An artificially constructed radiation-responsive promoter is activated by doxorubicin.

We previously developed an artificially constructed promoter that was activated in response to X-ray irradiation in LNCap, a prostate cancer cell line. Anticancer drugs were examined to see whether some of them could stimulate the activity of the promoter. It was found that doxorubicin (Dox) treatment to LNCap transfected with a gene cassette of the luciferase gene under control of the promoter-enhanced luciferase activity in a dose-dependent manner, indicating that the promoter could be controlled by Dox. When the luciferase gene was replaced with the fcy::fur gene whose product facilitates conversion of 5-fluorocytosine into 5-fluorouracil that is highly toxic, Dox stimulated the expression of the gene product, resulting in facilitation of cell killing effect in the presence of 5-fluorocytosine. These results suggest that therapeutic gene expression controlled with an anticancer drug may lead to a more effective cancer therapy with less hazardous side effects.

Analysis of gene expression in apoptosis of human lymphoma U937 cells induced by heat shock and the effects of alpha-phenyl N-tert-butylnitrone (PBN) and its derivatives.

Hyperthermia, a modality of cancer therapy, has been known as a stress to induce apoptosis. However, the molecular mechanism of heat shock-induced apoptosis, especially on roles of intracellular oxidative stress, is not fully understood. First, when human lymphoma U937 cells were treated with heat shock (44 degrees C, 30 min), the fraction of apoptosis, revealed by phosphatidylserine externalization, increased gradually and peaked at 6 hr after the treatment. In contrast, intracellular superoxide formation increased early during the heat shock treatment and peaked at 30 min after the treatment. When the cells were treated with heat shock in the presence of alpha -phenyl-N-tert-butylnitrone (PBN) and its derivatives, which are potent antioxidants, the DNA fragmentation was inhibited in an order according to the agents' hydrophobicity. PBN showing the highest inhibitory effects suppressed not only intracellular superoxide formation but also various apoptosis indicators. cDNA microarray was employed to analyze gene expression associated with heat shock-induced apoptosis, and the time-course microarray analysis revealed 5 groups showing changes in their pattern of gene expression. Among these genes, c-jun mRNA expression showed more than 40 fold increase 2 hr after heat treatment. The expression level of c-jun mRNA verified by quantitative real-time PCR was about 20 fold increase, and c-jun expression was similarly suppressed by PBN and its derivatives. These results suggest that the change of c-jun expression is an excellent molecular marker for apoptosis mediated by intracellular oxidative stress induced by heat shock.

Effects of antioxidants on X-ray- or hyperthermia-induced apoptosis in human lymphoma U937 cells.

Hydroxyl radicals (.OH) and superoxide anion radicals (O2.-) are known to play cardinal roles in cell killing and various types of cell damage. In order to elucidate the mechanism of the involvement of both free radicals on apoptosis, the correlation between anti-apoptotic effects and free radical scavenging abilities of anti-oxidants was studied. As an indicator of anti-apoptotic effects, C1/2 (antioxidant concentration to inhibit DNA fragmentation by 50%) was evaluated in human lymphoma cell line U937 cells 6 hr after X-ray (10 Gy) or hyperthermia (44 degrees C, 30 min) treatment. Rate constants of the reactions between antioxidants and .OH or O2.- were calculated as the scavenging ability of the antioxidants with graded concentration estimated by EPR spectroscopy. No apparent correlation between C1/2 obtained in apoptosis induced by X-rays or hyperthermia and the rate constants of antioxidants for .OH or O2.- was observed. On the other hand, the partition coefficients in 1-octanol/water of the antioxidants, an indicator of hydrophobicity, revealed a correlation with the C1/2 of the agents with hyperthermia, but not with X-ray irradiation. These results indicate that the prevention of apoptosis by an antioxidant is not simply associated with its scavenging ability for .OH or O2.-. The hydrophobicity of the antioxidant, among other possible factors, is involved in the inhibition of hyperthermia- induced apoptosis.

Enhanced ultrasound-induced apoptosis and cell lysis by a hypotonic medium.

PURPOSE: To test the hypothesis that non-lethal hypotonia will enhance ultrasound-induced cell killing in vitro and that the mechanism is mechanical in nature. MATERIALS AND METHODS: Hypotonic RPMI medium (146 mOsm) was used to induce non-lethal osmotic swelling of human myelomonocytic leukaemia U937 cells. Hypotonia for 10 min was started just before exposure to 1 MHz ultrasound at 0.5 or 1.0 Wcm(-2) for 10 min, or 5 min before exposure to 2.0 Wcm(-2) for 1 min. Surviving intact cells were then determined by the trypan blue dye exclusion test immediately after treatment. After 6-h incubation of the treated cells, early apoptosis and secondary necrosis were measured using a flow cytometer. Intracellular free calcium ion imaging by Fura-2 fluorescence and cellular ion scanning using a secondary ion mass spectrometer were also performed. RESULTS: Enhancement of ultrasound-induced cell lysis was observed at all intensities, and most prominently at 2.0 Wcm(-2), while apoptosis induction was significantly enhanced at intensities of 0.5 and 1.0 Wcm(-2), but not at 2.0 Wcm(-2). The enhanced cell lysis is attributed to the increased susceptibility of the cells to mechanical damage. This is consistent with previous reports describing the effects of mechanical stresses on cell membranes. Cellular ion scanning images also suggest that hypotonia has an effect on the membrane damage-and-repair mechanism of the cells. CONCLUSIONS: The results support the hypothesis that non-lethal hypotonia can enhance ultrasound-induced cell killing. These findings also suggest the 'sonomechanical' nature of the effects on the cells.

A lipophilic free radical initiator, 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) enhances caspase-dependent apoptosis induced by hyperthermia.

PURPOSE: A free radical initiator, 2,2'-azobis (2-amidinopropane) dehydrochloride (AAPH), was previously found to enhance apoptosis by hyperthermia. Here, but more lipophilic free radical initiator, 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) was investigated for its effects as a possible heat sensitizer. MATERIALS AND METHODS: Human myelogenous monocytic leukaemia U937 cells were treated with hyperthermia combined with a various concentration of AMVN for investigating its ability to induce apoptosis and various parameters to identify the pathway. RESULTS: Combined treatment of hyperthermia and AMVN induced DNA fragmentation markedly, while hyperthermia or AMVN alone induced marginal DNA fragmentation. Fractions of cells showed low mitochondrial membrane potential and increased superoxide production after the combined treatment. Experiments using various caspase inhibitors and a fluorogenic monitor of caspase 3 activities indicated that caspase acts both up- and down-stream of mitochondria. CONCLUSIONS: AMVN is suggested to be a potential heat sensitizer effective at a lower concentration than AAPH. The possible mechanism is discussed.