Receiver operating characteristic analysis: a general tool for DNA array data filtration and performance estimation.

A critical step for DNA array analysis is data filtration, which can reduce thousands of detected signals to limited sets of genes. Commonly accepted rules for such filtration are still absent. We present a rational approach, based on thresholding of intensities with cutoff levels that are estimated by receiver operating characteristic (ROC) analysis.

Vascular endothelial growth factor enhances endothelial cell survival and tumor radioresistance.

Purpose: Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and survival. The purpose of the present studies was to investigate the role of VEGF in the tumor response to ionizing radiation.

Methods: Two ras-transformed murine fibrosarcoma cell lines, VEGF+/+ and VEGF-/- were exposed to ionizing radiation (0, 1, 3, 5, 7 or 9 Gy) in vitro, and clonogenic survival was determined.

Dose-dependent and independent temporal patterns of gene responses to ionizing radiation in normal and tumor cells and tumor xenografts.

U87 cells derived from human malignant gliomas and growtharrested human embryonic lung (HEL) fibroblasts were examined with respect to their response to ionizing radiation by profiling their RNAs. In the first series of experiments, cells grown in vitro were harvested and the RNAs were extracted 5 h after exposure to 1, 3, or 10 Gy. In the second series of experiments the U87 tumors were implanted in nude mice and subjected to the same doses of irradiation.

Angiostatin effects on endothelial cells mediated by ceramide and RhoA.

Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death.

Angiostatin induces mitotic cell death of proliferating endothelial cells.

Angiostatin is an inhibitor of tumor angiogenesis that induces regression of experimental tumors and enhances the antitumor effects of radiation therapy. We report that the cytotoxic effects of angiostatin are restricted to the proliferating endothelial cell population. In addition, angiostatin and ionizing radiation (IR) interact by inducing death of dividing endothelial cells.

Down-regulation of ceramide production abrogates ionizing radiation-induced cytochrome c release and apoptosis.

Previous work has demonstrated that down-regulation of ceramide production after selection of cells with N-oleoylethanolamine (OE), an inhibitor of ceramidase, results in resistance to DNA damage-induced apoptosis. We report here that acute exposure of WEHI-231 cells (murine B-cell lymphoma) to OE activates neutral sphingomyelinase, induces ceramide production and increases intracellular reactive oxygen species. OE exposure also induces mitochondrial permeability, cytochrome c release, and apoptosis.

Modulation of apoptotic response of a radiation-resistant human carcinoma by Pseudomonas exotoxin-chimeric protein.

Strategies to sensitize human tumors that are resistant to apoptosis have been clinically unsuccessful. We demonstrate that a structurally modified chimeric Pseudomonas exotoxin, PEdelta53L/TGF-alpha/KDEL, with binding specificity for the epidermal growth factor receptor, markedly enhances sensitivity of human xenografts to radiation killing. Exposure to PEdelta53L/TGF-alpha/KDEL decreases the apoptotic threshold through protein synthesis inhibition and simultaneous production of ceramide in tumor cells that lack functional p53 protein.

Human papillomavirus expression and p53 gene mutations in squamous cell carcinoma.

Objectives: To determine the incidence of human papillomavirus (HPV) infection and p53 gene mutation expression in squamous cell carcinomas (SCCs) of the oral cavity and tonsils, to correlate the presence of HPV and p53 gene mutation with known clinical and pathological features of SCC, and to determine whether infection with HPV or the presence of p53 gene mutations are independent prognosticators of patient survival.

Design: To accomplish this goal, 58 patients with SCCs of the oral cavity and 42 patients with SCCs of the tonsils were randomly examined. The cases examined met the criteria of 5-year clinical follow-up, availability of complete staging information and treatment history, and the presence of paraffin-embedded tumor specimens.

Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation.

Approximately 30% of cancer deaths result from the failure to control local and regional tumors. The goal of radiotherapy is to maximize local and regional tumor cell killing while minimizing normal tissue destruction. Attempts to enhance radiation-mediated tumor cell killing using halogenated pyrimidines, antimetabolites, and other DNA-damaging agents or sensitizers of hypoxic tumor cells have met with only modest clinical success.

Loss of ceramide production confers resistance to radiation-induced apoptosis.

Ionizing radiation mediates cell death, in part, through chromosomal damage following one or more cell divisions. X-rays also induce programmed cell death (apoptosis) in some cell types both in vitro and in vivo. Both neutral and acidic sphingomyelinases, which generate the lipid second messenger ceramide, are reported to induce apoptosis following ionizing radiation and other death signals such as tumor necrosis factor alpha and Fas ligand.

Protein kinase C inhibition induces apoptosis and ceramide production through activation of a neutral sphingomyelinase.

We report that WEHI-231 undergo apoptosis following exposure to the protein kinase C inhibitors chelerythrine chloride and calphostin C. Following the addition of chelerythrine or calphostin C to WEHI-231 cells, ceramide production increased over baseline levels with a concurrent decrease in sphingomyelin. More detailed examinations determined that the ceramide accumulation resulted from activation of neutral, but not acidic, sphingomyelinase.

Human papilloma virus and p53 in head and neck cancer: clinical correlates and survival.

Recent studies have shown that p53 mutations are frequently found in cancer of the head and neck, whereas others have indicated that human papilloma virus (HPV) infection may be involved. Thus far, no studies have examined both p53 and HPV in the same patient population and correlated the results with clinical characteristics and outcome. The purpose of this study was to examine any interrelationship between p53 and HPV in patients with squamous cell carcinoma (SCC) of the head and neck.

Cross-talk between ceramide and PKC activity in the control of apoptosis in WEHI-231.

WEHI-231, a murine B-cell lymphoma, readily undergoes programmed cell death following surface immunoglobulin (Ig) cross-linking [1]. Ceramide has been shown to induce apoptosis in WEHI-231 following its exposure to anti-lg antibodies, dexamethasone, and irradiation [2]. Recently, Haimovitz-Friedman et al.

Ribonucleotide reductase gene expression during cyclic AMP-induced cell cycle arrest in T lymphocytes.

In both 3T3 mouse fibroblasts and S49 mouse T lymphocytes the genes encoding both subunits of ribonucleotide reductase are expressed beginning in late G1 phase. In studies reported here, we compared the expression of the genes that code for the M1 and M2 subunits of ribonucleotide reductase in S49 cells, which are arrested in G1 phase by agents that increase cyclic AMP, with those from CEM human T lymphoma cells that are unaffected by exposure to dibutyryl cyclic AMP. Dibutyryl cyclic AMP treatment results in a prompt steady diminution of M2 mRNA concentration to levels at or below that of elutriated G1 cell-cycle-specific populations in S49 cells, in contrast to CEM cell M2 mRNA, which is unchanged.

Deoxyadenosine- and cyclic AMP-induced cell cycle arrest and cytotoxicity.

We compared deoxyadenosine (AdR)- and cyclic AMP (cAMP)-induced cell cycle arrest and cytotoxicity in wild type and mutant S49 cells to determine whether they resulted from the same or different mechanisms. Cyclic AMP and deoxyadenosine are synergistic rather than additive in cytotoxicity assays, suggesting different mechanisms of toxicity. Although cyclic AMP causes cell death after 72 h, in concentrations sufficient to result in cell cycle arrest it is reversible with virtually no cytotoxicity for at least 24 h, whereas AdR-induced cell cycle arrest is lethal and irreversible.

Effect of cyclic AMP on the cell cycle regulation of ribonucleotide reductase M2 subunit messenger RNA concentrations in wild-type and mutant S49 T lymphoma cells.

Ribonucleotide reductase activity in S49 T lymphoma cells is cell cycle regulated by de novo protein synthesis of the M2 subunit. There is maximal enzyme activity in S and G2/M phase with low activity and low concentrations of the M2 subunit in G1 phase. Pharmacologic concentrations of cyclic AMP arrest S49 cells in the G1 phase of the cell cycle.

The dose dependent effect of cyclic AMP on ribonucleotide reductase in mitogen stimulated mononuclear cells.

Cyclic adenosine monophosphate arrests proliferating T lymphocytes in the G1 phase of the cell cycle. Here we demonstrate that exogenous and endogenous elevations in cyclic AMP concentration result in diminished mitogen stimulation, cell cycle arrest, and decreased ribonucleotide reductase messenger RNA concentrations in peripheral blood mononuclear cells. At lower concentrations (less than 1mM) of dibutyryl cyclic AMP that do not generate cell cycle arrest there is inhibition of ribonucleotide reductase activity without decreased messenger RNA concentration for the M2 subunit of ribonucleotide reductase.

M2 subunit of ribonucleotide reductase is a target of cyclic AMP-dependent protein kinase.

Cyclic AMP arrests T lymphocytes in the G1 phase of the cell cycle, and prolonged exposure results in cytolysis. Both of these effects require cyclic AMP-dependent protein kinase. We recently observed that some S49 mouse T lymphoma cell lines selected for hydroxyurea resistance were not arrested in G1 by cyclic AMP.

Deoxyadenosine toxicity and cell cycle arrest in hydroxyurea-resistant S49 T-lymphoma cells.

Hydroxyurea-resistant S49 T-lymphoma cells have increased ribonucleotide reductase activity and deoxyribonucleoside triphosphate pools when compared with wild-type cultures. If ribonucleotide reductase inhibition is the mechanism by which deoxyadenosine is cytotoxic, then hydroxyurea (HU)-resistant S49 cells might be more resistant to deoxyadenosine toxicity when adenosine deaminase is inhibited than wild-type cells. Five S49 cell lines resistant to varying concentrations of HU were compared with wild-type cells by measuring CDP reductase activity, deoxyribonucleoside triphosphate pools, and deoxyadenosine toxicity.

Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea-resistant S49 T-lymphoma cells.

We investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea-resistant (HYUR) murine S49 T-lymphoma cell lines. Cell lines 10- to 40-fold more hydroxyurea-resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5- to 8-fold) and increased dNTP pools (up to 5-fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase.