Recombinant adenovirus expressing wild-type p53 is antiangiogenic: a proposed mechanism for bystander effect.
Angiogenesis is required for the growth and progression of malignancies. Recent studies have demonstrated that genetic alterations may accompany acquisition of the angiogenic phenotype. The tumor suppressor gene p53 is most frequently mutated in human cancers and is also known to be a transcriptional regulator of a variety of genes. Here, we investigated the antiangiogenic effect of the wild-type p53 (wt-p53) gene transfer on a human non-small cell lung cancer cell line. Mutant p53-expressing H226Br non-small cell lung cancer cells were transduced with the wt-p53 gene using a recombinant adenoviral vector (Ad5CMVp53) and applied to semiquantitative reverse transcription-PCRs for the detection of altered mRNA expression of angiogenic and/or antiangiogenic factors. In vivo neovascularization assay of Ad5CMVp53-infected cells was then performed using a membrane-diffusion chamber system s.c. transplanted in nu/nu mice. We also evaluated the effect of Ad5CMVp53-infected H226Br cells on nontransduced tumor cells in vivo by s.c. inoculating mixture of cells into nu/nu mice. Ad5CMVp53 infection markedly inhibited the expression of an angiogenic factor, vascular endothelial growth factor, and increased the expression of a novel antiangiogenic factor, brain-specific angiogenesis inhibitor 1, resulting in reduced neovascularization in vivo. Mixing experiments showed that tumor cells transduced with the wt-p53 gene inhibited the in vivo tumor growth of adjacent nontransduced cells. Our data suggest that a recombinant adenovirus expressing the wt-p53 gene is antiangiogenic, which may explain, in part, the mechanism of the bystander effect induced by the wt-p53 gene transfer on adjacent tumor cells. (+info
Inhibition of angiogenesis and tumour growth by VEGF121-toxin conjugate: differential effect on proliferating endothelial cells.
Vascular endothelial growth factor (VEGF) plays an important role in tumour angiogenesis. VEGF binds to tyrosine kinase receptors, which are expressed almost exclusively on tumour endothelium. Therefore, VEGF can be used to target toxin molecules to tumour vessels for anti-angiogenic therapy. However, recent evidence suggests that VEGF can also bind in an isoform-specific fashion to a newly identified neuropilin-1 (NP-1) receptor. NP-1 is widely expressed in normal tissue and presents a potential target for unwanted toxicity. As a consequence, we investigated whether the VEGF121 isoform, which lacks the NP-1 binding domain, could be used to target toxin polypeptides to tumour vasculature. Treatment of endothelial cells with a VEGF121-diphtheria toxin (DT385) conjugate selectively inhibited proliferating endothelial cells, whereas confluent cultures were completely resistant to the construct. In addition, VEGF121-DT385 conjugate treatment completely prevented tumour cell induced angiogenesis in vivo. Most importantly, the conjugate inhibited tumour growth in athymic mice and induced tumour-specific vascular damage. There was also no apparent toxicity associated with the treatment. Our results suggest that proliferating endothelial cells are highly sensitive to VEGF121-toxin conjugates and that the binding to NP-1 receptors is not necessary for efficient inhibition of tumour growth. (+info
Significant correlation between interleukin 10 expression and vascularization through angiopoietin/TIE2 networks in non-small cell lung cancer.
The expression of interleukin 10 (IL-10) is correlated with clinical prognosis in non-small cell lung cancer [NSCLC (H. Hatanaka et al., ANN: ONCOL:, 11: 815--819, 2000)]. However, the effects of IL-10 expression on vascularization in NSCLC are not apparent. We examined the gene expression of IL-10/IL-10 receptor and various angiogenic/angioinhibitory factors in 95 NSCLC samples to determine the correlation between IL-10 production and vascularization. Vascular endothelial growth factor, angiopoietin [Ang (Ang-1 and Ang-2)], thrombospondin, brain-specific angiogenesis inhibitor 1, vascular endothelial growth factor receptors (KDR and flt-1), and Ang receptor (TIE2) gene expression were evaluated by reverse transcription-PCR. The cellular localization of these factors and vascularity in the cancer stroma were examined immunohistochemically. Seventy-eight (82.1%) and 93 (97.9%) of these 95 NSCLCs were positive for IL-10 and IL-10 receptor, respectively. Ang-1, Ang-2, and TIE2 gene expression was seen in 76 (97.4%), 73 (93.6%), and 78 (100%) of 78 IL-10-positive NSCLCs, respectively, and was significantly correlated with IL-10 gene expression (P < 0.0088, <0.0008, and 0.0305, respectively; Fisher's exact method). The localizations of Ang-1, Ang-2, and TIE2 were confirmed within tumor cells immunohistochemically. Vascular number and measurement area were significantly higher in the IL-10-positive NSCLCs (33.500 +/- 9.299/microm(2) and 4.742 +/- 1.287%) as compared with IL-10-negative NSCLCs (10.611 +/- 2.839/microm(2) and 0.718 +/- 0.331%; Mann-Whitney U test, P = 0.0039). The IL-10 expression did not show any significant correlation with the expression of other factors. These results suggested that tumor-produced IL-10 promotes stromal vascularization through expression of Ang-1, Ang-2, and TIE2. (+info
Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis.
The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 "functional" binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules. (+info
Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and synapse-associated protein 102.
PDZ domains typically interact with the very carboxyl terminus of their binding partners. Type 1 PDZ domains usually require valine, leucine, or isoleucine at the very COOH-terminal (P(0)) position, and serine or threonine 2 residues upstream at P(-2). We quantitatively defined the contributions of carboxyl-terminal residues to binding selectivity of the prototypic interactions of the PDZ domains of postsynaptic density protein 95 (PSD-95) and its homolog synapse-associated protein 90 (SAP102) with the NR2b subunit of the N-methyl-d-aspartate-type glutamate receptor. Our studies indicate that all of the last five residues of NR2b contribute to the binding selectivity. Prominent were a requirement for glutamate or glutamine at P(-3) and for valine at P(0) for high affinity binding and a preference for threonine over serine at P(-2), in the context of the last 11 residues of the NR2b COOH terminus. This analysis predicts a COOH-terminal (E/Q)(S/T)XV consensus sequence for the strongest binding to the first two PDZ domains of PSD-95 and SAP102. A search of the human genome sequences for proteins with a COOH-terminal (E/Q)(S/T)XV motif yielded 50 proteins, many of which have not been previously identified as PSD-95 or SAP102 binding partners. Two of these proteins, brain-specific angiogenesis inhibitor 1 and protein kinase Calpha, co-immunoprecipitated with PSD-95 and SAP102 from rat brain extracts. (+info
Brain angiogenesis inhibitor 1 is differentially expressed in normal brain and glioblastoma independently of p53 expression.
Brain angiogenesis inhibitors (BAI) are putative transmembrane proteins containing an extracellular domain with thrombospondin type-1 repeats which can exhibit anti-angiogenic activity. BAI1 mRNA is expressed mainly in the brain, while BAI2 and BAI3 mRNAs are more widely expressed. We hypothesized that the BAI family might have anti-tumoral properties and studied the expression of BAI1 protein in normal human brain and in glioblastoma multiforme. We generated an anti-BAI1 antibody and showed that BAI1 was widely expressed in normal brain but was absent in 28 glioma cell lines and in the majority of human glioblastoma investigated. BAI1 expression did not correlate with TP53 status and we did not confirm previous findings that p53 regulates BAI1 mRNA expression in glioma cells. The finding that expression of BAI proteins may be lost during tumor formation is of special interest as restoration of their function in tumors may be of therapeutic benefit. (+info
Vascular endothelial growth factor principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis.
Vascular endothelial growth factor (VEGF)-mediated angiogenesis is essential for bone formation. However, the effect of VEGF on osteoblastic cells during osteoblastogenesis is still controversial. The aim of this study was to clarify the relationship between osteoblastic cells derived from human mesenchymal stem cells (MSCs) and VEGF in the early stage of osteoblastic differentiation. Continuous dexamethasone treatment with a low concentration stimulated osteoblastogenesis of MSCs and the expression of VEGF121 mRNA. The VEGF secretion from osteoblastic cells also increased along with osteoblastogenesis. Neuropilin-1, which mainly binds VEGF165, was detected at all stages during early osteoblastogenesis, but VEGF receptor-1 and -2 were not detected on RT-PCR analyses. In this study, VEGF had no direct effect on the proliferation of osteoblastic cells. However, the secreted VEGF in the conditioned medium of osteoblastic cells exhibited high angiogenic power as to endothelial cell proliferation. Our findings indicated that VEGF121 principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis. The present study also demonstrated the differential expression of VEGF121 during osteoblastogenesis. The increase of VEGF in the early stage might be a useful marker of induction of bone formation due to human MSCs. (+info
Circulating proangiogenic cytokines and angiogenesis inhibitor endostatin in untreated patients with chronic lymphocytic leukemia.
The serum concentration of two pro-angiogenic cytokines: basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1), and anti-angiogenic factor endostatin in the serum of 80 never treated B-cell chronic lymphocytic leukemia (CLL) patients and 27 healthy volunteers was measured using an enzyme linked immunosorbent assay. The serum levels of both bFGF and TGF-beta1 were found to be significantly higher in the CLL group (median 40.5 pg/ml and 38.6 ng/ml respectively) when compared to the control group (median 9.4 pg/ml and 18.9 ng/ml, respectively) (p<0.001). The levels of endostatin were not significantly different in CLL and control groups (median 12.3 ng/ml and 8.4 ng/ml, respectively) (p=0.09). In the group of CLL patients the level of bFGF was significantly higher in patients with progressive disease as compared with patients with stable disease (median 90.5 pg/ml and 40.5 pg/ml respectively) (p<0.001). Patients in Rai stage III and IV also had significantly higher levels of bFGF than patients in Rai stage 0-II (median 100.1 pg/ml and 29.3 pg/ml respectively) (p<0.001). The levels of both TGF-beta1 and endostatin were lower in patients in Rai stage III and IV (median 28.9 ng/ml and 9.1 ng/ml respectively) than in patients in Rai stage 0-II (42.8 ng/ml and 13.1 ng/ml respectively) (p<0.001 and p=0.002 respectively). The level of endostatin was also lower in the group of CLL patients with progressive disease (median 10.0 ng/ml) as compared to patients with stable disease (median 20.5 ng/ml) (p=0.008). In conclusion, the disturbance in the balance between pro- and anti-angiogenic factors may have an important influence on the course of CLL. (+info