Germline Variation in Cancer-Susceptibility Genes in a Healthy,Ancestrally Diverse Cohort: Implications for Individual Genome Sequencing

Technological advances coupled with decreasing costs are bringing whole genome and whole exome sequencing closer to routine clinical use. One of the hurdles to clinical implementation is the high number of variants of unknown significance. For cancer-susceptibility genes, the difficulty in interpreting the clinical relevance of the genomic variants is compounded by the fact that most of what is known about these variants comes from the study of highly selected populations, such as cancer patients or individuals with a family history of cancer. The genetic variation in known cancer-susceptibility genes in the general population has not been well characterized to date. To address this gap, we profiled the nonsynonymous genomic variation in 158 genes causally implicated in carcinogenesis using high-quality whole genome sequences from an ancestrally diverse cohort of 681 healthy individuals. We found that all individuals carry multiple variants that may impact cancer susceptibility, with an average of 68 variants per individual. Of the 2,688 allelic variants identified within the cohort, most are very rare, with 75% found in only 1 or 2 individuals in our population. Allele frequencies vary between ancestral groups, and there are 21 variants for which the minor allele in one population is the major allele in another. Detailed analysis of a selected subset of 5 clinically important cancer genes, BRCA1, BRCA2, KRAS, TP53, and PTEN, highlights differences between germline variants and reported somatic mutations. The dataset can serve a resource of genetic variation in cancer-susceptibility genes in 6 ancestry groups, an important foundation for the interpretation of cancer risk from personal genome sequences.     

Mangiferin attenuates oxidative stress induced renal cell damage through activation of PI3K induced Akt and Nrf-2 mediated signaling pathways

BackgroundMangiferin is a polyphenolic xanthonoid with remarkable antioxidant activity. Oxidative stress plays the key role in tert-butyl hydroperoxide (tBHP) induced renal cell damage. In this scenario, we consider mangiferin, as a safe agent in tBHP induced renal cell death and rationalize its action systematically, in normal human kidney epithelial cells (NKE).MethodsNKE cells were exposed to 20 µM mangiferin for 2 h followed by 50 µM tBHP for 18 h. The effect on endogenous ROS production, antioxidant status (antioxidant enzymes and thiols), mitochondrial membrane potential, apoptotic signaling molecules, PI3K mediated signaling cascades and cell cycle progression were examined using various biochemical assays, FACS and immunoblot analyses.ResultstBHP exposure damaged the NKE cells and decreased its viability. It also elevated the intracellular ROS and other oxidative stress-related biomarkers within the cells. However, mangiferin dose dependently, exhibited significant protection against this oxidative cellular damage. Mangiferin inhibited tBHP induced activation of different pro-apoptotic signals and thus protected the renal cells against mitochondrial permeabilization. Further, mangiferin enhanced the expression of cell proliferative signaling cascade molecules, Cyclin d1, NFκB and antioxidant molecules HO-1, SOD2, by PI3K/Akt dependent pathway. However, the inhibitor of PI3K abolished mangiferin's protective activity.ConclusionsResults show Mangiferin maintains the intracellular anti-oxidant status, induces the expression of PI3K and its downstream molecules and shields NKE cells against the tBHP induced cytotoxicity.General significanceMangiferin can be indicated as a therapeutic agent in oxidative stress-mediated renal toxicity. This protective action of mangiferin primarily attributes to its potent antioxidant and antiapoptotic nature.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

Alternative models of cancer stem cells: The stemness phenotype model, 10 years later

The classical cancer stem cell (CSCs) theory proposed the existence of a rare but constant subpopulation of CSCs. In this model cancer cells are organized hierarchically and are responsible for tumor resistance and tumor relapse. Thus, eliminating CSCs will eventually lead to cure of cancer. This simplistic model has been challenged by experimental data. In 2010 we proposed a novel and controversial alternative model of CSC biology (the Stemness Phenotype Model, SPM). The SPM proposed a non-hierarchical model of cancer biology in which there is no specific subpopulation of CSCs in tumors. Instead, cancer cells are highly plastic in term of stemness and CSCs and non-CSCs can interconvert into each other depending on the microenvironment. This model predicts the existence of cancer cells ranging from a pure CSC phenotype to pure non-CSC phenotype and that survival of a single cell can originate a new tumor. During the past 10 years, a plethora of experimental evidence in a variety of cancer types has shown that cancer cells are indeed extremely plastic and able to interconvert into cells with different stemness phenotype. In this review we will (1) briefly describe the cumulative evidence from our laboratory and others supporting the SPM; (2) the implications of the SPM in translational oncology; and (3) discuss potential strategies to develop more effective therapeutic regimens for cancer treatment.     

Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization

It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research.     

Comparative genomics and structural biology of the molecular innovations of eukaryotes

Eukaryotes encode numerous proteins that either have no detectable homologs in prokaryotes or have only distant homologs. These molecular innovations of eukaryotes may be classified into three categories: proteins and domains inherited from prokaryotic precursors without drastic changes in biochemical function, but often recruited for novel roles in eukaryotes; new superfamilies or distinct biochemical functions emerging within pre-existing protein folds; and domains with genuinely new folds, apparently 'invented' at the outset of eukaryotic evolution. Most new folds emerging in eukaryotes are either alpha-helical or stabilized by metal chelation. Comparative genomics analyses point to an early phase of rapid evolution, and dramatic changes between the origin of the eukaryotic cell and the advent of the last common ancestor of extant eukaryotes. Extensive duplication of numerous genes, with subsequent functional diversification, is a distinctive feature of this turbulent era. Evolutionary analysis of ancient eukaryotic proteins is generally compatible with a two-symbiont scenario for eukaryotic origin, involving an alpha-proteobacterium (the ancestor of the mitochondria) and an archaeon, as well as key contributions from their selfish elements.     

Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth

We have studied the effects of overexpression of superoxide dismutase (SOD), a tumor suppressor protein that dismutes superoxide radical to H2O2, on breast cancer cell growth in vitro and xenograft growth in vivo. No previous work has directly compared the growth-suppressive effects of manganese SOD (MnSOD) and copper-zinc SOD (CuZnSOD). We hypothesized that either adenoviral MnSOD (AdMnSOD) or adenoviral CuZnSOD (AdCuZnSOD) gene therapy would suppress the growth of human breast cancer cells. After determining the antioxidant profiles of three human breast cell lines, MCF 10A, MDA-MB231, and MCF-7, we measured the effects of MnSOD or CuZnSOD overexpression on cell growth and survival in vitro and in vivo. Results demonstrated that infection with AdMnSOD or AdCuZnSOD increased the activity of the respective enzyme in all three cell lines. In vitro, overexpression of MnSOD or CuZnSOD decreased not only cell growth but also clonogenic survival in a dose- and transgene-dependent manner. In vivo, treatment of tumors with AdMnSOD or AdCuZnSOD decreased xenograft growth compared to controls. The first direct comparison of MnSOD to CuZnSOD overexpression indicated that CuZnSOD and MnSOD were similarly effective at suppressing cancer cell growth.