SRC family kinase inhibitor SU6656 enhances antiangiogenic effect of irradiation.

PURPOSE: Src family kinases (SFK) have been identified as molecular targets. SU6656 is a small-molecle indolinone that specifically inhibits this family of kinases. METHODS AND MATERIALS: Human umbilical vein endothelial cells were used to study the effects of SFK inhibition. Western blot analysis was performed to determine the effect of SFK inhibition on the PI3K/Akt pathway and caspase cleavage. Apoptosis was studied by propidium iodide staining of nuclei. Angiogenesis was examined using capillary tubule formation in Matrigel. Tumor response was further studied in vivo using Lewis lung carcinoma cells implanted into the dorsal skin fold of mice in the window model and in the hind limb in the tumor volume model. RESULTS: Clonogenic survival of endothelial cells was decreased after the combined therapy of SU6656 and radiation compared with radiotherapy alone. Furthermore, SFK inhibition by SU6656 attenuated radiation-induced Akt phosphorylation and increased radiation-induced apoptosis and vascular endothelium destruction. In vivo, SU6656 administered before irradiation significantly enhanced radiation-induced destruction of blood vessels within the tumor windows and enhanced tumor growth delay when administered during fractionated irradiation. CONCLUSIONS: This study demonstrates the potential use of SFK inhibition to enhance the effects of ionizing radiation during radiotherapy.

The controversial abscopal effect.

The abscopal effect is potentially important for tumor control and is mediated through cytokines and/or the immune system, mainly cell-mediated immunity. It results from loss of growth stimulatory and/or immunosuppressive factors from the tumor. Until recently, the abscopal effect referred to the distant effects seen after local radiation therapy. However, the term should now be used interchangeably with distant bystander effect. Through analysis of distant bystander effects of other local therapies, we discuss the poorly understood and researched radiation-induced abscopal effect. Although the abscopal effect has been described in various malignancies, it is a rarely recognized clinical event. The abscopal effect is still extremely controversial with known data that both support and refute the concept.

Recapitulation of germ cell- and pituitary-specific expression with 1.6 kb of the cystatin-related epididymal spermatogenic (Cres) gene promoter in transgenic mice.

The Cres (cystatin-related epididymal spermatogenic) gene encodes the defining member of a new subgroup within the family 2 cystatins of cysteine protease inhibitors. Cres expression is highly tissue- and cell-specific, with messenger RNA (mRNA) present in the testicular round/elongating spermatids, proximal caput epididymal epithelium, gonadotroph cells in the anterior pituitary gland, and corpus luteum of the ovary. To begin to elucidate the molecular mechanisms controlling the tissue- and cell-specific expression of the Cres gene, transgenic mice were generated containing 1.6 kilobases (kb) of the mouse Cres promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. A CAT enzyme-linked immunosorbent assay detected CAT protein in the testis, epididymis, isolated cauda epididymal spermatozoa, and anterior pituitary gland from mice heterozygous and homozygous for the transgene. However, reverse transcription (RT)-PCR did not detect CAT mRNA in any regions of the epididymis, suggesting that the CAT protein detected in the epididymis was from spermatozoa. RT-PCR also did not detect CAT mRNA in the ovary. RT-PCR analysis of the testes from mice of different postnatal ages showed CAT mRNA first detected at day 22, which correlated with the first appearance of Cres mRNA and with the presence of round spermatids. These studies demonstrate that 1.6 kb of Cres promoter contains the DNA elements necessary for germ cell and pituitary gland-specific expression but lacks critical sequences necessary for expression in the epididymis and ovary.

p19ARF directly and differentially controls the functions of c-Myc independently of p53.

Increased expression of the oncogenic transcription factor c-Myc causes unregulated cell cycle progression. c-Myc can also cause apoptosis, but it is not known whether the activation and/or repression of c-Myc target genes mediates these diverse functions of c-Myc. Because unchecked cell cycle progression leads to hyperproliferation and tumorigenesis, it is essential for tumour suppressors, such as p53 and p19ARF (ARF), to curb cell cycle progression in response to increased c-Myc (refs 2, 3). Increased c-Myc has previously been shown to induce ARF expression, which leads to cell cycle arrest or apoptosis through the activation of p53 (ref. 4). Here we show that ARF can inhibit c-Myc by a unique and direct mechanism that is independent of p53. When c-Myc increases, ARF binds with c-Myc and dramatically blocks c-Myc's ability to activate transcription and induce hyperproliferation and transformation. In contrast, c-Myc's ability to repress transcription is unaffected by ARF and c-Myc-mediated apoptosis is enhanced. These differential effects of ARF on c-Myc function suggest that separate molecular mechanisms mediate c-Myc-induced hyperproliferation and apoptosis. This direct feedback mechanism represents a p53-independent checkpoint to prevent c-Myc-mediated tumorigenesis.

A specific antagonist of the p110delta catalytic component of phosphatidylinositol 3'-kinase, IC486068, enhances radiation-induced tumor vascular destruction.

The phosphatidylinositol 3'-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival and function during oxidative stress. The role of the PI3k/Akt signaling pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was rapidly phosphorylated in response to radiation in primary culture endothelial cells (human umbilical vascular endothelial cells) in the absence of serum or growth factors. PI3k consists of p85 and p110 subunits, which play a central upstream role in Akt activation in response to exogenous stimuli. The delta isoform of the p110 subunit is expressed in endothelial cells. We studied the effects of the p110delta specific inhibitor IC486068, which abrogated radiation-induced phosphorylation of Akt. IC486068 enhanced radiation-induced apoptosis in endothelial cells and reduced cell migration and tubule formation of endothelial cells in Matrigel following irradiation. In vivo tumor growth delay was studied in mice with Lewis lung carcinoma and GL261 hind limb tumors. Mice were treated with daily i.p. injections (25 mg/kg) of IC486068 during 6 days of radiation treatment (18 Gy). Combined treatment with IC486068 and radiation significantly reduced tumor volume as compared with either treatment alone. Reduction in vasculature was confirmed using the dorsal skinfold vascular window model. The vascular length density was measured by use of the tumor vascular window model and showed IC486068 significantly enhanced radiation-induced destruction of tumor vasculature as compared with either treatment alone. IC486068 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. These findings suggest that p110delta is a therapeutic target to enhance radiation-induced tumor control.