Flow Cytometry Detection of Sperm DNA Fragmentation and Apoptotic Markers in the Semen of Infertile Males.

The effect of sperm molecular defects on fertilization and pregnancy outcome after assisted reproductive therapy (ART) is widely documented by both research and clinical societies. Sperm DNA fragmentation and abnormal chromatin condensation represent critical causes of male infertility. Advanced androgenic techniques for accurately identifying molecular defects help in selecting an appropriate treatment strategy.

NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring.

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described.

Histone deacetylase inhibition sensitizes osteosarcoma to heavy ion radiotherapy.

Background: Minimal improvements in treatment or survival of patients with osteosarcoma have been achieved during the last three decades. Especially in the case of incomplete tumor resection, prognosis remains poor. Heavy ion radiotherapy (HIT) and modern anticancer drugs like histone deacetylase inhibitors (HDACi) have shown promising effects in osteosarcoma in vitro.

Concomitant expression of far upstream element (FUSE) binding protein (FBP) interacting repressor (FIR) and its splice variants induce migration and invasion of non-small cell lung cancer (NSCLC) cells.

Transcription factors integrate a variety of oncogenic input information, facilitate tumour growth and cell dissemination, and therefore represent promising therapeutic target structures. Because over-expression of DNA-interacting far upstream element binding protein (FBP) supports non-small cell lung cancer (NSCLC) migration, we asked whether its repressor, FBP-interacting repressor (FIR) is functionally inactivated and how FIR might affect NSCLC cell biology. Different FIR splice variants were highly expressed in the majority of NSCLCs, with the highest levels in tumours carrying genomic gains of chromosome 8q24.

The HMGB1 protein sensitizes colon carcinoma cells to cell death triggered by pro-apoptotic agents.

The HMGB1 protein has multiple functions in tumor biology and can act both as a transcription factor and as a cytokine. HMGB1 is released during cell death, and in our previous studies we demonstrated that HMGB1 induces a distinct, necrosis-like cell death in glioblastoma. In epithelial malignant tumors such as colorectal cancer (CRC), the HMGB1-dependent effects show cross-talk with apoptotic signal transduction.

FOXP1 inhibits cell growth and attenuates tumorigenicity of neuroblastoma.

Background: Segmental genomic copy number alterations, such as loss of 11q or 3p and gain of 17q, are well established markers of poor outcome in neuroblastoma, and have been suggested to comprise tumor suppressor genes or oncogenes, respectively. The gene forkhead box P1 (FOXP1) maps to chromosome 3p14.1, a tumor suppressor locus deleted in many human cancers including neuroblastoma.

Protumorigenic role of Timeless in hepatocellular carcinoma.

The mammalian timeless (TIM) protein interacts with proteins of the endogenous clock and essentially contributes to the circadian rhythm. In addition, TIM is involved in maintenance of chromosome integrity, growth control and development. Thus, we hypothesized that TIM may exert a potential protumorigenic function in human hepatocarcinogenesis.

p19-INK4d inhibits neuroblastoma cell growth, induces differentiation and is hypermethylated and downregulated in MYCN-amplified neuroblastomas.

Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas.

Prosurvival function of the cellular apoptosis susceptibility/importin-α1 transport cycle is repressed by p53 in liver cancer.

Unlabelled: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC.

Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas.

Background: High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear.

Results: We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines.

High SIRT1 expression is a negative prognosticator in pancreatic ductal adenocarcinoma.

Background: Several lines of evidence indicate that Sirt1, a class III histone deacetylase (HDAC) is implicated in the initiation and progression of malignancies and thus gained attraction as druggable target. Since data on the role of Sirt1 in pancreatic ductal adenocarcinoma (PDAC) are sparse, we investigated the expression profile and prognostic significance of Sirt1 in vivo as well as cellular effects of Sirt1 inhibition in vitro.

Methods: Sirt1 expression was analyzed by immunohistochemistry in a large cohort of PDACs and correlated with clinicopathological and survival data.

Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.

Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes.

Flow cytometric characterization of tumor subpopulations in three sublines of the Dunning R3327 rat prostate tumor model.

Background: Subsets of tumor cells were characterized by mapping DNA ploidy patterns in correlation with established cell surface markers in three non-treated sublines of the Dunning R3327 prostate tumor system representing different progressional stages.

Methods: Flow cytometry was used to analyze DNA-index, cell cycle distribution as well as multiparametric aquisition of single and combined cell surface markers in single cell suspensions of frozen tumor tissues.

Results: The three Dunning prostate tumor sublines clearly differ in their ploidy status.

Evidence for angiogenesis-independent contribution of VEGFR1 (FLT1) in gastric cancer recurrence.

Angiogenesis plays an important role in cancer progression and involves activation of multiple signaling cascades. This study investigated the relationships between microvessel density, expression of VEGF and VEGFR1 (FLT1), and gastric cancer (GC) recurrence. Twenty-nine surgically treated GC cases with similar initial clinical presentation were selected for the study; 11 of these cases recurred within 3 years, while the remaining 18 did not.

CDK4 inhibition restores G(1)-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage.

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G(1)-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations.

Sensitivity of malignant peripheral nerve sheath tumor cells to TRAIL is augmented by loss of NF1 through modulation of MYC/MAD and is potentiated by curcumin through induction of ROS.

Malignant peripheral nerve sheath tumor (MPNST) is a rare aggressive form of sarcoma often associated with the tumor syndrome neurofibromatosis type 1 (NF1). We investigated the effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on NF1 associated MPNST and determinants of TRAIL sensitivity. MPNST cell lines with complete neurofibromin deficiency were sensitive to apoptotic cell death induced by TRAIL whereas MPNST cells with retained neurofibromin expression or normal human Schwann cells were resistant.

Low p14ARF expression in neuroblastoma cells is associated with repressed histone mark status, and enforced expression induces growth arrest and apoptosis.

The TP53 tumor suppressor pathway is abrogated by TP53 mutations in the majority of human cancers. Increased levels of wild-type TP53 in aggressive neuroblastomas appear paradox but are tolerated by tumor cells due to co-activation of the TP53 ubiquitin ligase, MDM2. The role of the MDM2 antagonist, p14(ARF), in controlling the TP53-MDM2 balance in neuroblastoma is unresolved.

Binding of the phage display derived peptide CaIX-P1 on human colorectal carcinoma cells correlates with the expression of carbonic anhydrase IX.

Phage display represents an attractive screening strategy for the identification of novel, specific binding ligands that could be used for tumor targeting. Recently, a new peptide (CaIX-P1) with affinity for human carbonic anhydrase IX (CAIX) was identified and evaluated. The aim of the present study is to characterize the properties of CaIX-P1 for targeting human colorectal carcinoma and investigate the correlation of peptide binding with the expression of carbonic anhydrase IX.

MYCN-mediated overexpression of mitotic spindle regulatory genes and loss of p53-p21 function jointly support the survival of tetraploid neuroblastoma cells.

High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN, and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remain unclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, especially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using a functional RNAi screening approach and live-cell imaging, we identified a group of genes, including MAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutated neuroblastoma cells.

Methylome analysis and integrative profiling of human HCCs identify novel protumorigenic factors.

Unlabelled: To identify new tumor-suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome-wide methylation profiling and vertical integration with array-based comparative genomic hybridization (aCGH), as well as expression data from a cohort of well-characterized human hepatocellular carcinomas (HCCs). Bisulfite-converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy-number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation.

In vivo efficacy of the histone deacetylase inhibitor suberoylanilide hydroxamic acid in combination with radiotherapy in a malignant rhabdoid tumor mouse model.

Purpose: Histone deacetylase inhibitors are promising new substances in cancer therapy and have also been shown to sensitize different tumor cells to irradiation (XRT). We explored the effect as well as the radiosensitizing properties of suberoylanilide hydroxamic acid (SAHA) in vivo in a malignant rhabdoid tumor (MRT) mouse model.

Methods And Material: Potential radiosensitization by SAHA was assessed in MRT xenografts by analysis of tumor growth delay, necrosis (HE), apoptosis (TUNEL), proliferation (ki-67) and γH2AX expression as well as dynamic 18F-Fluorodeoxyglucose Positron Emission Tomography (18F-FDG -PET) after treatment with either SAHA alone, single-dose (10 Gy) or fractionated XRT (3 × 3Gy) solely as well as in combination with SAHA compared to controls.

Theranostic cRGD-BioShuttle Constructs Containing Temozolomide- and Cy7 For NIR-Imaging and Therapy.

Innovative and personalized therapeutic approaches result from the identification and control of individual aberrantly expressed genes at the transcriptional and post-transcriptional level. Therefore, it is of high interest to establish diagnostic, therapeutic and theranostic strategies at these levels. In the present study, we used the Diels-Alder Reaction with inverse electron demand (DAR(inv)) click chemistry to prepare a series of cyclic RGD-BioShuttle constructs.

Expression and therapeutic relevance of heat-shock protein 90 in pancreatic endocrine tumors.

Pancreatic endocrine tumors (PET) represent a heterogenous group of neoplasms. Although surgical resection is considered a safe and effective treatment for many PET, therapeutic options for inoperable and progressive PET are limited. The expression of heat-shock protein (HSP) 90 was investigated in 120 clinically and pathomorphologically well-characterized PET from 84 patients using immunohistochemistry.

Down-regulation of CXCL1 inhibits tumor growth in colorectal liver metastasis.

As part of ongoing studies to obtain a global picture of invasion related events in colorectal liver metastases, here, we report our findings on gene expression of the pro-angiogenic subgroup of chemokines, the CXCL-ELR+ chemokines. Apart from their pro-angiogenic and chemoattractant function, these chemokines appear to also contribute to tumor cell transformation, growth and invasion. In our nude mouse model of colorectal liver metastases, we found CXCL1,2,3,5 and 8 (IL-8) to be up-regulated in the tumor cells of the invasion front as compared to the tumor cells in the inner parts of the tumor.