PubMed Health⌕ Search

Biomedical subjects

S Kong

Publications and source records attributed to S Kong.

At least 19 recordsLinked to original sources
PubMed2005

Association between health-related quality of life and clinical efficacy endpoints in rheumatoid arthritis patients after four weeks treatment with anti-inflammatory agents.

BACKGROUND: Improvement of health-related quality of life (QoL) is increasingly recognized as a maj or treatment goal for patients with rheumatoid arthritis (RA). There are several measures of general health status and of physical functioning for assessing treatment effects on QoL in patients with RA, however, the relationship between QoL outcomes and conventional clinical efficacy endpoints is not completely understood. OBJECTIVE: To describe the association between changes in QoL and changes in other efficacy measures, among patients with RA after four weeks of treatment with etoricoxib, naproxen or placebo, and to explore differences in the association of changes in efficacy and changes in QoL parameters across treatment groups. METHODS: The study used data from 1684 patients with RA enrolled in two identical clinical trials (one US and one multinational). Patients were randomized to placebo, etoricoxib 90 mg once daily, or naproxen 500 mg twice daily in a 2 : 2: 1 allocation ratio. Primary efficacy endpoints were tender joint count, swollen joint count, patient global assessment of disease activity (100 mm VAS), and investigator global assessment of disease activity (0 - 4 Likert scale). QoL assessments were based on the Health Assessment Questionnaire (HAQ) and the Medical Outcomes Survey Short Form 36 (SF-36). Mean differences between baseline and week four were calculated for each parameter studied. Linear regression analysis was performed to assess the association between changes in clinical efficacy and changes in QoL parameters, adjusted for covariates. RESULTS: The degree of association between changes in tender or swollen joint counts and changes in QoL variables was low, explaining less than 10% of the variability for most QoL variables, except bodily pain (SF-36). In contrast, changes in patient global assessment of disease activity explained 33% of the variability in the overall HAQ score, and in the physical component score (SF-36; adjusted regression models). Values for investigator global assessment of disease activity were below those for patient global assessment but above joint count measures. Results were similar between the etoricoxib, naproxen and placebo groups in the degree of association between changes in efficacy and QoL variables. CONCLUSION: Currently used efficacy endpoints are less than ideal predictors of change in QoL. There is no evidence from this study that the association between changes in CE endpoints and QoL was different across treatments. Our results highlight the need to assess both conventional efficacy measures and QoL in clinical trials of RA treatments.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2002

Gamma-linolenic acid in borage oil reverses epidermal hyperproliferation in guinea pigs.

As dietary sources of gamma-linolenic acid [GLA; 18:3(n-6)], borage oil (BO; 24-25 g/100 g GLA) and evening primrose oil (PO; 8-10 g/100 g GLA) are efficacious in treating skin disorders. The triglycerol stereospecificity of these oils is distinct, with GLA being concentrated in the sn-2 position of BO and in the sn-3 position of PO. To determine whether the absolute level and/or the triglycerol stereospecificity of GLA in oils affect biological efficacy, epidermal hyperproliferation was induced in guinea pigs by a hydrogenated coconut oil (HCO) diet for 8 wk. Subsequently, guinea pigs were fed diets of PO, BO or a mixture of BO and safflower oil (SO) for 2 wk. The mixture of BO and SO (BS) diet had a similar level of GLA as PO but with sn-2 stereospecificity. As controls, two groups were fed SO and HCO for 10 wk. Epidermal hyperproliferation was reversed by all three oils in the order of BO > BS > PO. However, proliferation scores of group PO were higher than of the normal control group, SO. The accumulations of dihomo-gamma-linolenic acid [DGLA; 20:3(n-6)], an elongase product of GLA, into phospholipids and ceramides, of 15-hydroxyeicosatrienoic acid (15-HETrE), the potent antiproliferative metabolite of DGLA, and of ceramides, the major lipid maintaining epidermal barrier, in the epidermis of group BO were greater than of groups BS and PO. Group BS had higher levels of DGLA, 15-HETrE and ceramides than group PO. With primary dependence on absolute levels, our data demonstrate that the antiproliferative efficacy of GLA in the epidermis is preferably exerted from sn-2 stereospecificity of GLA in BO.

Acanthosis Nigricans↗
PubMed2001

Efficient delivery of Antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8+ T cells.

The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.

Animals↗
PubMed2001

Oxazole yellow homodimer YOYO-1-labeled DNA: a fluorescent complex that can be used to assess structural changes in DNA following formation and cellular delivery of cationic lipid DNA complexes.

To improve transfection efficiency following delivery of plasmid expression vectors using lipid-based carriers, it is crucial to define structural characteristics of the lipid/DNA complexes that optimize transgene expression. Due to its strong affinity for DNA and high quantum yield, the fluorescent DNA intercalator YOYO-1 was used as a tool to assess changes in DNA that occur following lipid binding and cell delivery. In this study, the stability of the dye/DNA complex following binding of poly-L-lysine or monocationic lipids is characterized. More than 98% of the fluorescence measured for a defined DNA/YOYO-1 complex was lost when DNA was condensed using poly-L-lysine. This loss in fluorescence could be attributed to displacement of bound dye. In contrast, more than 30% of the fluorescence of the dye-labeled DNA was retained after formation of cationic lipid/DNA complexes. Significantly, the results illustrate differences in structural changes cationic lipids and PLL exert on plasmid DNA. The fluorescent lipid/DNA complex was used to assess DNA delivery to murine B16/BL6 cells in vitro. An assay relying on fluorescence resonance energy transfer between bound YOYO-1 and propidium iodide was used to distinguish between DNA attached to the cell surface and internalized DNA.

Benzoxazoles↗
PubMed2001

Age-associated risk of cancer among individuals with N-acetyltransferase 2 (NAT2) mutations and mutations in DNA mismatch repair genes.

Mutations in N-acetyltransferase 2 (NAT2), a highly polymorphic enzyme involved in the metabolism of xenobiotics and carcinogens, may affect risk for colorectal cancer (CRC), especially among individuals with germ-line mutations in DNA mismatch repair genes. We determined the NAT2 genotypes and allele frequencies for 86 individuals with CRC who had mutations in hMLH1, hMSH2, or hPMS1. No significant difference in time to onset was observed between rapid (NAT2*4) and slow (NAT2*5, NAT2*6, and NAT2*7) acetylators. However, when individuals were stratified separately by NAT2 polymorphism (NAT2*5, NAT2*6, and NAT2*7), those who were heterozygous at the mutant locus NAT2*7 after adjustment for the NAT2 mutant loci NAT2*5 and NAT2*6 had a significantly higher risk of CRC (hazard ratio, 2.96; P = 0.012) and all of the cancers (hazard ratio, 3.37; P = 0.00004) than individuals homozygous for wild type at the NAT2*7 allele. These findings suggest that NAT2 genotype may be an important factor in tumorigenesis of CRC and cancers related to hereditary nonpolyposis CRC among individuals with mismatch repair defects.

Acetylation↗
PubMed2001

Alosetron improves quality of life in women with diarrhea-predominant irritable bowel syndrome.

OBJECTIVES: The aim of this study was to assess the impact of alosetron, a treatment recently approved in the United States for irritable bowel syndrome in diarrhea-predominant female patients, on health-related quality of life. METHODS: Quality of life was assessed as part of two 12-wk randomized, double-blind, placebo-controlled irritable bowel syndrome studies comparing alosetron 1 mg b.i.d. with placebo (S3BA3001 and S3BA3002). Patients completed a validated disease-specific quality of life questionnaire, the Irritable Bowel Syndrome Quality of Life Questionnaire (IBSQOL), at baseline and at the 12-wk or final visit. The clinical relevance of data were also evaluated by a minimal meaningful difference instrument. RESULTS: A total of 626 and 647 patients were enrolled in studies S3BA3001 and S3BA3002, respectively. Approximately 70% of patients in each study had diarrhea-predominant IBS. In diarrhea-predominant patients enrolled in S3BA3001, statistically significant (p < 0.05) improvements with alosetron versus placebo were observed on all nine IBSQOL scales (emotional health, mental health, sleep, energy, physical functioning, food/diet, social functioning, role-physical, and sexual relations) and for all but one scale (mental health) in S3BA3002. In both studies, a significantly greater percentage of patients treated with alosetron (p < 0.05) experienced clinically meaningful improvement on three of the nine IBSQOL scales (food/diet, social functioning, and role-physical) compared with patients treated with placebo. Patients treated with alosetron did not show worsening in any quality of life domain compared with patients treated with placebo. CONCLUSIONS: These results in women with diarrhea-predominant IBS demonstrate that alosetron significantly improves health-related quality of life.

Carbolines↗
PubMed2000

Efficacy and safety of alosetron in women with irritable bowel syndrome: a randomised, placebo-controlled trial.

BACKGROUND: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder with symptoms of abdominal pain, discomfort, and altered bowel function. Antagonists of the type 3 serotonin receptor (5-HT3) have shown promising results in the relief of IBS-associated symptoms. We aimed to confirm these findings by doing a randomised, placebo-controlled trial. METHODS: We studied 647 female IBS patients with diarrhoea-predominant or alternating bowel patterns (diarrhoea and constipation). 324 patients were assigned 1 mg alosetron and 323 placebo orally twice daily for 12 weeks, followed by a 4-week post-treatment period. Adequate relief of abdominal pain and discomfort was the primary endpoint; secondary endpoints included improvements in urgency, stool frequency, and stool consistency. Analysis was by intention to treat. FINDINGS: 79 (24%) of patients in the alosetron group and 53 (16%) in the placebo group dropped out. The difference in the drop-out rate between groups was mainly due to a greater occurrence of constipation in the alosetron group. A greater proportion of alosetron-treated patients than placebo-treated patients (133 [41%] vs 94 [29%], respectively) reported adequate relief for all 3 months of treatment (difference 12% [4.7-19.2]). Alosetron also significantly decreased urgency and stool frequency, and increased stool firmness. Constipation occurred in 30% and 3% of patients in the alosetron and placebo groups, respectively. INTERPRETATION: Alosetron was well tolerated and clinically effective in alleviating pain and bowel-related symptoms in this population of women with IBS.

Administration, Oral↗
PubMed2000

Effects of cyclin D1 polymorphism on age of onset of hereditary nonpolyposis colorectal cancer.

A common polymorphism in the cyclin D1 gene enhances the gene's alternate splicing. The alternatively spliced product encodes an altered protein that does not contain sequences involved in the turnover of the protein. We found that hereditary nonpolyposis colorectal carcinoma patients who were homozygous or heterozygous for the mutant allele developed colorectal cancer an average of 11 years earlier than patients who were homozygous for the normal alleles. This is the first report indicating that the cyclin D1 polymorphism influences age of onset of cancer. Because cyclin D1 plays an important role in the G1 to S phase transition of the cell cycle, our findings suggest that cells with the mutant allele accumulate mutations as a result of defective mismatch repair and may also bypass the G1-S checkpoint of the cell cycle more easily than in cells not carrying the polymorphism. The polymorphism has a dominant phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver.

We have examined hepatic levels of microsomal lauric acid hydroxylase activity and cyanide-insensitive palmitoyl coenzyme A oxidative activity in koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii) and compared our results to those determined in rat. Microsomal lauric acid hydroxylation was significantly higher in koala than in tammar wallaby or rat. However, cyanide-insensitive palmitoyl-CoA oxidation was absent in the koala. We have also determined the hepatic nicotinamide cofactors in these species. Hepatic nicotinamide-adenine dinucleotide (NAD) and the ratio of NAD/nicotinamide-adenine dinucleotide phosphate (NADP) were higher in koala than in tammar wallaby and rat liver. Reverse transcription of koala liver mRNA, followed by polymerase chain reaction using primers based on highly conserved areas in the CYP4A family led to the cloning of a partial, near full length, cDNA clone with approximately 70% nucleotide and deduced amino acid sequence identity to human CYP4A11. The CYP has been named CYP4A15.

Amino Acid Sequence↗
PubMed2000

Testosterone dehydrogenase activity in koala liver: characterisation of cofactor and steroid substrate differences.

We have studied the hepatic microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD) capacity of koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii). A detailed comparison of the activity in hepatic fractions from koala and rat was made. Hepatic microsomal NADP-supported 17beta-HSD activity was significantly higher in koala (11.64+/-3.35 nmoles/mg protein/min), (mean+/-S.D.) than in tammar wallaby liver (1.52+/-0.79 nmoles/mg protein/min). However, when NAD was utilised as cofactor the activity was similar in both marsupial species (2.83+/-2.03 nmoles/mg protein/min, koala; 0.70+/-0.71 nmoles/mg protein/min, tammar wallaby). Data for rat indicated a cofactor preference for NAD rather than NADP (17.94+/-6.40 nmoles/mg protein/min, NAD; 2.18+/-1.04 nmoles/mg protein/min, NADP). Michaelis-Menten parameters for the kinetics of 17beta-HSD testosterone oxidation by NADP and NAD were determined in the koala. The Km for testosterone was of the order of 10.0-24.0 microM (n = 6) irrespective of the cofactor used, whilst the Km for NADP was 0.28-0.43 microM (n = 2) and for NAD was 13.9-18.5 microM (n = 2). 17beta-estradiol was found to be an inhibitor of both NAD- and NADP- supported 17beta-HSD activity. These findings indicate that NADP-mediated, but not NAD-mediated testosterone dehydrogenation is a major pathway of steroid biotransformation in koala liver; the reaction is less extensive in fractions from wallaby, human and rat. Such species-related differences in cofactor preference may contribute along with species differences in gene expression to observed rates of 17beta-HSD activity in mammals.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Hydrogen peroxide decomposition in model subsurface systems.

Rates of hydrogen peroxide decomposition, hydroxyl radical production, and oxygen evolution were investigated in silica sand-goethite slurries using unstabilized and stabilized hydrogen peroxide formulations. The goethite-catalyzed decomposition of unstabilized hydrogen peroxide formulations resulted in more rapid hydrogen peroxide loss and oxygen evolution relative to systems containing a highly stabilized hydrogen peroxide formulation. Systems at neutral pH and those containing higher goethite concentrations were characterized by higher rates of hydrogen peroxide decomposition and by more oxygen evolution. The stabilized hydrogen peroxide formulation showed greater hydroxyl radical production relative to the unstabilized formulations. Furthermore, hydroxyl radical production rates were greater at neutral pH than at the acidic pH regimes. The results suggest that when stabilized hydrogen peroxide is injected into the subsurface during in situ bioremediation, naturally occurring minerals such as goethite may initiate Fenton-like reactions. While these reactions may prove to be toxic to microorganisms, they have the potential to chemically oxidize contaminants in soils and groundwater.

Biodegradation, Environmental↗
PubMed1999

Biological barriers to cellular delivery of lipid-based DNA carriers.

Although lipid-based DNA delivery systems are being assessed in gene therapy clinical trials, many investigators in this field are concerned about the inefficiency of lipid-based gene transfer technology, a criticism directed at all formulations used to enhance transfer of plasmid expression vectors. It is important to recognize that many approaches have been taken to improve transfection efficiency, however because of the complex nature of the formulation technology being developed, it has been extremely difficult to define specific carrier attributes that enhance transfection. We believe that these optimization processes are flawed for two reasons. First, a very defined change in formulation components affects the physical and chemical characteristics of the carrier in many ways. As a consequence, it has not been possible to define structure/activity relationships. Second, the primary endpoint used to assess plasmid delivery has been transgene expression, an activity that is under the control of cellular processes that have nothing to do with delivery. Gene expression following administration of a plasmid expression vector involves a number of critical steps: (i) DNA protection, (ii) binding to a specific cell population, (iii) DNA transfer across the cell membrane, (iv) release of DNA into the cytoplasm, (v) transport through the cell and across the nuclear membrane as well as (vi) transcription and translation of the gene. The objective of this review is to describe lipid-based DNA carrier systems and the attributes believed to be important in regulating the transfection activity of these formulations. Although membrane destabilization activity of the lipid-based carriers plays an important role, we suggest here that a critical element required for efficient transfection is dissociation of lipids bound to the plasmid expression vector following internalization.

Journal Article↗
PubMed1999

Hepatic microsomal enzyme activity in the koala and tammar wallaby: high 17beta-hydroxysteroid oxidoreductase activity in koala liver microsomes.

We have studied the hepatic microsomal xenobiotic metabolising capacity of koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii). Total cytochrome P450 content in hepatic microsomes from koala (0.87 +/- 0.18 nmol/mg protein, n = 4, mean (S.D.) and rat were comparable while tammar wallaby displayed reduced P450 content (0.24 +/- 0.04 nmol/mg protein). Associated microsomal activities (NADPH cytochrome P450 reductase, aminopyrine N-demethylation, aniline hydroxylation, and androstenedione 6beta- and 16alpha-hydroxylation) in koala liver were similar to or reduced relative to rat. Hepatic microsomal NADPH-supported 17beta-hydroxysteroid oxidoreductase (17beta-HSOR) activity was significantly higher in koala (9.99+/-3.08 nmol/mg protein/min) than in tammar wallaby liver (0.86 +/- 0.16 nmol/mg protein/min). However, when NADH was utilised as cofactor the activity was similar in both marsupial species (koala, 1.44 +/- 0.84 nmol/mg protein/min; tammar wallaby, 1.52 +/- 0.44 nmol/mg protein/min). Michaelis-Menten parameters for the kinetics of 17beta-HSOR androstenedione reduction by NADPH and NADH were determined in the koala. The Km for androstenedione was of the order of 1.9-4 microM (n = 4) irrespective of the cofactor used, whilst the Km for NADPH was 0.04-0.05 microM (n = 2) and for NADH was 134-430 microM (n = 2). Potential inhibitors were evaluated for their effects on NADPH-mediated 17beta-HSOR activity with menadione and, to lesser extents, menthone, benzaldehyde and metyrapone eliciting significant inhibition. From detailed kinetic studies menthone was found to be an uncompetitive inhibitor of the activity in koala liver (Ki 220 microM).

Animals↗
PubMed1999

Improvement in pain and bowel function in female irritable bowel patients with alosetron, a 5-HT3 receptor antagonist.

BACKGROUND: No currently available treatment provides consistent relief of irritable bowel syndrome. Colonic sensory and motor function are modulated partly through 5HT3-receptors. AIM: To evaluate effects of the 5HT3-receptor antagonist, alosetron, in irritable bowel syndrome. METHODS: Randomized, double-blind, placebo-controlled, dose-ranging (1, 2, 4, 8 mg b.d. alosetron), 12-week trial in 370 patients with diarrhoea-predominant or alternating constipation and diarrhoea irritable bowel syndrome. Weekly measurement of adequate relief was the key end-point; other irritable bowel syndrome symptoms were collected daily using an electronic phone system. RESULTS: Alosetron (1 mg or 2 mg b.d.) significantly (P < 0.05 vs. placebo) increased the proportion of females, but not males, reporting adequate relief. Stool consistency, frequency and percentage days with urgency improved over placebo (P < 0.05) within the first month with all doses of alosetron, and persisted throughout the trial with all doses in female patients. With 1 mg b.d. alosetron, females had improved stool consistency and urgency within the first week, and adequate relief and improved stool frequency within the first 2 weeks. There was no consistent improvement in bowel function among male patients. CONCLUSION: In female irritable bowel syndrome patients with predominant diarrhoea or alternating constipation and diarrhoea, alosetron is effective in treatment of abdominal pain and discomfort and bowel-related symptoms.

Abdominal Pain↗
PubMed1999

Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates.

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

Animals↗
PubMed1999

Preliminary results of heart-beating and non-heart-beating donor kidney transplants--the Singapore experience.

Renal transplantation is the ideal renal replacement therapy for treatment of end stage kidney failure but its limitation is the constant shortage of cadaveric donors. While the traditional source of cadaveric kidneys is procurement from heart-beating donors, organ procurement from non-heart-beating donors (NHBD) would help in coping with the rising demand for organs. However various important legal, logistic and technical constraints have limited the implementation of such a procurement programme. Nevertheless, prolonged warm ischaemic times in procuring kidneys from NHB donors may affect the viability of these organs and adversely affect overall outcome after transplant. Fifty-three consecutive patients who underwent renal allotransplantation (Tx) between January and December 1994 at the Singapore General Hospital were retrospectively reviewed. Outcomes after renal transplantation among 25 heart-beating donor (HBD) and 28 NHBD were compared. Despite significant differences in donor age (23.7 +/- 11.0 and 34.1 +/- 7.9, P = 0.001), first warm (0 and 22.2 +/- 9.0 minutes) and cold ischaemic times (12.8 +/- 6.7 and 5.2 +/- 5.0 hours, P = 0.001) between the HBD and NHBD groups, the two-year patient and graft survivals were very similar in both groups (100% versus 98% and 98% versus 96%). The incidence of delayed graft function (DF) was also comparable between the HBD (41%) and the NHBD (50%) Tx. These results suggest that NHBD is a viable source for kidney transplants and comparable patient and graft survivals can be achieved if the first warm and cold ischaemic times are kept to the minimum.

Adolescent↗
PubMed1997

Analysis of cationic liposome-mediated interactions of plasmid DNA with murine and human melanoma cells in vitro.

Lipid-based DNA transfer formulations are typically selected on the basis of in vitro transfection studies where the activity of specific formulations is defined by transgene expression. It is unclear, however, whether expression is directly related to the efficiency of DNA transfer. In an attempt to correlate DNA transfer with transgene expression, we used a simple assay consisting of measuring DNA (3H-plasmid encoding for beta-galactosidase) binding to murine (B16/BL6) and human (KZ) melanoma cells in vitro at 4 and 37 degrees C. The difference in cell association at these temperatures was assumed to be a consequence of DNA uptake, an assumption that was confirmed by protease removal of cell surface-associated DNA. DNA associated with B16/BL6 melanoma cells (up to 30 ng or 12% of the added DNA) following incubation with dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (DOPE) liposome-DNA aggregates was comparable to that achieved with 1,2-dioleoyloxypropyl-3-trimethylammonium bromide/DOPE or dimethyldioctadecylammonium bromide/DOPE liposomes; however, transgene expression was 2- and 5-fold less for the latter two formulations, respectively. Similarly, equivalent amounts of DNA delivery were achieved with B16/BL6 and KZ melanoma cells, yet the level of transgene expression in the KZ cells was undetectable. It was demonstrated that the lack of transgene expression was not a consequence of cell-specific differences in DNA degradation.

Animals↗