170 research outputs found
Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies
IntroductionMost human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated beta-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth.MethodsPre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or gamma-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated beta-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses.ResultsCultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated beta-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism.ConclusionsOur studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential
The Mechanisms of DNA Replication
DNA replication is a fundamental part of the life cycle of all organisms. Not surprisingly many aspects of this process display profound conservation across organisms in all domains of life. The chapters in this volume outline and review the current state of knowledge on several key aspects of the DNA replication process. This is a critical process in both normal growth and development and in relation to a broad variety of pathological conditions including cancer. The reader will be provided with new insights into the initiation, regulation, and progression of DNA replication as well as a collection of thought provoking questions and summaries to direct future investigations
Grand Celebration: 10th Anniversary of the Human Genome Project
In 1990, scientists began working together on one of the largest biological research projects ever proposed. The project proposed to sequence the three billion nucleotides in the human genome. The Human Genome Project took 13 years and was completed in April 2003, at a cost of approximately three billion dollars. It was a major scientific achievement that forever changed the understanding of our own nature. The sequencing of the human genome was in many ways a triumph for technology as much as it was for science. From the Human Genome Project, powerful technologies have been developed (e.g., microarrays and next generation sequencing) and new branches of science have emerged (e.g., functional genomics and pharmacogenomics), paving new ways for advancing genomic research and medical applications of genomics in the 21st century. The investigations have provided new tests and drug targets, as well as insights into the basis of human development and diagnosis/treatment of cancer and several mysterious humans diseases. This genomic revolution is prompting a new era in medicine, which brings both challenges and opportunities. Parallel to the promising advances over the last decade, the study of the human genome has also revealed how complicated human biology is, and how much remains to be understood. The legacy of the understanding of our genome has just begun. To celebrate the 10th anniversary of the essential completion of the Human Genome Project, in April 2013 Genes launched this Special Issue, which highlights the recent scientific breakthroughs in human genomics, with a collection of papers written by authors who are leading experts in the field
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The cellular impact of diminished DNA origin licensing capacity and its potential therapeutic exploitation
Genomic instability underlies various diseases including cancer. The maintenance of
genomic stability requires accurate replication of the genome, proper segregation of
duplicated DNA to progeny cells, and the capacity to respond effectively to DNA
damage. Early sections of this thesis focus on the response to DNA double-strand
breaks (DSBs) within compact regions of chromatin (heterochromatin). Here,
methodology was optimised for monitoring the repair of site-specific DSBs within
regions likely to be enriched for heterochromatin. This system was exploited to examine
the function of the Artemis endonuclease in heterochromatic DSB repair. Later sections
focus on factors involved in DNA replication and the response to replication stress.
Among the various mechanisms involved in the DNA damage response (DDR) to
replication stress, the licensing of excess origins of replication has been proposed to
safeguard against replication failure. Here, the impact of diminished origin licensing
capacity on the response to replication stress was compared in tumour and non-tumour
cell lines. I present findings demonstrating that depletion of origin licensing factors
causes hypersensitisation of tumour-derived but not non-tumour cell lines to replication
stress-inducing agents. Further, combining diminished origin licensing capacity with
depletion of the tumour suppressor, p53, or overexpression of the c-Myc oncogene
impairs viability under conditions of replication stress in non-tumour fibroblasts. These
findings suggest that tumour cells have a greater reliance on origin licensing capacity,
raising the possibility that licensing factors might represent suitable targets for drugbased
cancer therapy. Factors involved in replication origin licensing have also been
implicated in the establishment of heterochromatin. Here, I examined higher-order
chromatin structure and the ionizing radiation (IR)-induced DDR in cells from patients
harbouring mutations in origin licensing factors. Findings from these studies provide
evidence for the first time that origin licensing complex (ORC)-deficient Meier-Gorlin
Syndrome (MGS) may be classified as a disordered chromatin syndrome
RNA, the Epicenter of Genetic Information
The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers
Evolution of the germline mutation rate across vertebrates
The germline mutation rate determines the pace of genome evolution and is an evolving parameter itself1. However, little is known about what determines its evolution, as most studies of mutation rates have focused on single species with different methodologies2. Here we quantify germline mutation rates across vertebrates by sequencing and comparing the high-coverage genomes of 151 parent–offspring trios from 68 species of mammals, fishes, birds and reptiles. We show that the per-generation mutation rate varies among species by a factor of 40, with mutation rates being higher for males than for females in mammals and birds, but not in reptiles and fishes. The generation time, age at maturity and species-level fecundity are the key life-history traits affecting this variation among species. Furthermore, species with higher long-term effective population sizes tend to have lower mutation rates per generation, providing support for the drift barrier hypothesis3. The exceptionally high yearly mutation rates of domesticated animals, which have been continually selected on fecundity traits including shorter generation times, further support the importance of generation time in the evolution of mutation rates. Overall, our comparative analysis of pedigree-based mutation rates provides ecological insights on the mutation rate evolution in vertebrates
Liposomes encapsulating catechins: a biophysical approach for skin cancer therapy
Every year, a large number of skin cancer cases caused by a prolonged ultraviolet
radiation exposure, are diagnosed around the world. Epigallocatechin–3–
gallate (EGCG) derived from green tea leaves, display protective effect against
oxidative stress which reduce the risk of contracting skin cancer. However, frequently,
the antioxidant and anti–inflammatory activities of EGCG in are compromised
because this molecule is extremely unstable and rapidly degraded in
physiological conditions. Considering these issues, the main goal of this thesis
was developed a stable liposomal nanocarrier for topical/transdermal delivery of
EGCG, firstly, to increase its bioavailability and, secondly, to offer an desirable
skin protection against harmful effects of UV radiation. Primarily, the molecular
mechanisms between EGCG and different phospholipids were studied using
Langmuir experiments, revealling the affinity and localization of EGCG on each
lipidic membrane, which according to the results depends on the molecular organization
of lipidic monolayer (functional groups anchored at headgroup) and of
the degree of protonation of EGCG. EGCG establishes electrostatic and hydrogenbonding
interactions with zwitterionic (DMPC, DPPC) and anionic (DPPG and
DPPS) phospholipids, which condense the monolayers and alter the membrane’s
potential and compressibility. Regarding the irradiation experiments, the results
indicated that EGCG efficiently slows down the oxidant events in monolayers and
in lipid bilayers, which were produced by blue and ultraviolet radiation exposure,
respectively. Lastly, the nanofibers meshes containing EGCG-loaded liposomes
are biocompatible, support human fibroblasts adhesion and scavenge the oxidant
species generated by UV radiation, which guarantees a higher cell survival
Not only P-glycoprotein: amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins
The development of drug resistance continues to be a dominant hindrance toward curative cancer treatment.
Overexpression of a wide-spectrum of ATP-dependent efflux pumps, and in particular of ABCB1 (P-glycoprotein
or MDR1) is a well-known resistance mechanism for a plethora of cancer chemotherapeutics including for example
taxenes, anthracyclines, Vinca alkaloids, and epipodopyllotoxins, demonstrated by a large array of published
papers, both in tumor cell lines and in a variety of tumors, including various solid tumors and hematological
malignancies. Upon repeated or even single dose treatment of cultured tumor cells or tumors in vivo with
anti-tumor agents such as paclitaxel and doxorubicin, increased ABCB1 copy number has been demonstrated,
resulting from chromosomal amplification events at 7q11.2-21 locus, leading to marked P-glycoprotein overexpression,
and multidrug resistance (MDR). Clearly however, additional mechanisms such as single nucleotide
polymorphisms (SNPs) and epigenetic modifications have shown a role in the overexpression of ABCB1 and of
other MDR efflux pumps. However, notwithstanding the design of 4 generations of ABCB1 inhibitors and the
wealth of information on the biochemistry and substrate specificity of ABC transporters, translation of this vast
knowledge from the bench to the bedside has proven to be unexpectedly difficult.
Many studies show that upon repeated treatment schedules of cell cultures or tumors with taxenes and anthracyclines
as well as other chemotherapeutic drugs, amplification, and/or overexpression of a series of genes
genomically surrounding the ABCB1 locus, is observed. Consequently, altered levels of other proteins may
contribute to the establishment of the MDR phenotype, and lead to poor clinical outcome. Thus, the genes
contained in this ABCB1 amplicon including ABCB4, SRI, DBF4, TMEM243, and RUNDC3B are overexpressed in
many cancers, and especially in MDR tumors, while TP53TG1 and DMTF1 are bona fide tumor suppressors. This
review describes the role of these genes in cancer and especially in the acquisition of MDR, elucidates possible
connections in transcriptional regulation (co-amplification/repression) of genes belonging to the same ABCB1
amplicon region, and delineates their novel emerging contributions to tumor biology and possible strategies to
overcome cancer MDR
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