There are times when Professor Carlos Menck teases his students with the following question. Which appeared first: the DNA molecule, the carrier of the genetic material of living beings, or the DNA repair mechanism? Questions like this one do not only intrigue and amuse this researcher from the Institute of Biological Sciences (ICB) of the University of São Paulo. They have also led him to achieve conceptual and applied advances: they have helped to elucidate the mechanisms for cell survival and, at the same time, showed ways of correcting genetic defects that arise in some cells when the repair devices fail – and cancer sets in as a result. As a result of the findings, there was a series of recent articles, published in magazines with a high scientific impact, and, something rare for a Brazilian researcher: an invitation to write a commentary about research into repair genes carried out by a Dutch group, which came out in the November issue of Nature Genetics.
Contained in the nucleus of all plant and animal cells, the deoxyribonucleic acid molecule is not that resistant. At every movement, when the cells multiply in so order to allow the replacement of skin burnt by the sun or the growth of leaves, the DNA can break up or be copied in a different way from the original recipe. It is also altered by the action of sunlight, by chemical reactions, or changes in the chemical balance in the inside of the cell. But luckily there is a strict quality control: as soon as the DNA is being replicated, some proteins – the repair enzymes – check if the copy has come out according to the original, like a spell checker that replaces the mistaken letters as soon as the words finish being written. Other enzymes stay on the alert so as to mend , like tinsmiths, the DNA at the points where it breaks. They get no time off. In the human organism, just one kind of lesion, brought about by the heat of the body itself, at 37º Celsius, happens some 10,000 times a day in each one of the almost 100 trillion cells.
Using one of these repair genes, Menck managed something that he had wanted for 5 years: to correct the genetic fault in cells taken out of victims of xeroderma pigmentosum (XP), a rare kind of skin cancer. More common in Japan and in the north of Africa, this tumor affects about 100 people in Brazil and obliges its sufferers to avoid direct exposure to the sun and to wear dark glasses and long clothes, to prevent lesions to the skin and to the eyes. Accordingly, the team from USP has opened up a path for researching genetic therapy against this disease – an unprecedented approach, because nobody had managed to manipulate this gene, also called XP because of the problem it causes, using an adenovirus.
The discoveries by the team from USP may also make it easier to diagnose the disease and to characterize more precisely each one of the eight forms in which xeroderma shows itself – some with neurological complications, such as mental retardation and muscular spasms. Melissa Armelini and Ricardo Leite, members of this group from the ICB, are working to produce a test to make it possible to diagnose the disease using cells from the blood – a possible alternative to the technique in use, biopsy, which consists of removing a piece of skin.
Making it easier to diagnose, indeed, is an extremely important step towards preventing the appearance of the disease, according to one of the greatest specialists on the subject, researcher James Cleaver, from the university of California, in San Francisco, in the United States, who discovered the cause of the disease in 1968. Soon after the reports of the first cases, Cleaver saw that xeroderma pigmentosum would appear when there was a defect in the repair gene – today, one knows that eight genes may fail in varied combinations and generate the different forms of the disease.
When undergoing mutations, these genes become incapable of mending the damage that the sun’s ultraviolet light causes the DNA. As a consequence, chemical substances set off a red alert and the cell commits suicide, in a programmed cell death called apoptosis – a last resort activated in the case of imminent peril. Apoptosis is also an essential process of cleaning up, to prevent errors from propagating to the following generations and intensifying to the point of leading to cancer. In people with xeroderma, the repair failure increases the frequency of tumors in the skin, in regions exposed to sunlight.
Viral vectors
Menck’s teams have succeeded in mending the DNA of a group of cells in vitro, using a modified adenovirus, incapable of replicating itself in the cell. The adenovirus works like a vector for two genes that set off different forms of the disease, XPA and XPC, found also in other animals and in fungi. As a result, between 90% and 100% of the cells infected with the adenovirus went back to correcting the DNA damaged by the ultraviolet sunlight, while only 9% of the non-infected ones managed to reestablish this capacity. Described in the October issue of the Human Gene Therapy magazine, these results also represent a methodological advance, for having been obtained with an adenovirus, more effective that the vectors used previously, the retroviruses, – today, a secondary choice also because they insert their genetic material into the genome of the cells and can reproduce or activate cells that induce the appearance of cancer.
“We were the first to produce an adenovirus containing the XPA gene”, Menck says. “When the first results came up, I didn’t believe in the efficiency of these vectors”. With time, the researchers also managed to add other genes to the genetic material of the virus. In conjunction with Armando Moraes Ventura, from the ICB’s viral vector laboratory, they set the virus to carry a suicide gene, which calls to itself specific kinds of medicines and can therefore be used to eliminate tumor cells.
In January, a researcher in the team, Maria Carolina Marchetto, embarks for the University of Texas, in the United States, with plans for carrying out the first in vivo tests of the use of these XP viruses on mice with xeroderma pigmentosum. If the viruses succeed in protecting the skin of these mice from ultraviolet light, the possibility will be opened up for improving the lives of patients with this disease, through genetic therapy.
Controlled death
The team from USP made another important discovery: it just takes small doses of the XPA protein, produced from the gene of the same name, to prevent cells from dying when exposed to ultraviolet radiation. It really is a very small quantity, equivalent to 20% of what is normally found in human cells. “The protein is probably very stable and not easily degraded”, comments Melissa. In an article published in March in Carcinogenesis, Alysson Muotri, from the same team, demonstrated that the XPA protein is safe and does not affect the cell functioning, even when produced in high quantities – the so-called overexpression that occurs when the gene is led by the adenovirus.
But it should not be thought that the results only apply to xeroderma pigmentosum. In first place, because there are other diseases caused by problems in the DNA repair mechanisms and, when one of them is understood, it becomes easier to understand the others, not least because the role of the XP genes has been reasonably well clarified: they are provenly essential for the cell to mend the damaged DNA and to acquire resistance to ultraviolet light. They also act as protagonists in several mechanisms for mending DNA.
Furthermore, studies by the DNA repair laboratory are giving support for research in other areas, by revealing nuances of how apoptosis works. In an article published, also in October, in Cell Death and Differentiation, Menck and another researcher from the ICB, Vanessa Chiganças, associated apoptosis – probably for the first time – to specific kinds of lesions in DNA. The defects brought about by ultraviolet light prevent a correct reading of this molecule by another, RNA (ribonucleic acid), in the preliminary stages of the process of the proteins production that form any living being – and the cell that cannot read the DNA to transform it into RNA goes into a process of cell death. “The XP genes are important in the process of signaling that leads to apoptosis”, says Menck.
Solar protection
The results that Menck’s team has been obtaining may also help, even though it may take some time, people who have fair skin and dream of going to the beach without worrying about the sun. Besides XP, the team from the ICB is working with the gene that contains the recipe for producing photolyase, another enzyme that repairs the damage caused in the DNA by ultraviolet light. It is a gene that is common in bacteria, plants, insects and fish, but rare amongst mammals: it only occurs in marsupials (without a placenta), but not in the placentiferous, the group that the human species is part of. “A gene that we do not have any more, but which solves a problem that we still do”, says Menck. According to the researcher, the photolyase gene got lost some 170 million years ago, during the evolutionary process that was to lead to human beings.
Vanessa managed to implant a photolyase gene taken from a marsupial – a rat kangaroo (Potorous tridactylus) – in normal human cells. Afterwards, she exposed the cells to intense ultraviolet light irradiation, and next to visible light, to activate the photolyase. Few of them died, in an indication that they could achieve an extra mechanism for undoing and avoiding the damage caused by radiation, as reported in May 2000 in the prestigious Cancer Research magazine. Now, Menck’s team is at the final stages of producing an adenovirus carrying the photolyase gene. If this vector works with in vitro cell cultures, they will be able to be tested in vivo, infecting mice, in a similar way to what will be done by Maria Carolina in the United States with adenovirus and XP genes. The mice could thus gain an extra protection against solar radiation.
Even though it is not easy, the task does not seem impossible, since the groups led by Jan Hoeijmakers and Gijsbertus van der Horst, from Erasmus University Rotterdam, in Holland, have achieved transgenic mice that produce photolyase in cells from all over the body, strengthening the DNA’s capacity for getting mended – this was the work that Menck analyzed in two pages of November’s Nature Genetics. This time, though, it is not any longer a question of correcting a genetic deficiency, but of strengthening a cell repair mechanism.
When submitting the transgenic mice, with their backs shaved, to intense doses of ultraviolet radiation and to visible light, the Dutch found that the animals developed neither any wounds nor burns on their skin, characteristic of people unwittingly exposed to intense insolation. In view of the results, the idea is taking shape of a solar protector based on photolyase, to be used in the form of a cream both by ordinary people – above all, the fair-skinned, who suffer more in the summer – and by victims of xeroderma pigmentosum, who could then hide themselves less from the sun. Menck comments cautiously: “For the time being, I would prefer the chemical protectors, which are still safer”.
In another, more recent, experiment, Menck’s group is using the adenovirus vectors as a strategy for accompanying, step by step, the mending of the DNA molecule. The researchers from USP put together an adenovirus with a repair gene (photolyase or XP) and added a sort of tail, a gene that leads to the production of GFP, a Green Fluorescent Protein. With this structure, they observed the genes, now green, migrating to the nucleus of the cell and putting the DNA in order. More elegant and practical than the technique in use, this method has already indicated that XPA proteins appear quickly, in less than one hour, to mend the DNA – the lesions are known to take a few hours to be removed. “We have opened up excellent prospects for work, because this technique can be applied to any kind of human cell”, explains Menck.
The DNA repair mechanisms that Menck has been studying for so long are common to animals and plants – recently, by the way, Keronninn de Lima, from his group, discovered in sugarcane the gene of a photolyase that acts in a different way from all the others now known. For being extremely well preserved – almost immutable – the repair genes contain precious information on the origin and differentiation of living beings.
Replying now to the question at the beginning of this article, Menck believes that the DNA repair mechanisms arose even before DNA itself and prepared the chemical bases for the emergence of life on the planet, some 3.8 billion years ago, from the RNA molecule. According to him, a broken or incomplete DNA would not manage to go very far. It would be like a car without wheels: it would hardly have originated even the first cell on the primitive Earth.
The Project
DNA Repair and Biological Consequences; Modality Thematic project;
Coordinator Carlos Frederico Martins Menck – ICB/USP; Investment R$ 818,618.78
