Rodrig Marculescu

V(D)J-mediated Translocations in Lymphoid Neoplasms: A Functional Assessment of Genomic Instability by Cryptic Sites

Most lymphoid malignancies are initiated by specific chromosomal translocations between immunoglobulin (Ig)/T cell receptor (TCR) gene segments and cellular proto-oncogenes. In many cases, illegitimate V(D)J recombination has been proposed to be involved in the translocation process, but this has never been functionally established. Using extra-chromosomal recombination assays, we determined the ability of several proto-oncogenes to target V(D)J recombination, and assessed the impact of their recombinogenic potential on translocation rates in vivo. Our data support the involvement of 2 distinct mechanisms: translocations involving LMO2, TAL2, and TAL1 in T cell acute lymphoblastic leukemia (T-ALL), are compatible with illegitimate V(D)J recombination between a TCR locus and a proto-oncogene locus bearing a fortuitous but functional recombination site (type 1); in contrast, translocations involving BCL1 and BCL2 in B cell non-Hodgkins lymphomas (B-NHL), are compatible with a process in which only the IgH locus breaks are mediated by V(D)J recombination (type 2). Most importantly, we show that the t(11;14)(p13;q32) translocation involving LMO2 is present at strikingly high frequency in normal human thymus, and that the recombinogenic potential conferred by the LMO2 cryptic site is directly predictive of the in vivo level of translocation at that locus. These findings provide new insights into the regulation forces acting upon genomic instability in B and T cell tumorigenesis.

Identification of Heme Oxygenase-1 As a Novel BCR/ABL-Dependent Survival Factor in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL promotes the survival of leukemic cells. Heme oxygenase-1 (HO-1) is an inducible stress protein that catalyzes the degradation of heme and has recently been implicated in the regulation of growth and survival of various neoplastic cells. In the present study, we analyzed the expression and role of HO-1 in CML cells. As assessed by Northern and Western blot analysis as well as immunostaining, primary CML cells were found to express HO-1 mRNA and the HO-1 protein in a constitutive manner. Exposure of these cells to the BCR/ABL tyrosine kinase inhibitor STI571 resulted in decreased expression of HO-1 mRNA and protein. In addition, BCR/ABL was found to up-regulate HO-1 promoter activity, mRNA levels, and protein levels in Ba/F3 cells. To investigate the role of HO-1 for survival of primary CML cells, the HO-1 inducer hemin was used. Hemin-induced expression of HO-1 was found to protect CML cells from STI571-induced cell death. In addition, inhibition of HO-1 by zinc-(II)-deuteroporphyrin-IX-2,4-bisethyleneglycol resulted in a substantial decrease of cell viability. Furthermore, overexpression of HO-1 in the CML-derived cell line K562 was found to counteract STI571-induced apoptosis. Together, our data identify HO-1 as a novel BCR/ABL-driven survival molecule and potential target in leukemic cells in patients with CML. The pathogenetic and clinical implications of this observation remain to be elucidated.

Renal Elimination of Sclerostin Increases With Declining Kidney Function

CONTEXT Sclerostin serum levels are increased in patients with chronic kidney disease (CKD). Osteoporosis and CKD often occur simultaneously. Currently antisclerostin antibodies are in clinical development for the treatment of osteoporosis. OBJECTIVE The objective of this study was to study the renal handling of sclerostin. DESIGN This was a cross-sectional study. SETTING The study was conducted at a university hospital and outpatient renal clinic. PATIENTS One hundred twenty men and women with CKD stage 1-5 participated in the study. INTERVENTION Measurements of sclerostin in urine and serum (ELISA), renal function [estimated glomerular filtration rate (eGFR)], electrolytes, α1-microglobulin, PTH, vitamin D, and markers of bone turnover were conducted. Eight human kidney biopsies were stained for sclerostin using immunohistochemistry. MAIN OUTCOME MEASURE Urinary excretion of sclerostin was measured. RESULTS Urinary sclerostin excretion increased with declining eGFR (R=-0.75, P<.001), from 10.4 (±12.7) pmol/L in patients with eGFR greater than 90 mL/min per 1.73 m2 (CKD stage 1) to 117.9 (±65.4) pmol/L in patients with eGFR less than 15 mL/min per 1.73 m2 (CKD stage 5, P<.001). Fractional excretion of sclerostin increased with declining eGFR (R=-0.83, P<.001) from 0.45% (±0.6%) in CKD 1 to 26.3% (±17.6%) in CKD 5 (P<.001). Fractional excretion of sclerostin correlated with fractional excretion of α1-microglobulin (R=0.82, P<.001). No association between serum sclerostin and fractional excretion of phosphorus was found in a multivariate analysis. Sclerostin was detected in proximal tubular cells, showing a diffuse cytoplasmic staining pattern. CONCLUSION Increased sclerostin serum levels in CKD patients are not due to decreased renal elimination. On the contrary, renal elimination increases with declining kidney function. Whether this has consequences on antisclerostin antibody dosing, efficacy, or safety in patients with CKD remains to be determined.

A microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with increased bilirubin and HDL levels but not with coronary artery disease

Heme oxygenase 1 (HO-1) is involved in the generation of the endogenous anti-oxidant bilirubin which exerts beneficial effects against arteriosclerosis. A (GT) repeat polymorphism in the HO-1 promoter region modulates HO-1 expression in response to oxidative stress. Recently, this polymorphism has been reported to protect from coronary artery disease in Orientals. We intended to confirm this observation in Caucasians. We studied 649 individuals with myocardial infarction (n=258), stable coronary artery disease (n=180) and controls without coronary artery disease (n=211). Carriers of short alleles (<25 repeats) had higher bilirubin levels (median 0.66 mg/dL, IQR 0.49 to 0.91) compared to non-carriers (median 0.61 mg/dL, IQR 0.45 to 0.82; p=0.03) and a more favourable lipid profile (HDL median 47 mg/dL, IQR 40 to 50 vs. median 45, IQR 37 to 55, p=0.01; triglycerides median 118 mg/dL, IQR 87 to 174 vs. median 132, IQR 97 to 191, p=0.03). However, no significant differences of the genotype distribution were observed between the three groups in this Caucasian study population (p=0.94). Although potentially beneficial effects of the short HO-1 allele on lipid profile and serum bilirubin were observed, in contrast to Orientals, the HO-1 genotype was not associated with coronary artery disease in Caucasians.

Polymorphisms of the inflammatory system and risk of ischemic cerebrovascular events.

Abstract Background: Chronic and acute infections are associated with an increased risk of stroke. The inflammatory response can be influenced by functional polymorphisms in components of the immune system. We hypothesized that these polymorphisms may also modulate the risk of ischemic cerebrovascular events. Methods: We determined the frequency of polymorphisms in tumor necrosis factor-α[(TNF-α) G(–376)A, G(–244)A, G(–238)A, G(–308)A], Toll-like receptor 4 [(TLR4) Gly299Asp and Thr399Ile], interleukin-1-receptor antagonist [(IL-1-RA) intron 2 variable-number tandem repeat], monocyte differentiation antigen CD14 receptor C(–260)T, and interleukin-6 [(IL-6) G(–174)C] genes in 404 patients with acute stroke or transient ischemic attack before the age of 60years and in 415 healthy individuals. We also tested for interactions between genotypes, recent febrile episodes and stroke risk. Results: None of the polymorphisms was associated with an increased risk of stroke after adjustment for age and gender. Following multivariate adjustment, carriers of the TNF-α (–308)A allele, the IL-1-RA 2* allele or the IL-6 (–174)C allele appeared to have an increased risk of stroke in association with a febrile episode prior to strokes. Conclusion: In our study none of the investigated polymorphisms of the inflammatory system was associated with the risk of acute cerebrovascular events before the age of 60years. However, post-hoc analyses indicate that some polymorphisms seem to contribute to the risk of stroke in combination with fever. Clin Chem Lab Med 2006;44:918–23.

Protection from age-related increase in lipid biomarkers and inflammation contributes to cardiovascular protection in Gilbert's syndrome

Recent epidemiological and clinical data show protection from CVD (cardiovascular disease), all-cause mortality and cancer in subjects with GS (Gilberts syndrome), which is characterized by a mildly elevated blood bilirubin concentration. The established antioxidant effect of bilirubin, however, contributes only in part to this protection. Therefore we investigated whether mildly elevated circulating UCB (unconjugated bilirubin) is associated with altered lipid metabolism. The study was performed on GS and age- and gender-matched healthy subjects (n=59 per group). Full lipoprotein profile, TAG (triacylglycerols), Apo (apolipoprotein)-A1, Apo-B, lipoprotein(a), the subfractions of LDL (low-density lipoprotein) and selected pro-inflammatory mediators were analysed. A hyperbilirubinaemic rodent model (Gunn rats, n=40) was investigated to further support the presented human data. GS subjects had significantly (P<0.05) improved lipid profile with reduced total cholesterol, LDL-C (LDL-cholesterol), TAG, low- and pro-atherogenic LDL subfractions (LDL-1+LDL-2), Apo-B, Apo-B/Apo-A1 ratio and lower IL-6 (interleukin 6) and SAA (serum amyloid A) concentrations (P=0.094). When the control and GS groups were subdivided into younger and older cohorts, older GS subjects demonstrated reduced lipid variables (total cholesterol and LDL-C, TAG and LDL-C subfractions, Apo-B/Apo-A1 ratio; P<0.05; Apo-B: P<0.1) compared with controls. These data were supported by lipid analyses in the rodent model showing that Gunn rat serum had lower total cholesterol (2.29±0.38 compared with 1.27±0.72 mM; P<0.001) and TAG (1.66±0.67 compared with 0.99±0.52 mM; P<0.001) concentrations compared with controls. These findings indicate that the altered lipid profile and the reduced pro-inflammatory status in hyperbilirubinaemic subjects, particularly in the older individuals, probably contribute additionally to the commonly accepted beneficial antioxidant effects of bilirubin in humans.

The gallium complex KP46 exerts strong activity against primary explanted melanoma cells and induces apoptosis in melanoma cell lines

The antineoplastic properties of gallium are well documented. Owing to their robust accumulation of gallium, melanoma cells should be amenable to gallium-based anticancer drugs. With the aim of improving the disappointingly low activity of inorganic gallium salts, we have developed the orally bioavailable gallium complex KP46 [tris(8-quinolinolato)gallium(III)] that had already been successfully studied in a phase I clinical trial. To assess its therapeutic potential in malignant melanoma, its antiproliferative effects were investigated in series of human cell lines and primary explanted melanoma samples by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and the Human Tumor Cloning Assay, respectively. When compared with other cell lines, the majority of melanoma cells rank among the KP46-sensitive cell lines (50% inhibitory concentration values: 0.8–3.7 μmol/l). Clinically achievable concentrations of KP46 proved to be highly effective in melanoma cells from primary explants of cutaneous and lymph node metastases. Colony growth was inhibited in 10 of 10 specimens by 5 μmol/l KP46 (corresponding to the steady-state plasma concentration measured earlier in a study patient) and in four of 10 specimens by 0.5 μmol/l KP46. In-vitro potency of KP46 is higher than that of dacarbazine or fotemustine and comparable with that of cisplatin. The effects induced by KP46 in melanoma cell lines involve cell-cycle perturbations (S-phase arrest) and apoptosis (activation of caspase-9, PARP [poly(ADP-ribose) polymerase] cleavage, formation of apoptotic bodies). No effects on DNA secondary structure could be observed in an electrophoretic mobility shift assay using double-stranded plasmid DNA. Thus, further studies on the therapeutic applicability of KP46 in malignant melanoma are warranted.

Routinely available biomarkers improve prediction of long-term mortality in stable coronary artery disease: the Vienna and Ludwigshafen Coronary Artery Disease (VILCAD) risk score.

AIMS Previous risk assessment scores for patients with coronary artery disease (CAD) have focused on primary prevention and patients with acute coronary syndrome. However, especially in stable CAD patients improved long-term risk prediction is crucial to efficiently apply measures of secondary prevention. We aimed to create a clinically applicable mortality prediction score for stable CAD patients based on routinely determined laboratory biomarkers and clinical determinants of secondary prevention. METHODS AND RESULTS We prospectively included 547 patients with stable CAD and a median follow-up of 11.3 years. Independent risk factors were selected using bootstrapping based on Cox regression analysis. Age, left ventricular function, serum cholinesterase, creatinine, heart rate, and HbA1c were selected as significant mortality predictors for the final multivariable model. The Vienna and Ludwigshafen Coronary Artery Disease (VILCAD) risk score based on the aforementioned variables demonstrated an excellent discriminatory power for 10-year survival with a C-statistic of 0.77 (P < 0.001), which was significantly better than an established risk score based on conventional cardiovascular risk factors (C-statistic = 0.61, P < 0.001). Net reclassification confirmed a significant improvement in individual risk prediction by 34.8% (95% confidence interval: 21.7-48.0%) compared with the conventional risk score (P < 0.001). External validation of the risk score in 1275 participants of the Ludwigshafen Risk and Cardiovascular Health study (median follow-up of 9.8 years) achieved similar results (C-statistic = 0.73, P < 0.001). CONCLUSION The VILCAD score based on a routinely available set of risk factors, measures of cardiac function, and comorbidities outperforms established risk prediction algorithms and might improve the identification of high-risk patients for a more intensive treatment.

Cross-Regulation of Carbon Monoxide and the Adenosine A2a Receptor in Macrophages

Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5′-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-α production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-α. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.

Serum Levels of Sclerostin and Dickkopf-1: Effects of Age, Gender and Fracture Status

Background: Fragility fractures, especially hip fractures, are a very common complication of osteoporosis in elderly subjects. Sclerostin (SOST) and dickkopf-1 (DKK-1) are inhibitors of the canonical wnt signalling pathway and thus could be involved in the pathogenesis of age-related bone fragility. Objective: To investigate SOST and DKK-1 in a large group of geriatric patients with hip fractures and to relate the wnt inhibitors to age and gender. Methods: This was a cross-sectional study carried out in a department of acute geriatric care in a district hospital in Upper Austria and a hospital in Vienna, Austria. A total of 256 geriatric patients (172 women and 84 men) and 67 young control subjects were selected after exclusion. Medical history was obtained, a comprehensive geriatric assessment was performed and serum levels of SOST, DKK-1 and bone formation markers were analysed. Results: DKK-1 levels increased with age and in the presence of hip fractures. In contrast, SOST levels were lower in patients with hip fractures. When compared to women, men had higher SOST levels but lower DKK-1 levels. Conclusion: Serum levels of the inhibitors of the canonical wnt signalling pathway reflect different biological events and are useful for the study of bone fragility in geriatric patients.