Chemotherapy-associated angiogenesis in neuroblastoma tumors

The influences of cytotoxic drugs on endothelial cells remain incompletely understood. Herein, we examined the effects of chemotherapeutic agents in experimental angiogenesis models and analyzed vessel densities in clinical neuroblastoma tumor samples. Cisplatin (20 to 500 ng/mL), doxorubicin (4 to 100 ng/mL), and vincristine (0.5 to 4 ng/mL), drugs commonly involved in neuroblastoma therapy protocols, induced pro-angiogenic effects in different angiogenesis models. They enhanced endothelial cell tube formation, endothelial cell sprouting from spheroids, formation of tip cells in the sprouting assay, expression of αvβ3 integrin, and vitronectin binding. All three drugs increased global cellular kinase phosphorylation levels, including the angiogenesis-relevant molecules protein kinase Cβ and Akt. Pharmacological inhibition of protein kinase Cβ or Akt upstream of phosphatidylinositol 3-kinase reduced chemotherapy-induced endothelial cell tube formation. Moreover, the investigated chemotherapeutics dose dependently induced vessel formation in the chick chorioallantoic membrane assay. Tumor samples from seven high-risk patients with neuroblastoma were analyzed for vessel density by IHC. Results revealed that neuroblastoma samples taken after chemotherapy consistently showed an enhanced microvessel density compared with the corresponding samples taken before chemotherapy. In conclusion, our data show that chemotherapy can activate endothelial cells by inducing multiple pro-angiogenic signaling pathways and exert pro-angiogenic effects in vitro and in vivo. Moreover, we report a previously unrecognized clinical phenomenon that might, in part, be explained by our experimental observations: chemotherapy-associated enhanced vessel formation in tumors from patients with neuroblastoma.     

Psychiatric disease as a risk factor in fast-track hip and knee replacement: An overview of the literature

Recent studies suggest that patients with psychiatric disorders tend to do worse than patients without a psychiatric diagnosis when undergoing total hip arthroplasty (THA) or total knee arthroplasty (TKA). Whether this is due to their psychiatric condition, pharmacological treatment, a combination of the two, or something else has not been thoroughly analyzed—and there are no internationally accepted guidelines for perioperative management of psychiatric patients. This overview summarizes our current knowledge on perioperative risks in patients with preoperative psychiatric disorders and the possible role of psychotropic drugs in the perioperative course. This will be useful when planning future strategies for improvement of surgical outcome following hip and knee arthroplasty.     

Functional role of inositol-1,4,5-trisphosphate-3-kinase-A for motility of malignant transformed cells

Cell migration is one of the hallmarks of metastatic disease and thus identification of migration promoting proteins is crucial for the understanding of metastasis formation. Here we show that the neuron-specific, F-actin bundling inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) is ectopically expressed in tumor cells and critically involved in migration. Down-regulation of ITPKA expression in transformed cell-lines with ectopic expression of ITPKA significantly decreased migration and the number of linear and branched cell protrusion. Conversely, up-regulation of ITPKA in tumor cell lines with low endogenous ITPKA expression increased migration and formation of cell processes. In vitro, ITPKA alone induced the formation of linear actin filaments, whereas ITPKA mediated formation of branched protrusions seems to result from interaction between ITPKA and the F-actin cross-linking protein filamin C. Based on these actin-modulating and migration-promoting effects of ITPKA we examined its expression in clinical samples of different tumor entities, starting with the analysis of multiple tumor tissue arrays. As in lung adenocarcinoma specimens, the highest ITPKA expression rate was found, this tumor entity was examined in more detail. ITPKA was expressed early in adenocarcinoma progression (pN0) and was largely maintained in invasive and metastatic tumor cell populations (pN1/2, lymph node metastases). Together with our result that high expression of ITPKA increases motility of tumor cells we conclude that the observed expression of ITPKA early in tumor development increases the metastatic potential of lung adenocarcinoma cells. Therefore, we suggest that ITPKA may be a promising therapeutic molecular target for anti metastatic therapy of lung cancer.     

Zerebrale lymphomatoide Granulomatose

We present the case of a 57-year-old patient who was admitted with fever and disorientation. The cerebrospinal fluid showed a mild pleocytosis and increased protein content. MR imaging revealed multiple lesions, particularly in the subcortical white matter, with spot like central contrast enhancement. The diagnosis of lymphomatoid granulomatosis was finally made through open lung biopsy. Despite treatment with rituximab and, in the later course of the disease, additionally with cyclophosphamide, the patient died 3 months after the diagnosis was made.     

Opposite roles of FOXA1 and NKX2-1 in lung cancer progression

Gene copy number profiles of primary lung tumors were screened for high-level amplifications. We detected 22 high-level amplifications in various loci, including 14q13. This locus is known to harbor the adenocarcinoma (AC) lineage-specific target gene NKX2-1, which is not expressed in squamous cell carcinoma (SCC). As the 14q amplification was also found in SCC, we investigated whether or not FOXA1 might be the corresponding target gene for SCC. Focusing on these two target genes, we assessed gene amplifications and protein expression of NKX2-1 and FOXA1 in primary lung tumors (n = 554) and brain metastases (n = 68). Primary AC (n = 194) showed positive protein expression of NKX2-1 in 58.2% of the samples compared with 4.2% of primary SCC samples (n = 212). Positive staining for FOXA1 was seen in 34.7% of the SCC samples, which was comparable with 39.6% in the AC samples. For brain metastases, FOXA1 expression was slightly higher in the SCC samples (55.6%) compared with the non-matched primary SCC tumor samples (43.4%), whereas NKX2-1 expression was comparable in both primary tumors and brain metastases. Positive FOXA1 and NKX2-1 expression was associated with a gain or amplification in 34.6% and 28.6% of cases, respectively. The expression of NKX2-1 was associated with early stage and grade among the AC cases. In contrast, FOXA1 expression in SCC was associated with distant metastases as well as an unfavorable survival rate (P = 0.039). These results suggest that both FOXA1 and NKX2-1 may act as lineage-specific target genes within the 14q amplicon with opposite functions in lung cancer.     

Pattern of MUC6 expression across 119 different tumor types: A tissue microarray study on 15 412 tumors

Mucin 6 (MUC6) is a secreted gel-forming mucin covering the surfaces of gastrointestinal and other tissues. Published work demonstrates that MUC6 can also be expressed in several cancer types and can aid in the distinction of different tumor entities. To systematically analyze MUC6 expression in normal and cancerous tissues, a tissue microarray containing 15 412 samples from 119 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. At least a weak MUC6 positivity was seen in 50 of 119 (42%) tumor entities. Thirty-three tumor entities included tumors with strong positivity. MUC6 immunostaining was most frequent in mucinous carcinomas of the breast (44%), adenocarcinomas of the stomach (30%–40%) and esophagus (35%), and neuroendocrine carcinomas of the colon. Strong MUC6 staining was linked to advanced pT stage (p = 0.0464), defective mismatch repair status and right-sided tumor location (p < 0.0001 each) in colorectal cancer, as well as to high tumor grade (p = 0.0291), nodal metastasis (p = 0.0485), erb-b2 receptor tyrosine kinase 2 positivity (p < 0.0001) and negative estrogen receptor (p = 0.0332)/progesterone receptor (p = 0.0257) status in breast carcinomas of no special type. The broad range of tumor types with MUC6 expression limits the utility of MUC6 immunohistochemistry for the distinction of different tumor types.     

The zona pellucida 'receptors' ZP1, ZP2 and ZP3.

The male component that is necessary for successful reproduction depends on a large variety of biological processes working in concert. The sperm-egg interaction occurs through complementary molecules and is an obligatory process for successful fertilization. However, this complex phenomenon and its molecular mechanisms remain to be fully understood. The oocyte is protected by the zona pellucida, a network of various proteins which encloses the oocyte. Depending on the species, the zona pellucida consists of different glycoproteins that are proposed to function as 'receptors' for spermatozoa. In the mouse, ZP1 is the homodimeric filament crosslinker, held together by intermolecular disulphides. ZP2 is the 'secondary receptor', which is cleaved by egg proteases after egg activation. The mouse ZP3 protein appears to be the 'primary receptor', which is responsible for species-specific binding of spermatozoa to the oocyte and the induction of the acrosome reaction. To localize zona pellucida protein and to evaluate the function of ZP2 and ZP3, polyclonal antisera were raised against synthetic ZP2 or ZP3 peptides which are specific for human or for mouse zona pellucida proteins. It could be demonstrated that anti-synthetic peptide antisera detected their respective zona pellucida proteins in immunoblots, ovary sections and native hemizonae pellucidae. Functional assays with anti-ZP3 synthetic peptide antibodies revealed that the antisera did not inhibit sperm-zona pellucida binding, whereas one of the antisera against synthetic ZP2 peptides significantly inhibited binding of spermatozoa to the zona pellucida.     

人卵透明带蛋白huZP3a~(22～176)和huZP3b~(177～348)肽段在大肠杆菌中的表达及其纯化

目的:探索将huZP3a22~348蛋白编码基因拆分成两段,分别表达的可行性,并研究表达产物的免疫原性。方法:用PCR技术, 将去N-端信号肽和C-端跨膜区的huZP322~348蛋白编码基因拆成大小相近的两段, 以完整阅读框的形式分别重组插入热诱导型pBV221的多克隆区。结果:大肠杆菌分别特异地表达了可单独或复合应用的huZP3a22~176和huZP3b177~348,在经过抗人ZP3不同抗原区合成肽抗体的蛋白印迹鉴定后, 用改良的制备性PAGE方法分离纯化这两种表达产物。同时用兔抗猪ZP IgGs蛋白印迹试验表明, huZP3a和pZP3b的几个共同线性抗原表位都存在于其肽链的前半区域。结论:通过基因重组技术可获得足够量的huZP3a和huZP3b,这为开展huZP3a和huZP3b的免疫原性以及huZP3诱发人精子顶体胞吐的功能域等研究奠定了基础。     

G-protein alpha-subunits in cytosolic and membranous fractions of human neutrophils

In plasma membranes of human neutrophils, we identified two major pertussis toxin substrates of 40 kDa Mr with pI values of 5.30 and 5.37. Only the acidic of the two substrates was also present in neutrophil cytosol. Two-dimensional tryptic peptide maps revealed a high degree of homology of cytosolic and particulate substrates. Purified G-protein beta gamma-complex stimulated pertussis toxin-catalyzed [32P]ADP-ribosylation of membranous and cytosolic substrates of neutrophils less than 2-fold and 6-fold, respectively. Hydrodynamic properties of the cytosolic substrate strongly suggested that it exists as a monomer. Purified G-protein beta gamma-complex increased the s20,w value of the cytosolic substrate from 3.3 S to 4.0 S. The GTP analogue, guanosine 5'-O-(3-thiotriphosphate), promoted the release of pertussis toxin substrates from plasma membranes. An antiserum raised against a sequence specific for the Gi2 alpha-subunit reacted with 39-40 kDa proteins in plasma membranes and with an apparently single 40 kDa protein in cytosol. We conclude that neutrophil cytosol contains monomeric Gi2 alpha-subunits which--by interacting with hydrophobic beta gamma-complexes--may reversibly bind to the plasma membrane.