"two-hit" hypothesis, carcinogenesis, heterozygosity, Knudson, proto-oncogenes, RB1, restriction fragment length polymorphism RFLP, retinoblastoma, tumor suppressor genes TSG
The hypothesis: that cancer is the result of accumulated mutations to a cell´s DNA. It was first proposed by Carl O. Nordling in 1953, and later formulated by Alfred G. Knudson in 1971.
The multi-mutation theory on cancer was proposed by Nordling in the British Journal of Cancer in 1953: “the outbreak of cancer requires the accumulations of six consecutive mutations”. Knudson performed a statistical analysis on cases of retinoblastoma, a tumor of the retina that occurs both as an inherited disease and sporadically.
Knudson suggested that multiple “hits” to DNA were necessary to cause cancer. In the children with inherited retinoblastoma, the first insult was inherited in the DNA, and any second insult would rapidly lead to cancer. In non-inherited retinoblastoma, two “hits” had to take place before a tumor could develop, explaining the age difference.
It was later found that:
- carcinogenesis (development of cancer)
- depended both on the activation of proto-oncogenes (genes that stimulate cell proliferation) and on the desactivation of tumor suppressor genes (TSG), which are genes that keep proliferation in check.
- Knudson´s hypothesis refers specifically, however, to the heterozygosity of tumor suppressor genes. A mutation in both alleles is required, as a single functional TSG is usually sufficient.
Tumor suppressor genes act as “brakes” to stop cells before they can travel down the road to cancer. A loss of function mutation in these genes can be disastrous. Some of these genes are involved in DNA repair processes, which help prevent the accumulation of mutations in cancer-related genes. Tumor suppressor genes act as “brakes” to stop cells in their tracks before they can take the road to cancer. Given this situation, loss of tumor suppressor gene function can be disastrous, and it often puts once-normal cells on the fast track to disease.
Knudson hypothesis (The Two-Hit Hypothesis or Multiple-hit hypothesis)
It was first proposed by geneticist Alfred Knudson in 1971. The two-hit hypothesis arose of out Knudson´s interest in the genetic mechanisms underlying retinoblastoma, a childhood form of retinal cancer.
Knudson studied 48 patients with retinoblastoma who had been admitted to the hospital between 1944 and 1969. Suggested that:
- An individual could inherit a germ-line mutation but not have disease
- While the majority of children with an affected parent had bilateral tumors (25-30%) some had only unilateral tumors (10-15%).
Furthermore, he determined that approximately 60% of retinoblastoma cases in the U.S. were unilateral and were not associated with a family history of the disease.
HEREDITARY: Bilateral (25-30%), Unilateral (10-15%) : 35-45%
NONHEREDITARY: Unilateral (55 -65%): 55-65%
Knudson examined the age at which the children in these two groups were diagnosed with retinoblastoma: without an inherited mutation, the same cell would need to accumulate two mutations- one in each allele of the gene-and this process would be much slower.
The rate of diagnosis for unilateral nonhereditary retinoblastoma was delayed relative to that bilateral cases and showed a curve consistent with a two-mutation process.
Knudson concluded that retinoblastoma was caused by two mutations: one in each copy of a single tumor suppressor gene (RB1). He also estimated that each of the two mutations would occur at a rate of 2 x 10 -7 per year. Patients who inherited an RB1 mutation would develop tumors earlier, inherit a mutation would almost always be affected by a single tumor. This statement, which Knudson called the two-mutation hypothesis, is now known as the two-hit hypothesis.
“Loss of heterozygosity” is often used to describe the process that leads to the inactivation of the second copy of a tumor suppressor gene. A heterozygous cell receives a second hit in its remaining functional copy of the tumor suppressor gene, thereby becoming homozygous for the mutated gene. Mutations that inactivate tumor suppressor genes, called loss-of-function mutations, are often point mutations or small deletions that disrupt the function of the protein that is encoded by the gene; chromosomal deletions or breaks that delete the tumor suppressor gene; or instances of somatic recombination during which the normal gene copy is replaced with a mutant copy.
Knudson developed the two-hit hypothesis long before the human genome was sequenced, and the RB1 gene was itself discovered in 1986. Researches notice that: some cases of retinoblastoma were associated with a deletion of chromosome band 13q14 and then used restriction fragment length polymorphism (RFLP) analysis to isolate the RB1 gene (Friend et al 1986).
RB1 function has been shown to be inactivated by four distinct mechanisms:
- Genetic inactivation
- Sequestration of the RB1 –encoded protein by viral oncoproteins
- Phosphorylation of the RB1 –encoded protein
- And degradation of the RB1 –encoded protein
RB1 is one gene among a growing list of tumor suppressor genes. According to the American Cancer Society (2005): at least 30 different tumor suppressor genes have been identified:
- RB1 Retinoblastoma: cell division, DNA replication, cell death
- TP53 Li-Fraumeni syndrome (brain tumors, sarcomas, leukemia):cell division, DNA repair, cell death
- CDKN2A Melanoma: cell division, cell death
- APC Colorectal cancer (due to familial polyposis): cell division, DNA damage, cell migration, cell adhesion, cell death.
- MLH1, MSH2, MSH6 Colorectal cancer (without polyposis): DNA mismatch repair, cell cycle regulation
- BRCA1, BRCA2 Breast and/or ovarian cancer: Repair of double-stranded DNA breaks, cell division, cell death
- WT1, WT2 Wilm´s tumor: Cell division, transcriptional regulation
- NF1, NF2 Nerve tumors (including brain): RAS-mediated signal transduction, cell differentiation, cell division, developmental processes.
- VHL Kidney cancer: Cell division, cell death, cell differentiation, response to cell stress.
Alfred George Knudson, Jr (1922-2016) was an American physician and geneticist specializing in cancer genetics. Knudson was born in Los Angeles, California in 1922, his M.D. from Columbia University in 1947 and his Ph.D. from California Institute of Technology in 1956. From 1970 to 1976, Knudson served as the Dean of Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston in the Texas Medical Center. He has been affiliated with the Fox Chase Cancer Center in Philadelphia from 1976 until his death in 2016.
Knudson is best known for his “two-hit hypothesis” explaining the incidence of hereditary cancers, such as retinoblastoma.
Our increasing knowledge of tumor suppressor gene function will continue to enhance our ability to diagnose and more effectively treat cancers at the molecular level in the years to come.
- American Cancer Society 2005
- British Journal of Cancer 1953 7, 68–72. doi:10.1038/bjc.1953.8 http://www.bjcancer. com … A New Theory on the Cancer-inducing Mechanism. C O Nordling
- Nature Publishing Group Knudson, A. “Two genetic hits (more or less) to cancer. Nature Reviews Cancer 1, 160 – 2001.
Tumor Suppressor Genes (Retinoblastoma and the two hit hypothesis, p
Dr. Al Knudson discusses the «Two-Hit» Theory