Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells.
We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required. (+info)
The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice.
Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis. (+info)
Mutational inactivation of the xeroderma pigmentosum group C gene confers predisposition to 2-acetylaminofluorene-induced liver and lung cancer and to spontaneous testicular cancer in Trp53-/- mice.
Mice that are genetically engineered to mimic the human hereditary cancer-prone DNA repair-defective disease xeroderma pigmentosum (XP) are highly predisposed to UV radiation-induced skin cancer. It is not clear, however, whether XP mice or humans are predisposed to cancers in other tissues associated with exposure to environmental carcinogens. To test the importance of nucleotide excision repair in protection against chemical carcinogenesis in internal organs, we treated XPC mutant (XPC-/-) mice with 2-acetylaminofluorene and NOH-2-acetylaminofluorene. We observed a significantly higher incidence of chemically induced liver and lung tumors in XPC-/- mice compared with normal and heterozygous littermates In addition, the progression of liver tumors in XPC-/- Trp53+/- mice is accelerated compared with XPC-/- Trp53+/+ animals. Finally, we demonstrate a higher incidence of spontaneous testicular tumors in XPC-/- TrpS3-/- double mutant mice compared with XPC+/+ Trp53-/- mice. (+info)
Possible carcinogenic effects of X-rays in a transgenerational study with CBA mice.
A lifetime experiment using 4279 CBA/J mice was carried out to investigate whether the pre-conceptual exposure of sperm cells to X-ray radiation or urethane would result in an increased cancer risk in the untreated progeny, and/or increased susceptibility to cancer following exposure to a promoting agent. The study consisted of four main groups, namely a control group (saline), a urethane group (1 mg/g body wt) and two X-ray radiation groups (1 Gy, 2 Gy). At 1, 3 and 9 weeks after treatment, the males of these four parental groups were mated with untreated virgin females. The offspring of each parental group was divided into two subgroups: one received s.c. urethane (0.1 mg/g body wt once) as a promoter, the other saline, at the age of 6 weeks. All animals were evaluated for the occurrence of tumours. K-ras oncogene and p53 tumour suppressor gene mutations were investigated in frozen lung tumour samples. The female offspring of male parents exposed to X-rays 1 week before their mating showed a trend towards a higher tumour incidence of the haematopoietic system than the F1 controls. In addition, a higher percentage of bronchioloalveolar adenocarcinomas in male offspring born to irradiated paternals mated 1 week after X-ray treatment points to a plausible increased sensitivity of post-meiotic germ cell stages towards transgenerational carcinogenic effects. On the other hand, no increased tumour incidence and malignancy were observed in the offspring born to irradiated paternals mated 3 and 9 weeks after X-ray treatment. Paternal urethane treatment 1, 3 and 9 weeks prior to conception did not result in significantly altered incidence or malignancy of tumours of the lung, liver and haematopoietic tissue in the offspring. K-ras mutations increased during tumour progression from bronchioloalveolar hyperplasia to adenoma. Codon 61 K-ras mutations were more frequent in lung tumours of urethane-promoted progeny from irradiated parents than from control parents. P53 mutations were absent from these lung alterations. (+info)
Alterations of lipid and cholesterol metabolism in cachectic tumor-bearing rats are prevented by insulin.
The ascites hepatoma Yoshida AH130 causes in the host a rapid and progressive body weight loss, associated with reduced food intake, and protein and lipid hypercatabolism. Because insulin regulates glucose as well as lipid and protein metabolism, we suggest that the observed alterations are at least in part secondary to hypoinsulinemia and/or to the increase of counterregulatory hormones in AH130-bearing rats. To verify this hypothesis, controls with free access to food (n = 4), controls with free access to food plus insulin (107 micromol. kg body wt-1. d-1) (n = 4), controls pair-fed to the tumor-bearing rats (n = 4), pair-fed controls treated with insulin (n= 4), tumor hosts (n = 9), and tumor hosts treated with insulin (n = 6) were used. The Yoshida ascites hepatoma cells ( approximately 10(8) cells/rat) were inoculated intraperitoneally. Daily food intake and body weight were measured; insulin was injected starting the day of tumor implantation for 6 d. The metabolism of both cholesterol and lipids was investigated in tumor cells, and ascitic fluid and blood serum were investigated at the end of treatment. Insulin prevented the reduction of food intake (19 +/- 0.6 vs. 13 +/- 0.4 g/d, P < 0.01; AH130 hosts treated and not treated with insulin, respectively), the loss of body weight (202 +/- 12 vs. 135 +/- 9 g, P < 0.01), lowered the circulating triglycerides (48.3 +/- 4.9 vs. 84.5 +/- 7.1 mmol/L, P < 0.01), and free fatty acids (561 +/- 47 vs. 989 +/- 54 mmol/L (P < 0.01), while corrected the decrease of adipose lipoprotein lipase activity (1,240 +/- vs. 300 +/- pmol FA, P < 0.01) observed in AH130 hosts. Moreover, insulin prevented the decrease in HDL cholesterol (13.2 +/- 0.8 vs. 9.3. +/- 0.7 mmol/L, P < 0.01) and significantly increased hepatic cholesterol synthesis as evaluated by 14C-acetate incorporation into cholesterol, in both liver (3,337 +/- 245 vs. 830 +/- 115 Bq/g, P < 0.01) and AH130 cells (11,676 +/- 1,693 vs. 4,196 +/- 527 Bq/10(6) cells, P < 0.01). Thus insulin treatment ameliorated many metabolic derangements, with a lengthening of rats survival time (7 +/- 1 vs. 11 +/- 1 d, P < 0.05) without significantly stimulating tumor growth. These data, together with our previous observations on the effectiveness of insulin on protein turnover perturbations, suggest that many metabolic alterations occurring during cancer cachexia can be avoided by the administration of this hormone. (+info)
Rapid induction of cytokine and E-selectin expression in the liver in response to metastatic tumor cells.
The cytokine-inducible endothelial cell adhesion receptor E-selectin has been implicated in cancer metastasis. Previously, we reported that experimental liver metastasis of Lewis lung carcinoma subline H-59 cells could be abrogated in animals treated with an anti-E-selectin antibody. To gain further insight into the functional relevance of E-selectin expression to liver colonization, we investigated here the time course of cytokine and hepatic E-selectin expression after the intrasplenic/portal inoculation of H-59 cells by using a combination of reverse transcription-PCR, Northern blot analysis, immunohistochemistry, and in situ hybridization. In parallel, we analyzed cytokine induction in response to the injection of Lewis lung carcinoma subline M-27 and murine melanoma B16-F1 cells, which do not spontaneously metastasize to the liver. In livers derived from normal or saline-injected mice, only minimal basal levels of TNF-alpha and IL-1 mRNA were detectable by RT-PCR. Rapid cytokine mRNA induction was noted within 30-60 min of H-59 injection, reaching maximal levels at 4-6 h. This was followed by the appearance of E-selectin mRNA, which was detectable at 2 h after injection and reached maximal levels at 6-8 h, declining to basal levels by 24 h. In situ hybridization analysis and immunohistochemistry localized E-selectin mRNA and protein, respectively, to the sinusoidal endothelium. M-27 cells failed to induce cytokine or E-selectin expression, whereas B-16 cells elicited a delayed and more short-lived response. The results demonstrate that upon entry into the hepatic circulation, tumor cells can rapidly trigger a molecular cascade leading to the induction of E-selectin expression on the sinusoidal endothelium and suggest that E-selectin induction may contribute to the liver-colonizing potential of tumor cells. (+info)
Structural and mechanistic aspects of transcriptional induction of cytochrome P450 1A1 by benzimidazole derivatives in rat hepatoma H4IIE cells.
The effect of several structurally different benzimidazole compounds on CYP1A1 expression at the transcriptional, mRNA and protein levels was investigated in the rat hepatoma H4IIE cell line. Omeprazole, thiabendazole, carbendazim, 2-mercaptobenzimidazole and 2-mercapto-5-methoxybenzimidazole caused a dose-dependent increase in CYP1A1 protein levels that reached maximum effect at 250 microm, as measured by Western blot. In addition, hydroxyomeprazole, 2-aminobenzimidazole and 2-mercapto-5-nitro-benzimidazole caused a notable increase in CYP1A1 protein expression, whereas 5-O-desmethylomeprazole, 2-hydroxybenzimidazole, 2-benzimidazole propionic acid and 5-benzimidazole carboxylic acid were ineffective. Thus, benzimidazole substituted with a thiol or an amino group in the 2-position were active inducers. Northern blot analysis confirmed an extensive increase of CYP1A1 mRNA induced by omeprazole and 2-mercapto-5-methoxybenzimidazole which was 32% and 49% of maximal induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) respectively, whereas thiabendazole and carbendazim showed approximately 15% increase as compared to TCDD. Transient transfection of H4IIE cells, with a XRE-pGL3 reporter gene construct revealed a 2.3-4.3-fold induction by carbendazim, thiabendazole, and 2-mercapto-5-methoxybenzimidazole as compared to a 3.3- and 23-fold induction by omeprazole and TCDD, respectively. Thus, these data indicate that the benzimidazoles utilize the aryl hydrocarbon receptor-arnt-XRE-mediated signal-transduction pathway for induction of the CYP1A1 gene. (+info)
Bystander killing of tumour cells by antibody-targeted enzymatic activation of a glucuronide prodrug.
RHI-betaG-PEG, formed by linking poly(ethylene glycol)-modified beta-glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS-30D rat hepatoma cells. Sequential treatment of cells with 10 microg ml(-1) RH1-betaG-PEG and 20 microM BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-30D and N1S1 cells in BALB/c nu/nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-betaG-PEG in solid AS-30D and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection. Intravenous treatment with RH1-betaG-PEG and BHAMG cured seven of seven nude mice bearing solid s.c. AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo. (+info)