Evaluation of cationic liposome suitable for gene transfer into pregnant animals.

Cationic liposome-mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the beta-galactosidase gene through intravenous injection into pregnant animals. Transduction efficiency was assessed by Southern blot analysis and expression of the transferred gene was evaluated by enzymatic demonstration of beta-galactosidase activity. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE-C reagent, a liposome formulation of the cationic lipid DMRIE (1, 2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol in membrane-filtered water met our requirements. When the plasmid DNA/DMRIE-C complexes were administered intravenously into pregnant mice at day 11.5 post coitus (p.c.), transferred genes were observed in several organs in dams and were expressed. Furthermore, although the copy numbers transferred into embryos were low, we observed reporter gene expression in the progeny.

Influence of ethyl 3-(4'-geranyloxy-3'-methoxyphenyl)-2-propenoate (EGMP) on early stage colon carcinogenesis in rats treated with azoxymethane (AOM).

The influence of ethyl 3-(4'-geranyloxy-3'-methoxyphenyl)-2-propenoate (EGMP) on the initiation and post-initiation stages of colon carcinogenesis was investigated in male F344 rats treated with azoxymethane (AOM). In experimental protocol 1, EGMP was given in the diet at 0.1 or 0.2% for 1 week together with two s.c. 15 mg/kg body weight injections of AOM on days 1 and 7 (initiation period). In protocol 2, the test compound was administered starting at week 3(post initiation stage), and in protocol 3, the test compound was given throughout the experimental period(whole stage). Sacrifice and quantitation of aberrant crypt foci (ACF) was performed at the end of week 5. Dose-dependent decreases in numbers of ACF were noted with both cases of post-initiation and whole period exposure (protocol 2 and 3), large size lesions considered most likely to be precursor lesions also being significantly reduced in the protocol 2(4-9 crypt size total with the 0.2% dose group, 48.9% and 59.6% of control values, respectively). No effects on body or liver weights were evident. The present results thus suggest that EGMP might find application as a chemopreventive agent against colon tumor development.

Inhibition of azoxymethane initiated colon tumor and aberrant crypt foci development by bovine lactoferrin administration in F344 rats.

The influence of bovine lactoferrin (bLf) on colon carcinogenesis was investigated in male F344 rats treated with azoxymethane (AOM). In experiment I, 2% and 0.2% bLf, and Bifidobacterium longum (B. longum) as a positive control at 3% were given in the diet for 4 weeks, along with two s.c. 15 mg/kg injections of AOM on days 1 and 8. The numbers of aberrant crypt foci (ACF) were decreased by both treatments. Similar results were obtained in experiment II of 13 weeks duration. In experiment III, animals were given three weekly injections of AOM and then received 2 or 0.2% bLf, 2% bLf-hydrolysate, or 0.1% bovine lactoferricin (bLfcin) for 36 weeks. No effects indicative of toxicity were noted, but significant reduction in both the incidence and number of adenocarcinomas of the large intestine was observed with almost all the treatments. Thus, the incidences of colon adenocarcinomas in the groups receiving 2 or 0.2% bLf, 2% bLf-hydrolysate, or 0.1% bLfcin were 15%, 25%, 26.3% and only 10%, respectively, in contrast to the 57.5% control value (p < 0.01). ACF values also exhibited reduced development. Investigation of beta-glucuronidase revealed decrease in the cecal contents of animals receiving bLf. In addition, demonstration of enhancement of NK activity by bLf indicated that its inhibitory effects could have been related to elevated immune cytotoxicity.

Molecular cloning and functional analysis of cDNA encoding a rat leukemia inhibitory factor: towards generation of pluripotent rat embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo. Maintenance of the stem-cell phenotype of ES cells in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) such as leukemia inhibitory factor (LIF). Here we report the cloning of complete rat LIF cDNA and its nucleotide sequence so as to facilitate studies of rat ES cell technologies on tumor biology. The nucleotide sequence of the rat LIF cDNA indicated that the rat LIF has 91% amino acid sequence identity with murine LIF. The cloned rat LIF cDNA has a putative biological activity as a differentiation-inducing factor on the murine myeloid leukemia cell line M1 cells. Culture supernatant of the rat LIF cDNA-transduced rat fibroblast cell line could maintain the stem-cell phenotype of rat ES cells which showed alkaline phosphatase activity, and this effect was much stronger than that by murine LIF. The availability of rat LIF which shows DIA will assist the in vitro analysis of rat ES cells, and culture of these cells is a route for the generation of gene targeting in rat.

p16 gene overexpression in mouse bladder carcinomas.

Deletion of 9p21 has frequently been observed in human bladder carcinomas. A candidate target suppressor gene, p16, was recently identified within this deleted region. In this study, we therefore investigated the loss of heterozygosity (LOH) of the p16 gene which is located on mouse chromosome 4, as well as its expression in mouse bladder carcinomas. We also studied the effects of normal cell contamination on LOH analysis using xenografts in CD-1(ICR) nude mice from B6C3F1 bladder carcinomas. We could not detect any LOH at the p16 locus in the mouse primary bladder carcinomas and xenografts. Surprisingly, overexpression of p16 was found in all primary mouse bladder carcinomas. Using microsatellite polymorphisms, a distinction could be made between PCR products derived from B6C3F1 and CD-1(ICR) nude mice. It was thereby confirmed that effects of normal cell contamination on LOH analysis are negligible when only tumor tissue is carefully sampled. The results suggest that abnormalities of p16 expression may be involved in mouse bladder carcinogenesis, but that gene deletion is not involved.

Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation.

The present study was carried out to examine the chemopreventive effects of carotenoids such as fucoxanthin, lycopene and lutein as well as curcumin and its derivative, tetrahydrocurcumin (THC), on development of putative preneoplastic aberrant crypt foci (ACF) in colons of mice initiated with 1,2-dimethylhydrazine dihydrochloride (DMH). Influence on proliferation of colonic crypt epithelial cells was also assessed in terms of 5-bromo-2'-deoxyuridine (BrdU) incorporation. Five-week-old B6C3F1 male mice were divided into three groups, groups 1 and 2 being given DMH (20 mg/kg body wt, s.c.) twice a week for 3 weeks. Animals of group 1 were then treated with one of the test compounds, lycopene (0.005% and 0.0025%) or fucoxanthin (0.01%) in the drinking water and lutein (0.05%), curcumin (0.5%) or THC (0.5% and 0.2%) in the diet from weeks 5-12. Group 2 served as a carcinogen alone control and group 3 mice were given test compounds alone. All animals were killed at week 12. Numbers of ACF/mouse in the group 1 treated with fucoxanthin (47.1 +/- 13.7), lutein (42.6 +/- 19.6) or 0.5% THC (46.6 +/- 17.7) were significantly decreased as compared to the control group 2 value (63.3 +/- 19.4) (P < 0.01). Numbers of aberrant crypts (ACs)/mouse were also significantly lower after treatment with lutein (79.9 +/- 34.7) or 0.5% THC (81.8 +/- 32.5) than in the control group (115.1 +/- 37.1) (P < 0.01). BrdU labeling indices (LI) in mice treated with lutein and 0.5% THC were significantly decreased in both upper and lower half compartments of colonic crypts as compared to the controls (P < 0.05 and 0.01, respectively), especially the upper half data corresponding to reduction of ACs/mouse. The results thus suggest that fucoxanthin, lutein, and THC may have potential as chemopreventive agents against colon carcinogenesis.

Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats.

The influence of bovine lactoferrin (bLF) on colon carcinogenesis was investigated in male F344 rats treated with azoxymethane (AOM). Following three weekly injections of AOM, the animals received 2 or 0.2% bLF for 36 weeks. No effects indicative of toxicity were noted, but significant reduction in both the incidence and number of adenocarcinomas of the large intestine was observed with both doses. Thus, the incidences of adenocarcinomas in the groups receiving 2% and 0.2% bLF were 15% and 25%, respectively, in contrast to the 57.5% control value (P < 0.01 and P < 0.05, respectively). The results indicate that bLF might find application for chemoprevention of colon cancer.

Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane.

The influence of concomitant administration of bovine lactoferrin (bLF) on induction of aberrant crypt foci (ACF) by azoxymethane was investigated in male F344 rats. Two percent bLF and 3% Bifidobacterium longum (B. longum), as a positive control, significantly decreased the numbers of ACF as well as the total numbers of aberrant crypts reproducibly in three independent studies (2% bLF, P < 0.01; 3% B. longum, P < 0.05). Most importantly large size foci composed of four or more crypts were always significantly decreased by 2% bLF (P < 0.05). Additional investigation of the natural killer activity of spleen cells demonstrated enhancement by bLF (P < 0.01) and B. longum (P < 0.01) in line with the levels of influence on foci induction, indicating a possible role for elevated immune cytotoxicity in the observed inhibition.

Decreased connexin32 and a characteristic enzyme phenotype in clofibrate-induced preneoplastic lesions not shared with spontaneously occurring lesions in the rat liver.

Two different types of focal preneoplastic lesions, tentatively named Type I and II lesions, were recognized in the liver of rats chronically treated with clofibrate for 104 weeks. Type I lesions were characterized by mostly negative glucose-6-phosphate dehydrogenase (G6PD) activity (6 out of 10, 60%) and positive expression of succinate dehydrogenase (10 out of 10, 100%), in addition to the previously documented complete lack of expression of glutathione S-transferase, placental form (GST-P) and gamma-glutamyl transpeptidase (GGT). Furthermore, most importantly, Type I lesions exhibited a clear decrease in immunohistochemically demonstrated connexin32 (Cx32) spot counts on their hepatocyte membranes, similarly to nitrosamine-induced lesions. In contrast, Type II lesions, mostly small in size and positively expressing GST-P and/or GGT and G6PD, similarly to their previously reported nitrosamine-induced counterparts, did not exhibit a significant decrease in Cx32 count. In addition, spontaneously occurring lesions, again sharing the same enzyme phenotype, did not show a decrease in Cx32. The results indicate that: (i) a clear distinction between the two lesions, with Type I being involved in clofibrate-induced tumors and Type II being more likely to be spontaneous in nature; (ii) a decrease in Cx32 is closely linked to lesion development and possibly stage of progression, irrespective of the enzyme phenotype and the applied carcinogen; (iii) the unaltered condition of Cx32 may suggest a slow growing or non-progressive nature.

Decreased levels of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts in rats treated with beta-carotene, alpha-tocopherol and freeze-dried aloe.

To assess mechanisms of chemoprevention of hepatocarcinogenesis by trans-beta-carotene (beta-C), DL-alpha-tocopherol (alpha-T), and freeze-dried whole leaves of Kidachi aloe (Aloe), formation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adducts was measured by 32P-post-labeling analysis, and CYP1A1 and CYP1A2 protein levels were analyzed by ELISA. Group 1 rats were fed diet containing 0.02% beta-C, 1.5% alpha-T or 30% Aloe over an 8-day period, while group 2 was given basal diet alone. On day 7, all animals were subjected to two-thirds partial hepatectomy (PH). Twelve hours after PH, they received a single dose of the carcinogenic food pyrolysate IQ (100 mg/kg) intragastrically, to initiate hepatocarcinogenesis. Rats were killed 6, 12, 24 and 48 h after IQ administration. The levels of adducts, expressed as relative adduct labeling values in rats treated with beta-C, alpha-T and Aloe, were decreased as compared with the control group at hour 24 (36 h after PH), with a significant difference in the case of the beta-C group (46.4% of the control value). Similarly, all showed a tendency for decrease at hour 48. Furthermore, the levels of CYP1A2, known to be responsible for activation of IQ, showed a significant reduction at hour 24. It is concluded that beta-C, and possibly also alpha-T and Aloe, have the potential to reduce IQ-DNA adduct formation, presumably as a result of decreased formation of active metabolites. The results may explain, at least in part, the previously observed inhibitory effects of these compounds on induction of preneoplastic hepatocellular lesions.

Modifying influence of swine-serum-induced liver fibrosis on development of preneoplastic lesions in rat liver.

Modifying effects of fibrosis or a cirrhotic state, caused by treatment with swine serum (SS), on the induction of preneoplastic focal lesions were assessed in a rat medium-term liver bioassay model for the detection of environmental carcinogens, in which the test compound is administered during the promotion phase after initiation with diethylnitrosamine. In experiment I, repeated intraperitoneal administration of SS concomitantly with the hepatopromoting agent deoxycholic acid (DCA) or phenobarbital (PB) resulted in a cirrhotic state and a significant increase in the number or size of preneoplastic glutathione S-transferase placental form (GST-P)-positive liver cell foci as compared to the corresponding DCA or PB alone groups. In experiment II, SS was given prior to commencement of the same medium-term bioassay system, in which a known hepatopromoting agent, DCA, 17-alpha-ethynylestradiol, or 2-acetylaminofluorene, was applied. In this case, the liver did not show obvious cirrhotic change and, rather than any enhancement, slight inhibition of promotion occurred. The results indicate that a coexisting, but not a pre-existing, cirrhotic condition acts to increase growth pressure on GST-P+ preneoplastic foci, and suggest that concomitant administration of SS with the promoting agent could be applied to improve the sensitivity of the assay protocol.

Demonstration of organotropic effects of chemopreventive agents in multiorgan carcinogenesis models.

Organotropic chemopreventive effects of three (pro)vitamins and three unsaturated fatty acids were examined using mouse and rat multiorgan carcinogenesis models. For the study of (pro)vitamins, male and female B6C3F1 mice were treated with N,N-diethylnitrosamine (DEN) and N-methyl-N-nitrosourea (MNU) during the first 11 weeks, then from weeks 12 to 32 they received alpha-carotene (0.4 mg/mouse), beta-carotene (0.4 mg/mouse) or alpha-tocopherol (40 mg/mouse) three times a week by gavage; control mice received vehicle alone. In male mice, alpha-carotene significantly reduced liver weights, representing a reduced tumour mass (P < 0.001), and alpha-carotene, beta-carotene and alpha-tocopherol significantly reduced the numbers of liver tumours (adenomas and carcinomas combined) (P < 0.001-0.01) as compared with control mice, the effects being greatest with alpha-carotene. In female mice, alpha-carotene significantly decreased the number of liver tumours (P < 0.001). In the lung, alpha-carotene and alpha-tocopherol reduced the area of lesions (hyperplasias and adenomas combined) only in males (P < 0.05). For the study of unsaturated fatty acids, F344 male rats were treated with DEN, MNU, N-butyl-N-hydroxybutylnitrosamine (BBN), 1,2-dimethylhydrazine (DMH) and N,N-bis(2-hydroxy)propylnitrosamine during the first 5 weeks, then from weeks 6 to 36 they were given docosahexaenoic acid (C22:6), eicosapentaenoic acid (C20:5) or linoleic acid (C18:2) at 1.0 g/rat, three times a week by gavage; control rats were treated with oleic acid (C18:1) using the same protocol. All animals were fed a low linoleic acid and calorie-adjusted basal diet during fatty acid administration. Docosahexaenoic acid and linoleic acid reduced tumours in the large and small intestines, respectively. However, they did not influence the yield of preneoplastic liver, lung, kidney, forestomach and urinary bladder lesions. The data thus provide evidence for organotropic effects of carotenoids and unsaturated fatty acids on carcinogenesis.

Clofibrate-induced neoplastic development in the rat liver is associated with decreased connexin 32 expression but not with a co-ordinated shift in expression of marker enzymes.

Altered enzyme phenotype and expression of connexin 32 (Cx32), a gap junction protein were studied during the development of rat liver tumors induced by the non-genotoxic carcinogen, clofibrate. (1) In contrast to previous findings for nitrosamine-induced lesions, preneoplastic enzyme-altered foci (EAF) and neoplastic nodules (NN) lacked any clear association with degree of altered enzyme expression because of an almost complete negativity for GST-P and GGT. (2) Immunohistochemically demonstrated Cx32 spots on the hepatocyte membranes showed a clear decrease in clofibrate-induced lesions. (3) Naturally occurring EAF demonstrating GST-P and/or GGT positivity did not show a significant decrease of Cx32 counts suggesting a reversible nature. Therefore, the Cx32 decrease appears closely linked to progression of hepatocarcinogenesis irrespective of the enzyme phenotype of neoplastic focal lesions and the carcinogens used for their induction.