Biomedical subjects

Jason H Bielas

Publications and source records attributed to Jason H Bielas.

9 recordsLinked to original sources

PubMed2006

Human cancers express a mutator phenotype.

Cancer cells contain numerous clonal mutations, i.e., mutations that are present in most or all malignant cells of a tumor and have presumably been selected because they confer a proliferative advantage. An important question is whether cancer cells also contain a large number of random mutations, i.e., randomly distributed unselected mutations that occur in only one or a few cells of a tumor. Such random mutations could contribute to the morphologic and functional heterogeneity of cancers and include mutations that confer resistance to therapy. We have postulated that malignant cells exhibit a mutator phenotype resulting in the generation of random mutations throughout the genome. We have recently developed an assay to quantify random mutations in human tissue with unprecedented sensitivity. Here, we report measurements of random single-nucleotide substitutions in normal and neoplastic human tissues. In normal tissues, the frequency of spontaneous random mutations is exceedingly low, less than 1 x 10(-8) per base pair. In contrast, tumors from the same individuals exhibited an average frequency of 210 x 10(-8) per base pair, an elevation of at least two orders of magnitude. Our data document tumor heterogeneity at the single-nucleotide level, indicate that accelerated mutagenesis prevails late into tumor progression, and suggest that elevation of random mutation frequency in tumors might serve as a novel prognostic indicator.

DNA, Single-Stranded↗

PubMed2006

Non-transcribed strand repair revealed in quiescent cells.

Stem cells, one of the progenitors of cancer, exist predominately in a quiescent state. Thus, understanding the mechanisms of DNA repair and mutagenesis in such arrested cells may help unravel the complex process of tumorigenesis. Two major nucleotide excision repair (NER) pathways are known to remove bulky physical or chemical lesions from DNA. Transcription-coupled repair (TCR) acts solely on the transcribed strand of expressed genes, while global genomic repair (GGR) is responsible for the ubiquitous repair of the genome. Indirectly, it has been shown that while TCR functions in quiescent cells GGR does not. To explicitly elucidate this phenomenon, we adapted a quantitative PCR (QPCR) assay to study UV-damage repair via TCR and GGR in quiescent and proliferating cells. We present evidence that repair of untranscribed silent regions of the genome and repair of the non-transcribed strand of active genes proceeds by two discrete mechanisms in quiescent cells; rather than by GGR, which was believed to encompass both. Thus, our findings suggest the existence of an alternate NER pathway in quiescent cells. The proposed subcategories of NER are as follows: (i) TCR, responsible for maintenance of transcribed strands; (ii) GGR, responsible for ubiquitous genome repair; and (iii) non-transcribed strand repair (NTSR), predominantly responsible for the repair of the NTS in arrested cells. In quiescent cells, it is evident that TCR and NTSR function and GGR are arrested. As a consequence, mutation accumulation at temporally silent genes and incomplete or imperfect repair of transcribed genes, in quiescent stem cells, may provide a source of cancer causing mutations.

Animals↗

PubMed2005

Generation of mutator mutants during carcinogenesis.

Mutations are rare in normal cells. In contrast, multiple mutations are characteristic in most tumors. Previously we proposed a "mutator phenotype" hypothesis to explain how pre-cancer cells may acquire large number of mutations during carcinogenesis. Here we extend the "mutator phenotype" hypothesis considering recently discovered biochemical activities whose aberrant expression may result in genome-wide random mutations. The scope of this article is to emphasize that simple random point mutations can drive carcinogenesis and highlight new emerging pathways that generate these mutations. We focus specifically on random point mutations generated by replication errors, oxidative base damage, covalent base modifications by enzymes, and spontaneously generated abasic sites as a source of mutator mutants.

Animals↗

PubMed2005

Quantification of random genomic mutations.

Cancer cells contain numerous clonal mutations. It has been theorized that malignant cells sustain an elevated mutation rate and, as a consequence, harbor yet larger numbers of random point mutations. Testing this hypothesis has been precluded by lack of an assay to measure random mutations-that is, mutations that occur in only one or a few cells of a population. We have established a method that has permitted us to detect and identify rare random mutations in human cells, at a frequency of 1 per 10(8) base pairs. The assay is based on gene capture, by hybridization with a uracil-containing probe, followed by magnetic separation. Mutations that render the mutational target sequence non-cleavable by a restriction enzyme are quantified by dilution to single molecules and real-time quantitative PCR amplification. The assay can be extended to quantify mutation in any DNA-based organism, at different sites in the genome, in introns and exons, in unselected and selected genes, and in proliferating and quiescent cells.

Chromosome Mapping↗

PubMed2004

Quiescent murine cells lack global genomic repair but are proficient in transcription-coupled repair.

The majority of the cells in the body, including stem cells, exist in a quiescent state, so it is in quiescent cells where most DNA damage occurs. It has been uncertain whether or not this damage is repaired or fixed into mutations during quiescence or if proliferation is required for both. Prior to the development of transgenic mice, it was difficult to distinguish between these two possibilities, as cells had to proliferate to form colonies before mutations could be detected. Transgenes, however, can be shuttled out of quiescent mouse cells directly, and the level of DNA damage and mutation can be measured. Such measurements show that both mutation and repair are absent at a non-transcribed transgene in quiescent cells, although both are initiated when these cells are induced to proliferate. Conversely, the repair of transcriptionally active genes proceeds independently of proliferation in the same cells, as shown by the differential survival of wild-type and XPA-/- cells. We infer from these results that global genomic DNA repair (GGR) is not active during cellular quiescence but that transcription-coupled repair (TCR) is, suggesting that GGR is restricted to S, whereas TCR remains active throughout the cell cycle.

Alkylating Agents↗

PubMed2003

Elevated mutagenesis and decreased DNA repair at a transgene are associated with proliferation but not apoptosis in p53-deficient cells.

p53, the most commonly mutated gene in human tumors, is believed to play a crucial role in the prevention of cancer by protecting cells from mutation, a theory commonly known as the "Guardian of the Genome" hypothesis. There are two hypotheses as to how this can occur. In the first, p53 protects the genome by retarding the cell cycle, thus allowing more time for DNA repair. In the second, p53 reduces cancer by initiating apoptosis in damaged cells, thus making it impossible for these cells to become carcinogenic. This study directly tested these two theories in primary murine embryonic fibroblasts on a common genetic background with and without p53, using a lacI transgene as a mutational target. The data demonstrate that, as a direct consequence of cell cycle delay, p53 slowed the induction of mutations and decreased their frequency but had little effect on the frequency of apoptosis. This indicates that the function of p53 in cell cycle control is more important than the role of p53 in apoptosis, for mutation prevention, in any uniform cell population. Moreover, p53-mediated protection is further improved in slowly dividing cells, suggesting that p53 may be particularly important in protecting stem cells from mutation. The role of apoptosis in vivo, however, may be to remove whole tissue subpopulations that can be renewed by less sensitive stem cells.

Animals↗

PubMed2002

A more efficient Big Blue protocol improves transgene rescue and accuracy in a adduct and mutation measurement.

Transgenic mutational systems have provided researchers with an invaluable tool, allowing the measurement of both spontaneous and induced mutations. The Big Blue transgenic rodent mutagenesis system developed by Stratagene (La Jolla, CA) uses a lambda shuttle vector carrying lacI as the mutational target gene. A common criticism of the Big Blue system is that it relies on visual screening to detect mutants rather than positive selection, which is employed in more recently developed systems. The lack of positive selection, however, has provided the Big Blue system with a unique advantage, as it allows for the dynamic quantification of mutation fixation, repair, and adduct stability, since both pre-mutagenic DNA adducts and mutations can readily be quantified [Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 11391]. Improvements to the standard Big Blue assay protocol are required for the visualization of mutant plaques resulting from pre-mutagenic damage, as these can appear much lighter in color than the lightest color control mutant (CM0). This increase in detection has been achieved by the development of a protocol that now permits the effective measurement of repair and mutation fixation utilizing the Big Blue system. This new protocol has also addressed efficiency, allowing for a two-fold increase in the number of plaques produced per packaging reaction and a decrease in both phage migration and plaque size, permitting a greater than three-fold increase in plating density. The implementation of this protocol will make the Big Blue assay more economical and less demanding than before, while providing researchers with an efficient means to measure both repair and mutation in this system.

Animals↗

PubMedDate not supplied

Mutator phenotype in cancer: timing and perspectives.

Normal human cells replicate their DNA with exceptional accuracy. During every division cycle, each daughter cell receives a full and accurate complement of genetic information. It has been estimated that approximately one error occurs during DNA replication for each 10(9) to 10(10) nucleotides polymerized. Stem cells, the cells that are progenitors of cancer, may replicate their genes even more accurately. In contrast, the malignant cells that constitute a tumor are markedly heterogeneous and exhibit multiple chromosomal abnormalities and alterations in the nucleotide sequence of DNA. To account for the disparity between the rarity of mutations in normal cells and the large numbers of mutations present in cancer, we initially hypothesized that during tumor progression, cancer cells must exhibit a mutator phenotype. In this perspective, we summarize the evidence supporting a mutator phenotype in human cancer, analyze recent measurements of mutations in human cancer, consider the timing for the expression of a mutator phenotype, and focus on the important consequences of large numbers of random mutations in human tumors.

Disease Progression↗

PubMedDate not supplied

Unifying concept of DNA repair: the polymerase scanning hypothesis.

According to a series of experiments on untransformed mouse embryonic fibroblasts, quiescent mouse cells lack global genomic repair (GGR) of premutagenic DNA damage. The gene used to assess mutation and premutagenic DNA damage was the lacI transgene incorporated permanently in the DNA in a lambda shuttle vector. The transgene lacks mammalian transcription signals and thus is unexpressed in the cells. Although the cells conducted transcription-coupled repair (TCR) of UV damage, the transgene was not repaired over a 4-day interval. These cells are not terminally differentiated and can readily be induced to resume cellular division. In this article, we discuss the interpretation of these results and suggest a new hypothesis for DNA scanning, the mechanism by which cells discover DNA damage and initiate DNA repair. Our hypothesis, which we call the polymerase scanning hypothesis, is that GGR is initiated in very much the same way as TCR, by a polymerase complex encountering the damage. We call the two together polymerase-coupled repair (PC repair). In the case of GGR, it would be the DNA replication complex during the S-phase. This is, we suggest, the dominant mechanism of repair of DNA at low doses for untranscribed genes. Evidence contrary to this hypothesis exists, which we discuss, but it should be noted that existing hypotheses about DNA scanning and DNA repair cannot account for the results that we have obtained.

Animals↗