Stefan Seibold

Identification of a new tumor suppressor gene located at chromosome 8p21.3-22

Transformation of normal cells into malignant tumor cells, a process termed carcinogenesis, depends on progressive acquisition of genetic alterations. These result in activation of protooncogenes or inactivation of tumor suppressor genes responsible for the loss of proliferative control in tumor cells and the failure to undergo cellular differentiation. The aim of our study was the identification of molecular regulators of carcinogenesis by studying gene expression during induction of cellular differentiation and quiescence in a three‐dimensional (3D) cell culture model. Here, we report the discovery of a tumor suppressor gene located at chromosome 8p21.3–22 near marker D8S254. It is ubiquitously expressed in normal tissue and transiently up‐regulated during initiation of cellular differentiation and quiescence in 3D cell culture. In contrast, mRNA expression was not detectable in tissue from pancreatic tumor and the pancreatic tumor cell line MIA PaCa‐2. Recombinant expression in the tumor cell line MIA PaCa‐2 inhibited proliferation, as shown by a 30% reduction of BrdU uptake after recombinant expression. Immunocytochemistry and Western blot analysis of subcellular fractions demonstrated a mitochondrial localization for the mature protein. In conclusion, we identified a tumor suppressor gene at chromosome 8p21.3–22, encoding a mitochondrial protein, controlling cellular proliferation.

Oxidized LDL Induces Proliferation and Hypertrophy in Human Umbilical Vein Endothelial Cells via Regulation of p27Kip1 Expression: Role of RhoA

Oxidized LDL (OxLDL) induces proliferation in human umbilical vein endothelial cells (HUVEC). The influence of OxLDL on the cyclin-dependent kinase inhibitor p27(Kip1), on the activity of the small GTPase RhoA as a known regulator of p27(Kip1), and on resulting cell proliferation and hypertrophy was studied. HUVEC were stimulated with OxLDL (1 to 50 mug/ml). Proliferation was quantified by (3)H-thymidine incorporation, colorimetric 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazolium bromide assay, and cell count and was compared with proliferation of HUVEC that were transfected with dominant negative RhoA or treated with the Rho-kinase inhibitor Y27632. Hypertrophy was quantified by (3)H-leucine incorporation and by planimetry. p27(Kip1) expression was determined by Western blot analysis. p27(Kip1) was downregulated by transient transfection with antisense oligonucleotides. Low concentrations of OxLDL induced proliferation of HUVEC, paralleled by a persistent decrease of p27(Kip1) expression. With the use of antisense oligonucleotides, further downregulation of p27(Kip1) expression enhanced the OxLDL-induced proliferative response. High concentrations of OxLDL resulted in cellular hypertrophy and caused a delayed increase in p27(Kip1) expression after initial downregulation. Concomitant, OxLDL caused a significant activation of the small GTPase RhoA. In cells that were transfected with dominant negative RhoA, the effect of OxLDL on p27(Kip1) expression and on cellular proliferation was abolished. HUVEC that were preincubated with the Rho-kinase inhibitor Y27632 also showed a significantly decreased proliferative response to OxLDL stimulation. In summary, OxLDL has a dual effect on cell-cycle progression via regulation of p27(Kip1) expression, resulting in cellular proliferation and hypertrophy, involving activation of RhoA. OxLDL may importantly contribute to vascular hyperplasia in atherosclerosis and other diseases associated with increased levels of OxLDL.

Oxidized LDL and its Compound Lysophosphatidylcholine Potentiate AngII-Induced Vasoconstriction by Stimulation of RhoA

RhoA stimulates vascular tone by increasing smooth muscle Ca(2+) sensitivity, e.g., in atherosclerosis. This study was an investigation of the influence of oxidized LDL (OxLDL), which accumulates in atherosclerotic plaques, on vascular tone induced by angiotensin II (AngII), with particular emphasis on the RhoA pathway. OxLDL had no influence on unstimulated vascular tone of isolated rabbit aorta, but it potentiated contractile responses induced by AngII. The Ca(2+)-antagonist felodipin partially prevented potentiation of contractile responses, whereas the AT(1) receptor antagonist losartan blunted AngII responses in presence and in absence of OxLDL. Rho-kinase inhibition by Y27632 abolished potentiation of contractile responses, and RhoA inhibition by C3-like transferase partially prevented it, suggesting that OxLDL activated RhoA. Activation of RhoA was further analyzed by detection of its translocation to the cell membrane after stimulation with OxLDL. Western blot analysis of aorta homogenates, as well as direct visualization in cultured smooth muscle cells using confocal laser scan microscopy, revealed that OxLDL potently activated RhoA. The effect of OxLDL was mimicked by its compound lysophosphatidylcholine, and C3 inhibited both lysophosphatidylcholine and OxLDL-induced RhoA stimulation. In conclusion, OxLDL stimulates the RhoA pathway, resulting in potentiation of AngII-induced vasoconstriction. Lysophosphatidylcholine mimics the OxLDL effect, consistent with a causal role of this OxLDL compound. Stimulation of RhoA by OxLDL may contribute to vasospasm in atherosclerotic arteries.

LOH and copy neutral LOH (cnLOH) act as alternative mechanism in sporadic colorectal cancers with chromosomal and microsatellite instability

BACKGROUND AND AIMS Tumor suppressor genes are often located in frequently deleted chromosomal regions of colorectal cancers (CRCs). In contrast to microsatellite stable (MSS) tumors, only few loss of heterozygosity (LOH) studies were performed in microsatellite instable (MSI) tumors, because MSI carcinomas are generally considered to be chromosomally stable and classical LOH studies are not feasible due to MSI. The single nucleotide polymorphism (SNP) array technique enables LOH studies also in MSI CRC. The aim of our study was to analyse tissue from MSI and MSS CRC for the existence of (frequently) deleted chromosomal regions and tumor suppressor genes located therein. METHODS AND RESULTS We analyzed tissues from 32 sporadic CRCs and their corresponding normal mucosa (16 MSS and 16 MSI tumors) by means of 50K SNP array analysis. MSS tumors displayed chromosomal instability that resulted in multiple deleted (LOH) and amplified regions and led to the identification of MTUS1 (8p22) as a candidate tumor suppressor gene in this region. Although the MSI tumors were chromosomally stable, we found several copy neutral LOHs (cnLOH) in the MSI tumors; these appear to be instrumental in the inactivation of the tumor suppressor gene hMLH1 and a gene located in chromosomal region 6pter-p22. DISCUSSION Our results suggest that in addition to classical LOH, cnLOH is an important mutational event in relation to the carcinogenesis of MSS and MSI tumors, causing the inactivation of a tumor suppressor gene without copy number alteration of the respective region; this is crucial for the development of MSI tumors and for some chromosomal regions in MSS tumors.

Inflammation in uremic patients: what is the link?

Uremic patients suffer to an extremely high degree from cardiovascular disease. Cardiovascular disease results mainly from atherosclerotic remodeling of the arterial system. Inflammation is considered to contribute significantly to development of atherosclerosis, and albeit many different factors may lead to inflammation, generation of enhanced oxidative stress is believed to be an important common feature of pro-inflammatory causes. Studies in the general population without renal disease could clearly show that markers of inflammation, in particular C-reactive protein, predict the cardiovascular risk. In this review article, we discuss the presence and the predictive value of inflammation in patients with end-stage renal disease, and analyze whether uremic patients are exposed to specific pro-inflammatory and pro-oxidative conditions. Particular emphasis is set on oxidative stress induced by oxidatively modified lipoproteins and angiotensin II. Based on pathophysiological considerations valid for uremic patients, we discuss therapeutical options that might help to reduce cardiovascular disease in uremic patients.

L-Arginine does not affect renal morphology and cell survival in ischemic acute renal failure in rats.

Background:L-Arginine (L-Arg), a substrate of nitric oxide synthases, improves renal function in ischemic acute renal failure (iARF). We evaluated whether L-Arg improves renal morphology and cell survival in the course of iARF. Methods and Results: iARF was induced in rats by bilateral clamping of renal arteries for 45 min. L-Arg was applied intraperitoneally during clamping, and orally during 14 days of follow-up. Morphology and cell survival of renal cortical and medullar tissue was analyzed on days 1, 3, 7, and 14 of follow-up, using toluidine blue staining and immunohistochemistry of perfusion-fixated tissue, and Western blot analysis of tissue homogenate. Renal tubular injury showed typical features of necrosis and was most severe on days 1 and 3 after clamping, predominantly in S3 segments, with almost complete recovery by day 14. Enhanced medullar monocyte infiltration, determined by ED-1 expression as well as by immunohistochemistry, and enhanced expression of proliferating cell nuclear antigen (PCNA), indicative of proliferation and regeneration, accompanied these morphological changes. Compared to controls, L-Arg had no impact on renal morphology, ED-1, and PCNA expression. Furthermore, expression of markers of apoptosis Bcl-2, Bax, and cleaved caspase-3 was only slightly increased in iARF rats, compared to sham-operated animals, and was also not influenced by L-Arg. Conclusion: Despite its repeatedly reported positive impact on renal function as also shown in our model, L-Arg does not alter cell death and proliferation in the course of iARF in our model. Thus, different mechanisms have to be considered, in particular improved intrarenal hemodynamics.

Microtubule associated tumor suppressor 1 deficient mice develop spontaneous heart hypertrophy and SLE-like lymphoproliferative disease

The microtubule associated tumor suppressor gene 1 (MTUS1) is a recently published tumor suppressor gene, which has also been shown to act as an early component in the growth inhibitory signaling cascade of the angiotensin II type 2 receptor (AT2R). In this study we report the generation of MTUS1 knock-out (KO) mice, which develop normally but reveal higher body weights and slightly decreased blood pressure levels. Twenty-eight percent of the studied MTUS1 KO mice also developed heart hypertrophy and 12% developed nephritis, independent of blood pressure levels. Forty-three percent of the MTUS1 KO mice revealed lymphoid hyperplasia affecting spleen (20%), kidney (37%), lung (23%), lymph nodes (17%), and liver (17%) accompanied with leukocytosis, lymphocytosis, and mild anemia. One animal (3%) developed a marginal zone B-cell lymphoma affecting submandibular salivary gland and regional lymph nodes. The symptoms of all mentioned animals are consistent with a B-cell lymphoproliferative disease with features of systemic lupus erythematosus. In addition, body weight of the MTUS1 KO mice was significantly increased and isolated skin fibroblasts showed increased cell proliferation and decreased cell size, compared to wild-type (WT) fibroblasts in response to depleted FCS concentration and lack of growth factors. In conclusion we herein report the first generation of a MTUS1 KO mouse, developing spontaneous heart hypertrophy and increased cell proliferation, confirming once more the anti-proliferative effect of MTUS1, and a SLE-like lymphoproliferative disease suggesting crucial role in regulation of inflammation. These MTUS1 KO mice can therefore serve as a model for further investigations in cardiovascular disease, autoimmune disease and carcinogenesis.

Oxidative Stress, Lipoproteins and Angiotensin II

Renal fibrosis usually indicates irreversible tissue damage, irrespective of the initial cause. Thus, it is most relevant to understand mechanisms leading to renal fibrosis. Oxidative stress has emerged as an important factor contributing to tissue damage, and oxidative stress is enhanced in a variety of inflammatory disease states relevant for the kidney. It is therefore the purpose of this chapter to discuss the role of oxidative stress in the development of renal fibrosis. Inflammation is generally associated with enhanced oxidative stress, and since multiple factors contribute to inflammation [such as cytokines (e.g., interleukin-6, tumor necrosis factor α), infection, ischemia reperfusion injury, homocysteine, advanced glycation end products, atherogenic lipoproteins, or angiotensin II], multiple factors can cause enhanced oxidative stress. Here we will focus on the role of atherogenic lipoproteins, particularly oxidized low density lipoproteins, and the activated renin angiotensin system, for several reasons: firstly, these factors are well characterized as proinflammatory and as stimulators of superoxide-generating enzymes; secondly, the contribution of these factors to tubulointerstitial fibrosis has frequently been described; and thirdly, we already possess pharmacological tools to efficiently lower their activity. Thus, this chapter highlights the interplay of oxidative stress, atherogenic lipoproteins, and the renin angiotensin system in the pathophysiology of renal fibrosis and discusses potential treatment options.