Major Research Interests

Our Group has contributed some key observations regarding aging and cancer  in several related areas noted below which include:

Cell Cycle Regulation. The frequently cited Riabowol et al., Cell (1989) provided the first evidence that cell cycle regulatory mechanisms have diverged significantly from lower to higher eukaryotes and prompted further examination of the similarities and differences in cyclin and CDK function between yeast and mammalian cells. Although our report was followed by several from different groups that attempted to show that mammalian cdc2 (now called CDK1) activity was required at both the G1/S and G2/M checkpoints as in yeast, our initial observations that cdc2 was only required for completion of mitosis have now been corroborated by a number of different approaches including a temperature-sensitive cdc2 mutant cell line and has helped shape how we view the cell cycle.

 

Transcriptional Regulation in Cell Aging.  After we published the first report that the activity of the c-Fos transcription factor is repressed in senescing cells (Riabowol et al., Proc. Natl. Acad. Sci. USA, 1992) our further work showed that unlike results reported before in which phosphorylation activated the factor, hyperphosphorylation of the serum response factor (SRF) that regulated c-fos expression inactivated it in senescing cells (Atadja et al., Mol. Cell Biol., 1994).  We have extended this observation to other genes regulated by the SRF including a study by Keith Wheaton which has shown that hyperactivation of Protein Kinase C delta by proteolysis produces high levels of an active catalytic fragment in senescing cells that inactivates SRF and prevents immediate early gene expression, blocking cell growth. This paper (Wheaton & Riabowol, MCB, 2004) earned Keith a CIHR AGE+ prize.

 

Aging and Telomere Dynamics. Our study of accelerated aging of the immune system in patients with chronic viral (HIV) infection (Bestilny et al., AIDS 14: 771-80, 2000) led to our landmark discovery that telomere length in children, and hence replicative capacity, is linked to the age of the father at conception, explaining how telomere length is regulated in human populations (Unryn et al., Aging Cell, 2005, cited by the Faculty of 1000). We have followed this study with the novel and clinically relevant observation that chemotherapy-induced telomere loss is exacerbated in the elderly (Unryn et al. Clin Can. Res., 2006).

Tumour suppressor activation in senescing cells.  Atadja et al., Proc. Natl. Acad. Sci. USA, 1995) represents the first demonstration of increased activity of a tumour suppressor (p53) during the aging of normal human fibroblasts, a concept now widely accepted for several different tumour suppressors both in vitro anfd in vivo.  This observation led us to develop a method to isolate additional negative regulators of growth, resulting in our discovery of the ING family of type II tumour suppressors.

 

Discovery of a new family of type II tumour suppressors.  Possibly our most significant publications to date describe the isolation, cloning and ongoing characterization of a novel type II tumour suppressor that we have called ING1 for INhibitor of Growth as outlined in (Garkavtsev et al., Nature Genetics, 1996).

   

This initial description of ING1 was followed by our studies showing ING roles in senescence (Garkavtsev & Riabowol, MCB 17: 2014-19, 1997), apoptosis (Helbing et al., Can Res., 1997), tumorigenesis (Toyama et al. Oncogene, 1999), DNA repair (Scott et al., J. Cell Sci., 2001), chromatin remodeling (Vieyra et al. JBC, 2002), regulating gene expression (Feng et al., MCB, 2006) and p53 homolog activity in the C. elegans genetic model (Liu et. al., Genetics, in press, 2008). An interesting link between ING1 & ING2, both of which reside in histone acetylation/deacetylation complexes, and the induction of HSP70 suggested a potential mechanism by which ING proteins affect apoptosis.  This was independent of their ability to interact with methylated histones since the effect did not require the PHD region of the INGs that interacts with histone H3K4-Me3.

 

Since their initial discovery in 1996, we now know that the ING genes in mammals encode a number of different isoforms generated by alternative splicing, internal initiation or alternative promoter usage in several of the genes (He et al., 2005; Soliman & Riabowol, TiBS, 2007).  Sequence analysis followed by construction of a radial plot of the known ING genes in all species, showed that the ING proteins are highly conserved, being present in yeast through humans, with three ING genes in lower organisms corresponding to three groups containing 5 genes in mammals.  Sequence analysis is reflected in functional groupings with ING1 and ING2 being found in HDAC complexes, ING4 and ING5 being found in similar HAT complexes and ING3 being found in a distinct HAT complex.

 

ING1 family members have now been included in a number of diagrams outlining players in the process of cell cycle regulation, chromatin remodeling and at least one textbook describing growth regulators, and recent reports link the PHD domains of ING proteins to reading the histone code, particularly through their interactions with methylated histone H3.  Linkage between the PHD domains of ING proteins and methylated histones, and the interaction between ING proteins and HAT and HDAC complexes have resulted in models being developed for how this family of chromatin regulators might contribute to chromatin structure (Soliman & Riabowol, TiBS 2007).

 

A new region of the ING proteins that is unique to them in the human proteome was described recently as shown in the diagram below.  It was found that the strongest interaction with this region was with the lamin A protein, thus we have called this unique sequence the lamin interacting domain or LID.  Mutation of lamin A is known to result in several laminopathies including a form of accelerated premature aging called Hutchinson-Gilford Progeria Syndrome.  Children with HGPS caused by a de novo mutation of one allele of the lamin A gene, typically die of "old age" ~13-16 years after birth and exhibit a number of features closely resembling normal human aging.  Cells from HGPS patients show abnormalities in the nuclear envelope, alterations in chromatin distribution, altered gene expression and a slightly increased sensitivity to apoptosis.  How mutation of one lamin A allele elicits these effects is not well understood but likely involves a response to reduced amounts of functional lamin A and/or interference in normal lamin A function by the mutated form of lamin A called progerin.

Another consequence of lamin A loss is the decreased expression and mislocalization of ING1 as shown below in lamin A knockout cells.  This may contribute to the altered

distribution of chromatin seen in both lamin A knockout, and HGPS cells since interference in the ability of ING1 to bind to lamin A by various methods results in cell phenotypes similar to those seen in HGPS (Han et al., Nature Cell Biology 2008).

The increasing study of the INGs by many groups (Soliman & Riabowol, Trends in Biochem. Sci. 2007) suggests that discovery of the ING1 family may have continued impact upon the relevant research communities examining cell aging and cancer. The range of activities through which this family of chromatin regulators contributes to cell aging and cell transformation during oncogenesis continues to be a major focus of our group, and is summarized in the general diagram of cell aging and immortalization shown below.

Current interests.  The major interests of the laboratory are to understand the biochemical & genetic mechanisms that enforce replicative senescence in normal human diploid fibroblasts and to determine how cells are able to evade them in becoming immortal cancer cells. A number of gene products such as the tumor suppressors p53 and p105Rb, the D-type cyclins and the cyclin-dependent kinase inhibitors p16 and p21 increase in activity and/or amount as cells reach the end of their replicative lifespan. Conversely, expression of the immediate-early genes encoding c-Fos and Egr-1 that normally promote cell growth is progressively lost as cells become senescent. Different members of the ING1 family of alternatively spliced growth and chromatin regulators also show changes in levels and localization that are consistent with contributing to the phenotypes of both senescence and tumorigenesis. How these changes in gene expression are related to the erosion of telomeric DNA that is very strongly associated with replicative senescence, and how the different ING family members function in the processes of cell aging and immortalization are also major areas of investigation.