ERBB2 gene amplification increases during the transition of proximal EGFR(+) to distal HLA-G(+) first trimester cell column trophoblasts.

INTRODUCTION: Although, DNA copy-number alterations (CNAs) have been well documented in a number of adverse phenotypic conditions, accumulating data suggest that CNAs also occur during physiological processes. Interestingly, extravillous trophoblasts induce the expression of the transforming, proto-oncogene ERBB2, which is frequently amplified in human cancer. However, no data are available to address whether trophoblast-related ERBB2 expression might also be linked to genomic amplification. METHODS: Dual color silver as well as fluorescence in situ hybridization analyses were carried out to evaluate frequency and degree of ERBB2 gene and chromosome 17 copy numbers in first trimester placental cell columns and isolated trophoblasts. Proliferative EGFR(+) and differentiated HLA-G(+) trophoblasts were identified or separated by means of in situ immunofluorescence co-stainings and magnetic beads cell isolation, respectively. RESULTS: ERBB2 gene amplification is detected in approximately 40% of isolated HLA-G(+) trophoblasts. Although already detectable in EGFR(+) cells, the percentage and extent of ERBB2 amplification was markedly increased in HLA-G(+) trophoblasts in situ and after isolation. Accordingly, HLA-G(+) trophoblasts highly express ERBB2 on protein level. Finally, ERBB2 copy number variations occur independently of aneuploidy as the majority of ERBB2 amplifying cells were cytogenetically diploid for chromosome 17. DISCUSSION: ERBB2 gene amplification is a frequent event during EVT differentiation. This finding challenges the long standing paradigm, which associates gene amplification with pathological conditions and further supports recent evidences suggesting that CNAs are a normal feature of developmental processes.

Intratumoral expression profiling of genes involved in angiogenesis in colorectal cancer patients treated with chemotherapy plus the VEGFR inhibitor PTK787/ZK 222584 (vatalanib).

The phase III CONFIRM clinical trials demonstrated that metastatic colorectal cancer patients with elevated serum lactate dehydrogenase (LDH) had improved outcome when the vascular endothelial growth factor receptor (VEGFR) inhibitor PTK/ZK (Vatalanib) was added to FOLFOX4 chemotherapy. We investigated the hypothesis that high intratumoral expression of genes regulated by hypoxia-inducible factor-1 alpha (HIF1α), namely LDHA, glucose transporter-1 (GLUT-1), VEGFA, VEGFR1, and VEGFR2, were predictive of outcome in CONFIRM-1. Tumor tissue was isolated by laser-capture microdissection from 85 CONFIRM-1 tumor specimens; FOLFOX4/placebo n=42, FOLFOX4/PTK/ZK n=43. Gene expression was analyzed using quantitative RT-PCR. In univariate analyses, elevated mRNA expression of LDHA, GLUT-1, and VEGFR1 were associated with response to FOLFOX4/PTK/ZK. In univariate and multivariate analyses, elevated LDHA and VEGFR1 mRNA levels were associated with improved progression-free survival in FOLFOX4/PTK/ZK patients. Furthermore, increased HIF1α and VEGFR2 mRNA levels were associated with decreased survival in FOLFOX/placebo patients but not in patients who received FOLFOX4/PTK/ZK. These are the first data suggesting intratumoral mRNA expression of genes involved in angiogenesis/HIF pathway may predict outcome to VEGFR-inhibitors. Biomarkers that assist in directing VEGFR-inhibitors toward patients with an increased likelihood of benefit will improve the cost-effectiveness of these promising agents.

Vascular density analysis in colorectal cancer patients treated with vatalanib (PTK787/ZK222584) in the randomised CONFIRM trials.

BACKGROUND: Pharmacological inhibitors of vascular endothelial growth factor (VEGF) receptors, like vatalanib, have been tested in randomised trials (CONFIRM (Colorectal Oral Novel therapy For the Inhibition of Angiogenesis and Retarding of Metastases) 1 and 2) in colorectal cancer showing activity in a subgroup of patients with high serum LDH expression. In the current study, we assessed the predictive role of vascular density (VD) in patients treated in the above trials. METHODS: Paraffin-embedded materials from 141 patients were analysed with immunohistochemistry for the expression of the CD31 (pan-endothelial cell marker) and of phosphorylated pVEGFR2/KDR on endothelial cells. The VD was correlated with response to therapy and with progression-free (PFS) and overall survival (OS). RESULTS: A significant association of pVEGFR2/KDR+ VD with poor response in the placebo group was noted (response rates (RRs) 15% (3/20) when high VD vs 52% (26/50) when low VD; P=0.006). The RR increased from 15 (3/20) to 50% (11/22) in tumours with high VD when vatalanib was added to chemotherapy (P=0.02). A significantly improved PFS was noted in patients with high pVEGFR2/KDR+ VD when treated with vatalanib (P=0.002). A similar effect was also noted in patients with high CD31+ VD (P=0.07). Overall survival was marginally improved (P=0.07). CONCLUSION: Assessment of the activated vessel density may allow the stratification of patients recruited in randomised trials with VEGFR-targeting anti-angiogenic agents, unmasking their therapeutic potential and enabling their introduction in the clinical practice for the benefit of specific patient subgroups, at the same time reducing the cost of therapy.

Expression, regulation and functional characterization of matrix metalloproteinase-3 of human trophoblast.

MMP-3 has been detected in human placenta and reduced expression of the enzyme was observed in invasive trophoblasts of patients with severe preeclampsia. However, detailed expression pattern, regulation and biological properties of the placental protease have not been elucidated so far. RT-PCR analyses, Western blotting and enzyme activity assays revealed that pro- and active form of MMP-3 were predominantly expressed in purified first trimester villous trophoblasts, in invasive cytotrophoblasts of differentiating explant cultures and in trophoblastic SGHPL-4 cells. Accordingly, immunofluorescene of first trimester placental tissues detected MMP-3 mainly in villous and extravillous cytotrophoblasts. IL-1beta, an inducer of MMP-3 in decidual cells, increased secretion and activity of the protease in trophoblast supernatants in a dose- and time-dependent manner. IL-1beta-stimulated production of the enzyme was suppressed in the presence of inhibitors of MAPK and AKT signalling. Similar to recombinant MMP-3, MMP-3 in supernatants of IL-1beta-stimulated decidual stromal or SGHPL-4 cells degraded IGFBP-1 in vitro resulting in the appearance of cleavage products at approximately 25, 22, 17, 14 and 11kD. However, cleavage assays using recombinant MMP-2 suggested that the gelatinase may contribute to IGFBP-1 degradation in trophoblast supernatants. Despite its effects on MMP-3 expression IL-1beta failed to significantly alter invasion of SGHPL-4 cells through Matrigel-coated transwells. In conclusion, the data suggest that invasive trophoblast cell models secrete bioactive MMP-3. Inducible expression of the protease involves MAPK and AKT signalling. In addition to the decidua, MMP-3 of trophoblasts may contribute to the regulation of the IGF system by degrading IGFBP-1.

Coronary vasospasm as the underlying cause for chest pain in patients with PVB19 myocarditis.

OBJECTIVE: To evaluate the incidence of coronary vasospasm as a possible pathophysiological mechanism causing chest pain symptoms in patients with clinically suspected myocarditis. DESIGN AND SETTING: Prospective study in a teaching hospital. PATIENTS: 85 patients who presented at hospital with atypical chest pain and demonstrated clinical signs suggestive of myocarditis. MAIN OUTCOME MEASURES: Incidence of coronary vasospasm demonstrated by intracoronary acetylcholine (ACh) testing. METHODS: The combined procedure of intracoronary ACh testing and endomyocardial biopsy (EMB) was performed after ruling out significant coronary artery disease (CAD). EMBs were analysed for myocardial inflammation by immunohistological methods and for virus genome persistence. RESULTS: Pathological biopsy results, including myocardial inflammation or detection of viral genomes, or both, were found in 55 (64.7%) patients while 30 (35.3%) patients showed neither cardiac inflammation nor viral genomes and were defined as the control group. Coronary vasospasm was demonstrated in 39/55 (70.9%) patients with pathological results compared with only 12/30 (40.0%) with normal biopsy results (p = 0.01). Patients with isolated PVB19 infection (n = 22) demonstrated a significantly higher incidence of coronary vasospasm than both those with isolated HHV6 infection (86.4% vs 46.7%; p = 0.025) and those with normal biopsy results (86.4% vs 40.0%; p<0.001). Univariate and multivariate logistic regression analysis showed that only PVB19 infection was independently correlated with coronary vasospasm (OR = 4.9, 95% CI 1.56 to 15.28, p = 0.006). CONCLUSIONS: Coronary vasospasm is one of the main reasons for atypical chest pain in patients with clinical signs of myocarditis and biopsy-proven PVB19 myocarditis in the absence of significant CAD.

Biomarker analysis in polycythemia vera under interferon-alpha treatment: clonality, EEC, PRV-1, and JAK2 V617F.

Three consecutive polycythemia vera (PV) patients were analyzed before and during pegylated-interferon (rIFNalpha) treatment for the following markers: (1) granulocyte and CD34(+) cell clonality, (2) Jak2V617F expression, (3) PRV-1 mRNA overexpression, and (4) Epo-independent colony (EEC) growth. Before rIFNalpha therapy, all patients displayed clonal hematopoiesis, 100% Jak2V617F expression as well as PRV-1 overexpression, and EEC growth. After rIFNalpha treatment, all three patients demonstrated polyclonal hematopoiesis. Nonetheless, Jak2V617F expression, PRV-1 overexpression, and EEC-growth remained detectable, albeit at lower levels. We conclude that reemergence of polyclonal hematopoiesis after rIFNalpha treatment may be achieved in a substantial proportion of patients. However, this does not constitute elimination of the PV clone. These data demonstrate the usefulness of novel markers in monitoring minimal residual disease and caution against discontinuation of rIFNalpha treatment after hematologic remission. Long-term follow-up of large patient cohorts is required to determine whether rIFNalpha treatment can cause complete molecular remissions in PV.

Evidence for cell adhesion-mediated drug resistance of multiple myeloma cells in vivo.

BACKGROUND/AIMS: Multiple myeloma is an incurable disease and patients eventually die of disease progression due to drug resistance. VLA-4 (very late antigen 4), VCAM (vascular adhesion molecule), LFA-1 (leukocyte function-associated antigen 1), and ICAM-1 (intercellular adhesion molecule 1)-mediated adhesion of myeloma cells to bone marrow stromal cells induces primary multidrug resistance in vitro. Based on these preclinical data we hypothesized that myeloma cells with strong adhesion - due to strong expression of adhesion molecules on the cell surface - are selected by chemotherapy in patients. To prove this hypothesis we determined the expression levels of adhesion molecules in 31 multiple myeloma patients by flow cytometry. METHODS: A 3-color stain with CD38, CD138 and antibodies against VLA-4, ICAM-1, LFA-1, and VCAM was performed. The patients were either at diagnosis (chemo-naive; n=17) or at relapse (pre-treated; n=15). Furthermore, the response to the next chemotherapy of chemo-naive patients was correlated with the expression levels of adhesion molecules. RESULTS: ICAM-1, VLA-4, and VCAM expression was higher in pre-treated patients than in chemo-naive patients and the expression levels increased with the number of chemotherapy regimens. Primarily multidrug-resistant patients had significantly higher expression levels of VLA-4 and ICAM-1 than responders. CONCLUSION: This study suggests that multiple myeloma cells expressing high levels of VLA-4 and ICAM-1 are drug resistant and that such a subpopulation of cells is selected by chemotherapy.

Vascular ectasia of the whole intestine as a cause of recurrent gastrointestinal bleeding after high-dose chemotherapy.

We present the first case in the literature of vascular ectasia of the whole intestine as a cause of recurrent and profuse gastrointestinal bleeding in a patient with relapsing Hodgkin's disease. The 17-year-old patient experienced early relapse of his Hodgkin's disease after first-line chemotherapy. Salvage chemotherapy was followed by high-dose chemotherapy and autologous stem cell transplantation. Complete remission was achieved after another relapse by means of a second transplant. The patient presented with profuse gastrointestinal bleeding 5 months later, however. Gastric antral vascular ectasia following hematopoietic stem cell transplantation was diagnosed by endoscopy, with histological confirmation. Similar lesions were found in the duodenum, the ileum, and throughout the entire colon. In conclusion, vascular ectasia of the whole intestine should be considered as cause of acute gastrointestinal bleeding after stem cell transplantation. Physicians should be aware of this complication because its onset is typically delayed. Importantly, this disease is not limited to patients who have undergone allogeneic transplantation, but can also occur after autologous transplantation.

Cell-cell contact mediated signalling - no fear of contact.

Cancer research with sole focus on the cancer cell and possibly growth factors cannot faithfully reproduce the environmental interaction, such as adhesion of tumor cells to e.g. stromal cells, which may determine the response of these tumors to therapy. Methodologically cell adhesion studies are often difficult since complete but careful detachment is the prerequisite for most signal transduction assays. We describe for the first time an alternative method for the co-incubation of multiple myeloma cells on long term primary bone marrow stromal cultures using the bone marrow stromal cell line HS-5. The methods are precisely described, advantages and disadvantages are discussed, and troubleshooting advises are given.

[Enlarged lymph nodes--rapid diagnosis of infectious and neoplastic causes]. / Entscheidungshilfe bei vergrösserten Lymphknoten. Aggressiv abklären oder ruhig abwarten?

Although lymphadenopathy is usually an expression of a harmless, self-limiting immune reaction, it may nevertheless also signify the presence of a malignant tumor or relevant infectious disease--in particular when the swelling is sizeable, rapidly progressive, and associated symptoms are present. If, however, unfavorable criteria are definitely absent (Table 1), a wait-and-see policy with re-evaluation in three to four weeks may initially be adopted.

Tissue-specific and ubiquitous basic helix-loop-helix transcription factors in human placental trophoblasts.

Tissue-specific class B basic helix-loop-helix (bHLH) transcription factors, dimerising with ubiquitously produced class A bHLH proteins, play a major role in murine trophoblast development. Here, we investigated expression patterns of class A and B bHLH factors in the human placenta and different trophoblast culture systems. Semi-quantitative RT-PCR and RNase protection assay revealed expression of the tissue-restricted factors Hash-2, I-mfa and Stra13 in placentae of early and late pregnancy, in purified villous trophoblasts as well as in invasive trophoblasts isolated from first trimester villous explant cultures. Accordingly, RNA in situ hybridisation localised Hash-2, I-mfa and Stra13 to the trophoblast epithelium, cell columns and extravillous trophoblasts invading maternal decidua. Villous stromal cells in situ and cultivated placental fibroblasts also produced I-mfa and Stra13 but failed to express Hash-2. The widely expressed class A proteins, E12/E47 were absent from all placental cell types while ITF-2 was restricted to placental stromal cells of early and late gestation. In contrast, HEB was identified in all trophoblast cell types using RT-PCR, Western blotting and immunohistochemistry. The negative HLH-regulators Id-1 and Id-2 lacking the DNA-binding domain, were detected in villous stromal cells and different cytotrophoblast subtypes but were absent from the syncytium. The data suggest that a complex interplay of activators (Hash-2, HEB) and repressors (Stra13, I-mfa) could be involved in extravillous trophoblast differentiation whereas downregulation of Id proteins could play a role in syncytialisation.

Modulation of malignant B cell activation and apoptosis by bcl-2 antisense ODN and immunostimulatory CpG ODN.

Inhibition of bcl-2 expression by antisense oligodeoxynucleotides (ODN) might render bcl-2 overexpressing malignant B cells more susceptible to chemotherapy. ODN containing unmethylated CG dinucleotides (CpG) are known to activate B cells. We studied the effects of two bcl-2 antisense ODN, with (G3139) or without CG dinucleotides (NOV 2009) within the sequence, and the effects of a nonantisense, CpG-containing ODN (ODN 2006) on activation and apoptosis of malignant B cell lines and primary B-CLL cells. Without cationic lipids, no antisense-mediated inhibition of bcl-2 synthesis was achieved with G3139 and NOV 2009. Instead, G3139, but not NOV 2009, induced similar changes as ODN 2006 in proliferation, expression of costimulatory and antigen-presenting molecules, as well as in bcl-2 and bcl-xL levels of primary B-CLL cells. G3139 and ODN 2006 inhibited in vitro, spontaneous apoptosis in B-CLL cells of patients with high serum thymidine kinase activity (s-TK, marker for proliferative activity of malignant B cells), whereas in patients with low s-TK activity, apoptosis was induced. In conclusion, our results suggest that modulation of malignant B cell apoptosis by G3139 depends on its immunostimulatory properties rather than on antisense-mediated reduction of bcl-2 expression. Immunostimulatory CpG ODN may have a therapeutic potential in patients with B-CLL, especially those with low s-TK activity.

Phosphodiesterase type 4 inhibitor suppresses expression of anti-apoptotic members of the Bcl-2 family in B-CLL cells and induces caspase-dependent apoptosis.

B cell chronic lymphocytic leukemia (B-CLL) is an incurable clonal disease which shows initial responsiveness to a number of chemotherapeutic drugs. However, in most patients the disease becomes resistant to treatment. Rolipram, a specific inhibitor of phosphodiesterase (PDE) type 4, the PDE predominantly expressed in B-CLL cells, has been shown to induce cAMP-dependent apoptosis in these cells. In the present study, we demonstrate that the extent of rolipram-induced apoptosis is similar to fludarabine-induced apoptosis in vitro. The combination of rolipram and fludarabine results in an enhancement in the number of apoptotic cells compared to apoptosis induced by either agent alone. Second, rolipram suppresses the expression of anti-apoptotic members of the Bcl-2 family and induces the pro-apoptotic protein Bax, thereby shifting the balance between pro- and anti-apoptotic members of the Bcl-2 family towards a pro-apoptotic direction. Finally rolipram-induced apoptosis is caspase-dependent. PDE 4 inhibitors are currently under investigation for chronic obstructive pulmonary disease and asthma in phase III clinical trials showing promising results with tolerable side-effects. In conclusion, by inducing apoptosis, by enhancing apoptosis induced by fludarabine, by suppressing Bcl-2, Bcl-X and by inducing Bax expression, PDE 4 inhibitors may add a new therapeutic option for patients with B-CLL.

STAT3 is constitutively active in some patients with Polycythemia rubra vera.

OBJECTIVE: Polycythemia vera is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. While it has been shown that progenitor cells of P. vera patients are hypersensitive to several growth factors including erythropoietin, insulin-like growth factor-1, thrombopoietin, interleukin-3, and granulocyte/monocyte colony-stimulating factor, the molecular pathogenesis of this disease remains unknown. Growth factor hypersensitivity could be mediated by changes in signal transduction pathways. We therefore investigated a common downstream effector of cytokines, the signal transducers and activators of transcription (STATs). A constitutive activation of STAT factors could explain the increased proliferation of P. vera cells even in the absence of growth factor stimulation. METHODS: Peripheral granulocytes from patients with P. vera and from healthy volunteers were assayed for STAT1, 3, and 5 DNA binding by electrophoretic mobility shift assay. RESULTS: Four of 14 P. vera patients analyzed showed constitutive STAT3 DNA binding in unstimulated peripheral granulocytes, while none of the 17 healthy volunteers tested did. None of the subjects showed constitutive STAT1 or STAT5 activity. Western blotting demonstrated that, in the three patients, STAT3 is constitutively phosphorylated on Tyr 705, whereas it is unphosphorylated in the other patients and in controls. Interestingly, constitutive STAT3 activity did not correlate with the duration of disease or the treatment regimen. It was observed in a recently diagnosed patient and in two patients treated only with phlebotomy. CONCLUSION: Our data suggest that constitutive phosphorylation and activation of STAT3 is not a secondary event induced by mutagenizing agents or by prolonged hyperproliferation of hematopoietic cells, but rather represents a primary molecular aberration. Constitutively active STAT3 may contribute to the growth factor hypersensitivity of P. vera cells.

CpG DNA increases primary malignant B cell expression of costimulatory molecules and target antigens.

Multiple factors, including expression of costimulatory molecules, antigen-presenting molecules, and target antigens, likely impact the efficacy of antibody therapy and other approaches to the immunotherapy of B cell malignancy. Unmethylated CpG-dinucleotides in select base contexts ("CpG motifs") that resemble sequences found in bacterial DNA are potent immunostimulatory agents capable of inducing a complex immune response, including a strong B cell stimulus. We examined the effect of a potent human CpG oligonucleotide (CpG ODN 2006) on different types of primary human malignant B cells and reactive follicular hyperplasia. CpG oligodeoxynucleotide (CpG ODN), but not control (non-CpG ODN), increased the expression of costimulatory molecules (CD40, CD80, CD86, CD54) on malignant B cells without altering the phenotype of B cells obtained from reactive follicular hyperplasia. CpG ODN also enhanced expression of class I and class II MHC in most samples. CD20 expression was increased in response to CpG ODN, most notably in B-CLL and marginal zone lymphoma. An inverse correlation was found between baseline expression of CD20 and CD40 and their expression after exposure to CpG ODN, thus the most significant increase in expression of these molecules was found in those samples that had the lowest baseline levels. In conclusion, CpG ODN can lead to increasing expression of molecules involved in costimulation, antigen presentation, and as targets for antibody-based therapy and deserve further evaluation as potential immunotherapeutic agents for B cell malignancy.

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis.

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways.

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.

Cloning of PRV-1, a novel member of the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera.

Polycythemia vera (PV) is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. Although it has been shown that progenitor cells of patients with PV are hypersensitive to several growth factors, the molecular pathogenesis of this disease remains unknown. To investigate the molecular defects underlying PV, we used subtractive hybridization to isolate complementary DNAs (cDNAs) differentially expressed in patients with PV versus normal controls. We isolated a novel gene, subsequently named PRV-1, which is highly expressed in granulocytes from patients with PV (n = 19), but not detectable in normal control granulocytes (n = 21). Moreover, PRV-1 is not expressed in mononuclear cells from patients with chronic myelogenous leukemia (n = 4) or acute myelogenous leukemia (n = 5) or in granulocytes from patients with essential thrombocythemia (n = 4) or secondary erythrocytosis (n = 4). Northern blot analysis showed that PRV-1 is highly expressed in normal human bone marrow and to a much lesser degree in fetal liver. It is not expressed in a variety of other tissues tested. Although PRV-1 is not expressed in resting granulocytes from normal controls, stimulation of these cells with granulocyte colony-stimulating factor induces PRV-1 expression. The PRV-1 cDNA encodes an open reading frame of 437 amino acids, which contains a signal peptide at the N-terminus and a hydrophobic segment at the C-terminus. In addition, PRV-1 contains 2 cysteine-rich domains homologous to those found in the uPAR/Ly6/CD59/snake toxin-receptor superfamily. We therefore propose that PRV-1 represents a novel hematopoietic receptor. (Blood. 2000;95:2569-2576)

Signaling defect in the activation of caspase-3 and PKCdelta in human TUR leukemia cells is associated with resistance to apoptosis.

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-beta-d-arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G0/G1 of the cell cycle and an accumulation of a population in the sub-G1 phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measured in vitro by enhanced metabolization of a fluorescence substrate and in vivo by cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cdelta. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.

Molecular pathogenesis of chronic lymphocytic leukemia: factors and signaling pathways regulating cell growth and survival.

Chronic lymphocytic leukemia is a malignant disease characterized by clonal expansion of relatively mature B-lymphocytes with a high percentage of cells arrested in the nonproliferative G0/G1 cell cycle phase. Possibly reflecting the clinical heterogeneity observed in patients, various signaling pathways may become affected during the initiation and course of this disease. This review discusses frequent alterations concerning proliferative, differentiation-inducing, and apoptotic pathways elucidated in the recent years that have improved our understanding of this disease.