Two recent studies by CRG researchers delve into the role of chromatin modifying enzymes and transcription factors in tumour cells.

In one, published on September 9 in Genes & Development, it was found that the PARP1 enzyme activated by kinase CDK2 is necessary to induce the genes responsible for the proliferation of breast cancer cells in response to progesterone. In addition, extensive work has been undertaken to identify those genes activated by the administration of progesterone in breast cancer, the sequences that can be recognised and how these genes are induced. This work will be published on November 21 in the journal Molecular Cell.

Cancer is a complex set of diseases and only thanks to advances in genomic techniques have researchers begun to understand, at a cellular and molecular level, the mechanisms which are disrupted in cancer cells, a prerequisite for developing effective strategies to treat these diseases.

One clear example of this is breast cancer. It has long been known that hormones such as estrogen and progesterone encourage the proliferation of cancer cells. Because of this, one of the most common treatments is the administration of hormone receptor blockers. The block, however, affects all the cells of the body not only the cancer cells, and causes a number of side effects in patients. Additionally, most cancers develop resistance after a time and continue to grow despite anti-hormone therapy. To treat these patients it is necessary to understand the mechanisms that trigger the proliferation, which will allow their direct inhibition.

The scientists from the Cromatin and Gene Expression lab at the CRG, led by Miguel Beato, are dedicated to understanding how hormones activate cell division in breast cancer, focusing on regulating the expression of the genes that control the cell cycle.

Hormone receptors are transcription factors that bind to DNA sequences in the vicinity of the genes they regulate. But the DNA of the genes is packed into a dense structure known as "chromatin," which is considered a barrier preventing the access of transcription factors to genes. Therefore the chromatin must be decompacted for the transcription factors to activate the target genes expressed in RNA and subsequently translate them into proteins that stimulate cell proliferation. This is where the progesterone, via its receptor, activates various enzymes initiating chromatin opening.

In the study published on September 1 in the journal Genes & Development, the researchers looked at the role of an enzyme, PARP-1, which is primarily responsible for the repair of cuts in DNA. "It was not known how PARP-1 is activated and we found that it happens via the activation of another enzyme, CDK2, which phosphorylates and activates PARP-1, which in turn modifies the histone H1 and the chromatin displacement. And if PARP1 does not do this, many of the progesterone's target genes are not regulated," explains Roni Wright, first author of the study. Wright is a postdoctoral researcher in Beato's lab. She believes that much remains to be discovered in this area of research. "This experiment was conducted on cell lines, but now we have to do it on real, patient cells to see if their behaviour is the same," adds the researcher.

How do we know how the proliferation of cancer cells is controlled?

Gene regulation (how genes activate and deactivate) is the key to the overall understanding of how our genome works and when this function is altered. "It is important to discover the mechanism by which genes are activated around chromatin," explains Miguel Beato, head of the Chromatin and Gene Expression group. The chromatin packs the DNA at several levels, the first being the "nucleosomes," which help stabilise the DNA chain. "It was thought that the chromatin structure was not relevant to explaining how genes turn on and off, but we have discovered that it is crucial," adds Beato.

The second study, published online on November 21, 2012 in the journal Molecular Cell, addresses this issue. Firstly, all the genes that progesterone activates or represses in breast cancer cells were identified. Then the researchers identified which DNA sequences recognise the progesterone receptor in the genome. They found that these represented only a small proportion of the possibilities, making them think that interaction with DNA was not sufficient. It was necessary for the sequences which bind to the receptor to be incorporated into nucleosomes, which also provide interaction sites. "It seems that chromatin has a lot to do with determining which genes are activated and which are not," says Cecilia Ballaré, first author of this second paper.

The researchers believe that the only way to create increasingly specific and effective cancer treatments is by studying the role of all the elements that regulate gene expression and cell proliferation in response to hormones. "Knowing the exact way progesterone affects the proliferation of cancer cells may help develop more specific treatments that fight only cancer cells and thus produces fewer side effects," adds Ballaré.

Researchers have for the first time produced human embryonic stem cells (hESCs) using somatic nuclear transfer (SCNT), a method in which the nucleus of a donor cell—in this case a skin cell or fibroblast—is transferred to an egg cell whose own nucleus has been removed.

The work, published in Cell, opens up the possibility of an alternative source of patient-specific stem cells to help scientists understand disease and develop personalized cell-based therapies. What’s more, hESCs produced via nuclear transfer (NT-hESCs) may not have the genetic and epigenetic abnormalities found in induced pluripotent stem cells (iPSCs), made by adding key genes to reprogram adult cells.

“I think it is a beautiful piece of work,” said George Daley of Boston Children’s Hospital and the Harvard Stem Cell Institute, who was not involved in the research, in an email to The Scientist. “This group has become remarkably proficient at a very technically demanding procedure and has shown that SCNT-ESCs may in fact be a practical source of cells for regenerative medicine.”

SCNT has previously been used to clone animals and to successfully reprogram somatic cells into ESCs is mice and primates, but little is known about how it works and which factors in the egg cell are responsible stimulating the reversion of the implanted mature nucleus to a pluripotent state.

Moreover, all previous attempts to produce NT-ESCs have failed. Researchers have been unable to get human SCNT embryos to progress past the 8-cell stage, never mind to the 150-cell blastocyte stage from which hESCs can be plucked. The causes of the roadblock are not clear, but likely involve certain key embryonic genes from the donor cell nucleus that could not be activated.

To overcome these obstacles, Shoukhrat Mitalipov of Oregon Health and Science University and colleagues first examined failed attempts with human cells and successful work in rhesus macaques to identify factors that could be responsible.

The researchers evaluated various activation and culture protocols that led to successful SCNT reprogramming in monkeys, and set about testing various combinations on human oocytes. They found that the optimized protocols that worked in monkeys also worked in humans. In particular, the incorporation of caffeine into the cocktails of chemicals used during host nucleus removal and donor transplantation and the use of electrical pulses to activate embryonic development in the recipient egg improved cellular reprograming and blastocyte development, allowing human SCNT embryos to reach a stage that yielded hESCs.

“[The researchers] worked diligently to overcome the early embryo blockade that we and others have confronted as a barrier to human SCNT,” said Daley. “Their distinct culture media, which was supplemented with caffeine, and their optimized activation protocol appears to have been the needed breakthrough.”

“It was a huge battery of changes to the protocols over a number of different steps,” said Mitalipov. “I was worried that we might need a couple of thousand eggs to make all these optimizations, to find that winning combination. But it actually took just 128 [eggs], which is a surprisingly low number to make 6 [hESC] lines.”

The researchers then analyzed four of these cell lines and found that their NT-hESCs could successfully differentiate into beating heart cells in vitro and into a variety of cell types in teratoma tumors on live mice. The cells also closely resembled those derived from fetal fibroblasts, had no chromosomal abnormalities, and displayed fewer problematic epigenetic leftovers from parental somatic cells than are typically seen in iPSCs. Mitalipov said more comparisons are required, however.

“We are now left to analyze the detailed molecular nature of SCNT-ES cells to determine how closely they resemble embryo-derived ES cells and whether they have any advantages over iPS cells,” added Daley. “iPS cells are easier to produce and have wide applications in research and regenerative medicine, and it remains to be shown whether SCNT-ES cells have any advantages.”

But Milatipov pointed out one fundamental difference: while their nuclear genome comes from the donor cell, NT-hESCs contain mitochondrial DNA (mtDNA) from the egg cell. So unlike in iPSCs, nuclear transfer not only reprograms the cell but also corrects any mtDNA mutations that the donor may carry, meaning that patient-specific NT-hESCs could be used to treat people with diseases caused by mitochondrial mutations. “That’s one of the clear advantages with SCNT,” Milatipov said.

A new approach mimicking the body’s natural defenses could help treat a therapy-resistant breast cancer

The quest for longer and healthier life, if not immortality, has been part of the human experience since we evolved the ability to recognize the total annihilation of individual death. Our understanding of the biology of aging at the molecular level is advancing so rapidly that it appears inevitable that another decade or two of life will be enabled before long. A new step in what may be the right direction has just been published by researchers at the University of California, Berkeley.

The ravages of aging appear to be related to oxidative stress combined with telomere exhaustion, along with many other known and unknown factors. The subject of the new Berkeley study is a class of proteins called sirtuins that are known to play a central role in regulating aging and longevity in many non-human models (such as mice).

There is good evidence that these proteins also play a similar role in humans. For example, research has shown that, of two variants of the SIRT3 sirtuin protein (known to have strong anti-oxidant properties), humans who live past 90 years of age only have one of the variants in their bodies, the variant that enhances production of SIRT3. The difference between the two variants results from a change of one gene by one mutation, and appears to be sufficient to significantly affect an organism's longevity. This suggests a strong link between SIRT3 and longevity.

The genetics of longevity are quite interesting, but still more interesting would be finding an approach to offset the hand you were dealt at birth, or better yet, to stack the deck. The authors of the Berkeley study decided to see if SIRT3 could rejuvenate blood stem cells extracted from old mice.

Their first step was to see what happened as mice, which did not possess the SIRT3 gene, aged. When young, these "knockout" mice followed the same course of aging as did a set of normal control mice. However, when the mice were two years old (about an average lifespan for a lab mouse), the knockout mice had far fewer blood stem cells than did the control group.

What causes the difference in the course of aging? Young cells have low levels of oxidative stress (the generation of reactive oxygen species during metabolism), low enough that the body's normal anti-oxidants can keep up with the resulting damage. When they get older, they can't keep up, and need a boost of SIRT3 to help them. When there is no SIRT3, the progress of old age occurs sooner and more rapidly.

“When we get older, our system doesn’t work as well, and we either generate more oxidative stress or we can’t remove it as well, so levels build up,” said Chen. “Under this condition, our normal anti-oxidative system can’t take care of us, so that’s when we need SIRT3 to kick in to boost the anti-oxidant system. However, SIRT3 levels also drop with age, so over time, the system is overwhelmed.”

So it appears that age-related degeneration speeds up in the absence of SIRT3 in the system – at least among mice. The Berkeley team decided to see if increasing SIRT3 levels could rehabilitate the blood stem cells. This was done by infusing the blood stem cells with the SIRT3 protein, following which their ability to make new blood cells did indeed return.

Further studies will address if SIRT3-induced rejuvenation will apply to whole organisms, so that they might live longer when so treated, even after experiencing normal aging events.