Markus Duechler

Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia

Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110β, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2.

Recently, proteasome inhibitors (PI) have attracted interest as novel anticancer agents in B-cell chronic lymphocytic leukemia (B-CLL). A prominent feature of B-CLL cells is the high expression of CD23, which is closely related to cell survival and is regulated by Notch2. Since several components of the Notch signaling cascade are tightly regulated by proteasomal degradation, we studied the effect of PI on Notch2 activity and CD23 expression. Exposure of B-CLL cells to PI led to induction of apoptosis, a time- and dose-dependent downregulation of CD23 expression and a decline in DNA binding of transcriptionally active Notch2. In contrast, the transcription factor NF-AT and its putative target gene CD5, which is highly expressed in B-CLL cells, were unaffected. When the late phase of PI-induced apoptosis was arrested by inhibition of caspase 3, the reduction of Notch2 activity was still observed, indicating that reduction of active Notch2 took place already during an earlier phase of apoptosis. Enforced expression of constitutively active Notch2 decreased PI-mediated apoptosis in a human B-cell line. These data indicate that downregulation of CD23 and loss of Notch2 activity are early steps in PI-induced apoptosis of B-CLL lymphocytes and may be part of the full apoptotic response.

TGF-β1 induces bone marrow reticulin fibrosis in hairy cell leukemia

The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-β1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-β1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-β1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-β1. Active TGF-β1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-β1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti–TGF-β1 antibodies. These results show, for the first time to our knowledge, that TGF-β1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.

MiRNA in melanoma-derived exosomes.

Proteins, RNAs and viruses can be spread through exosomes, therefore transport utilizing these nanovesicles is of the great interest. MiRNAs are common exosomal constituents capable of influencing expression of a variety of target genes. MiRNA signatures of exosomes are unique in cancer patients and differ from those in normal controls. The knowledge about miRNA profiles of tumor-derived exosomes may contribute to better diagnosis, determination of tumor progression and response to treatment, as well as to the development of targeted therapies. We summarize the current knowledge with regard to miRNAs that are found in exosomes derived from tumors, particularly from melanoma.

In vitro cytotoxic effect of proteasome inhibitor bortezomib in combination with purine nucleoside analogues on chronic lymphocytic leukaemia cells

Abstract:  Objective: The anti‐tumour in vitro activity of proteasome inhibitor bortezomib (PS‐341, VELCADE) in combination with purine nucleoside analogues, cladribine (2‐CdA) and fludarabine (FA) was tested in lymphocytes derived from 26 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL). Methods: Cell viability was assessed by propidium iodide staining, and apoptosis by annexin‐V and caspase activation flow cytometry assays. Additionally, expression of the apoptosis‐regulating proteins Bax, Bak, Bid, Bcl‐w, Bcl‐2, XIAP and Mcl‐1 was evaluated in B‐CLL lymphocytes. Results: Bortezomib alone induced significant, dose‐dependent cytotoxicity starting from the low concentration 2.5 nm, inducing apoptosis of B‐CLL cells. Combination of this agent with 2‐CdA or FA resulted in an increase of cytotoxicity when compared with that mediated by single drugs. The observed increase was especially evident when 5 nm of bortezomib were combined with suboptimal doses of 2‐CdA or FA. The combination index (CI) was 0.87 for bortezomib + 2‐CdA and 0.82 for bortezomib + FA, indicating an evident additive effect of these combinations. Moreover, B‐CLL cells were more sensitive to proteasome inhibitor used alone or combined with 2‐CdA or FA comparing to CD3+ lymphocytes. Corresponding to enhanced apoptosis, the expression levels of several apoptosis‐regulating proteins were altered. The most pronounced changes were down‐regulation of XIAP and up‐regulation of Bid proteins by the combination of bortezomib with either 2‐CdA or FA. Conclusions: This study suggest that the in vitro cytotoxic effect through proteasome inhibition by bortezomib can be increased substantially with low doses of the purine nucleoside analogues, 2‐CdA and FA, and that this effect on B‐CLL cell is selectively higher than on normal, CD3‐positive lymphocytes.

Nucleoside modifications in the regulation of gene expression: focus on tRNA.

Both, DNA and RNA nucleoside modifications contribute to the complex multi-level regulation of gene expression. Modified bases in tRNAs modulate protein translation rates in a highly dynamic manner. Synonymous codons, which differ by the third nucleoside in the triplet but code for the same amino acid, may be utilized at different rates according to codon–anticodon affinity. Nucleoside modifications in the tRNA anticodon loop can favor the interaction with selected codons by stabilizing specific base pairs. Similarly, weakening of base pairing can discriminate against binding to near-cognate codons. mRNAs enriched in favored codons are translated in higher rates constituting a fine-tuning mechanism for protein synthesis. This so-called codon bias establishes a basic protein level, but sometimes it is necessary to further adjust the production rate of a particular protein to actual requirements, brought by, e.g., stages in circadian rhythms, cell cycle progression or exposure to stress. Such an adjustment is realized by the dynamic change of tRNA modifications resulting in the preferential translation of mRNAs coding for example for stress proteins to facilitate cell survival. Furthermore, tRNAs contribute in an entirely different way to another, less specific stress response consisting in modification-dependent tRNA cleavage that contributes to the general down-regulation of protein synthesis. In this review, we summarize control functions of nucleoside modifications in gene regulation with a focus on recent findings on protein synthesis control by tRNA base modifications.

Sphere formation and self-renewal capacity of melanoma cells is affected by the microenvironment.

Melanomas contain subsets of cancer stem-like cells with tumor-initiating capacity. The frequency of these cells in the tumor is still a topic of debate. We investigated the phenotypic plasticity of cancer cells grown as melanospheres to elucidate the influence of the microenvironment on some features of melanoma stem-like cells. Cells from surgical specimens of nodular melanoma were grown as anchorage-independent melanospheres in a stem cell medium and as adherent monolayer cultures in the presence of serum. Proliferation and viability were measured by cell counting and an acid phosphatase assay; surface marker expression was evaluated by flow cytometry, and the clonogenic potential of single cells was assessed by growth in soft agar. Patient-derived melanoma cells could be maintained in cell culture for more than 16 months when grown as melanospheres. In the presence of serum, melanospheres completely changed their growth characteristics and formed adherent monolayers. The transition from melanospheres to monolayers was accompanied by an apparent loss of clonogenic potential, an increased proliferation rate, and altered expressions of cell surface markers ABCB5, CD133, and CD49f. These changes, however, were reversible. Compared with adherent monolayer cultures, melanospheres are enriched in cells with clonogenic potential, reflecting the self-renewing capacity of cancer stem-like cells. This clonogenic potential can be lost and regained depending on the growth conditions. Our results demonstrate how easily melanoma cells change their function upon exposure to external stimuli and suggest that the frequency of melanoma stem-like cells strongly depends on the microenvironment.

Targeting NF-κB and HIF-1 Pathways for the Treatment of Cancer: Part I

The process of chronic inflammation is a common link which connects different kinds of environmental pollutants and infections with tumorigenesis. Transcription factor NF-κB is a common final target for many inflammatory and cell proliferation pathways, independent of the source of stimuli (e.g., cytokines, growth factors, environmental carcinogens, radiation, hypoxia, bacteria, and viruses). Over-activation of NF-κB has been confirmed in many tumors, resulting in worse prognosis for patient survival. Therefore, inhibition of cellular pathways for NF-κB activation is nowadays considered as a promising anti-cancer therapy and is extensively studied in clinical trials, or even has been adopted as an approved therapy in some kinds of cancer.

Rhinoviral receptor discrimination : mutational changes in the canyon regions of human rhinovirus types 2 and 14 indicate a different site of interaction

Amino acid sequence comparisons between the capsid proteins of several human rhinovirus (HRV) serotypes identified residues potentially involved in the discrimination between the major and the minor group receptors. Amino acids conserved within minor group HRVs were substituted in a full-length cDNA clone of HRV2 for those found at equivalent positions in major group HRVs. Transfection of HeLa cells with RNAs transcribed from seven individual mutated cDNAs gave rise to only two viable viruses; growth characteristics and affinity for the minor group receptor of both were unchanged compared to wild-type. Similar mutations in HRV14 were previously shown to alter the affinity for its receptor; the contact sites between the minor group viruses and the respective receptor may therefore be different.

The heterogeneous immune microenvironment in breast cancer is affected by hypoxia-related genes

The immune system constitutes an important first-line defence against malignant transformation. However, cancer mediated immunosuppression inactivates the mechanisms of host immune surveillance. Cancer cells shut down anti-cancer immunity through direct cell-cell interactions with leukocytes and through soluble factors, establishing an immunosuppressive environment for unimpeded cancer growth. The composition of the immunosuppressive microenvironment in breast tumours is not well documented. To address this question, selected immunosuppressive factors were analyzed in tumour specimens from 33 breast cancer patients after surgery. The mRNA expression of selected genes was quantified in fresh tumour samples. Tumour infiltrating leukocytes were characterized by flow cytometry to identify regulatory T cells, myeloid derived suppressor cells, and type 2 macrophages. Statistical analysis revealed several interesting correlations between the studied parameters and clinical features. Overall, a surprisingly high degree of heterogeneity in the composition of the immunosuppressive environment was found across all breast cancer samples which adds to the complexity of this disease. The influence of the hypoxia inducible factors (HIFs) on the immune microenvironment was also addressed. The level of HIFs correlated with hormone receptor status and the expression of several immunosuppressive molecules. Targeting HIFs might not only sensitize breast tumours for radiation and chemotherapies but also interfere with cancer immunosuppression.