No description available.

<p>The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc.</p><p>Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies.</p><p>Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-<i>myc</i> amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. </p><p>In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.</p>

The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc. Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies. Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-myc amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.

No description available.

Das Ziel dieser Studie war es, die Beteiligung der nichtmuskulären schweren Myosinketten an der Kontraktion der glatten Muskeln unter physiologischen Bedingungen zu untersuchen. Als Versuchsmodell wurde die Harnblase von neugeborenen Wildtyp und transgenen Mäusen verwendet, bei denen das Gen für die glattmuskelspezifischen schweren Myosinketten durch "Gene Targeting" funktionell eliminiert wurde (Knock-Out). Das Fehlen der Expression der glattmuskelspezifischen schweren Myosinketten wurde durch Elektrophorese und Immunfärbung bestätigt. Im Gegensatz dazu blieb die Expression der nichtmuskulären schweren Myosinketten unverändert. Die mechanische Analyse des glatten Muskels wurde mit intakten Muskelpräparaten aus der Harnblase durchgeführt. Das Muskelpräparat wurde in KCl-Lösung oder mit Phorbolester stimuliert. Die Aktivierung mittels depolarisierender KCl-Lösung führte bei neugeborenen Wildtyp Mäusen zuerst zu einer transienten Kontraktion (Phase 1) mit hoher Kraftentwicklung und maximaler Verkürzungsgeschwindigkeit, und danach zu einer tonischen Kontraktion (Phase 2) mit niedrigerer Kraftentwicklung und maximaler Verkürzungsgeschwindigkeit. Blasenpräparate neugeborener Knock-Out Mäuse dagegen zeigten keine Phase 1, sondern nur eine tonische Kontraktion, die mit Wildtyp Mäusen vergleichbar war. Daher scheint nichtmuskuläres Myosin an der tonischen Kontraktion des glatten Muskels beteiligt zu sein. Durch Stimulierung mit Phorbolester waren ähnliche tonische Muskelkontraktionen der Blasenpräparate sowohl bei Wildtyp als auch bei Knock-Out Mäusen zu beobachten. Vermutlich wird also das nichtmuskuläre Myosin durch Stimulierung mit Phorbolester aktiviert. Intrazelluläre Filamente wurden durch Immunfluoreszenz mit einem spezifischen Antikörper gegen nichtmuskuläre schwere Myosinketten in kultivierten primären glatten Muskelzellen untersucht. Dabei zeigten die Muskelzellen sowohl von Wildtyp als auch von Knock-Out Mäusen intrazelluläre dicke Myosinfilamente, was für die Beteiligung des nichtmuskulären Myosins an der glatten Muskelkontraktion spricht. Entsprechend wurden intrazelluläre Filamente mit einem Antikörper gegen glattmuskelspezifische schwere Myosinketten in kultivierten primären glatten Muskelzellen untersucht. Wie erwartet, konnten nur in glatten Muskelzellen von Wildtyp Mäusen intrazelluläre Filamente nachgewiesen werden, nicht aber in denen von Knock-Out Mäusen. In dieser Arbeit konnte zum ersten Mal gezeigt werden, dass nichtmuskuläres Myosin zumindest an der tonischen Kontraktion glatter Muskelzellen beteiligt sein kann. / The aim of the present study was to investigate the involvement of non-muscle myosin heavy chain in smooth muscle contraction under physiological conditions. As an experimental model urinary bladder from neonatal wild-type mice as well as from neonatal mice with disrupted smooth muscle myosin heavy chain expression was used. This animal model was established through gene targeting technology, resulting in complete elimination of the expression of smooth muscle myosin heavy chains. The lack of expression of smooth muscle myosin heavy chains was confirmed by electrophoresis and immunoblotting. On the other hand, non-muscle myosin heavy chain expression remained normal, as verified by Western blot analysis. The mechanical analysis of smooth muscle was performed with intact urinary bladder preparations, stimulated using prolonged KCl depolarization or with phorbol ester. Prolonged activation by KCl depolarization of intact bladder preparations from wild-type neonatal mice produced an initial transient state (phase 1) of high force generation and maximal shortening velocity, followed by a sustained state (phase 2) with lower force generation and maximal shortening velocity. In contrast, bladder preparations from homozygous knockout neonatal mice did not exhibit phase 1, but phase 2 could be observed, i.e. a similar isometric force and maximal shortening velocity, compared to wild-type phase 2. Thus, non-muscle myosin appears to be recruited in the sustained phase of smooth muscle contraction during prolonged KCl depolarization in the animal model used. Upon stimulation with phorbol ester a similar sustained contraction was observed in both wild-type and knockout smooth muscle preparations. Therefore, non-muscle myosin may also be recruited during phorbol ester stimulation in both wild-type and knockout muscle preparations. The participation of non-muscle myosin in smooth muscle contraction was further supported by the finding of longitudinally arranged intracellular filaments in cultivated smooth muscle cells from both wild-type and knockout mice by immunofluorescence microscopy, using a specific antibody raised against non-muscle myosin heavy chain. In a similar way, smooth muscle myosin heavy chain structures were investigated in cultivated smooth muscle cells. As expected, longitudinally arranged intracellular filamentous structures of smooth muscle myosin were observed in wild-type smooth muscle cells, but not in smooth muscle cells from knockout mice. In conclusion, in neonatal smooth muscle the initial phase of contraction elicited by KCl depolarization is generated by smooth muscle myosin heavy chain recruitment. Upon prolonged KCl depolarization non-muscle myosin is recruited in the sustained phase of contraction, as well as upon stimulation with phorbol ester. Thus, it was possible, for the first time, to verify the involvement of the non-muscle myosin in smooth muscle contraction in vivo. The results of the present study contribute to the understanding of the regulatory mechanisms of smooth muscle contraction.