Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids.

We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtk-expressing LM05e, LM3 and B16 spheroids were 16-, three- and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight- and five-fold more efficiently. Mixed populations of HSVtk- and their respective beta gal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk-expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the beta gal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis.

Lipofection of early passages of cell cultures derived from murine adenocarcinomas: in vitro and ex vivo testing of the thymidine kinase/ganciclovir system.

Early passages of cultured cells derived from four spontaneous Balb/c murine adenocarcinomas were used to explore the feasibility of a nonviral HSVtk-based suicide gene therapy system. After lipofection with pCMVtk, the transiently HSVtk expressing P07 (lung), M3, M05, and M38 (mammary gland) cells were, respectively, about 130-, 30-, 120-, and 170-fold more sensitive to ganciclovir (GCV) in vitro than their respective controls. Eighty percent of Balb/c mice subcutaneously inoculated with ex vivo pCMVtk-lipofected P07 cells, followed by intraperitoneal GCV injection for 7 days, displayed a complete inhibition of tumor growth for over 70 days. Control animals started to display tumors 13 days after inoculation. We present evidence showing that early passages of cultured tumor cells can efficiently express lipofected genes and that they are sensitive to the lipoplex-mediated HSVtk/GCV system.

Enhanced uptake of antisense oligonucleotides using cationic liposomes and the apoptotic effect of idarubicin in K-562 cell line.

We have evaluated the apoptotic and DNA damaging activity of Idarubicin (IDA) on K-562 cells alone and following the uptake of modified antisense-oligodeoxynucleotides (AS-ODNs) targeting b3a2 mRNA of bcr/abl hybrid gene, after treatment with AS-ODNs/DCChol-DOPE (liposomes) complexes. The uptake of FITC-labeled oligonucleotide-liposomes complexes (FITC-ODNs/DCChol-DOPE) was analyzed by flow cytometry and fluorescence microscopy. Both techniques indicated cytoplasmic accumulation of labeled liposome complexes following 24h of exposure. In absence of liposomes, AS-ODNs uptake was minimal. Pre-treatment of cells with AS-ODNs/DCChol-DOPE increased the capability of IDA to induce apoptosis as determined by morphology and the comet assay. In contrast, the use of a non-sense oligodeoxynucleotide conjugated with liposomes, in the presence of IDA, did not increase K-562 cell apoptosis. Nevertheless, DNA damage in IDA treated cells was not related to ODNs/liposomes pre-treatment, as determined by the comet assay. Our data suggests that DCChol-DOPE increases the uptake of ODNs in K-562 cells, and these modified AS-ODNs increase IDA induced apoptosis by decreasing p210(bcr/abl) levels in K-562 cells.

Alpha-melanocyte-stimulating hormone through melanocortin-4 receptor inhibits nitric oxide synthase and cyclooxygenase expression in the hypothalamus of male rats.

There is evidence that alpha-melanocyte-stimulating hormone (alpha-MSH) has immunomodulatory and anti-inflammatory actions within the brain. In this study, we tested whether these actions are due to inhibition of the synthesis of nitric oxide (NO) and prostaglandins induced by lipopolysaccharide (LPS). Since melanocortin subtype MC4 receptor has been detected in the hypothalamus, we investigated the effect of central administration of alpha-MSH and HS024 (a selective MC4 receptor antagonist) on the gene expression of inducible, neuronal and endothelial NO synthase (iNOS, nNOS and eNOS) and on cyclooxygenase (COX-1 and COX-2) expression in the mediobasal hypothalamus (MBH) of LPS-treated male Wistar rats. Peripheral administration of LPS (250 microg/rat, 3 h) induced iNOS and COX-2 gene expression in the MBH. This stimulatory effect was reduced by alpha-MSH (3 nmol/rat) injected 30 min before LPS. alpha-MSH and HS024 (1 nmol/rat) alone had no effect on iNOS and COX-2 expression. The action of alpha-MSH on LPS-induced iNOS and COX-2 mRNA levels was not observed in the presence of HS024, suggesting that MC4-R may be involved in the modulatory effect of alpha-MSH. None of these treatments produced any modifications in nNOS, eNOS and COX-1 expression in MBH. The increase in serum corticosterone levels induced by LPS was attenuated by alpha-MSH. Both LPS and alpha-MSH decreased serum LH and prolactin levels. HS024 failed to modify the inhibitory effects of LPS and alpha-MSH on prolactin release but reverted the effect of LPS on LH secretion, indicating that MC4-R activation may be involved in the effects of alpha-MSH on LH secretion in male rats. When we examined the in vitro effect of LPS (10 microg/ml) and LPS plus interferon-gamma (IFN-gamma, 100 ng/ml) on iNOS expression in MBH, an increase in iNOS mRNA levels was observed only in the presence of LPS + IFN-gamma. This stimulatory effect was attenuated in the presence of alpha-MSH (5 microM), which by itself had no effect. No changes were found in nNOS, eNOS, COX-1 or COX-2 expression. These results indicate that alpha-MSH reduces the induction of iNOS and COX-2 gene expression at the hypothalamic level during endotoxemia and suggest that endogenous alpha-MSH may exert an inhibitory tone on iNOS and COX-2 transcription via MC4 receptors acting as a local anti-inflammatory agent within the hypothalamus.