21. Along with a protein-encoding region, genes include regulatory sequences called promoters and enhancers that control where and when that gene is expressed. Promoters of genes that encode proteins are typically located immediately “upstream” on the DNA. An enhancer activates the use of a promoter, thereby controlling the rate of gene expression. Most genes require enhancers to be expressed. Enhancers have been called “the major determinant of differential transcription in space (cell type) and time”; and any given gene can have several different enhancer sites linked to it (S. F. Gilbert, Developmental Biology, 6th ed. [Sunderland, Mass.: Sinauer Associates, 2000]; available online at www.ncbi.nlm. nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=.0BpKYEB-SPfx18nm8Q OxH).
By binding to enhancer or promoter regions, transcription factors start or repress the expression of a gene. New knowledge of transcription factors has transformed our understanding of gene expression. Per Gilbert in the chapter “The Genetic Core of Development: Differential Gene Expression”: “The gene itself is no longer seen as an independent entity controlling the synthesis of proteins. Rather, the gene both directs and is directed by protein synthesis. Natalie Anger (1992) has written, ‘A series of discoveries suggests that DNA is more like a certain type of politician, surrounded by a flock of protein handlers and advisors that must vigorously massage it, twist it and, on occasion, reinvent it before the grand blueprint of the body can make any sense at all.’ ”
22. Bob Holmes, “Gene Therapy May Switch Off Huntington’s,” March 13, 2003, http://www.newscientist.com/news/news.jsp?id=ns99993493. “Emerging as a powerful tool for reverse genetic analysis, RNAi is rapidly being applied to study the function of many genes associated with human disease, in particular those associated with oncogenesis and infectious disease.” J. C. Cheng, T. B. Moore, and K. M. Sakamoto, “RNA Interference and Human Disease,” Molecular Genetics and Metabolism 80.1–2 (October 2003): 121–28. RNAi is a “potent and highly sequence-specific mechanism.” L. Zhang, D. K. Fogg, and D. M. Waisman, “RNA Interference-Mediated Silencing of the S100A10 Gene Attenuates Plasmin Generation and Invasiveness of Colo 222 Colorectal Cancer Cells,” Journal of Biological Chemistry 279.3 (January 16, 2004): 2053–62.
23. Each chip contains synthetic oligonucleotides that replicate sequences that identify specific genes. “To determine which genes have been expressed in a sample, researchers isolate messenger RNA from test samples, convert it to complementary DNA (cDNA), tag it with fluorescent dye, and run the sample over the wafer. Each tagged cDNA will stick to an oligo with a matching sequence, lighting up a spot on the wafer where the sequence is known. An automated scanner then determines which oligos have bound, and hence which genes were expressed. . . .” E. Marshall, “Do-It-Yourself Gene Watching,” Science 286.5439 (October 15, 1999): 444–47.
24. Ibid.
25. J. Rosamond and A. Allsop, “Harnessing the Power of the Genome in the Search for New Antibiotics,” Science 287.5460 (March 17, 2000): 1973–76.
26. T. R. Golub et al., “Molecular Classification of Cancer: Class Discovery and Class Prediction by Gene Expression Monitoring,” Science 286.5439 (October 15, 1999): 531–37.
27. Ibid., as reported in A. Berns, “Cancer: Gene Expression in Diagnosis,” Nature 403 (February 3, 2000): 491–92. In another study, 1 percent of the genes studied showed reduced expression in aged muscles. These genes produced proteins associated with energy production and cell building, so a reduction makes sense given the weakening associated with age. Genes with increased expression produced stress proteins, which are used to repair damaged DNA or proteins. J. Marx, “Chipping Away at the Causes of Aging,” Science 287.5462 (March 31, 2000): 2390.
As another example, liver metastases are a common cause of colorectal cancer. These metastases respond differently to treatment depending on their genetic profile. Expression profiling is an excellent way to determine an appropriate mode of treatment. J. C. Sung et al., “Genetic Heterogeneity of Colorectal Cancer Liver Metastases,” Journal of Surgical Research 114.2 (October 2003): 251.
As a final example, researchers have had difficulty analyzing the Reed-Sternberg cell of Hodgkin’s disease because of its extreme rarity in diseased tissue. Expression profiling is now providing a clue regarding the heritage of this cell. J. Cossman et al., “Reed-Sternberg Cell Genome Expression Supports a B-Cell Lineage,” Blood 94.2 (July 15, 1999): 411–16.
28. T. Ueland et al., “Growth Hormone Substitution Increases Gene Expression of Members of the IGF Family in Cortical Bone from Women with Adult Onset Growth Hormone Deficiency—Relationship with Bone Turn-Over,” Bone 33.4 (October 2003): 638–45.
29. R. Lovett, “Toxicologists Brace for Genomics Revolution,” Science 289.5479 (July 28, 2000): 536–37.
30. Gene transfer to somatic cells affects a subset of cells in the body for a period of time. It is theoretically possible also to alter genetic information in egg and sperm (germ-line) cells, for the purpose of passing on those changes to the next generations. Such therapy poses many ethical concerns and has not yet been attempted. “Gene Therapy,” Wikipedia, http://en.wikipedia.org/wiki/Gene_therapy.
31. Genes encode proteins, which perform vital functions in the human body. Abnormal or mutated genes encode proteins that are unable to perform those functions, resulting in genetic disorders and diseases. The goal of gene therapy is to replace the defective genes so that normal proteins are produced. This can be done in a number of ways, but the most typical way is to insert a therapeutic replacement gene into the patient’s target cells using a carrier molecule called a vector. “Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove the disease-causing genes and insert therapeutic genes” (Human Genome Project, “Gene Therapy,” http://www.ornl.gov/TechResources/Human_Genome/medicine/gene therapy.html). See the Human Genome Project site for more information about gene therapy and links. Gene therapy is an important enough area of research that there are currently six scientific peer-reviewed gene-therapy journals and four professional associations dedicated to this topic.
32. K. R. Smith, “Gene Transfer in Higher Animals: Theoretical Considerations and Key Concepts,” Journal of Biotechnology 99.1 (October 9, 2002): 1–22.
33. Anil Ananthaswamy, “Undercover Genes Slip into the Brain,” March 20, 2003, http://www.newscientist.com/news/news.jsp?id=ns99993520.
34. A. E. Trezise et al., “In Vivo Gene Expression: DNA Electrotransfer,” Current Opinion in Molecular Therapeutics 5.4 (August 2003): 397–404.
35. Sylvia Westphal, “DNA Nanoballs Boost Gene Therapy,” May 12, 2002, http://www.newscientist.com/news/news.jsp?id=ns99992257.
36. L. Wu, M. Johnson, and M. Sato, “Transcriptionally Targeted Gene Therapy to Detect and Treat Cancer,” Trends in Molecular Medicine 9.10 (October 2003): 421–29.
37. S. Westphal, “Virus Synthesized in a Fortnight,” November 14, 2003, http://www. newscientist.com/news/news.jsp?id=ns99994383.
38. G. Chiesa, “Recombinant Apolipoprotein A-I(Milano) Infusion into Rabbit Carotid Artery Rapidly Removes Lipid from Fatty Streaks,” Circulation Research 90.9 (May 17, 2002): 974–80; P. K. Shah et al., “High-Dose Recombinant Apolipoprotein A-I(Milano) Mobilizes Tissue Cholesterol and Rapidly Reduces Plaque Lipid and Macrophage Content in Apolipoprotein e-Deficient Mice,” Circulation 103.25 (June 26, 2001): 3047–50.
39. S. E. Nissen et al., “Effect of Recombinant Apo A-I Milano on Coronary Atherosclerosis in Patients with Acute Coronary Syndromes: A Randomized Controlled Trial,” JAMA 290.17 (November 5, 2003): 2292–2300.
40. A recent phase 2 study reported “markedly increased HDL cholesterol levels and also decreased LDL cholesterol levels.” M. E. Brousseau et al., “Effects of an Inhibitor of Cholesteryl Ester Transfer Protein on HDL Cholesterol,??
? New England Journal of Medicine 350.15 (April 8, 2004): 1505–15, http://content.nejm. org/cgi/content/abstract/350/15/1505. Global phase 3 trials began in late 2003. Information on Torcetrapib is available on the Pfizer site: http://www.pfizer.com/are/investors_reports/annual_2003/
review/p2003ar14_15.htm.
41. O. J. Finn, “Cancer Vaccines: Between the Idea and the Reality,” Nature Reviews: Immunology 3.8 (August 2003): 630–41; R. C. Kennedy and M. H. Shearer, “A Role for Antibodies in Tumor Immunity,” International Reviews of Immunology 22.2 (March–April 2003): 141–72.
42. T. F. Greten and E. M. Jaffee, “Cancer Vaccines,” Journal of Clinical Oncology 17.3 (March 1999): 1047–60.
43. “Cancer ‘Vaccine’ Results Encouraging,” BBCNews, January 8, 2001, http://news. bbc.co.uk/2/hi/health/1102618.stm, reporting on research by E. M. Jaffee et al., “Novel Allogeneic Granulocyte-Macrophage Colony-Stimulating Factor-Secreting Tumor Vaccine for Pancreatic Cancer: A Phase I Trial of Safety and Immune Activation,” Journal of Clinical Oncology 19.1 (January 1, 2001): 145–56.
44. John Travis, “Fused Cells Hold Promise of Cancer Vaccines,” March 4, 2000, http://www.sciencenews.org/articles/20000304/fob3.asp, referring to D. W. Kufe, “Smallpox, Polio and Now a Cancer Vaccine?” Nature Medicine 6 (March 2000): 252–53.
45. J. D. Lewis, B. D. Reilly, and R. K. Bright, “Tumor-Associated Antigens: From Discovery to Immunity,” International Reviews of Immunology 22.2 (March–April 2003): 81–112.
46. T. Boehm et al., “Antiangiogenic Therapy of Experimental Cancer Does Not Induce Acquired Drug Resistance,” Nature 390.6658 (November 27, 1997): 404–7.
47. Angiogenesis Foundation, “Understanding Angiogenesis,” http://www.angio.org/understanding/content_understanding.html; L. K. Lassiter and M. A. Carducci, “Endothelin Receptor Antagonists in the Treatment of Prostate Cancer,” Seminars in Oncology 30.5 (October 2003): 678–88. For an explanation of the process, see the National Cancer Institute Web site, “Understanding Angiogenesis,” http:// press2.nci.nih.gov/sciencebehind/angiogenesis/angio02.htm.
48. I. B. Roninson, “Tumor Cell Senescence in Cancer Treatment,” Cancer Research 63.11 (June 1, 2003): 2705–15; B. R. Davies et al., “Immortalization of Human Ovarian Surface Epithelium with Telomerase and Temperature-Sensitive SV40 Large T Antigen,” Experimental Cell Research 288.2 (August 15, 2003): 390–402.
49. See also R. C. Woodruff and J. N. Thompson Jr., “The Role of Somatic and Germline Mutations in Aging and a Mutation Interaction Model of Aging,” Journal of Anti-Aging Medicine 6.1 (Spring 2003): 29–39. See also notes 18 and 19.
50. Aubrey D. N. J. de Grey, “The Reductive Hotspot Hypothesis of Mammalian Aging: Membrane Metabolism Magnifies Mutant Mitochondrial Mischief,” European Journal of Biochemistry 269.8 (April 2002): 2003–9; P. F. Chinnery et al., “Accumulation of Mitochondrial DNA Mutations in Ageing, Cancer, and Mitochondrial Disease: Is There a Common Mechanism?” Lancet 360.9342 (October 26, 2002): 1323–25; A. D. de Grey, “Mitochondrial Gene Therapy: An Arena for the Biomedical Use of Inteins,” Trends in Biotechnology 18.9 (September 2000): 394–99.
51. “The notion of ‘vaccinating’ individuals against a neurodegenerative disorder such as Alzheimer’s disease is a marked departure from classical thinking about mechanism and treatment, and yet therapeutic vaccines for both Alzheimer’s disease and multiple sclerosis have been validated in animal models and are in the clinic. Such approaches, however, have the potential to induce unwanted inflammatory responses as well as to provide benefit” (H. L. Weiner and D. J. Selkoe, “Inflammation and Therapeutic Vaccination in CNS Diseases,” Nature 420.6917 [December 19–26, 2002]: 879–84). These researchers showed that a vaccine in the form of nose drops could slow the brain deterioration of Alzheimer’s. H. L. Weiner et al.,“Nasal Administration of Amyloid-beta Peptide Decreases Cerebral Amyloid Burden in a Mouse Model of Alzheimer’s Disease,” Annals of Neurology 48.4 (October 2000): 567–79.
52. S. Vasan, P. Foiles, and H. Founds, “Therapeutic Potential of Breakers of Advanced Glycation End Product-Protein Crosslinks,” Archives of Biochemistry and Biophysics 419.1 (November 1, 2003): 89–96; D. A. Kass, “Getting Better Without AGE: New Insights into the Diabetic Heart,” Circulation Research 92.7 (April 18, 2003): 704–6.
53. S. Graham, “Methuselah Worm Remains Energetic for Life,” October 27, 2003, www.sciam.com/article.cfm?chanID=sa003&articleID=000C601F-8711-1F99-86FB83414B7F0156.
54. Ron Weiss’s home page at Princeton University (http://www.princeton.edu/~ rweiss) lists his publications, such as “Genetic Circuit Building Blocks for Cellular Computation, Communications, and Signal Processing,” Natural Computing, an International Journal 2.1 (January 2003): 47–84.
55. S. L. Garfinkel, “Biological Computing,” Technology Review (May–June 2000), http://static.highbeam.com/t/technologyreview/may012000/
biologicalcomputing.
56. Ibid. See also the list of current research on the MIT Media Lab Web site, http://www.media.mit.edu/research/index.html.
57. Here is one possible explanation: “In mammals, female embryos have two X-chromosomes and males have one. During early development in females, one of the X’s and most of its genes are normally silenced or inactivated. That way, the amount of gene expression in males and females is the same. But in cloned animals, one X-chromosome is already inactivated in the donated nucleus. It must be reprogrammed and then later inactivated again, which introduces the possibility of errors.” CBC News online staff, “Genetic Defects May Explain Cloning Failures,” May 27, 2002, http://www.cbc.ca/stories/2002/05/27/cloning_errors020527. That story reports on F. Xue et al., “Aberrant Patterns of X Chromosome Inactivation in Bovine Clones,” Nature Genetics 31.2 (June 2002): 216–20.
58. Rick Weiss, “Clone Defects Point to Need for 2 Genetic Parents,” Washington Post, May 10, 1999, http://www.gene.ch/genet/1999/Jun/msg00004.html.
59. A. Baguisi et al., “Production of Goats by Somatic Cell Nuclear Transfer,” Nature Biotechnology 5 (May 1999): 456–61. For more information on the partnership between Genzyme Transgenics Corporation, Louisiana State University, and Tufts University School ofMedicine that produced this work, see the April 27, 1999, press release, “Genzyme Transgenics Corporation Announces First Successful Cloning of Transgenic Goat,” http://www.transgenics.com/pressreleases/pr042799.html.
60. Luba Vangelova, “True or False? Extinction Is Forever,” Smithsonian, June 2003, http://www.smithsonianmag.com/smithsonian/issues03/jun03/
phenomena.html.
61. J. B. Gurdon and A. Colman, “The Future of Cloning,” Nature 402.6763 (December 16, 1999): 743–46; Gregory Stock and John Campbell, eds., Engineering the Human Germline: An Exploration of the Science and Ethics of Altering the Genes We Pass to Our Children (New York: Oxford University Press, 2000).
62. As the Scripps Research Institute points out, “The ability to dedifferentiate or reverse lineage-committed cells to multipotent progenitor cells might overcome many of the obstacles associated with using ESCs and adult stem cells in clinical applications (inefficient differentiation, rejection of allogenic cells, efficient isolation and expansion, etc.). With an efficient dedifferentiation process, it is conceivable that healthy, abundant and easily accessible adult cells could be used to generate different types of functional cells for the repair of damaged tissues and organs” (http://www.scripps.edu/chem/ding/sciences.htm).
The direct conversion of one differentiated cell type into another—a process referred to as transdifferentiation—would be beneficial for producing isogenic [patient’s own] cells to replace sick or damaged cells or tissue. Adult stem cells display a broader differentiation potential than anticipated and might contribute to tissues other than those in which they reside. As such, they could be worthy therapeutic agents. Recent advances in transdifferentiation involve nuclear transplantation, manipulation of cell culture conditions, induction of ectopic gene expression and uptake of molecules from cellular extracts. These appro
aches open the doors to new avenues for engineering isogenic replacement cells. To avoid unpredictable tissue transformation, nuclear reprogramming requires controlled and heritable epigenetic modifications. Considerable efforts remain to unravel the molecular processes underlying nuclear reprogramming and evaluate stability of the changes in reprogrammed cells.
Quoted from P. Collas and Anne-Mari Håkelien, “Teaching Cells New Tricks,” Trends in Biotechnology 21.8 (August 2003): 354–61; P. Collas, “Nuclear Reprogramming in Cell-Free Extracts,” Philosophical Transactions of the Royal Society of London, B 358.1436 (August 29, 2003): 1389–95.
63. Researchers have converted human liver cells to pancreas cells in the laboratory: Jonathan Slack et al., “Experimental Conversion of Liver to Pancreas,” Current Biology 13.2 (January 2003): 105–15. Researchers reprogrammed cells to behave like other cells using cell extracts; for example, skin cells were reprogrammed to exhibit T-cell characteristics. Anne-Mari Håkelien et al., “Reprogramming Fibro-blasts to Express T-Cell Functions Using Cell Extracts,” Nature Biotechnology 20.5 (May 2002): 460–66; Anne-Mari Håkelien and P. Collas, “Novel Approaches to Transdifferentiation,” Cloning Stem Cells 4.4 (2002): 379–87. See also David Tosh and Jonathan M. W. Slack, “How Cells Change Their Phenotype,” Nature Reviews Molecular Cell Biology 3.3 (March 2002): 187–94.
64. See the description of transcription factors in note 21, above.
65. R. P. Lanza et al., “Extension of Cell Life-Span and Telomere Length in Animals Cloned from Senescent Somatic Cells,” Science 288.5466 (April 28, 2000): 665–69. See also J. C. Ameisen, “On the Origin, Evolution, and Nature of Programmed Cell Death: A Timeline of Four Billion Years,” Cell Death and Differentiation 9.4 (April 2002): 367–93; Mary-Ellen Shay, “Transplantation Without a Donor,” Dream: The Magazine of Possibilities (Children’s Hospital, Boston), Fall 2001.