Tak W Mak, PhD

Apoptosis is the process of programmed cell death essential for normal embryonic development, resolution of immune responses, and defence against tumourigenesis. We have generated mice with defects in several molecules associated with apoptotic pathways, including TRAF-2, FADD, Apaf-1, caspase-3 and caspase-9.

Analyses of these mutant animals have revealed roles for these molecules in lymphocyte ontogeny, activation, effector function and cell death, as well as for susceptibility to autoimmune diseases. In addition, it has become clear that multiple apoptotic pathways exist that are stimulus- and tissue-specific. These pathways show differential requirements for Apaf-1 and the caspases. Study of mice deficient for tumour necrosis factor receptor (TNFR) revealed that, depending on the adaptors recruited to the cytoplasmic tail of this receptor, signalling for either survival or apoptosis can be transduced. We have also examined molecules involved in the regulation of apoptosis. SMAC/DIABLO binds the 'inhibitor of apoptosis' proteins and thus is thought to have an anti-apoptotic function. However, mice lacking this molecule show no phenotypic defects. These animals demonstrate the redundancy built into essential pathways governing cellular homeostasis.

Genetic susceptibility factors are associated with most types of cancer, but few of these genes have been identified. We have generated mutant mouse strains with engineered modifications of genes shown to be strongly linked to cancer. In addition to mice with null mutations, we have used the Cre-loxP or FLP systems to generate mice with conditional mutations. We have also adopted DNA microarray screening and a Drosophila screening method to isolate novel genes that interact with genes linked to cancer.

One of our earliest mutants was a mouse lacking MSH2, a component of the DNA mismatch repair machinery. MSH2-deficient mice spontaneously develop tumours early in life, making them a useful model for the study of tumorigenesis, carcinogens and anti-cancer agents.

More recently we have concentrated on tumour suppressor genes (TSGs). Studies of mice lacking the breast cancer susceptibility genes Brca1 or Brca2 suggest that these proteins have roles in pathways controlling DNA damage repair and the maintenance of genome stability. Loss of Brca1/2 function in cells that have already acquired one tumourigenic 'hit' may lead to uncontrolled proliferation of cells with damaged DNA, ultimately leading to oncogenesis. The phenotypes of mice bearing tissue-specific mutations of Brca1/2 have generally supported this hypothesis. Similarly, null mutation of the SMAD4 gene in mice leads to dysregulation of apoptosis, an event that likely contributes to tumourigenesis. At least some IRFs may also act as TSGs under certain conditions. Functional inactivation of IRF1 renders a cell highly prone to transformation.

One of the most important TSGs for human cancer is p53, a multi- functional protein that induces apoptosis or cell cycle arrest in cells with damaged DNA. We identified Chk2 as a kinase that phosphorylates p53 and stabilizes it, allowing it to carry out its anti-tumorigenic functions. We also generated mice lacking HIPK1, a kinase that binds to p53 and appears to modulate its transcriptional activity. HIPK1 may promote oncogenesis if dysregulated. Functional inactivation of the TSG tsg101 leads to an accumulation of p53 that blocks embryonic development.

Our most recent TSG work has focussed on PTEN and genes with which it interacts. Studies of mice lacking PTEN indicated that PTEN negatively regulates the PKB/AKT cell survival pathway. PTEN-deficient cells are resistant to agents inducing apoptosis, becoming immortal and thus susceptible to acquiring additional mutations that lead to tumorigenesis. Loss of heterozygosity for PTEN in heterozygous mice results in a high incidence of tumours, especially T cell lymphomas.

We have undertaken the analysis of gene expression in the absence of PTEN function using DNA microarray screening and phenotype rescue experiments in Drosophila to identify novel genes acting either upstream or downstream of PTEN. The functions of these newly isolated genes are under investigation in the hopes of identifying new targets for cancer drug therapy.