Inhibition of clonogenic tumor growth: a novel function of Smac contributing to its antitumor activity

While second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis, its effect on cell viability in the absence of apoptotic stimuli has remained unclear. Here, we report that Smac inhibits clonogenic tumor growth by blocking random migration and proliferation and by enhancing apoptosis in a cell density and cell type dependent manner in SH-EP neuroblastoma cells. Inhibition of clonogenic survival by overexpression of full-length or processed Smac strictly depended on low cell density, and was reversible by replatement at high density. We discovered that Smac inhibits cell motility and random migration at low cell density. In addition, Smac enhanced apoptosis and inhibited protein, but not mRNA expression of XIAP, survivin and other short-lived proteins (FLIP, p21), indicating that Smac may globally inhibit protein expression. Also, Smac inhibited proliferation and increased polynucleation with no evidence for polyploidy, cell cycle arrest or senescence indicating that Smac impaired cell division. Interestingly, inhibition of clonogenic capacity by Smac occurred independent of its apoptosis promoting activity. By demonstrating that Smac restrains clonogenic tumor growth, our findings may have important implications for control of tumor growth and/or its metastatic spread. Thus, Smac agonists may be useful in cancer therapy, for example, for tumor control in minimal residual disease.Oncogene (2005) 24, 7190-7202. doi:10.1038/sj.onc.1208876; published online 8 August 2005.     

Inhibition des doxorubicininduzierten Zelltods in Herzmuskelzellen durch Dexrazoxan

ZusammenfassungHintergrund Doxorubicin ist ein wichtiges Chemotherapeutikum für die Behandlung krebskranker Kinder und fester Bestandteil der meisten Therapieprotokolle sowohl für akute Leukämien als auch für solide Tumoren. Der Einsatz wird dadurch eingeschränkt, dass Doxorubicin zu schweren Herzschädigungen führen kann, auch noch Jahre nach der erfolgreichen Heilung eines tumorkranken Kindes. Die Substanz Dexrazoxan (ICFR 187/Zinecard 7) vermindert die Herzschädigung von Doxorubicin über noch unbekannte Mechanismen.Diskussion Wir haben untersucht, ob das Apoptosesystem CD95/APO-1/FAS die protektive Wirkung von Dexrazoxan auf die Herzschädigung durch Doxorubicin vermittelt. Doxorubicin löst auf primären neonatalen Rattenkardiomyozyten Apoptose aus, was durch die Freisetzung des Todesliganden CD95L in den Zellkulturüberstand begleitet wird. Die zusätzliche Gabe von Dexrazoxan vermindert sowohl die Freisetzung von CD95L als auch den doxorubicininduzierten Zelltod von Herzmuskelzellen. Das molekulare Ausschalten des CD95-Apoptosesystems durch die stabile Transfektion von dysfunktionalem FADD hat jedoch keinen Einfluss auf die Herzschädigung durch Doxorubicin. CD95 ist somit nicht ursächlich an der protektiven Wirkung von Dexrazoxan auf den doxorubicininduzierten Herzschaden beteiligt.Diese Arbeit wurde von der Wilhelm-Sander-Stiftung sowie der Bettina-Bräu-Stiftung Mehr Leben für krebskranke Kinder gefördert.     

Impact of diet on stool signal in dark lumen magnetic resonance colonography

PURPOSE: To examine the magnetic resonance (MR) properties of different foods and their effect on the colonic stool signal to potentially support fecal tagging strategies for dark lumen MR colonography (MRC). MATERIALS AND METHODS: T1 relaxation times of 120 different foods (partially diluted with sufficient water) were determined by use of a multi-flip-angle two-dimensional gradient echo (GRE) sequence and correlated to the foods' signal in a three-dimensional GRE volumetric interpolated breath-hold examination (VIBE) sequence. Different dilutions of six foods were examined. VIBE stool signal was determined in six volunteers under two different conditions: after a three-day diet of short T1 food and of long T1 food, respectively. RESULTS: Most foods exhibit short to very short T1 relaxation times. T1 correlates well with the fat-saturated VIBE signal except for fatty products. Diluted food exhibits T1 times similar to water; concentrated food strongly varies according to their T1 values. No significant difference in stool signal could be found in the in vivo examination comparing the two diets. CONCLUSION: According to our results, a restricted diet strategy to reduce fecal signal for dark lumen MRC is unlikely to be successful. Moreover, the stool signal reduction found in the other fecal tagging studies can be explained at least to a great extent by the relative content of other material with long T1 relaxation times, such as water or oral barium.     

Effect of dialysate calcium concentrations on intradialytic blood pressure course in cardiac-compromised patients

To prevent hypercalcemia in the treatment of secondary hyperparathyroidism, low calcium (L-Ca) dialysate is advocated. However, changes in ionized calcium (i-Ca) levels have a pivotal role in myocardial contraction and could influence blood pressure stability during dialysis. Recently, our group found in patients with normal cardiac function a significant decrease in blood pressure (decrease in systolic blood pressure [DSBP]: -13 mm Hg and decrease in mean arterial pressure [DMAP]: -7 mm Hg) during dialysis with L-Ca dialysate compared with high calcium (H-Ca) dialysate, and this was mainly related to a decreased left ventricular contractility with use of L-Ca dialysate. On the basis of these data, it could be expected that changes in i-Ca levels during dialysis are of more clinical importance in cardiac-compromised patients (CCpts), New York Heart Association classifications III and IV. In this study, the effects of L-Ca dialysate (1.25 mmol/L) and H-Ca dialysate (1.75 mmol/L) on arterial blood pressure parameters (systolic [SBP], diastolic [DBP], and mean arterial blood pressure [MAP]), heart rate, stroke distance (SDist), and minute distance (MDist) during 3 hours of a standardized ultrafiltration/hemodialysis (UF+HD) in nine CCpts was investigated. i-Ca levels increased significantly with H-Ca dialysate UF+HD, whereas there was no change with L-Ca dialysate. SBP, DBP, and MAP decreased statistically and clinically significantly during UF+HD with L-Ca dialysate and were significantly lower with the use of L-Ca dialysate compared with H-Ca dialysate. SDist and MDist decreased significantly with L-Ca dialysate, whereas there were no changes in SDist and MDist with H-Ca dialysate. The predialysis and postdialysis index of systemic vascular resistance (SVRI) was similar between L-Ca dialysate and H-Ca dialysate use. Between the two groups, there were no significant differences in changes in SVRI. From this study, we can conclude that changes in i-Ca levels are a very important determinant of the blood pressure response during UF+HD in CCpts, and this response is mediated by changes in myocardial contractility.     

Insulin sensitivity and beta-cell secretion in thalassaemia major with secondary haemochromatosis: assessment by oral glucose tolerance test

Diabetes mellitus in patients with thalassaemia major is caused by secondary haemochromatosis due to transfusional iron overload. The pathogenetic mechanisms leading from siderosis to diabetes are still poorly understood. This study aimed at assessing the influence of insulin resistance and insulin deficiency on that process. Glucose, insulin and C-peptide levels during oral glucose tolerance tests (OGTT) from 36 thalassaemic patients with normal ( n=23), impaired ( n=6), or diabetic glucose tolerance ( n=7) and 32 control subjects were examined. Insulin secretion and insulin sensitivity were assessed by established calculated indices. Fasting, 2h and integrated glucose concentration were significantly increased in thalassaemic patients with normal glucose tolerance compared to controls (5.01/4.59 mmol/l, 6.33/5.17 mmol/l, and 844.2/739.3 mmol/l per min, respectively; all P<0.03). Patients with impaired glucose tolerance presented hyperinsulinaemia and delayed peak insulin during OGTT. The C-peptide/insulin ratio was decreased in patients with abnormal glucose tolerance compared to controls (5.85/7.33 x 10(3)pmol/l per min, P<0.03). It was negatively correlated with age in patients ( r=-0.45, P<0.01), but positively in controls ( r=0.43, P<0.03). Insulin sensitivity was significantly reduced in patients with impaired glucose tolerance or diabetes compared to controls. In addition, a significant decrease in patients with normal glucose tolerance was shown by two insulin sensitivity indices (all P<0.05). In thalassaemia patients, insulin sensitivity was negatively correlated with age. Insulin secretion capacity according to the homeostasis assessment model was significantly reduced in patient groups compared to controls (Kruskal-Wallis-test, P<0.004). CONCLUSION: Insulin resistance is of central importance for the development of diabetes mellitus in patients with secondary haemochromatosis. An additional early defect in beta-cell secretion cannot be excluded.     

Inhibition of JNK signaling diminishes early but not late cellular stress-induced apoptosis

The human leukemic T-cell line Jurkat was used to define the role of the cellular stress pathway with its key player kinase JNK in cancer therapy-induced apoptosis. JNK activity was inhibited by stable transfection with a dominant negative mutant of the upstream kinase JNKK/MKK4 or with the novel, potent and selective JNK1, -2 and -3 inhibitor SP600125. Inhibition of JNK activity delayed the onset of apoptosis induced by cisplatin, doxorubicin, gamma-irradiation and CD95-L but did not prevent apoptosis per se. Early events during apoptosis such as induction of CD95-L, activation of caspase-8 and exposure of phosphatidylserine on the cell surface were strongly inhibited. Also, at early time points of apoptosis, loss of the mitochondrial membrane potential and release of cytochrome c were markedly impaired. However, late signaling events during apoptosis such as cleavage of PARP and DNA fragmentation apoptosis were only marginally affected. These findings are in accordance with the activity of initiator and effector caspases. Whereas activity of the initiator caspase-8 was strongly inhibited early and late after induction, an inhibition of caspase-3 activity was only observed early after induction of apoptosis. We therefore suggest that cellular stress signaling contributes to the initiation of apoptosis, whereas it might be dispensable for the progression of apoptosis. Dysfunction of this pathway under pathological conditions might contribute to therapy resistance of cancer cells.     

Apoptosis pathways in neuroblastoma therapy

Apoptosis, the cell's intrinsic death program, plays a crucial role in the regulation of tissue homeostasis, and an imbalance between cell death and proliferation may result in tumor formation. Also, killing of tumor cells by diverse cytotoxic approaches such as anticancer drugs, gamma-irradiation, suicide genes or immunotherapy, is predominantly mediated through induction of apoptosis. Failure to activate apoptotic pathways in response to drug treatment may lead to resistance of neuroblastoma cells to anticancer therapies. Understanding the molecular events that regulate apoptosis induced by cytotoxic therapies and how neuroblastoma cells evade apoptotic events may provide a new paradigm for neuroblastoma therapy. Thus, novel strategies targeting resistance of neuroblastoma cells will be based on insights into the molecular mechanisms of apoptosis as well as other forms of cell death.     

MR venography using an intravascular contrast agent: results from a multicenter phase 2 study of dosage

OBJECTIVE: The objective of this study was to determine the optimal dose of the iron oxide contrast agent feruglose for contrast-enhanced MR venography of the abdominopelvic and lower extremity veins and to evaluate its safety and tolerability in patients with deep venous thrombosis. SUBJECTS AND METHODS: We enrolled in our study a total of 45 patients at six centers who had lower extremity deep venous thrombosis documented on radiographic venography. Forty-four patients received the study drug; 39 completed the study. Each patient received three sequential IV injections of feruglose at doses of 0.75, 1.25, and 3.0 mg Fe/kg body weight. MR venography at 1.5 T was repeated at three levels after each dose. Safety was evaluated. RESULTS: The agreement between contrast-enhanced MR venography and radiographic venography with regard to deep venous thrombosis above the knee was zero at the lowest dose (0.75 mg Fe/kg body weight), 43% at the dose 2.0 mg Fe/kg body weight, and 49% at the dose 5.0 mg Fe/kg body weight. No significant difference was seen between the two highest doses. The highest cumulative dose provided the greatest diagnostic usefulness score. No serious adverse events occurred. CONCLUSION: The two highest doses of feruglose showed the best agreement between contrast-enhanced MR venography and radiographic venography for deep venous thrombosis above the knee. The safety and tolerability of feruglose were confirmed.     

TSH receptor mutation V509A causes familial hyperthyroidism by release of interhelical constraints between transmembrane helices TMH3 and TMH5

Mutations of the human thyrotrophin receptor (TSH-R) are a cause of thyroid adenomas and hyperthyroidism. Here we study mechanisms of receptor activation in a genomic TSH-R variant V509A located in transmembrane helix (TMH) 3, which we identify in a family with congenital hyperthyroidism, multiple adenomas and follicular thyroid cancer. Using molecular modelling and dynamic simulation, we predicted the release of amino acid residue A593 (located opposite in domain TMH5) from a tight 'knob-and-hole' interaction with TMH3, physiologically constrained in the native receptor state by the bulky side chain of V509. To experimentally validate this concept, we generated mutant TSH-R expression constructs for functional in vitro studies. TSH-R mutant V509A showed a 2.8-fold increase in basal cAMP production, confirming constitutive TSH-R activation. The addition of a second site suppressor mutant A593V to TSH-R V509A resulted in the normalization of basal cAMP release, and the dose-responsiveness to TSH ligand was maintained. These data thus demonstrate that TSH-R V509A activation is caused by the release of TMH3-TMH5 interhelical constraints, while the native TSH-R conformation is re-stabilized by the introduction of a spacious valine residue at position 593. In conclusion, we delineate a novel mechanism of constitutive TSH-R activation, leading to thyroid hyperfunction and neoplasia.