Hypoxia-inducible factor-1␣ (HIF-1␣) is an important transcriptional factor that is activated when mammalian cells experience hypoxia, a tumor microenvironmental condition that plays pivotal roles in tumor progression and treatment. In this study, we examined the idea of downregulating HIF-1␣ in tumor cells for therapeutic gain. We show that the expression levels of HIF-1␣ can be significantly attenuated by use of the recently established small interfering RNA technology in combination with adenovirus-mediated gene transfer. Down-regulation of the HIF-1␣ protein enhanced hypoxia-mediated tumor cell apoptosis in vitro. Subcutaneous tumor growth was also prevented from cells with attenuated HIF-1␣ expression. In addition, intratumoral injection of adenovirus encoding the HIF-1␣-targeted small interfering RNA had a small but significant effect on tumor growth when combined with ionizing radiation. Therefore, our results provide proof of HIF-1␣ as an effective target for anticancer therapy. They also suggest that an adenovirus-based small interfering RNA gene transfer approach may be a potentially effective adjuvant strategy for cancer treatment.

Systemic virus dissemination is a potential problem during local gene delivery in solid tumours. However, the kinetics and pathways of the dissemination have not been well characterised during the first 24 h after the infusion is started. To this end, we infused adenoviral vectors for luciferase or enhanced green fluorescence protein into three different tumour models in mice. During and/or after the infusion, we determined the amount of adenoviruses in the tumour, blood, and liver, and examined the transgene expression in the liver, lung, blood, and tumour. In addition, we intravenously injected tumour cells expressing luciferase and examined the biodistribution of these cells in the body. We observed transgene expression in the liver and tumour at 24 h after the infusion, but could not detect transgene expression in the blood and lung. The peak concentration of viral vectors in the plasma occurred during the intratumoral infusion. At 10 min after the infusion, few viral vectors remained in the blood and the ratio of copy numbers of adenoviruses between liver and tumour was 42 in 80% and X10 in 40% of the mice. Most tumour cells injected intravenously accumulated in the lung within the first 24 h. Taken together, these data indicated that systemic virus dissemination occurred mainly during the first 10 min after the intratumoral infusion was started, and that the dissemination was due to infusion-induced convective transport of viral vectors into leaky tumour microvessels.

Citrin, encoded by SLC25A13, constitutes the malate-aspartate shuttle, the main NADH-shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Citrin deficiency is predicted to impair hepatic glycolysis and de novo lipogenesis, resulting in hepatic energy deficit. Secondary decrease in hepatic argininosuccinate synthetase (ASS1) expression has been considered a cause of hyperammonemia in CTLN2. We previously reported that medium-chain triglyceride (MCT) supplement therapy with a low-carbohydrate formula was effective in CTLN2 to prevent a relapse of hyperammonemic encephalopathy. We present the therapy for six CTLN2 patients. All the patients' general condition steadily improved and five patients with hyperammonemic encephalopathy recovered from unconsciousness in a few days. Before the treatment, plasma glutamine levels did not increase over the normal range and rather decreased to lower than the normal range in some patients. The treatment promptly decreased the blood ammonia level, which was accompanied by a decrease in plasma citrulline levels and an increase in plasma glutamine levels. These findings indicated that hyperammonemia was not only caused by the impairment of ureagenesis at ASS1 step, but was also associated with an impairment of glutamine synthetase (GS) ammonia-detoxification system in the hepatocytes. There was no decrease in the GS expressing hepatocytes. MCT supplement with a low-carbohydrate formula can supply the energy and/or substrates for ASS1 and GS, and enhance ammonia detoxification in hepatocytes. Histological improvement in the hepatic steatosis and ASS1-expression was also observed in a patient after long-term treatment.

A 46-year-old man presented with severe tension pneumocephalus triggered by mild head injury 7 years after craniotomy. He had a history of subarachnoid hemorrhage due to ruptured anterior communicating artery aneurysm, coating of the aneurysm performed via a craniotomy, and a ventriculoperitoneal (VP) shunt inserted. He fell from bed in a rehabilitation hospital. Eight hours after the injury, he became comatose and suffered general convulsion. He was then transferred to our hospital. Radiography and computed tomography (CT) revealed a large amount of intracranial air and a widely opened frontal sinus. On the day of admission, the shunt tube was ligated. Surgery was performed to repair the dura mater and close the frontal sinus. Postoperative CT revealed reduction in the amount of air and frontal sinus obstruction. The patient had a good postoperative course without meningitis. Tension pneumocephalus may occur as a complication several years after a craniotomy because of the chronic lowering of intracranial pressure induced by a VP shunt. Complete frontal sinus repair is important during the initial craniotomy.

Cultured human cervical cancer (HeLa) and rat mammary carcinoma (R3230Ac) cells were transfected with vectors encoding green fluorescent protein (GFP) under the control of hsp70B promoter. Aliquots of 10-μl transfected cells (5 × 10 7 cells/ml) were placed in 0.2-ml thin-wall polymerase chain reaction tubes and exposed to 1.1-MHz high intensity focused ultrasound (HIFU) at a peak negative pressure P − =2.68 MPa. By adjusting the duty cycle of the HIFU transducer, the cell suspensions were heated to a peak temperature from 50 to 70 °C in 1-10 s. Exposure dependent cell viability and gene activation were evaluated. For a 5-s HIFU exposure, cell viability dropped from 95% at 50 °C to 13% at 70 °C. Concomitantly, gene activation in sublethally injured tumor cells increased from 4% at 50 °C to 41% at 70 °C. A similar trend was observed at 60 °C peak temperature as the exposure time increased from 1 to 5 s. Further increase of exposure duration to 10 s led to significantly reduced cell viability and lower overall gene activation in exposed cells. Altogether, maximum HIFU-induced gene activation was achieved at 60 °C in 5 s. Under these experimental conditions, HIFU-induced gene activation was found to be produced primarily by thermal rather than mechanical stresses.

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Tumor-derived glucose-regulated protein 94 (GRP94/gp96) has shown great promise as a tumor vaccine. However, current protein-based approaches require the availability of large quantities of tumor tissue, which are often not possible. In addition, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of a combined GRP94/gp96-based genetic immunotherapy and radiation therapy strategy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding a modified, secretable form of GRP94 gene (AdsGRP94) was constructed and evaluated in various antitumor experiments. Lethally irradiated, virus-infected cells were used as vaccines. Adenoviral vectors were also injected directly into tumors in conjunction with tumor irradiation. Vaccination with lethally irradiated, AdsGRP94-infected 4T1 cells completely prevented subsequent tumor growth from challenge inoculations of as many as 10 7 cells per mouse. In established tumor models, vaccinations alone had minimal effect on local and metastatic tumor growth. However, when vaccination was combined with radiation therapy and i.t. AdsGRP94 injections, local tumor growth and pulmonary metastasis were markedly inhibited. In some cases, complete tumor regression was observed. In these cases, the mice were resistant to subsequent tumor challenge and remain tumor free up to 10 months after initial therapy. Our results indicate that combined AdsGRP94-based immunotherapy and radiation therapy may be a potentially effective strategy for cancer treatment. (Cancer Res 2005; 65(20): 9126-31)