Nonmyeloablative stem cell transplantation in metastatic renal cell carcinoma: delayed graft-versus-tumor effect is associated with chimerism conversion but transplantation has high toxicity.

Seven out of 29 patients with metastatic renal cell carcinoma (RCC) considered eligible for allogeneic stem cell transplantation underwent nonmyeloablative stem cell transplantation (NST) from HLA-identical donors. Conditioning comprised cyclophosphamide, fludarabine and antithymocyte globulin. Prolonged mixed chimerism (MC) after engraftment converted to complete donor chimerism (CC) after infusion of donor lymphocytes and/or graft-versus-host disease (GvHD) in six patients. Five patients developed severe GvHD. Two of seven patients had a delayed tumor response after conversion to CC. After a median follow-up of 10 months (4-24 months), 5/7 patients are alive, one in very good partial remission (PR), one with stable and three with progressive disease. One of the seven patients died from sepsis in PR and 1/7 died from rapid tumor progression after sustained MC. None of the 22 nontransplanted patients responded to further therapies. Survival after 1 year was 59% in transplanted and 66% in nontransplanted patients (n.s.). A pooled data analysis from the literature suggests a graft-versus-tumor effect after transplant in patients with metastatic RCC, which becomes effective after chimerism conversion. Available data demonstrate high nonrelapse mortality in these patients. NST in RCC still has to be regarded as an investigational approach requiring careful patients' selection and longer follow-up within clinical studies.

Infectious complications after high-dose chemotherapy and autologous stem cell transplantation: comparison between patients with lymphoma or multiple myeloma and patients with solid tumors.

From November 1994 to May 1998, 117 patients (66 with solid tumor, 36 with lymphoma, 14 with multiple myeloma, one with acute leukemia) underwent 178 cycles of high-dose chemotherapy and autologous stem cell transplantation (ASCT) at our institution. We retrospectively analyzed the infectious complications that occurred after ASCT. Median duration of neutropenia (granulocyte count <0.5 x 10(9)/l ) was 8 days, the overall incidence of fever requiring antimicrobial treatment was 63%. 35.4% of patients had fever of unknown orign (FUO), whereas primary bacteremia occurred in 21.3%, pneumonia in 3.4% and severe skin infection in 1.1% of patients. Invasive fungal infections occurred in three, and enterocolitis in one patient. Infection was fatal in three patients (2.6%), in each case due to septic shock. The most frequently isolated pathogens were Gram-positive cocci. Median time to defervescence with antimicrobial therapy was 4 days (6 days in patients with bacteremia or other severe infection, and 3 days in patients with FUO). First-line antimicrobial therapy was successful in 65% of patients with FUO and 30.6% of patients with documented infections. With respect to the incidence, type and clinical course of infection, no significant differences between patients with lymphoma or multiple myeloma and those with solid tumors were detected.

Induction of stable long-term mixed hematopoietic chimerism following nonmyeloablative conditioning with T cell-depleting antibodies, cyclophosphamide, and thymic irradiation leads to donor-specific in vitro and in vivo tolerance.

BACKGROUND: Successful transplantation of solid organs relies on long-term immunosuppression for the prevention of graft rejection. Donor-specific tolerance without the need for continuous immunosuppression can be observed after allogeneic BMT. However, its routine use for tolerance induction has been precluded so far by the high conditioning-related toxicity of standard BMT regimens. Our laboratory has recently established a cyclophosphamide (CTX) plus thymic irradiation (TI)-based nonmyeloablative conditioning protocol for the treatment of hematologic malignancies. We have recently described the successful clinical application of this approach for the induction of donor-specific tolerance in a patient receiving a living-related kidney transplant, which resulted in graft acceptance without long-term immunosuppression. The aim of this study was to evaluate the induction and maintenance of host-versus-graft tolerance following this CTX-plus-TI-based regimen in a mouse model. METHODS: Induction of mixed hematopoietic chimerism and development of donor-specific tolerance following the CTX-based nonmyeloablative conditioning regimen (200 mg/kg CTX, in vivo T-cell depletion [anti-CD4 monoclonal antibody (MoAb) GK1.5 and anti-CD8 MoAb 2.43], and 7 Gy TI) was studied in the fully major histocompatibility complex (MHC)-mismatched B10.A (H2a)-->B6 (H2b) strain combination. RESULTS: The conditioning regimen allowed allogeneic bone marrow engraftment and persistent (>30 weeks) mixed lymphohematopoietic chimerism in almost all recipients. TI was essential to allow engraftment and development of tolerance, which was evident in all lasting chimeras. Compared to animals receiving a similar TBI-based conditioning regimen, overall levels of chimerism were significantly lower in the CTX-plus-TI-conditioned animals. However, donor-specific tolerance in vitro and in vivo was evident in CTX-plus-TI-conditioned chimeras. Tolerance was associated with the presence of donor-type MHC class II+ cells in the thymus and deletion of donor-reactive cells, as determined by Mtv-8 and Mtv-9 superantigen-mediated deletion of Vbeta11+ and Vbeta5/1.2+ T cells. CONCLUSION: Engraftment, long-term chimerism, and induction of donor-specific tolerance can be achieved using a nonmyeloablative CTX-based conditioning regimen in fully MHC-mismatched BMT recipients without the induction of GVHD.

Anti-CD20- and B-cell receptor-mediated apoptosis: evidence for shared intracellular signaling pathways.

Clinical administration of the anti-CD20 antibody IDEC-C2B8 can induce remission of low-grade B-cell lymphoma. Whereas it has been suggested that the main mechanisms of action are complement-mediated and antibody-dependent cell-mediated cytotoxicity, we demonstrate that monoclonal antibody IDEC-C2B8 is a strong inducer of apoptosis in CD20-positive B-cell lymphoma cell lines reflecting different stages of lymphomagenesis. Thus, CD20-dependent apoptosis was inducible in human surface IgM-positive Burkitt's lymphoma cell lines as well as in more mature surface IgM-negative B-cell lymphoma cell lines carrying the t(14;18) translocation. Furthermore, in Burkitt's lymphoma cell lines, we observed a striking correlation between anti-CD20- and B-cell receptor-mediated apoptosis with regard to sensitivity toward the apoptotic stimuli and the execution of the apoptotic pathway. Thus, induction of anti-CD20- or B-cell receptor-mediated apoptosis involved rapid up-regulation of the proapoptotic protein Bax. In addition, we show similar changes in the mRNA expression level of two early response genes, c-myc and Berg36, as well as activation of the mitogen-activated protein kinase family members p44 (extracellular signal-regulated kinase 1) and p42 (extracellular signal-regulated kinase 2) and activation of activator protein 1 (AP-1) DNA binding activity. These data support our hypothesis that both pathways are mediated in part by the same signal-transducing molecules. These results might help explain the resistance and regression of lymphomas to IDEC-C2B8 and give new insights in the signaling cascade after CD20 ligation.

Successful peripheral blood stem cell mobilization with etoposide (VP-16) in patients with relapsed or resistant lymphoma who failed cyclophosphamide mobilization.

High-dose chemotherapy (HDCT) followed by autologous blood stem cell transplantation is considered the treatment of choice for patients with relapsed or resistant aggressive non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD). However, several authors report failure of standard mobilization regimens in 29% to 56% of these patients making the completion of HDCT impossible and as a result, negatively influencing long-term outcome. Thus, effective new regimens for patients failing initial mobilization are needed. Here we report the results of using etoposide as a mobilizing agent in 16 patients with primary resistant or relapsed malignant lymphoma who had failed prior mobilization of peripheral blood stem cells (PBSC) with cyclophosphamide (4 g/m2) followed by G-CSF. The use of etoposide 500 mg/m2 (days 1-4) + G-CSF resulted in the successful collection of adequate numbers of PBSC with a median harvest of 3.6 x 10(6)/kg (range 2.2-12.6) CD34+ cells in all 16 patients. In 7/16 (44%) patients, the target yield of at least 2.0 x 10(6) CD34+ cells was harvested by a single apheresis and the maximum number of separations for all patients was two. No excessive toxicities appeared, allowing all patients to proceed to myeloablative chemotherapy. In addition, median peak values of circulating CD34+ cells were significantly higher after etoposide as compared to cyclophosphamide (49.2/microl vs 4.7/microl; P = 0.0004). These results indicate that etoposide + G-CSF is a highly effective mobilization regimen in patients who have failed cyclophosphamide mobilization.

Combined positive/negative purging and transplantation of peripheral blood progenitor cell autografts in breast cancer patients: a pilot study.

This trial studied the feasibility and efficiency of a novel procedure of double purging to eliminate tumor cells from leukapheresis products of stage IV breast cancer patients. After induction and mobilization therapy, 35 leukapheresis products from 16 breast cancer patients were subjected to CD34+ enrichment (i.e., positive selection) with the Isolex 300 device and subsequent immunomagnetic depletion of tumor cells (i.e., negative selection) using a cocktail of three monoclonal antibodies directed against epithelial antigens. Patients with clinical response to induction chemotherapy proceeded to tandem high-dose chemotherapy, which consisted of melphalan (140 mg/m2) followed by retransfusion of the purged graft. After hematologic recovery, patients received ifosfamide 14 g/m2, carboplatin 1.5 g/m2, and etoposide 1.5g/m2 (ICE), again followed by autografting. After positive selection, a median purity of 96.6% CD34+ cells (range 48.4-99.2%) and a recovery of 56.8% (range 25.8-92.6%) were achieved. Subsequent negative purging resulted in a median CD34+ purity of 97.2%. Overall CD34+ recovery after both purging procedures was 51.1% (range 18.5-82.4%). Tumor cells were detectable in 8 of 16 (50%) starting fractions before purging. After both purging cycles, only 1 of 16 autografts remained positive for tumor cells compared to 3 of 16 after CD34+ selection. A calculated purging efficiency of 2 to >4 log was achieved. Engraftment was rapid, reaching > or =500/microL neutrophils on day +10 after melphalan and on day +9 after ICE. A platelet count of > or =20.000/microL was reached on day +12 after melphalan and on day +11 after ICE. Thus, combining positive and negative purging is feasible, further enhances purging efficiency, and does not compromise the quality of the graft, leading to rapid engraftment after high-dose chemotherapy.

Detection of tumor cells in peripheral blood samples from patients with germ cell tumors using immunocytochemical and reverse transcriptase-polymerase chain reaction techniques.

The aim of this study was to establish sensitive techniques for the detection of residual germ cell tumor cells in peripheral blood and progenitor cell harvests. For this purpose, we used immunocytochemical staining for cytokeratin filaments and reverse transcriptase-polymerase chain reaction (RT-PCR) for epidermal growth factor receptor (EGF-R) and germ cell alkaline phosphatase (GCAP). Germ cell tumor lines Tera-1 and Tera-2 were titrated into normal peripheral blood. Immunocytochemical staining allowed detection of one cytokeratin-positive tumor cell in 10(5) cells. RT-PCR for EGF-R revealed one tumor cell in 10 cells for Tera-1 and one tumor cell in 10(3) cells for Tera-2, while RT-PCR for GCAP displayed a sensitivity of one tumor cell in 10(6) cells for Tera-1, one tumor cell in 10(4) cells for Tera-2, and no positivity in normal mononuclear cells (n = 20) and progenitor cell harvests (n = 5). The analysis of peripheral blood and progenitor cell harvests from 20 patients with germ cell tumors revealed tumor cell contamination in three patients using immunocytochemical staining and in seven patients by RT-PCR for GCAP. We conclude that RT-PCR for GCAP appears to be suitable for the sensitive detection of residual germ cell tumor cells in peripheral blood and progenitor cell harvests.

Growth and differentiation of human stem cell factor/erythropoietin-dependent erythroid progenitor cells in vitro.

Stem cell factor (SCF) and erythropoietin (Epo) effectively support erythroid cell development in vivo and in vitro. We have studied here an SCF/Epo-dependent erythroid progenitor cell from cord blood that can be efficiently amplified in liquid culture to large cell numbers in the presence of SCF, Epo, insulin-like growth factor-1 (IGF-1), dexamethasone, and estrogen. Additionally, by changing the culture conditions and by administration of Epo plus insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34(+) peripheral blood stem cells and human bone marrow and can be isolated, amplified, and differentiated in vitro under the same conditions. Thus, highly homogenous populations of SCF/Epo-dependent erythroid progenitors can be obtained in large cell numbers that are most suitable for further biochemical and molecular studies. We demonstrate that such cells express the recently identified adapter protein p62(dok) that is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21(cip1) and p27(kip1) that are highly induced when cells differentiate. Thus, the in vitro system described allows the study of molecules and signaling pathways involved in proliferation or differentiation of human erythroid cells.

Ex vivo breast cancer cell purging by adenovirus-mediated cytosine deaminase gene transfer and short-term incubation with 5-fluorocytosine completely prevents tumor growth after transplantation.

Peripheral blood progenitor harvests of breast cancer patients are contaminated with tumor cells, suggesting a potential role for these cells in the relapse after high-dose chemotherapy. Whereas physical purging methods do not eliminate contaminating tumor cells completely, pharmacological purging, although highly efficient, is hampered by a strong nonspecific toxicity toward hematopoietic progenitor cells. Taking advantage of the high efficiency of adenovirus-mediated gene transfer to epithelial cells, we selectively loaded breast cancer cells in vitro with a cytotoxic drug by gene transfer of the prodrug-converting enzyme cytosine deaminase (AdCMV.CD) and 5-fluorocytosine (5-FC). Despite the low dose of vector administered, limited exposure to 5-FC, and transplantation only of viable tumor cells into SCID mice, all animals that received cells treated in vitro with AdCMV.CD plus 5-FC were completely free of tumor development. These data show that the selective loading of tumor cells with AdCMV.CD/5-FC might be useful for purging of autografts.

Inhibition of CPP32 blocks surface IgM-mediated apoptosis and D4-GDI cleavage in human BL60 Burkitt lymphoma cells.

Apoptosis (programmed cell death) is instrumental in the process of controlling lymphocyte growth and selection. Negative selection, mediated by surface IgM (sIgM) signaling after encountering self antigen, eliminates autoreactive B cells. To identify proteins which are potentially involved in anti-IgM-mediated apoptosis, we used an anti-IgM-sensitive subclone of the human Burkitt lymphoma cell line BL60. After anti-IgM treatment and separation of apoptosis-committed cells, we performed high resolution two-dimensional gel electrophoresis (2-DE). Comparison of the 2-DE protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots. Subsequent analysis of these proteins was performed by mass spectrometry and Edman microsequencing. We report that one of these spots which disappears after sIgM cross-linking turned out to be D4-GDI. D4-GDI is an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPase. D4-GDI was rapidly truncated to a 23-kDa fragment in BL60 cells. By using a Rho-GDI-specific antiserum, which cross-reacts with D4-GDI, we observed the onset of cleavage after 8 h of stimulation with anti-IgM. Cleavage and apoptosis could be completely inhibited by z-DEVD-fmk, a selective irreversible inhibitor of CPP32 (caspase-3), whereas ac-YVAD-cmk, an inhibitor for interleukin-1beta-converting enzyme-like proteases, did not block cleavage of D4-GDI or apoptosis. Our results revealed the functional importance of caspases and a new target protein in the process of anti-IgM-mediated apoptosis.

Overexpression of the death-promoting gene bax-alpha sensitizes human BL-41 Burkitt lymphoma cells for surface IgM-mediated apoptosis.

Major regulators of programmed cell death, or apoptosis, are the members of the bcl-2 gene family. Recently, we reported that surface(s) IgM triggering of the human B lymphoma cell line BL-41 led to strong induction of bax-alpha, a death-promoting member of the bcl-2 family, and subsequently to induction of apoptosis, suggesting a potential regulatory role of bax-alpha in sIgM-mediated cell death. In contrast, apoptosis-resistant subclones of this cell line showed only weak bax-alpha expression, which was not inducible by sIgM cross-linking. In this study, we were able to demonstrate the functional significance of this observation. We stably transfected bax-alpha into a BL-41 subline resistant against sIgM-mediated apoptosis. Several bax-alpha overexpressing clones could be selected, which all showed enhanced sensitivity for sIgM-mediated apoptosis. In contrast, no sensitive clone could be identified in a large number of mock controls. This clearly indicates that induction of bax-alpha is a critical regulatory step, which sensitizes B cells for sIgM-mediated apoptosis.

Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression.

Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.

Activation and activation-induced death of human tonsillar B cells and Burkitt lymphoma cells: lack of CD95 (Fas/APO-1) ligand expression and function.

Activation of T cells was shown to up-regulate the Fas ligand (FasL) which binds to the CD95 (APO-1/Fas) antigen and mediates activation-induced cell death (AICD) of activated T cells and T lymphoma cells. A recent report showed that mouse B cells express the FasL upon activation with lipopolysaccharide (LPS). We therefore asked whether activation of human B cells induces expression of FasL and whether AICD is mediated, as in T cells, through autocrine production of the FasL. We used human tonsillar B cells and Burkitt lymphoma cell lines which were activated by CD40 ligand, surface (s)IgM cross-linking, or LPS. Northern and Western blot analysis failed to detect FasL during B cell activation or AICD of both normal and malignant B cells. Low-level expression of FasL was detected by reverse transcriptase-polymerase chain reaction. Functional experiments, however, showed that FasL is not functionally expressed upon activation. IgM-mediated AICD in the tonsillar or Burkitt lymphoma B cells could not be inhibited by FasL blocking. Thus, our data show that, in contrast to T cells, activation of normal or malignant human B cells does not lead to functional FasL expression.

Monitoring of tumor cell purging after highly efficient immunomagnetic selection of CD34 cells from leukapheresis products in breast cancer patients: comparison of immunocytochemical tumor cell staining and reverse transcriptase-polymerase chain reaction.

We studied the efficiency of indirect tumor cell purging via enrichment of CD34+ hematopoietic progenitor cells from leukapheresis products (LP) in breast cancer patients based on immunomagnetic selection of CD34+ cells. Detection of tumor cells was made by immunocytochemical staining. In addition, we evaluated the capacity of cytokeratin 19 (CK19)- and a novel epidermal growth factor receptor (EGF-R)-specific reverse transcriptase-polymerase chain reaction (RT-PCR) for monitoring tumor cell depletion. LP from 13 breast cancer patients were analyzed. Twenty-three CD34 selection procedures were performed. A median of 1.4 x 10(10) total nucleated cells ([TNC] range, 0.88 to 3.5 x 10(10)) with a median CD34 purity of 2.5% (range, 0.4% to 6.3%) were entered into the selection procedure. Immunomagnetic CD34 enrichment resulted in a median purity of 83.3% (range, 45% to 95.4%) and a median recovery of 73.2% (range, 22% to 95%). Retransfusion of CD34-selected cells after high-dose chemotherapy resulted in a rapid and sustained hematologic recovery, reaching an absolute neutrophil count of 500/microL at day +10 and platelet count of 20,000/microL at day +11. Tumor cell depletion was quantified by immunocytochemical detection of CK19-positive cells. By this method, a median tumor cell depletion of 1.9 log (range, 0.7 to > 3 log) could be demonstrated. Immunocytochemical detection of tumor cells was more sensitive than RT-PCR, yielding positive results in 81% of LP (17 to 21) versus 58% positive LP (10 of 17). However, EGF-R-based RT-PCR was much more sensitive than CK19-based RT-PCR (10 of 17 v 1 of 17). Despite highly efficient CD34 selection, tumor cells were still detectable after CD34 enrichment using immunocytochemistry and EGF-R-specific RT-PCR. Thus, this novel EGF-R-specific RT-PCR appears to be of value as an additional method to detect contaminating breast cancer cells within LP.

Reverse transcriptase-polymerase chain reaction (RT-PCR)-controlled immunomagnetic purging of breast cancer cells using the magnetic cell separation (MACS) system: a sensitive method for monitoring purging efficiency.

A modified reverse transcriptase-polymerase chain reaction (RT-PCR) technique was established with the aim of monitoring the tumor cell contamination in peripheral blood stem cells harvested from breast cancer patients. In an experimental approach, single cell suspensions of different breast cancer cell lines were mixed to normal peripheral blood mononuclear cells in order to 1) determine the sensitivity of tumor cell detection within PBMC and 2) compare polymerase chain reaction in its capacity of monitoring the efficiency of immunomagnetic purging using the magnetic cell separation (MACS) system to immunocytochemical staining. Several target sequences were assessed for their indicative potential and specificity allowing the detection of breast cancer cells by RT-PCR. Among the sequences evaluated, epithelial growth factor receptor (EGF-R) mRNA and Cytokeratin 19 mRNA were shown to be highly specific and sensitive markers for the detection of breast cancer cells within normal peripheral blood mononuclear cells and for the evaluation of the efficiency in immunomagnetic purging. In addition, we were able to show that the MACS is a potent and efficient tool for the selection of tumor cells from peripheral blood mononuclear cells, thus establishing its value for clinical scale immunomagnetic purging.

Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells.

The pathogenesis and etiology of Hodgkin's disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkin's disease and for developing new therapeutic strategies against it.

Induction of the death-promoting gene bax-alpha sensitizes cultured breast-cancer cells to drug-induced apoptosis.

Resistance to apoptosis plays an important role in malignancies that are refractory to chemotherapy treatment. Recently we have shown that the expression of bax-alpha, a death-promoting member of the bcl-2 family, is down-regulated in breast cancer and have provided evidence that low bax expression might contribute to the pathogenesis of breast cancer. In this study we were able to demonstrate the role of this gene in chemotherapy-induced apoptosis. We transfected bax-alpha into the breast-cancer cell lines R30C and MCF-7 under the control of an inducible tetracycline-dependent expression system. Induction of bax-alpha expression did not affect viability by itself but strongly increased chemosensitivity to epirubicin. We were able to demonstrate that this sensitization is due to apoptosis. These data might explain the recently published observation that reduced expression of bax is associated with poor response rates to chemotherapy in breast cancer.

Overexpression of the death-promoting gene bax-alpha which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.

We have studied the expression of members of the bcl-2 family in human breast cancer. The expression pattern of these genes in breast cancer tissue samples was compared with the expression pattern in normal breast epithelium. No marked difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and cancer tissue. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined so far. Of interest, downregulation of bax-alpha was found in different histological subtypes. Furthermore, we transfected bax-alpha into breast cancer cell lines under the control of a tetracycline-dependent expression system. We were able to demonstrate for the first time that induction of bax expression in breast cancer cell lines restores sensitivity towards both serum starvation and APO-I/Fas-triggered apoptosis and significantly reduces tumor growth in SCID mice. Therefore, we propose that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the bcl-2 gene family.

High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/Reed-Sternberg cells.

There is a considerable lack of understanding about the common molecular defects that form the basis for the occurrence of Hodgkin's lymphoma. Despite a number of molecular tools used thus far in immunophenotypic and karyotypic studies, it has not been possible to establish a single common trait among various Hodgkin (H)-cell lines or primary tumor cells that would allow classification into a particular hematopoietic lineage. With this study, we demonstrate that a characteristic expression pattern of transcription factors provides a unifying principle. Seven different cell lines derived from patients with Hodgkin's disease (HD), as well as primary H/Reed-Sternberg (RS) cells isolated from the pericardial fluid of a patient with HD, were compared with a number of hematopoietic and nonhematopoietic cell lines for the expression of Oct-2, a tissue-specific transcription factor normally restricted to B cells, and nuclear factor kappa B (NF-kappa B), an inducible transcription factor. Regardless of the heterogeneous phenotypes and genotypes of the H cell lines, which varied inconsistently between B-cell-, T-cell-, or monocyte-like properties, all H cells tested displayed expression of Oct-2 protein at levels comparable to those seen in B cells. Furthermore, all cell lines showed an abundant constitutive nuclear NF-kappa B activity. Interestingly, anaplastic large-cell lymphoma (ALCL) cell lines, which have many features in common with H/RS cells, were devoid constitutive nuclear NF-kappa B activity. Unlike the constitutive NF-kappa B activity known for B cells, which mainly consists of the p50 and c-Rel or RelB subunits, we demonstrate by antibody supershifting experiments that H cells contain constitutive nuclear p50 and p65, the dimeric NF-kappa B normally observed only for limited time intervals after stimulation with diverse inducers. Additionally, some H-cell lines also displayed nuclear c-Rel activity, whereas RelB or p52 were not detected as part of the constitutive activity. The expression pattern of Oct-2 and NF-kappa B appears to be a unifying and characteristic property of H cells and might explain the deregulated expression of various cytokines leading to the clinical and pathologic manifestations of HD.