Research IndicatorsGraph generated 25 June 2015 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 25 June, 2015 using data from PubMed, MeSH and CancerIndex
Specific Cancers (4)
Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.
Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
Search the Epigenomics database and view relevant gene tracks of samples.
Latest Publications: APOB (cancer-related)
PURPOSE OF REVIEW: Several mutations in the apoB, proprotein convertase subtilisin/kexin type 9 (PCSK9), and MTP genes result in low or absent levels of apoB and LDL-cholesterol in plasma, which cause familial hypobetalipoproteinemia and abetalipoproteinemia. Mutations in the ANGPTL3 gene cause familial combined hypolipidemia. Clinical manifestations range from none to severe, debilitating, and life-threatening disorders. This review summarizes recent genetic, metabolic, and clinical findings and presents an update on management strategies.
RECENT FINDINGS: Cases of cirrhosis and hepatocellular carcinoma have now been identified in heterozygous familial hypobetalipoproteinemia probably because of decreased triglyceride transport capacity from the liver. ANGPTL3 mutations cause low levels of LDL-cholesterol and low HDL-cholesterol in compound heterozygotes and homozygous individuals, decrease reverse cholesterol transport, and lower glucose levels. The effect on atherosclerosis is unknown; however, severe fatty liver has been identified. Loss-of-function mutations in PCSK9 cause familial hypobetalipoproteinemia, which appears to lower risk for coronary artery disease and has no adverse sequelae. Phase III clinical trials are now underway examining the effect of PCSK9 inhibitors on cardiovascular events in combination with statin drugs.
SUMMARY: Mutations causing low LDL-cholesterol and apoB have provided insight into lipid metabolism, disease associations, and the basis for drug development to lower LDL-cholesterol in disorders causing high levels of cholesterol. Early diagnosis and treatment are necessary to prevent adverse sequelae from familial hypobetalipoproteinemia and abetalipoproteinemia.
He X, Wang Y, Zhang W, et al.Screening differential expression of serum proteins in AFP-negative HBV-related hepatocellular carcinoma using iTRAQ -MALDI-MS/MS.
Neoplasma. 2014; 61(1):17-26 [PubMed
] Related Publications
Hepatocellular carcinoma(HCC) is serious condition associated with a high morbidity and mortality. Therefore is an urgent need to develop novel noninvasive techniques for early diagnosis, particularly for patients with AFP-negative [AFP(-)] HCC. In this study, iTRAQ-MALDI-MS/MS was used to identify differentially expressed proteins in AFP(-) HBV-related HCC compared with non-cancerous hepatitis B virus (HBV) and healthy controls subjects.Serum was obtained from 18 patients with AFP(-) HBV-related HCC, 18 matched patients with HBV without HCC and 18 healthy control subjects. High abundance proteins were removed from serum and the differentially expressed proteins from the three groups were screened out using iTRAQ-MALDI-MS/MS. The Gene Ontology (GO) function and the interaction networks of differentially expressed proteins were then analyzed. A total of 24 expressed differential proteins associated with AFP(-) HBV-related HCC were screened out, 15 proteins were up-regulated and 9 down-regulated. The most common molecular function of the 24 differentially expressed proteins was enzyme inhibition. Interaction network of the 24 differentially expressed proteins showed that 14 proteins (C5, KNG1, FN1, LRG1, HRG, SERPINC1, CRP, APOB, SAA1, APCS, C4BPA, CFI, CFB and GSN) were central to the functional network. The expression levels of the GSN protein were down-regulated in AFP(-) HBV-related HCC subjects compared with healthy controls and the HBV group (p<0.01), consistent with the iTRAQ results.The 14 proteins from the serum of AFP(-) HBV-related HCC appeared at the fulcrum of the functional network and were differentially expressed compare to HBV and healthy controls suggesting a possible association with HCC progression.
Cefalù AB, Pirruccello JP, Noto D, et al.A novel APOB mutation identified by exome sequencing cosegregates with steatosis, liver cancer, and hypocholesterolemia.
Arterioscler Thromb Vasc Biol. 2013; 33(8):2021-5 [PubMed
] Free Access to Full Article Related Publications
OBJECTIVE: In familial hypobetalipoproteinemia, fatty liver is a characteristic feature, and there are several reports of associated cirrhosis and hepatocarcinoma. We investigated a large kindred in which low-density lipoprotein cholesterol, fatty liver, and hepatocarcinoma displayed an autosomal dominant pattern of inheritance.
APPROACH AND RESULTS: The proband was a 25-year-old female with low plasma cholesterol and hepatic steatosis. Low plasma levels of total cholesterol and fatty liver were observed in 10 more family members; 1 member was affected by liver cirrhosis, and 4 more subjects died of either hepatocarcinoma or carcinoma on cirrhosis. To identify the causal mutation in this family, we performed exome sequencing in 2 participants with hypocholesterolemia and fatty liver. Approximately 22 400 single nucleotide variants were identified in each sample. After variant filtering, 300 novel shared variants remained. A nonsense variant, p.K2240X, attributable to an A>T mutation in exon 26 of APOB (c.6718A>T) was identified, and this variant was confirmed by Sanger sequencing. The gentotypic analysis of 16 family members in total showed that this mutation segregated with the low cholesterol trait. In addition, genotyping of the PNPLA3 p.I148M did not show significant frequency differences between carriers and noncarriers of the c.6718A>T APOB gene mutation.
CONCLUSIONS: We used exome sequencing to discover a novel nonsense mutation in exon 26 of APOB (p.K2240X) responsible for low cholesterol and fatty liver in a large kindred. This mutation may also be responsible for cirrhosis and liver cancer in this family.
BACKGROUND: Gallstones (GS) is the major manifestation of gallbladder disease, and is the most common risk factor for gallbladder cancer (GBC). Previous studies investigating the association between ApoB-100 gene polymorphisms and the risks of GS and GBC have yielded conflicting results. Therefore, we performed a meta-analysis to clarify the effects of ApoB-100 gene polymorphisms on the risks of GS and GBC.
METHODS: A computerized literature search was conducted to identify the relevant studies from PubMed and Embase. Fixed or random effects model was selected based on heterogeneity test. Publication bias was estimated using Begg's funnel plots and Egger's regression test.
RESULTS: A total of 10, 3, and 3 studies were included in the analyses of the association between ApoB-100 XbaI, EcoRI, or insertion/deletion (ID) polymorphisms and the GS risks, respectively, while 3 studies were included in the analysis for the association between XbaI polymorphism and GBC risk. The combined results showed a significant association in Chinese (X+ vs. X-, OR = 2.37, 95%CI 1.52-3.70; X+X+/X+X- vs. X+X+, OR = 2.47, 95%CI 1.55-3.92), but not in Indians or Caucasians. Null association was observed between EcoRI or ID polymorphisms and GS risks. With regard to the association between XbaI polymorphism and GBC risk, a significant association was detected when GBC patients were compared with healthy persons and when GBC patients were compared with GS patients. A significant association was still detected when GBC patients (with GS) were compared with the GS patients (X+X+ vs. X-X-, OR = 0.33, 95%CI 0.12-0.90).
CONCLUSION: The results of this meta-analysis suggest that the ApoB-100 X+ allele might be associated with increased risk of GS in Chinese but not in other populations, while the ApoB-100 X+X+ genotype might be associated with reduced risk of GBC. Further studies with larger sample sizes are needed to confirm these results.
Liu X, Wang Y, Qu H, et al.Associations of polymorphisms of rs693 and rs1042031 in apolipoprotein B gene with risk of breast cancer in Chinese.
Jpn J Clin Oncol. 2013; 43(4):362-8 [PubMed
] Related Publications
BACKGROUND: Lipid synthesis is an integrated result of genetic, epigenetic and environmental factors, and also can promote growth and survival of cancer cells. Apolipoprotein B plays a central role in lipid metabolism as the major protein component of very-low-density lipoprotein and low-density lipoprotein.
METHODS: We investigated the associations of polymorphisms of rs693 (-7673C>T) and rs1042031 (-12669 G>A) in the APOB gene with risk of breast cancer in 675 blood-unrelated Chinese patients with breast cancer and 712 healthy controls.
RESULTS: Polymorphisms of -12669 G>A and -7673C>T in the APOB gene were significantly associated with an increased risk of breast cancer (P = 0.000), especially for postmenopausal women (P = 0.000, 0.023). The positive associations still remained after further analysis of the two polymorphisms' distribution according to body mass index. However, no statistical associations were found between -12669 G>A and -7673C>T polymorphisms and other clinical characteristics, including tumor size, lymph node metastasis, histological grade, estrogen and progesterone receptor status and Her-2 status.
CONCLUSIONS: rs693 and rs1042031 polymorphisms in the APOB gene increased the risk of breast cancer in Chinese, and this role of the two polymorphisms in connection with breast cancer was not dependent on body mass index.
Sperm abnormalities are one of the main factors responsible for male infertility; however, their pathogenesis remains unclear. The role of microRNAs in the development of sperm abnormalities in infertile men has not yet been investigated. Here, we used human induced pluripotent stem cells to investigate the influence of miR-122 expression on the differentiation of these cells into spermatozoa-like cells in vitro. After induction, mutant miR-122-transfected cells formed spermatozoa-like cells. Flow cytometry of DNA content revealed a significant increase in the haploid cell population in spermatozoa-like cells derived from mutant miR-122-transfected cells as compared to those derived from miR-122-transfected cells. During induction, TNP2 and protamine mRNA and protein levels were significantly higher in mutant miR-122-transfected cells than in miR-122-transfected cells. High-throughput isobaric tags for relative and absolute quantification were used to identify and quantify the different protein expression levels in miR-122- and mutant miR-122-transfected cells. Among all the proteins analyzed, the expression of lipoproteins, for example, APOB and APOA1, showed the most significant difference between the two groups. This study illustrates that miR-122 expression is associated with abnormal sperm development. MiR-122 may influence spermatozoa-like cells by suppressing TNP2 expression and inhibiting the expression of proteins associated with sperm development.
Zhang J, Fan P, Liu H, et al.Apolipoprotein A-I and B levels, dyslipidemia and metabolic syndrome in south-west Chinese women with PCOS.
Hum Reprod. 2012; 27(8):2484-93 [PubMed
] Related Publications
STUDY QUESTION: What are the relationships between apolipoprotein (apo) A-I and apoB concentrations, the apoB/apoA-I ratio and the prevalences of dyslipidemia and metabolic syndrome (MS) in south-west Chinese women with polycystic ovary syndrome (PCOS).
SUMMARY ANSWER: There is a relatively high incidence of dyslipidemia and MS in south-west Chinese women with PCOS, especially in patients without hyperandrogenism. Patients with dyslipidemia are more obese, and have a more adverse glucose and lipid metabolic profile and higher apoB levels and apoB/apoA-I ratio. The increased apoB levels and apoB/A1 ratio and the MS are strongly associated with PCOS, suggesting that there is an increased risk of cardiovascular diseases in these patients.
WHAT IS KNOWN AND WHAT THIS PAPER ADDS: Dyslipidemia and MS have been widely studied in women with PCOS, but to date no data from south-west Chinese subjects have been available. The apoB/apoA-I ratio has been reported to be strongly associated with MS and insulin resistance (IR) and to be a reliable parameter that reflects lipid disturbances and the potential to develop atherosclerosis, but its relationship with PCOS is unclear. DESIGN This case-control study included 406 patients with PCOS and 342 control women between 17 and 40 years of age from a population in south-west China during 2006-2011.
PARTICIPANTS AND SETTING: The diagnosis of PCOS was based on the revised 2003 Rotterdam criteria. The control group, consisting of women with infertility due to a Fallopian obstruction or the husband's infertility, women undergoing a pre-pregnancy check and healthy volunteers, was recruited from the same hospital during the same period. All women were not taking any medication known to affect carbohydrate or lipid or hormone metabolism for at least 3 months prior to the study, and were studied during the follicular phase of their menstrual cycle. MS was assessed by the National Cholesterol Education Program-Adult treatment Panel (NCEP-ATP) III criteria modified for Asian populations. Dyslipidemia was defined by one or more of the following conditions: fasting total cholesterol≥5.7 mmol/l, fasting triglycerides (TG)≥1.7 mmol/l, fasting high-density lipoprotein cholesterol (HDL-C)<1.29 mmol/l or fasting low-density lipoprotein cholesterol (LDL-C)≥3.6 mmol/l.
MAIN RESULTS AND THE ROLE OF CHANCE: The prevalence of dyslipidemia in patients with PCOS was 52.96%, about two times than that in the controls, 28.95%. The most common components of dyslipidemia in patients with PCOS were decreased HDL-C (41.13%) and increased TG (24.14%). PCOS patients with dyslipidemia had significantly higher TG/HDL-C ratios, and lower HDL-C and apoA-I levels when compared with the controls or patients without dyslipidemia, and had significantly higher BMIs, fasting insulin concentrations, 2-h insulin and glucose levels, homeostatic model assessment IR, TG levels, LDL-C levels, atherogenic indexes, apoB concentrations and apoB/apoA-I ratios when compared with all of the control women, with or without dyslipidemia and patients without dyslipidemia. The frequency of MS in patients with PCOS was 25.62%, more than five times than that in the controls. The main two risk factors were increased waist circumference and low HDL-C levels. In the four PCOS phenotypes based on the Rotterdam criteria, the oligo- and/or anovulation+PCO presented the highest prevalence of dyslipidemia (66.14%) and MS (34.65%). Binary logistic regression analysis showed that increased apoB levels, an increased apoB/apoA-I ratio and MS was strongly associated with PCOS (odds ratio=17.41, 27.16 and 7.66, 95% confidence interval: 6.93-43.74, 9.46-77.93 and 4.32-13.57, respectively) after adjustment for age.
BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION: The relatively minor limitations of this study are discussed within the paper. GENERALISABILITY TO OTHER POPULATIONS: The metabolic patterns found in south-west Chinese with PCOS are compared with that of other populations.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Chinese National Natural Science Foundation (81070463), Program for Changjiang Scholars and Innovative Research Team in University (IRT0935), and Research Seed Fund from West China Second Hospital of Sichuan University (to H.B.). There are no any competing interests.
TRIAL REGISTRATION NUMBER: N/A.
Prediction of the responses to neoadjuvant chemotherapy (NACT) can improve the treatment of patients with advanced breast cancer. Genes and proteins predictive of chemoresistance have been extensively studied in breast cancer tissues. However, noninvasive serum biomarkers capable of such prediction have been rarely exploited. Here, we performed profiling of N-glycosylated proteins in serum from fifteen advanced breast cancer patients (ten patients sensitive to and five patients resistant to NACT) to discover serum biomarkers of chemoresistance using a label-free liquid chromatography-tandem MS method. By performing a series of statistical analyses of the proteomic data, we selected thirteen biomarker candidates and tested their differential serum levels by Western blotting in 13 independent samples (eight patients sensitive to and five patients resistant to NACT). Among the candidates, we then selected the final set of six potential serum biomarkers (AHSG, APOB, C3, C9, CP, and ORM1) whose differential expression was confirmed in the independent samples. Finally, we demonstrated that a multivariate classification model using the six proteins could predict responses to NACT and further predict relapse-free survival of patients. In summary, global N-glycoproteome profile in serum revealed a protein pattern predictive of the responses to NACT, which can be further validated in large clinical studies.
Zaiden N, Yap WN, Ong S, et al.Gamma delta tocotrienols reduce hepatic triglyceride synthesis and VLDL secretion.
J Atheroscler Thromb. 2010; 17(10):1019-32 [PubMed
] Related Publications
AIM: Present study aimed to elucidate the suppression of serum lipids by gamma- and delta-tocotrienol (γδT3).
METHODS: The lipid-lowering effects of γδT3 were investigated using HepG2 liver cell line, hypercholesterolemic mice and borderline-high cholesterol patients.
RESULTS: In-vitro results demonstrated two modes of action. First, γδT3 suppressed the upstream regulators of lipid homeostasis genes (DGAT2, APOB100, SREBP1/2 and HMGCR) leading to the suppression of triglycerides, cholesterol and VLDL biosyntheses. Second, γδT3 enhanced LDL efflux through induction of LDL receptor (LDLr) expression. Treatment of LDLr-deficient mice with 1 mg/day (50 mg/kg/day) γδT3 for one-month showed 28%, 19% reduction in cholesterol and triglyceride levels respectively, whereas HDL level was unaltered. The lipid-lowering effects were not affected by alpha-tocopherol (αTP). In a placebo-controlled human trial using 120 mg/day γδT3, only serum triglycerides were lowered by 28% followed by concomitant reduction in the triglyceride-rich VLDL and chylomicrons. In contrast, total cholesterol, LDL and HDL remained unchanged in treated and placebo groups. The discrepancies between in-vitro, in-vivo and human studies may be attributed to the differential rates of post-absorptive γδT3 degradation and LDL metabolism.
CONCLUSION: Reduction in triglycerides synthesis and transport may be the primary benefit caused by ingesting γδT3 in human.
AIM: To prospectively investigate the association between the XbaI polymorphisms of apolipoprotein B (APOB) gene and gallstone formation following gastrectomy.
METHODS: The study was conducted between January 2005 and December 2006. A total of 186 gastric cancer patients who had undergone radical gastrectomy were grouped according to XbaI polymorphisms of APOB gene (X(+)X(-) group, n = 24 and X(-)X(-) group, n = 162) and compared. The XbaI polymorphisms of APOB gene were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
RESULTS: The incidence of gallstone was significantly higher in the X(+)X(-) group than in the X(-)X(-) group [54.2% vs 9.3%, RR = 5.85 (2.23-15.32), P < 0.001]. The serum levels of total cholesterol (TC) and low-density lipoprotein (LDL) were higher in the X(+)X(-) than in the X(-)X(-) group (4.02 +/- 1.12 vs 3.48 +/- 0.88, P = 0.004 before surgery and 3.88 +/- 1.09 vs 3.40 +/- 0.86, P = 0.008 after surgery). LDL was 2.21 +/- 0.96 vs 1.89 +/- 0.84 (P = 0.042) before surgery and 2.09 +/- 0.95 vs 1.72 +/- 0.85 (P = 0.029) after surgery in the two groups. No relationship was found between XbaI polymorphisms and gallbladder motility.
CONCLUSION: In Chinese patients after radical gastrectomy, X(+) allele of APOB gene is another risk factor for the development of gallstone besides the gallbladder motility disorder after surgery.
AIM: To determine the effects of genetic variants associated with gallstone formation and capsaicin (a pungent component of chili pepper) metabolism on the risk of gallbladder cancer (GBC).
METHODS: A total of 57 patients with GBC, 119 patients with gallstones, and 70 controls were enrolled in this study. DNA was extracted from their blood or paraffin block sample using standard commercial kits. The statuses of the genetic variants were assayed using Taqman SNP Genotyping Assays or Custom Taqman SNP Genotyping Assays.
RESULTS: The non-ancestral T/T genotype of apolipoprotein B rs693 polymorphism was associated with a decreased risk of GBC (OR: 0.14, 95% CI: 0.03-0.63). The T/T genotype of cholesteryl ester transfer protein (CETP) rs708272 polymorphism was associated with an increased risk of GBC (OR: 5.04, 95% CI: 1.43-17.8).
CONCLUSION: Genetic variants involved in gallstone formation such as the apolipoprotein B rs693 and CETP rs708272 polymorphisms may be related to the risk of developing GBC in Chilean women.
In eukaryotes mRNA transcripts are extensively processed by different post-transcriptional events such as alternative splicing and RNA editing in order to generate many different mRNAs from the same gene, increasing the transcriptome and then the proteome diversity. The most frequent RNA editing mechanism in mammals involves the conversion of specific adenosines into inosines by the ADAR family of enzymes. This editing event can alter the sequence and the secondary structure of RNA molecules, with consequences for final proteins and regulatory RNAs. Alteration in RNA editing has been connected to tumor progression and many other important human diseases. Analysis of many editing sites in various cancer types is expected to provide new diagnostic and prognostic markers and might contribute to early detection of cancer, the monitoring of response to therapy, and to the detection of minimal residual disease.
Mochizuki Y, Maebuchi M, Kohno M, et al.Changes in lipid metabolism by soy beta-conglycinin-derived peptides in HepG2 cells.
J Agric Food Chem. 2009; 57(4):1473-80 [PubMed
] Related Publications
In this study, HepG2 cells were treated with short peptides (7S-peptides) derived from highly purified soybean beta-conglycinin (7S), which was free from lipophilic protein, and the effect of the peptide treatment on lipid metabolism was determined. 7S-peptide treatment suppressed the secretion of apolipoprotein B-100 from HepG2 cells into the medium. The 7S-peptides also suppressed the incorporation of (3)H-glycerol and (14)C-acetate into triacylglyceride but not into major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. Additionally, the synthesis of cholesterol esters was dramatically decreased for 2 h after the addition of the 7S-peptides, whereas the synthesis of cholesterol remained unchanged by 4 h and increased by 8 h after the addition of the 7S-peptides. The cleaved nuclear form of SREBP-2 increased 8 h after the addition of the 7S peptides, suggesting a decrease in intracellular cholesterol levels. Analysis of changes in mRNA expression after 7S-peptide treatment suggested that the 7S-peptides lower the level of cholesterol in the endoplasmic reticulum, increase the mRNA of genes related to beta-oxidation of fatty acids, and increase the synthesis of cholesterol. From these results, it may be concluded that the peptides derived from 7S altered the lipid metabolism to decrease secretion of apolipoprotein B-100-containing lipoprotein from HepG2 cells.
Han CZ, Du LL, Jing JX, et al.Associations among lipids, leptin, and leptin receptor gene Gin223Arg polymorphisms and breast cancer in China.
Biol Trace Elem Res. 2008; 126(1-3):38-48 [PubMed
] Related Publications
We evaluated the relationship among the leptin receptor (LEPR) gene Gln223Arg polymorphism, body mass index (BMI), waist and hip circumference ratio (WHR), dietary structure, lifestyle, and other biomarkers with breast cancer and determined whether they could be effective for the prevention and treatment of breast cancer. The Gln223Arg polymorphisms in the LEPR gene were investigated in blood deoxyribonucleic acid (DNA) available for 240 breast cancer cases and 500 controls. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. Leptin, insulin were determined by enzyme-linked immunosorbent assays. We found that the serum levels of leptin, insulin, triglyceride (TG), free cholesterol (FCH), apolipoprotain (APO) A1, and BMI were significantly higher in breast cancer cases than the controls, while physical activity was clearly less in breast cancer cases (P < 0.02 approximately P < 0.001, respectively). Moreover, there were significant association between the Gln223Arg genotype and breast cancer risk; homozygotes for AA and heterozygotes for AG,AG + GG genotypes had been proved to increase the risk of breast cancer, and their corresponding odds ratio were 7.14 (95% confidence interval [CI] = 1.92-25.64), 1.33(95% CI = 1.03-2.70), and 2.04 (95% CI = 1.09-3.82). Interestingly, logistic regression analysis showed that LEPR gene Gln223Arg polymorphism and elevated leptin, insulin, TG, FCH, APOA1, WHR, and reduced APOB increased the risk of developing breast cancer, respectively. And, it also suggested that LEPR gene Gln223Arg polymorphisms, elevated leptin, insulin, TG, FCH, APOA1, WHR, and reduced APOB should play a major role in the development of breast cancer.
Kamdem LK, Hamilton L, Cheng C, et al.Genetic predictors of glucocorticoid-induced hypertension in children with acute lymphoblastic leukemia.
Pharmacogenet Genomics. 2008; 18(6):507-14 [PubMed
] Related Publications
OBJECTIVE: Glucocorticoids are used universally in the remission induction therapy for acute lymphoblastic leukemia (ALL). One of the adverse effects of glucocorticoids is hypertension. Our aim was to define the frequency of and clinical and genetic risk factors for steroid-induced hypertension.
METHODS: We determined the genotypes for 203 candidate polymorphisms in genes previously linked to hypertension or to the pharmacokinetics or pharmacodynamics of antileukemic agents. Hypertension was defined according to the guidelines of the American Academy of Pediatrics; patients were evaluated during the 28-day period of prednisone at 40 mg/m2/day during remission induction of childhood ALL.
RESULTS: Of the 602 children with newly diagnosed ALL who were normotensive pretherapy, 270 (45%) developed hypertension during remission induction. None of the putative risk factors (age, sex, race, white blood cell count, risk group, body mass index, or serum creatinine) was associated with hypertension. Among the polymorphisms genotyped, we identified eight genes (CNTNAP2, LEPR, CRHR1, NTAN1, SLC12A3, ALPL, BGLAP, and APOB) containing variants that were associated with hypertension (chi2 P values 0.002-0.048), several of which interact with the hypothalamus-pituitary-adrenal axis. Polymorphisms in CYP3A4 and CYP3A5 were not associated with hypertension.
CONCLUSION: Hypertension is common during ALL remission induction and is related to germline genetic variation.
Nahmias Y, Goldwasser J, Casali M, et al.Apolipoprotein B-dependent hepatitis C virus secretion is inhibited by the grapefruit flavonoid naringenin.
Hepatology. 2008; 47(5):1437-45 [PubMed
] Related Publications
UNLABELLED: Hepatitis C virus (HCV) infects over 3% of the world population and is the leading cause of chronic liver disease worldwide. HCV has long been known to associate with circulating lipoproteins, and its interactions with the cholesterol and lipid pathways have been recently described. In this work, we demonstrate that HCV is actively secreted by infected cells through a Golgi-dependent mechanism while bound to very low density lipoprotein (vLDL). Silencing apolipoprotein B (ApoB) messenger RNA in infected cells causes a 70% reduction in the secretion of both ApoB-100 and HCV. More importantly, we demonstrate that the grapefruit flavonoid naringenin, previously shown to inhibit vLDL secretion both in vivo and in vitro, inhibits the microsomal triglyceride transfer protein activity as well as the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and acyl-coenzyme A:cholesterol acyltransferase 2 in infected cells. Stimulation with naringenin reduces HCV secretion in infected cells by 80%. Moreover, we find that naringenin is effective at concentrations that are an order of magnitude below the toxic threshold in primary human hepatocytes and in mice.
CONCLUSION: These results suggest a novel therapeutic approach for the treatment of HCV infection.
Andreotti G, Chen J, Gao YT, et al.Polymorphisms of genes in the lipid metabolism pathway and risk of biliary tract cancers and stones: a population-based case-control study in Shanghai, China.
Cancer Epidemiol Biomarkers Prev. 2008; 17(3):525-34 [PubMed
] Free Access to Full Article Related Publications
Biliary tract cancers, encompassing the gallbladder, extrahepatic bile duct, and ampulla of Vater, are uncommon yet highly fatal malignancies. Gallstones, the primary risk factor for biliary cancers, are linked with hyperlipidemia. We examined the associations of 12 single nucleotide polymorphisms of five genes in the lipid metabolism pathway with the risks of biliary cancers and stones in a population-based case-control study in Shanghai, China. We included 235 gallbladder, 125 extrahepatic bile duct, and 46 ampulla of Vater cancer cases, 880 biliary stone cases, and 779 population controls. Subjects completed an in-person interview and gave blood. Genotyping was conducted by TaqMan assay using DNA from buffy coats. The effects of APOE IVS1+69 (rs440446) and APOB IVS6+360C>T (rs520354) markers were limited to men. Men carrying the G allele of APOE IVS1+69 had a 1.7-fold risk of stones [95% confidence interval (95% CI), 1.2-2.4], a 1.8-fold risk of gallbladder cancer (95% CI, 1.0-3.3), a 3.7-fold risk of bile duct cancer (95% CI, 2.0-7.0), and a 4-fold risk of ampullary cancer (95% CI, 1.4-12.4). Male carriers of the T allele of APOB IVS6+360C>T had a 2-fold risk of bile duct cancer (95% CI, 1.2-3.4). The APOB T-T haplotype (APOB IVS6+360C>T, EX4+56C>T) was associated with a 1.6-fold risk of bile duct cancer (95% CI, 1.1-2.3). Male and female carriers of the T allele of LDLR IVS9-30C>T (rs1003723) had a 1.5-fold risk of bile duct cancer. Our findings suggest that gene variants in the lipid metabolism pathway contribute to the risk of biliary tract stones and cancers, particularly of the bile duct.
Swagell CD, Henly DC, Morris CPRegulation of human hepatocyte gene expression by fatty acids.
Biochem Biophys Res Commun. 2007; 362(2):374-80 [PubMed
] Related Publications
It is known that fatty acids (FAs) regulate transcription through a number of FA responsive transcription factors. In order to investigate the effect of FAs on gene regulation in cultured human hepatocytes we examined the effect of palmitate on hepatic glucokinase (GK) promoter activity and expression of transcription factors that regulate GK expression. GK promoter activity was increased in constructs lacking a cAMP response element (CRE), while palmitate incubation decreased GK promoter activity in CRE-negative constructs. Cells exposed to palmitate showed increased levels of PPARalpha apolipoprotein-AII and -B100 mRNA and decreased levels of SREBP-1c mRNA but there was no effect on LXRalpha and HNF-4alpha mRNA. In addition, cDNA microarray analysis of short-term (1.5h) transcriptional regulation by palmitate, oleate, and eicosapentaenoic acid (EPA) identified that oleate and EPA initiated similar changes in the pattern of hepatic gene regulation, whereas palmitate had little effect.
UNLABELLED: Betaine-homocysteine methyltransferase (BHMT) regulates homocysteine levels in the liver. We previously reported that the alteration of BHMT is associated with alcoholic liver steatosis and injury. In this study, we tested whether BHMT protects hepatocytes from homocysteine-induced injury and lipid accumulation. Both BHMT transfectants of HepG2 cells and primary mouse hepatocytes with suppressed BHMT were generated. Comparisons were made between the cell models with respect to their response to homocysteine treatments. Homocysteine metabolism was impaired in HepG2 cells, and the expression of BHMT in HepG2 cells ameliorated the impairment and stabilized the levels of intracellular homocysteine after the addition of exogenous homocysteine. BHMT expression inhibited homocysteine-induced glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP) and homocysteine-induced cell death. A betaine treatment protected primary mouse hepatocytes from a homocysteine-induced increase in GRP78 and cell death but not a tunicamycin-induced increase. Homocysteine induced greater CHOP expression (2.7-fold) in BHMT small interfering RNA (siRNA)-transfected cells than in a control (1.9-fold). Homocysteine-induced cell death was increased by 40% in the siRNA-treated cells in comparison with the control. Apolipoprotein B (apoB) expression was higher and triglycerides and cholesterol were lower in HepG2 expressing BHMT. In primary mouse hepatocytes, homocysteine induced the accumulation of triglycerides and cholesterol, which was reduced in the presence of betaine. Betaine partially reduced homocysteine-induced sterol regulatory element binding protein 1 expression in HepG2 cells and increased S-adenosylmethionine in primary mouse hepatocytes.
CONCLUSION: The BHMT/betaine system directly protects hepatocytes from homocysteine-induced injury but not tunicamycin-induced injury, including an endoplasmic reticulum stress response, lipid accumulation, and cell death. This system also exhibits a more generalized effect on liver lipids by inducing ApoB expression and increasing S-adenosylmethionine/S-adenosylhomocysteine.
Pandey SN, Srivastava A, Dixit M, et al.Haplotype analysis of signal peptide (insertion/deletion) and XbaI polymorphisms of the APOB gene in gallbladder cancer.
Liver Int. 2007; 27(7):1008-15 [PubMed
] Related Publications
PURPOSE: The incidence of gallbladder cancer (GBC) is usually paralleled by the prevalence of gallstone disease, and genes of cholesterol metabolism have been implicated in gallstone disease. The XbaI and insertion/deletion (ins/del) polymorphism of Apolipoprotein B (APOB) appears to influence cholesterol homoeostasis and possibly risk for gallstone disease. We examined the effect of these polymorphisms individually as well as their haplotypes on GBC and gallstone patients in North Indian population.
METHODS: The study comprises 123 consecutive cases of proven GBC, 172 cases of gallstone and 232 healthy subjects of similar age and sex. The genomic DNA was extracted from peripheral blood leucocytes and genotyping was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism.
RESULTS: In a case-control study, APOB XbaI and ins/del polymorphisms were not significantly associated with risk of GBC. Using the expectation maximization algorithm, four haplotypes were obtained, and haplotype X(+),D was found to be significantly higher in GBC patients without stone in comparison with healthy subjects [odds ratio (OR) 2.9, 95% confidence interval 1.2-6.6 P=0.012].
CONCLUSIONS: The X(+),D haplotype of APOB is associated with increased risk for development of GBC and the risk is not modified in the presence of gallstones.
Sidiropoulos KG, Zastepa A, Adeli KTranslational control of apolipoprotein B mRNA via insulin and the protein kinase C signaling cascades: evidence for modulation of RNA-protein interactions at the 5'UTR.
Arch Biochem Biophys. 2007; 459(1):10-9 [PubMed
] Related Publications
The link between hepatic insulin signaling and apolipoprotein B (apoB) production has important implications in understanding the etiology of metabolic dyslipidemia commonly observed in insulin-resistant states. Recent studies have revealed important translational mechanisms of apoB mRNA involving the 5' untranslated region (5'UTR) and insulin-mediated translational suppression via an insulin-sensitive RNA binding protein. Here, we have investigated the role of the protein kinase C (PKCs) signaling cascade in the regulation of apoB mRNA translation, using a series of chimeric apoB UTR-luciferase constructs, in vitro translation of UTR-luciferase cRNAs, and metabolic labeling of intact HepG2 cells. The PKC activator, phorbol 12-myristate 13-acetate (PMA), increased luciferase expression of constructs containing the apoB 5' UTR whereas treatment with Bis-I, a general PKC inhibitor or Go6976, a more specific PKC alpha/beta inhibitor, decreased expression, under both basal and insulin-treated conditions. These effects were confirmed to be translational in nature based on in vitro translation studies of T7 apoB UTR-luciferase constructs transcribed and translated in vitro in the presence of HepG2 cytosol treated with insulin or signaling modulators. Mobility shift experiments using cytosol treated with either PKC inhibitor (Bis-I) or activator (PMA) showed parallel changes between translation of apoB 5'UTR-luciferase constructs and the binding of a protein(s) complex migrating around 110 kDa to the apoB 5' UTR. ApoB mRNA levels were unaltered under these conditions based on real-time PCR analysis. Bis-I and Go6976 were both able to significantly decrease newly synthesized apoB100 protein in the presence or absence of insulin. Overall, the data suggests that PKC activation may induce increased mRNA translation and synthesis of apoB100 protein through a mechanism involving the interaction of trans-acting factors with the apoB 5'UTR. We postulate potential links between PKC activation as seen in insulin-resistant/diabetic states, enhanced translation of apoB mRNA, and hepatic VLDL-apoB overproduction.
BACKGROUND: Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels.
RESULTS: We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26-27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27.
CONCLUSION: The induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48.
Tsai J, Qiu W, Kohen-Avramoglu R, Adeli KMEK-ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-ApoB100 particle assembly.
Arterioscler Thromb Vasc Biol. 2007; 27(1):211-8 [PubMed
] Related Publications
OBJECTIVE: Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway.
METHODS AND RESULTS: HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins.
CONCLUSIONS: Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.
Leong WF, Chow VTTranscriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection.
Cell Microbiol. 2006; 8(4):565-80 [PubMed
] Related Publications
Insights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host- and virus-mediated transcriptional responses. An oligo-based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real-time reverse transcription polymerase chain reaction (RT-PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two-dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis-associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase-like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real-time RT-PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.
Liver plays a key role in the metabolism of plasma apolipoproteins, endogenous lipids and lipoproteins. Hepatocellular carcinoma (HCC) is one of the most common fatal malignant tumors in China and in other Southeast Asian countries. This has been attributed to the high incidence of hepatitis B infection. Hepatitis B proteins, such as the hepatitis B X protein (HBx) that is large hepatitis B surface protein could regulate transcription of many candidate genes for liver carcinogenesis. It has known that patients who suffered from acute hepatitis B could have lipid disorders such as decreased plasma level of high-density lipoproteins (HDL). Furthermore, aberrations of lipid metabolism are often seen in the chronic hepatitis B infection. Plasma lipid profiles could be changed under HCC. In majority of the reports in HCC, plasma levels of triglycerides (TG), cholesterol, free fatty acids (FFA), HDL, low-density lipoproteins (LDL), lipoprotein (a) (Lp(a)), apolipoprotein AI (apoAI) and apoB were slight to significantly decreased, however, in some cases plasma levels of TG and Lp(a) might be increased. It has been suggested that analysis of plasma levels of lipids, lipoproteins and apolipoproteins in the patients suffered from HCC reflects on the hepatic cellular impairment status. Studies revealed that alterations seen in the plasma levels of lipids, lipoproteins and apolipoproteins reflecting patients' pathologic conditions. Decreased serum levels of cholesterol and apoAI may indicate a poor prognosis. Human leukaemic cells and certain tumor tissues have a higher receptor-mediated uptake of HDL and LDL than the corresponding normal cells or tissues. LDL and HDL have therefore been proposed as a carrier for the water-insoluble anti-cancer agents.
Li J, Wang F, Mamon H, et al.Antiprimer quenching-based real-time PCR and its application to the analysis of clinical cancer samples.
Clin Chem. 2006; 52(4):624-33 [PubMed
] Related Publications
BACKGROUND: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality.
METHODS: We modified one of the 2 PCR primers by use of an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3' quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer.
RESULTS: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r(2) >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens.
CONCLUSION: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.
Jackson KG, Maitin V, Leake DS, et al.Saturated fat-induced changes in Sf 60-400 particle composition reduces uptake of LDL by HepG2 cells.
J Lipid Res. 2006; 47(2):393-403 [PubMed
] Related Publications
The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of 125I-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of 125I-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (Sf) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of 125I-labeled LDL compared with PUFA- and MUFA-rich particles (P < 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of 125I-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.
Domitrovich AM, Felmlee DJ, Siddiqui AHepatitis C virus nonstructural proteins inhibit apolipoprotein B100 secretion.
J Biol Chem. 2005; 280(48):39802-8 [PubMed
] Related Publications
Host genes involved in lipid metabolism are differentially regulated during the early stages of hepatitis C virus (HCV) infection. The majority of lipids synthesized in the liver are exported to other tissues in the form of lipoproteins. The formation of these lipoproteins is dependent upon the association of triglycerides with apolipoprotein B100. Using the HCV subgenomic replicon expression system, we show that secretion of apoB100 is significantly reduced. Inhibition of apoB100 degradation by ALLN did not improve secretion. Triglyceride levels as well as microsomal triglyceride transfer protein mRNA and activity levels were reduced in replicon-expressing cells, indicating potential reasons for the observed decrease. Further evidence is presented for the interaction between the HCV nonstructural protein 5A and apoB100. These results provide further insight into the alteration of lipid metabolism by HCV.
Sidiropoulos KG, Pontrelli L, Adeli KInsulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein.
Biochemistry. 2005; 44(37):12572-81 [PubMed
] Related Publications
Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.
Lemken ML, Wybranietz WA, Schmidt U, et al.Expression liver-directed genes by employing synthetic transcriptional control units.
World J Gastroenterol. 2005; 11(34):5295-302 [PubMed
] Related Publications
AIM: To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems.
METHODS: Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting.
RESULTS: A new bifunctionally-enhanced green fluorescent protein (EGFP)/neo(r) fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis.
CONCLUSION: Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.