Timothy M. Block

Human Urine Contains Small, 150 to 250 Nucleotide-Sized, Soluble DNA Derived from the Circulation and May Be Useful in the Detection of Colorectal Cancer

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.

In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal gangliaof latently infected mice

The presence of herpes simplex virus (HSV-1) DNA in the trigeminal ganglia of latently infected mice was detected by an in situ DNA polymerase chain reaction (PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). These results were compared to the number of neurons possessing the HSV-1 latency associated transcript (LAT), as detected by in situ RNA hybridization with LAT probes. Sensitivity assays were shown to detect a single copy cellular gene in 48% of neuronal cell bodies. The results suggest that in situ PCR is an effective method to locate and detect HSV-1 within latently infected neurons. Moreover, the number of neurons found to be harboring HSV-1, by the method of in situ PCR, which does not depend upon virus gene expression, is within threefold of the number detected by in situ hybridization for LAT. Therefore, this report describes the first detection of HSV-1 DNA in latently infected murine trigeminal ganglia by the method of indirect in situ PCR, and compares the findings to the number of neurons expressing LAT, as assessed by conventional in situ hybridization.

Transrenal DNA as a Diagnostic Tool: Important Technical Notes

Abstract: 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.

The combination of interferon α-2b and n-butyl deoxynojirimycin has a greater than additive antiviral effect upon production of infectious bovine viral diarrhea virus (BVDV) in vitro: implications for hepatitis C virus (HCV) therapy

Interferon alpha-2b (IFN) alone or in combination with Ribavirin is approved in the United States for the treatment of chronic hepatitis C virus (HCV) infection. We have previously reported that the glucosidase inhibitor, n-butyl deoxynojirimycin (nB-DNJ) inhibits the production of infectious bovine diarrhea virus (BVDV) (Proc. Natl. Acad. Sci. 96 (1999) 11878). Since BVDV has been used as a model for HCV and grows productively in tissue culture, and IFN and glucosidase inhibitors are thought to act at different steps in the virus life cycle, it was of interest to determine the antiviral impact of combining nB-DNJ with IFN. Using plaque reduction and single-step growth analyses of the cytopathic BVDV strain NADL, data are presented that shows human IFN inhibited BVDV production in a dose dependent manner, with 3 IU/ml inhibiting 50% of the yield of virus (IC50) when added within 1 h post infection. Under the same conditions, the glucosidase inhibitors nB-DNJ and castanospermine (CST) also prevented BVDV production in a dose dependent manner with IC50s of 226 microM and 47 microM, respectively. In combination with 138 microM nB-DNJ the apparent IC50 for IFN was 0.056 IU/ml. This 54-fold increase in IFN potency suggests that nB-DNJ can synergize with IFN. Two additional independent analyses were performed to measure combination effects which demonstrated that the combined antiviral effect of nB-DNJ and IFN were greater than would be expected for a simple additivity. These data are consistent with an interpretation that glucosidase inhibitors and IFN have a synergistic antiviral effect in tissue culture. The relevance of these finding to treatment of HCV infection is discussed.

Imino sugars that are less toxic but more potent as antivirals, in vitro, compared with N-n-nonyl DNJ

Imino sugar glucosidase inhibitors have selective antiviral activity against certain enveloped, mammalian viruses. Deoxynojirimycins (DNJs) modified by N-alkylation to contain a nine carbon atom side chain (N-n-nonyl-deoxynojirimycin; N-nonyl-DNJ, NN-DNJ) were shown to be, for example, at least 20 times more potent in inhibiting hepatitis B virus (HBV) and bovine viral diarrhoea virus (BVDV) in cell based assays than the non-alkylated DNJ. These data suggested that modification of the alkyl side chain could influence antiviral activity. Previous work has focused on varying side chain length. In this report, the influence of side chain branching and cyclization upon toxicity and antiviral activity was explored. Briefly, using a virus secretion assay for HBV and a single step growth (yield reduction) assay for BVDV, 14 different DNJ-based sugars, possessing various N-alkyl substitutions, were tested for antiviral activity. Of the series, N-methoxy-nonyl-DNJ and N-butyl-cyclohexyl DNJ were determined to have the best selectivity index against BVDV and HBV, with the N-methoxy analogue being the most potent with micromolar antiviral activity. The results of this antiviral survey and the implications for the mechanism of action and ultimate therapeutic potential of the DNJ-based imino sugars is provided and discussed.

Stability and circularization of herpes simplex virus type 1 genomes in quiescently infected PC12 cultures

Herpes simplex virus type 1 (HSV-1) DNA has been shown to exist as a linear, double-stranded molecule in the virion and as a non-linear (endless), episomal, nucleosomal form in latently infected trigeminal ganglia. The kinetics of the formation and appearance of endless viral genomes and the stability of linear genomes in neuronal cells are not well understood. Nerve growth factor (NGF)-differentiated PC12 cells can sustain long-term, quiescent infections with HSV-1. In this report, the structure and stability of HSV-1 viral DNA in NGF-differentiated PC12 cells was studied as a function of time following infection using both wild-type and replication-defective virus. Unexpectedly, unencapsidated linear genomes were stable in the nucleus of NGF-differentiated PC12 cells for up to 2-3 weeks following infection, beyond the period at which there is no detectable viral gene expression. However, following infection with wild-type HSV, the majority of quiescent viral genomes were in an endless form after 3-4 weeks. These data suggest that the stability and fate of HSV-1 DNA in non-mitotic neuronal-like cells is different from that in productively infected cells and thus there is a significant cellular role in this process. The relevance to the virus life-cycle in neurones in vivo is discussed.

Long term herpes simplex virus type 1 infection of nerve growth factor-treated PC12 cells

The behaviour of herpes simplex virus type 1 (HSV-1) strain 17 in tissue cultures of PC12 cells treated with nerve growth factor (NGF) was studied. PC12 cells respond to NGF by ceasing to proliferate and extending long neurites. After differentiation with NGF, cultures were infected with HSV-1 and maintained in the presence of the hormone for several weeks. These long-term infected cultures were tested for HSV DNA, transcripts and the ability to produce virus, before and after NGF removal. Before NGF removal, the cultures were characterized by little or no virus production and the presence of HSV-1 DNA in a predominantly endless form. In situ analysis of long-term infected cultures revealed latency-associated transcript expression in only a portion of the cells. However, as shown by an infectious centre assay, virus was present in almost all cells in the population. Moreover, removal of NGF from long-term cultures resulted in the appearance of significantly increased amounts of virus in the media. The degree to which this system resembles HSV latency in vivo is discussed.

A Herpes simplex virus type 1 mutant with a deletion immediately upstream of the LAT locus establishes latency and reactivates from latently infected mice with normal kinetics

The latency associated transcripts (LATs) are the only abundant viral gene products detected during latent herpes simplex virus (HSV) infection of peripheral nerves in animals and people. A LAT promoter has been identified and mutant viruses with lesions removing the promoter and surrounding region have been observed to reactivate slowly from trigeminal ganglia (TG) explanted from latently infected mice. Previous work has shown that most mutants with lesions limited to regions downstream of the LAT promoter reactivate normally. Therefore, to help map the boundaries of the slow reactivation phenotype, a mutant virus with lesions located immediately upstream of the LAT promoter was constructed and called 17 delta S/N. 17 delta S/N contains a 437 nucleotide (nt) deletion 332 nts upstream of the TATAA box of the LAT promoter. In productively infected cells, 17 delta S/N failed to synthesize detectable amounts of the 1.1 and 1.8 kb transcripts which are produced during wild-type infections and are specified by a region just upstream of the LAT promoter. However, 17 delta S/N did produce normal amounts of LAT in tissue culture as well as in neurons derived from latently infected cells, as ascertained by Northern blot and in situ hybridization analysis. Moreover, in latently infected mice, 17 delta S/N established and maintained infection in as many neurons as did wild type virus, as determined by in situ polymerase chain reaction (PCR) to detect viral DNA. Finally, the virus reactivated from TG derived from latently infected mice with kinetics indistinguishable from those of wild-type virus. Therefore, reactivation from latency, in this model system, does not appear to require function from the viral genomic region located immediately upstream of the LAT promoter.

Novel bacteriological assay for detection of potential antiviral agents.

A prototype assay for the initial screening of potential antiviral agents that uses bacterial growth on selective media is described. The human immunodeficiency virus (HIV) protease recognition sequence was inserted into the tetracycline resistance (Tet) protein encoded by plasmid pBR322 of Escherichia coli. Expression of both the HIV protease and the modified Tet protein prevented growth in the presence of tetracycline. However, inhibition of the HIV protease restored tetracycline resistance. Thus, potential HIV protease inhibitors can be identified by their ability to confer tetracycline resistance to this bacterial strain. The assay is simple, rapid, and inexpensive, and this concept can be applied to the search for inhibitors of other viral proteases. Images

Inhibition of transient gene expression with plasmids encoding herpes simplex virus type 1 UL55 and alpha genes

Herpes simplex virus type 1 (HSV-1) subgenomic sequences from 0.743 to 0.782 map units have been molecularly cloned as plasmid AT1 and shown to inhibit stable DNA-mediated gene transformation of Ltk- cells with the HSV-1 thymidine kinase (tk) gene. Here it is shown that AT1 also inhibits transient gene expression. Expression from the chloramphenicol acetyltransferase (CAT) gene under the control of either the HSV-1 tk gene or the Rous sarcoma virus (RSV) promoter was inhibited when cotransfected into Ltk- and CV-1 cells with equimolar amounts of AT1. AT1 was subcloned as three overlapping plasmids called AT1a, alpha 27 and AT1b. The alpha 27 plasmid encodes the HSV-1 immediate early gene, alpha 27; AT1a possesses sequences that specify an open reading frame in HSV-1 strain KOS used in these studies, although the significance of this open reading frame is unknown; AT1b possesses the sequences for UL55 and UL56, also genes for which no function has been reported. No single subclone or pair of subclones demonstrated significant inhibition of transient gene expression. Cotransfection of all three subclones did result in inhibition of RSV-CAT gene expression, suggesting that information from each subclone is necessary. One of the three subclones, alpha 27, contains the HSV-1 immediate early gene, alpha 27, so the possibility that other immediate early genes could substitute for alpha 27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an alpha 0- or alpha 4-containing plasmid, suggesting that the role of the alpha 27-containing plasmid can be replaced by other alpha genes with trans-regulating capability. Finally, AT1a and AT1b linker insertion mutants have been constructed and used to study the role these plasmids play in mediating inhibition. These results suggest that AT1 contains HSV-1 functions in addition to that of alpha 27 that interfere with gene expression.