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| This article will appear in a forthcoming issue of Trends in Biotechnology. | |
Abstract
Most of us have had the experience of asking a student or coworker what they did to make the experiment finally work and being answered with a laundry list of modifications. More often than not the further query: "and which one made the crucial difference?" will elicit the answer: "I was desperate so I tried everything I could think of at once." Although some of us are gifted with sufficient intuition to succeed in this manner, it is subsequently necessary to go back and test individual variables until the causative agent is found. For the less precocious (or lucky) the tried and tested method is to vary one parameter at a time.
The paper by the Kinzler and Vogelstein group [1] takes an important step towards a more rational and controlled approach towards cancer drug screening. They started with a human colon cancer cell line (DLD-1) in which the Shirasawa group had previously disrupted an activated Ki-Ras R13W allele using gene targeting [2]. They went on to stably modify the Ki-Ras +/- cell line with blue fluorescent protein (BFP) and its parental Ki-Ras +/(R13W) cell line with yellow fluorescent protein (YFP). This allowed the co-culture of the two cell lines and a real-time, non-invasive assessment of the relative composition of the cultures using simple spectral analysis. Thus, if a drug were added that differentially affected one or the other cell line, the co-culture would change color. This clever method provided not only an internal control but also lent itself easily to scale-up for a high throughput drug screen.
It is important to note that Torrance et al. chose ablation of an endogenously activated Ras allele rather than introducing a mutant Ras gene into a cell that previously lacked it. Although conceptually similar, the latter approach can be confounded by the fact that excessive expression of mutant Ras genes can trigger growth inhibitory effects [3]. Thus, although engineering gain-of-function mutations is an important and powerful tool, investigators must be mindful of maintaining physiological levels as well as patterns of expression. In this regard "knock-in" gene targeting interventions would be the ideal way to proceed.
Careful attention to such details should enable the eventual "construction" of human tumor cells from normal cells [4] by recreating a clinically relevant series of genetic events. Multiple genetic interventions, either to reconstruct or deconstruct a cancer cell, would also be desirable in studies aimed at discovering drugs that impair one or more precursor cell types without affecting the ultimate normal precursor. Although this is conceptually similar to current methods of screening tumor versus normal cells, it is to be expected that complete genetic control over the evolution of a cancer cell would lead to significantly more sophisticated drug screens to target the individual steps.
Although in this study only a single genetic locus was chosen for modification, the cells eventually subjected to the drug screen might not have been truly isogenic. This is because of the well-known genetic instability of many human cell lines and the extensive scale-up inherent in a large drug screen. In cases where the introduced genetic change elicits growth impairment, genetic (as well as epigenetic) plasticity is likely to be a significant problem. We should be reminded that even a subtle phenotype could lead to adaptation during extended culture.
Torrance et al. wisely chose a genetic intervention without an apparent growth phenotype but this might not be possible in many cases of interest to cancer biologists. A significant differential in growth, even if genetically stable, would limit the period of co-culture and thus further complicate the analysis. In such cases it might be necessary to use growth conditions, treatments, or further genetic interventions to equalize the growth of the two cell lines.
Drug discovery by rational design based on structural considerations of known - or suspected - targets (the "bottom-up" approach) has not yet yielded its promised fruits. Likewise, "rational drug screening" based on superior genetic manipulation of the cellular substrates is likely to be complicated by the complexities of signal transduction pathways. In the Torrance et al. study, the analysis of several known effectors in the known Ras pathways failed to reveal the direct target of the discovered drug, which remains elusive. I am sure that there are many people of the opinion that the conventional screening regimens in current use have given us important discoveries and are unlikely to become obsolete in the near future. Although obviously correct, I for one would welcome a shift to a more genetically defined and thus rational approach to the problem. This study represents a key proof-of-principle and thus an important early step into the future of rational screening.
Novel Anticancer Drug Discovery - reviews recent advances. From Current Opinion in Chemical Biology, 1999, 3:500-509. Full text available from BioMedNet.
Scoring a Bull's-eye Against Cancer Genome Targets - reviews advances in new drugs acting on a range of new genome-based molecular targets. From Current Opinion in Pharmacology, 2001, 1:4:342-352. Full text available from BioMedNet.
Pharmacogenetic Determinants of Anti-cancer Drug Activity and Toxicity - focuses on the application of pharmacogenetics in the characterization of differences in the pharmacokinetics and pharmacodynamics of anti-cancer agents among individuals. From Trends in Pharmacological Sciences, 2001, 22:8:420-426. Full text available from BioMedNet.
National Cancer Institute - a prime jumping-off point for information. NCI also runs CancerNet, a site updated monthly that provides information geared to three categories: patients and the public; health professionals; and basic researchers.
OncoLink - a sprawling multimedia site. Maintained by the University of Pennsylvania Cancer Center.
Cancer Genome Anatomy Project - provides information on genes expressed during the development of human tumors and on determinants of cancer pathways in mice. It includes a cDNA Library Browser, a Tumor Suppressor and Oncogene Directory, and much more.
Cancer Related Links - a comprehensive list of cancer-related Web sites. Covers educational institutions, government servers, and more.
CancerWEB - information on cancer investigation and treatment. Includes links to resources that are organized for patients and their families, health-care professionals, or scientific researchers.
Medscape: Hematology-Oncology - information for health-care professionals. Provides conference summaries, news, patient resources, and much more.
Oncology Tools - cancer-related information from the U.S. Food and Drug Administration. Contains information on approved cancer-drug therapies, a patient-liaison program, regulatory tools, and other resources.
TeleSCAN - the European Internet service for cancer. Supplies links to information resources and services for patients, medical professionals, and cancer researchers.
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