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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 agonistic antibody, HeFi-1. Proliferative responses, CD30+ cutaneous lymphoproliferative disorders char- mitogen-activated kinase, and nuclear factor acterized by spontaneous tumor regression. The kB activities were determined with and without CD30 mechanism(s) of regression is unknown. In a recent activation. Mac-1 and Mac-2 A showed increased pro- study, a selective increase in CD30 expression in liferative responses to incubation with CD30 activating regressing lesions of lymphomatoid papulosis and antibody, HeFi-1. Inhibition of the mitogen-activated cutaneous CD30+ anaplastic large cell lymphoma was protein kinase activity caused growth inhibition of the shown, suggesting that activation of the CD30 signal- Mac-1, Mac-2 A, and JK cell lines. Activation of the ing pathway may be responsible for tumor regression, Fas pathway induced in all three cell lines. whereas no difference in Fas/ expression Taken together, these ®ndings suggest that resistance was found between regressing and nonregressing to CD30-mediated growth inhibition provides a lesions. Therefore we tested the effects of CD30 and possible mechanism for escape of cutaneous anaplastic Fas activation on three CD30+ cutaneous lymphoma large cell lymphoma from tumor regression. Mitogen- cell lines (Mac-1, Mac-2 A, JK) derived from non- activated protein kinase inhibitors are potential thera- regressing tumors of two patients who had progressed peutic agents for the treatment of advanced cutaneous from lymphomatoid papulosis to systemic anaplastic anaplastic large cell lymphoma. Key words: CD30/ large cell lymphoma. To evaluate the effects of CD30 cutaneous anaplastic large cell lymphoma/MAPK/NF-kB. signaling, the cell lines were incubated with a CD30 J Invest Dermatol 115:1034±1040, 2000

rimary cutaneous CD30+ lymphoproliferative disorders mycosis fungoides, CD30-negative T cell lymphomas of the skin (LPD) are recognized as a distinct biologic entity in the tend to have a more aggressive course and a poorer prognosis than European Organization for Research and Treatment of CD30+ cutaneous LPD. Cancer classi®cation of cutaneous lymphomas and the Members of the (TNF) super- Pforthcoming World Health Organization classi®cation of family, such as Fas (CD95), TNF receptors types I and II, CD27, lymphomas (Willemze et al, 1997; Jaffe et al, 1999). CD30+ CD30, CD40, DR3, DR4, and DR5 play an important role in cutaneous LPD are associated with frequent spontaneous regression immune regulation. These members of the TNF receptor family (Cabanillas et al, 1995; Paulli et al, 1995). Although cutaneous contain a ``death domain'' that triggers CD30+ LPD generally have a good prognosis, certain cases when activated by the appropriate ligand (Ashkenazi and Dixit, progress to aggressive systemic lymphomas and become resistant 1998). Some members of the TNF receptor and ligand superfamily to chemotherapy (Willemze and Beljaards, 1993; Paulli et al, 1995; are implicated in the pathogenesis of immune disorders and Willemze et al, 1997; Bekkenk et al, 2000). lymphomas (Gruss and Dower, 1995). An important function of CD30 antigen expression is a favorable prognostic factor in the TNF members is regulation of growth and elimination of cutaneous T cell lymphomas (Beljaards et al, 1993; Willemze et al, peripheral T cells during their expansion in response to antigenic 1993; Paulli et al, 1995; Bekkenk et al, 2000). With the exception of 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 Manuscript received May 5, 2000; revised July 28, 2000; accepted for regulated by Fas and TNFRI (Lenardo et al, 1999). The speci®city publication August 22, 2000. of T cell elimination by this pathway is partly due to a requirement Reprint requests to: Dr. Marshall E. Kadin, Director of Hemato- for simultaneous activation of T cell antigen receptor and the TNF pathology, Department of Pathology, YA 309, Beth Israel-Deaconess or Fas receptor (Lenardo et al, 1999). Medical Center, 330 Brookline Avenue, Boston, MA 02215. Email: The second mechanism of T cell elimination is cytokine- [email protected] Abbreviations: ALCL, anaplastic large cell lymphoma; LPD, lympho- withdrawal-induced cell death. In this pathway, T cells are proliferative disorders; LyP, lymphomatoid papulosis; MAPK, mitogen- deprived of the cytokine support that is associated with the activated protein kinase; NF-kB, nuclear factor kB; TNF, tumor necrosis antigen-induced activation state (Telford et al, 1997; Lenardo et al, factor. 1999). This mechanism of apoptosis is prevented by the presence of

0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 1034 VOL. 115, NO. 6 DECEMBER 2000 CD30 SIGNALING IN CUTANEOUS ALCL 1035 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 Figure 1. Immunohistochemical staining of CD30L. Immunohisto- that express CD30 in response to antigen stimulation and CD30 chemistry with antibody M80 reveals staining of Reed-Sternberg-like cells costimulation. and some smaller cells in LyP lesions of patients from whom Mac-1 and As CD30+ cutaneous LPD represents clonal expansion of an Mac-2 A cells (a and b) and JK cells (c and d) were derived. 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 for SN50. U0126 is a selective inhibitor of MEK1 and MEK2 (Favata et al, seen in these lesions is unknown. We believe that Fas and CD30 are 1998). All three chemicals were purchased from Calbiochem (San Diego, CA). Anti-human Fas antibody with agonistic properties was obtained from two signaling pathways that can mediate regression. The tumor R&D Systems, Minneapolis, MN (clone DX2.1). cells in CD30+ cutaneous LPD have been shown to express Fas (Paulli et al, 1998; Mori et al, 1999). CD30L expression shows a 3H-Thymidine incorporation assay Tumor cells were grown in correlation with regression of the lesions suggesting a role for CD30 round-bottomed 96 well plates in triplicate. Following incubation with activation in the usual regression of cutaneous CD30+ lymphomas soluble antibodies and 10% fetal bovine serum in RPMI, the wells were (Mori et al, 1999). pulsed with 1 mCi 3H-thymidine during the ®nal 12 h. Tumor cells were Until now, there has not been a functional study of the responses harvested with the aid of a cell harvester (PhD Systems, Cambridge, MA) of tumor cells from cutaneous CD30+ LPD to the activation of and radioactivity was measured with a scintillation counter (Wallac 1409; TNF receptor superfamily members including Fas and CD30, due Wallac, Gaithersburg, MD). to the lack of tumor cell lines. We developed three cell lines from Electrophoretic mobility shift experiments Nuclear extracts from cutaneous tumors and circulating tumor cells of two patients who tumor cell lines were obtained before and after stimulation with HeFi-1 had lymphomatoid papulosis (LyP) and then progressed to fatal antibody for 1 h. The extracts were incubated with 32P end-labeled NF-kB systemic lymphomas during the course of their disease (Davis et al, binding site oligoprobes (5¢-AGCTTGGGGTATTTCCAGCCG-3¢), 1992; Kadin et al, 1994; Schiemann et al, 1999). We investigated mutant oligoprobes (5¢-AGCTTGGCATAGGTCCAGCCG-3¢), and the responses of these cell lines to Fas and CD30 activation in vitro. excess cold oligoprobes, and were run on a nondenaturing 6% We also investigated the intracellular signaling mechanisms polyacrylamide gel. Gels were dried and exposed to ®lm (NF-kB/Rel T associated with CD30 activation, including nuclear factor kappa cell activation Gelshift , Geneka, Montreal, Canada). Nuclear extracts B (NF-kB) and mitogen-activated protein kinase (MAPK), in order were prepared as follows: 10 3 106 cells were incubated with HeFi-1 for to understand the mechanism of disease progression and to develop 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 novel therapies targeting these pathways in advanced disease dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl ¯uoride (PMSF), pH originating from cutaneous CD30+ LPD. 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 re- MATERIALS AND METHODS suspended in 40 ml buffer B (20 mM HEPES, 25% glycerol, 420 mM NaCl, Cell lines JK, Mac-1, and Mac-2 A are cutaneous ALCL cell lines (Kadin 1.5 mM MgCl2, 0.2 mM ethylenediamine tetraacetic acid, 0.5 mM DTT, et al, 1994). The JK cell line was developed from an advanced skin tumor of 0.2 mM PMSF) and incubated on ice for 20 min. Nuclei were pelleted by a patient who progressed over a 10 y period from LyP to ALCL spinning for 3 min at 4°C. Supernatants were aliquoted and stored at (Schiemann et al, 1999); the clonal relationship of LyP and ALCL lesions ±80°C. 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 MAPK kinase assay MAPK activity was determined by the MAPK clonally related cell lines derived from a CD30+ cutaneous T cell ALCL kinase assay kit according to the manufacturer's recommended protocols that had progressed from LyP over a 17 y period. Details of the cell lines (New England Biolabs, Beverly, MA). Brie¯y, whole cell lysates were have been reported elsewhere (Davis et al, 1992; Kadin et al, 1994). immunoprecipitated with an anti-MAPK antibody. The immuno- precipitated were used in a nonradioactive in vitro kinase assay. Immunohistochemistry Immunohistochemical staining for CD30L A Gst-Elk1 fusion peptide was used as a substrate for the detection of kinase was performed on 4 mm sections of formalin-®xed paraf®n-embedded activity in the immunoprecipitates. Following the kinase assay, material as previously described (Mori et al, 1999). Sections were stained phosphorylation of the Gst-Elk1 substrate was measured by Western with the M80 antibody against CD30L kindly provided by Immunex, blots using an antiphopho-Elk1 antibody for detection. Seattle, WA. RESULTS Antibodies and inhibitors HeFi-1 (NCI, Frederick, MD) is a mouse Immunohistochemical staining for CD30L Immuno- monoclonal IgG1 antibody raised against human CD30 (Hecht et al, 1985). histochemical staining con®rmed the presence of CD30L in tissue HeFi-1 has an agonistic effect on the CD30 pathway mimicking the effects sections from the LyP mainly in the cytoplasm of 10%-20% of large of CD30L. Mouse IgG1 (Sigma, St. Louis, MO) is used as an isotype- speci®c control. SN50 is a synthetic peptide that is cell permeable and atypical cells, including Reed-Sternberg-like cells, and some inhibits NF-kB by competing with the nuclear localization sequence of smaller cells (Fig 1). Staining of CD30L appeared to be associated NF-kB (p50) (Lin et al, 1995). SN50M is a synthetic peptide mutated at the with degenerative changes in some cells of the case from which nuclear localization sequence site so that it can be used as a negative control Mac-1 and Mac-2 A cell lines were derived. 1036 LEVI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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 isotype- speci®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. 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). 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, Figure 3. Fas-mediated growth inhibition of the cell lines. Effects of and TRAF-5 are known to bind the intracytoplasmic domain of Fas activation on the cell lines was determined by 3H-thymidine CD30 (Arch et al, 1998). TRAF-2 is known to activate NF-kB and incorporation assays following incubation with a Fas agonist antibody for the c-jun N-terminal kinase (JNK) pathways (Song et al, 1997). In 24 h. The counts are shown as percentages compared to controls (mean addition, in some cell lines MAPK is activated by CD30 signaling 6 SD). The experiments were performed in triplicate and repeated at least (Wendtner et al, 1995). As NF-kB and MAPK pathways are three times. A representative experiment is shown. *p < 0.05. important for numerous cell functions including cell survival, proliferation, and cytokine secretion, we thought these two Effects of CD30 activation on cell proliferation In order to pathways would be most important to explain the proliferative analyze the functional status of the CD30/CD30L signaling responses observed by CD30 activation (Hunter, 1995; Barnes and pathway in the cell lines representing advanced cutaneous ALCL, Karin, 1997; Sonenshein, 1997). As the JNK pathway is usually we used a , HeFi-1 (Hecht et al, 1985), that associated with stress responses and apoptosis, we did not pursue recognizes the CD30L binding domain of CD30 antigen and leads that pathway (Ip and Davis, 1998). The downstream signaling to activation of the CD30 signaling cascade. We and others have pathways associated with CD30 signaling and the sites of action of previously shown that this antibody mimics the action of CD30L the inhibitors used in the study are illustrated in Fig 4. and is an effective growth inhibitor of tumor cells of systemic We measured the nuclear NF-kB activity of cell lines Mac-1 and CD30+ ALCL, both in vitro and in vivo (Tian et al, 1995; Pfeifer Mac-2 A before and after CD30 activation. Nuclear extracts were obtained from cells that were incubated with antibody HeFi-1. The et al, 1999). Upon activation of the CD30 signaling pathway by 32 HeFi-1, Mac-1 and Mac-2 A cell lines showed proliferative nuclear extracts were incubated with P-labeled oligoprobes responses whereas the JK cell line did not change its proliferative representing NF-kB binding sites and run on a polyacrylamide activity, as measured in a 3H-thymidine incorporation assay (Fig 2). gel. Gelshift experiments showed minimal constitutive NF-kB We also tested the effects of CD30 activation of the Karpas 299 cell activity in Mac-1 and Mac-2 A cell lines. In contrast, following line, a systemic ALCL cell line with the translocation t(2; 5)(p23; incubation with HeFi-1 for 30 min, both cell lines showed strong q35), and observed a 50% growth inhibition as previously reported NF-kB activation (Fig 5). The speci®city of the binding was tested (Pfeifer et al, 1999) (not shown). by addition of excess cold probe and excess cold mutant probe. We then investigated the NF-kB pathway by use of a speci®c Effects of Fas activation on cell growth In order to assess inhibitor of NF-kB, SN50 (Lin et al, 1995). Mac-1 and Mac-2 A their sensitivity to Fas-mediated apoptosis in vitro, the cell lines cell lines demonstrated inhibition of cell growth within 24 h when were incubated with a Fas agonistic antibody (clone DX2.1, R&D incubated with the NF-kB inhibitor SN50 at 50 mg per ml systems) for 24 h, and then pulsed with 3H-thymidine for 12 h. All concentration in the presence of HeFi-1 (1 mg per ml). The JK three cell lines demonstrated signi®cant growth inhibition in cell line showed no response to the NF-kB inhibitor (Fig 6). As a response to Fas activation whereas control cells incubated with an negative control for SN50, we incubated the cells with SN50M, isotype-speci®c mouse IgG were unaffected (Fig 3). Cell viability which is a synthetic peptide that differs from SN50 in just two was independently assessed by trypan blue vital staining and cell 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 7. Activation of MAPK by CD30 signaling. MAPK activity Figure 5. Activation of NF-kB by CD30 activation. Nuclear extracts was measured after CD30 activation for 10 min, and control cells were obtained from Mac-1 and Mac-2 A cell lines were incubated with 32P- incubated with isotype-speci®c mouse IgG. CD30 activation was achieved labeled oligoprobes (H), (H) + excess cold oligoprobes (C), and (H) + by HeFi-1 antibody. Mac-2 A cell line was also incubated with the MEK1 excess cold mutant oligoprobes (M), with or without HeFi-1 for 1 h. The inhibitor U0126 for 10 min and the MAPK activity was measured. Equal arrows show the speci®c bands, which are retarded due to the binding of loading of wells was con®rmed by measuring the protein concentration of the probes to nuclear proteins, and the unbound probes at the bottom of the cell lysates before immunoprecipitation, and staining the blotted the gel. The remaining bands are due to nonspeci®c binding of the probe to membranes for protein. 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.

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 Figure 8. Inhibition of MAPK activity causes growth inhibition. that was slightly increased by CD30 activation (Fig 7). Growth responses of the cell lines to inhibition of MEK1 were tested at We also tested the effects of MAPK inhibition on the two different doses of the MEK1 and MEK2 inhibitor, U0126.The proliferative properties of the cell lines by using a recently inhibitor was added 30 min before the addition of HeFi-1. Responses are described speci®c MEK1 inhibitor, U0126 (Favata et al, 1998). shown as percentages of the control values (mean 6 SD). The experiments The cells were incubated with 1 or 10 mM of the inhibitor for were performed in triplicate and repeated at least three times. A 30 min, and then HeFi-1 was added. We measured 3H-thymidine representative experiment is shown. *p < 0.05. incorporation after 24 h. At 1 mM, all cell lines demonstrated signi®cant growth inhibition. When 10 mM of the inhibitor was cells in the presence of recombinant IL-2 (Leca et al, 1994). Those added, the cell lines were almost completely growth inhibited cells were not transformed, however, and cell growth arrest (Fig 8). occurred upon removal of the antibody. In contrast, our cell lines express an ab rather than gd phenotype and do not undergo DISCUSSION growth arrest upon removal of the CD30 activating antibody, In this study we demonstrate that activation of CD30 in cutaneous HeFi-1. Cutaneous and systemic CD30+ ALCL cell lines appear to CD30+ ALCL cell lines does not induce growth inhibition; instead have opposite growth responses to CD30 activation as CD30 it causes proliferation of the Mac-1 and Mac-2 A cell lines. Others activation is an inhibitory signal for systemic ALCL cell lines (Gruss have shown that activation of CD30 by a monoclonal antibody et al, 1994). Cutaneous and systemic ALCL also have distinct could induce sustained proliferation of human autoreactive gd T pathways of pathogenesis. Systemic ALCL cell lines harbor the 1038 LEVI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 9. Hypothesis for mechanisms of pro- gression 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 pro- gression 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 tumorigenicity (Bargou et al, 1997). We were interested to NPM/ALK fusion protein (ALK) leading to the unregulated compare cutaneous ALCL cell lines with Hodgkin's lymphoma as growth of tumor cells (Morris et al, 1994). ALK is characteristically these diseases have similar morphologic and immunohistochemical absent in cutaneous ALCL (DeCoteau et al, 1996). Therefore, features (Davis et al, 1992; Kadin, 1993). In addition, patients with cutaneous and systemic ALCLs have been identi®ed as separate LyP sometimes develop Hodgkin's lymphoma (Davis et al, 1992; entities in the new lymphoma classi®cations (Jaffe et al, 1999). Wang et al, 1992; Cabanillas et al, 1995). Our study shows that, There is limited information regarding the etiology and biology unlike Hodgkin's lymphoma, cutaneous ALCL cell lines do not of CD30+ cutaneous ALCL. In general, the primary cutaneous have high constitutive NF-kB activity. LPD constitute a spectrum of diseases ranging from LyP to The ERK (MAPK) pathway is also known to be activated by cutaneous ALCL, which have an excellent prognosis (Paulli et al, members of the TNF receptor family, including CD30 (Wendtner 1995). A small percentage of these diseases (approximately 5%), et al, 1995; Wallach et al, 1999). In a previous study by Wendtner however, progress to fatal systemic lymphomas (Beljaards and et al (1995), CD30 signaling led to MAPK activation in the T-cell- Willemze, 1992). The cell lines we used in our study are type Hodgkin's lymphoma cell line HDLM-2, but not in the representative of such uncommon cases that ®rst present as LyP systemic ALCL cell line Karpas 299 nor in the B-cell-type and later progress to fatal systemic lymphomas. Hodgkin's lymphoma cell line KMH2. Interestingly, the HDLM-2 A well-known phenomenon associated with cutaneous cell line showed increased proliferation in response to CD30 CD30+ LPD is the frequent occurrence of spontaneous regression activation whereas Karpas 299 was growth inhibited. Here, we (Willemze et al, 1993). In a recent study, a positive correlation was demonstrate that the MAPK (ERK) pathway is activated by CD30 demonstrated between regression and CD30L expression levels signaling in cutaneous T cell ALCL cell lines Mac-1 and JK as well, measured by semiquantitative reverse transcriptase polymerase whereas Mac-2A has high constitutive expression of MAPK; chain reaction in cases of LyP and primary cutaneous ALCL, inhibition of MAPK is an effective way of inhibiting growth of suggesting that CD30 activation may play a role in the spontaneous these cell lines. regression of such lesions (Mori et al, 1999). In this study, we The mechanism of altered responsiveness to CD30 activation in con®rmed the presence of CD30L in the LyP skin lesions of each the cell lines from advanced cutaneous ALCL is unclear. We have patient by immunohistochemistry. Our results raise the possibility shown that, in the systemic ALCL cell line Karpas 299, CD30 that the development of resistance to the CD30L-mediated growth activation causes cell cycle arrest despite activation of the NF-kB inhibitory effects, or even transformation of the tumor response to pathway (Levi et al, submitted for publication). The proliferative one of cellular proliferation in response to CD30 activation, may response seen in the Mac-1 and Mac-2 A cell lines was at least partly provide a mechanism for tumor cell escape from the normal growth due to MAPK and NF-kB activation by CD30 signaling. In this regulatory responses and support progression of LyP to ALCL. study, we demonstrated that MAPK activation is one of the major Recently, Fas expression was demonstrated in cutaneous effectors of CD30 signaling in these cutaneous ALCL cell lines. CD30+ LPD (Paulli et al, 1998; Mori et al, 1999). A functional It has been shown that mammalian cells can be transformed by role for Fas-mediated apoptosis in the regression of these lesions, transfection with constitutively active MAPK (Mansour et al, however, has not been shown. We investigated the susceptibility of 1994). In addition, inhibition of the MAPK activity in mice our cutaneous ALCL lines to Fas-induced apoptosis. As all three xenografted with colon tumors causes inhibition of tumor growth cell lines were sensitive to Fas-induced apoptosis, we conclude that (Sebolt-Leopold et al, 1999). We believe that inhibitors of the the Fas signaling pathway is unlikely to play a role in the MAPK pathway can become useful in the treatment of various progression of LyP to ALCL. cancers as the Ras pathway, which is upstream of the MAPK, is In order to gain insight into the downstream effects of CD30 frequently mutated in human cancers (Macara et al, 1996; Hunter, activation, we investigated some pathways that are involved in the 1997). CD30/CD30L signaling system. CD30 activation is known to Based on this study and previous work, we propose a model of cause upregulation of NF-kB, utilizing the TRAFs as adapter progression of LyP to systemic lymphoma by several steps that molecules (Duckett et al, 1997; Song et al, 1997). Constitutive abolish the physiologic inhibitory mechanisms that are important activation of NF-kB has been demonstrated in Hodgkin's for the regulation of the immune system such as transforming lymphoma cell lines and has been implicated in their survival and growth factor beta (TGF-b) and CD30 (Fig 9). According to this VOL. 115, NO. 6 DECEMBER 2000 CD30 SIGNALING IN CUTANEOUS ALCL 1039 model, an unknown antigenic stimulus triggers clonal expansion of favorable prognosis: a European multicenter study on 47 patients. Cancer T cells, which form cutaneous in®ltrates that manifest as multiple 71:2097±2104, 1993 Cabanillas F, Armitage J, Pugh WC, Weisenburger D, Duvic M: Lymphomatoid regressing skin lesions of LyP. The LyP cells are usually eliminated papulosis: a T cell dyscrasia with a propensity to transform into malignant by growth inhibitory mechanisms such as Fas, CD30, and TGF-b. lymphoma. Ann Intern Med 122:210±217, 1995 Therefore, the lesions spontaneously regress in weeks to months. Chiarle R, Podda A, Prolla G, Podack ER, Thorbecke GJ, Inghirami G: CD30 After recurrent episodes of spontaneously regressing LyP lesions, a overexpression enhances negative selection in the thymus and mediates programmed cell death via a Bcl-2 sensitive pathway. J Immunol 163:194±205, small number of patients will develop large lesions that require 1999 treatment. A few of these patients will have extracutaneous spread Chott A, Vonderheid EC, Olbricht S, Miao N-N, Balk SP, Kadin ME: The of the disease, which may be resistant to chemotherapy. We dominant T cell clone is present in multiple regressing skin lesions and hypothesize that in these patients disease progression from LyP to associated T cell lymphomas of patients with lymphomatoid papulosis. J Invest Dermatol 106:696±700, 1996 ALCL is associated with impairment of the normal T cell growth Davis TH, Morton CC, Miller-Cassman R, Balk SP, Kadin ME: Hodgkin's disease, inhibitory mechanisms that would otherwise cause regression of lymphomatoid papulosis and cutaneous T cell lymphoma derived from a these lesions. In these and prior experiments, we tested three of the common T cell clone. N Eng J Med 326:1115±1122, 1992 best characterized inhibitory mechanisms that regulate the T cell DeCoteau JF, Butmarc JR, Kinney MC, Kadin ME: The t (2; 5) chromosomal translocation is not a common feature of primary cutaneous CD30+ arm of the immune system - Fas, CD30, and TGF-b - and lymphoproliferative disorders: comparison with anaplastic large cell demonstrated a selective role for CD30 in the context of escape lymphoma of nodal origin. Blood 87:3437±3441, 1996 from growth inhibitory mechanisms. In the case of TGF-b, Duckett CS, Gedrich RW, Gil®an MC, Thompson CB: Induction of nuclear factor resistance was shown to be mediated by mutations of the type I (JK) kB by the CD30 receptor is mediated by TRAF1 and TRAF2. Mol Cell Biol 17:1535±1542, 1997 and type II (Mac-2 A) receptors (Knaus et al, 1996; Schiemann et al, Falini B, Pileri S, Pizzolo G, et al: CD30 (Ki-1) molecule: a new of 1999). These mutations were due to a deletion that eliminates the the tumor necrosis factor receptor superfamily as a tool for diagnosis and initiating methionine required for translation of the type I receptor immunotherapy. Blood 85:1±14, 1995 in the JK cell line, and due to a single nucleotide substitution (A to Favata MF, Horiochi KY, Manos EJ, et al: Identi®cation of a novel inhibitor of mitogen-activated protein kinase kinase. 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J Immunol 134:4231±4236, 1985 respond to HeFi-1. At present no cell lines from regressing lesions Hunter T: Protein kinases and phosphatases: the Yin and Yang of protein are available for comparison but this is a long-term goal. The fact phosphorylation and signaling. Cell 80:225±236, 1995 Hunter T: Oncoprotein networks. Cell 88:333±346, 1997 that both TGF-b and CD30 signaling systems were noninhibitory Ip TY, Davis RJ: by the c-jun N-terminal kinase (JNK) - from suggests that abrogation of both pathways may be required for the in¯ammation to development. Curr Opin Cell Biol 10:205±219, 1998 progression of these lymphomas to fatal systemic disease. Jaffe ES, Harris NL, Diebold J, Muller-Hermelink HK: WHO classi®cation of In summary, we have demonstrated that in addition to evading neoplastic diseases of the hematopoietic and lymphoid tissues. A progress report. 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