A team of researchers at the University of Toronto has unravelled the function of an important gene that causes cells to grow abnormally.

They hope their research will have powerful implications for reshaping our knowledge of cancer and other diseases caused by cells growing out of control.

Published recently in the journal Nature Genetics, the discovery involves a gene called PTEN. By deleting (or “knocking out”) the PTEN gene in the brains of laboratory mice, the scientists showed that it regulates cell size and cell division in mammals, including humans.

The paper’s lead author is Stéphanie Backman, a graduate student in the Department of Medical Biophysics at U of T. “We study PTEN because it has been found to be mutated in a wide spectrum of cancers, including brain, breast, endometrial (uterus) and prostate cancers,” she explained. By better understanding what PTEN does, scientists should be able to find better cancer treatments.

PTEN is involved in controlling the growth, division and death of cells. Prior work with fruit flies has shown that when PTEN is missing, some types of cells become larger or more numerous than normal, creating oversized wings and eyes. “Our paper shows that regulation of organ size by PTEN is also relevant in mammals,” says Backman.

To study the mammalian effects of PTEN mutations, Backman’s group deleted the gene in the brain cells of the mice using a technique called the Cre-loxP system. This technology allows scientists to delete a gene from a specific tissue in an animal’s body and leave it intact everywhere else.

The technique relies on genetically engineered mice. These so-called Gfap-cre mice produce the Cre enzyme in their brains. This enzyme acts on DNA to delete sections of the molecule found between special loxP marker genes. Any genetic information in the mouse that is marked by loxP will be “knocked out,” but only in brain cells where the Cre enzyme is produced (see sidebar).

For this study, the researchers engineered normal mice so that the loxP marker flanked their PTEN genes. Then these mice were crossbred with the Gfap-cre mice, so that their offspring have both the marked PTEN gene and the Cre enzyme to necessary to delete it. The result was “PTEN knockout mice” which express PTEN normally in all tissues except their brains.

As the PTEN knockout mice developed, they suffered from seizures and poor muscle control. They did not get tumours, but their brains grew much larger than in healthy mice. Neurons kept growing far beyond their normal size, and their nervous systems malformed and failed to work properly.

Those symptoms of overgrown neurons are the same as in humans with LDD, or Lhermitte-Duclos disease. LDD is a sub-type of Cowden’s disease, which involves overgrowth in many different tissues. And the crucial link is that Cowden’s patients have PTEN mutations.

“Prior to our paper, there was limited evidence implicating PTEN in LDD and Cowden’s disease,” says Backman. With these new results, PTEN knockout mice can be used as a model for LDD so that the disease can be better studied and new treatments tested. Backman is now working to extend her research on the PTEN gene. It is not yet clear why mice in this study had overgrown brain cells but no tumours. PTEN probably does not act in isolation, so by studying what happens when the gene is deleted from different tissues, those other factors may become clearer.

In one new experiment with tissue-specific deletion, Backman has knocked out PTEN in the skin cells of mice. These mutants develop thickened skin and unusual wavy hair. “Whether this abnormality is a result of increased cell division, decreased cell death or increased size of epidermal cells is one of the things I’m investigating now,” she said Although deleting PTEN from the brain or skin only causes abnormal cell growth, deleting it from other tissues might create cancerous tumours. Commenting on the significance of her research, Backman said: “[Our work] makes the identification of other genes involved in regulating cell size potentially important for cancer.”

What a knockout!
How scientists delete genes to discover what they do

1.Mouse embryonic stem cells (ES cells) are isolated from mouse embryos. These cells have the potential to divide and grow into any tissue in the animal’s body. The ES cells are kept alive in a petri dish.

2.Scientists use genetic engineering techniques to insert a short piece of DNA called a loxP sequence on either side of the gene they want to delete in the chromosomes of the ES cells.

3.Meanwhile, mice are mated to produce a fertilized embryo. These are flushed from the female before they implant in the uterus. This early embryo is called a blastocyst, which is hollow, save for a small clump of cells called the inner cell mass (ICM). The ICM divides to form every cell in a newborn mouse

4.The genetically engineered ES cells are injected into the blastocyst, where they will join with the ICM. The blastocysts are then inserted into surrogate mothers where they develop to term

5.Some of these developing mice will be derived only from injected ES cells (blue). The loxP sequence will be in every cell of these mice once they are born.

6.After birth, researchers treat the engineered mice with an enzyme that chops out the gene from between the loxP sites. The animals will have that gene “knocked out” from that point on in their development.