Mutation Rate In Cells Research Articles

The effect of cigarette smoking on lung cancer evolution.

The aim of this paper is to elucidate the risk of lung carcinogenesis from cigarette smoking among current and former smokers. To achieve this goal, we have set up a stochastic three-stage model to fit the data of Surveillance, Epidemiology, and End Results (SEER) program besides the data set of smoking derived from the Nurses' Health Study cohort of females (NHS) and the Health Professionals Follow up Study cohort of men (HPFS). The calculations are performed by considering both mutation and clonal expansion rates as parameters in each compartment. For current smokers, our findings show that cigarette smoking has more significant impact on the mutation rates of cells than the clonal expansion rates of premalignant cells among men and women. In particular, for male patients, cigarette smoking affects the mutation in normal cells and the transformation from premalignant cells to malignant ones in the optimal model. In addition, cigarette smoking induces only the initial mutation rates in normal cells among American women. For current and former smokers, cigarette smoking stimulates only the clonal expansion rate of the first premalignant cells in both sexes. However, we find that the impact of cigarette smoking is minimal in former smokers who have stopped smoking for over ten years among men and women in US.

Goal programming model for predicting the parameters involved in growth of cancer cells

Some nonlinear goal programming problems are formulated for predicting the decision parameters involved in the cancer and related cell growths such growth rate of normal and malignant cells, death rate of normal and malignant cells and mutation rate of normal cells. An application of preemptive and non-preemptive goal programming problem, some optimisation programming problems are constructed using the statistical moments derived from two state cancer cell growths model and solved using the optimisation LINGO software.

Aberrant DNA Damage Response and DNA Repair Pathway in High Glucose Conditions

Higher cancer rates and more aggressive behavior of certain cancers have been reported in populations with diabetes mellitus. This association has been attributed in part to the excessive reactive oxygen species generated in diabetic conditions and to the resulting oxidative DNA damage. It is not known, however, whether oxidative stress is the only contributing factor to genomic instability in patients with diabetes or whether high glucose directly also affects DNA damage and repair pathways. Normal renal epithelial cells and renal cell carcinoma cells are more chemo- and radiation resistant when cultured in high concentrations of glucose. In high glucose conditions, the CHK1-mediated DNA damage response is not activated properly. Cells in high glucose also have slower DNA repair rates and accumulate more mutations than cells grown in normal glucose concentrations. Ultimately, these cells develop a transforming phenotype. In high glucose conditions, defective DNA damage responses most likely contribute to the higher mutation rate in renal epithelial cells, in addition to oxidative DNA damage. The DNA damage and repair are normal enzyme dependent mechanisms requiring euglycemic environments. Aberrant DNA damage response and repair in cells grown in high glucose conditions underscore the importance of maintaining good glycemic control in patients with diabetes mellitus and cancer.

Heterogeneity in untreated, stressed and drug-tolerant cells: insights into the evolution of cancer resistance

The evolution of cancer is a Darwinian process, the composition of cancer cell population varying with time, especially after chemotherapy. Such changes occurring in the genetic landscape of tumours are possibly responsible for the chemotherapeutic resistance. Due to the recent advances in single cellular sequencing, the evolution of cancers can be monitored at the single cellular resolution. We reanalysed a previously published dataset composed of single cell RNAseq data, collected before, during and after the establishment of Paclitaxel tolerance, to identify single cell heterogeneity. We used gene expression analysis, gene expression rank correlation analysis, pathway analysis and mutation analysis to identify within group variations of cancer cells after chemotherapy. We identified a decrease in cancer cell within group diversity during the transition to drug-tolerance. Additionally, we observed high mutation rate in stressed single cells, which suggests genetic instability of cancer cells that could ultimately result in the development of drug resistance. Our analysis carries significant implications for developing personalised and efficient therapeutics against cancer.

Abstract 514: Genetic load in cancer cell populations

Abstract Populations accumulate deleterious mutations that have yet to be removed by natural selection. This mutated portion of the population that does not contribute to its fitness is called the genetic load and is proportional to the deleterious mutation rate. Given the genomic instability of cancer cells, the genetic load could be substantial if the instability indeed leads to loss of cell fitness. We hypothesize that because aneuploidy and copy number variations (CNVs) are the most common forms of genome instability, their fitness consequences may determine the genetic load in cancer cells. To test this hypothesis, we randomly selected single cells from a HeLa cell line and measured the cell growth rate via daily cell counting and an MTT cell proliferation assay. We then measured the genetic changes and resulting growth rate variation. We observed rapid generation of heterogeneity in the growth rate within the population. The growth rate of different progeny cells was stable indicating that it is genetically determined. Next, using whole genome sequencing of recently descended clones of a single HeLa cell, we found that cells with fewer newly acquired CNVs have higher proliferation rates vice versa. This indicated that CNV accumulation decreased cell fitness systematically. We next estimated the rate of CNV mutation by measuring the growth rate of two daughter cells from single proliferation and found that there is approximately 1 deleterious mutation in every 4 cell divisions. We speculated that due to a high, deleterious CNV mutation rate, tumor cells inevitably accumulate deleterious CNVs and a large percentage of tumor cells are genetically defective. Accordingly, we observed that the average growth rate of tumor cell populations decreased in the short term since defective cells accumulated in the population and the variation of cellular growth rates within the population increased. By modeling the process of mutation accumulation and measuring cell growth rate, we estimated that the deleterious mutation rate in HeLa cells is about 0.26-0.31 per cell division, and that HeLa cells reduce roughly 5%-6% of fitness for every cell division. The observations of a high proliferation rate and high genetic load in this representative tumor cell line indicates a “high risk, high reward” evolution strategy for tumor cells and suggests that increasing the level of genomic instability may cause the meltdown of tumor cell population by forcing cells to accelerate the cell cycle. Citation Format: Yuezheng Zhang, Xu Shen, Yawei Li, Tianqi Zhu, Yong Tao, Tao Li, Di Wang, Xueying Li, Qin Ma, Xuemei Lu, Hurng-Yi Wang, Chung-I Wu. Genetic load in cancer cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2017-514

Expression of progerin does not result in an increased mutation rate

In the premature ageing disease Hutchinson-Gilford progeria syndrome (HGPS), the underlying genetic defect in the lamin A gene leads to accumulation at the nuclear lamina of progerin—a mutant form of lamin A that cannot be correctly processed. This has been reported to result in defects in the DNA damage response and in DNA repair, leading to the hypothesis that, as in normal ageing and in other progeroid syndromes caused by mutation of genes of the DNA repair and DNA damage response pathways, increased DNA damage may be responsible for the premature ageing phenotypes in HGPS patients. However, this hypothesis is based upon the study of markers of the DNA damage response, rather than measurement of DNA damage per se or the consequences of unrepaired DNA damage—mutation. Here, using a mutation reporter cell line, we directly compared the inherent and induced mutation rates in cells expressing wild-type lamin A or progerin. We find no evidence for an elevated mutation rate in progerin-expressing cells. We conclude that the cellular defect in HGPS cells does not lie in the repair of DNA damage per se.

P53 alteration in morphologically normal/benign breast luminal cells in BRCA carriers with or without history of breast cancer

Germline mutations in BRCA genes have been shown to predispose patients to breast cancer. Studies have suggested that p53 alteration is a necessary step in tumorigenesis in BRCA carriers. Our previous study showed p53 alteration in morphologically normal/benign breast luminal cells in sporadic breast cancer patients, the so-called breast p53 signature. Here, we studied p53 status in 66 BRCA1/2 carriers' breasts: 29 patients with breast carcinoma (2 patients with bilateral breast carcinomas) and 37 without. Seven of the 12 (58%) triple-negative breast carcinomas in BRCA carriers were positive for p53 alteration (immunohistochemical stain and/or sequencing), the same frequency as in sporadic triple-negative breast carcinomas. Focal p53 positivity in adjacent normal/benign luminal cells was identified in 4 of the 7 cases with p53-positive carcinomas but not in breasts with p53-negative carcinomas, indicating that p53 positivity in normal/benign breast luminal cells is not a random event. Furthermore, in BRCA carriers' prophylactic mastectomies, 12 of the 94 (12.77%) breasts had focal p53 positivity in normal/benign luminal cells, with 2 cases in bilateral breasts, significantly higher than in previously studied mammoplasty specimens (0%). Our study suggests that germline BRCA gene mutations could result in genomic instability and an elevated gene mutation rate (such as the p53 gene) in breast luminal cells compared with the general population, predisposing BRCA carriers to develop p53-positive/triple-negative breast carcinomas.

Cloning of CHO Cells, Productivity and Genetic Stability—A Discussion

While many perceive mammalian cell culture-based manufacturing for biopharmaceuticals an established technology, numerous open questions remain to be solved. Genetic diversity and mutation rates in CHO cells have been underestimated since progeny of a clonal CHO cell become genetically diverse with each cell division. This is an important issue since products are made in bioreactors containing up to 1014 cells that have divided for weeks. Regulatory interest in “Proof of Clonality” is a misguided and misunderstood concern in this context. We revisit decades of research on scope and rate of genetic changes in CHO cells and suggest approaches to minimize trends for genomic instability when establishing reliable manufacturing processes. A concept is proposed for transfection-derived cell populations containing relatively stable (The term “stable” will always be used in a context of relative stability—considering time frames of weeks to months at best.) “CHO master sequence” genomes (containing the desired DNAs of interest). Stable cell populations are to be selected for and maintained for the various phases of manufacturing under specific culture conditions reducing trends for the selection of diverse subpopulations. Such conditions are based on insights gained from population genetics, evolutionary landscape fitness principles, and a 40-year old model for evolution of error prone replicating systems—the Quasi-Species concept.

Single, long molecule PCR for the detection of rare mutations in Mitochondrial DNA

IntroductionOocytes contain the whole mitochondrial genomes of mammals, and the inheritance pattern of mitochondrial DNA in oocytes do not follow Mendel's rules of heredity. Mitochondrial DNA is maternally inherited in the oocytes, and only mother's mitochondrial genome can be passed to the next generation. The mitochondrial genome is a closed, circular piece of DNA which has a length of about 16kb, and there are hundreds to hundreds of thousands mitochondrial DNA in a single cell. Mitochondrial DNA mutation rate is generally difficult to detect and is often over‐estimated. This research is focusing on developing a sequencing strategy to accurately get mitochondrial DNA mutation rate of single, long molecules from a wild type mouse germline.Research ObjectivesThe research goal is to develop an efficient and accurate way to assess the mutation rate of mitochondrial DNA from wild type mouse oocytes.MethodsWe isolated and individually lysed single oocytes from a wild type female mouse. A serial dilution of the DNA sample was made to find its working concentration for generating single, long molecules, and prepared a PCR reaction by using the specific DNA sample concentration. A high‐fidelity DNA polymerase was used to generate 16kb PCR products originating from single template molecules. Sanger sequencing was used to sequence these DNA molecules.ResultsWe used single molecule PCR to amplify the mitochondrial DNA from three wild type mouse oocytes and sequenced all the PCR products, yielding a total coverage about 330,392bp. Twenty‐two mitochondrial genomes from three oocytes were sequenced, with an average length of about 15,017bp. Mouse mitochondrial DNA length is 16,299kb, so each of our mitochondrial DNA molecule has an average coverage about 92.13% of the whole mitochondrial DNA genome. Only two mutations were found in two separate mitochondrial genomes, and the resulting mutation rate of mitochondrial DNA in the oocytes was 6.05×10^‐6. This is a very low mutation rate across over 300,000bp.ConclusionThe mutation rate of mitochondrial DNA in wild type mouse germline is remarkable low in our research by using single, long molecule PCR. This PCR method allows for the accurate detection of mutations in the mouse mitochondrial DNA in oocytes. Our data indicates that, by using single, long molecule PCR combined with Sanger sequencing, we can accurately get informations from a molecule that has the most coverage of its genome. Additionally, we used a high‐fidelity enzyme to serve as DNA polymerase in the PCR reaction, thus preventing the chance of artificial mutations from the beginning of the PCR reaction. With the right procedure of our sequencing strategy, the rare mutations and the mutation rate in single germline cells can be accurately determined.Support or Funding InformationEllison Medical Foundation; NIH R37‐AG012279

Parameter estimation for multistage clonal expansion models from cancer incidence data: A practical identifiability analysis.

Many cancers are understood to be the product of multiple somatic mutations or other rate-limiting events. Multistage clonal expansion (MSCE) models are a class of continuous-time Markov chain models that capture the multi-hit initiation–promotion–malignant-conversion hypothesis of carcinogenesis. These models have been used broadly to investigate the epidemiology of many cancers, assess the impact of carcinogen exposures on cancer risk, and evaluate the potential impact of cancer prevention and control strategies on cancer rates. Structural identifiability (the analysis of the maximum parametric information available for a model given perfectly measured data) of certain MSCE models has been previously investigated. However, structural identifiability is a theoretical property and does not address the limitations of real data. In this study, we use pancreatic cancer as a case study to examine the practical identifiability of the two-, three-, and four-stage clonal expansion models given age-specific cancer incidence data using a numerical profile-likelihood approach. We demonstrate that, in the case of the three- and four-stage models, several parameters that are theoretically structurally identifiable, are, in practice, unidentifiable. This result means that key parameters such as the intermediate cell mutation rates are not individually identifiable from the data and that estimation of those parameters, even if structurally identifiable, will not be stable. We also show that products of these practically unidentifiable parameters are practically identifiable, and, based on this, we propose new reparameterizations of the model hazards that resolve the parameter estimation problems. Our results highlight the importance of identifiability to the interpretation of model parameter estimates.

Mitochondrial diseases with the main manifestations of cardiomyopathy and respiratory muscle damage

Objective The 3243A>G mutation in mitochondrial DNA is a common cause of the classical mitochondrial diseases characterized by neuro-muscular disorders.This study reports a rare case with the main manifestations of mitochondrial disease in children of mitochondrial cardiomyopathy and respiratory muscle damage. Methods The clinical characteristics, diagnosis and treatment, biochemical, pathological and genetic features of a 10-year-old girl were studied. Results The girl was admitted because of heart failure and respiratory failure at the age of 10.Ragged red fibers in skeletal muscles had been noticed.On her mitochondrial gene, 3243A>G mutation, Leu tRNA (UUR), was detected.The mutation rate in the peripheral blood cells was 94%.After the treatment with a high dose of creatine phosphate sodium, coenzyme Q10 and L-carnitine with assisted ventilation, the patient improved rapidly.The child was followed up for 2 years without recurrence.Meanwhile the growth, development and daily life were normal. Conclusions Cardiac and respiratory muscle impairments that appeared at the same time as the first manifestations of the children's mitochondrial disease is not common, and it is rare to have cardiomyopathy based mitochondrial gene 3243A>G mutation is seldom seen clinically.Skeletal muscle biopsy and genetic test is the key for accurate diagnosis.Improving mitochondrial metabolism and assisted ventilation appear to be helpful treatments. Key words: Cardiomyopathy; Mitochondrial disease; Ragged red fiber; Mitochondrial gene; 3243A>G

Antibiotic treatment enhances the genome-wide mutation rate of target cells

Although it is well known that microbial populations can respond adaptively to challenges from antibiotics, empirical difficulties in distinguishing the roles of de novo mutation and natural selection have left several issues unresolved. Here, we explore the mutational properties of Escherichia coli exposed to long-term sublethal levels of the antibiotic norfloxacin, using a mutation accumulation design combined with whole-genome sequencing of replicate lines. The genome-wide mutation rate significantly increases with norfloxacin concentration. This response is associated with enhanced expression of error-prone DNA polymerases and may also involve indirect effects of norfloxacin on DNA mismatch and oxidative-damage repair. Moreover, we find that acquisition of antibiotic resistance can be enhanced solely by accelerated mutagenesis, i.e., without direct involvement of selection. Our results suggest that antibiotics may generally enhance the mutation rates of target cells, thereby accelerating the rate of adaptation not only to the antibiotic itself but to additional challenges faced by invasive pathogens.

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that several SAMHD1 mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of one SAMHD1 allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associated SAMHD1 mutations increase mutation rates in cancer cells.

Abstract LB-307: Computational modeling of serous ovarian carcinoma dynamics: Implications for screening and therapy

Abstract High-grade serous ovarian carcinoma (HG-SOC) is the most common subtype of ovarian cancer and has the worst prognosis. There is intense controversy on whether the temporal order of cytoreductive surgery and chemotherapy affects treatment outcome, with the main options being primary debulking surgery with adjuvant chemotherapy (PDS) versus neo-adjuvant chemotherapy with interval debulking surgery (NACT). Although some studies report that PDS-treated patients survive significantly longer than those receiving NACT, other reports showed no significant difference in patient outcome. To address this question in an unbiased way, we used computational modeling to simulate HG-SOC progression dynamics with different treatments. We adapted a mathematical framework to predict the evolution of chemotherapy resistance, and populated our model with survival data from >300 patients receiving PDS or NACT. After estimating the mutation rate of carcinoma cells and cancer cell number at diagnosis, we determined that most HG-SOC patients likely harbor chemotherapy-resistant cancer cells at diagnosis. Furthermore, we predicted the effects of PDS and NACT on the number of sensitive and resistant cells, as well as patient survival following treatment, and found that our model closely recapitulated clinical observations in both training and test sets. Based on our results, we predict that PDS with optimal debulking (<1mm residual tumor) has the potential to be curative because surgery can sometimes remove all chemo-resistant cells, while adjuvant chemotherapy depletes the remaining chemo-sensitive cells. By contrast, NACT is unlikely to cure the disease because neo-adjuvant chemotherapy depletes chemo-sensitive cells that can mark the location of accompanying “passenger” chemo-resistant cells. As a result, it leads to extensive enrichment of chemo-resistant cells before surgery, but the deposits of such cells are typically too small to be visualized at eventual surgery. Our model also predicts that PDS should have a better outcome than NACT, when controlled for residual tumor size. Finally, we evaluated the potential benefits of early diagnosis of naïve or relapsed HG-SOC. We recapitulated the clinical finding that CA125-based earlier diagnosis of relapsed cancer does not improve survival. We also predict that more sensitive detection methods (such as ctDNA-based diagnosis) are unlikely to improve survival post-relapse with current chemotherapy, because earlier diagnosis does not decrease the number of chemo-resistant cells, which are already enriched at recurrence. By contrast, our model predicts that with sufficiently sensitive assays, early detection could improve survival time and increase chances of cure. Note: This abstract was not presented at the meeting. Citation Format: Shengqing Gu, Liat Hogen, Stephanie Lheureux, Azin Sayad, Iryna Vyarvelska, Paulina Cybulska, Marcus Bernardini, Barry Rosen, Amit Oza, Benjamin G. Neel. Computational modeling of serous ovarian carcinoma dynamics: Implications for screening and therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-307. doi:10.1158/1538-7445.AM2015-LB-307

Yeast 2-Cys peroxiredoxin Tsa1 protects cells from DNA damage-induced reactive oxygen species through peroxidase activity

Peroxiredoxins (Prxs) belong to a family of thiol-specific peroxidases that scavenge reactive oxygen species (ROS). Yeast Prx thiol-specific antioxidant (Tsa1) protects cells from oxidative stress and maintains genome stability. In this study, we investigated the role of Tsa1 in the response to DNA damage. We observed that Tsa1 among all five Prxs of yeast is the most responsive in cell viability and mutation rate assays. The Δtsa1 mutant is hypersensitive to DNA-damaging agents such as hydroxyurea, methyl methane sulfonate, and camptothecin. The Δtsa1 cells lead to reduced cell viability and cell growth after DNA damage. The Δtsa1-expressing wild-type Tsa1 rescues the sensitivity of Δtsa1 cells to DNA damage, but not in Δtsa1 cells carrying the active-site mutant Tsa1-C47S. We found that the level of ROS in Δtsa1 cells was increased more than in wild-type cells by DNA damage. Tsa1 completely restored the production of DNA damage-induced ROS in Δtsa1 cells, but not Tsa1-C47S. Finally, DNA damage-induced ROS resulted in a switch of Tsa1 from a low-molecular-weight to a high-molecular-weight complex. Taken together, these findings suggest that Tsa1 protects yeast cells from DNA damage by regulating the homeostasis of ROS through peroxidase activity.

Abstract A68: Hypoxia-induced CHK1 repression may enhance the mutator phenotype of pancreatic cancer cells

Abstract Background: Dysregulation of DNA repair mechanisms are known to play a major role in tumor progression. Moreover, the tumor microenviroment itself has been shown to enhance this phenomenon. For example, studies using reporter genes and endogenous loci have demonstrated increased mutation rates in cells grown in tumors compared to the identical cells grown in culture, or a higher frequency of point mutations in cancer cells cultured under hypoxic conditions, a common feature during tumor growth. An accepted mechanism for these observations is hypoxia-induced down-regulation of MLH1, MSH2 or MSH6 leading to an increased mutation rate, or to hypoxia-induced repression of DNA double strand break repair. Methods: Expression microarrays were performed using RNA isolated from nine pancreatic cancer cell lines cultured under normoxic (Nx, 20% O2) or hypoxic conditions (Hx, 1% O2) for 24 hours to identify differentially expressed genes. Retroviruses encoding oligos for HIF-1 or HIF-2 knockdown were used to generate stable pancreatic cancer cell lines in which HIF-1 or HIF-2, respectively, expression was blocked. Alternatively, Hx was induced by culture of cells inside an air-tight chamber with inflow and outflow valves that was infused with a mixture of 1% O2, 5% CO2 and 94% N2. Protein levels of HIF-1, HIF-2, MLH1, MSH2, MSH6 and CHK1 were determined by western blotting. Cell proliferation was measured by BrdU incorporation and cell cycle analysis by flow cytometry of propidium iodide stained cells. G2/M check point was studied analyzing Histone H3 phosphorylation by flow cytometry and DNA damage was determined by immunofluorescence of cells stained with anti phospho γ-H2AX antibody (γ-H2AX foci formation) Results: We first compared the gene expression profiles of 9 different pancreatic cancer cell lines each grown under normoxic versus hypoxic conditions. As previously described, hypoxia led to down-regulation of the mRNA levels of the mismatch repair proteins MLH1, MSH2 and MSH6 as well as down-regulation of several genes involved in the DNA damage response to double strand breaks such as Brca1, Rad51, Ku70 and Ku80. Of interest, a novel downregulated gene identified was CHK1, a master coordinator of DNA repair following genotoxic stress. Growth of pancreatic cancer cells under hypoxic conditions was confirmed to decrease CHK1 mRNA and protein levels and stable knockdown of HIF-1a but not HIF-2 abolished these effects. In contrast to what have been previously reported in other cancer cell types, hypoxia had no effects on cell proliferation in pancreatic cancer cells as assessed by cell cycle analysis or by BrdU incorporation.To determine if hypoxic down-regulation of CHK1 protein levels has functional effects, we determined the ability of pancreatic cancer cells to arrest in the G2/M checkpoint after ionizing radiation. We found that cells cultured under hypoxia were significantly less arrested on G2/M check point after radiation compared to the same irradiated cells grown under normoxic conditions. This hypoxic effect on G2/M checkpoint was abolished on HIF-1 knockdown cells. Finally, pancreatic cancer cells grown under hypoxic conditions accumulated more DNA damage 24 hours after radiation compared to the same irradiated cells grown under normoxic conditions. Conclusion: Hypoxic conditions decrease CHK1 mRNA and protein levels in pancreatic cancer cells in a HIF1-dependent manner. Impairment on CHK1 function may promote the mutator phenotype of pancreatic cancer cells during tumor progression by allowing unchecked cell divisions despite ongoing DNA damage. This finding also has significance for the use of CHK1 inhibitors for this tumor type. Citation Format: Salvador Naranjo-Suarez, Elsa Callen, Yi Zhong, Alvin Makohon-Moore, Andre Nussenzweig, Christine Iacobuzio-Donahue. Hypoxia-induced CHK1 repression may enhance the mutator phenotype of pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A68.

A computational model incorporating neural stem cell dynamics reproduces glioma incidence across the lifespan in the human population.

Glioma is the most common form of primary brain tumor. Demographically, the risk of occurrence increases until old age. Here we present a novel computational model to reproduce the probability of glioma incidence across the lifespan. Previous mathematical models explaining glioma incidence are framed in a rather abstract way, and do not directly relate to empirical findings. To decrease this gap between theory and experimental observations, we incorporate recent data on cellular and molecular factors underlying gliomagenesis. Since evidence implicates the adult neural stem cell as the likely cell-of-origin of glioma, we have incorporated empirically-determined estimates of neural stem cell number, cell division rate, mutation rate and oncogenic potential into our model. We demonstrate that our model yields results which match actual demographic data in the human population. In particular, this model accounts for the observed peak incidence of glioma at approximately 80 years of age, without the need to assert differential susceptibility throughout the population. Overall, our model supports the hypothesis that glioma is caused by randomly-occurring oncogenic mutations within the neural stem cell population. Based on this model, we assess the influence of the (experimentally indicated) decrease in the number of neural stem cells and increase of cell division rate during aging. Our model provides multiple testable predictions, and suggests that different temporal sequences of oncogenic mutations can lead to tumorigenesis. Finally, we conclude that four or five oncogenic mutations are sufficient for the formation of glioma.

Modeling age-dependent radiation-induced second cancer risks and estimation of mutation rate: an evolutionary approach.

Although the survival rate of cancer patients has significantly increased due to advances in anti-cancer therapeutics, one of the major side effects of these therapies, particularly radiotherapy, is the potential manifestation of radiation-induced secondary malignancies. In this work, a novel evolutionary stochastic model is introduced that couples short-term formalism (during radiotherapy) and long-term formalism (post-treatment). This framework is used to estimate the risks of second cancer as a function of spontaneous background and radiation-induced mutation rates of normal and pre-malignant cells. By fitting the model to available clinical data for spontaneous background risk together with data of Hodgkin's lymphoma survivors (for various organs), the second cancer mutation rate is estimated. The model predicts a significant increase in mutation rate for some cancer types, which may be a sign of genomic instability. Finally, it is shown that the model results are in agreement with the measured results for excess relative risk (ERR) as a function of exposure age and that the model predicts a negative correlation of ERR with increase in attained age. This novel approach can be used to analyze several radiotherapy protocols in current clinical practice and to forecast the second cancer risks over time for individual patients.

Fastest time to cancer by loss of tumor suppressor genes.

Genetic instability promotes cancer progression (by increasing the probability of cancerous mutations) as well as hinders it (by imposing a higher cell death rate for cells susceptible to cancerous mutation). With the loss of tumor suppressor gene function known to be responsible for a high percentage of breast and colorectal cancer (and a good fraction of lung cancer and other types as well), it is important to understand how genetic instability can be orchestrated toward carcinogenesis. In this context, this paper gives a complete characterization of the optimal (time-varying) cell mutation rate for the fastest time to a target cancerous cell population through the loss of both copies of a tumor suppressor gene. Similar to the (one-step) oncogene activation model previously analyzed, the optimal mutation rate of the present two-step model changes qualitatively with the convexity of the (mutation rate-dependent) cell death rate. However, the structure of the Hamiltonian for the new model differs significantly and intrinsically from that of the one-step model, and a completely new approach is needed for the solution of the present two-step problem. Considerable insight into the biology of optimal switching (between corner controls) is extracted from numerical results for cases with nonconvex death rates.

Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells.

Decitabine (5-aza-2′-deoxycytidine) is a DNA methyltransferase inhibitor and an archetypal epigenetic drug for the therapy of myeloid leukemias. The mode of action of decitabine strictly depends on the incorporation of the drug into DNA. However, DNA incorporation and ensuing genotoxic effects of decitabine have not yet been investigated in human cancer cell lines or in models related to the approved indication of the drug. Here we describe a robust assay for the quantitative determination of decitabine incorporation rates into DNA from human cancer cells. Using a panel of human myeloid leukemia cell lines we show appreciable amounts of decitabine incorporation that closely correlated with cellular drug uptake. Decitabine incorporation was also detectable in primary cells from myeloid leukemia patients, indicating that the assay is suitable for biomarker analyses to predict drug responses in patients. Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level. Interestingly, this approach failed to reveal significant changes in the rates of point mutations and genome rearrangements in myeloid leukemia cell lines. These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.