Scientists pinpoint key proteins in blood stem cell replication

A family of cancer-fighting molecules helps blood stem cells in mice decide when and how to divide, say medical school researchers. Blocking the molecules' function spurs the normally resting cells to begin proliferating strangely - making too much of one kind of cell and not enough of another. Many types of human blood cancers involve a similar disruption in the expression of that same family of molecules.

The blood stem cells' misguided enthusiasm also inhibits their ability to repopulate the immune system of a recipient animal after a bone marrow transplant - a common leukemia treatment.

The discovery is the first to directly link the notorious members of the retinoblastoma family of proteins to the cellular production factories responsible for churning out all the blood and immune cells in the body. 'This is an important step in understanding the initiation of human cancer at a cellular level,' said Patrick Viatour, PhD, a postdoctoral scholar who performed the research in the laboratory of Julien Sage, PhD.

Sage, assistant professor of pediatrics and of genetics, received a SEED grant from the California Institute of Regenerative Medicine to investigate how the retinoblastoma, or Rb, proteins affect human embryonic stem cells. Viatour is the first author of the research, published Oct. 9 in Cell Stem Cell.

'These studies, and additional experiments from our lab in other tissues and organs, indicate that Rb proteins play a critical role in suppressing tumors originating in adult stem cells populations,' said Sage, who is also a member of the Stanford Cancer Center.

The first retinoblastoma protein, pRb, was identified through studies of retinal cancer arising in children in whom the protein is missing or mutated. Since then, Rb proteins have been shown to be involved in preventing many types of human cancers. Further study showed that pRb stops a cell from dividing before it has appropriately duplicated and segregated its genetic material - coordinating the complex series of events like a traffic light at a busy intersection.

The protein doesn't work alone, however. Two other family members, p107 and p130, also help. Their ability to fill in for one another makes it difficult to parse out exactly what the proteins are doing at a molecular level. Unfortunately, lab animals missing one or two family members die soon after birth.

Viatour and Sage devised a way to inhibit, or knock out, the function of all three proteins in adult mice. They genetically engineered animals in which the p107 gene is deleted and the pRb and p130 genes are flanked by pieces of DNA that are recognized and cleaved by a specialized protein, Cre recombinase. When expressed in blood stem cells, the recombinase snips out the Rb and p130 genes, leaving these stem cells and their progeny - that is, the entire blood system - without any functional Rb family members.

The researchers found that blood, or hematopoetic, stem cells, which usually hang around quietly waiting to be called into action, began proliferating when Rb family members were missing. While unmodified blood stem cells give rise to two main groups of cells - myeloid and lymphoid - the cells missing the Rb family strongly favored the myeloid lineage. 'The differentiation of these hematopoetic stem cells is clearly defective,' said Viatour, who also collaborated with bioinformatician and pediatrician Atul Butte, MD, PhD, and other Stanford researchers. Butte, assistant professor of medicine and pediatrics, helped to investigate the gene expression profiles of the blood stem cells.

'We found that key myeloid genes were upregulated in the cells, and that lymphoid-associated genes were downregulated,' said Viatour. In contrast, the ability of the stem cells to make more of themselves seems unimpaired.

Finally, in an experiment mimicking human bone marrow transplantation, hematopoetic stem cells from the mice missing the Rb family members were no longer able to repopulate the immune systems of animals that had received a lethal dose of radiation.

'It's been known that resting, or quiescent, stem cells are more likely to be successful candidates for transplantation in both humans and mice than are actively dividing cells,' said Viatour. 'We now have a good model for understanding why that is.' The researchers plan to continue investigating how the Rb family members affect these stem cells. One challenge will be to develop an organ-specific way to knock out Rb family function. They'd also like to find out why differentiated myeloid cells don't also proliferate inappropriately, since they too are missing Rb family members.

Other co-authors are postdoctoral scholars Tim Somervaille, MD, PhD, and Shivkumar Venkatasubrahmanyam, PhD, and the director of the Stem Cell Biology and Regenerative Medicine Institute, Irving Weissman, MD. Emmanuelle Passegue, PhD, a postdoctoral scholar in Weissman's lab when the work was conducted, is now at UC-San Francisco. The research was supported by the National Institutes of Health, the Lucile Packard Foundation for Children's Health, the Damon Runyon Cancer Foundation, the California Institute for Regenerative Medicine, the Human Frontier Science Program, the European Molecular Biology Organization, the Fonds de la Recherche Scientifique, the Leon Fredericq Foundation and the Leukemia and Lymphoma Society.