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We have previously demonstrated that mutated DNA derived from the circulation can be detected in urine and predominantly exists as DNA fragments <1 kb. To preferentially isolate the trans-renal DNA from urine, we developed a method using carboxylated beads to separate high-MW (1 kb or larger) from low-MW DNA in urine. A primer set for 18s rRNA (generating a PCR product of 872 bp) was designed and optimized for real-time PCR quantification of high-MW DNA templates. To evaluate the method, urine samples from 5 volunteers with no known diseases and 36 patients with various colorectal diseases were collected and tested. It was found that the average removal efficiency of high-MW DNA from total urine DNA using carboxylated beads is 92.72%+/- 1.42%. Furthermore, compared with using total urine DNA, our method provides a greater ability to detect mutated K-ras in the urine of colorectal cancer patients. The concurrence of K-ras mutations detected in disease tissue and the corresponding urine specimen is significantly higher (P= 0.0015) when the samples were enriched in low-MW DNA.

Our previous studies demonstrated that urine contains DNA derived from the circulation and that this DNA originated, in part, from organ sites and tumors distal to the urinary tract. To explore the potential use of DNA from urine as compared to other body fluids as a source for circulating DNA for cancer detection, the DNA concentration and the frequency of detection of mutated Kristin-ras (K-ras) DNA in serum, plasma, and urine were examined. The concentration of DNA in the urine was similar to that in the serum, but the DNA concentration in plasma was significantly lower than in either urine or serum (P < 0.05). When DNA derived from 10 muL of body fluid was used in each mutation assay, the detection frequency of mutated K-ras DNA was comparable among serum, plasma, and urine. However, when DNA derived from 200 muL of body fluid was used, the incidence of detecting mutated K-ras DNA in urine was significant higher (95%) than in either serum (35%) or plasma (40%) (P < 0.0005), suggesting that inhibitory factors in serum/plasma may be more limiting than in urine. The use and practicality of urine as a source of circulating DNA for cancer detection are discussed.

A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal-size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr-DNA) belongs to a relatively low molecular-weight (150-250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular-weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular-weight DNA. As a result, sensitivity in detection of K-RAS mutations in serum of patients with colorectal tumors is significantly higher with DNA isolated with the GITC/WR method than with the QIAamp kit. Amplicon size is also extremely important in analysis of Tr-DNA, because the shorter the amplicon, the higher is the sensitivity of biomarker detection in Tr-DNA. One hundred fifty-seven and 87 bp amplicons were employed for detection of mutant K-RAS in DNA isolated from 0.1 mL of urine obtained from 15 patients with pancreatic cancer. Mutant K-RAS was found in Tr-DNA of 3 and 10 patients with the long and short amplicons, respectively. The sensitivity and specificity of detection of mutant sequences are reduced in the presence of high excess of a respective wild-type allele, but they can be significantly increased through application of enriched polymerase chain reaction (PCR), peptide nucleic acid (PNA) clamped PCR, and/or stencil-aided mutation analysis (SAMA), based on selective pre-PCR elimination of wild-type sequences.

Human urine has been shown to possess submicrogram per milliliter amounts of DNA. We show here that DNA isolated from human urine resolves into two size categories: the large species, greater than 1 kb, being predominantly cell associated and heterogeneous in size, and the smaller, between 150 to 250 bp, being mostly non-cell associated. We showed that the low molecular weight class of urine DNA is derived from the circulation, by comparing the mutated K-ras sequences present in DNA isolated from tumor, blood, and urine derived from an individual with a colorectal carcinoma (CRC) containing a mutation in codon 12 of the K-ras proto-oncogene. In the urine, mutated K-ras sequences were abundant in the low molecular weight species, but far less abundant in the large molecular weight-derived DNA. Finally, the possibility that detection of mutant K-ras sequences in DNA derived from the urine correlates with the occurrence of a diagnosis of CRC and polyps that contain mutant K-ras was explored in a blinded study. There was an 83% concurrence of mutated DNA detected in urine and its corresponding disease tissue from the same individuals, when paired urine and tissue sections from 20 subjects with either CRC or adenomatous polyps were analyzed for K-ras mutation. The possibility that the source of the trans renal DNA is apoptotic cells, and the potential use of this finding for cancer detection and monitoring is discussed.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general. [ABSTRACT FROM AUTHOR]

Hypermethylation of the glutathione S-transferase p 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite-PCR sequencing. We found that the 59 region of the position 248 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 39 region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 39 region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 59 region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 39 region, we found that the methylation of the 59-end of the GSTP1 promoter was significantly more specific than that of the 39-end (97.1% vs. 60%, p,0.0001 by Fisher's exact test) for distinguishing HCC (n = 120) from hepatitis (n = 35) and cirrhosis (n = 35). Encouragingly, 33.8% of the AFP-negative HCC contained the methylated GSTP1 gene. This study clearly demonstrates the importance of the location of CpG site methylation for HCC specificity and how liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC. [ABSTRACT FROM AUTHOR]

Abstract: Although alpha interferon (IFN-α) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-α inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-α. We found that IFN-α efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin–Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-α did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-α treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-α-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections. [Copyright &y& Elsevier]

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is formed by conversion of capsid-associated relaxed circular DNA (rcDNA) via unknown mechanisms and exists in the nucleus of the infected hepatocyte as a minichromosome that serves as the transcription template for viral RNAs. To study the molecular pathway of cccDNA formation and its regulation by viral and cellular factors, we have established a cell line that supports the replication of an envelope protein-deficient HBV genome in a tetracycline-inducible manner. Following induction of HBV replication, the cells accumulate higher levels of cccDNA as well as larger amounts of deproteinized rcDNA (DP-rcDNA) than cells that replicate wild-type HBV genomes. These results indicate that HBV envelope proteins negatively regulate cccDNA formation, and conversion of DP-rcDNA into cccDNA is a rate-limiting step of cccDNA formation in HepG2 cells. Detailed analyses reveal the following: (i) DP-rcDNA exists in both cytoplasm and nucleus; (ii) while nuclear DP-rcDNA is sensitive to DNase I digestion, a small fraction of cytoplasmic DP-rcDNA is DNase I resistant; (iii) both DNase I-sensitive and -resistant cytoplasmic DP-rcDNAs cosediment with capsids and can be immunoprecipitated with HBV core antibody; and (iv) a primer extension assay maps the 5' end of the minus strand of DP-rcDNA at the authentic end of virion rcDNA. Hence, our results favor a hypothesis that the removal of viral polymerase protein covalently linked to the 5' end of the minus-strand DNA occurs inside the capsid in the cytoplasm and most possibly via a reaction that cleaves the phosphodiester bond between the tyrosine of the polymerase and the 5' phosphoryl group of minus-strand DNA. [ABSTRACT FROM AUTHOR]

Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an "endless" or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain d120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant d120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the d120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant d120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the d120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the d120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in trans. [ABSTRACT FROM AUTHOR]