p16INK4a and the control of cellular proliferative life span

Lily I. Huschtscha and Roger R. Reddel1

Children's Medical Research Institute, 214 Hawkesbury Rd, Westmead, Sydney, New South Wales 2145, Australia


    Abstract
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
Normal somatic cells have a limited proliferative capacity in vitro: after a finite number of cell divisions they eventually enter a non-proliferative state referred to as senescence. Senescence is thought to be a major tumor suppressor mechanism, and many cancers contain cells that have escaped from senescence and become immortalized. The role of telomerase activation in immortalization is currently attracting considerable attention, but immortalization is often associated with other changes including loss of normal function of the tumor suppressor locus, INK4a/ARF. Two proteins, p16INK4a and p14ARF, are encoded by this locus. Here we focus on p16INK4a and review accumulating evidence that loss of p16INK4a function may be involved in escape from the normal limits on cellular proliferative life span.

Abbreviations: cdk, cyclin-dependent kinase; HMEC, human mammary epithelial cells; HPV, human papillomavirus; LFS, Li–Fraumeni syndrome; MTS-1 gene, multiple tumor suppressor-1 gene; PD, population doubling; Rb, retinoblastoma; SV40, simian virus 40.


    Introduction
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
When normal human cells are cultured in vitro, they proliferate only a limited number of times before entering a state of permanent growth arrest, referred to as senescence, in which they remain alive and metabolically active but are completely refractory to mitogenic stimuli (1). The population doubling (PD) level at which senescence occurs is known as the Hayflick limit. Various factors (reviewed in ref. 2) including {gamma} irradiation (3), oxidative stress (47) and an activated Ha-ras or raf oncogene (810) are able to induce a senescence-like state (`premature senescence') at a PD level below the Hayflick limit.

Many cancers contain cell populations that have escaped from the normal limitations on proliferative potential, and it is possible that senescence is a major tumor suppressor mechanism which must be overcome during tumorigenesis (11,12). The extensive molecular changes that characterize senescence are still being catalogued (13), and the mechanisms of these changes are very poorly understood. One change that is commonly seen in senescence is an elevated level of the p16INK4a protein (1419). Conversely, p16INK4a function is commonly lost in immortalized cells (18,2025). These observations suggest that p16INK4a may be involved in senescence, and that its loss is causally related to escape from the normal controls on proliferative life span. Here we review evidence for this hypothesis.


    Immortalization is often a multi-step process
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
The most detailed studies of immortalization have involved normal human cells transduced with the oncogenes of DNA tumor viruses, especially those of simian virus 40 (SV40) and the oncogenic human papillomaviruses (HPVs). Cells expressing these oncogenes continue proliferating beyond the PD level at which their untreated counterparts become senescent (Figure 1AGo), but they eventually cease proliferating in a state referred to as crisis (26). The nature of the crisis growth-arrest state and its relationship to senescence is poorly understood. A small number of cells within the population may acquire the ability to escape from crisis and form an immortalized cell line (2628). In all such cell lines examined, escape from crisis has been shown to be associated with activation of a telomere maintenance mechanism, either telomerase (2931) or a non-telomerase alternative mechanism (32).



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Fig. 1. Modes of escape from senescence. (A) Normal cells proliferate a finite number of times before entering senescence, a viable state of permanent growth arrest. Various DNA tumor viruses encoding oncoproteins that interact with p53 and pRb permit a temporary escape from senescence that ends in crisis. Rare cells may activate a telomere maintenance mechanism, escape from crisis, and become immortalized. (B) Spontaneous loss of wild-type p53 in Li–Fraumeni syndrome cells is associated with a temporary extension of life span that ends in permanent growth arrest (p53-minus growth arrest; p53– GA). When p53 and pRb are both inactivated by viral oncoproteins, there is a greater extension of life span, but it is also finite and ends in crisis. Spontaneous loss of both p53 and p16INK4a expression results in a greater life span extension than the loss of p53 alone, but eventually these cells also undergo growth arrest (p53– p16– GA). (C) HMECs grown in serum-free conditions cease dividing at `selection'. Cells that escape from selection have spontaneously lost p16INK4a expression due to methylation of the p16INK4a CpG island, and exhibit a finite period of post-selection growth that ends in a senescence-like state. (D) hTERT transfection immortalizes or substantially increases the life span of human fibroblasts or retinal pigment epithelial cells. Other cell types require the disruption of the Rb pathway as well as expression of hTERT for enhanced growth.

 
The SV40 and HPV oncoproteins interact with a variety of cellular proteins. The regions of these oncoproteins that interact with and presumably inactivate the protein products of the p53 and retinoblastoma (Rb) genes are required for temporary extension of proliferative life span (reviewed in ref. 33). Thus, immortalization, at least in these in vitro model systems, involves multiple steps, including alterations in p53 and pRb function and activation of a telomere maintenance mechanism.

Most human tumors are not caused by DNA tumor viruses, so an obvious question is whether immortalization of tumor cells involves alterations equivalent to those seen in DNA tumor virus-induced immortalization. Like cell lines immortalized in vitro, most human tumors express telomerase activity (reviewed in ref. 34) or, less commonly, an alternative telomere maintenance mechanism (35). p53 mutations, which occur commonly in a wide variety of human cancers (36,37), may contribute to immortalization in a manner analogous to the interaction of viral oncoproteins with p53. Evidence for the role of p53 in senescence and immortalization has been reviewed by Wynford-Thomas (38). In human tumors, mutations in the Rb gene itself are less common than p53 mutations, but loss of normal p16INK4a expression occurs commonly and the evidence summarized below suggests that this may be functionally equivalent to pRb inactivation by viral oncoproteins.


    One locus, two pathways
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
Understanding the function of p16INK4a is complicated by the observation that the INK4a/ARF locus has an important role in both the p53 and pRb pathways. This locus is very unusual in that it encodes two distinct proteins translated from alternatively spliced mRNAs, each regulated by its own promoter (39). p16INK4a is specified by the {alpha} transcript (containing exons 1{alpha}, 2 and 3), and the alternative, or ß transcript contains exons 1ß, 2 and 3 encoding p19ARF in the mouse (40) and p14ARF in humans (4143). The primary amino acid sequences of the ARF and p16INK4a proteins are completely different because the common exon 2 sequences are translated in different reading frames. As described below p16INK4a is a key component of the Rb pathway, but p14ARF has a major role in the p53 pathway. p14ARF acts by binding specifically to MDM2, resulting in stabilization of both p53 and MDM2. p14ARF-induced growth arrest is therefore p53 dependent (44,45).

Deletion events affecting p16INK4a expression may also affect p14ARF, so it is important to keep in mind that in studies carried out before the identification of p14ARF, the effects of losing both proteins may have been attributed incorrectly to loss of p16INK4a alone. There is also recent evidence that expression of both of these proteins is coordinately regulated by the Polycomb-group transcriptional repressor, bmi-1 (46). Thus, although in this review we concentrate on the role of p16INK4a in controlling proliferative life span, the potential involvement of p14ARF must also be recognized.


    p16INK4a function is frequently lost in immortal cells
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
In its physiologically active state as an inhibitor of progression through the cell cycle, pRb is hypophosphorylated (47,48). To permit cell cycling, pRb is phosphorylated by a holoenzyme complex containing a cyclin D and a cyclin-dependent kinase (cdk4 or cdk6) (Figure 2Go). p16INK4a was first identified as an inhibitor of this complex (49), hence the designation of this gene as CDKN2A. Thus, the function of p16INK4a is to maintain pRb in its active state. Additional members of the INK4 family of cyclin D–cdk4 inhibitors have subsequently been identified: p15INK4b, p18INK4c and p19INK4d (50,51). Like p16INK4a (49), the other INK4 proteins also contain four to five tandemly repeated ankyrin motifs (usually involved in protein–protein interactions), each ~32 amino acids in length. A study using peptide-mediated inhibition studies suggested that p16INK4a binds cdk4 and cdk6 through its third ankyrin repeat (52). Crystallographic studies indicate that the region of interaction is more extensive (53,54). p16INK4a (and the related p19INK4d protein) bind adjacent to the ATP-binding site of the cdk6 catalytic cleft, distorting the cleft and preventing binding of ATP. Prevention of cyclin binding appears to be indirect via structural changes that propagate to the cyclin-binding site (53,54).



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Fig. 2. p16INK4a and the `Rb pathway'. Phosphorylation of pRb by a complex containing a cyclin D and either cdk4 or cdk6 results in release of transcription factors including members of the E2F family from binding to pRb, and allows cell cycle progression through G1 into S phase. pRb phosphorylation and, thus, cell cycle progression, is inhibited by p16INK4a.

 
Like pRb, p16INK4a is itself a tumor suppressor gene, and it is sometimes referred to as the multiple tumor suppressor-1 (MTS-1) gene. The human INK4a/ARF locus is located on chromosome 9p21, a region that sustains allelic loss in many human tumors (55,56). Homozygous INK4a deletions occur predominantly in bladder carcinomas, gliomas, T-cell acute lymphoblastic leukemias, melanomas and renal cell carcinomas (for a review, see ref. 57). As an alternative to gene deletion, p16INK4a is also commonly inactivated in head and neck, lung, brain, breast, colon, esophageal and bladder cancers by methylation of its promoter, which prevents transcription and results in loss of p16INK4a protein expression (58,59). Treatment of tumor cell lines with the demethylating agent, 5-aza-2'-deoxycytidine, resulted in significant decrease in p16INK4a promoter methylation, re-expression of p16INK4a protein and growth inhibition (60). Less commonly, point mutations of the INK4a/ARF gene are found in cancers, including pancreatic and esophageal carcinomas (61,62). Most tumor-specific mutations affect the stability of the tertiary structure of p16INK4a (63,64) and map to the surface involved in contact with cdk molecules (53,54). Germline INK4a/ARF point mutations co-segregate with familial melanoma in some kindreds (6568), and are involved in some cancers of the pancreas and liver (6971).

The earliest indication that disruption of p16INK4a function may be associated with immortalization came from the observation that deletion of p16INK4a is common in immortalized tumor cell lines (20). Tumor cell lines differ from normal cells in many more ways than immortalization alone, but the link between p16INK4a loss and immortalization was strengthened by the finding that several non-tumorigenic in vitro immortalized cell lines also lack functional p16INK4a protein (18,2125). In support of the hypothesis that loss of p16INK4a function may have a causal role in immortalization, it has been found that cells from mice which are made nullizygous for INK4a by targeted deletion undergo immortalization more readily than normal control cells (72), although it must be noted that these mice appear to have no functional p19ARF. Furthermore, expression of p16INK4a in immortalized human fibroblasts induced a senescence-like growth arrest (23).


    p16INK4a loss is associated with finite extension of life span
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
In view of this evidence linking loss of p16INK4a function with immortalization, and the observation described above that immortalization is a multi-step process, the question arises whether p16INK4a loss is involved in escape from crisis or in some earlier step. In Li–Fraumeni syndrome (LFS) fibroblasts, which became immortalized spontaneously without a period of crisis, it was found that INK4a/ARF gene deletion preceded activation of a telomere maintenance mechanism; this suggested that loss of p16INK4a occurs earlier than full immortalization (22). In another culture of the same LFS fibroblasts, spontaneous loss of the wild-type p53 allele and deletion of both copies of the INK4a/ARF gene resulted in a large, but finite, extension of proliferative life span. The cells entered a permanent growth arrest (crisis) from which no cells escaped (22; Figure 1BGo). Thus, p16INK4a loss was shown to be associated with the life span extension phase and to precede escape from crisis.

These data are consistent with the concept that loss of p16INK4a function is analogous to inactivation of pRb in DNA tumor virus-induced immortalization. This is perhaps not surprising, given that the action of p16INK4a is to inhibit the inactivation of pRb by cdks. Thus, loss of functional p16INK4a might be expected to have consequences similar to the loss of functional pRb. In accord with this expectation, loss of function of pRb or p16INK4a, but not both, occurs in most immortalized cell lines (20,21).

To further emphasize the parallels between virus-induced immortalization and the genetic changes in spontaneously immortalized LFS fibroblasts, loss of wild-type p53 and loss of p16INK4a had an additive effect in extending the proliferative life span of the LFS cells (Figure 1BGo). The life span extension in cells that lost both p53 and p16INK4a (22) was greater than that seen in cells that lost p53 but retained p16INK4a (73) and similar to that seen in cells transfected with SV40 (74) or HPV (K.Maclean and R.Reddel, unpublished data) oncogenes that interact with both p53 and pRb.

The association between loss of p16INK4a function and limited increase in proliferative life span was strengthened recently by three groups who independently observed spontaneous loss of p16INK4a function in normal human mammary epithelial cells (HMECs) (7577). Under standard growth conditions in serum-free medium, HMECs proliferate a small number of times after growing out from explanted tissue and then enter a senescence-like state, referred to as selection (7880). Small colonies of dividing cells may then appear, and these post-selection cells proliferate for an additional 40–50 PDs before entering permanent growth arrest (Figure 1CGo). It has been shown that the post-selection growth of HMECs is associated with loss or reduction of p16INK4a expression due to methylation of its gene's CpG island (7577). Treatment of these cells with the methylation inhibitor 5-aza-2'-deoxycytidine resulted in restoration of p16INK4a expression and growth arrest with expression of senescence-associated ß-galactosidase (75). The selection growth arrest can also be bypassed by transducing pre-selection HMECs with the HPV E7 gene (encoding an oncoprotein that inactivates pRb) (81), or with a mutant SV40 large T antigen that contains a pRb-binding domain but is unable to bind p53 (L.Huschtscha, unpublished data).


    p16INK4a levels increase as cells become senescent
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
In HMECs it was shown that the p16INK4a level increases as cells approach the senescence-like selection growth-arrest state (75). Other types of human cells have also been shown to accumulate p16INK4a as they approach senescence, including fibroblasts (1416), epithelial cells (17,18) and T lymphocytes (19). Senescent fibroblasts may contain p16INK4a levels at least 40-fold greater than early passage cells (16). It is of particular interest that cells expressing SV40 oncoproteins continue to accumulate p16INK4a during precrisis growth at the same rate as normal cells prior to senescence, suggesting that the upregulation of p16INK4a is not a consequence of entry into senescence but rather it is directly correlated with the number of PDs (15). This suggests the possibility that accumulation of p16INK4a may be involved in triggering the onset of senescence.

According to the telomere hypothesis of senescence, it is telomere shortening in normal somatic cells that triggers the onset of senescence (82,83). It is possible that the increasing levels of p16INK4a may be due to progressive telomere shortening. However, another possibility might be that some other `clock' mechanism induces the rise in p16INK4a levels. It has been proposed elsewhere that there may be more than one mitotic clock mechanism, any one of which may trigger senescence (2).


    Loss of pRb/p16INK4a may be required for telomerase-induced life span extension
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
As predicted by the telomere hypothesis of senescence, prevention of telomere shortening by expression of telomerase in normal fibroblasts and retinal pigment epithelial cells transduced with the telomerase catalytic subunit (hTERT) cDNA results in a substantial extension of proliferative life span (8487). It is not yet clear whether these cells are immortalized, or whether they will eventually become senescent after a greatly extended life span (Figure 1DGo). In contrast to the fibroblasts and the retinal epithelial cells, hTERT cDNA did not extend the proliferative capacity of keratinocytes or HMECs unless either pRb was inactivated by a viral oncoprotein or p16INK4a expression was decreased due to methylation, respectively (88). The significance of this is not clear. It is possible that disruption of the Rb pathway is required for the cells to overcome limitations on in vitro growth posed by the standard culture conditions. It is also possible that there are multiple pathways to senescence, or multiple mitotic clocks, each of which must be inactivated before cells permanently escape from limited proliferative life span (2).


    p16INK4a and premature senescence
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
Several studies have shown that expression of ras or raf oncogenes in normal human fibroblasts induces accumulation of p16INK4a and p53 and a state of premature senescence that has its onset within a few days (9,10,89). These oncogenes signal premature senescence through activation of the ras/raf/MEK/MAP kinase cascade (for a review of this cascade, see ref. 90), as a pharmacological inhibitor of MEK kinase prevented growth arrest and also the accumulation of p16INK4a. Experimentally induced expression of p16INK4a, or the p53 effector molecule p21, elicited cell-cycle arrest and senescence in human fibroblasts (10,23). Therefore, the induction of p53 and p16INK4a expression by the ras/raf/MEK/MAP kinase cascade seems likely to have a causal role in premature senescence. Induction of premature senescence presumably provides the cell with a protective mechanism against neoplastic transformation when the ras signaling pathway is inappropriately activated.

Two additional observations strengthen the association between increased p16INK4a expression and premature senescence. First, it has been shown that induction of DNA double-strand breaks results in increased levels of p16INK4a and in premature senescence (91). Secondly, it was found that cdk inhibitors induce premature senescence in human fibroblasts (92).

Ras-induced premature senescence has many features similar to cell aging, including expression of senescence-associated ß-galactosidase activity (93). There are some differences, however. Most cells rendered senescent by raf are in G2/M and not G1, and their morphology is typically small, rounded and refractile (10). The state of premature senescence is apparent within a few days of expression of the activated oncogene, whereas prolonged growth in culture is required before diploid cells become senescent at the Hayflick limit. The relationship between cellular senescence and premature senescence is therefore unclear. It is currently unknown whether premature senescence is only quantitatively different from senescence (i.e. a rapid movement of the mitotic clock to midnight due to a very intense signal), or whether there are also some qualitative differences (e.g. induction of a different arrest state).


    Perspectives
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 
p16INK4a is implicated in the control of cellular proliferative life span in vitro, on the basis of the observations that elevated levels of p16INK4a accompany cellular senescence, that loss of p16INK4a expression is associated with a finite increase in proliferative life span, and that experimental induction of p16INK4a expression results in senescence. Given the postulated importance of senescence as a barrier to tumorigenesis, it may be expected that inactivation of p16INK4a would be an important step in the process of tumorigenesis.

The function of the accumulated p16INK4a at senescence requires investigation. Much of the p16INK4a is present in an active free state in normal cells (94). It needs to be determined whether p16INK4a may have some function(s) at senescence other than inhibition of cyclin D–cdk-4 or cdk-6 complexes.

The mechanism of p16INK4a accumulation in cells approaching senescence is unknown and requires elucidation. It also needs to be determined whether the MEK/MAP kinase pathway is involved in increased p16INK4a expression at the Hayflick limit. According to the telomere hypothesis, senescence is due to telomere shortening that accompanies the cell division cycle in telomerase-negative normal cells. It will therefore be of considerable interest to determine whether p16INK4a accumulation at senescence is due to telomere shortening or whether there is some other mechanism. The possibility of mitotic clocks other than progressive telomere shortening cannot be excluded at present.

The current evidence suggests that induction of telomerase activity in otherwise normal fibroblasts by expression of hTERT cDNA from a heterologous promoter without inactivation of p16INK4a or other components of the Rb pathway results in a very large extension of proliferative life span. Although it is possible that these cells may eventually senesce despite well-maintained telomeres, this observation raises important questions as to the role of p16INK4a inactivation in the immortalization process. Maybe loss of p16INK4a function facilitates the eventual upregulation of telomerase expression, for example, by extending proliferative life span sufficiently to allow the accumulation of genetic changes that result in derepression of hTERT expression. In keratinocytes and HMECs transduced with an hTERT expression vector, loss of p16INK4a or pRb activity seems to be a pre-requisite for any hTERT effect. In addition to the possibility that there are other mitotic clocks in which p16INK4a is involved, we need to know whether this is a cell-type specific response to hTERT expression, or whether p16INK4a loss interferes with terminal differentiation or some process other than senescence which would otherwise limit the apparent in vitro life span.


    Acknowledgments
 
The authors thank Lindy Hodgkin for assistance with the manuscript, and Drs Steve Linke, Gordon Peters and Manuel Serrano for their helpful comments. Work in the authors' laboratory was supported by grants from the National Health and Medical Research Council of Australia, the Kathleen Cuningham Foundation for Breast Cancer Research and the Carcinogenesis Fellowship of the New South Wales Cancer Council.


    Notes
 
1 To whom correspondence should be addressed at: Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia Email: rreddel{at}cmri.usyd.edu.au Back


    References
 Top
 Abstract
 Introduction
 Immortalization is often a...
 One locus, two pathways
 p16INK4a function is frequently...
 p16INK4a loss is associated...
 p16INK4a levels increase as...
 Loss of pRb/p16INK4a may...
 p16INK4a and premature...
 Perspectives
 References
 

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Received December 21, 1998; accepted January 20, 1999.