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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Cancer cells' DNA repair disrupted to increase radiation sensitivity

ScienceDaily (Dec. 1, 2011) — Shortening end caps on chromosomes in human cervical cancer cells disrupts DNA repair signaling, increases the cells' sensitivity to radiation treatment and kills them more quickly, according to a study in Cancer Prevention Research.

Researchers would to like see their laboratory findings -- published in the journal's Dec. 5 print edition -- lead to safer, more effective combination therapies for hard-to-treat pediatric brain cancers like medulloblastoma and high-grade gliomas. To this end, they are starting laboratory tests on brain cancer cells.

"Children with pediatric brain cancers don't have very many options because progress to find new treatments has been limited the last 30 years," said Rachid Drissi, PhD, principal investigator on the study and a researcher in the Division of Oncology at Cincinnati Children's. "The ability to make cancer cells more sensitive to radiation could allow physicians to use lower radiation doses to lessen side effects. Too many children with brain cancer can develop disabilities or die from treatment."

Before treating cells with ionizing radiation, the researchers blocked an enzyme called telomerase, found in over 90 percent of cancer cells but barely detectable in most normal human cells. In cancer cells, telomerase helps maintain the length of caps on the ends of chromosomes called telomeres. This helps cancer cells replicate indefinitely, grow and spread, Drissi said.

Unraveling DNA stability

Found on chromosomes in both cancerous and normal cells, telomeres are analogous to plastic caps that keep shoestring ends from unraveling. Telomeres help preserve DNA stability in cells by containing genetic miscues. This helps explain why cells with maintained or long telomeres appear to be more resistant to radiation.

In normal cells lacking the telomerase enzyme, telomeres get shorter each time cells divide. They continue doing so until normal cells stop dividing, reaching a condition called senescence. If this first cell-cycle "stop sign" is bypassed, cells continue dividing until telomeres become critically short and reach a second stopping point, when most cells die. In rare instances, cells bypass this second "stop sign" and survive. This survival is often associated with telomerase activation and the onset of cancer.

This was the basis for experiments Drissi and his colleagues conducted to compare the radiation sensitivity and survivability of cells based on telomere length. They also monitored DNA repair responses in the cells by looking for specific biochemical signs that indicate whether the repair systems are working.

The tests involved normal human foreskin cells -- called fibroblasts -- and human cervical carcinoma cells. They exposed the cells to ionizing radiation and analyzed DNA repair responses as telomeres became progressively shorter. In the cervical cancer cells, researchers blocked the telomerase enzyme before radiation treatment to induce progressively shorter telomeres.

Both late-stage noncancerous cells with shorter telomeres, and cancer cells with induced shorter telomeres, were more radiosensitive and died more quickly, according to the study.

Among cancer cells with maintained telomere length, close to 10 percent receiving the maximum dose of ionizing radiation used in the study (8 Gy, or Gray Units) survived the treatment. None of the cancer cells with the shortest telomeres survived that exposure.

Researchers said the cancer cells became more radiosensitive because material inside the chromosomes -- called chromatin -- compacted as telomeres became shorter. Compacted chromatin then disrupted the biochemical signaling of a protein called ATM (ataxiatelangeietasia mutated).

ATM is a master regulator of DNA repair and cell division. It sends signals to activate other biochemical targets (H2AX, SMC1, NBS1 and p53) that help direct DNA repair and preserve genetic stability. In telomere-shortened cancer cells, the compacted chromatin inhibited ATM signaling to all of the chromatin-bound targets tested in the study. This disrupted DNA repair responses and increased radiation sensitivity.

Testing brain cancer cells

The researchers are now testing their findings in cells from hard-to-treat pediatric brain tumors. These tests begin as Drissi's laboratory also leads correlative cancer biology studies of tumor samples from a current clinical trial. The trial is evaluating telomere shortening as a stand-alone therapy for pediatric cancer.

Managed through the National Institutes of Health's Children's Oncology Group (COG), the multi-institutional Phase 1 trial is testing the safety and tumor response capabilities of the drug Imetelstat, which blocks telomerase in cancer cells. Drissi serves on the clinical trial committee along with Maryam Fouladi, MD, MSc, and medical director of Neuro-Oncology at Cincinnati Children's. She leads the medical center's clinical participation in the trial.

Drissi and Fouladi are starting preparatory work to develop, and seek approvals for, a possible clinical trial to test telomere shortening and radiation treatment as a safer, more effective treatment for pediatric brain tumors.

Funding support for the current study in Cancer Prevention Research -- published by the American Society for Cancer Research -- came from the National Institutes of Health, the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital and Cincinnati Children's Hospital Medical Center. Also collaborating were researchers from Children's National Medical Center in Washington, D.C., and from St. Jude. Funding support for the Drissi lab's correlative studies on the COG clinical trial comes from CancerFree Kids Pediatric Cancer Research Alliance and from Children's Cancer Research Fund.

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Journal Reference:

R. Drissi, J. Wu, Y. Hu, C. A. Bockhold, J. S. Dome. Telomere shortening alters the kinetics of the DNA damage response after ionizing radiation in human cells. Cancer Prevention Research, 2011; DOI: 10.1158/1940-6207.CAPR-11-0069

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Natural killer cells could be key to anthrax defense

ScienceDaily (Oct. 27, 2011) — One of the things that makes inhalational anthrax so worrisome for biodefense experts is how quickly a relatively small number of inhaled anthrax spores can turn into a lethal infection. By the time an anthrax victim realizes he or she has something worse than the flu and seeks treatment, it's often too late; even the most powerful antibiotics may be no help against the spreading bacteria and the potent toxins they generate.

Now, though, University of Texas Medical Branch at Galveston researchers have found new allies for the fight against anthrax. Known as natural killer cells, they're a part of the immune system normally associated with eliminating tumor cells and cells infected by viruses. But natural killer cells also attack bacteria -- including anthrax, according to the UTMB group.

"People become ill so suddenly from inhalational anthrax that there isn't time for a T cell response, the more traditional cellular immune response," said UTMB assistant professor Janice Endsley, lead author of a paper now online in the journal Infection and Immunity. "NK cells can do a lot of the same things, and they can do them immediately."

In test-tube experiments, a collaborative team led by Endsley and Professor Johnny Peterson profiled the NK cell response to anthrax, documenting how NK cells successfully detected and killed cells that had been infected by anthrax, destroying the bacteria inside the cells along with them. Surprisingly, they found that NK cells were also able to detect and kill anthrax bacteria outside of human cells.

"Somehow these NK cells were able to recognize that there was something hostile there, and they actually caused the death of these bacteria," Endsley said.

In further experiments, the group compared the anthrax infection responses of normal mice and mice that were given a treatment to remove NK cells from the body. All the mice died with equal rapidity when given a large dose of anthrax spores, but the non-treated (NK cell-intact) mice had much lower levels of bacteria in their blood. "This is a significant finding," Endsley said. "Growth of bacteria in the bloodstream is an important part of the disease process."

The next step, according to Endsley, is to apply an existing NK cell-augmentation technique (many have already been developed for cancer research) to mice, in an attempt to see if the more numerous and active NK cells can protect them from anthrax. Even if the augmented NK cells don't provide enough protection by themselves, they could give a crucial boost in combination with antibiotic treatment.

"We may not be able to completely control something just by modulating the immune response," Endsley said. "But if we can complement antibiotic effects and improve the efficiency of antibiotics, that would be of value as well."

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Journal Reference:

C. M. Gonzales, C. B. Williams, V. E. Calderon, M. B. Huante, S. T. Moen, V. L. Popov, W. B. Baze, J. W. Peterson, J. J. Endsley. Antibacterial Role for Natural Killer Cells in Host Defense to Bacillus Anthracis. Infection and Immunity, 2011; DOI: 10.1128/IAI.05439-11

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Lung stem cells offer therapeutic clues

ScienceDaily (Oct. 27, 2011) — Guided by insights into how mice recover after H1N1 flu, researchers at Harvard Medical School and Brigham and Women's Hospital, together with researchers at A*STAR of Singapore, have cloned three distinct stem cells from the human airways and demonstrated that one of these cells can form into the lung's alveoli air sac tissue. What's more, the researchers showed that these same lung stem cells are rapidly deployed in a dynamic process of lung regeneration to combat damage from infection or chronic disease.

"These findings suggest new cell- and factor-based strategies for enhancing lung regeneration following acute damage from infection, and even in chronic conditions such as pulmonary fibrosis," said Frank McKeon, professor of cell biology at Harvard Medical School. Other senior authors on the paper include Wa Xian of the Institute of Medical Biology in Singapore and Brigham and Women's Hospital, and Christopher Crum, Director of Women's and Perinatal Pathology at Brigham and Women's Hospital. The researchers worked as part of an international consortium involving scientists from Singapore and France.

The findings will be reported in the Oct. 28 issue of Cell.

For many years, clinicians have observed that patients who survive acute respiratory distress syndrome (ARDS), a form of airway damage involving wholesale destruction of large regions of lung tissue, often recover considerable pulmonary function within six to 12 months. But researchers did not know whether that recovery was due to lung regeneration or to some other kind of adaptive remodeling.

"This study helps clear up the uncertainty," said McKeon. "We have found that the lungs do in fact have a robust potential for regeneration, and we've identified the specific stem cells responsible."

To probe the potential for lung regeneration, Xian, McKeon and colleagues infected mice with a sublethal dosage of a virulent strain of H1N1 influenza A virus. After two weeks of infection, these mice showed a loss of nearly 60 percent of tissue in the lung air sacs after two weeks of infection, but -- remarkably -- by three months, the lungs appeared completely normal by all histological criteria.

These findings demonstrated true lung regeneration, but raised the question of the nature of the stem cells underlying this regenerative process.

Adapting the methods for cloning epidermal skin stem cells pioneered by Howard Green, the George Higginson Professor of Cell Biology at HMS and the 2010 Warren Alpert Foundation Prize recipient, the researchers cloned stem cells from the lung airway in a dish and watched as they differentiated to unusual structures with gene profiles similar to alveoli, the cells in the lung's air sacs.

"This was startling to us," Xian said, "and even more so as we observed the same stem cell populations involved in alveoli formation during the peak of H1N1 infections in mice." The researchers genetically traced the formation of new alveoli to a discrete population of stem cells in the fine endings of the conducting airways that rapidly divide in response to infection and migrate to sites of lung damage.

The scientists were intrigued when molecular dissection of these incipient alveoli revealed the presence of an array of signaling molecules known to control cell behavior, suggesting the possibility that these molecules coordinate the regeneration process itself.

Currently the team is testing the possibility that the secreted factors they observed might promote regeneration, suggesting a therapeutic approach for conditions such as chronic obstructive pulmonary disease and even asthma. They also foresee the possibility that these distal airway stem cells could contribute to repairing lungs scarred by irreversible fibrosis, conditions resistant to present therapies.

This work was supported by the National Heart, Lung, and Blood Institute, the Institute of General Medical Sciences, the National Cancer Institute, and the Defense Advanced Research Projects Agency.

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Journal Reference:

Pooja A. Kumar, Yuanyu Hu, Yusuke Yamamoto, Neo Boon Hoe, Tay Seok Wei, Dakai Mu, Yan Sun, Lim Siew Joo, Rania Dagher, Elisabeth M. Zielonka, De Yun Wang, Bing Lim, Vincent T. Chow, Christopher P. Crum, Wa Xian, Frank McKeon. Distal Airway Stem Cells Yield Alveoli In Vitro and during Lung Regeneration following H1N1 Influenza Infection. Cell, 28 October 2011 DOI: 10.1016/j.cell.2011.10.001

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