INTRODUCTION

TOP ABSTRACT INTRODUCTION CELL CYCLE CONTROL EPITHELIAL CELL RESPONSE TO... CONCLUSION REFERENCES

Keywords: lung; epithelium; oxidant; cyclins; cell cycle

There is now overwhelming evidence of the importance of oxidative stress in a number of pathological situations, and among the various organ structures the epithelium represents a major target of oxidant injury (1). This applies to the respiratory system, which is continuously exposed to many forms of oxidants from either exogenous or endogenous sources.

The mechanism of injury by oxidants relies on the ability of these components to interact with and modify a spectrum of biomolecules including DNA. An adequate response of cells to DNA-damaging agents requires a complex integration of different molecular events. After DNA damage, signals are generated that converge to either cell cycle arrest or apoptosis (5). Control of cell cycle progression in response to oxidative stress is linked to activation of a checkpoint mechanism operating before entry into the S phase of the cell cycle. This checkpoint is critical to maintain genomic integrity (5). The current understanding of the mechanisms regulating cell cycle progression in situations of oxidative stress is reviewed, and their involvement in the response of epithelial cells to oxidants is discussed.

	CELL CYCLE CONTROL

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The cell cycle machinery is controlled by external signals such as growth factors and antimitogens in order to integrate cell division with environmental and developmental stimuli. As cells progress through the cell cycle, they undergo several transitions that can be defined as unidirectional change of state to shift their activities to perform a different set of processes (10).

Cell cycle checkpoints are regulatory pathways that control the sequence and timing of cell cycle transitions, and ensure that critical events such as DNA replication and chromosome segregation are completed with fidelity (13). Numerous studies have documented that oxidants inhibit proliferation through activation of cell cycle checkpoints. In higher eukaryotic cells, such as mammalian cells, signals that arrest the cycle usually act at a G1 checkpoint. Much has been learned about the cell cycle checkpoint that controls G1-S transition and the onset of DNA replication. Progression of cells through the G1 phase and the G1-S transition involves sequential assembly and activation of key regulators of the cell cycle machinery, the cyclin-dependent kinases (CDKs).

Various G1 CDKs and Their Activation

The first CDK to be identified was the product of the CDC2 gene (34 kDa) in fission yeast, the homolog of which in mammalian cells is referred to as CDK1. A number of other protein kinases were subsequently identified and designated as CDKs on their ability to bind to a cyclin. The CDK family shares high homology within the catalytic core, which ensures binding to the cyclins. To date, nine different proteins have been characterized and have been designed CDK1 to CDK9. One defining characteristic of the CDKs is that they are inactive as monomers and become active only after binding to a cyclin partner (16).

The cyclin family is also expanding (19). These proteins were originally characterized by their level of expression, which is tied to the cell cycle, and are now identified on the basis of their sequence homology. They share a 150-amino acid region of structural homology called the cyclin box, which is responsible for binding to the CDK subunit. It is now well demonstrated that different cyclins can bind to the same CDKs. This flexibility allows the CDKs to perform distinct functions during progression through the cell cycle as well as in processes that do not relate to proliferation. Therefore, the specificity of the cyclin-CDK complex relies on each of the two proteins that constitute the complex.

The G1 CDKs include CDK2, CDK3, CDK4, CDK5 and CDK6. These enzymes form complexes with the G1 cyclins, mostly cyclins D, E, and A, at the end of G1 phase. Activity of the complexes appears to be regulated by several mechanisms (19, 20). The levels of cyclins are controlled by synthesis and ubiqutin-mediated proteolysis (21). Cell cycle kinases and phosphatases can either activate or inhibit the complex by modulating phosphorylation of the CDKs. The activatory site of phosphorylation is at a threonine. Phosphorylation of this residue by a kinase termed CAK increases the affinity of cyclin binding and converts the CDK into an active enzyme. The catalytic subunit can also be inhibited through either dephosphorylation by a phosphatase (Kap1) at the activating threonine residue or phosphorylation of another residue (Tyr-14) (22).

Activity of cyclin-CDK complexes can be negatively regulated by a group of proteins termed CDK inhibitory proteins (CKIs) (11, 17, 20, 23). CKIs have been reported to associate with either the CDK complex, the cyclin, or the catalytic subunit. Several CKIs have been identified on the basis of their homology and are currently divided into two groups that were classified after the first members were cloned: p16 and p21. The p16 family consists of p15^Ink4B, p16^Ink4, p18^Ink4C, and p19^Ink4D. The p21 family includes p21^CIP1 (also known as WAF1), p27^KIP1, and p57^KIP2. The proteins of the p16 family display a certain degree of affinity for CDK4 and CDK6, preventing the association of the CDKs with cyclin D. The p21 family proteins act on a wide range of cyclin-CDK complexes. In addition, p21^CIP1 has been reported to bind to proliferating cell nuclear antigen (PCNA) and to inhibit PCNA-dependent replication in vitro.

Studies have begun to provide some insights into the mechanisms by which CKIs inhibit the cyclin-CDKs. p21^CIP1 has two cyclin-binding sites termed Cy1 and Cy2, and one site for CDK named the K site. p21^CIP1 utilizes either the Cy1 or the Cy2 site for association with cyclin E and the K site for association with CDK2. Chen and coworkers showed that the Cy sites stabilize the interaction of the K site and directly inhibit kinase activity. Such simultaneous interaction with cyclin and CDK seems to be essential for optimal kinase inhibition (24). By contrast, isolated cyclin D or CDK4 does not associate with p21^CIP1. Also, the cyclin-CDK4 complex exclusively utilizes the Cy1 site for its interaction with p21^CIP1. In this situation, only one site is used to interact with p21^CIP1, which will result in partial inhibition of the kinase.

G1 Checkpoint

The G1 phase is a determinant phase of the cell cycle. During this phase, cells respond to extracellular signals and decide either to progress through the cell cycle and advance toward another division, or to withdraw from the cell cycle into a quiescent state also called G0. G1 events appear to occur sequentially, leading to a split of the G1 phase into subphases depending on the requirement and effects of specific growth factors, nutrients, and inhibitors. Late in G1, a critical time point has been individualized and termed the restriction point. It is now believed that the decision for a cell to divide occurs at this level. Before the restriction point, cells are dependent on signals provided by their environment. Once they pass the restriction point, they become refractory to these signals and follow an autonomous program that leads to mitosis (15, 25).

Passage through the restriction point and progression toward the S phase appear to be controlled by the G1 cyclins and their CDKs. Findings have led investigators to hypothesize a sequence for the involvement of these factors (13, 14, 16, 25, 26). The D-type cyclins (D1, D2, and D3) are induced before cyclins E and A. When cells transit from G0 to G1 in response to growth factor stimulation, synthesis of the D-type cyclins and assembly of these proteins with their CDKs (mostly CDK4 and CDK6) occur. Mitogenic stimulation plays a central role in the activation of the D-type cyclin-CDK complex. Indeed, D-type cyclins remain expressed as long as the growth factors are present in the cell environment (27, 28). Unlike the D-type cyclins, expression of cyclin E is normally periodic and maximal at the G1-S transition. This induction is associated with the formation of active cyclin E-CDK2 complexes. Once cells enter the S phase, cyclin E is degraded and CDK2 associates with cyclin A. The onset of cyclin A synthesis is observed late in G1 and seems to be essential for the beginning of DNA synthesis, as a defect in the formation of cyclin A-CDK2 complexes has been reported to inhibit S phase entry (11, 20).

Critical substrates of the G1 cyclin-CDK complexes are the retinoblastoma protein Rb and the related proteins p107 and p130 (29, 30). Unlike cyclin E, the D-type cyclins can directly bind to Rb and target CDK4 to this protein. In its hypophosphorylated form, Rb binds to a family of heterodimeric transcription factors called E2Fs. Phosphorylation of Rb releases E2Fs, enabling them to activate genes that are required for DNA replication including thymidine kinase, thymidylate synthetase, DNA polymerase , CDC2, as well as cyclin E and E2F. Rb phosphorylation appears to be triggered first by the D-type cyclin-CDK complexes and then by the cyclin E-CDK2 complexes. The onset of Rb phosphorylation induces an upregulation in E2F and cyclin E synthesis, which in turn leads to an increase in cyclin E-CDK2 complexes. Therefore, progression through the restriction point late in G1 is characterized by Rb phosphorylation, which shifts from being mitogen dependent and controlled by D-type cyclins to mitogen independent through the control of cyclin E-CDK2 complexes.

CONTROL OF G1 PROGRESSION IN SITUATIONS OF OXIDATIVE STRESS

The major threat posed by oxidant injury is, at the cellular level, damage of the genetic material. Repair of DNA damage is therefore critical in order to prevent accumulation of mutations that can disrupt normal cell growth control and lead to gross chromosomal aberrations in daughter cells (1, 7, 31, 32). To counter these threats to genomic integrity, cells can induce mechanisms that arrest the cell cycle, allowing DNA repair to take place in order to prevent replication of damaged genetic templates. In addition to cell cycle arrest, DNA damage may also induce apoptosis in order to eliminate cells in which damage is beyond repair.

From a number of studies, it is now well established that p53 plays a central role in response to DNA damage by its involvement in cell cycle arrest as well as in apoptosis (33, 34). The p53 gene encodes a nuclear phosphoprotein involved in the control of cell growth. p53 is one of the most commonly mutated genes found in human tumors. It can function as a sequence-specific DNA-binding protein that positively regulates gene expression (33). Products of genes transcriptionally activated by p53 include p21^CIP1; GADD45, which is induced on DNA damage and is involved directly in DNA nucleotide excision repair; Mdm2, which inactivates p53-mediated transcription and therefore forms an autoregulatory loop with p53 activity; cyclin G, which is a novel cyclin of unknown function with no cell cycle fluctuation levels; and Bax, which is a member of the BCL2 family that promotes apoptosis (35).

In situations of DNA damage, there is a rapid increase in the level of p53. As mentioned above, one target gene for p53 is the p21^CIP1 gene. Indeed, in response to various forms of DNA damage, the activated p53 turns on the p21 gene (33, 39). Activation of p21^CIP1 inhibits the activities of cyclin-CDK4 and -CDK2 complexes, preventing phosphorylation and activation of Rb, and consequently inhibiting the release of transcription factor E2F. In addition, p21^CIP1 binds to PCNA. The formation of p21^CIP1-PCNA complexes blocks PCNA activity as a DNA polymerase processivity factor in DNA replication, leaving its activity in DNA repair intact (35, 40). p21^CIP1 is not the sole protein involved in the downstream events in response to p53. As a matter of fact, fibroblasts derived from mice deficient in the p21 gene (null phenotype) display some ability to arrest in G1 in response to DNA damage. Such an observation has led investigators to suggest the existence of p21-independent pathways. One potential candidate for p53-induced G1 arrest is GADD45. Evidence suggests that GADD45 can interact directly with PCNA. Such interaction subsequently blocks DNA replication and possibly coordinately enhances nucleotide excision repair of damaged DNA.

Study of the mechanisms leading to p53 accumulation in response to DNA damage indicates that no de novo transcription is required. Indeed, use of cycloheximide in experiments does not prevent accumulation of p53 protein and partially abrogates the G1 arrest of cells exposed to DNA-damaging agents. These results indicate that the increase in p53 protein may be due in part to enhanced translation of pre-existing p53 transcripts (41). Another mechanism includes stabilization of p53. Normally, the p53 protein is present at low concentration due to its relatively short half-life, which is approximately 15 min. Such a half-life has been reported to increase by as much as 7-fold in cells after exposure to irradiation. Activation of p53 occurs within minutes of exposure to oxidative stress. This activation is achieved through posttranscriptional modification of p53 (42). In addition to changes in its levels, p53 may be modulated by functional modifications. As an example, p53 binding activity has been shown to be subject to redox regulation, the reduced form of p53 displaying increased binding capacity (43, 44).

In response to DNA damage, some signals ultimately lead to cell cycle arrest or to apoptosis. Data suggest a role for the ATM (ataxia telangiectasia mutated) gene product in the choice between these two pathways. Ataxia telangiectasia (A-T) is an autosomal recessive disease that is characterized by a variety of clinical and cellular defects and that appears to be associated with an altered response to DNA damage. Cloning of the ATM gene suggests that ATM plays a role in signal transduction (45, 46). Analysis of its sequence indicates homology with a family of proteins that include phosphoinositide 3-kinase, which strongly suggests a possible protein kinase activity for ATM (47). On the basis of various reports, the following model has emerged. Low levels of DNA damage are sensed by a pathway that involves the ATM protein, which may itself be the damage sensor. ATM might be required for a rapid induction of an active form of p53, most likely through phosphorylation, which leads to cell cycle arrest. By contrast, response to high levels of damage can occur in the absence of ATM through a less specific mechanism that is associated with a continuous induction of p53, which would trigger apoptosis. Such a model is consistent with the observation that in A-T cells, X-irradiation does not induce cell cycle arrest, yet triggers p53-dependent apoptosis (48).

	EPITHELIAL CELL RESPONSE TO OXIDANTS AND THE CYCLIN SYSTEM

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Studies of the mechanisms controlling G1 progression in situations of oxidative stress have been performed mainly in cellular systems other than epithelial cells. Several works using epithelial cells have provided evidence that p21^CIP1 plays a central role cellular response to injury (49). In lung epithelial cells, a dramatic increase in p21^CIP1 was observed on oxygen exposure, at both the levels of mRNA and protein (8). Furthermore, such induction appeared to be reversible in cells allowed to resume proliferation, with a rapid decrease in p21^CIP1 expression. One major target of p21^CIP1 action appeared to be the cyclin E-CDK2 complexes. The involvement of p21^CIP1 in the response of epithelial cells to oxidative stress was confirmed by the group of O'Reilly in lung epithelial cells and in experimental models (32, 53). In mice exposed to hyperoxia, analysis of total lung protein revealed a dramatic increase in the expression of p21^CIP1 after 48 h of oxygen exposure. Immunohistochemical staining showed that p21^CIP1 accumulated in the bronchial and alveolar epithelium. In contrast, expression of another CKI of the CIP/KIP family, p27^KIP1, was not altered by hyperoxia. The role of p21^CIP1 in the response of lung to oxidative stress has been confirmed by the use of p21^CIP1-deficient mice (54).

Mechanisms that regulate p21^CIP1 expression in situations of oxidative stress are likely to be numerous. At the posttranscriptional level, changes in p21^CIP1 mRNA stability through activation of the MAP kinase pathway have been reported (55). At the transcriptional level, the regulatory mechanisms include p53 as well as p53-independent pathways. The involvement of p53-independent mechanisms in the activation of p21^CIP1 is supported by several reports. Embryo fibroblasts obtained from p21^CIP1-deficient mice displayed impaired ability to undergo G1 arrest after DNA damage (56). Deng and coworkers observed in p21^CIP1-deficient mice that p53 apoptosis remains unchanged (57). In addition, in several cell systems, it has been shown that p21^CIP1 could mediate growth arrest in the absence of p53 (58).

The p21^CIP1 gene can be regulated by a rapidly growing list of physiological and pathological factors other than p53, including differentiation factors, growth factors, and cytokines. In situations of oxidative stress, the role of transforming growth factor  (TGF-) in p21^CIP1 induction and consequently in the inactivation of G1 cyclin-CDK activity should be considered (8). Much has been learned about the mechanisms by which TGF- propagates its signal. On direct phosphorylation by type I TGF- receptor, Smad2 or Smad3 binds to Smad4 to form a complex, which then translocates into the nucleus. In turn, the Smad complex activates transcription of target genes through interaction with other transcription factors or direct binding to DNA. It has been proposed that Smad complex can participate in the transcriptional regulation of p21^CIP1 through direct binding to Smad-binding sequences present in the promoter of the p21^CIP1 gene, and through interaction with the transcription factor Sp1. Using several cell systems, Pardali and coworkers recently provided data demonstrating physical association of Smads with Sp1 (59). One consequence of such interactions is the increased affinity of Sp1 for its cognate DNA element. Finally, the protein-protein interactions between Smads and Sp1 can account for the synergistic regulation of the p21^CIP1 promoter by these factors in response to TGF-. Whether interactions between Smads and Sp1 are involved in the regulation of p21^CIP1 in lung epithelial cells exposed to oxidants remains to be determined.

	CONCLUSION

TOP ABSTRACT INTRODUCTION CELL CYCLE CONTROL EPITHELIAL CELL RESPONSE TO... CONCLUSION REFERENCES

An appropriate response of epithelial cells to oxidative stress involves activation of checkpoint proteins that regulate cell cycle progression. Among these proteins, p53 plays a critical role in cell cycle arrest, apoptosis, and genome stability. Studies have also established the importance of p21^CIP1 in orchestrating the epithelial cell response to oxidant injury.