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. Additionally, specific markers of apoptosis are increasingly important in predicting male infertility. The ability of flow cytometry to estimate the quantity of sperm with DNA fragmentation or damage and multifactor measurements in immotile sperm have made this developed technique essential in fertility centers. The study is aimed at assessing the level of DNA fragmentation and apoptosis by measuring flow cytometry using new techniques. Flow cytometry analysis revealed a varying degree of DNA damage. It was able to quantify the degree of impairment even in samples with minimal DNA fragmentation. DNA damage was observed even in samples that were considered normal with a routine semen analysis. Flow cytometry was sensitive to changes in sperm apoptosis. Elevated p53 activity levels were associated with high DNA fragmentation. Meanwhile, B-cell lymphoma 2 (Bcl-2) activities showed a different pattern. In conclusion, flow cytometry for sperm DNA fragmentation and markers of apoptosis can be a valuable tool in assisted reproductive centers.

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. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

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 8q24.3, which contained the FIR gene locus. Nuclear FIR expression was significantly enriched at the invasion front of primary NSCLCs, but this did not correlate with tumour cell proliferation. FIR accumulation was associated with worse patient survival and tumour recurrence; in addition, FIR over-expression significantly correlated with lymph node metastasis in squamous cell carcinomas (SCCs). In vitro, we applied newly developed methods and modelling approaches for the quantitative and time-resolved description of the pro-migratory and pro-invasive capacities of SCC cells. siRNA-mediated silencing of all FIR variants significantly reduced the speed and directional movement of tumour cells in all phases of migration. Furthermore, sprouting efficiency and single cell invasiveness were diminished following FIR inhibition. Interestingly, the silencing of FIR isoforms lacking exon 2 (FIR(Δexon2)) alone was sufficient to reduce lateral migration and invasion. In summary, by using scale-spanning data derived from primary human tissues, quantitative cellular analyses and mathematical modelling, we have demonstrated that concomitant over-expression of FIR and its splice variants drives NSCLC migration and dissemination.

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. In this study, we tested the effect of HIT and the combination of HIT and the HDACi suberoylanilide hydroxamic acid (SAHA) in a xenograft mouse model. METHODS: Osteosarcoma xenografts were established by subcutaneous injection of KHOS-24OS cells and treated with either vehicle (DMSO), SAHA, HIT or HIT and SAHA. Tumor growth was determined and tumor necrosis, proliferation rate, apoptotic rate as well as vessel density were evaluated. RESULTS: Here, we show that the combination of HIT and SAHA induced a significant delay of tumor growth through increased rate of apoptosis, increased expression of p53 and p21(Waf1/Cip1), inhibition of proliferation and angiogenesis compared to tumors treated with HIT only. CONCLUSION: HIT and in particular the combination of HIT and histone deacetylase inhibition is a promising treatment strategy in OS and may be tested in clinical trials.

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. Treatment of CRC cells with low concentrations of recombinant HMGB1 results in dose-dependent cytotoxicity which is morphologically characterized by the formation of giant mitochondria and does not share features of apoptosis. HMGB1-triggered cell death is associated with intracellular ROS release, and overexpression of Bcl-2 blocks both the increase of ROS as well as HMGB1-dependent cell death. Importantly, treatment with recombinant HMGB1 or overexpression of endogenous HMGB1 strongly sensitizes CRC cells to the cytotoxic activity of the pro-apoptotic death ligand TRAIL as well as the small molecule Bcl-2 family inhibitor ABT­737. Moreover, treatment of CRC cells with TRAIL or ABT­737 induces a release of endogenous HMGB1 into the extracellular space, and preincubation with glycyrrhizin, an HMGB1 inhibitor, significantly inhibits induction of cell death by TRAIL and ABT­737, suggesting that HMGB1 functionally contributes to the execution of cell death triggered by pro-apoptotic agents. Finally, we investigated the expression of HMGB1 in human CRC tumor samples and found that loss of HMGB1 expression is associated with a more aggressive phenotype and a more advanced stage of disease in patients with CRC. Altogether, our findings demonstrate a functional link between cytotoxic signaling cascades triggered by HMGB1 and pro-apoptotic agents leading to an HMGB1-dependent sensitization to CRC cell death. Thus, a further evaluation of recombinant HMGB1 as part of an experimental combination treatment of CRC seems warranted.

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. TIM was overexpressed in a subset of human HCCs both at the mRNA and the protein level. siRNA-mediated knockdown of TIM reduced cell viability due to the induction of apoptosis and G2 arrest. The latter was mediated via CHEK2 phosphorylation. In addition, siRNA-treated cells showed a significantly reduced migratory capacity and reduced expression levels of various proteins. Mechanistically, TIM directly interacts with the eukaryotic elongation factor 1A2 (EEF1A2), which binds to actin filaments to promote tumor cell migration. siRNA-mediated knockdown of TIM reduced EEF1A2 protein levels thereby affecting ribosomal protein biosynthesis. Thus, overexpression of TIM exerts oncogenic function in human HCCs, which is mediated via CHEK2 and EEF1A2.

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. FoxP1 belongs to a family of winged-helix transcription factors that are involved in processes of cellular proliferation, differentiation and neoplastic transformation. METHODS: Microarray expression profiles of 476 neuroblastoma specimens were generated and genes differentially expressed between favorable and unfavorable neuroblastoma were identified. FOXP1 expression was correlated to clinical markers and patient outcome. To determine whether hypermethylation is involved in silencing of FOXP1, methylation analysis of the 5' region of FOXP1 in 47 neuroblastomas was performed. Furthermore, FOXP1 was re-expressed in three neuroblastoma cell lines to study the effect of FOXP1 on growth characteristics of neuroblastoma cells. RESULTS: Low expression of FOXP1 is associated with markers of unfavorable prognosis like stage 4, age >18 months and MYCN amplification and unfavorable gene expression-based classification (P < 0.001 each). Moreover, FOXP1 expression predicts patient outcome accurately and independently from well-established prognostic markers. Array-based CGH analysis of 159 neuroblastomas revealed that heterozygous loss of the FOXP1 locus was a rare event (n = 4), but if present, was associated with low FOXP1 expression. By contrast, DNA methylation analysis in 47 neuroblastomas indicated that hypermethylation is not regularly involved in FOXP1 gene silencing. Re-expression of FoxP1 significantly impaired cell proliferation, viability and colony formation in soft agar. Furthermore, induction of FOXP1 expression led to cell cycle arrest and apoptotic cell death of neuroblastoma cells. CONCLUSIONS: Our results suggest that down-regulation of FOXP1 expression is a common event in high-risk neuroblastoma pathogenesis and may contribute to tumor progression and unfavorable patient outcome.

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. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk 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. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. CONCLUSION: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.

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. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability. CONCLUSIONS: Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas.

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. Furthermore, we investigated the impact of overexpression and small molecule inhibition on Sirt1 in pancreatic cancer cell culture models including combinatorial treatment with chemotherapy and EGFR-inhibition. Cellular events were measured quantitatively in real time and corroborated by conventional readouts including FACS analysis and MTT assays. RESULTS: We detected nuclear Sirt1 expression in 36 (27.9%) of 129 PDACs. SIRT1 expression was significantly higher in poorly differentiated carcinomas. Strong SIRT1 expression was a significant predictor of poor survival both in univariate (p = 0.002) and multivariate (HR 1.65, p = 0.045) analysis. Accordingly, overexpression of Sirt1 led to increased cell viability, while small molecule inhibition led to a growth arrest in pancreatic cancer cells and impaired cell survival. This effect was even more pronounced in combinatorial regimens with gefitinib, but not in combination with gemcitabine. CONCLUSIONS: Sirt1 is an independent prognosticator in PDACs and plays an important role in pancreatic cancer cell growth, which can be levered out by small molecule inhibition. Our data warrant further studies on SIRT1 as a novel chemotherapeutic target in PDAC.

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. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.

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. In addition each tumor subline displays a characteristic cell surface marker profile, which is correlated with the cell cycle phase and the amount of genomic alterations. CONCLUSIONS: In a feasibility study we have shown that cross-reacting antibodies to human cell surface markers stain discrete tumor subpopulations in three sublines of the Dunning tumor model. Although it remains presently uncertain, which cell surface markers are most suitable for cell sorting to display cancer initiating (CIC) properties following subcutaneous or orthotopic grafting, the model may be useful for mechanistic investigations of putative stem-like tumor subpopulations and their significance in response to radio- or chemotherapy.

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. Microvessel density correlated with VEGF mRNA content, but neither of these parameters was associated with the disease outcome. When tumors were ranked according to the level of expression of angiogenic molecules, 9 out of 10 cases with the highest VEGFR1 expression belonged to the recurrence group, while none of the 10 GC with the lowest content of VEGFR1 mRNA had the disease relapse (p = 0.000). VEGFR1 expression did not show even a trend to correlation with the level of cancer tissue vascularization. Immunofluorescent staining by anti-VEGFR1 antibody revealed VEGFR1 expression in tumor cells but not in other cell types. Our data provide indirect support to the evidence for a non-angiogenic contribution of VEGFR1 in cancer pathogenesis.

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. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19(INK4D) overexpression partly restored G(1)-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19(INK4D), but not p16(INK4A), in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G(1)-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G(1) checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G(1)-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.

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. Increased sensitivity to TRAIL was associated with overexpression of death receptors, especially DR5. Re-expression of the GAP related domain of neurofibromin (NF1-GRD) suppressed DR5 expression and decreased sensitivity to TRAIL. We show that death receptor expression and TRAIL sensitivity critically depend on c-MYC and that c-MYC amounts are increased by MEK/ERK and PI3K/AKT signalling pathways which are suppressed by neurofibromin. Furthermore PI3K/AKT signalling strongly suppresses the MYC-antagonist MAD1 which significantly contributes to TRAIL sensitivity. Re-expression of the NF1-GRD decreased c-MYC and increased MAD1 amounts suggesting that neurofibromin influences TRAIL sensitivity at least in part by modulating the MYC/MAX/MAD network. The phytochemical curcumin further increased the sensitivity of neurofibromin deficient MPNST cells to TRAIL. This was presumably mediated by ROS, as it correlated with increased ROS production, was blocked by N-acetylcysteine and mimicked by exogenous ROS.

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. In the present study, we show that conditional p14(ARF) expression substantially suppresses viability, clonogenicity and anchorage-independent growth in p14(ARF)-deficient or MYCN-amplified neuroblastoma cell lines. Furthermore, ectopic 14(ARF) expression induced accumulation of cells in the G1 phase and apoptosis, which was paralleled by accumulation of TP53 and its targets. Comparative genomic hybridization analysis of 193 primary neuroblastomas detected one homozygous deletion of CDKN2A (encoding both p14(ARF) and p16(INK4A)) and heterozygous loss of CDKN2A in 22% of tumors. Co-expression analysis of p14(ARF) and its transactivator, E2F1, in a set of 68 primary tumors revealed only a weak correlation, suggesting that further regulatory mechanisms govern p14(ARF) expression in neuroblastomas. Intriguingly, analyses utilizing chromatin immunoprecipitation revealed different histone mark-defined epigenetic activity states of p14(ARF) in neuroblastoma cell lines that correlated with endogenous p14(ARF) expression but not with episomal p14(ARF) promoter reporter activity, indicating that the native chromatin context serves to epigenetically repress p14(ARF) in neuroblastoma cells. Collectively, the data pinpoint p14(ARF) as a critical factor for efficient TP53 response in neuroblastoma cells and assign p14(ARF) as a neuroblastoma suppressor candidate that is impaired by genomic loss and epigenetic repression.

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. We found that MYCN/MYC-mediated overactivation of the metaphase-anaphase checkpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuroblastoma cells.

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. Human colorectal carcinoma HCT116 and HT29 cells were investigated for CAIX expression using Western Blot analysis. Binding and competition studies of 125I-radiolabeled CaIX-P1 were performed on HCT116 cells in vitro. FACS analysis and fluorescence microscopy studies were carried out after cell incubation with fluorescein-labeled CaIX-P1 and rhodamine-labeled anti-human CAIX-mAb. Our studies revealed an enhanced in vitro expression of carbonic anhydrase IX in HCT116 and HT29 cells with increasing cell density. Binding of 125I-labeled-CaIX-P1 on HCT116 cells increased with increasing cell density and correlated to the CAIX expression. FACS analysis demonstrated a correlation of cell labeling between FITC-CaIX-P1 and rhodamine-labeled anti-CAIX-mAb in both HCT116 and HT29 cells. The results of our study indicate that the phage display identified peptide CaIX-P1 might be an attractive candidate for the development of a ligand targeting CAIX in colorectal cancer.