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Philip J Tofilon

Publications and source records attributed to Philip J Tofilon.

28 records · Page 2Linked to original sources
PubMed2004

In vivo tumor imaging in mice with near-infrared labeled endostatin.

Endostatin is a potent inhibitor of angiogenesis currently in phase I clinical trials. Imaging technologies that use near-infrared fluorescent probes are well suited to the laboratory setting. The goal of this study was to determine whether endostatin labeled with a near-infrared probe (Cy5.5) could be detected in an animal and whether it would selectively localize to a tumor. Endostatin was conjugated to Cy5.5 monofunctional dye and injected into mice bearing Lewis lung carcinoma tumors (350 mm2). Mice were imaged at various time points while under sedation using a lightproof box affixed to a fluorescent microscope mounted with a filter in the near-infrared bandwidth consistent with Cy5.5 fluorescence. After i.p. injection, endostatin-Cy5.5 was absorbed producing a near-infrared fluorescent image within the tumors at 18 h reaching a maximum at 42 h after injection. No signal was emitted from mice injected with unlabeled endostatin or Cy5.5 dye alone or those that received no injection. Further results show that a dose response exists with injection of endostatin-Cy5.5. Mimicking the clinical route of administration, an i.v. injection had a peak signal emission at 3 h but also persisted to 72 h. Finally, to determine the intratumoral binding site for endostatin, we performed immunofluorescence on tumor specimens and demonstrated that endostatin binds to tumor vasculature and colocalizes with platelet/endothelial cell adhesion molecule 1 expression. This study demonstrates that endostatin covalently bound to Cy5.5 will migrate from a distant i.p. injection site to a tumor. These data indicate that endostatin-Cy5.5 is appropriate for selectively imaging tumors in uninjured experimental animals.

Animals↗
PubMed2004

Transcriptional signature of flavopiridol-induced tumor cell death.

Flavopiridol has been shown to inhibit the proliferation of a variety of human tumor cells and is currently undergoing clinical evaluation in cancer treatment. Although the antiproliferative effect of flavopiridol has been attributed to the inhibition of cyclin-dependent kinases 2 and 4, recent reports indicate that the mechanism responsible for the cell death induced by this agent is more complex. To provide insight into the molecular processes mediating flavopiridol-induced cytotoxicity and to investigate the availability of markers indicative of its activity, we have applied cDNA microarray technology. Gene expression profiles were determined for four human tumor cell lines (prostate carcinomas PC3 and DU145 and gliomas SF359 and U251) following exposure to selected concentrations of flavopiridol. Treatment of these cell lines with a concentration of flavopiridol sufficient to reduce survival to 10% resulted in the identification of a set of 209 genes, the expression of which were altered in each of the cell lines. This common set of 209 gene expression changes suggested that flavopiridol-induced cell death can be defined in terms of a specific transcriptome. The flavopiridol death transcriptome consisted primarily of down-regulated genes; however, there were also a significant number of genes with increased expression. Whereas causal relationships were not established, these data suggest molecular events/processes that may be associated with flavopiridol-induced tumor cell death. Moreover, the identification of a set of gene expression changes in four human tumor cell lines suggests that such a transcriptome may be applicable to investigations of flavopiridol pharmacodynamics.

Antineoplastic Agents↗
PubMed2003

Gleevec-mediated inhibition of Rad51 expression and enhancement of tumor cell radiosensitivity.

Rad51 is an essential component of the homologous DNA repair pathway and has been implicated as a determinant of cellular radiosensitivity. Gleevec is a relatively specific inhibitor of c-Abl, a tyrosine kinase that can play a role in the regulation Rad51. The aim of this study was to determine the effects of Gleevec on Rad51 levels and the radiosensitivity of two human glioma cell lines and a nonimmortalized normal human fibroblast cell line. Exposure of both glioma cell lines to radiation resulted in an increase in Rad51 expression; Gleevec treatment alone reduced Rad51 expression. When glioma cells were pretreated with Gleevec, radiation-induced Rad51 expression and nuclear foci formation were reduced. Accordingly, pretreatment of the glioma cells with Gleevec resulted in an enhancement in their radiosensitivity. These data indicate that Gleevec enhances radiation-induced tumor cell killing and suggest that the mechanism involves the reduction in Rad51 levels. In contrast to the glioma cell lines, radiation or Gleevec treatments had no effect on Rad51 expression or foci formation in the normal fibroblast cells. Consistent with these observations, Gleevec did not modify the radiosensitivity of the normal cell line. These results suggest that Rad51 expression is subject to different regulatory processes in the glioma and normal cell lines and further suggest that Rad51 may be an appropriate target for selectively enhancing the radiosensitivity of brain tumor cells.

Benzamides↗
PubMed2003

Enhanced cell killing induced by the combination of radiation and the heat shock protein 90 inhibitor 17-allylamino-17- demethoxygeldanamycin: a multitarget approach to radiosensitization.

PURPOSE: Current strategies for tumor cell radiosensitization focus on a target-based approach. However, the radioresponse of a tumor cell is influenced by a wide variety of signaling molecules existing in a number of different survival pathways. Therefore, in an attempt to increase the probability and/or degree of radiosensitization, we have begun to investigate a multitarget approach using the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG). EXPERIMENTAL DESIGN: The effect of 17AAG on the levels of three proteins (Raf-1, ErbB2, and Akt) previously implicated in the regulation of radiosensitivity was determined in four human tumor cell lines. Tumor cell survival after exposure to corresponding concentrations of 17AAG combined with clinically relevant doses of X-rays was then evaluated using a clonogenic assay. The radiosensitivity of a nonimmortalized, normal fibroblast cell line was also determined after exposure to 17AAG. RESULTS: Exposure to nanomolar concentrations of 17AAG reduced the levels of the three radiosensitivity-associated proteins in a cell type manner. Using corresponding concentrations, 17AAG enhanced the radiosensitivity of each of the tumor cell lines with enhancement factors ranging from 1.3 to 1.7. The enhancement appeared to be related to the number of radioresponse-regulatory proteins affected. In contrast to the tumor cell lines, 17AAG had no effect on the radiosensitivity of a normal, nonimmortalized human fibroblast cell line. CONCLUSIONS: These data suggest that heat shock protein 90 may be an appropriate target for selectively enhancing the radiosensitivity of tumor cells over normal cells. Furthermore, they illustrate the potential of a multitarget approach to radiosensitization.

Benzoquinones↗
PubMed2003

Ibuprofen-mediated reduction of hypoxia-inducible factors HIF-1alpha and HIF-2alpha in prostate cancer cells.

PURPOSE: Hypoxia-inducible factors HIF-1alpha and HIF-2alpha are considered to be potential targets for antineoplastic therapy because they regulate the expression of genes that contribute to tumor cell survival, aggressiveness, and angiogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) have gained considerable interest as anticancer agents because of their cytotoxic and antiangiogenic properties. The aim of this study was to investigate whether NSAIDs inhibit HIFs and HIF-regulated gene expression in prostate cancer cells. EXPERIMENTAL DESIGN: PC3 and DU-145 cells were treated with ibuprofen (Ibu) and other NSAIDs under normoxic and hypoxic (95% N(2), 5% CO(2); <10 ppm O(2)) conditions. The effect of NSAIDs on HIF proteins was analyzed by Western blot analysis. HIF-regulated proteins, vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1), were analyzed by ELISA and Western blot analysis, respectively. RESULTS: Exposure of PC3 and DU-145 cells to hypoxic condition up-regulated HIF-1alpha and HIF-2alpha proteins. Treatment with Ibu under normoxic and hypoxic conditions reduced the level of HIF-1alpha and HIF-2alpha. Ibu-mediated down-regulation of HIFs was associated with down-regulation of HIF-regulated proteins VEGF and Glut-1 in cells exposed to hypoxia. Other nonspecific NSAIDs, diclofenac and ketorolac, also inhibited HIF-1alpha and HIF-2alpha. The reduction in HIFs was observed in PC3 cells that expressed cyclooxygenase-2 (COX-2) protein as well as in DU-145 cells, which did not express COX-2 protein. COX-2-specific inhibitor NS-398 did not inhibit HIF-1alpha or VEGF and GLUT-1. CONCLUSIONS: These data indicate that one of the effects of NSAIDs is to reduce HIF protein levels. The inhibition of HIFs by NSAIDs was COX-2 independent.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2003

Molecular and cellular biology of moderate-dose (1-10 Gy) radiation and potential mechanisms of radiation protection: report of a workshop at Bethesda, Maryland, December 17-18, 2001.

Exposures to doses of radiation of 1-10 Gy, defined in this workshop as moderate-dose radiation, may occur during the course of radiation therapy or as the result of radiation accidents or nuclear/radiological terrorism alone or in conjunction with bioterrorism. The resulting radiation injuries would be due to a series of molecular, cellular, tissue and whole-animal processes. To address the status of research on these issues, a broad-based workshop was convened. The specific recommendations were: (1) RESEARCH: Identify the key molecular, cellular and tissue pathways that lead from the initial molecular lesions to immediate and delayed injury. The latter is a chronic progressive process for which postexposure treatment may be possible. (2) Technology: Develop high-throughput technology for studying gene, protein and other biochemical expression after radiation exposure, and cytogenetic markers of radiation exposure employing rapid and accurate techniques for analyzing multiple samples. (3) Treatment strategies: Identify additional biological targets and develop effective treatments for radiation injury. (4) Ensuring sufficient expertise: Recruit and train investigators from such fields as radiation biology, cancer biology, molecular biology, cellular biology and wound healing, and encourage collaboration on interdisciplinary research on the mechanisms and treatment of radiation injury. Communicate knowledge of the effects of radiation exposure to the general public and to investigators, policy makers and agencies involved in response to nuclear accidents/events and protection/treatment of the general public.

Animals↗
PubMed2002

Inhibition of radiation-induced nuclear factor-kappaB activation by an anti-Ras single-chain antibody fragment: lack of involvement in radiosensitization.

We have shown previously that the transduction of a number of human tumor cell lines with an adenovirus (AV1Y28) expressing a single-chain antibody fragment (scFv) directed against Ras proteins results in radiosensitization. Because Ras is involved in the regulation of a number of transcription factors, we have determined the effects of this adenovirus on the activation of nuclear factor-kappaB (NF-kappaB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-kappaB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IkappaB kinase activity and IkappaBalpha phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-kappaB. Whereas AV1Y28 inhibited NF-kappaB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-alpha-induced NF-kappaB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-kappaB. In additional cell lines radiosensitized by AV1Y28, radiation-induced NF-kappaB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-kappaB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-kappaB activation. However, inhibition of NF-kappaB activation via the expression of a dominant-negative form of IkappaBalpha in U251 cells had no effect on radiation-induced cell killing and did not influence AV1Y28-mediated radiosensitization. Therefore, whereas AV1Y28 inhibits radiation-induced NF-kappaB activation, this process does not appear to play a direct role in its radiosensitizing actions.

DNA-Binding Proteins↗
PubMed2002

Apoptosis induced by adenovirus-mediated p53 gene transfer in human glioma correlates with site-specific phosphorylation.

Therapeutic replacement of the p53 gene using an adenovirus vector (Ad-p53) may be an effective alternative to conventional therapies for the treatment of glioma. We have previously demonstrated that the introduction of Ad-p53 into glioma cells containing mutant p53 induces apoptosis, whereas glioma cells containing wild-type p53 are resistant. However, Ad-p53 will enhance the radiosensitivity of wild-type p53 glioma cells by increasing their tendency for apoptosis. The mechanism underlying these different responses to Ad-p53 has not been elucidated to date. Because phosphorylation of p53 at serines 15, 20, and 392 may play a role in regulating p53-mediated apoptotic activity, we determined the phosphorylation status of exogenous p53 in mutant and wild-type gliomas after Ad-p53 transfer. Monolayer cultures of glioma cell lines expressing mutant p53 (U251 and U373) or wild-type p53 (U87 and D54) were infected with Ad-p53 and analyzed by Western blotting. High levels of exogenous p53 were detected in both cell lines after Ad-p53 transfer. However, only apoptotic mutant p53 cells expressed high levels of phospho-Ser15-p53 and phospho-Ser20-p53. The levels of phospho-Ser15-p53 and phospho-Ser20-p53 were very low in wild-type p53 cells after Ad-p53 infection alone. When wild-type p53 glioma cells were exposed to radiation after Ad-p53 infection, phospho-Ser15-p53 and phospho-Ser20-p53 were detected at high levels, and the cells subsequently underwent apoptosis; no change in serine 392 was detected. The induction of apoptosis and the expression of phospho-Ser15 and phospho-Ser20 in these cells were also enhanced by the combination of Ad-p53 and other DNA-damaging agents such as cisplatin and bichloroethyl nitrosourea. Furthermore, the expression of phospho-Ser15-p53 and phospho-Ser20-p53 correlated with the amount of apoptosis; the apoptotic activity of p53 in glioma cells was partially inhibited by a mutation of p53 at serine 15. These results suggest that phosphorylation of p53 at serine 15 and serine 20 is critical for apoptosis induction in p53 gene therapy for gliomas.

Adenoviridae↗
PubMed2002

Radiation-induced activation of nuclear factor-kappaB involves selective degradation of plasma membrane-associated I(kappa)B(alpha).

In contrast to nuclear factor-kappaB (NF-kappaB) activation by tumor necrosis factor-alpha (TNF-alpha), the specific processes involved in the activation of this transcription factor by ionizing radiation (IR) have not been completely defined. According to the classical paradigm, a critical event in NF-kappaB activation is the degradation of I(kappa)B(alpha). Data presented herein show that, in contrast to treatment with TNF-alpha, IR-induced NF-kappaB activation was not accompanied by degradation of I(kappa)B(alpha) in the U251 glioblastoma cell line as determined in whole cell lysates. However, treatment with the proteosome inhibitor MG-132 inhibited NF-kappaB activation induced by IR, suggesting that I(kappa)B(alpha) degradation was a critical event in this process. To reconcile these results, U251 cell lysates were separated into soluble and insoluble fractions and I(kappa)B(alpha) levels evaluated. Although I(kappa)B(alpha) was found in both subcellular fractions, treatment with IR resulted in the degradation of I(kappa)B(alpha) only in the insoluble fraction. Further subcellular fractionation suggested that the IR-sensitive, insoluble pool of I(kappa)B(alpha) was associated with the plasma membrane. These data suggest that the subcellular location of I(kappa)B(alpha) is a critical determinant in IR-induced NF-kappaB activation.

Cell Fractionation↗