Search

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.

A trans-splicing ribozyme which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful agent for tumor-targeted gene therapy. In this study, we evaluated in vivo function of the hTERT-targeting trans-splicing ribozymes by employing the molecular analysis of expression level of genes affected by the ribozyme delivery into peritoneal carcinomatosis mice model. To this effect, we constructed adenoviral vector encoding the specific ribozyme. Noticeably, more than four-fold reduction in the level of hTERT RNA was observed in tumor nodules by the systemic infection of the ribozyme-encoding virus. Such hTERT RNA knockdown in vivo induced changes in the global gene expression profile, including the suppression of specific genes associated with anti-apoptosis including bcl2, and genes for angiogenesis and metastasis. In addition, specific trans-splicing reaction with the targeted hTERT RNA took place in the tumors established as peritoneal carcinomatosis in mice by systemic delivery of the ribozyme. In conclusion, this study demonstrates that an hTERT-specific RNA replacement approach using trans-splicing ribozyme represents a potential modality to treat cancer.

Imatinib mesylate is a selective Bcr/Abl kinase inhibitor and an effective anticancer agent for Bcr/Abl-positive chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Mutations within the BCR/ABL kinase domain are the most commonly identified mechanism associated with relapse. To overcome the imatinib resistance in CML, many investigators have tried to clarify molecular mechanism for imatinib resistance in cells of patients who failed to respond to imatinib. Our aim was to invesitigate underlying mechanism for imatinib resistance in SR-1 cells, which were derived from a CML patient in blast crisis. We detected the new mutation of BCR/ABL, resulting in premature termination and loss of BCR/ABL fusion protein expression, which might be possible mechanism for the resistance to imatinib in SR-1 cells.

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21(Waf1), p27(Kip1) and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21(Waf1) was increased, while p27(Kip1) and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21(Waf1) over-expression.

Repetitive low dose thioacetamide (TA) treatment of hepatocytes was found to induce cells in G2 arrest. In the present study, an attempt was made to investigate alterations in expression of cell cycle regulators after G1 progression in the same repetitive low dose TA treated hepatocytes system and to define the determinators involved in G2 arrest. TA was daily administered intraperitoneally, with a dose of 50 mg/kg for 7 days. Expression levels of cyclin E and CDK2 were similar, increased at day 1 and reached a peak at day 2. And they recycled from day 3 reaching a second peak at day 5. Expression level of cyclin A was similar to p27(Kip1) and p57(Kip2) but not to CDK2 and increased to a peak level at day 2. Expression levels of cyclin B1 and cdc2 were similar although the cyclin B1 level was generally low, decreased from day 1 to basal levels at day 3 and persisted at a low level till day 7. The expression level of cyclin G1 was similar to p53 that peaked at day 3 and again at day 6 elevated over basal level. BrdU-labeled hepatocytic nuclei increased from 12 h, reached a peak at day 2, then decreased, and were not detectable from day 6. The number of PCNA-labeled nuclei increased immediately, peaked at day 2, and maintained till day 7. These results suggest that G2 arrest induced by repeated TA treatment might be p53-dependent, via activation of cyclin G1, rather than inhibition of cyclin B1- cdc2 complex, and inhibitors holding S phase progression might be p27(Kip1) and p57(Kip2).

Since there have been very few studies on nucleolar signaling, an attempt was made to establish nucleolar signal pathways which link the cell membrane to the nucleolus for the transfer of extracellular signals. Two pathways were studied. One was the G alpha s mediated cAMP pathway where two signal molecules were yielded, including RII and protein kinase A. The other was the G alpha q mediated DAG/IP3 pathway which yields two signals including protein kinase C and IP3/Ca2+. By the studying isolated nucleoli from resting liver, regenerating liver or weak carcinogen thioacetamide treated liver, it was possible to detect protein kinase A (PKA), protein kinase C (PKC) and RII subunits. In addition, CK2 was detected. It was found that external signals transmitted through G protein coupled receptors could reach into the nucleolus and that physical translocation of signal molecules was an integral step involved in membrane-nucleolus linked pathways. When an in vitro assay of the above signal molecules was carried out using [gamma-32P]-ATP, most kinase dependent phosphorylation was via the major CK2 (more than 95%). Therefore, it is suggested that the major CK2 dependent pathway is involved in 'house keeping' for nucleolar integrity and the minor pathways, dependent on PKA, PKC and others, are involved in subtle regulatory mechanisms such as 'extra-house-keeping' activities by nucleolar chromosomal remodeling.