With the help of special glass sheets with the approximate dimensions of a index finger e – the so-called microarrays or DNA chips -, the researcher Sergio Verjovski-Almeida, of the Chemical Institute of the São Paulo University (IQ/USP), has found six genes never before described in medical literature that could be related to prostrate cancer. It is the second most frequent type of tumor found in men here in Brazil, where annually almost twenty one thousand new cases of the illness are registered. If their link is proven with this type of cancer, the genes – whose names and location are still being held in secret – could become an important tool to help in the precocious diagnosis of this disease or of its clinical evolution.
“We still have to carry out more studies, with more patients, in order to be certain of the possible implication of these genes with prostrate cancer”, explains Verjovski, the coordinator of one of seven sub-projects of the institute that make up part of Cage – Cooperation for the Analysis of Gene Expression. Besides deepening the research with prostrate tissue, the biochemist carried out a similar study when looking for genes linked to lung cancer.
The mastery of the complete process of construction, experimentation and analysis of microarrays – Cage’s main objective – allows for making up chips on request. This may lead to interesting results, even more so when one has on hand differentiated raw material to put in these chips – genes or potential genes still not studied in relation to determined pathologies or situations. This was the case of Verjovski’s team. By using the laboratory equipment of microarrays provided through the Cage program, inaugurated at the Chemical Institute in December of 2000, the researchers constructed a DNA chipunheard-of in the world. In a microarray, where thousands of genetic sequences can be held, they deposited four thousand genes.
Half of these genes were already known, many of them with involvement in some types of cancer. The other half was composed of two thousand ESTs (Expressed Sequencing Tags, regions of the genome that are candidates to be genes), generated by the institute itself through the Human Genome Cancer Project, a partnership initiative between FAPESP and the branch of the Ludwig Cancer Institute in São Paulo. In these two thousand ESTs obtained through genomic research in São Paulo and not available in the commercial DNA chips sold in the world, is one of the great secrets of the good results achieved by Verjovski’s microarray. “It would be difficult for anybody else in the world to have a chip the same as ours”, emphasized the researcher.
An important detail: the chips and their experiments have to be carried out in an extremely aseptic and controlled environment, in order to not to imperil the trustworthiness of the data. For microarrays, the so-called clean room must have a temperature of around 22º C, relative air humidity of between 45% and 53% and at the maximum 10,000 dust particles per cubic meter of air, a standard a thousand times more severe than the sterilized operating room of a hospital. “To set up and keep a laboratory of this type is not a trivial operation”, says Hugo Armelin, also of the Chemical Institute and the general coordinator of the Cage program. The microarray laboratory can simultaneously manufacture thirty six DNA chips in four hours.
How are the genes placed on the sheet? A description of what was done at the Chemical Institute gives one a notion of the process. Kept in freezers beside the Cage laboratory, clones of the ESTs from the Human Cancer Genome Project were enlarged through a method called PCR (polymerase chain reaction) and, with the help of a robot, deposited one by one in microscopic receptacles (tip or circumference shaped) of the chip. Something similar was done with the other two thousand already known genes. Afterwards, cellular material of normal tissue and with cancer – taken out form from sixty patients at the Albert Einstein Hospital in São Paulo, and at the National Cancer Institute Hospital of Rio de Janeiro, with prostrate tumors in intermediary stage – were added to the chip with 4,000 genes, in a process named hybridization. The healthy tissues were marked with fluorescent green coloring and those with cancer, red.
This done, the microarray is ready to provide indicators as to which genes are more or less expressed (used up) by healthy cells and by those with the tumors. The expression of the genes in the two types of tissue is measured with the help of a scanner, laser and software. The results are set out as published below. Each dot represents a gene. The green color means that the gene in question is more expressed in normal tissue than in tumors. The red states the contrary. And, the yellow signifies that the expression is the same both with one or other type of tissue. After treatment and computer modeling, the expression of the genes in the two types of tissue is compared and analyzed.
Every cell of an organism, normal or with cancer, has the same DNA, the same genes. However, each cell according to its function and other parameters, expresses (uses), with greater or lesser intensity, certain genes in certain moments, while the others remain inactive. Nevertheless, in spite of the availability of the same DNA, a normal cell exhibits a standard of gene expression distinct from a tumor cell. The use of microarrays allows one to know which genes are used (and to what intensity) in an infinity of situations.
Mammals and oranges
Besides prostate cancer, six Cage projects are working with DNA chip technology, analyzing the expression of genes linked to different processes or pathologies in distinct organisms. For example, Armelin is studying the cellular cycles in mammals, and for this he is developing a microarray starting with 33,000 ESTs of mice.
Another group, coordinated by Suely Lopes Gomes, has managed to place in a chip more than 90% of the close to 2,800 genes of the bacterium Xylella fastidiosa, whose genome was sequenced by the Organization of Nucleotide Sequencing Analysis (Onsa), a network of laboratories created by FAPESP. The main goal of the researchers is to compare the expression of genes in distinct parts of the bacterium, which causes Citric Variegated Chlorosis (CVC), the popularly named yellowing disease, harmful to orange trees. “We’re going to try to understand why one of these strains cannot be modified through genetic engineering while the other accepts these alterations”, says Aline Maria da Silva, of the Biochemistry Department who participated in the sub-project, and is one of those responsible for the microarray laboratory. “In one month we should be able to manage to place all of the genes of Xylella on the chip and have a final version of the microarray.”
Another initiative of the Cage project is to construct DNA chips to study the genetic expression of different strains and evolutionary stages of the parasite Trypanosoma cruzi , the protozoan that causes Chagas disease. The central objective of the work is to eventually identify genes or groups of genes that can be determined as causing the degree of severity of the illness. “Close to 60% of the carriers of the parasite don’t develop the illness”, says Bianca Zingales, the coordinator of this subproject, whose first results with the microarray are forecast for next year.
“However, 30% develop serious cardiac illnesses and a further 10% present digestive problems.” Still in the remit of the Cage project, there are three other sub-projects that are studying gene expressions in distinct micro organisms: the ameba Dictyostelium discoideum (whose genes, though in lesser number by a factor of ten, are similar to the grouping of human genes), the bacterium Xanthomonas campestris (that brings on illnesses in vegetal species) and the yeast Saccharomyces cerevisiae .
Furthermore, the Cage program can also count upon the participation of a research group from the Mathematics and Statistics Institute (IME) of USP, which is working in harmony with the Chemistry Institute researchers in the development of a series of data banks, software and computer models for the analysis of the expression of genes in DNA chips. This is a strategic area in the studies with microarrays, which generates an enormous quantity of information and needs efficient programs and mathematical models to carry out the so called mining of data – to separate out what is statistically important from what is secondary.
There are commercial programs or those of public knowledge which do this, but they have limitations. “Many are simply a black box”, tells Junior Barrera, the coordinator of the Bio Information Center of the IME and a member of Cage. “We don’t know exactly how they function nor are we certain what they measure.” As well as this, some softwareneeds an interaction so intense by the user that two researchers, using the same program, can arrive at different results. The São Paulo bioinformation experts, who have already published at least three articles in international magazines because of their work in the Cage, are attempting to minimize these problems. In some cases, they obtained good results: in conjunction with colleagues of the National Institutes of Health (NIH) of the United States, have created a software to visualize images. Shortly the program will be available for downloading on the Internet site of the NIH.
Cage – Cooperation for Analysis of Gene Expression (nº 99/07390-0); Modality Thematic Project; Coordinator Hugo Aguirre Armelin – Chemical Institute of USP; Investment R$ 1,973,072.96