The etiology of human hepatitis B virus-related hepatocellular carcinoma

In this thesis, the p53 tumor suppressor gene in 10 different human hepatoma cell lines (Chapter 5) and in 38 (at least 17 of 38 are HBV serum-positive) primary hepatocellular carcinomas have been examined (Chapters 5 & 6) at the DNA, RNA, and protein levels. Chapter 5. The p53 alleles in the hepatoma cell lines appear to be a frequent target of mutation as demonstrated by Southern and Northern blotting, immunoprecipitation and Western blot analysis. In general, the steady state level of p53 specific RNA or protein in these hepatoma cell lines is higher than in normal liver. However, in three out of ten cell lines, normal-sized p53 mRNAs are not detectable. In contrast, the involvement of the p53 allele in HBV serum-positive primary hepatocellular carcinomas appears to be an exceedingly rare event. In Chapter 5, 18 different primary HCCs (status of HBV-positivity unknown) were analysed. Steady state levels of p53-specific RNA in primary hepatomas are practically indistinguishable from those in normal liver. Using the polymerase chain reaction technique, we amplified and subcloned exons 5, 6, 7, and 8 of p53 from 9 different hepatoma samples. No apparent structural alterations of p53 were found. In Chapter 6, a total of 20 different HCC samples (17 were HBV serum-positive) and their non-tumorous tissues, have been further examined using restriction fragment length polymorphism (RFLP) analyses. Loss of heterozygosity (LOH) was found in only 3 out of 20 samples (3 out of 17 HBV serum-positive). These three samples were also found to carry a point mutation within the remaining nondeleted p53 allele. One point mutation of p53 was found to be at a novel position at codon 180. All three point mutations are of somatic origin. Ten samples, randomly chosen from the remaining 17 LOH negative HCC tumors, were analysed further by DNA sequencing and Western blot analyses. No point mutations in p53 were found from these ten samples. None of the point mutations reported in this thesis were at amino acid 249, the proposed aflatoxin-induced mutational hotspot. We conclude that p53 mutation occurs infrequently, only approximately 18% in HBV serum-positive primary hepatomas, and only 10% in total primary hepatomas from Taiwan. (Abstract shortened with permission of author.

Sproutv1, a wt1 target gene, anagonizes the action of, receptor tyrosine kinases

WT1 is a tumor suppressor gene with an essential role in the development of the kidney. In addition WT1 in expressed in hematopoietic progenitor cells and can inhibit hematopoietic cell growth. In a model system for WT1 action we found that in addition to inhibiting cell growth.WTl could induce features of epithelial differentiation in NIH 3T3 fibroblasts. Using this cell line model we performed representative difference analysis to isolate genes regulated by WT1. The genes isolated were then tested for their ability to be induced after transient transduction of naive 3T3 cells with a WT1-harboring retrovirus, their expression in a podocyte and mesonephric cell line and finally for their expression in the developing murine kidney. Among the genes meeting all these criteria was sprouty 1. Sprouty was originally isolated in Drosophila as an inhibitor of FGF signaling and branching morphogensis of the respiratory system of the fly. In order to understand the potential function of sprouty in organogenesis and tumor development, we established cells that conditionally expressed sprouty. Sproutyl as well as sprouty 2 inhibited growth of NIH 3T3 cells. Sprouty inhibited the activation of ERK by FGF and PDGF and inhibited the ability of growth factors to activate the ELK1, API and NFkB transcription factors. Inhibition of NFkB activity was specific to the PDGF and not the TNF pathway. While the RAS/RAF/MEK pathway was inhibited by sprouty, activation of AKT through the PI3 kinase pathway was not, again indicating a specific point of action of sprouty downstream of the receptor tyrosine kinase. In this regard, sprouty inhibited the activation of ERK, MEK and inhibited the binding of RAS to RAF. This suggest that sprouty either prevents the accumulation of GTP-RAS or inhibts the RAS/RAF interaction. These data indicate that sprouty is growth inhibitor downstream of WT1 whose expression in response to WT1 and growth factor signaling may limit proliferation during organ development and hematopoiesis

Unbiased Whole-Genome Amplification Directly From Clinical Samples

Preparation of genomic DNA from clinical samples is a bottleneck in genotyping and DNA sequencing analysis and is frequently limited by the amount of specimen available. We use Multiple Displacement Amplification (MDA) to amplify the whole genome 10,000-fold directly from small amounts of whole blood, dried blood, buccal cells, cultured cells, and buffy coats specimens, generating large amounts of DNA for genetic testing. Genomic DNA was evenly amplified with complete coverage and consistent representation of all genes. All 47 loci analyzed from 44 individuals were represented in the amplified DNA at between 0.5- and 3.0-fold of the copy number in the starting genomic DNA template. A high-fidelity DNA polymerase ensures accurate representation of the DNA sequence. The amplified DNA was indistinguishable from the original genomic DNA template in 5 SNP and 10 microsatellite DNA assays on three different clinical sample types for 20 individuals. Amplification of genomic DNA directly from cells is highly reproducible, eliminates the need for DNA template purification, and allows genetic testing from small clinical samples. The low amplification bias of MDA represents a dramatic technical improvement in the ability to amplify a whole genome compared with older, PCR-based methods

A Highly Sensitive, High-Throughput Assay for the Detection of Turner Syndrome

Turner syndrome is detected in the clinical setting by high-throughput pyrosequencing using buccal swab and other DNA samples, providing an alternative to karyotype testing