Discovery of a Gene That Suppresses Retinoblastoma

Date October, 1986

When Thaddeus P. Dryja and his associates identified and isolated the retinoblastoma gene, their work stimulated renewed interest in retinoblastoma research.

Locale Boston, Massachusetts

Key Figures

• Thaddeus P. Dryja (b. 1940), American medical scientist
• Alfred G. Knudson, Jr. (b. 1922), American geneticist

Summary of Event

Retinoblastoma, a malignant tumor of the eye that arises from the immature retina, occurs in one in fifteen thousand to one in thirty thousand live births and represents approximately 2 percent of childhood malignancies. The disease may be inherited or the result of a new germinal mutation. About 10 percent of patients have a family history of retinoblastoma and another 30 percent have bilateral disease. All of these (that is, 40 percent of patients) will pass the trait to their children as an autosomal dominant. The remaining 60 percent of patients have unilateral and nonheritable disease. A small portion of the cases have a deletion involving chromosome 13q14, but all heritable cases carry a mutant gene.

In 1971, Alfred G. Knudson, Jr., developed a model explaining how both genetics and environment may cause retinoblastoma and Wilms’ tumor, another rare childhood cancer. Subsequent studies verified Knudson’s theory that there exist certain anticancer genes, or antioncogenes, that protect against disease. (Genes that play a role in the development of cancer are called oncogenes.) The lack of, or destruction or damage of, antioncogenes can cause cancers such as retinoblastoma. Knudson also examined why some families seem to be prone to cancers and worked to identify genetic factors that may relate to other forms of cancer.

In December, 1988, it was reported that researchers had for the first time succeeded in causing cancer cells growing in the laboratory to revert to normal by replacing a defective gene with a healthy one. The gene involved was named Rb. When it is defective, retinoblastoma can develop. The Rb gene, discovered in October, 1986, by researchers at the Massachusetts Eye and Ear Infirmary in Boston, was the first of the anticancer genes to be found. Antioncogenes apparently protect against cancer by preventing adult cells from proliferating.

Normally, an individual receives two Rb genes at conception, one from the father and one from the mother. If both are healthy, a tumor can arise later only if both genes in one cell are disabled by a chemical or a virus. If a child inherits one defective Rb gene, however, only the one healthy gene must be disabled for cancer to occur. Many children who inherit a defective Rb gene suffer from multiple eye tumors.

The isolation of the Rb gene by Thaddeus P. Dryja and his colleagues required the creation of a lambda phage library that contained inserted fragments from human chromosome 13. One of the inserts detected a corresponding fragment that was deleted in two of thirty-seven retinoblastoma tumor DNAs (deoxyribonucleic acid). This suggested that the probed segment was linked to the Rb genes. A nearby probe detected not only the human sequence but also a mouse homologue to a somatic cell hybrid carrying human chromosome 13. The latter probe was used for RNA (ribonucleic acid) analysis to detect any transcripts in a retinal cell line. The analysis showed a 4.7-kilobase transcript in the tumor cell line but not in several retinoblastomas. The latter probe also was used to analyze DNA from a large group of retinoblastomas and osteosarcomas (secondary tumors of mesenchymal origin) and found gross changes in genetic structure in approximately 30 percent of the tumors’ DNAs. The researchers mapped the boundaries of homozygously deleted fragments. In their analysis of an osteosarcoma and a retinoblastoma, they discovered that the end points of the deletion were within the confines of the genetic unit defined by the probe. This indicated that the target of inactivation was the segment under study and not a neighboring DNA sequence.

Three groups of researchers reported in mid-1986 that a defective Rb gene also is present in cells from about 20 percent of breast tumors, more than half of small-cell cancers, and most bone tumors. The successful conversion of cancerous cells into normal ones was carried out by scientists at the University of California at San Diego. They used a specially engineered virus to insert the healthy Rb gene into retinoblastoma and bone cancer cells. Once the gene had been inserted into the cells, the cells immediately stopped proliferating. When the engineered cells were injected into special laboratory mice that had no immune systems, no tumors formed, indicating that the cells were no longer malignant. The researchers noted that the technique might eventually be useful in treating genetically linked conditions such as cancers and Alzheimer’s disease.

Significance

Dryja’s research had an almost immediate impact on retinoblastoma research in general, reinvigorating this area of study. In 1986, Emil Bogen Mann studied retinoblastoma cell differentiation in culture. As little was known about the biology of retinoblastoma in vitro because of the lack of adequate culture systems, and only a few retinoblastoma cell lines had been established in the past, Mann developed a culture system using rat smooth muscle cells as feeder layers that allowed for routine growth of primary retinoblastomas and/or their metastases. These cells were routinely grown up to passages and, in some cultures, spontaneous formulation of Flexner-Wintersteiner rosettes was observed.

In April, 1986, Jack Rootman and his colleagues studied the ocular penetration, toxicity, and radiosensitizing properties of two new nitroimidazoles. To determine the highest level of the drug that is relatively nontoxic for subconjunctival administration, the investigators assessed the effect of varying concentrations of both nitroimidazoles. A dose of 100 milligrams of each drug in 0.5 milliliter of normal saline was the maximum acceptable toxicity as determined by these studies. There was no significant difference in response to injection of either drug. High-pressure liquid chromatographic analysis was performed on test samples from plasma, urine, and ocular compartments. Chinese hamster ovary cells were irradiated (X rays) at a dose of 150 rads per minute. After radiation, cells were washed free of the drug and plated. After seven days of incubation, colonies were stained and scored. Colonies containing fifty cells or more were scored as survivors.

In May, 1986, Yenyun Wang and associates compared diploid fibroblast cell lines derived from two hereditary retinoblastoma patients with those of three normal persons of comparable ages for their sensitivity to ionizing radiation, induced transformation to anchorage independence. The target cells were exposed to cobalt 60, allowed to undergo an expression period, and assayed for ability to form colonies in 0.33 percent agar. There was no detectable difference between the Rb cells and the normal cells in response to the transforming action of cobalt 60. Concomitantly, the gene for esterase D (ESD) is known to be tightly linked to the retinoblastoma locus (Rbl) in the q14.l band of chromosome 13. Jeremy Squire and colleagues were able to clone the ESD gene from a human cDNA library by using oligonucleotides specific for a partial amino acid sequence of the purified enzyme to provide a genetic marker for further studies on retinoblastoma. The putative ESD gene coded for a message of 1.2 kilobases, which was present on all cell types examined and mapped to 13q14.1, thus confirming that it was the ESD gene.

In October, 1987, Wen-hwa Lee discovered that a null allele of esterase D was a marker for genetic events in retinoblastoma formation. Using a rabbit anti-esterase D antibody and the esterase D cDNA probe, Lee and associates found that low but detectable quantities of esterase D protein and enzymatic activity were present in tumor cells from a patient with bilateral retinoblastoma; fibroblasts from this patient contained two copies of the esterase D gene, indicated by heterozygosity at a polymorphic site within this gene; and tumor cells from the same patient were homozygous at this site, indicating loss and reduplication of the esterase D locus. The results demonstrated that one of the two esterase D alleles in this patient acted as a “null,” or silent, allele.

Since Dryja’s work in 1986, numerous studies on genetic, cytogenetic, and molecular genetic approaches have improved the understanding of the biological events that lead to the occurrence of retinoblastoma and have provided tools for the enhanced assessment of risk for some individuals. In the case of retinoblastoma, gene probes have even been found to identify potentially affected newborns.

Bibliography

Buchanan, Janet, and Cavenee Webster. “Genetic Markers for Assessment of Retinoblastoma Predisposition.” Disease Markers 5 (February, 1987): 141-152. Review article provides a conceptual framework for the application of current approaches to the analysis of retinoblastoma in the clinical setting. Discusses the progress made in elucidating the basis of retinoblastoma.

Cowell, John, and Jon Pritchard. “The Molecular Genetics of Retinoblastoma and Wilms’ Tumor.” CRC Critical Reviews in Oncology/Hematology 7, no. 2 (1987): 153-168. Presents a description of cancers in adults and a discussion of probable causes of certain types of cancer, such as exposure to environmental carcinogens.

Fanciulli, Maurizio, ed. Rb and Tumorigenesis. New York: Springer, 2006. Nine chapters by geneticists and molecular biologists address how the tumor-suppressing gene and its encoding protein work.

Singh, Arun D., ed. Ophthalmic Oncology. Philadelphia: W. B. Saunders, 2005. Presents a thorough, technical overview of all eye cancers, including retinoblastoma.

Watson, James. Molecular Biology of the Gene. 5th ed. San Francisco: Pearson/Benjamin Cummings, 2004. Designed to give interested readers the rigor and the perspective needed to bridge the gap between an understanding of the single cell and an understanding of the complexities of higher organisms. Presents fundamentals of biochemistry, molecular genetics, and cytology.

Winter, Jens. “In Vitro and In Vivo Growth of an Intraocular Retinoblastoma-like Tumour in F-344 Rats.” Acta Ophthalmologia 64 (April, 1986): 657-663. Informative article describes the growth of a transplantable retinoblastoma-like tumor in the eyes of rats and in cell cultures.