The mitogen-activated protein kinase (MAPK) (also called extracellular signal-regulated kinase [ERK]) pathway has been implicated in malignant transformation and in the regulation of cellular growth and proliferation of several tumor types, but its expression and function in Hodgkin disease (HD) are unknown. We report here that the active phosphorylated form of MAPK/ERK is aberrantly expressed in cultured and primary HD cells. Inhibition of the upstream MAPK kinase (also called MEK) by the small molecule UO126 inhibited the phosphorylation of ERK and demonstrated a dose-and time-dependent antiproliferative activity in HD cell lines. UO126 modulated the levels of several intracellular proteins including B-cell lymphoma protein 2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1) and caspase 8 ho-molog FLICE-inhibitory protein (cFLIP), and induced G 2 M cell-cycle arrest or apo-ptosis. Furthermore, UO126 potentiated the activity of apoliprotein 2/tumor necro-sis factor-related apoptosis-inducing li-gand (APO2L/TRAIL) and chemotherapy-induced cell death. Activation of CD30, CD40, and receptor activator of nuclear kappa (RANK) receptors in HD cells by their respective ligands increased ERK phosphorylation above the basal level and promoted HD cell survival. UO126 inhibited basal and ligand-induced ERK phosphorylation, and inhibited ligand-induced cell survival of HD cell lines. These findings provide a proof-of-principle that inhibition of the MEK/ERK pathway may have therapeutic value in HD.

Summary. T-cell cytotoxicity is primarily mediated by two cell surface proteins, Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and intracellular perforin and granzyme granules. FasL-deficient and perforin-deficient T lymphocytes maintain cytotoxicity but fail to induce graft-versus-host disease (GVHD) when transplanted into mice, suggesting that GVHD and graftversus-tumour (GVT) effects can be dissociated, and that TRAIL is not involved in the pathogenesis of GVHD. Because TRAIL could mediate a favourable GVT effect it became important to study the spectrum of its activity and to investigate factors that can dissociate its expression from FasL. TRAIL induced apoptosis in 11/41 (27%) tumour specimens of haematological origin compared to 16/41 (39%) induced by FasL. Although eight specimens were sensitive to both FasL and TRAIL, no synergism was observed between these two ligands. TRAIL induced apoptosis in a dose and time dependent manner with an ED 50 of 0 . 5 mg/ml and ED max of 1 mg/ml. TRAIL activity was not reduced by the over-expression of the multidrug resistant (MDR) protein, and was not enhanced by 9-cis retinoic acid (RA), which can down-regulate bcl-2 protein. Both ligands were simultaneously up-regulated in normal peripheral blood lymphocytes in response to IL-2, IL-15 and anti-CD3 antibody, whereas IL-10 had no effect. Together, our data show that (1) TRAIL can mediate cell death in a variety of human haematological malignancies, (2) resistance to TRAIL is not mediated by MDR protein, (3) the lack of synergy between TRAIL and FasL suggests that either one is sufficient to mediate T-cell cytotoxicity, and (4) within the panel of cytokines tested, the expression of TRAIL and FasL could not be dissociated.

The expression of Bcl-2 family members was examined in normal and leukemic hematopoietic cells. Immature hematopoietic progenitor cells (CD34 + /33 − /13 − ) did not express Bcl-2 but Bcl-X L , the majority of CD34 cells expressed Bcl-2, Bcl-X L and BAD, and normal promyelocytes (CD34 − /33 + ) lacked expression of both Bcl-2 and Bcl-X L , while leukemic CD34 + progenitors and promyelocytes expressed these anti-apoptotic proteins. In AML, Bcl-2 expression was higher on CD34 + than on all AML cells, however, expression of Bcl-2 or Bcl-X L did not predict achievement of complete remission. Surprisingly, low Bcl-2 content was associated with poor survival in a group of patients with poor prognosis cytogenetics. The anti-apoptotic BAD protein was found to be expressed in AML, but was phosphorylated in 41/42 samples. Phosphorylation was found at both sites, Ser 112 and Ser 136. During induction chemotherapy, Bcl-2 levels of CD34 cells increased significantly. In the context of evidence for small numbers of leukemic CD34 + cells expressing very high levels of Bcl-2 prior to therapy, this finding is interpreted as a survival advantage of Bcl-2 overexpressing progenitors and rapid elimination of cells with low Bcl-2. Bcl-2 and Bcl-X L were both expressed in minimal residual disease cells. Downregulation of Bcl-2 mRNA and protein was observed by ATRA and the combination of Ara-C, followed by ATRA, resulted in markedly increased cytotoxicity in HL-60 cells, as compared to Ara-C alone or ATRA followed by Ara-C. Implications of these findings for the development of new therapeutic strategies for AML are discussed.

Summary. In acute myeloid leukaemia (AML), cell kinetic quiescence has been postulated to contribute to drug resistance. As the anti-apoptotic genes Bcl-2 and Bcl-X L have been implicated in cell cycle regulation, we investigated the expression of these genes in non-proliferating (Q) and proliferating (P) AML and normal CD34 + progenitor cells. Using reverse transcription polymerase chain reaction, Bcl-X L and Bcl-2 were overexpressed in Q versus P AML cells, whereas no difference in Bcl-X S and Bax expression was found. Furthermore, the Bcl-X L /X S but not the Bcl-2/Bax ratio was higher in Q AML compared with normal CD34 + Q cells (P ¼ 0AE001). An inverse correlation between Bcl-2 expression of leukaemic Q cells and their ability to enter the cell cycle was found. Treatment with all-trans retinoic acid (ATRA) reduced Bcl-2 and Bcl-X L expression in the leukaemic Q cells, and enhanced their chemosensitivity to cytosine arabinoside (ara-C). These findings demonstrate overexpression of the anti-apoptotic proteins Bcl-X L and Bcl-2 in quiescent CD34+ AML cells and suggest their involvement in the chemoresistance. The observed inverse correlation between Bcl-2 and proliferation suggests a role for Bcl-2 in the cell cycle regulation of AML. These findings could be used in the development of therapies that selectively induce apoptosis in quiescent leukaemic progenitor cells.