Research Program for Web

Research in the Loeb Lab is focused on the role of mutations in the initiation and progression of human cancers. These studies in the lab are centered on determining the accuracy of DNA synthesis by biochemical and genetic assays, or on measuring mutations in normal cells and in human tumors. We wish to identify the sources of spontaneous mutations and the role of mutations in driving tumor progression. Is the mutation rate in cancers greater than in normal cells and does increased mutation frequency drive the evolution of human tumors? That is to say, do cancer cells express a mutator phenotype and does this phenotype allow them to overcome environmental limitations? Does increased mutation frequency allow tumors to divide where they ought not to, to invade, and to metastasize? Our current research efforts are in the following areas:

1)    Human cancers express a mutator phenotype: For many years we have advanced the hypothesis that the mutation frequency in human tumors is greater than in corresponding normal cells- we proposed that cancers exhibit a mutator phenotype. The basic concept is that normal mutation rates are very low and insufficient to account for the large numbers of mutations in tumors. Mathematical models indicate that mutator pathways are the most efficient paths to cancer and are most effective when they are expressed early. Until recently, this hypothesis was ignored. However, evidence by DNA sequencing now verifies that human tumors have thousands of clonal mutations. We have established several assays to measure rare random mutations and subclonal mutations in human tumors that are being used to address fundamental aspects of how cancers evolve.

2)    Fidelity of DNA replication: We are continuing to study the mechanism(s) by which DNA polymerases can copy DNA with phenomenally high accuracy, approaching 1 error in every 10 million nucleotides polymerized.  Our approach is to copy DNA in vitro with purified polymerases, transform bacteria with reaction products, and assay for the frequency and spectrum of mutations produced by the polymerase. Recently, we have extended this approach to measure mutation frequencies by different polymerases in mammalian cells. An analysis of mutations produced by polymerases is key to evaluating the contribution of DNA replication to spontaneous mutations and to determine if reduced fidelity of DNA replication is causally associated with cancer.

3)    Lethal mutagenesis: Increasing the mutation rate of RNA viruses invariably induces an error catastrophe since the error rate exceeds the threshold for viability. We have measured the enhancement of mutagenesis in HIV-infected cells in culture by mutagenic nucleoside analogs and one of these is now in Phase 2 clinical trials. We currently desire to determine if mutagenic nucleoside analogs will also generate an error catastrophe in human cancer cells, particularly in patients who have an increased elevation of mutations due to treatment by chemotherapeutic agents.

4)    Mutations in aging: DNA is under constant bombardment by environmental agents and endogenous reactive chemical metabolites. As a result, mutations increase in an age-dependent manner. We have focused our experiments on measuring mutations in mitochondrial DNA, particularly in age-dependent neurological diseases including Alzheimer and Parkinson disease. In addition, we are interested in Werner syndrome, a rare inherited disease caused by mutations in a DNA helicase/exonuclease and manifested by premature aging and a high incidence of specific cancers. We desire to determine the function of this helicase in human cells. We have carried out studies on functional genetics by determining whether nucleotide variations in the Werner helicase gene found in human populations reduce its function. We are interested in identifying individuals with these variants and determining if they exhibit premature manifestations of aging or have increased predisposition to developing cancers prevalent in Werner syndrome.

5)    Duplex DNA Sequencing: We have established a new method for DNA sequencing that offers unprecedented sensitivity and accuracy for detecting rare mutations in large population of heterogeneous DNAs.  Our approach takes advantage of the complementary inherent in Watson-Crick DNA.  Using linkers with complementary unique identifiers we can sequence each of two strands of a DNA duplex.  True mutations would be present opposite each other and would be complementary.  This approach has a theoretical background mutation frequency of less than one mutation per billion nucleotides sequenced.

6) Applied molecular evolution: This project constantly interdigitates with many of the experiments that are carried out in our laboratory. We have developed protocols to construct vast populations of oligonucleotides containing random sequences, insert them into plasmids, and after transforming the plasmid constructs into bacteria assay for mutant enzymes coded for by the random sequences. We have obtained active herpes thymidine kinase, DNA polymerases, thymidine synthase, and DNA repair enzymes with properties that differ from those that nature has selected. These preternatural genes have the potential for use in industrial processes and gene therapy. For us, they provide variations on the enzyme variants to explore structure functional relationships.