Feb. 27, 2003 COLUMBUS, Ohio – Scientists have discovered that the genetic mutation that causes the childhood cancer retinoblastoma routinely triggers fetal death and miscarriage in laboratory animals by disrupting the normal functions of the placenta, a finding that may force researchers to reevaluate the powerful Rb gene and the role it plays in causing cancer.
“This is one of those discoveries where our team was in the right place at the right time and we ran with a hunch,” says Gustavo Leone, a geneticist at The Ohio State University Comprehensive Cancer Center-Arthur G. James Cancer Hospital and Richard J. Solove Research Institute and senior author of the study.
Twenty years ago, the Rb gene was the first to be identified as a tumor suppressor gene – meaning when it functions normally, it is able to keep cells from growing out of control. Scientists believe it is a critical player in the cell cycle, turning “on” and “off” at key stages in cell division. It is also believed to be linked to two key processes that frequently malfunction when cancer begins – proliferation (cell growth), and apoptosis (cell death).
“There is some sort of problem in the Rb pathway in virtually every cancer we know,” says Leone.
Although Rb mutation is most readily linked with retinoblastoma, Leone and his colleagues had been following its impact in laboratory animals. Scientists have known for several years that mice with two defective Rb genes invariably develop massive neural and structural problems and die half way through their gestational period. But the question has always remained: why those particular defects? What was going on?
As it turns out, the answer eluded them because they had simply been looking in the wrong place.
Writing in the February 27 issue of Nature, Leone and colleagues from the United States and Canada demonstrated that fetal death is actually due to mutation-induced structural changes in the placenta caused by the gene mutation.
“The evidence lay right in front of us the whole time, but it was a matter of shifting focus,” says Leone.
The discovery can be traced back to a casual but critical observation by a member of the research team that had been working with mice genetically engineered to lack a protein called E2F3, a key link in the downstream Rb pathway. Alain de Bruin, a veterinary pathologist working in Leone’s laboratory, noticed that the tiny mouse placentas with the E2F3 deficiency looked different. Rather than dismiss it, he shared his observation with Leone.
“ It would have been so easy to ignore something that you don’t know very much about,” says Leone, “but I thought the consequences could be significant.”
Leone had been studying the Rb pathway for some time, and knew that deletion of E2F3 as well as two of the proteins that direct Rb function ( p57 and p27) both led to cell proliferation and defects in the placenta. He wondered if the absence of Rb itself would lead to the same thing.
To test their hypothesis, they compared the placentas of normal mice with those carrying Rb mutation. Normal placentas are composed of three distinct layers: the labyrinth, spongiotrophoblast and trophoblast giant cell layers. While all are important in terms of fetal development, the labyrinth may be the most critical; that is the place where the most intimate exchange of nutrients, oxygen and waste takes place between the fetus and the mother.
They found that in normal placentas, the labyrinth is a neat and well-ordered environment, with maternal blood sinusoids and trophoblasts evenly dispersed among fetal blood cells. In the Rb defective placentas, however, the trophoblasts had grown wildly out of control, clumping together and disrupting the smooth and even matrix of fetal and maternal cells necessary for proper embryonic growth and development (see illustration).
Further tests revealed more discrete differences between the two sets of mice. In the Rb deficient group, proliferation of the trophoblasts reduced by almost 40 percent the area of nutrient transfer surface between mother and fetus, a finding reinforced by tests that showed a marked decrease in the amount of essential fatty acids in the mutated mice compared with the normal ones. The fatty acids – essential for growth – cannot be synthesized by the fetus and must be provided by the mother through the placenta.
“ It was pretty clear that the lack of Rb had crippled normal placental function,” says Leone. “The embryos died from anemia and massive cell death in the nervous system around two weeks into their gestational period. What we needed to do at that point was to prove that it was due to placental malfunctioning. In other words, could we create an Rb mutated mouse with a normal placenta?”
It turns out they could.
Through two separate and very different experimental approaches, they demonstrated that Rb deficient embryos with normal placentas were able to be carried to term and be born alive. In addition, those embryos lacked many of the neurological or developmental problems associated with Rb mutation. These “rescued” mice had been largely cured of anemia and showed no abnormal cell death in the brain.
Through embryonic manipulation, Michael Robinson, a member of the OSUCCC as well as a professor in the department of pediatrics at Ohio State, created a tetraploid embryo – one which had twice the number of normal chromosomes – with with normal Rb function that, when aggregated with an Rb mutant diploid embryo, generated a fetus that was Rb deficient but supplied by a normal placenta (see graphic).
“ These animals were able to be brought to term, because the normal placenta –with normal Rb functioning - helped them grow properly,” says Robinson. “This experiment was the first that gave us evidence that embryos lacking a functional Rb gene can be brought to birth if given a normal placenta.”
The research team came to the same conclusion in another experiment where they were able to isolate the Rb-placental-defect relationship through genetic engineering.
Still, even though the researchers were able to successfully rescue the mutated mice, Leone points out that all the mice brought to term died shortly after birth because developmental defects in their skeletal muscle and nervous systems prevented them from breathing properly.
“ This leads us to conclude that there are some very complex relationships between mutation and growth that we still need to explore,” says Leone. “It may be that Rb is not the mastermind of all of the functions we originally thought. I believe we may have uncovered strong evidence to suggest that Rb function is critical to the proliferative process, but may be more loosely aligned with apoptosis, or cell death. We may want to re-examine how Rb works in other areas. What we have learned is that in cancer, as in many areas of our lives, while we have to work with very tiny structures, we cannot forget the big picture.”
The research included work from Ohio State collaborators Lizhao Wu, Harold Saavedra, Anthony Trimboli, Jana Opavska, Pamela Wilson, Michael Ostrowski, Thomas Rosol and Michael Weinstein, as well as support from faculty at the University of Cincinnati and the University of Calgary.
The project was funded by grants from the National Cancer Institute, the National Institutes of Health, the National Center for Research Resources and the Canadian Institutes of Health Research.
The Ohio State University Comprehensive Cancer Center (OSUCCC) is a network of interdisciplinary research programs comprising over 200 investigators in 12 colleges and the Arthur G. James Cancer Hospital and Richard J. Solove Research Institute on the Ohio State campus. OSUCCC members conduct research on the prevention, detection, diagnosis and treatment of cancer, generating over $75 million annually in external funding.
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