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Genetics

Lightproof

Enzyme reduces the loss of healthy cells exposed to ultraviolet radiation

MIGUEL BOYAYANExtra power: photolyase helps to repair the genetic damage cause by excessive sunMIGUEL BOYAYAN

Those who had children or were a child in the 1980’s will certainly remember a video game that was famous those days, Pac-Man. The personage, a famished yellow ball, would set out to eat the pills he found ahead of him in a labyrinth full of ghosts.  The game was won by whoever managed to gobble up more pills and escape from the little monsters. In the human body, something similar happens when some genes are altered. Old cells or have damaged genetic material disintegrate and are swallowed up by neighboring cells or from the immune system. This mechanism of programmed cell death, or apoptosis, is a normal process for renewing the cells. But, when it occurs in an uncontrolled manner, it causes damage to the organism – including tumors.

This is what happens with the sufferers from xeroderma pigmentosum (XP), a rare genetic disease that affects one in every 250,000 Americans and, it is estimated, almost a thousand Brazilians – although only some hundred cases have been identified in the country. Because of alterations in the DNA, the cells of the skin of these people show a defective form of a certain kind of protein – the repair enzymes, which detect and mend the alterations in the structure of the DNA caused by ultraviolet rays, after repeated exposure to the sun.

As these cells lose the capacity for correcting these defects, chemical messengers give the order for cell suicide, preventing the transmission of defective genes to their descendents. The victims of this disease are born with a high sensitivity to sunlight – that is why they are obliged to use protective creams, dark glasses and long clothes – and run a risk 2,000 greater than the population at large of getting skin cancer, because some of the cells that ought to die possibly do not follow the orders and do not commit suicide.

Well, another kind of light has helped the researchers from the Biomedical Sciences Institute of the University of São Paulo (USP) to repair lesions in the DNA of cells removed from the skin of people with xeroderma pigmentosum and to reduce by half the death of healthy cells exposed to ultraviolet radiation, creating a technique that may come to be used in the treatment of other kinds of skin cancer. Using an adenovirus, the team led by Carlos Menck introduced, for the first time with success, the gene of the photolyase enzyme in cells taken from people with xeroderma. Activated by visible light and found in bacteria, plants and animals, but not in placental mammals like human beings, this enzyme restored in less than an hour the most numerous lesions, called cyclobutane pyrimidine dimer (CPD), generally corrected very slowly. The natural repair system in humans – the XP proteins – manages to repair, in four hours, only part of these lesions generated by the ultraviolet rays.

Gene therapy
“We gave the cells the extra capacity for repairing lesions. Those that already had repair systems managed to mend the DNA better. In those that showed a defect, the photolyase enzyme extracted from kangaroos solved the problem of the CPD lesions”, Menck declares. In July, he and Vanessa Chiganças published an article about the study in the Journal of Cell Science, in collaboration with the team led by Alain Sarasin, from the Institut Gustave-Roussy, in France. The results make it possible to understand better how the repair system in human cells works and the consequences of the lesions caused by ultraviolet light. And although there are still several stages left – tests on animals and clinical testing with humans -, the data from this work is opening up prospects for gene therapy. “If they are successful, it will be possible to think about using photolyase in future as a prevention for any skin cancer”, Vanessa explains.

To see whether photolyase was capable of putting in order the DNA of the cells of xeroderma sufferers – in 2000, the researchers showed that, with this protein, it was possible to repair the genetic material of normal human cells -, the team from USP used a virus that is harmless to health as a sort of cell postman, which carried the gene capable of repairing the defect and a green fluorescent protein from jellyfish, which made it possible to map the path followed by the gene of the photolyase. Then they exposed the cells to ultraviolet radiation. Some of them were also submitted to visible light, to activate the enzyme.

Vanessa divided the cells into four groups: the first, in which the gene of the photolyase was implanted, was kept in white light after the exposure to the ultraviolet rays; the second was given photolyase, but remained in the dark. In the third and the fourth, also exposed to radiation, the gene was not implanted, and one of the groups remained in the dark, and the other, in the light. The mortality rate amongst the cells exposed to the ultraviolet light, which received the photolyase enzyme or not and remained in the dark, varied from 55% to 68%. The death of the cells with the defective XPA repair gene in which the photolyase was implanted and which were kept in the presence of light fell by half: between 25% and 29%. “I eliminated the lesion, which is one of the signs of cell apoptosis, and thereby prevented their death from being induced”, Vanessa explains.

Preliminary data from another study by Menck’s team points to a way of protecting even more the cells of the skin of sufferers from xeroderma pigmentosum. An adenovirus containing a new gene of the photolyase is under test, this time extracted from the Arabidopsis thaliana plant, to correct another kind of lesion, called photoproducts 6-4. This genetic fault was always regarded as inoffensive, as it appears in a smaller quantity and it is repaired quickly. But it seems that the damage that it can cause the cells was underestimated. “Although it arises in a lower proportion, this lesion is just as important as the other for inducing cell death”, explains Keronninn de Lima, the author of the study under way. She exposed the cells to ultraviolet radiation and inserted the new repair gene, specific for this kind of lesion. The result: also an increase in the survival of the cells.

The team from USP has also been achieving success with the use of the XPD gene, which is a component of the natural repair system in humans. By means of an adenovirus, a normal version of this gene was introduced into skin cells taken from people with xeroderma and two other diseases caused by defects in the proteins that repair the alterations in the DNA generated by ultraviolet rays – Cockayne syndrome and trichothiodystrophy. “While none of the cells exposed to radiation, but which were given the repair gene, died, only 10% of the cells without the XPD protein survived”, says Melissa Armelini, whose work should be published shortly in Cancer Gene Therapy. It is if the tables had been turned in the Pac-Man game, and the pills had found a way of not being swallowed up by the yellow ball.

The Project
DNA repair genes: functional analysis and evolution
Modality
Thematic Project
Coordinator
Carlos Menck – ICB/USP
Investment
R$ 1,059,975.46 (FAPESP)

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