First analysis of tumor-suppressor interactions with whole genome in normal human cells reveals key differences with cancer cells

ScienceDaily (Nov. 30, 2011) — Scientists investigating the interactions, or binding patterns, of a major tumor-suppressor protein known as p53 with the entire genome in normal human cells have turned up key differences from those observed in cancer cells. The distinct binding patterns reflect differences in the chromatin (the way DNA is packed with proteins), which may be important for understanding the function of the tumor suppressor protein in cancer cells.

The study was conducted by scientists at the U.S. Department of Energy's (DOE) Brookhaven National Laboratory and collaborators at Cold Spring Harbor Laboratory, and is published in the December 15 issue of the journal Cell Cycle.

"No other study has shown such a dramatic difference in a tumor suppressor protein binding to DNA between normal and cancer-derived cells," said Brookhaven biologist Krassimira Botcheva, lead author on the paper. "This research makes it clear that it is essential to study p53 functions in both types of cells in the context of chromatin to gain a correct understanding of how p53 tumor suppression is affected by global epigenetic changes -- modifications to DNA or chromatin -- associated with cancer development."

Because of its key role in tumor suppression, p53 is the most studied human protein. It modulates a cell's response to a variety of stresses (nutrient starvation, oxygen level changes, DNA damage caused by chemicals or radiation) by binding to DNA and regulating the expression of an extensive network of genes. Depending on the level of DNA damage, it can activate DNA repair, stop the cells from multiplying, or cause them to self-destruct -- all of which can potentially prevent or stop tumor development. Malfunctioning p53 is a hallmark of human cancers.

Most early studies of p53 binding explored its interactions with isolated individual genes, and all whole-genome studies to date have been conducted in cancer-derived cells. This is the first study to present a high-resolution genome-wide p53-binding map for normal human cells, and to correlate those findings with the "epigenetic landscape" of the genome.

"We analyzed the p53 binding in the context of the human epigenome, by correlating the p53 binding profile we obtained in normal human cells with a published high-resolution map of DNA methylation -- a type of chemical modification that is one of the most important epigenetic modifications to DNA -- that had been generated for the same cells," Botcheva said.

Key findings

In the normal human cells, the scientists found p53 binding sites located in close proximity to genes and particularly at the sites in the genome, known as transcriptions start sites, which represent "start" signals for transcribing the genes. Though this association of binding sites with genes and transcription start sites was previously observed in studies of functional, individually analyzed binding sites, it was not seen in high-throughput whole-genome studies of cancer-derived cell lines. In those earlier studies, the identified p53 binding sites were found not close to genes, and not close to the sites in the human genome where transcription starts.

Additionally, nearly half of the newly identified p53 binding sites in the normal cells (in contrast to about five percent of the sites reported in cancer cells) reside in so-called CpG islands. These are short DNA sequences with unusually high numbers of cytosine and guanine bases (the C and G of the four-letter genetic code alphabet, consisting of A, T, C, and G). CpG islands tend to be hypo- (or under-) methylated relative to the heavily methylated mammalian genome.

"This association of binding sites with CpG islands in the normal cells is what prompted us to investigate a possible genome-wide correlation between the identified sites and the CpG methylation status," Botcheva said.

The scientists found that p53 binding sites were enriched at hypomethylated regions of the human genome, both in and outside CpG islands.

"This is an important finding because, during cancer development, many CpG islands are subjected to extensive methylation while the bulk of the genomic DNA becomes hypomethylated," Botcheva said. "These major epigenetic changes may contribute to the differences observed in the p53-binding-sites' distribution in normal and cancer cells."

The scientists say this study clearly illustrates that the genomic landscape -- the DNA modifications and the associated chromatin changes -- have a significant effect on p53 binding. Furthermore, it greatly extends the list of experimentally defined p53 binding sites and provides a general framework for investigating the interplay between transcription factor binding, tumor suppression, and epigenetic changes associated with cancer development.

This research, which was funded by the DOE Office of Science, lays groundwork for further advancing the detailed understanding of radiation effects, including low-dose radiation effects, on the human genome.

The research team also includes John Dunn and Carl Anderson of Brookhaven Lab, and Richard McCombie of Cold Spring Harbor Laboratory, where the high-throughput Illumina sequencing was done.

Methodology

The p53 binding sites were identified by a method called ChIP-seq: for chromatin immunoprecipitation (ChIP), which produces a library of DNA fragments bound by a protein of interest using immunochemistry tools, followed by massively parallel DNA sequencing (seq) for determining simultaneously millions of sequences (the order of the nucleotide bases A, T, C and G in DNA) for these fragments.

"The experiment is challenging, the data require independent experimental validation and extensive bioinformatics analysis, but it is indispensable for high-throughput genomic analyses," Botcheva said. Establishing such capability at BNL is directly related to the efforts for development of profiling technologies for evaluating the role of epigenetic modifications in modulating low-dose ionizing radiation responses and also applicable for plant epigenetic studies.

The analysis required custom-designed software developed by Brookhaven bioinformatics specialist Sean McCorkle.

"Mapping the locations of nearly 20 million sequences in the 3-billion-base human genome, identifying binding sites, and performing comparative analysis with other data sets required new programming approaches as well as parallel processing on many CPUs," McCorkle said. "The sheer volume of this data required extensive computing, a situation expected to become increasingly commonplace in biology. While this work was a sequence data-processing milestone for Brookhaven, we expect data volumes only to increase in the future, and the computing challenges to continue."

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Journal References:

Krassimira Botcheva, Sean R. McCorkle, W.R. McCombie, John J. Dunn, Carl W. Anderson. Distinct p53 genomic binding patterns in normal and cancer-derived human cells. Cell Cycle, 2011; 10 (24) [link]William A. Freed-Pastor, Carol Prives. Dissimilar DNA binding by p53 in normal and tumor-derived cells. Cell Cycle, 2011; 10 (24) [link]

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Trail of 'stone breadcrumbs' reveals the identity of one of the first human groups to leave Africa

ScienceDaily (Nov. 30, 2011) — A series of new archaeological discoveries in the Sultanate of Oman, nestled in the southeastern corner of the Arabian Peninsula, reveals the timing and identity of one of the first modern human groups to migrate out of Africa, according to a research article published in the open-access journal PLoS ONE.

An international team of archaeologists and geologists working in the Dhofar Mountains of southern Oman, led by Dr. Jeffrey Rose of the University of Birmingham, report finding over 100 new sites classified as "Nubian Middle Stone Age (MSA)." Distinctive Nubian MSA stone tools are well known throughout the Nile Valley; however, this is the first time such sites have ever been found outside of Africa. According to the authors, the evidence from Oman provides a "trail of stone breadcrumbs" left by early humans migrating across the Red Sea on their journey out of Africa. "After a decade of searching in southern Arabia for some clue that might help us understand early human expansion, at long last we've found the smoking gun of their exit from Africa," says Rose. "What makes this so exciting," he adds, "is that the answer is a scenario almost never considered." These new findings challenge long-held assumptions about the timing and route of early human expansion out of Africa.

Using a technique called Optically Stimulated Luminescence (OSL) to date one of the sites in Oman, researchers have determined that Nubian MSA toolmakers had entered Arabia by 106,000 years ago, if not earlier. This date is considerably older than geneticists have put forth for the modern human exodus from Africa, who estimate the dispersal of our species occurred between 70,000 and 40,000 years ago. Even more surprising, all of the Nubian MSA sites were found far inland, contrary to the currently accepted theory that envisions early human groups moving along the coast of southern Arabia. "Here we have an example of the disconnect between theoretical models versus real evidence on the ground," says co-author Professor Emeritus Anthony Marks of Southern Methodist University. "The coastal expansion hypothesis looks reasonable on paper, but there is simply no archaeological evidence to back it up.

Genetics predict an expansion out of Africa after 70,000 thousand years ago, yet we've seen three separate discoveries published this year with evidence for humans in Arabia thousands, if not tens of thousands of years prior to this date." The presence of Nubian MSA sites in Oman corresponds to a wet period in Arabia's climatic history, when copious rains fell across the peninsula and transformed its barren deserts to sprawling grasslands. "For a while," remarks Rose, "South Arabia became a verdant paradise rich in resources -- large game, plentiful freshwater, and high-quality flint with which to make stone tools." Far from innovative fishermen, it seems that early humans spreading from Africa into Arabia were opportunistic hunters traveling along river networks like highways. Whether or not these pioneers were able to survive in Arabia during the hyperarid conditions of the Last Ice Age is another matter -- a mystery that will require archaeologists to continue combing the deserts of southern Arabia, hot on the trail of stone breadcrumbs.

The Dhofar Archaeological Project is conducted under the auspices of the Ministry of Heritage and Culture in Oman. The team is composed of an interdisciplinary group of researchers from the University of Birmingham and Oxford Brookes University, UK; Arizona State University and Southern Methodist University, USA; Institute of Archaeology, National Academy of Sciences, Ukraine; Institute of Archaeology of the Academy of Science, Czech Republic; University of Tübingen, Germany, and the University of Wollongong, Australia. The project is funded by research grants from the UK Arts and Humanities Research Council and the Australian Research Council.

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Jeffrey I. Rose, Vitaly I. Usik, Anthony E. Marks, Yamandu H. Hilbert, Christopher S. Galletti, Ash Parton, Jean Marie Geiling, Viktor Cerný, Mike W. Morley, Richard G. Roberts. The Nubian Complex of Dhofar, Oman: An African Middle Stone Age Industry in Southern Arabia. PLoS ONE, 2011; 6 (11): e28239 DOI: 10.1371/journal.pone.0028239

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Snakes with big hearts may help yours: Fatty acids involved in python heart growth could benefit diseased human heart

ScienceDaily (Oct. 27, 2011) — Identification of three fatty acids involved in the extreme growth of Burmese pythons' hearts following large meals could prove beneficial in treating diseased human hearts, according to research co-authored by a University of Alabama scientist and publishing in the Oct. 28 issue of Science.

Growth of the human heart can be beneficial when resulting from exercise -- a type of growth known as physiological cardiac hypertrophy -- but damaging when triggered by disease -- growth known as pathological hypertrophy. The new research shows a potential avenue by which to make the unhealthy heart growth more like the healthy version.

"We may later be able to turn the tables, in a sense, in the processes involved in pathological hypertrophy by administering a combination of fatty acids that occur in very high concentrations in the blood of digesting pythons," said Dr. Stephen Secor, associate professor of biological sciences at UA and one of the paper's co-authors. "This could trigger, perhaps, something more akin to the physiological form of hypertrophy."

The research, conducted in collaboration with multiple researchers at the University of Colorado working in the lab of Dr. Leslie Leinwand, identified three fatty acids, myristic acid, palmitic acid and palmitoleic acid, for their roles in the snakes' healthy heart growths following a meal.

Researchers took these fatty acids from feasting pythons and infused them into fasting pythons. Afterward, those fasting pythons underwent heart-rate growths similar to that of the feasting pythons. In a similar fashion, the researchers were able to induce comparable heart-rate growths in rats, indicating that the fatty acids have a similar effect on the mammalian heart.

The paper, whose lead author was Dr. Cecilia Riquelme of the University of Colorado, also showed that the pythons' heart growth was a result of the individual heart cells growing in size, rather than multiplying in number.

By studying gene expression in the python hearts -- which genes are turned on following feasting -- the research, Secor said, shows that the changes the pythons' hearts undergo is more like the positive changes seen in a marathon runner rather than the types of changes seen in a diseased, or genetically altered, heart.

"Cyclists, marathon runners, rowers, swimmers, they tend to have larger hearts," Secor said. "It's the heart working harder to move blood through it. The term is 'volume overload,' in reference to more blood being pumped to tissues. In response, the heart's chambers get larger, and more blood is pushed out with every contraction, resulting in increased cardiac performance."

However, the time-frame of this increased heart performance of a python blows away even the most physically-fit distance runner, Secor said.

"Instead of experiencing elevated cardiac performance for several hours with running, the Burmese python is maintaining heightened cardiac output for five to six days, non-stop, while digesting their large meal."

Another interesting finding of the research, Secor said, is even with the increased volume of triglycerides circulating in the snakes after feeding, those lipids are not remaining within the snakes' hearts or vascular systems after the completion of digestion.

"The python hearts are using the circulating lipids to fuel the increase in performance."

Traditionally, mice have been the preferred animal model used to study the genetic heart disease known as hypertrophic cardiomyopathy, characterized by heart growth and contractile dysfunction. However, the snakes' unusual physiological responses render them more insightful models, in some cases, Secor said.

Pythons are infrequent feeders, sometimes eating only once or twice a year in the wild. When they do eat, they undergo extreme physiologic and metabolic changes that include increases in the size of the heart, along with the liver, pancreas, small intestine and kidney. Three days after a feeding, a python's heart mass can increase as much as 40 percent, before reverting to its pre-meal size once digestion is completed, Secor said.

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Cecilia A. Riquelme, Jason A. Magida, Brooke C. Harrison, Christopher E. Wall, Thomas G. Marr, Stephen M. Secor, Leslie A. Leinwand. Fatty Acids Identified in the Burmese Python Promote Beneficial Cardiac Growth. Science, 2011; 334 (6055): 528-531 DOI: 10.1126/science.1210558

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Python study may have implications for human heart health

ScienceDaily (Oct. 27, 2011) — A surprising new University of Colorado Boulder study shows that huge amounts of fatty acids circulating in the bloodstreams of feeding pythons promote healthy heart growth, results that may have implications for treating human heart disease.

CU-Boulder Professor Leslie Leinwand and her research team found the amount of triglycerides -- the main constituent of natural fats and oils -- in the blood of Burmese pythons one day after eating increased by more than fiftyfold. Despite the massive amount of fatty acids in the python bloodstream there was no evidence of fat deposition in the heart, and the researchers also saw an increase in the activity of a key enzyme known to protect the heart from damage.

After identifying the chemical make-up of blood plasma in fed pythons, the CU-Boulder researchers injected fasting pythons with either "fed python" blood plasma or a reconstituted fatty acid mixture they developed to mimic such plasma. In both cases, the pythons showed increased heart growth and indicators of cardiac health. The team took the experiments a step further by injecting mice with either fed python plasma or the fatty acid mixture, with the same results.

"We found that a combination of fatty acids can induce beneficial heart growth in living organisms," said CU-Boulder postdoctoral researcher Cecilia Riquelme, first author on the Science paper. "Now we are trying to understand the molecular mechanisms behind the process in hopes that the results might lead to new therapies to improve heart disease conditions in humans."

The paper is being published in the Oct. 28 issue of the journal Science. In addition to Leinwand and Riquelme, the authors include CU postdoctoral researcher Brooke Harrison, CU graduate student Jason Magida, CU undergraduate Christopher Wall, Hiberna Corp. researcher Thomas Marr and University of Alabama Tuscaloosa Professor Stephen Secor.

Previous studies have shown that the hearts of Burmese pythons can grow in mass by 40 percent within 24 to 72 hours after a large meal, and that metabolism immediately after swallowing prey can shoot up by fortyfold. As big around as telephone poles, adult Burmese pythons can swallow prey as large as deer, have been known to reach a length of 27 feet and are able to fast for up to a year with few ill effects.

There are good and bad types of heart growth, said Leinwand, who is an expert in genetic heart diseases including hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes. While cardiac diseases can cause human heart muscle to thicken and decrease the size of heart chambers and heart function because the organ is working harder to pump blood, heart enlargement from exercise is beneficial.

"Well-conditioned athletes like Olympic swimmer Michael Phelps and cyclist Lance Armstrong have huge hearts," said Leinwand, a professor in the molecular, cellular and developmental biology department and chief scientific officer of CU's Biofrontiers Institute. "But there are many people who are unable to exercise because of existing heart disease, so it would be nice to develop some kind of a treatment to promote the beneficial growth of heart cells."

Riquelme said once the CU team confirmed that something in the blood plasma of pythons was inducing positive cardiac growth, they began looking for the right "signal" by analyzing proteins, lipids, nucleic acids and peptides present in the fed plasma. The team used a technique known as gas chromatography to analyze both fasted and fed python plasma blood, eventually identifying a highly complex composition of circulating fatty acids with distinct patterns of abundance over the course of the digestive process.

In the mouse experiments led by Harrison, the animals were hooked up to "mini-pumps" that delivered low doses of the fatty acid mixture over a period of a week. Not only did the mouse hearts show significant growth in the major part of the heart that pumps blood, the heart muscle cell size increased, there was no increase in heart fibrosis -- which makes the heart muscle more stiff and can be a sign of disease -- and there were no alterations in the liver or in the skeletal muscles, he said.

"It was remarkable that the fatty acids identified in the plasma-fed pythons could actually stimulate healthy heart growth in mice," said Harrison. The team also tested the fed python plasma and the fatty acid mixture on cultured rat heart cells, with the same positive results, Harrison said.

The CU-led team also identified the activation of signaling pathways in the cells of fed python plasma, which serve as traffic lights of sorts, said Leinwand. "We are trying to understand how to make those signals tell individual heart cells whether they are going down a road that has pathological consequences, like disease, or beneficial consequences, like exercise," she said.

The prey of Burmese pythons can be up to 100 percent of the constricting snake's body mass, said Leinwand, who holds a Marsico Endowed Chair of Excellence at CU-Boulder. "When a python eats, something extraordinary happens. Its metabolism increases by more than fortyfold and the size of its organs increase significantly in mass by building new tissue, which is broken back down during the digestion process."

The three key fatty acids in the fed python plasma turned out to be myristic acid, palmitic acid and palmitoleic acid. The enzyme that showed increased activity in the python hearts during feeding episodes, known as superoxide dismutase, is a well-known "cardio-protective" enzyme in many organisms, including humans, said Leinwand.

The new Science study grew out of a project Leinwand began in 2006 when she was named a Howard Hughes Medical Institute Professor and awarded a four-year, $1 million undergraduate education grant from the Chevy Chase, Md.-based institute. As part of the award Leinwand initiated the Python Project, an undergraduate laboratory research program designed to focus on the heart biology of constricting snakes like pythons thought to have relevance to human disease.

Undergraduates contributed substantially to the underpinnings of the new python study both by their genetic studies and by caring for the lab pythons, said Leinwand. While scientists know a great deal about the genomes of standard lab animal models like fruit flies, worms and mice, relatively little was known about pythons. "We have had to do a lot of difficult groundwork using molecular genetics tools in order to undertake this research," said Leinwand.

CU-Boulder already had a laboratory snake facility in place, which contributed to the success of the project, she said.

"The fact that the python study involved faculty, postdoctoral researchers, a graduate student and an undergraduate, Christopher Wall, shows the project was a team effort," said Leinwand. "Chris is a good example of how the University of Colorado provides an incredible educational research environment for undergraduates." Wall is now a graduate student at the University of California, San Diego.

Hiberna Corp., a Boulder-based company developing drugs based on natural models of extreme metabolic regulation, signed an exclusive agreement with CU's Technology Transfer Office in 2008, licensing technology developed by Leinwand based on the natural ability of pythons to dramatically increase their heart size and metabolism.

Directed by Nobel laureate and CU Distinguished Professor Tom Cech, the Biofrontiers Institute was formed to advance human health and welfare by exploring critical areas of biology and translating new knowledge into practical applications. The institute is educating a new generation of interdisciplinary scientists to work together on solutions to complex biomedical challenges and to expand Colorado's leadership in biotechnology. For more information on the Biofrontiers Institute visit cimb.colorado.edu .

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Journal Reference:

Cecilia A. Riquelme, Jason A. Magida, Brooke C. Harrison, Christopher E. Wall, Thomas G. Marr, Stephen M. Secor, Leslie A. Leinwand. Fatty Acids Identified in the Burmese Python Promote Beneficial Cardiac Growth. Science, 2011; 334 (6055): 528-531 DOI: 10.1126/science.1210558

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More clues to causes of breast cancer: Hyperactivation of Akt and overexpression of IKBKE observed in 50 percent of human cancers

ScienceDaily (Oct. 27, 2011) — Publishing in the current issue of The Journal of Biological Chemistry, researchers at Moffitt Cancer Center in Tampa, Fla., have discovered additional mechanisms of "Akt" activation and suggest a component of that activation mechanism -- inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) -- could be targeted as a therapeutic intervention for treating cancer.

Akt, also known as protein kinase B, is one of about 500 protein kinases in the human genome. Kinases are known to regulate the majority of cellular pathways. Akt modifies other proteins chemically and regulates cell proliferation.

"Recent evidence suggests that IKBKE is an oncogenic kinase that participates in malignant transformation and tumor development," said Moffitt senior researcher and lead author Jin Q. Cheng, Ph.D., M.D. "Our study identified Akt as a bona fide substrate of IKBKE and IKBKE direct activation of Akt independent PI3K and revealed a functional link between IKBKE and Akt activation in breast cancer."

Cheng's lab studies a variety of genetic alterations and their molecular mechanisms in both ovarian and breast cancer, particularly on their effect on the molecules that are regulated by Akt and the small molecule inhibitors of Akt.

"We found that inhibition of Akt suppresses IKBKE's oncogenic transformation," said Cheng. "This is significant because overexpression of IKBKE and activation of Akt has been observed in more than 50 percent of human cancers. Akt inhibitors targeting PH domain do not have inhibitory effect on IKBKE-induced Akt."

The researchers experimented with a variety of inhibitors currently being used in clinical trials.

The laboratory study utilized breast cancer cell lines from received from patient donors at Moffitt and cell lines received from Harvard University and Johns Hopkins University. The work was supported by a National Institutes of Health grant and a grant from the James and Esther King Biomedical Research Program.

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J.-P. Guo, D. Coppola, J. Q. Cheng. IKBKE Protein Activates Akt Independent of Phosphatidylinositol 3-Kinase/PDK1/mTORC2 and the Pleckstrin Homology Domain to Sustain Malignant Transformation. Journal of Biological Chemistry, 2011; 286 (43): 37389 DOI: 10.1074/jbc.M111.287433

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