Expression of XPG protein in human normal and tumor tissues.

XPG (Xeroderma pigmentosum group G complementing factor) is a protein associated with DNA repair and transcription. Point mutations in ERCC5, the gene coding for XPG, cause the cancer-prone disorder xeroderma pigmentosum (XP) while truncation mutations give rise to individuals with the combined clinical features of XP and Cockayne syndrome. Polymorphisms of ERCC5 or alterations in XPG mRNA expression were also associated to an increase risk of different cancers types and to prognosis of cancer patients. However, the expression of XPG protein in different normal or tumor human tissues is not well known. In the present work, we have validated an immunohistochemistry (IHC) assay for detection of expression levels of XPG protein in FFPE human tissue samples. We have also tested this IHC assay in different normal and tumor human tissues. On a microarray containing 28 normal cores, positive staining was observed in 60% of the samples. The highest staining was detected in adrenal gland, breast, colon, heart, kidney, thyroid and tongue. In tumors, positive staining was observed in 9 of 10 breast cancer samples and in all 5 ovarian cancer and 5 sarcomas samples. Subcellular localization was predominantly nuclear. The use of this validated methodology would help to interpret the role of XPG in tumorogenesis and its use as a possible prognostic or predictive factor.

Level of phosphohistone H3 among various types of human cancers.

OBJECTIVES: Anti-phosphorylated histone H3 (pHH3) antibodies specifically detect the core protein histone H3 only when phosphorylated at serine 10 (Ser10) or serine 28 (Ser28). Measurement of pHH3 levels can be used for quantifying mitosis and the effectiveness of mitotic inhibitors in early drug development. However, data on the expression level of pHH3 (Ser10) and pHH3 (Ser28) among different cancers are limited. This study was designed to investigate the expression levels of pHH3 across different types of cancers, using uniform techniques and assay platforms in a single laboratory. DESIGN: Retrospective study. SETTING: Single laboratory. SPECIMENS: Formalin-fixed, paraffin-embedded various human cancer specimens were provided by Mosaic Laboratories Tissue Bank. PRIMARY AND SECONDARY OUTCOME MEASURES: Using immunohistochemistry, pHH3 levels were measured using both pHH3 (Ser10) and (Ser28) antibodies among 10 human melanoma and 10 ovarian tumour samples. The samples were reviewed blindly by two reviewers. pHH3 (Ser10) was then selected to measure the pHH3 levels in cancers of breast, colorectal, oesophageal, gastric, head and neck and lung (n=5 for each cancer). RESULTS: The pHH3 (Ser10) expression was higher than pHH3 (Ser28) in both melanoma and ovarian cancers (p<0.01), with the mean (SD) levels of 1.28% (0.47%) for Ser10 and 0.53% (0.44%) for Ser28 among melanoma and 3.47% (3.51%) for Ser10 and 0.62% (0.68%) for Ser28 among ovarian cancers, respectively. No statistically significant differences were observed among different cancer types tested for pHH3 using Ser10 (p=0.197). No reviewer effect was identified. CONCLUSIONS: The pHH3 Ser10 was significantly higher than Ser28 and may serve as the more robust of two pHH3 assays for measuring mitotic index.

An anti-urokinase plasminogen activator receptor antibody (ATN-658) blocks prostate cancer invasion, migration, growth, and experimental skeletal metastasis in vitro and in vivo.

Urokinase plasminogen activator receptor (uPAR) is a multidomain protein that plays important roles in the growth, invasion, and metastasis of a number of cancers. In the present study, we examined the effects of administration of a monoclonal anti-uPAR antibody (ATN-658) on prostate cancer progression in vitro and in vivo. We examined the effect of treatment of ATN-658 on human prostate cancer cell invasion, migration, proliferation, and regulation of intracellular signaling pathways. For in vivo studies, PC-3 cells (1 x 10(6)) were inoculated into the right flank of male Balb C nu/nu mice through subcutaneous or through intratibial route (2 x 10(5)) of male Fox Chase severe combined immunodeficient mice to monitor the effect on tumor growth and skeletal metastasis. Treatment with ATN-658 resulted in a significant dose-dependent decrease in PC-3 cell invasion and migration without affecting cell doubling time. Western blot analysis showed that ATN-658 treatment decreased the phosphorylation of serine/threonine protein kinase B (AKT), mitogen-activated protein kinase (MAPK), and focal adhesion kinase (FAK) without affecting AKT, MAPK, and FAK total protein expression. In in vivo studies, ATN-658 caused a significant decrease in tumor volume and a marked reduction in skeletal lesions as determined by Faxitron x-ray and micro-computed tomography. Immunohistochemical analysis of subcutaneous and tibial tumors showed a marked decrease in the levels of expression of pAKT, pMAPK, and pFAK, consistent with the in vitro observations. Results from these studies provide compelling evidence for the continued development of ATN-658 as a potential therapeutic agent for the treatment of prostate and other cancers expressing uPAR.

Janus kinase 2 inhibitors. Synthesis and characterization of a novel polycyclic azaindole.

The synthesis and characterization of a novel polycyclic azaindole based derivative is disclosed, and its binding to JAK2 is described. The compound is further evaluated for its ability to block the EPO/JAK2 signaling cascade in vitro and in vivo.

Corpus luteum regression in the rat--in vivo and in vitro studies of apoptotic mechanisms.

Apoptosis has been implicated in corpus luteum regression. Prostaglandin F2 alpha (PGF2alpha) is a luteolytic agent known to induce corpus regression in the rat. We have shown previously that staurosporine, a protein kinase C inhibitor, induces apoptosis in corpora luteal cells. In this study, we hypothesize that PGF2alpha and staurosporine will induce apoptosis in the rat corpus luteum and this process is mediated through the mitochondrial phospholipid cardiolipin and activation of caspase-9. Female prepubertal rats were superovulated and luteal cells were analyzed for apoptosis after treatment with a luteolytic dose of PGF2alpha (in vivo study) or cultured and treated with 10(-5) M PGF2alpha or 1 alphaM staurosporine (in vitro studies). Apoptosis was measured by annexin V and propidium iodide staining, loss of fluorescence of 10-N-nonyl acridine orange (NAO), a cardiolipin specific fluorescent dye, and cleavage of pro-caspase-9 by western blot analysis. PGF2alpha treatment demonstrated evidence of apoptosis in vivo, while staurosporine treatment demonstrated evidence of apoptosis in vitro. We provide, for the first time in the rat ovary, evidence that apoptosis during corpus luteum regression is mediated in part by mitochondria and by cleavage activation of caspase-9.