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HSP90's are overexpressed in different cancer types and they probably are required to sustain aberrant signalling in malignant cells. Recently, pharmacological inhibition of HSP90 was found to suppress growth of myeloma cell lines and in primary myeloma cells. Therefore, we wanted to investigate the role of HSP90alpha and HSP90beta in the pathogenesis of malignant myeloma (MM) in more detail. Immunohistochemistry was employed to examine the expression of HSP90alpha and HSP90beta in MM. The importance of HSP90 for survival of MM -cells was investigated by SiRNA-mediated knockdown of HSP90 and blockade of the IL-6R/STAT3 and the MAPK signaling pathways in vitro. HSP90alpha and HSP90beta were overexpressed in majority of investigated MM cases, but not in MGUS or in normal plasma cells. SiRNA-mediated knockdown of HSP90 or treatment with the novel HSP90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. The knockdown of HSP90alpha was sufficient to induce apoptosis. This effect was strongly increased when both HSP90s were targeted, indicating a cooperation of both. HSP90 critically contributes to myeloma survival in the context of its microenvironment and therefore strengthen the potential value of HSP90 as a therapeutic target.

The bone marrow micro-environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL-6. Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL-6R antagonist SANT-7 on growth and survival of the IL-6--dependent MM cell lines INA-6 and XG-1 as well as primary MM cells from 7 patients co-cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT-7 enhances the growth-inhibitory effects of dexamethasone (Dex) and all-trans-retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT-7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT-7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell-cycle arrest and induction of apoptosis., (Copyright 2001 Wiley-Liss, Inc.)

Although bispecific antibodies directed against malignant lymphoma have been shown to be effective in vitro and in vivo, extended clinical trials so far have been hampered by the fact that conventional approaches to produce these antibodies suffer from low yields, ill-defined byproducts, or laborious purification procedures. To overcome this problem, we have generated a small, recombinant, lymphoma-directed, bispecific single-chain (bsc) antibody according to a novel technique recently described. The antibody consists of 2 different single-chain Fv fragments joined by a glycine-serine linker. One specificity is directed against the CD3 antigen of human T cells, and the other antigen-binding site engages the pan-B-cell marker CD19, uniformly expressed on the vast majority of B-cell malignancies. The construct was expressed in Chinese hamster ovary cells and purified by its C-terminal histioline tag. Specific binding to CD19 and CD3 was demonstrated by fluorescence-activated cell sorter analysis. By redirecting unstimulated primary human T cells derived from the peripheral blood against CD19-positive lymphoma cells, the bscCD19 x CD3 antibody showed significant cytotoxic activity at very low concentrations of 10 to 100 pg/mL and at effector to target cell ratios as low as 2:1. Moreover, strong lymphoma-directed cytotoxicity at low antibody concentrations was rapidly induced during 4 hours even in experiments without any T-cell prestimulation. Thus, this particular antibody proves to be much more efficacious than the bispecific antibodies described until now. Therefore, the described bscCD19 x CD3 molecule should be a suitable candidate to prove the therapeutic benefit of bispecific antibodies in the treatment of non-Hodgkin lymphoma. (Blood. 2000;95:2098-2103)

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.

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.

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.

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.

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.

Recently G alpha 16, a new guanosine triphosphate (GTP) binding protein alpha subunit has been described to be specifically expressed in human hematopoietic cells. Expression of G alpha 16 was observed in human cell lines of myelomonocytic and T-lymphocytic origin, but not in human B-cell lines Raji and IM9. We studied the expression of G alpha 16 in human B cells corresponding to different stages of B-cell differentiation by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The human Burkitt's lymphoma cell lines Raji, Ramos, BJAB, the lymphoblastoid cell line SKW6.4, and the plasmocytoma cell line U266 were devoid of G alpha 16. In contrast, G alpha 16 was detected in the human progenitor B cell lines Reh and Nalm-6. Using the mu+, k- cell line BLIN-1 (pre-B cell phenotype) and its derived subclone 1E8 (surface mu+, k+; B-cell phenotype) G alpha 16 expression was found to disappear on transition from pre-B to B-cell differentiation stage. The analysis of a broad panel of human neoplastic B lymphocytes ranging from progenitor B-acute lymphatic leukemia (pre-pre-B-ALL), common acute leukemias (cALL), pre-B-ALL, mature B-ALL to low grade B-cell lymphoma (chronic lymphocytic leukemia of B-cell type, leukemic centrocytic non-Hodgkins lymphoma [NHL], hairy cell leukemia) showed that G alpha 16 expression is limited to progenitor and pre-B-ALL cells. Therefore, we conclude that within B-cell differentiation, G alpha 16 is expressed solely during early B cell ontogeny and downregulated during differentiation. Thus, G alpha 16 might be an important regulator involved in signaling processes in progenitor B cells.

We have studied the expression of the apoptosis-regulating genes bcl-2, bcl-x, bax and APO-1/fas (CD95) in human breast cancer. The expression pattern of these genes in human breast-cancer tissues and breast-cancer-derived cell lines was compared to that seen in normal breast epithelium and breast epithelial cell lines. No difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and tumor tissue or breast cancer and non-malignant epithelial cell lines. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal cell lines and breast tissue, whereas only weak or no expression could be detected in cancer-cell lines and malignant tissue. In contrast to malignant cell lines, which express low levels of bax-alpha, non-malignant epithelial cell lines displaying high amounts of bax-alpha were highly sensitive to induction of programmed cell death by both serum starvation and APO-1/fas (CD95) triggering. We therefore propose that dysregulation of apoptosis contributes to the pathogenesis of breast cancer, at least in part, due to an imbalance between anti-apoptosis genes (such as bcl-2/bcl-x) and apoptosis-promoting genes (bax).

The members of the bcl-2 gene family are major regulators of programmed cell death, but their role in sIg-triggered apoptosis remains unclear. Using sensitive and resistant variants of the human B cell line BL-41, we studied the expression of the bcl-2 gene family during surface IgM-mediated apoptosis. We found constitutive Bcl-2 and Bcl-x expression, which remained unaltered after sIg cross-linking, in both resistant and sensitive cells. This and other experiments suggest that constitutive expression of Bcl-2 or Bcl-x alone is not sufficient to protect from activation-induced cell death in B cells. We therefore investigated Bax-alpha, the death-promoting splice variant of Bax, and found strong induction of both mRNA and protein upon sIg stimulation in sensitive cells. However, resistant subclones showed only weak expression, which was not inducible by sIg cross-linking. We provide evidence that up-regulation of Bax-alpha and the resulting imbalance of Bcl-2/Bax might be a major regulator of sIg-mediated apoptosis. Additionally, we found strong constitutive expression of Bcl-xs, the death promoting variant of Bcl-x, in sensitive cells, whereas resistant cells showed only weak Bcl-xs expression. Thus, we observed a much stronger expression of the death-promoting proteins Bax-alpha (inducible) and Bcl-xs (constitutive) in sensitive cells than in resistant cells. We therefore propose a potential role of the novel bcl-2 gene family members bcl-x and bax in surface IgM-triggered apoptosis.