unbound probes at the bottom of the gel. The remaining bands are due to nonspeci®c binding of the probe to other nuclear proteins. Unlike the speci®c bands, which disappear when excess cold unlabeled oligoprobes are added (C), these bands do not disappear. A representative experiment is shown.
Figure 7. Activation of MAPK by CD30 signaling. MAPK activity was measured after CD30 activation for 10 min, and control cells were incubated with isotype-speci®c mouse IgG. CD30 activation was achieved by HeFi-1 antibody. Mac-2 A cell line was also incubated with the MEK1 inhibitor U0126 for 10 min and the MAPK activity was measured. Equal loading of wells was con®rmed by measuring the protein concentration of the cell lysates before immunoprecipitation, and staining the blotted membranes for protein.
Incubation with SN50M caused no changes in the responses of the cells, con®rming the speci®city of responses to SN50 (not shown). Role of MAPK in CD30 signaling Another candidate pathway to explain the responses of the cell lines to CD30 activation is MAPK. As in the NF-kB experiments, we measured the activity of MAPK before and after CD30 activation by a kinase assay, and then tested the effects of inhibitors of the MAPK pathway. The cell lines were starved in serum-free media for 24 h before the experiments. CD30 activation was achieved by incubating the cell lines with the HeFi-1 antibody for 10 min. JK and Mac-1 cell lines had weak to minimal baseline MAPK activities, which were enhanced by CD30 activation, whereas Mac-2 A had a high constitutive MAPK activity that was slightly increased by CD30 activation (Fig 7). We also tested the effects of MAPK inhibition on the proliferative properties of the cell lines by using a recently described speci®c MEK1 inhibitor, U0126 (Favata et al, 1998). The cells were incubated with 1 or 10 mM of the inhibitor for 30 min, and then HeFi-1 was added. We measured 3H-thymidine incorporation after 24 h. At 1 mM, all cell lines demonstrated signi®cant growth inhibition. When 10 mM of the inhibitor was added, the cell lines were almost completely growth inhibited (Fig 8). DISCUSSION In this study we demonstrate that activation of CD30 in cutaneous CD30+ ALCL cell lines does not induce growth inhibition; instead it causes proliferation of the Mac-1 and Mac-2 A cell lines. Others have shown that activation of CD30 by a monoclonal antibody could induce sustained proliferation of human autoreactive gd T
Figure 8. Inhibition of MAPK activity causes growth inhibition. Growth responses of the cell lines to inhibition of MEK1 were tested at two different doses of the MEK1 and MEK2 inhibitor, U0126.The inhibitor was added 30 min before the addition of HeFi-1. Responses are shown as percentages of the control values (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
cells in the presence of recombinant IL-2 (Leca et al, 1994). Those cells were not transformed, however, and cell growth arrest occurred upon removal of the antibody. In contrast, our cell lines express an ab rather than gd phenotype and do not undergo growth arrest upon removal of the CD30 activating antibody, HeFi-1. Cutaneous and systemic CD30+ ALCL cell lines appear to have opposite growth responses to CD30 activation as CD30 activation is an inhibitory signal for systemic ALCL cell lines (Gruss et al, 1994). Cutaneous and systemic ALCL also have distinct pathways of pathogenesis. Systemic ALCL cell lines harbor the
1038
LEVI ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 9. Hypothesis for mechanisms of progression of cutaneous CD30+ LPD. The cartoon depicts the growth inhibitory effects of CD30L, Fas, and TGF-b on LyP in regressing skin lesions. Our studies indicate that tumor progression to ALCL is in part due to escape from growth inhibition through altered CD30 signaling and mutations of the TGF-b receptor (Knaus et al, 1996; Schiemann et al, 1999).
translocation t(2; 5)(p23; q35), which causes overexpression of the NPM/ALK fusion protein (ALK) leading to the unregulated growth of tumor cells (Morris et al, 1994). ALK is characteristically absent in cutaneous ALCL (DeCoteau et al, 1996). Therefore, cutaneous and systemic ALCLs have been identi®ed as separate entities in the new lymphoma classi®cations (Jaffe et al, 1999). There is limited information regarding the etiology and biology of CD30+ cutaneous ALCL. In general, the primary cutaneous LPD constitute a spectrum of diseases ranging from LyP to cutaneous ALCL, which have an excellent prognosis (Paulli et al, 1995). A small percentage of these diseases (approximately 5%), however, progress to fatal systemic lymphomas (Beljaards and Willemze, 1992). The cell lines we used in our study are representative of such uncommon cases that ®rst present as LyP and later progress to fatal systemic lymphomas. A well-known phenomenon associated with cutaneous CD30+ LPD is the frequent occurrence of spontaneous regression (Willemze et al, 1993). In a recent study, a positive correlation was demonstrated between regression and CD30L expression levels measured by semiquantitative reverse transcriptase polymerase chain reaction in cases of LyP and primary cutaneous ALCL, suggesting that CD30 activation may play a role in the spontaneous regression of such lesions (Mori et al, 1999). In this study, we con®rmed the presence of CD30L in the LyP skin lesions of each patient by immunohistochemistry. Our results raise the possibility that the development of resistance to the CD30L-mediated growth inhibitory effects, or even transformation of the tumor response to one of cellular proliferation in response to CD30 activation, may provide a mechanism for tumor cell escape from the normal growth regulatory responses and support progression of LyP to ALCL. Recently, Fas expression was demonstrated in cutaneous CD30+ LPD (Paulli et al, 1998; Mori et al, 1999). A functional role for Fas-mediated apoptosis in the regression of these lesions, however, has not been shown. We investigated the susceptibility of our cutaneous ALCL lines to Fas-induced apoptosis. As all three cell lines were sensitive to Fas-induced apoptosis, we conclude that the Fas signaling pathway is unlikely to play a role in the progression of LyP to ALCL. In order to gain insight into the downstream effects of CD30 activation, we investigated some pathways that are involved in the CD30/CD30L signaling system. CD30 activation is known to cause upregulation of NF-kB, utilizing the TRAFs as adapter molecules (Duckett et al, 1997; Song et al, 1997). Constitutive activation of NF-kB has been demonstrated in Hodgkin's lymphoma cell lines and has been implicated in their survival and
tumorigenicity (Bargou et al, 1997). We were interested to compare cutaneous ALCL cell lines with Hodgkin's lymphoma as these diseases have similar morphologic and immunohistochemical features (Davis et al, 1992; Kadin, 1993). In addition, patients with LyP sometimes develop Hodgkin's lymphoma (Davis et al, 1992; Wang et al, 1992; Cabanillas et al, 1995). Our study shows that, unlike Hodgkin's lymphoma, cutaneous ALCL cell lines do not have high constitutive NF-kB activity. The ERK (MAPK) pathway is also known to be activated by members of the TNF receptor family, including CD30 (Wendtner et al, 1995; Wallach et al, 1999). In a previous study by Wendtner et al (1995), CD30 signaling led to MAPK activation in the T-celltype Hodgkin's lymphoma cell line HDLM-2, but not in the systemic ALCL cell line Karpas 299 nor in the B-cell-type Hodgkin's lymphoma cell line KMH2. Interestingly, the HDLM-2 cell line showed increased proliferation in response to CD30 activation whereas Karpas 299 was growth inhibited. Here, we demonstrate that the MAPK (ERK) pathway is activated by CD30 signaling in cutaneous T cell ALCL cell lines Mac-1 and JK as well, whereas Mac-2A has high constitutive expression of MAPK; inhibition of MAPK is an effective way of inhibiting growth of these cell lines. The mechanism of altered responsiveness to CD30 activation in the cell lines from advanced cutaneous ALCL is unclear. We have shown that, in the systemic ALCL cell line Karpas 299, CD30 activation causes cell cycle arrest despite activation of the NF-kB pathway (Levi et al, submitted for publication). The proliferative response seen in the Mac-1 and Mac-2 A cell lines was at least partly due to MAPK and NF-kB activation by CD30 signaling. In this study, we demonstrated that MAPK activation is one of the major effectors of CD30 signaling in these cutaneous ALCL cell lines. It has been shown that mammalian cells can be transformed by transfection with constitutively active MAPK (Mansour et al, 1994). In addition, inhibition of the MAPK activity in mice xenografted with colon tumors causes inhibition of tumor growth (Sebolt-Leopold et al, 1999). We believe that inhibitors of the MAPK pathway can become useful in the treatment of various cancers as the Ras pathway, which is upstream of the MAPK, is frequently mutated in human cancers (Macara et al, 1996; Hunter, 1997). Based on this study and previous work, we propose a model of progression of LyP to systemic lymphoma by several steps that abolish the physiologic inhibitory mechanisms that are important for the regulation of the immune system such as transforming growth factor beta (TGF-b) and CD30 (Fig 9). According to this
VOL. 115, NO. 6 DECEMBER 2000
model, an unknown antigenic stimulus triggers clonal expansion of T cells, which form cutaneous in®ltrates that manifest as multiple regressing skin lesions of LyP. The LyP cells are usually eliminated by growth inhibitory mechanisms such as Fas, CD30, and TGF-b. Therefore, the lesions spontaneously regress in weeks to months. After recurrent episodes of spontaneously regressing LyP lesions, a small number of patients will develop large lesions that require treatment. A few of these patients will have extracutaneous spread of the disease, which may be resistant to chemotherapy. We hypothesize that in these patients disease progression from LyP to ALCL is associated with impairment of the normal T cell growth inhibitory mechanisms that would otherwise cause regression of these lesions. In these and prior experiments, we tested three of the best characterized inhibitory mechanisms that regulate the T cell arm of the immune system - Fas, CD30, and TGF-b - and demonstrated a selective role for CD30 in the context of escape from growth inhibitory mechanisms. In the case of TGF-b, resistance was shown to be mediated by mutations of the type I (JK) and type II (Mac-2 A) receptors (Knaus et al, 1996; Schiemann et al, 1999). These mutations were due to a deletion that eliminates the initiating methionine required for translation of the type I receptor in the JK cell line, and due to a single nucleotide substitution (A to G) in the kinase domain of the type II receptor in the Mac-2 A cell line, whereas Mac-1, which is inhibited by TGF-b, had no TGF-b receptor mutation. Both mutations were present in the corresponding tissue sections obtained from the patients. Preliminary data from our laboratory indicate the presence of mutations in CD30 that may alter CD30 signaling and could explain the proliferative responses of Mac-1 and Mac-2 A cells and failure of JK cells to respond to HeFi-1. At present no cell lines from regressing lesions are available for comparison but this is a long-term goal. The fact that both TGF-b and CD30 signaling systems were noninhibitory suggests that abrogation of both pathways may be required for the progression of these lymphomas to fatal systemic disease. In summary, we have demonstrated that in addition to evading TGF-b-mediated growth control, advanced CD30+ ALCL cell lines can be resistant to growth inhibition by CD30 signaling, but retain susceptibility to Fas-induced cell death. We also identi®ed the ERK (MAPK) pathway inhibitors as candidates for novel therapies against these advanced cutaneous ALCL. EL was a fellow of the Lymphoma Research Foundation of America. WP was a fellow of Deutsche Krebshilfe. TP-H was a fellow of the Cure for Lymphoma Foundation. WK is ®nancially supported by the Schweizerische Stiftung fuÈr Medizinisch-Biologische Stipendien, Basel, Switzerland. This study was supported by grants to Dr. Kadin from the American Cancer Society (ROG-98±125±01), the Leukemia and Lymphoma Society (178±98), and Merritt Care Foundation, Fargo, ND.
REFERENCES Amakawa R, Hakem A, Kundig TM, et al: Impaired negative selection of T cells in Hodgkin's disease antigen CD30-de®cient mice. Cell 84:551±562, 1996 Arch RH, Gedrich RW, Thompson CB: Tumor necrosis factor receptor-associated factors (TRAFs) - a family of adapter proteins that regulates life and death. Genes Dev 12:2821±2830, 1998 Ashkenazi A, Dixit VM: Death receptors: signaling and modulation. Science 281:1305±1326, 1998 Bargou RC, Emmerich F, Krappman D, et al: Constitutive nuclear factor kB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells. J Clin Invest 100:2961±2969, 1997 Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic in¯ammatory diseases. N Engl J Med 336:1066±1071, 1997 Bekkenk MW, Geelen FAMJ, van Vorst Vader PC, et al: Primary and secondary cutaneous CD30+ lymphoproliferative disorders: a report from the Dutch Cutaneous Lymphoma Group on the long-term follow-up data of 219 patients and guidelines for diagnosis and treatment. Blood 95:3653±3661, 2000 Beljaards RC, Willemze R: The prognosis of patients with lymphomatoid papulosis associated with malignant lymphomas. Br J Dermatol 126:596±602, 1992 Beljaards RC, Kaudewitz P, Berti E, et al: Primary cutaneous CD30-positive large cell lymphoma: de®nition of a new type of cutaneous lymphoma with a
CD30 SIGNALING IN CUTANEOUS ALCL
1039
favorable prognosis: a European multicenter study on 47 patients. Cancer 71:2097±2104, 1993 Cabanillas F, Armitage J, Pugh WC, Weisenburger D, Duvic M: Lymphomatoid papulosis: a T cell dyscrasia with a propensity to transform into malignant lymphoma. Ann Intern Med 122:210±217, 1995 Chiarle R, Podda A, Prolla G, Podack ER, Thorbecke GJ, Inghirami G: CD30 overexpression enhances negative selection in the thymus and mediates programmed cell death via a Bcl-2 sensitive pathway. J Immunol 163:194±205, 1999 Chott A, Vonderheid EC, Olbricht S, Miao N-N, Balk SP, Kadin ME: The dominant T cell clone is present in multiple regressing skin lesions and associated T cell lymphomas of patients with lymphomatoid papulosis. J Invest Dermatol 106:696±700, 1996 Davis TH, Morton CC, Miller-Cassman R, Balk SP, Kadin ME: Hodgkin's disease, lymphomatoid papulosis and cutaneous T cell lymphoma derived from a common T cell clone. N Eng J Med 326:1115±1122, 1992 DeCoteau JF, Butmarc JR, Kinney MC, Kadin ME: The t (2; 5) chromosomal translocation is not a common feature of primary cutaneous CD30+ lymphoproliferative disorders: comparison with anaplastic large cell lymphoma of nodal origin. Blood 87:3437±3441, 1996 Duckett CS, Gedrich RW, Gil®an MC, Thompson CB: Induction of nuclear factor kB by the CD30 receptor is mediated by TRAF1 and TRAF2. Mol Cell Biol 17:1535±1542, 1997 Falini B, Pileri S, Pizzolo G, et al: CD30 (Ki-1) molecule: a new cytokine receptor of the tumor necrosis factor receptor superfamily as a tool for diagnosis and immunotherapy. Blood 85:1±14, 1995 Favata MF, Horiochi KY, Manos EJ, et al: Identi®cation of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273:18623±18632, 1998 Gruss HJ, Dower SK: Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas. Blood 85:3378±3404, 1995 Gruss HJ, Boiani N, Williams DE, Armitage RJ, Smith CA, Goodwin RG: Pleiotropic effects of the CD30 ligand on CD30 expressing cells and lymphoma cell lines. Blood 83:2045±2056, 1994 Hecht TT, Longo DL, Cossman J, Bolen HB, Hsu SM, Israel M, Fisher RI: Production and characterization of a monoclonal antibody that binds ReedSternberg cells. J Immunol 134:4231±4236, 1985 Hunter T: Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signaling. Cell 80:225±236, 1995 Hunter T: Oncoprotein networks. Cell 88:333±346, 1997 Ip TY, Davis RJ: Signal transduction by the c-jun N-terminal kinase (JNK) - from in¯ammation to development. Curr Opin Cell Biol 10:205±219, 1998 Jaffe ES, Harris NL, Diebold J, Muller-Hermelink HK: WHO classi®cation of neoplastic diseases of the hematopoietic and lymphoid tissues. A progress report. Am J Clin Path 111 (Suppl. 1):S8±S12, 1999 Kadin ME: Lymphomatoid papulosis and associated lymphomas. How are they related? Arch Dermatol 129:351±352, 1993 Kadin ME, Cavaille-Coll MW, Gertz R, Massague J, Cheifetz S, George D: Loss of receptors for transforming growth factor beta in human T-cell malignancies. Proc Natl Acad Sci USA 91:6002±6006, 1994 Knaus PI, Lindemann D, DeCoteau JF, et al: A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T cell. Lymphoma Mol Cell Biol 16:3480±3489, 1996 Kurts C, Carbone FR, Krummel MF, Koch KM, Miller JFAP, Heath WR: Signaling through CD30 protects against autoimmune diabetes mediated by CD8 T cells. Nature 398:341±344, 1999 Leca G, Vita N, Maiza H, Fasseu M, Bensussan A: A monoclonal antibody to the Hodgkin's associated antigen CD30 induces activation and long-term growth of human autoreactive gd T cell clone. Cell Immunol 156:230±239, 1994 Lenardo M, Chan FKM, Hornung F, McFarland H, Siegel R, Wang J, Zheng L: Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment. Annu Rev Immunol 17:221±253, 1999 Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J: Inhibition of nuclear transcription factor NF-kB by a synthetic peptide containing a cell membranepermeable motif and nuclear localization sequence. J Biol Chem 270:14255± 14258, 1995 Macara IG, Lounsbury KM, Richards SA, McKiernan C, Bar-Sagi D: The Ras superfamily of GTPases. FASEB J 10:625±630, 1996 Mansour SJ, Matten WT, Hermann AS, et al: Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966±970, 1994 Mori M, Manuelli C, Pimpinelli N, et al: CD30±CD30L interaction in primary cutaneous CD30+ positive T cell lymphomas: a clue to the pathophysiology of clinical regression. Blood 94:3077±3083, 1999 Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL, Look AT: Fusion of a kinase gene ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263:1281±1284, 1994 Paulli M, Berti E, Rosso R, et al: CD30/Ki-1 positive lymphoproliferative disorders of the skin - clinicopathologic correlation and statistical analysis of 86 cases: a multicentric study from the European Organization for Research and Treatment of Cancer, Cutaneous Lymphoma Project Group. J Clin Oncol 13:1343±1354, 1995 Paulli M, Berti E, Boveri E, et al: Cutaneous CD30+ lymphoproliferative disorders: expression of bcl-2 and proteins of the tumor necrosis factor receptor superfamily. Human Pathol 29:1223±1230, 1998 Pfeifer W, Levi E, Petrogiannis-Haliotis T, Lehman L, Wang ZX, Kadin ME: A murine xenograft model for human CD30+ anaplastic large cell lymphoma: successful growth inhibition with an anti-CD30 antibody (HeFi-1). Am J Pathol 155:1353±1359, 1999
1040
LEVI ET AL
Schiemann W, Pfeifer WM, Levi E, Kadin ME, Lodish HF: A deletion in the gene for transforming growth factor B type I receptor abolishes growth regulation by TGF-b in a cutaneous T-cell lymphoma. Blood 94:2854±2861, 1999 Sebolt-Leopold JS, Dudley DT, Herrera R, et al: Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nature Med 5:810±816, 1999 Smith CA, Gruss HJ, Davis T, et al: CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand de®nes an emerging family of cytokines with homology to TNF. Cell 73:1349±1360, 1993 Sonenshein GE: Rel/NF-kB transcription factors and the control of apoptosis. Semin Cancer Biol 8:13±19, 1997 Song HY, Regnier CH, Kirschning CJ, Goeddel DV, Rothe M: Tumor necrosis (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kB and c-jun Nterminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc Natl Acad Sci 94:9792±9796, 1997 Telford WG, Nam SY, Podack ER, Miller RA: CD30-regulated apoptosis in murine CD8 T cells after cessation of TCR signals. Cellular Immunol 182:125±136, 1997
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Tian ZG, Longo DL, Funakoshi S, et al: In vivo antitumor effects of unconjugated CD30 monoclonal antibodies on human anaplastic large-cell lymphoma xenografts. Cancer Res 55:5335±5341, 1995 Wallach D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, Boldin MP: Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 17:331±367, 1999 Wang HH, Lach L, Kadin ME: Epidemiology of lymphomatoid papulosis. Cancer 70:2951±2957, 1992 Wendtner CM, Schmitt B, Gruss HJ, Druker BJ, Emmerich B, Goodwin RG, Hallek M: CD30 ligand signal transduction involves activation of a tyrosine kinase and of mitogen-activated protein kinase in a Hodgkin's lymphoma cell line. Cancer Res 55:4157±4161, 1995 Willemze R, Beljaards RC: Spectrum of primary cutaneous CD30 (Ki-1)-positive lymphoproliferative disorders. A proposal for classi®cation and guidelines for management and treatment. J Am Acad Dermatol 28:973±980, 1993 Willemze R, Kerl H, Sterry W, et al: EORTC classi®cation for primary cutaneous lymphomas: a proposal from the Cutaneous Lymphoma Study Group of the EORTC. Blood 90:354±371, 1997
Lymphomatoid papulosis is part of a spectrum of CD30+ cutaneous lymphoproliferative disorders characterized by spontaneous tumor regression. The mechanism(s) of regression is unknown. In a recent study, a selective increase in CD30 ligand expression in regressing lesions of lymphomatoid papulosis and cutaneous CD30+ anaplastic large cell lymphoma was shown, suggesting that activation of the CD30 signaling pathway may be responsible for tumor regression, whereas no difference in Fas/Fas ligand expression was found between regressing and nonregressing lesions. Therefore we tested the effects of CD30 and Fas activation on three CD30+ cutaneous lymphoma cell lines (Mac-1, Mac-2 A, JK) derived from nonregressing tumors of two patients who had progressed from lymphomatoid papulosis to systemic anaplastic large cell lymphoma. To evaluate the effects of CD30 signaling, the cell lines were incubated with a CD30
P
rimary cutaneous CD30+ lymphoproliferative disorders (LPD) are recognized as a distinct biologic entity in the European Organization for Research and Treatment of Cancer classi®cation of cutaneous lymphomas and the forthcoming World Health Organization classi®cation of lymphomas (Willemze et al, 1997; Jaffe et al, 1999). CD30+ cutaneous LPD are associated with frequent spontaneous regression (Cabanillas et al, 1995; Paulli et al, 1995). Although cutaneous CD30+ LPD generally have a good prognosis, certain cases progress to aggressive systemic lymphomas and become resistant to chemotherapy (Willemze and Beljaards, 1993; Paulli et al, 1995; Willemze et al, 1997; Bekkenk et al, 2000). CD30 antigen expression is a favorable prognostic factor in cutaneous T cell lymphomas (Beljaards et al, 1993; Willemze et al, 1993; Paulli et al, 1995; Bekkenk et al, 2000). With the exception of Manuscript received May 5, 2000; revised July 28, 2000; accepted for publication August 22, 2000. Reprint requests to: Dr. Marshall E. Kadin, Director of Hematopathology, Department of Pathology, YA 309, Beth Israel-Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. Email: [email protected] Abbreviations: ALCL, anaplastic large cell lymphoma; LPD, lymphoproliferative disorders; LyP, lymphomatoid papulosis; MAPK, mitogenactivated protein kinase; NF-kB, nuclear factor kB; TNF, tumor necrosis factor. 0022-202X/00/$15.00
agonistic antibody, HeFi-1. Proliferative responses, mitogen-activated protein kinase, and nuclear factor kB activities were determined with and without CD30 activation. Mac-1 and Mac-2 A showed increased proliferative responses to incubation with CD30 activating antibody, HeFi-1. Inhibition of the mitogen-activated protein kinase activity caused growth inhibition of the Mac-1, Mac-2 A, and JK cell lines. Activation of the Fas pathway induced apoptosis in all three cell lines. Taken together, these ®ndings suggest that resistance to CD30-mediated growth inhibition provides a possible mechanism for escape of cutaneous anaplastic large cell lymphoma from tumor regression. Mitogenactivated protein kinase inhibitors are potential therapeutic agents for the treatment of advanced cutaneous anaplastic large cell lymphoma. Key words: CD30/ cutaneous anaplastic large cell lymphoma/MAPK/NF-kB. J Invest Dermatol 115:1034±1040, 2000
mycosis fungoides, CD30-negative T cell lymphomas of the skin tend to have a more aggressive course and a poorer prognosis than CD30+ cutaneous LPD. Members of the tumor necrosis factor (TNF) receptor superfamily, such as Fas (CD95), TNF receptors types I and II, CD27, CD30, CD40, DR3, DR4, and DR5 play an important role in immune regulation. These members of the TNF receptor family contain a ``death domain'' that triggers programmed cell death when activated by the appropriate ligand (Ashkenazi and Dixit, 1998). Some members of the TNF receptor and ligand superfamily are implicated in the pathogenesis of immune disorders and lymphomas (Gruss and Dower, 1995). An important function of the TNF members is regulation of growth and elimination of peripheral T cells during their expansion in response to antigenic stimuli. T cell apoptosis occurs in two major forms: antigen driven and lymphokine withdrawal induced. Antigen-driven death is induced by the activation of the T cell antigen receptor, and is regulated by Fas and TNFRI (Lenardo et al, 1999). The speci®city of T cell elimination by this pathway is partly due to a requirement for simultaneous activation of T cell antigen receptor and the TNF or Fas receptor (Lenardo et al, 1999). The second mechanism of T cell elimination is cytokinewithdrawal-induced cell death. In this pathway, T cells are deprived of the cytokine support that is associated with the antigen-induced activation state (Telford et al, 1997; Lenardo et al, 1999). This mechanism of apoptosis is prevented by the presence of
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 1034
VOL. 115, NO. 6 DECEMBER 2000
cytokines such as interleukin 2 (IL-2), IL-4, and IL-7 (Lenardo et al, 1999). Telford et al (1997) have shown in a mouse model that antigen-withdrawal-induced apoptosis is independent of Fas and TNFRI but is regulated mainly by CD30 antigen. CD30 is normally detectable on a small number of immunoblasts located in the perifollicular region around germinal centers (Falini et al, 1995). In addition, CD30 is expressed by Reed-Sternberg cells in virtually all cases of classical Hodgkin's lymphoma and by tumor cells in anaplastic large cell lymphoma (ALCL) (Falini et al, 1995). CD30 ligand (CD30L) is expressed on neutrophils, histiocytes, eosinophils, and a small subset of activated T cells (Smith et al, 1993). Accumulating evidence suggests that CD30 acts as a negative regulator of T cells. CD30 knock out mice show thymic hyperplasia and a defect in negative selection of T cells responding to self-antigens in the thymus (Amakawa et al, 1996). In a murine model, CD30 antigen is protective against autoimmune type I diabetes mediated by autoreactive CD8+ cells (Kurts et al, 1999), and Chiarle et al (1999) demonstrated enhanced cell death of T cells that express CD30 in response to antigen stimulation and CD30 costimulation. As CD30+ cutaneous LPD represents clonal expansion of an abnormal T cell clone, the normal fate of these T cells should be elimination by one of the mechanisms mentioned above. The early lesions do regress, but the mechanism of spontaneous regression seen in these lesions is unknown. We believe that Fas and CD30 are two signaling pathways that can mediate regression. The tumor cells in CD30+ cutaneous LPD have been shown to express Fas (Paulli et al, 1998; Mori et al, 1999). CD30L expression shows a correlation with regression of the lesions suggesting a role for CD30 activation in the usual regression of cutaneous CD30+ lymphomas (Mori et al, 1999). Until now, there has not been a functional study of the responses of tumor cells from cutaneous CD30+ LPD to the activation of TNF receptor superfamily members including Fas and CD30, due to the lack of tumor cell lines. We developed three cell lines from cutaneous tumors and circulating tumor cells of two patients who had lymphomatoid papulosis (LyP) and then progressed to fatal systemic lymphomas during the course of their disease (Davis et al, 1992; Kadin et al, 1994; Schiemann et al, 1999). We investigated the responses of these cell lines to Fas and CD30 activation in vitro. We also investigated the intracellular signaling mechanisms associated with CD30 activation, including nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK), in order to understand the mechanism of disease progression and to develop novel therapies targeting these pathways in advanced disease originating from cutaneous CD30+ LPD. MATERIALS AND METHODS Cell lines JK, Mac-1, and Mac-2 A are cutaneous ALCL cell lines (Kadin et al, 1994). The JK cell line was developed from an advanced skin tumor of a patient who progressed over a 10 y period from LyP to ALCL (Schiemann et al, 1999); the clonal relationship of LyP and ALCL lesions has been demonstrated (Chott et al, 1996). JK cells have the same phenotype as the original tumor (CD30+, CD3+). Mac-1 and Mac-2 A are clonally related cell lines derived from a CD30+ cutaneous T cell ALCL that had progressed from LyP over a 17 y period. Details of the cell lines have been reported elsewhere (Davis et al, 1992; Kadin et al, 1994). Immunohistochemistry Immunohistochemical staining for CD30L was performed on 4 mm sections of formalin-®xed paraf®n-embedded material as previously described (Mori et al, 1999). Sections were stained with the M80 antibody against CD30L kindly provided by Immunex, Seattle, WA. Antibodies and inhibitors HeFi-1 (NCI, Frederick, MD) is a mouse monoclonal IgG1 antibody raised against human CD30 (Hecht et al, 1985). HeFi-1 has an agonistic effect on the CD30 pathway mimicking the effects of CD30L. Mouse IgG1 (Sigma, St. Louis, MO) is used as an isotypespeci®c control. SN50 is a synthetic peptide that is cell permeable and inhibits NF-kB by competing with the nuclear localization sequence of NF-kB (p50) (Lin et al, 1995). SN50M is a synthetic peptide mutated at the nuclear localization sequence site so that it can be used as a negative control
CD30 SIGNALING IN CUTANEOUS ALCL
1035
Figure 1. Immunohistochemical staining of CD30L. Immunohistochemistry with antibody M80 reveals staining of Reed-Sternberg-like cells and some smaller cells in LyP lesions of patients from whom Mac-1 and Mac-2 A cells (a and b) and JK cells (c and d) were derived. for SN50. U0126 is a selective inhibitor of MEK1 and MEK2 (Favata et al, 1998). All three chemicals were purchased from Calbiochem (San Diego, CA). Anti-human Fas antibody with agonistic properties was obtained from R&D Systems, Minneapolis, MN (clone DX2.1). 3H-Thymidine incorporation assay Tumor cells were grown in round-bottomed 96 well plates in triplicate. Following incubation with soluble antibodies and 10% fetal bovine serum in RPMI, the wells were pulsed with 1 mCi 3H-thymidine during the ®nal 12 h. Tumor cells were harvested with the aid of a cell harvester (PhD Systems, Cambridge, MA) and radioactivity was measured with a scintillation counter (Wallac 1409; Wallac, Gaithersburg, MD).
Electrophoretic mobility shift experiments Nuclear extracts from tumor cell lines were obtained before and after stimulation with HeFi-1 antibody for 1 h. The extracts were incubated with 32P end-labeled NF-kB binding site oligoprobes (5¢-AGCTTGGGGTATTTCCAGCCG-3¢), mutant oligoprobes (5¢-AGCTTGGCATAGGTCCAGCCG-3¢), and excess cold oligoprobes, and were run on a nondenaturing 6% polyacrylamide gel. Gels were dried and exposed to ®lm (NF-kB/Rel T cell activation Gelshift kit, Geneka, Montreal, Canada). Nuclear extracts were prepared as follows: 10 3 106 cells were incubated with HeFi-1 for 1 h, and then washed in phosphate-buffered saline and lyzed in 400 ml cold buffer A [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl ¯uoride (PMSF), pH 7.9]. Samples were kept on ice for 15 min, and then vortexed for 10 s. Nuclei were pelleted by microcentrifugation for 10 s. Pellets were resuspended in 40 ml buffer B (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM ethylenediamine tetraacetic acid, 0.5 mM DTT, 0.2 mM PMSF) and incubated on ice for 20 min. Nuclei were pelleted by spinning for 3 min at 4°C. Supernatants were aliquoted and stored at ±80°C. MAPK kinase assay MAPK activity was determined by the MAPK kinase assay kit according to the manufacturer's recommended protocols (New England Biolabs, Beverly, MA). Brie¯y, whole cell lysates were immunoprecipitated with an anti-MAPK antibody. The immunoprecipitated proteins were used in a nonradioactive in vitro kinase assay. A Gst-Elk1 fusion peptide was used as a substrate for the detection of kinase activity in the immunoprecipitates. Following the kinase assay, phosphorylation of the Gst-Elk1 substrate was measured by Western blots using an antiphopho-Elk1 antibody for detection.
RESULTS Immunohistochemical staining for CD30L Immunohistochemical staining con®rmed the presence of CD30L in tissue sections from the LyP mainly in the cytoplasm of 10%-20% of large atypical cells, including Reed-Sternberg-like cells, and some smaller cells (Fig 1). Staining of CD30L appeared to be associated with degenerative changes in some cells of the case from which Mac-1 and Mac-2 A cell lines were derived.
1036
LEVI ET AL
Figure 2. Proliferative responses to CD30 activation. Proliferative activity was measured by a 3H-thymidine incorporation assay. Cutaneous CD30+ ALCL cell lines JK, Mac-1, and Mac-2 A were incubated in 10% fetal bovine serum and RPMI in the presence of CD30 activating antibody HeFi-1 (1 mg per ml) for 48 h and pulsed with 3H-thymidine (1 mCi) during the last 12 h of incubation. Control cells were incubated with isotypespeci®c mouse IgG. The counts are shown as percentages compared to controls (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 4. Signaling pathways triggered by CD30 activation. This cartoon depicts intracellular signaling by CD30 activation through TRAFs, NF-kB, and MAPK. The sites of action of the inhibitors used in this study are shown. GFR, growth factor receptor.
death was con®rmed (not shown). As expected, the Karpas 299 cell line, which is known to be resistant to Fas-mediated apoptosis, did not show inhibition of cell growth upon Fas activation (not shown).
Figure 3. Fas-mediated growth inhibition of the cell lines. Effects of Fas activation on the cell lines was determined by 3H-thymidine incorporation assays following incubation with a Fas agonist antibody for 24 h. The counts are shown as percentages compared to controls (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
Effects of CD30 activation on cell proliferation In order to analyze the functional status of the CD30/CD30L signaling pathway in the cell lines representing advanced cutaneous ALCL, we used a monoclonal antibody, HeFi-1 (Hecht et al, 1985), that recognizes the CD30L binding domain of CD30 antigen and leads to activation of the CD30 signaling cascade. We and others have previously shown that this antibody mimics the action of CD30L and is an effective growth inhibitor of tumor cells of systemic CD30+ ALCL, both in vitro and in vivo (Tian et al, 1995; Pfeifer et al, 1999). Upon activation of the CD30 signaling pathway by HeFi-1, Mac-1 and Mac-2 A cell lines showed proliferative responses whereas the JK cell line did not change its proliferative activity, as measured in a 3H-thymidine incorporation assay (Fig 2). We also tested the effects of CD30 activation of the Karpas 299 cell line, a systemic ALCL cell line with the translocation t(2; 5)(p23; q35), and observed a 50% growth inhibition as previously reported (Pfeifer et al, 1999) (not shown). Effects of Fas activation on cell growth In order to assess their sensitivity to Fas-mediated apoptosis in vitro, the cell lines were incubated with a Fas agonistic antibody (clone DX2.1, R&D systems) for 24 h, and then pulsed with 3H-thymidine for 12 h. All three cell lines demonstrated signi®cant growth inhibition in response to Fas activation whereas control cells incubated with an isotype-speci®c mouse IgG were unaffected (Fig 3). Cell viability was independently assessed by trypan blue vital staining and cell
Role of NF-kB in CD30 signaling Following the assessment of the responses of the cell lines to CD30 and Fas activation, we wanted to analyze the downstream effectors of the CD30 signaling pathway. The immediate transducers of CD30 signaling are the TNF-associated factors (TRAFs). TRAF-1, TRAF-2, TRAF-3, and TRAF-5 are known to bind the intracytoplasmic domain of CD30 (Arch et al, 1998). TRAF-2 is known to activate NF-kB and the c-jun N-terminal kinase (JNK) pathways (Song et al, 1997). In addition, in some cell lines MAPK is activated by CD30 signaling (Wendtner et al, 1995). As NF-kB and MAPK pathways are important for numerous cell functions including cell survival, proliferation, and cytokine secretion, we thought these two pathways would be most important to explain the proliferative responses observed by CD30 activation (Hunter, 1995; Barnes and Karin, 1997; Sonenshein, 1997). As the JNK pathway is usually associated with stress responses and apoptosis, we did not pursue that pathway (Ip and Davis, 1998). The downstream signaling pathways associated with CD30 signaling and the sites of action of the inhibitors used in the study are illustrated in Fig 4. We measured the nuclear NF-kB activity of cell lines Mac-1 and Mac-2 A before and after CD30 activation. Nuclear extracts were obtained from cells that were incubated with antibody HeFi-1. The nuclear extracts were incubated with 32P-labeled oligoprobes representing NF-kB binding sites and run on a polyacrylamide gel. Gelshift experiments showed minimal constitutive NF-kB activity in Mac-1 and Mac-2 A cell lines. In contrast, following incubation with HeFi-1 for 30 min, both cell lines showed strong NF-kB activation (Fig 5). The speci®city of the binding was tested by addition of excess cold probe and excess cold mutant probe. We then investigated the NF-kB pathway by use of a speci®c inhibitor of NF-kB, SN50 (Lin et al, 1995). Mac-1 and Mac-2 A cell lines demonstrated inhibition of cell growth within 24 h when incubated with the NF-kB inhibitor SN50 at 50 mg per ml concentration in the presence of HeFi-1 (1 mg per ml). The JK cell line showed no response to the NF-kB inhibitor (Fig 6). As a negative control for SN50, we incubated the cells with SN50M, which is a synthetic peptide that differs from SN50 in just two amino acids that correspond to the nuclear localization sequence.
VOL. 115, NO. 6 DECEMBER 2000
CD30 SIGNALING IN CUTANEOUS ALCL
1037
Figure 6. Effects of NF-kB inhibition during CD30 activation. The NF-kB inhibitor SN50 was added 30 min before the addition of HeFi-1. 3H-Thymidine was added in the last 12 h of the 24 h incubation. The results are shown as percentages of the control values (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
Figure 5. Activation of NF-kB by CD30 activation. Nuclear extracts obtained from Mac-1 and Mac-2 A cell lines were incubated with 32Plabeled oligoprobes (H), (H) + excess cold oligoprobes (C), and (H) + excess cold mutant oligoprobes (M), with or without HeFi-1 for 1 h. The arrows show the speci®c bands, which are retarded due to the binding of the probes to nuclear proteins, and the unbound probes at the bottom of the gel. The remaining bands are due to nonspeci®c binding of the probe to other nuclear proteins. Unlike the speci®c bands, which disappear when excess cold unlabeled oligoprobes are added (C), these bands do not disappear. A representative experiment is shown.
Figure 7. Activation of MAPK by CD30 signaling. MAPK activity was measured after CD30 activation for 10 min, and control cells were incubated with isotype-speci®c mouse IgG. CD30 activation was achieved by HeFi-1 antibody. Mac-2 A cell line was also incubated with the MEK1 inhibitor U0126 for 10 min and the MAPK activity was measured. Equal loading of wells was con®rmed by measuring the protein concentration of the cell lysates before immunoprecipitation, and staining the blotted membranes for protein.
Incubation with SN50M caused no changes in the responses of the cells, con®rming the speci®city of responses to SN50 (not shown). Role of MAPK in CD30 signaling Another candidate pathway to explain the responses of the cell lines to CD30 activation is MAPK. As in the NF-kB experiments, we measured the activity of MAPK before and after CD30 activation by a kinase assay, and then tested the effects of inhibitors of the MAPK pathway. The cell lines were starved in serum-free media for 24 h before the experiments. CD30 activation was achieved by incubating the cell lines with the HeFi-1 antibody for 10 min. JK and Mac-1 cell lines had weak to minimal baseline MAPK activities, which were enhanced by CD30 activation, whereas Mac-2 A had a high constitutive MAPK activity that was slightly increased by CD30 activation (Fig 7). We also tested the effects of MAPK inhibition on the proliferative properties of the cell lines by using a recently described speci®c MEK1 inhibitor, U0126 (Favata et al, 1998). The cells were incubated with 1 or 10 mM of the inhibitor for 30 min, and then HeFi-1 was added. We measured 3H-thymidine incorporation after 24 h. At 1 mM, all cell lines demonstrated signi®cant growth inhibition. When 10 mM of the inhibitor was added, the cell lines were almost completely growth inhibited (Fig 8). DISCUSSION In this study we demonstrate that activation of CD30 in cutaneous CD30+ ALCL cell lines does not induce growth inhibition; instead it causes proliferation of the Mac-1 and Mac-2 A cell lines. Others have shown that activation of CD30 by a monoclonal antibody could induce sustained proliferation of human autoreactive gd T
Figure 8. Inhibition of MAPK activity causes growth inhibition. Growth responses of the cell lines to inhibition of MEK1 were tested at two different doses of the MEK1 and MEK2 inhibitor, U0126.The inhibitor was added 30 min before the addition of HeFi-1. Responses are shown as percentages of the control values (mean 6 SD). The experiments were performed in triplicate and repeated at least three times. A representative experiment is shown. *p < 0.05.
cells in the presence of recombinant IL-2 (Leca et al, 1994). Those cells were not transformed, however, and cell growth arrest occurred upon removal of the antibody. In contrast, our cell lines express an ab rather than gd phenotype and do not undergo growth arrest upon removal of the CD30 activating antibody, HeFi-1. Cutaneous and systemic CD30+ ALCL cell lines appear to have opposite growth responses to CD30 activation as CD30 activation is an inhibitory signal for systemic ALCL cell lines (Gruss et al, 1994). Cutaneous and systemic ALCL also have distinct pathways of pathogenesis. Systemic ALCL cell lines harbor the
1038
LEVI ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 9. Hypothesis for mechanisms of progression of cutaneous CD30+ LPD. The cartoon depicts the growth inhibitory effects of CD30L, Fas, and TGF-b on LyP in regressing skin lesions. Our studies indicate that tumor progression to ALCL is in part due to escape from growth inhibition through altered CD30 signaling and mutations of the TGF-b receptor (Knaus et al, 1996; Schiemann et al, 1999).
translocation t(2; 5)(p23; q35), which causes overexpression of the NPM/ALK fusion protein (ALK) leading to the unregulated growth of tumor cells (Morris et al, 1994). ALK is characteristically absent in cutaneous ALCL (DeCoteau et al, 1996). Therefore, cutaneous and systemic ALCLs have been identi®ed as separate entities in the new lymphoma classi®cations (Jaffe et al, 1999). There is limited information regarding the etiology and biology of CD30+ cutaneous ALCL. In general, the primary cutaneous LPD constitute a spectrum of diseases ranging from LyP to cutaneous ALCL, which have an excellent prognosis (Paulli et al, 1995). A small percentage of these diseases (approximately 5%), however, progress to fatal systemic lymphomas (Beljaards and Willemze, 1992). The cell lines we used in our study are representative of such uncommon cases that ®rst present as LyP and later progress to fatal systemic lymphomas. A well-known phenomenon associated with cutaneous CD30+ LPD is the frequent occurrence of spontaneous regression (Willemze et al, 1993). In a recent study, a positive correlation was demonstrated between regression and CD30L expression levels measured by semiquantitative reverse transcriptase polymerase chain reaction in cases of LyP and primary cutaneous ALCL, suggesting that CD30 activation may play a role in the spontaneous regression of such lesions (Mori et al, 1999). In this study, we con®rmed the presence of CD30L in the LyP skin lesions of each patient by immunohistochemistry. Our results raise the possibility that the development of resistance to the CD30L-mediated growth inhibitory effects, or even transformation of the tumor response to one of cellular proliferation in response to CD30 activation, may provide a mechanism for tumor cell escape from the normal growth regulatory responses and support progression of LyP to ALCL. Recently, Fas expression was demonstrated in cutaneous CD30+ LPD (Paulli et al, 1998; Mori et al, 1999). A functional role for Fas-mediated apoptosis in the regression of these lesions, however, has not been shown. We investigated the susceptibility of our cutaneous ALCL lines to Fas-induced apoptosis. As all three cell lines were sensitive to Fas-induced apoptosis, we conclude that the Fas signaling pathway is unlikely to play a role in the progression of LyP to ALCL. In order to gain insight into the downstream effects of CD30 activation, we investigated some pathways that are involved in the CD30/CD30L signaling system. CD30 activation is known to cause upregulation of NF-kB, utilizing the TRAFs as adapter molecules (Duckett et al, 1997; Song et al, 1997). Constitutive activation of NF-kB has been demonstrated in Hodgkin's lymphoma cell lines and has been implicated in their survival and
tumorigenicity (Bargou et al, 1997). We were interested to compare cutaneous ALCL cell lines with Hodgkin's lymphoma as these diseases have similar morphologic and immunohistochemical features (Davis et al, 1992; Kadin, 1993). In addition, patients with LyP sometimes develop Hodgkin's lymphoma (Davis et al, 1992; Wang et al, 1992; Cabanillas et al, 1995). Our study shows that, unlike Hodgkin's lymphoma, cutaneous ALCL cell lines do not have high constitutive NF-kB activity. The ERK (MAPK) pathway is also known to be activated by members of the TNF receptor family, including CD30 (Wendtner et al, 1995; Wallach et al, 1999). In a previous study by Wendtner et al (1995), CD30 signaling led to MAPK activation in the T-celltype Hodgkin's lymphoma cell line HDLM-2, but not in the systemic ALCL cell line Karpas 299 nor in the B-cell-type Hodgkin's lymphoma cell line KMH2. Interestingly, the HDLM-2 cell line showed increased proliferation in response to CD30 activation whereas Karpas 299 was growth inhibited. Here, we demonstrate that the MAPK (ERK) pathway is activated by CD30 signaling in cutaneous T cell ALCL cell lines Mac-1 and JK as well, whereas Mac-2A has high constitutive expression of MAPK; inhibition of MAPK is an effective way of inhibiting growth of these cell lines. The mechanism of altered responsiveness to CD30 activation in the cell lines from advanced cutaneous ALCL is unclear. We have shown that, in the systemic ALCL cell line Karpas 299, CD30 activation causes cell cycle arrest despite activation of the NF-kB pathway (Levi et al, submitted for publication). The proliferative response seen in the Mac-1 and Mac-2 A cell lines was at least partly due to MAPK and NF-kB activation by CD30 signaling. In this study, we demonstrated that MAPK activation is one of the major effectors of CD30 signaling in these cutaneous ALCL cell lines. It has been shown that mammalian cells can be transformed by transfection with constitutively active MAPK (Mansour et al, 1994). In addition, inhibition of the MAPK activity in mice xenografted with colon tumors causes inhibition of tumor growth (Sebolt-Leopold et al, 1999). We believe that inhibitors of the MAPK pathway can become useful in the treatment of various cancers as the Ras pathway, which is upstream of the MAPK, is frequently mutated in human cancers (Macara et al, 1996; Hunter, 1997). Based on this study and previous work, we propose a model of progression of LyP to systemic lymphoma by several steps that abolish the physiologic inhibitory mechanisms that are important for the regulation of the immune system such as transforming growth factor beta (TGF-b) and CD30 (Fig 9). According to this
VOL. 115, NO. 6 DECEMBER 2000
model, an unknown antigenic stimulus triggers clonal expansion of T cells, which form cutaneous in®ltrates that manifest as multiple regressing skin lesions of LyP. The LyP cells are usually eliminated by growth inhibitory mechanisms such as Fas, CD30, and TGF-b. Therefore, the lesions spontaneously regress in weeks to months. After recurrent episodes of spontaneously regressing LyP lesions, a small number of patients will develop large lesions that require treatment. A few of these patients will have extracutaneous spread of the disease, which may be resistant to chemotherapy. We hypothesize that in these patients disease progression from LyP to ALCL is associated with impairment of the normal T cell growth inhibitory mechanisms that would otherwise cause regression of these lesions. In these and prior experiments, we tested three of the best characterized inhibitory mechanisms that regulate the T cell arm of the immune system - Fas, CD30, and TGF-b - and demonstrated a selective role for CD30 in the context of escape from growth inhibitory mechanisms. In the case of TGF-b, resistance was shown to be mediated by mutations of the type I (JK) and type II (Mac-2 A) receptors (Knaus et al, 1996; Schiemann et al, 1999). These mutations were due to a deletion that eliminates the initiating methionine required for translation of the type I receptor in the JK cell line, and due to a single nucleotide substitution (A to G) in the kinase domain of the type II receptor in the Mac-2 A cell line, whereas Mac-1, which is inhibited by TGF-b, had no TGF-b receptor mutation. Both mutations were present in the corresponding tissue sections obtained from the patients. Preliminary data from our laboratory indicate the presence of mutations in CD30 that may alter CD30 signaling and could explain the proliferative responses of Mac-1 and Mac-2 A cells and failure of JK cells to respond to HeFi-1. At present no cell lines from regressing lesions are available for comparison but this is a long-term goal. The fact that both TGF-b and CD30 signaling systems were noninhibitory suggests that abrogation of both pathways may be required for the progression of these lymphomas to fatal systemic disease. In summary, we have demonstrated that in addition to evading TGF-b-mediated growth control, advanced CD30+ ALCL cell lines can be resistant to growth inhibition by CD30 signaling, but retain susceptibility to Fas-induced cell death. We also identi®ed the ERK (MAPK) pathway inhibitors as candidates for novel therapies against these advanced cutaneous ALCL. EL was a fellow of the Lymphoma Research Foundation of America. WP was a fellow of Deutsche Krebshilfe. TP-H was a fellow of the Cure for Lymphoma Foundation. WK is ®nancially supported by the Schweizerische Stiftung fuÈr Medizinisch-Biologische Stipendien, Basel, Switzerland. This study was supported by grants to Dr. Kadin from the American Cancer Society (ROG-98±125±01), the Leukemia and Lymphoma Society (178±98), and Merritt Care Foundation, Fargo, ND.
REFERENCES Amakawa R, Hakem A, Kundig TM, et al: Impaired negative selection of T cells in Hodgkin's disease antigen CD30-de®cient mice. Cell 84:551±562, 1996 Arch RH, Gedrich RW, Thompson CB: Tumor necrosis factor receptor-associated factors (TRAFs) - a family of adapter proteins that regulates life and death. Genes Dev 12:2821±2830, 1998 Ashkenazi A, Dixit VM: Death receptors: signaling and modulation. Science 281:1305±1326, 1998 Bargou RC, Emmerich F, Krappman D, et al: Constitutive nuclear factor kB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells. J Clin Invest 100:2961±2969, 1997 Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic in¯ammatory diseases. N Engl J Med 336:1066±1071, 1997 Bekkenk MW, Geelen FAMJ, van Vorst Vader PC, et al: Primary and secondary cutaneous CD30+ lymphoproliferative disorders: a report from the Dutch Cutaneous Lymphoma Group on the long-term follow-up data of 219 patients and guidelines for diagnosis and treatment. Blood 95:3653±3661, 2000 Beljaards RC, Willemze R: The prognosis of patients with lymphomatoid papulosis associated with malignant lymphomas. Br J Dermatol 126:596±602, 1992 Beljaards RC, Kaudewitz P, Berti E, et al: Primary cutaneous CD30-positive large cell lymphoma: de®nition of a new type of cutaneous lymphoma with a
CD30 SIGNALING IN CUTANEOUS ALCL
1039
favorable prognosis: a European multicenter study on 47 patients. Cancer 71:2097±2104, 1993 Cabanillas F, Armitage J, Pugh WC, Weisenburger D, Duvic M: Lymphomatoid papulosis: a T cell dyscrasia with a propensity to transform into malignant lymphoma. Ann Intern Med 122:210±217, 1995 Chiarle R, Podda A, Prolla G, Podack ER, Thorbecke GJ, Inghirami G: CD30 overexpression enhances negative selection in the thymus and mediates programmed cell death via a Bcl-2 sensitive pathway. J Immunol 163:194±205, 1999 Chott A, Vonderheid EC, Olbricht S, Miao N-N, Balk SP, Kadin ME: The dominant T cell clone is present in multiple regressing skin lesions and associated T cell lymphomas of patients with lymphomatoid papulosis. J Invest Dermatol 106:696±700, 1996 Davis TH, Morton CC, Miller-Cassman R, Balk SP, Kadin ME: Hodgkin's disease, lymphomatoid papulosis and cutaneous T cell lymphoma derived from a common T cell clone. N Eng J Med 326:1115±1122, 1992 DeCoteau JF, Butmarc JR, Kinney MC, Kadin ME: The t (2; 5) chromosomal translocation is not a common feature of primary cutaneous CD30+ lymphoproliferative disorders: comparison with anaplastic large cell lymphoma of nodal origin. Blood 87:3437±3441, 1996 Duckett CS, Gedrich RW, Gil®an MC, Thompson CB: Induction of nuclear factor kB by the CD30 receptor is mediated by TRAF1 and TRAF2. Mol Cell Biol 17:1535±1542, 1997 Falini B, Pileri S, Pizzolo G, et al: CD30 (Ki-1) molecule: a new cytokine receptor of the tumor necrosis factor receptor superfamily as a tool for diagnosis and immunotherapy. Blood 85:1±14, 1995 Favata MF, Horiochi KY, Manos EJ, et al: Identi®cation of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273:18623±18632, 1998 Gruss HJ, Dower SK: Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas. Blood 85:3378±3404, 1995 Gruss HJ, Boiani N, Williams DE, Armitage RJ, Smith CA, Goodwin RG: Pleiotropic effects of the CD30 ligand on CD30 expressing cells and lymphoma cell lines. Blood 83:2045±2056, 1994 Hecht TT, Longo DL, Cossman J, Bolen HB, Hsu SM, Israel M, Fisher RI: Production and characterization of a monoclonal antibody that binds ReedSternberg cells. J Immunol 134:4231±4236, 1985 Hunter T: Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signaling. Cell 80:225±236, 1995 Hunter T: Oncoprotein networks. Cell 88:333±346, 1997 Ip TY, Davis RJ: Signal transduction by the c-jun N-terminal kinase (JNK) - from in¯ammation to development. Curr Opin Cell Biol 10:205±219, 1998 Jaffe ES, Harris NL, Diebold J, Muller-Hermelink HK: WHO classi®cation of neoplastic diseases of the hematopoietic and lymphoid tissues. A progress report. Am J Clin Path 111 (Suppl. 1):S8±S12, 1999 Kadin ME: Lymphomatoid papulosis and associated lymphomas. How are they related? Arch Dermatol 129:351±352, 1993 Kadin ME, Cavaille-Coll MW, Gertz R, Massague J, Cheifetz S, George D: Loss of receptors for transforming growth factor beta in human T-cell malignancies. Proc Natl Acad Sci USA 91:6002±6006, 1994 Knaus PI, Lindemann D, DeCoteau JF, et al: A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T cell. Lymphoma Mol Cell Biol 16:3480±3489, 1996 Kurts C, Carbone FR, Krummel MF, Koch KM, Miller JFAP, Heath WR: Signaling through CD30 protects against autoimmune diabetes mediated by CD8 T cells. Nature 398:341±344, 1999 Leca G, Vita N, Maiza H, Fasseu M, Bensussan A: A monoclonal antibody to the Hodgkin's associated antigen CD30 induces activation and long-term growth of human autoreactive gd T cell clone. Cell Immunol 156:230±239, 1994 Lenardo M, Chan FKM, Hornung F, McFarland H, Siegel R, Wang J, Zheng L: Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment. Annu Rev Immunol 17:221±253, 1999 Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J: Inhibition of nuclear transcription factor NF-kB by a synthetic peptide containing a cell membranepermeable motif and nuclear localization sequence. J Biol Chem 270:14255± 14258, 1995 Macara IG, Lounsbury KM, Richards SA, McKiernan C, Bar-Sagi D: The Ras superfamily of GTPases. FASEB J 10:625±630, 1996 Mansour SJ, Matten WT, Hermann AS, et al: Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966±970, 1994 Mori M, Manuelli C, Pimpinelli N, et al: CD30±CD30L interaction in primary cutaneous CD30+ positive T cell lymphomas: a clue to the pathophysiology of clinical regression. Blood 94:3077±3083, 1999 Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL, Look AT: Fusion of a kinase gene ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263:1281±1284, 1994 Paulli M, Berti E, Rosso R, et al: CD30/Ki-1 positive lymphoproliferative disorders of the skin - clinicopathologic correlation and statistical analysis of 86 cases: a multicentric study from the European Organization for Research and Treatment of Cancer, Cutaneous Lymphoma Project Group. J Clin Oncol 13:1343±1354, 1995 Paulli M, Berti E, Boveri E, et al: Cutaneous CD30+ lymphoproliferative disorders: expression of bcl-2 and proteins of the tumor necrosis factor receptor superfamily. Human Pathol 29:1223±1230, 1998 Pfeifer W, Levi E, Petrogiannis-Haliotis T, Lehman L, Wang ZX, Kadin ME: A murine xenograft model for human CD30+ anaplastic large cell lymphoma: successful growth inhibition with an anti-CD30 antibody (HeFi-1). Am J Pathol 155:1353±1359, 1999
1040
LEVI ET AL
Schiemann W, Pfeifer WM, Levi E, Kadin ME, Lodish HF: A deletion in the gene for transforming growth factor B type I receptor abolishes growth regulation by TGF-b in a cutaneous T-cell lymphoma. Blood 94:2854±2861, 1999 Sebolt-Leopold JS, Dudley DT, Herrera R, et al: Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nature Med 5:810±816, 1999 Smith CA, Gruss HJ, Davis T, et al: CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand de®nes an emerging family of cytokines with homology to TNF. Cell 73:1349±1360, 1993 Sonenshein GE: Rel/NF-kB transcription factors and the control of apoptosis. Semin Cancer Biol 8:13±19, 1997 Song HY, Regnier CH, Kirschning CJ, Goeddel DV, Rothe M: Tumor necrosis (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kB and c-jun Nterminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc Natl Acad Sci 94:9792±9796, 1997 Telford WG, Nam SY, Podack ER, Miller RA: CD30-regulated apoptosis in murine CD8 T cells after cessation of TCR signals. Cellular Immunol 182:125±136, 1997
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Tian ZG, Longo DL, Funakoshi S, et al: In vivo antitumor effects of unconjugated CD30 monoclonal antibodies on human anaplastic large-cell lymphoma xenografts. Cancer Res 55:5335±5341, 1995 Wallach D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, Boldin MP: Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 17:331±367, 1999 Wang HH, Lach L, Kadin ME: Epidemiology of lymphomatoid papulosis. Cancer 70:2951±2957, 1992 Wendtner CM, Schmitt B, Gruss HJ, Druker BJ, Emmerich B, Goodwin RG, Hallek M: CD30 ligand signal transduction involves activation of a tyrosine kinase and of mitogen-activated protein kinase in a Hodgkin's lymphoma cell line. Cancer Res 55:4157±4161, 1995 Willemze R, Beljaards RC: Spectrum of primary cutaneous CD30 (Ki-1)-positive lymphoproliferative disorders. A proposal for classi®cation and guidelines for management and treatment. J Am Acad Dermatol 28:973±980, 1993 Willemze R, Kerl H, Sterry W, et al: EORTC classi®cation for primary cutaneous lymphomas: a proposal from the Cutaneous Lymphoma Study Group of the EORTC. Blood 90:354±371, 1997