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2004 Kyoto Prize Laureates 
Basic Sciences Category
Prize Field: Life Sciences (Molecular Biology, Cell Biology, Neurobiology) "For his seminal contribution to the establishment of the theory of the tumor suppressor gene in the mechanism of human carcinogenesis." Dr. Alfred George Knudson, Jr. (U.S.A., b. 1922) Geneticist and Physician Senior Advisor, Fox Chase Cancer Center Commentary on Achievements 1.Formulation of the Two-Hit Theory (See Glossary for the definition of terms marked with an asterisk) In a 1971 paper (Proc. Natl. Acad. Sci. USA 68-4: 820-823), Dr. Knudson put forward a "two-hit" hypothesis in which he applied statistical analysis to a comparison of patients with the hereditary and nonhereditary forms of retinoblastoma*, a rare tumor of the eye. Based upon the clinical records of 48 retinoblastoma patients admitted to the M. D. Anderson Hospital between 1944 and 1969, he found that the hereditary form (often develop bilateral tumors), and non-hereditary form (usually unilateral) fit into statistical prediction of single and double hits, respectively. Dr. Knudson tabulated the 48 cases with respect to age at diagnosis, unilaterality or bilaterality, family history, the number of tumors, and the number of retina cells, and conducted a statistical analysis (Poisson distribution*) 
Fig. 1 Patients with the hereditary form, with tumors in both eyes, follow the one-hit curve, whereas patients with the nonhereditary form, with tumors in one eye, follow the two-hit curve. Two lines are from the theoretical prediction based on one/two-hit hypothesis, and both lines match quite well with the actual data of patients. (from Fig.1 of the 1971 paper)
Fig. 2 Diagram of the "Two-Hit" hypothesis: Patients with the hereditary form had already incurred a mutation in one of the alleles, and the cancer is caused when another mutational event occurs, whereas patients with the nonhereditary form develop cancer after two mutational events in the alleles of somatic cell genes.
Fig. 3 Differences in DNA sequence show up in RFLP as differences in the band positions. In case of heterozygous normal cells, two bands appear as above (suppose the alleles a &b); in cancer cells, however, sometimes only one band is shown (aa or bb), indicating either a or b has been lost.
using the incidence of retinoblastoma per population and mutation rates. The analysis showed that, in the hereditary form, the fraction of cases of retinoblastoma not yet diagnosed decreased exponentially with age in months, which supported the prediction that one mutational event causes tumors (Fig. 1). Whereas in the nonhereditary form, the fraction distributed in a manner that two mutational events were involved. These findings led Dr. Knudson to infer that patients with the hereditary form of retinoblastoma inherit one mutated retinoblastoma gene and the cancer is caused when an additional mutational event occurs in cells of the eye (somatic cells) after birth. In the case of patients with the nonhereditary form, in contrast, he explained that the first mutational event occurs not in germinal cells but in cells of the eye focally, and that the cells that incurred a first hit accumulate a second hit, later triggering a tumor (Fig. 2). Dr. Knudson also considered the genetic significance of "two-hit" intracellularly, and predicted the involvement of the two alleles of a tumor suppressing recessive gene in these mutational events causing retinoblastoma (1973). In 1982, he dubbed these genes anti-oncogenes*. Later, it was proven that the protein these genes encode suppresses transformation in normal cells, but that cancer develops when both of the alleles are mutated and their protein production stops. 2. The History of Cancer Genetics Knudson's "two-hit" hypothesis was put forward as early as 1971, before scientists even understood exactly what oncogenes* were. By that time, the existence of RNA tumor viruses had been confirmed in chickens and mice through previous research on sarcoma and leukemia. Some researchers had postulated that a specific type of viral genes caused tumors. Similarly, it had been demonstrated that such DNA viruses as SV40, polyoma virus and adenovirus cause cell transformation in the test tube. Accordingly, the existence of oncogenes was predicted, which was proven later with the use of advanced recombinant DNA technology and sequencing. However, dominant* oncogenes were the cause of tumors in RNA and DNA viruses. Knudson's paper shed light for the first time on recessive* genes as causing tumors - in this case anti-oncogenes. It was not until 1976 that the existence of cellular proto-oncogenes* was advocated (Nature 260: 170-173, 1976). Based on the discovery that an oncogene of an RNA tumor virus was in fact an altered form of a gene derived from the host cell genome, Varmus and Bishop received the Nobel Prize. Another line of approach was led by Stanbridge and colleagues, who discovered that, based on the findings of their own and of Harris et al. certain chromosomes of normal cells can tame cancer cells like normal cells when cancer cells are fused with normal cells (Nature 260: 17-20, 1976). One of these tumor suppressor genes was later found to be an anti-oncogene WT (Wilm's tumor gene) (Science 254: 293-295, 1991). Judging from the chronology of these events, however, this was obviously an extension of Dr. Knudson's two-hit hypothesis. In 1983, restriction fraction length polymorphism (RFLP) analysis of polygenes on chromosomes led to demonstration at the genetic level that one of the alleles on the thirteenth chromosome is deleted in retinoblastoma patients. Specifically, in a heterozygous (DNA sequence differs in the alleles) normal cell, the alleles appear on an electropherogram as two bands, while in a cancer cell, only one band is evident, one of the alleles having been lost (Loss of Heterozygosity: LOH)(Fig. 3). This indicated that a tumor suppressor gene is encoded in a fraction of the deleted chromosome, establishing a retinoblastoma susceptibility gene (RB1) as a tumor suppressor gene (Cavenee and White team, Nature 305: 779-784, 1983). Cloning of RB1 was reported in 1986 by Dryja and Weinberg team (Nature 323: 643-646, 1986). Thereafter, many scientists were to elucidate the deep involvement of this Rb protein in the regulation of the cell cycle when combined with E1A proteins and E2F transcription factors of adenovirus oncogenes. Glossary <Anti-oncogene and tumor suppressor gene> Suppresses excessive cell proliferation in normal cells by controlling the cell cycle, inducing apoptosis, but also causes normal cells to change into cancer cells through its own inactivation. Dr. Knudson dubbed these "anti-oncogenes" in 1983, but, to avoid any misapprehension that they oppose oncogenes, they are now called tumor suppressor genes after their function. In many cases, tumor suppressor genes are recessive in somatic cell genetics, but the cancer caused by their mutations is dominant as a disease. <Dominant gene and recessive gene> In the case of dominant genes like oncogenes, cancer develops when one of the alleles becomes activated. In the case of recessive genes like tumor suppressor genes, on the other land, cancer does not develop even when one of the alleles is inactivated, but cancer develops when the other allele is mutated and the protein production ceases. <Oncogene> Genes that cause normal cells to transform into cancer cells. Genetically, they are dominant over the normal (wild-type) alleles. <Poisson distribution analysis> A statistical method for analyzing independent phenomena with small probability. <Proto oncogene> Genes that don't cause cancer by themselves, but can be mutated into oncogenes when gene mutation leads to increased or unregulated activity. When incorporated into RNA tumor virus, or stimulated by chemical carcinogens or chromosomal abnormality as amplification or translocation, they become oncogenes. In normal cells, they are involved in cell growth and thus are necessary for growth of individuals. <Retinoblastoma> A rare cancer affecting one in 20,000 children, with the ratio of bilateral to unilateral forms at 1:3. Approximately 60% of all retinoblastoma patients have a tumor in only one eye (unilateral), have the nonhereditary form, and develop retinoblastoma due to a mutational event. When tumors of the hereditary form occur in both eyes (bilateral), it is estimated that about 50% of the patients' offspring will inherit the disease. The average age for the onset of retinoblastoma is about one or two years old. Usually, by the age of three or four years, most young patients consult with an ophthalmologist about leukocoria (the pupil reflects a white or yellow color) or strabismus when their parents find these symptoms. |
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