Reinhard Depping

Lack of functional erythropoietin receptors of cancer cell lines

Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real‐time RT‐PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH‐SY5Y, MCF7, HepG2, U2‐OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross‐reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation.

Nuclear translocation of hypoxia-inducible factors (HIFs) : Involvement of the classical importin α/β pathway

Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.

Importin α3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication

ABSTRACT HIV-1 employs the cellular nuclear import machinery to actively transport its preintegration complex (PIC) into the nucleus for integration of the viral DNA. Several viral karyophilic proteins and cellular import factors have been suggested to contribute to HIV-1 PIC nuclear import and replication. However, how HIV interacts with different cellular machineries to ensure efficient nuclear import of its preintegration complex in dividing and nondividing cells is still not fully understood. In this study, we have investigated different importin α (Impα) family members for their impacts on HIV-1 replication, and we demonstrate that short hairpin RNA (shRNA)-mediated Impα3 knockdown (KD) significantly impaired HIV infection in HeLa cells, CD4+ C8166 T cells, and primary macrophages. Moreover, quantitative real-time PCR analysis revealed that Impα3-KD resulted in significantly reduced levels of viral 2-long-terminal repeat (2-LTR) circles but had no effect on HIV reverse transcription. All of these data indicate an important role for Impα3 in HIV nuclear import. In an attempt to understand how Impα3 participates in HIV nuclear import and replication, we first demonstrated that the HIV-1 karyophilic protein integrase (IN) was able to interact with Impα3 both in a 293T cell expression system and in HIV-infected CD4+ C8166 T cells. Deletion analysis suggested that a region (amino acids [aa] 250 to 270) in the C-terminal domain of IN is involved in this viral-cellular protein interaction. Overall, this study demonstrates for the first time that Impα3 is an HIV integrase-interacting cofactor that is required for efficient HIV-1 nuclear import and replication in both dividing and nondividing cells.

An alternative splice variant in Abcc6, the gene causing dystrophic calcification, leads to protein deficiency in C3H/He mice.

Dystrophic cardiac calcification (DCC) is an autosomal recessive trait characterized by calcium phosphate deposits in myocardial tissue. The Abcc6 gene locus was recently found to mediate DCC; however, at the molecular level the causative variants remain to be determined. Examining the sequences of Abcc6 cDNA in DCC-resistant C57BL/6 and DCC-susceptible C3H/He mice, we identified a missense mutation (Cys to Thr at codon 619, rs32756904) at the 3′-border of exon 14 that creates an additional donor splice site (GT). Accordingly, an alternative transcript variant was detected, lacking the last 5 bp of exon 14 (-AGG(C/T)GCTgtga-) in DCC-susceptible C3H/He mice that carry the Thr allele. The 5-bp deletion was found to result in premature termination at codon 684, in turn leading to protein deficiency in DCC-susceptible mouse tissue as well as in cells transfected with Abcc6 cDNA lacking the last 5 bp of exon 14. All mouse strains that were found to carry the Thr allele, including C3H/He, DBA/2J, and 129S1/SvJ, were also found to be positive for DCC. In summary, we identified a splice variant leading to a 5-bp deletion in the Abcc6 transcript that gives rise to protein deficiency both in vivo and in vitro. The fact that all mouse strains that carry the deletion also develop dystrophic calcifications further suggests that the underlying splice variant affects the biological function of MRP6 protein and is a cause of DCC in mice.

Trps1, a regulator of chondrocyte proliferation and differentiation, interacts with the activator form of Gli3

Trps1, the gene mutated in human Tricho-Rhino-Phalangeal syndrome, represents an atypical member of the GATA-family of transcription factors. Here we show that Trps1 interacts with Indian hedgehog (Ihh)/Gli3 signaling and regulates chondrocyte differentiation and proliferation. We demonstrate that Trps1 specifically binds to the transactivation domain of Gli3 in vitro and in vivo, whereas the repressor form of Gli3 does not interact with Trps1. A domain of 185aa within Trps1, containing three predicted zinc fingers, is sufficient for interaction with Gli3. Using different mouse models we find that in distal chondrocytes Trps1 and the repressor activity of Gli3 are required to expand distal cells and locate the expression domain of Parathyroid hormone related peptide. In columnar proliferating chondrocytes Trps1 and Ihh/Gli3 have an activating function. The differentiation of columnar and hypertrophic chondrocytes is supported by Trps1 independent of Gli3. Trps1 seems thus to organize chondrocyte differentiation interacting with different subsets of co-factors in distinct cell types.

Targeted Disruption of the Mouse PAS Domain Serine/Threonine Kinase PASKIN

ABSTRACT PASKIN is a novel mammalian serine/threonine kinase containing two PAS (Per-Arnt-Sim) domains. PASKIN is related to the Rhizobium oxygen sensor protein FixL and to AMP-regulated kinases. Like FixL, the sensory PAS domain of PASKIN controls the kinase activity by autophosphorylation in a (unknown) ligand-dependent manner. In Saccharomyces cerevisiae, the two PASKIN orthologues PSK1 and PSK2 phosphorylate three translation factors and two enzymes involved in glycogen synthesis, thereby coordinately regulating protein synthesis and glycolytic flux. To elucidate the function of mammalian PASKIN, we inactivated the mouse Paskin gene by homologous recombination in embryonic stem cells. Paskin−/− mice showed normal development, growth, and reproduction. The targeted integration of a lacZ reporter gene allowed the identification of the cell types expressing mouse PASKIN. Surprisingly, PASKIN expression is strongly upregulated in postmeiotic germ cells during spermatogenesis. However, fertility and sperm production and motility were not affected by the PASKIN knockout. The Ppp1r7 gene encoding Sds22, a regulatory subunit of protein phosphatase 1, shares the promoter region with the Paskin gene, pointing towards a common transcriptional regulation. Indeed, Sds22 colocalized with the cell types expressing PASKIN in vivo, suggesting a functional role of protein phosphatase-1 in the regulation of PASKIN autophosphorylation.

Curcumin Decreases Survival of Hep3B Liver and MCF-7 Breast Cancer Cells

Background:Curcumin, a commonly used spice, affects the activities of cytokines, enzymes, and transcription factors that are linked to inflammation. Furthermore, curcumin has been assigned tumor growth inhibiting effects, possibly mediated by promoting hypoxia-inducible factor (HIF) degradation. HIFs are transcription factors that play a central role in the adaptation and response to low oxygen levels in metazoan cells. However, curcumin also exhibits properties of an iron chelator indicating its potential of inhibiting HIF-α prolyl hydroxylase (PHD) activity.Methods:We were interested in clarifying these divergent actions of curcumin in due consideration of the effects on radio-therapy. Thus, concentration- and time-dependent effects of curcumin on HIF-α and -β protein levels and activity in hepatoma and breast carcinoma cell cultures under normoxic and hypoxic conditions were studied.Results:It was shown that HIF-1α accumulated in normoxia after the application of higher doses of the drug. Curcumin proved to lower HIF-1α and HIF-2α protein levels in hypoxia. HIF-1β (ARNT; arylhydrocarbon nuclear translocator) protein levels and HIF transcriptional activity were reduced in normoxia and hypoxia after 4 h and 24 h incubation periods. Furthermore, curcumin treatment negatively impacted on clonogenic cell survival of Hep3B hepatoma and MCF-7 breast carcinoma cells.Conclusion:Effects of curcumin on cell growth and survival factor expression suggest its potential benefit in the treatment of cancer without a direct radiosensitizing influence of curcumin on these cells.Hintergrund:Das traditionell verwendete Gewürz Kurkumin beeinflusst die Aktivität von Entzündungsvermittlern wie Zytokinen und Transkriptionsfaktoren. Weiterhin hat Kurkumin inhibierenden Einfluss auf das Tumorwachstum – möglicherweise hervorgerufen durch einen verstärkten Abbau des Hypoxie-induzierbaren Faktors (HIF). Dieser Transkriptionsfaktor spielt eine entscheidende Rolle bei der Anpassung an eine verminderte Sauerstoffverfügbarkeit im Organismus. Dem entgegen könnte Kurkumin aber auch – aufgrund seiner Struktur – Eisen-Ionen komplexieren und so die Aktivität HIF-inhibierender Prolylhydroxylasen (PHD) beeinflussen. Gegenstand dieser Untersuchungen war daher die Klärung dieser divergenten Mechanismen in Bezug auf die Bestrahlungseffizienz von Tumoren.Methoden:Hierfür wurden die Poteinmengen der HIF-Untereinheiten sowie die HIF-Aktivität nach Kurkumin-Behandlung von Leber- und Brusttumorzellen innerhalb festgelegter Zeit- und Konzentrationsfenster geprüft. Anschließend wurde der Einfluss von Kurkumin auf die Strahlensibilität der Zellen im Klonogenen Assay untersucht.Ergebnisse:Es zeigte sich eine konzentrationsabhängige leichte Stabilisierung von HIF-1α-Protein unter normoxischen sowie eine Abnahme von HIF-1α-, -2α- und -1β-(ARNT: Arylhydrocarbon Nuclear Translocator-)Proteinmengen unter hypoxischen Bedingungen nach 4 Stunden Kurkumin-Behandlung. Die Aktivität von HIF nahm ebenfalls konzentrationsabhängig nach 4 h und 24 h Kurkumin-Inkubation ab. Weiterhin hatte die Vorbehandlung mit Kurkumin einen negativen Einfluss auf das klonogene Wachstum der untersuchten Leber- und Brusttumorzellen.Schlussfolgerung:Die Untersuchungen zur Kurkumin-Wirkung auf Tumorzellen sind ein Indiz für einen potentiell positiven Einfluss von Kurkumin auf den Bestrahlungserfolg dieser Zellen.

Notch1 signaling is mediated by importins alpha 3, 4, and 7

The Notch signaling pathway is an important regulation system for the development and self-renewal of different tissues. A specific feature of this signaling cascade is the function of Notch as a surface receptor and regulator of gene expression. Hence, Notch activation and signal transduction requires the proteolytic release of the Notch intracellular domain (NICD), which activates the transcription of cell-specific genes after its transport into the nucleus. To date, little is known about the mechanisms that mediate NICD nuclear import. We here show that transport of NICD into the nucleus is mediated by the canonical importin α/β1 pathway. GST pull-down experiments revealed that NICD binds via one of its four potential nuclear localization signals to importins α3, α4, and α7, but not to α1 and α5. siRNA-mediated knockdown experiments showed that importins α3, α4 (and to a lesser extent, α7) mediate nuclear import of NICD and thus are directly involved in Notch signaling.

Curcumin decreases survival of Hep3B liver and MCF-7 breast cancer cells: the role of HIF.

Background:Curcumin, a commonly used spice, affects the activities of cytokines, enzymes, and transcription factors that are linked to inflammation. Furthermore, curcumin has been assigned tumor growth inhibiting effects, possibly mediated by promoting hypoxia-inducible factor (HIF) degradation. HIFs are transcription factors that play a central role in the adaptation and response to low oxygen levels in metazoan cells. However, curcumin also exhibits properties of an iron chelator indicating its potential of inhibiting HIF-α prolyl hydroxylase (PHD) activity.Methods:We were interested in clarifying these divergent actions of curcumin in due consideration of the effects on radio-therapy. Thus, concentration- and time-dependent effects of curcumin on HIF-α and -β protein levels and activity in hepatoma and breast carcinoma cell cultures under normoxic and hypoxic conditions were studied.Results:It was shown that HIF-1α accumulated in normoxia after the application of higher doses of the drug. Curcumin proved to lower HIF-1α and HIF-2α protein levels in hypoxia. HIF-1β (ARNT; arylhydrocarbon nuclear translocator) protein levels and HIF transcriptional activity were reduced in normoxia and hypoxia after 4 h and 24 h incubation periods. Furthermore, curcumin treatment negatively impacted on clonogenic cell survival of Hep3B hepatoma and MCF-7 breast carcinoma cells.Conclusion:Effects of curcumin on cell growth and survival factor expression suggest its potential benefit in the treatment of cancer without a direct radiosensitizing influence of curcumin on these cells.Hintergrund:Das traditionell verwendete Gewürz Kurkumin beeinflusst die Aktivität von Entzündungsvermittlern wie Zytokinen und Transkriptionsfaktoren. Weiterhin hat Kurkumin inhibierenden Einfluss auf das Tumorwachstum – möglicherweise hervorgerufen durch einen verstärkten Abbau des Hypoxie-induzierbaren Faktors (HIF). Dieser Transkriptionsfaktor spielt eine entscheidende Rolle bei der Anpassung an eine verminderte Sauerstoffverfügbarkeit im Organismus. Dem entgegen könnte Kurkumin aber auch – aufgrund seiner Struktur – Eisen-Ionen komplexieren und so die Aktivität HIF-inhibierender Prolylhydroxylasen (PHD) beeinflussen. Gegenstand dieser Untersuchungen war daher die Klärung dieser divergenten Mechanismen in Bezug auf die Bestrahlungseffizienz von Tumoren.Methoden:Hierfür wurden die Poteinmengen der HIF-Untereinheiten sowie die HIF-Aktivität nach Kurkumin-Behandlung von Leber- und Brusttumorzellen innerhalb festgelegter Zeit- und Konzentrationsfenster geprüft. Anschließend wurde der Einfluss von Kurkumin auf die Strahlensibilität der Zellen im Klonogenen Assay untersucht.Ergebnisse:Es zeigte sich eine konzentrationsabhängige leichte Stabilisierung von HIF-1α-Protein unter normoxischen sowie eine Abnahme von HIF-1α-, -2α- und -1β-(ARNT: Arylhydrocarbon Nuclear Translocator-)Proteinmengen unter hypoxischen Bedingungen nach 4 Stunden Kurkumin-Behandlung. Die Aktivität von HIF nahm ebenfalls konzentrationsabhängig nach 4 h und 24 h Kurkumin-Inkubation ab. Weiterhin hatte die Vorbehandlung mit Kurkumin einen negativen Einfluss auf das klonogene Wachstum der untersuchten Leber- und Brusttumorzellen.Schlussfolgerung:Die Untersuchungen zur Kurkumin-Wirkung auf Tumorzellen sind ein Indiz für einen potentiell positiven Einfluss von Kurkumin auf den Bestrahlungserfolg dieser Zellen.

A p38-p65 transcription complex induced by endothelin-1 mediates signal transduction in cancer cells ☆

Endothelin-1 is a powerful mitogen for various tumor and non-tumor cells. Its signaling cascade induces the inflammatory NF-kappaB complex, leading to expression of a number of target genes. In this context, MAPK p38 has been regarded as a potential phosphate donor for the p65 subunit of NF-kappaB. In the present study in HeLa cells, we have found that ET-1 induced signalling activates the NF-kappaB transcription complex (TC) in the nucleus at 6 h specifically via ET-A - but not ET-B receptor. The TC contains p65, p38 (alpha and beta) - binding to the NLS of p65 in the cytoplasm - as well as p50, but no IkappaBalpha. Specific p38 inhibition by SB203580 or by siRNA interferes markedly with gene expression of several target genes. Complex formation occurs in the cytoplasm, and both transcription factors transmigrate as a complex in the nucleus. Overexpression of p38, treatment with Chrysin, MG132, or dimethylformamide shows dependence of TC on p38 as partner. In other tumor cells lines studied, ET-1 activates TC, with p38 as an important complex partner of p65. TC-induction by ET-1 contains about twice the amount of p38 than by TNFalpha. Thus, p38 may be an additional therapeutic target to control inflammatory gene expression in tumor cells.