The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the α transcript, p16 INK4a , is a recognized tumour suppressor that induces a G 1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A β transcript, p14 ARF , activates a p53 response manifest in elevated levels of MDM2 and p21 CIP1 and cell cycle arrest in both G 1 and G 2 /M. As a consequence, p14 ARFinduced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14 ARF acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14 ARF expression and there is an inverse correlation between p14 ARF expression and p53 function in human tumour cell lines. However, p14 ARF expression is not involved in the response to DNA damage. These results place p14 ARF in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression.

The Krüppel-like factor (KLF) family of transcription factors regulates diverse biological processes that include proliferation, differentiation, growth, development, survival, and responses to external stress. Seventeen mammalian KLFs have been identified, and numerous studies have been published that describe their basic biology and contribution to human diseases. KLF proteins have received much attention because of their involvement in the development and homeostasis of numerous organ systems. KLFs are critical regulators of physiological systems that include the cardiovascular, digestive, respiratory, hematological, and immune systems and are involved in disorders such as obesity, cardiovascular disease, cancer, and inflammatory conditions. Furthermore, KLFs play an important role in reprogramming somatic cells into induced pluripotent stem (iPS) cells and maintaining the pluripotent state of embryonic stem cells. As research on KLF proteins progresses, additional KLF functions and associations with disease are likely to be discovered. Here, we review the current knowledge of KLF proteins and describe common attributes of their biochemical and physiological functions and their pathophysiological roles.

The Krüppel-like factors (KLFs) comprise a family of evolutionarily conserved zinc finger transcription factors that regulate numerous biological processes including proliferation, differentiation, development and apoptosis. KLF4 and KLF5 are two closely related members of this family and are both highly expressed in epithelial tissues. In the intestinal epithelium, KLF4 is expressed in terminally differentiated epithelial cells at the villus borders of the mucosa and inhibits cell growth, while KLF5 is expressed in proliferating epithelial cells at the base of the intestinal crypts and promotes cell growth. KLF4 and KLF5 respond to a myriad of external stress stimuli and are likely involved in restoring cellular homeostasis following exposure to stressors. Confirming their importance in maintaining tissue integrity, KLF4 and KLF5 are both dysregulated in various types of cancer. Here we review the recent advances in defining the physiological and pathobiological roles of KLF4 and KLF5, focusing on their functions in the intestinal epithelium.

To investigate the mode of action of the p16INK4a tumor suppressor protein, we have established U2-OS cells in which the expression of p16INK4a can be regulated by addition or removal of isopropyl-␤-D-thiogalactopyranoside. As expected, induction of p16INK4a results in a G 1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16INK4a also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27 KIP1 . Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16INK4a , this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16INK4a . Sequestration of CDK4 by p16 INK4a allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16 INK4a, p27KIP1 appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27 KIP1 and p21 CIP1 . Significantly, p16 INK4a itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.

After a limited number of population doublings (PDs), cultures of normal mammalian diploid cells undergo an irreversible growth arrest known as replicative senescence [1]. As well as contributing to cellular ageing, senescence is viewed as an important mechanism of tumour suppression by preventing the emergence of immortal cell clones [2-4]. Senescent cells have a number of characteristics that distinguish them from cycling or quiescent cells including elevated levels of two cyclin-dependent kinase (Cdk) inhibitors, p16INK4a and p21CIP1 [5-11]. Here, we demonstrate that both of these Cdk inhibitors, as well as other members of their protein families (the INK4 and CIP/KIP families, respectively [12]), induce several facets of the senescent phenotype when ectopically expressed in young human diploid fibroblasts. These include a reduced proliferative capacity, an altered size and shape, the presence of underphosphorylated retinoblastoma protein (pRb), increased expression of plasminogen activator inhibitor (PAI-1) and the appearance of senescence-associated beta-galactosidase (SA-beta-gal) activity [2,3,13-15]. A 20 amino acid peptide from p16INK4a that inhibits Cdks active in the G1 phase of the cell cycle [16] produces similar effects in a dose-dependent manner suggesting that, in primary fibroblasts, inhibition of G1-specific Cdk activity is sufficient to induce phenotypic changes that normally occur at the end of their finite lifespan.