Inactivation of DNA-PK by knockdown DNA-PKcs or NU7441 impairs non-homologous end-joining of radiation-induced double strand break repair.

The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in non-homologous end-joining (NHEJ) repair. We investigated the mechanism of NU7441, a highly selective DNA-PK inhibitor, in NHEJ-competent mouse embryonic fibroblast (MEF) cells and NHEJ-deficient cells and explored the feasibility of its application in radiosensitizing nasopharyngeal carcinoma (NPC) cells. We generated wild-type and DNA-PKcs-/- MEF cells.

Inhibiting DNA-PKcs in a non-homologous end-joining pathway in response to DNA double-strand breaks.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a distinct factor in the non-homologous end-joining (NHEJ) pathway involved in DNA double-strand break (DSB) repair. We examined the crosstalk between key proteins in the DSB NHEJ repair pathway and cell cycle regulation and found that mouse embryonic fibroblast (MEF) cells deficient in DNA-PKcs or Ku70 were more vulnerable to ionizing radiation (IR) compared with wild-type cells and that DSB repair was delayed. γH2AX was associated with phospho-Ataxia-telangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for the arrest of cell cycle through the G2/M phase.

Visualizing the antivascular effect of bortezomib on the hypoxic tumor microenvironment.

Bortezomib, a novel proteasome inhibitor, has been approved for treating multiple myeloma and mantle cell lymphoma and studied pre-clinically and clinically for solid tumors. Preferential cytotoxicity of bortezomib was found toward hypoxic tumor cells and endothelial cells in vitro. The purpose of this study is to investigate the role of a pretreatment hypoxic tumor microenvironment on the effects of bortezomib in vitro and ex vivo, and explore the feasibility of dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to noninvasively evaluate the biological effects of bortezomib.

Elevation of heat shock gene expression from static magnetic field exposure in vitro.

Previously, we found that extremely low frequency (ELF) electric fields were able to elicit an approximate 3.5-fold increase in heat shock gene expression, a response which may have applicability to cancer therapy. Based on recent studies demonstrating the ability of magnetic fields to influence gene expression, we hypothesized that low level static magnetic fields may be able to affect heat shock gene expression while avoiding some of the clinical difficulties that arise with electric fields.

Induction of heat shock gene expression in RAT1 primary fibroblast cells by ELF electric fields.

Recent studies have demonstrated that the Ku70 gene fragment can be placed in the anti-sense orientation under the control of a heat-inducible heat shock protein 70 (HSP70) promoter and activated through heat shock exposure. This results in attenuation of the Ku70 protein expression, inhibiting cellular repair processes, and sensitizing the transfected cells to exposures such as the ionizing radiation exposures used clinically. However, achieving the tissue temperatures necessary to thermally induce the HSP70 response presents significant limitations to the clinical application of this strategy.

The effect of DN (dominant-negative) Ku70 and reoxygenation on hypoxia cell-kill: evidence of hypoxia-induced potentially lethal damage.

Purpose: To study the effect of DN (dominant-negative) Ku70 and reoxygenation on the hypoxia-induced cell-kill.

Materials And Methods: Cell lines were human colorectal carcinoma HCT8 and HT29 cells and their respective derivatives, v-HCT8 and v-HT29 infected with DNKu70-containing adenovirus. Cells were plated in glass tubes and made hypoxic by flushing N(2) gas containing 0, 0.

Response to multiple radiation doses of human colorectal carcinoma cells infected with recombinant adenovirus containing dominant-negative Ku70 fragment.

Purpose: To investigate the effect of recombinant replication-defective adenovirus containing dominant-negative Ku70 fragment on the response of tumor cells to multiple small radiation doses. Our ultimate goal is to demonstrate the feasibility of using this virus in gene-radiotherapy to enhance the radiation response of tumor cells.

Methods And Materials: Human colorectal HCT8 and HT29 carcinoma cells were plated in glass tubes, infected with virus (25 multiplicity of infection), and irradiated with a single dose or zero to five doses of 3 Gy each at 6-h intervals.

The effect of mild temperature hyperthermia on tumour hypoxia and blood perfusion: relevance for radiotherapy, vascular targeting and imaging.

Clinically achievable mild temperature local hyperthermia (<43 degrees C) has been demonstrated to be an effective adjuvant to radiotherapy in pre-clinical and clinical studies. In this article, we briefly review the recent progress in the following areas: (1) the effect of mild temperature hyperthermia (MTH) on tumour hypoxia and blood perfusion as assessed by dual marker immunohistochemistry (IHC); (2) the kinetics of MTH induced changes in tumour hypoxia; (3) the potential role of heat-induced tumour reoxygenation on radio- and chemo-sensitisation; (4) the potential role of MTH in combination with vascular targeting agents (VTAs) on tumour response; and (5) non-invasive detection of changes in tumour oxygenation and blood perfusion. It is shown that MTH, by itself or in combination with VTAs, leads to changes in tumour perfusion and oxygenation with potential for radio- and chemo-sensitisation.

Hyperthermia classic article commentary: 'Re-induction of hsp70 synthesis: an assay for thermotolerance' by Gloria C. Li and Johnson Y. Mak, International Journal of Hyperthermia 1989;5:389-403.

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Specifically, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance in tumor cells in vitro and in animal tumor models.

Noninvasive molecular imaging of hypoxia in human xenografts: comparing hypoxia-induced gene expression with endogenous and exogenous hypoxia markers.

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells.

Non-invasive molecular and functional imaging of cytosine deaminase and uracil phosphoribosyltransferase fused with red fluorescence protein.

Introduction: Increased expression of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) may improve the antitumoral effect of 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC), and thereby enhance the potential of gene-directed enzyme prodrug therapy. For the applicability of gene-directed enzyme prodrug therapy in a clinical setting, it is essential to be able to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using magnetic resonance spectroscopy and optical imaging to measure non-invasively CD and UPRT expression and function.

Changes in tumor hypoxia induced by mild temperature hyperthermia as assessed by dual-tracer immunohistochemistry.

Purpose: To study the changes in hypoxia resulting from mild temperature hyperthermia (MTH) in a subcutaneous xenograft model using dual-tracer immunohistochemical techniques.

Materials And Methods: HT29 tumors were locally heated at 41 degrees C. Changes in tumor hypoxia were investigated by pimonidazole and EF5.

Response to multiple radiation doses of fibroblasts over-expressing dominant negative Ku70.

Purpose: To evaluate the response of cells over-expressing dominant negative (DN) Ku70 to single and multiple small radiation doses.

Methods And Materials: Clones of fibroblasts over-expressing DNKu70, DNKu70-7, DNKu70-11, and parental Rat-1 cells were irradiated under oxic or hypoxic conditions with single or multiple doses. Cells were trypsinized 0 or 6 h after irradiation to determine surviving fraction (SF).

Comparative effects of natural and synthetic diallyl disulfide on apoptosis of human breast-cancer MCF-7 cells.

The apoptotic effects of natural (n-) and synthetic (s-) DADS (diallyl disulfide; 3,3'-thiobisprop-1-ene) on human breast-cancer MCF-7 cells were investigated in vitro. 5-Fu (5-fluorouracil) and CTX (cyclophosphamide; Cytoxan) were used as comparative control anticancer agents. After MCF-7 cells had been treated with the drugs, cell viability, morphological change, apoptosis and changes in the cell cycle were measured.

Investigation of the mechanism of enhanced effect of EGCG on huperzine A's inhibition of acetylcholinesterase activity in rats by a multispectroscopic method.

The mechanism of enhanced effect of (-)-epigallocatechin-3-gallate (EGCG) on huperzine A's (HUP) inhibition of acetylcholinesterase (AChE) activity in rats was investigated. The inhibitory effects of HUP at 10 and 5 microg/kg on AChE activity were quite weak in the whole phase. In contrast, upon addition of EGCG (100 mg/kg) to the HUP 10 and 5 microg/kg groups, remarkably enhanced inhibitory effects with maximum inhibitory percentages of 90.

Comparison of Helzel and OxyLite systems in the measurements of tumor partial oxygen pressure (pO2).

Wen, B., Urano, M., Humm, J.

Adenovirus-mediated expression of a dominant negative Ku70 fragment radiosensitizes human tumor cells under aerobic and hypoxic conditions.

Ku70 is one component of a protein complex, the Ku70/Ku80 heterodimer, which binds to DNA double-strand breaks and activates DNA-dependent protein kinase (DNA-PK), leading to DNA damage repair. Our previous work has confirmed that Ku70 is important for DNA damage repair in that Ku70 deficiency compromises the ability of cells to repair DNA double-strand breaks, increases the radiosensitivity of cells, and enhances radiation-induced apoptosis. Because of the radioresistance of some human cancers, particularly glioblastoma, we examined the use of a radio-gene therapy paradigm to sensitize cells to ionizing radiation.

Induction of apoptosis and transient increase of phosphorylated MAPKs by diallyl disulfide treatment in human nasopharyngeal carcinoma CNE2 cells.

This study was undertaken to elucidate the effect of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, in suppressing human nasopharyngeal carcinoma cells. A potent increase (of at least 9-fold) in apoptotic cells has accompanied 1) a decrease in cell viability, 2) a increase of the fraction of S-phase cells by up to 63.8%, and 3) a transient increase of the phospho-p38 and phospho-p42/44 (phosphorylated p38 MAPK and phosphorylated p42/44 MAPK) in a time- and concentration-dependent manner.

Hyperthermia and gene therapy: potential use of microPET imaging.

In recent years, both hyperthermia and gene-therapy have been evaluated as approaches to improve cancer radiotherapy. In addition, potential exists to combine these approaches to increase the overall therapeutic efficacy. For example, it has been reported that adenovirus-mediated heat-inducible gene expression may reduce the normal tissue toxicity associated with constitutively controlled expression of therapeutic genes.

Modulation of DNA end joining by nuclear proteins.

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination and non-homologous end joining (NHEJ). NHEJ may either be error-free or mutagenic with deletions or insertions at the joint. Recent studies showed that DNA ends can also be joined via microhomologous sequences flanking the break point especially when proteins responsible for NHEJ, such as Ku, are absent.