Adenoviral mediated interferon-alpha 2b gene therapy suppresses the pro-angiogenic effect of vascular endothelial growth factor in superficial bladder cancer.

PURPOSE: Intravesical adenovirus mediated interferon-alpha gene transfer has a potent therapeutic effect against superficial human bladder carcinoma xenografts growing in the bladder of athymic nude mice. We determined whether the inhibition of angiogenesis might contribute to the antitumor effect. MATERIALS AND METHODS: We treated several human urothelial carcinoma cells with adenovirus mediated interferon-alpha 2b and monitored its effects on the production of angiogenic factors using real-time reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemical analysis and a gel shift based transcription factor array. To assess the role of adenovirus mediated interferon 2b in angiogenic activity we used in vitro invasion assays and evaluated the anti-angiogenic effects of adenovirus mediated interferon gene therapy in an orthotopic murine model of human superficial bladder cancer. RESULTS: In adenovirus mediated interferon-alpha infected 253J B-V cells vascular endothelial growth factor was decreased and anti-angiogenic interferon-gamma inducible protein 10 was up-regulated. In contrast, the addition of as much as 100,000 IU recombinant interferon had no apparent effect on vascular endothelial growth factor production. Conditioned medium derived from adenovirus mediated interferon 2b infected 253J B-V cells greatly decreased the invasive potential of human endothelial cells and down-regulated their matrix metalloproteinase 2 expression compared to controls. Furthermore, adenovirus mediated interferon 2b blocked pro-angiogenic nuclear signals, such as the transcription factors activating protein-1 and 2, stimulating protein-1, nuclear factor kappaB and c-myb. In vivo experiments revealed significant vascular endothelial growth factor down-regulation and decreased tumor vessel density in the adenovirus mediated interferon 2b treated group compared to controls. CONCLUSIONS: Treatment with adenovirus mediated interferon 2b increases the angiostatic activity of the bladder cancer microenvironment. This inhibition may prove beneficial for treating superficial bladder cancer with adenovirus mediated interferon-alpha and hopefully contribute to a decreased recurrence rate of this neoplasm.

Cyclophilin A is required for M-CSF-dependent macrophage proliferation.

The immunosuppressor sanglifehrin A (SfA) is a member of a family of immunophilin cyclophilin A-binding molecules and does not inhibit calcineurin activity. Sanglifehrin A inhibits M-CSF-dependent macrophage proliferation by arresting the G1 phase of the cell cycle but does not affect cell viability. This immunosuppressor exerts its action on proliferation by inactivating cyclin-dependent kinase 2 (Cdk2) activity. Moreover, c-myc expression is also repressed. In the early steps of M-CSF signaling, SfA inhibits the phosphorylation of Raf-1 and the external regulated kinases (ERK)1/2 and mitogen-activated protein kinase phosphatase-1, which are required for proliferation. The effects of SfA are not related to a block of the proteosome activity. These data show that immunophilin contributes to M-CSF-dependent proliferation through activation of the Raf-1/MEK/ERK pathway and the regulation of Cdk activities, which is required for cell cycle progression.

Different roles of p38 MAPK and ERK in STI571-induced multi-lineage differentiation of K562 cells.

STI571 is a specific tyrosine kinase inhibitor of Abl kinase. It was previously reported that STI571 induced hemoglobin synthesis in the chronic myelogenous leukemia (CML) cell line K562. However, its mechanisms remain unknown. In this study, we demonstrated that STI571 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and dephosphorylation of extracellular signal-regulated kinase (ERK) in K562 cells. In contrast, the phosphorylation of c-Jun N-terminal kinases (JNK) in K562 cells was not altered by STI571. We also found that STI571 induced all the myeloid (CD11b, CD13), megakaryocytic (CD41a, CD42), and erythroid (glycophorin-A) markers on K562 cells. A p38 MAPK-specific inhibitor, SB203580, inhibited the STI571-induced multi-lineage differentiation of K562 cells, indicating that p38 MAPK is crucial for this differentiation. In contrast, SB203580 did not overcome the inhibitory effect for proliferation of K562 cells, indicating that p38 MAPK activation by STI571 does not affect cell numbers. Among the hematopoietic transcription factors, the expression level of c-myb mRNA was clearly downregulated after incubation with STI571 in K562 cells. STI571-induced downregulation of c-myb mRNA was prevented by the pretreatment of K562 cells by SB203580. Our data provides insights into how p38 MAPK and ERK pathways are involved in STI571-induced differentiation of K562 cells.

Antisense oligodeoxynucleotide directed against c-myb has anticancer activity and potentiates the antiproliferative effect of conventional anticancer drugs acting by different mechanisms in human colorectal cancer cells.

The C-MYB proto-oncogene encodes a DNA-binding protein with transactivation properties that plays an important regulatory role in cell proliferation and differentiation. Overexpression of C-MYB in colonic tumors compared to normal mucosa suggests that c-myb may play a role in the malignant transformation of colonic mucosa and that inhibition of c-myb expression may suppress, to some extent, the proliferation of neoplastic cells. Complete suppression of tumor cell proliferation may require inhibition of multiple growth-promoting genes. Alternatively, the combination of oncogene-targeted oligodeoxynucleotides (ODNs) with standard cytotoxic agents might represent a useful therapeutic approach to improving cancer treatment. In the present study, we have investigated whether the inhibition of a growth-promoting gene, namely c-myb, affects the sensitivity of human colorectal cancer cells, in vitro, to conventional chemotherapeutic drugs: taxol, 5-fluorouracil, vinblastine and doxorubicin. We show that c-myb antisense phosphorothioate (S) ODN treatment induces growth arrest in the G(1)/G(2) phases of the cell cycle and inhibits cell proliferation in a dose- and time-dependent manner. Also, treatment with c-myb antisense (S)ODN decreases c-myb mRNA and protein expression. A greater inhibition of cell proliferation in vitro was obtained with the combination of c-myb (S)ODN and cytotoxic drugs. The combinations exerted additive and synergistic effects on human colorectal cancer cells. This study suggests that c-myb antisense (S)ODN might be useful in the therapy of colorectal cancer in combination with chemotherapeutic drugs.

Interleukin-7 and interleukin-15 regulate the expression of the bcl-2 and c-myb genes in cutaneous T-cell lymphoma cells.

Interleukin-7 (IL-7) and IL-15 have been recently identified as growth factors for cutaneous T-cell lymphoma (CTCL) cells, and they protect these cells from cell death. Using the CTCL cell line SeAx as a test system now shows that IL-7 and IL-15 are indeed necessary to maintain high levels of bcl-2. The up-regulation of bcl-2 was paralleled by increased DNA-binding activities of the transcription factors STAT2, STAT5, STAT6, and c-Myb to bcl-2 gene promoter-enhancer elements. Because STAT5 and c-Myb positively regulate bcl-2, IL-7 and IL-15 may mediate some of their effects on cell death survival gene expression through these 2 factors. Constitutive activities of the 3 STAT factors and c-Myb were found in the IL-7- and IL-15-independent CTCL cell lines HUT78 and MyLa 2059. The c-Myb protein was also present in CTCL cells of the skin lesions of all investigated patients. These results indicate that IL-7 and IL-15 may increase bcl-2 expression in CTCL cells by the activation of c-myb and STAT factors.

Intra- and intermolecular triplex DNA formation in the murine c-myb proto-oncogene promoter are inhibited by mithramycin.

Mithramycin inhibits transcription by binding to G/C-rich sequences, thereby preventing regulatory protein binding. However, it is also possible that mithramycin inhibits gene expression by preventing intramolecular triplex DNA assembly. We tested this hypothesis using the DNA triplex adopted by the murine c-myb proto-oncogene. The 5'-regulatory region of c-myb contains two polypurine:polypyrimidine tracts with imperfect mirror symmetry, which are highly conserved in the murine and human c-myb sequences. The DNA binding drugs mithramycin and distamycin bind to one of these regions as determined by DNase I protection assay. Gel mobility shift assays, nuclease and chemical hypersensitivity and 2D-gel topological analyses as well as triplex-specific antibody binding studies confirmed the formation of purine*purine:pyrimidine inter- and pyrimidine*purine:pyrimidine intra-molecular triplex structures in this sequence. Mithramycin binding within the triplex target site displaces the major groove-bound oligonucleotide, and also abrogates the supercoil-dependent H-DNA formation, whereas distamycin binding had no such effects. Molecular modeling studies further support these observations. Triplex-specific antibody staining of cells pretreated with mithramycin demonstrates a reversal of chromosomal triplex structures compared to the non-treated and distamycin-treated cells. These observations suggest that DNA minor groove-binding drugs interfere with gene expression by precluding intramolecular triplex formation, as well as by physically preventing regulatory protein binding.