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New Technologies Yield Insight Into How Cells Go Awry The discovery, so far, of approximately 50 oncogenes and tumor suppressor genes has wrought important, yet incremental victories in the war against cancer. The problem is that trying to understand such a complex disease in terms of individual genetic defects is like the five blind men seeking to comprehend an elephant. In what has been dubbed the postgenomics revolution, several technologies promise to allow researchers to view the entire elephant, instead of a leg here and a trunk there. Rather than searching for single genes in tumor cells, researchers will be able to make side by side comparisons of normal and cancerous cells, or of different stages of malignancy, to see which genes are expressed in one state while silent in the other. For example, "If you find a gene in normal epithelium that is absent in tumor, that might be a tumor suppressor gene," said Mark Adams, Ph.D., investigator at the Institute for Genomic Research, Gaithersburg, Md. New Insights More importantly, researchers believe that the patterns of gene expression in cancer cells will give them new insights into how and why cancer cells go awry, and how better to intervene. Two of the techniques are ways of detecting messenger RNA in samples of cellular extract. The third, known as proteome technology, is a modernized version of an old technique using 2-dimensional gels. 228 NEWS Researchers anticipate discovering a slew of cancer-related genes, from which may emerge the maps of the pathways of transformation. The techniques should also illuminate drug action mechanisms, and rationalize the search for new therapeutics. "If you treat a cancer cell with interferon or TNF, you are going to be able to see what the global effect on a large number of genes is," explained Paul Meltzer, M.D., Ph.D., head of the Section on Molecular Genetics, National Center for Human Genome Research. "That takes you towards a better understanding of how these important biological pathways in cell growth really work." "The greatest impact will be on development of complex diagnostics to guide therapeutic use," said Leigh Anderson, Ph.D., president of Large Scale Biology Corp., a biotechnology company in Rockville, Md. "Different tumors may be related to different patterns of oncogenes." "It might be possible to identify prognostic markers for drug sensitivity, resistance, and for metastatic and non-metastatic cancers," said Kenneth Kinzler, Ph.D., associate professor of oncology, Johns Hopkins Oncology Center, Baltimore. Working with one of the RNA techniques known as SAGE, he and Bert Vogelstein, M.D., also professor of oncology at Johns Hopkins, are characterizing expression patterns of colon and pancreatic cancer cells. Prognosis and diagnosis are only the beginning. The genomics revolution will refine oncologists' crude tools. A better understanding of how sex hormones influence cancer might permit doctors to prevent testosterone from fanning the flames of prostate cancer without turning off the sex drive and feminizing men, said Otis Brawley, M.D., senior investigator in the National Cancer Institute's Division of Cancer Prevention and Control. How Drugs Work Besides a better understanding of how cancer arises, researchers also expect to gain a clearer picture of how drugs work. This would greatly enhance the search for new drugs. Proteins that wax or wane when the cell is exposed to a drug are usually playing an important role, either therapeutically, or as part of toxic reactions, explained Anderson. "We studied chemopreventive compounds with Vernon Steele, [Ph.D., program director of the chemoprevention branch, NCI]," said Anderson. "A group of those compounds are analogs of the drug Oltipraz, which is known to protect against many carcinogens in rodent test systems. The potency [of the analogs] was very closely correlated with the appearance of certain proteins in the livers of the treated rats." "We then selected the protein that was most strongly induced," said Anderson. It turned out to be aflatoxin aldehyde reductase, an enzyme that metabolizes the eponymous fungus, and has proven protective against aflatoxin in animal studies. (Human trials are under way in China, where aflatoxin is implicated in liver cancer.) Journal of the National Cancer Institute, Vol. 88. No. 5, March 6, 1996 News News Studies of the antihistamine methapyrilene, and a benign analog, pyrilamine, further support the hypothesis that expression patterns have predictive value. In 1980, methapyrilene was pulled from pharmacy shelves after researchers discovered it caused cancer in 100% of exposed rats. "We looked at the effects of both of these compounds in rodent liver," said Anderson. "Methapyrilene causes significant changes in abundances of more than 100 proteins. Pyrilamine causes none." Improving Therapeutic Windows "One of the principal things to be learned is how to do the most good with the least harm," said Anderson. "You can get a lot of information just by comparing how many proteins are affected by a series of analogous drugs. If they all have similar potency, there is pretty good reason to believe the best one is the one that causes the fewest changes." In its quest for new drugs, NCI's Developmental Therapeutics Program has screened 55,725 new compounds against 60 cell lines from different types of cancer. Recently, the program's John Weinstein, M.D., Ph.D., has teamed up with Anderson in using the updated proteome technology to develop a database of expression patterns from each of the cell lines. This will make it possible to gain a better understanding of how anti-cancer compounds work. For example, a compound's impotence against all cell lines expressing the MDR-1 gene, which codes for a membrane pump that removes certain compounds from the cell, would reflect its removal by that pump. Expression studies also offer the opportunity to determine just how predic- tive particular in vitro and animal models really are, said Norman Anderson, Ph.D., chairman and chief scientist at Large Scale Biology, and father of Leigh. The greater the similarity of expression patterns, the more predictive the model, he said. For the animal and in vitro models to be predictive, their expression patterns should be similar to those in human tumors, particularly the expression patterns of proteins that are targets of drugs. But Weinstein and Leigh Anderson recently compared expression patterns of some of the cell lines used in the NCI screen. Expression patterns sometimes appeared more similar among different cancer types than among cells from one cancer type in different systems, leading Norman Anderson to question the predictive value of NCI's models. Edward A. Sausville, M.D., Ph.D., NCI's associate director for developmental therapeutics, asserted that the program's screening remains "a defensible first pass at looking at whether a compound may be active in a clinical situation." Of the thousands of compounds that have undergone initial screening to date, 31 compounds or classes of analogs have been selected for development, and Investigational New Drug applications have been filed for five of these. Resolving Theoretical Questions Nongenotoxic cancers are one of the mysteries of cancer research. These are cancers whose cells do not show any genetic mutation and which are caused by compounds that generally only promote cancer. Such cancers are well known in animal models, but to her knowledge, none has been proven to occur in humans, said Lucy Anderson, Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 Ph.D., chief of the perinatal carcinogenesis section of NCI. No one has yet determined the molecular biology of nongenotoxic carcinogenesis. A variety of simple mechanisms have been proposed, such as out-of-control cell division, or interference with programmed cell death, said Leigh Anderson, "but it's extremely unclear whether any of those simple mechanisms will explain the diverse effects of non-genotoxic carcinogens. Proteome studies show that nongenotoxic carcinogens cause far greater changes in gene expression than "almost all other classes of agents we've looked at," said Leigh Anderson, possibly due to the size of the disturbance. Beyond this question, the new technologies may open up whole new areas of inquiry, both in oncology specifically, and in molecular biology generally. For they allow researchers not only to determine which proteins are active, but where in the cell they are active, according to the Andersons. This can be done simply by dividing the cell into different component parts prior to conducting the studies. If sequencing the genome is like gathering anatomical data, then postgenomics can be likened to physiology, said Leigh Anderson. Understanding how it all fits together will require the kinds of skills that software designers have, he predicted, adding that they would contribute to this new era in the manner that physicists and chemists helped usher in molecular biology. Then, it will become possible to model how cells function, and to understand, at the deepest level, why they go awry. — David Holzman NEWS 229