Aurora-A kinase inhibition enhances the cytosine arabinoside-induced cell death in leukemia cells through apoptosis and mitotic catastrophe.

Aurora-A (Aur-A) is a centrosome-associated serine/threonine kinase that is overexpressed in various cancers and potentially correlated with chemoresistance. In the Ara-C-sensitive leukemia cell lines, silencing of Aur-A by small interfering RNA transfection led to a significant increase in the Ara-C-induced cell death rate through induction of mitochondria-mediated, caspase-dependent apoptosis. In contrast, combined treatment of the Ara-C-resistant leukemia cell lines with Aur-A siRNA and Ara-C remarkably enhanced the cell death rate via non-caspase-dependent mitotic catastrophe. Taken together, Aur-A inhibition was an effective treatment for both the Ara-C-sensitive and resistant leukemia cells by increasing apoptosis and mitotic catastrophe, respectively.

Gene transfer using liposome-complexed adenovirus seems to overcome limitations due to coxsackievirus and adenovirus receptor-deficiency of cancer cells, both in vitro and in vivo.

Use of adenoviruses as vehicle for gene therapy requires that target cells express appropriate receptors such as coxsakievirus and adenovirus receptor (CAR). We show here that CAR-deficiency in cancer cells, that limits adenoviral gene delivery, can be overcome by using adenovirus complexed with the liposome, Ad-PEGPE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]. We first confirmed that CT-26 mouse colon cancer cells are deficient in CAR by RT-PCR, and then showed that CT-26 cells infected with Ad-GFP/PEGPE exhibited highly enhanced expression of green fluorescent protein (GFP), compared with those infected with Ad-GFP. GFP expression depends on the dose of liposome and adenovirus. Luciferase expression in livers treated with Ad-luc/PEGPE was about 1,000-fold less than those infected with Ad-luc. In a liver metastasis mouse tumor model developed by intrasplenic injection of CT-26 cells, luciferase expression following i.v. injection of Ad-luc/PEGPE was significantly higher in tumors than in adjacent non-neoplastic liver. Following systemic administration of Ad-GFP/PEGPE, GFP expression increased in tumors more than in adjacent liver while the reverse was true following administration of Ad-GFP. In the latter case, GFP expression was higher in liver than in tumors. This study demonstrates that systemic delivery of PEGPE-adenovirus complex is an effective tool of adenoviral delivery as it overcomes limitation due to CAR deficiency of target cells while reducing hepatic uptake and enhancing adenoviral gene expression in tumors.

Antitumor effects of systemically delivered adenovirus harboring trans-splicing ribozyme in intrahepatic colon cancer mouse model.

PURPOSE: Our previous studies suggested that human telomerase reverse transcriptase (hTERT) RNA-targeting trans-splicing ribozyme could be a useful tool for cancer gene therapy. Here, we investigated whether adenoviruses harboring this ribozyme can be systemically delivered to mice, and whether they selectively mark tumors expressing hTERT and sensitize them to ganciclovir treatments. EXPERIMENTAL DESIGN: We constructed adenoviral vectors containing modified hTERT-targeting trans-splicing ribozyme with downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by a cytomegalovirus promoter. The tumor-specific trans-splicing reaction and the tumor-killing effect of adenoviruses harboring ribozyme were investigated both in vitro and in vivo using mice with intrahepatic colon cancer metastasis via systemic administration. The safety of systemic administration of the viruses was also evaluated. RESULTS: We showed that Ad-Ribo-LacZ, when injected i.v., performs a highly specific trans-splicing reaction on hTERT mRNA and that it selectively marks tumors expressing hTERT in mice. More importantly, i.v. injection of Ad-Ribo-HSVtk plus ganciclovir significantly reduced tumor burden, with minimal liver toxicity, in mice with metastatic liver cancer, compared with the untreated group (P = 0.0009). Moreover, animals receiving Ad-Ribo-HSVtk showed improved survival compared with controls (P < 0.0001). CONCLUSIONS: This study shows that systemically delivered adenovirus harboring trans-splicing ribozyme can recognize cancer-specific transcripts and reprogram them to combat the cancer cells. Use of trans-splicing ribozymes seems to be a potentially useful gene therapy for cancer.

In vivo reprogramming of hTERT by trans-splicing ribozyme to target tumor cells.

We have developed and validated a new tumor-targeting gene therapy strategy based upon the targeting and replacement of human telomerase reverse transcriptase (hTERT) RNA, using a trans-splicing ribozyme. By constructing novel adenoviral vectors harboring the hTERT-targeting trans-splicing ribozymes with the downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by the cytomegalovirus (CMV) promoter, we demonstrated that this viral system selectively marks tumor cells expressing hTERT or sensitizes tumor cells to prodrug treatments. We confirmed that Ad-Ribo-LacZ successfully and selectively delivered a ribozyme that performed a highly specific trans-splicing reaction into hTERT-expressing cancer cells, both in vitro and in a peritoneal carcinomatosis nude mouse model. We also determined that the hTERT-specific expression of the suicide gene in the Ad-Ribo-HSVtk, and treatment with the corresponding prodrug, reduced tumor progression with almost the same efficacy as the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and in nude mouse HT-29 xenografts. These observations provide the basis for a novel approach to cancer gene therapy, and demonstrate that trans-splicing ribozymes can be employed as targeting anti-cancer agents which recognize cancer-specific transcripts and reprogram them, thereby combating cancerous cells.

Molecular imaging of endogenous mRNA expression in a mouse tumor model by adenovirus harboring trans-splicing ribozyme.

We previously showed that a trans-splicing ribozyme reprograms tumor-related genes at the mRNA level, resulting in the expression of therapeutic genes and that this approach can be efficiently employed to target specific molecules. Here, we show that trans-splicing ribozyme technology can be applied in molecular imaging of specific RNA expression in living animals. We exemplify this concept successfully by imaging mouse cytoskeleton-associated protein 2 (mCKAP2) expression in intrahepatic tumor nodules using systemically delivered adenovirus harboring mCKAP2-specific trans-splicing ribozyme.

Differential thioredoxin reductase activity from human normal hepatic and hepatoma cell lines.

Thioredoxin reductase (TrxR), a component of the thioredoxin system, including thioredoxin (Trx) and NADPH, catalyzes the transfer of electrons from NADPH to Trx, acts as a reductant of disulfide-containing proteins and participates in the defense system against oxidative stresses. In this study, the regulation pattern of TrxR in the presence of various stressful reagents was compared between Chang (human normal hepatic cell) and HepG2 (human hepatoma cell) cell lines. Aluminum chloride (0.5 mM) and zinc chloride (0.5 mM) enhanced the TrxR activity in the Chang cell line to a higher degree than in the HepG2 cell line, but cupric chloride (0.2 mM) and cadmium chloride (0.1 mM) enhanced the TrxR activity in the HepG2 cell line to a greater degree. The TrxR activities in both Chang and HepG2 cell lines were similarly induced by treatment with sodium selenite (0.02 mM) and menadione (0.5 and 1.0 mM). Lipopolysaccharide (2 micro g/m1) increased the TrxR activity upto 4.02- and 2.2-fold in the Chang and HepG2 cell lines, respectively, in time-dependent manners. Hydrogen peroxide (5 mM) markedly enhanced the TrxR activity in the HepG2 cell line, but not in the Chang cell line. NO-generating sodium nitroprusside (3.0 and 6.0 mM) induced TrxR activities in both human liver cell lines. The TrxR activity was also induced in human liver cells under limited growth conditions by serum deprivation. These results imply that the TrxR activities in normal hepatic and hepatoma cell lines are subject to different regulatory responses to various stresses.

Age-related changes in the activity of antioxidant and redox enzymes in rats.

Cellular defense system, including glutathione, glutathione-related enzymes, and antioxidant and redox enzymes, may play crucial roles in the aging of aerobic organisms. To understand the physiological roles of these factors in the aging process, their levels were compared in the livers and brains of 5-week- and 9-month-old rats. GST activity was higher in livers and brains of 9-month-old rats than in those of 5-week-old rats, and brain catalase activity was about 2-fold higher. However, it was unchanged in the livers of the 9-month-old rats. gamma-Glutamylcysteine synthetase activity was about 2-fold higher in the brains of the older rats but again not in their livers. In contrast glutathione synthetase activity appeared to be lower in the livers of the older rats while GSH content did not change with age in livers and brains. Glutathione peroxidase activity was higher in 9-month-old rat brains, but lower in 9-month-old rat livers, while superoxide dismutase activity was higher in both tissues in the older rats. The activities of two redox enzymes, thiol-transferase and thioredoxin reductase, did not change with age, nor did that of glutathione reductase. These results indicate that levels of different cellular defense systems vary with age in an irregular manner.

Regulation of the manganese-containing superoxide dismutase gene from fission yeast.

The manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates a potentially toxic superoxide radical into hydrogen peroxide and dioxygen. To study the regulation of the Schizosaccharomyces pombe MnSOD gene, the 943 bp upstream region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357, which resulted in the fusion plasmid pMS14. Restriction mapping and nucleotide sequencing confirmed its construction. The synthesis of beta-galactosidase from the fusion plasmid was induced by aluminum chloride, menadione, cadmium chloride, manganese chloride, and hydrogen peroxide. It was also induced by NO-generating S-nitroso-N-acetylpenicillamine (SNAP). However, cupric chloride and zinc chloride did not affect the synthesis of beta-galactosidase from the fusion plasmid. The beta-galactosidase synthesis appeared to be independent of the Pap1 protein. These results suggest that some metals, oxidative stress, and nitric oxide regulate the S. pombe MnSOD gene.

Activities of antioxidant and redox enzymes in human normal hepatic and hepatoma cell lines.

The cellular defense system (including glutathione, glutathione-related enzymes, antioxidant and redox enzymes) plays a crucial role in cell survival and growth in aerobic organisms. To understand its physiological role in tumor cells, the glutathione content and related enzyme activities in the human normal hepatic cell line, Chang and human hepatoma cell line, HepG2, were systematically measured and compared. Superoxide dismutase, catalase, and glutathione peroxidase activities are 2.8-, 4.3-, and 2.9-fold higher in HepG2 cells than in Chang cells. Total glutathione content is also about 1.4-fold higher in HepG2, which is supported by significant increases in gamma-glutamylcysteine synthetase and glutathione synthetase activities. Two other glutathione-related enzymes, glutathione reductase and gamma-glutamyltranspeptidase, are upregulated in HepG2 cells. However, thioredoxin reductase and glutathione S-transferase activities are significantly lower in HepG2 cells. These results propose that defense-related enzymes are largely modulated in tumor cells, which might be linked to their growth and maintenance.

Regulation of Schizosaccharomyces pombe gene encoding copper/zinc superoxide dismutase.

Copper/zinc superoxide dismutase (Cu/Zn SOD) is an abundant enzyme that scavenges superoxide radicals. To independently examine the regulation of the Cu/Zn SOD gene of the fission yeast Schizosaccharomyces pombe, the 882 bp upstream region of the Cu/Zn SOD gene was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357R, which generated the fusion plasmid pSC601. Cupric chloride (4.5 microM), aluminum chloride (10 mM), cadmium chloride (30 microM, 50 microM), mercuric chloride (1 microM), zinc chloride (11 mM), and hydrogen peroxide (0.3 mM) enhanced the synthesis of beta-galactosidase from the fusion plasmid. These results indicate that the expression of the S. pombe Cu/Zn SOD gene is, therefore, regulated by various metal ions, however superoxide-generating menadione did not affect the expression of the S. pombe Cu/Zn SOD gene. The expression of the S. pombe Cu/Zn SOD gene is also regulated by the transcription factor Pap1.