Hyperoxia Induces S-Phase Cell-Cycle Arrest and p21-Independent Cdk2 Inhibition in Human Carcinoma T47D-H3 Cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O₂, 40–64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21^Cip1. The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21^Cip1 or p27^Kip1 in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21^Cip1/p27^Kip1-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.     

Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.     

Cooperation of p27(Kip1) and p18(INK4c) in progestin-mediated cell cycle arrest in T-47D breast cancer cells

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.     

Cyclin D1 overexpression induces progestin resistance in T-47D breast cancer cells despite p27(Kip1) association with cyclin E-Cdk2

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.     

Non-malignant and tumor-derived cells differ in their requirement for p27Kip1 in transforming growth factor-beta-mediated G1 arrest

Transforming growth factor beta (TGF-beta) induces G(1) arrest in susceptible cells by multiple mechanisms that inhibit the G(1) cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-beta treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27(Kip1) in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G(1) arrest by TGF-beta was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-beta-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G(1) phase. Maintenance of the G(1) arrest was accompanied by increased association of the Cdk inhibitor p21(WAF-1/Cip-1) and the retinoblastoma family member p130(Rb2) in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G(1) arrest by TGF-beta in two tumor-derived lines. ASp27 transfection into two TGF-beta-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G(1) arrest despite the continued presence of TGF-beta. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G(1)-to-S phase progression.     

Retinoic acid-mediated G1 arrest is associated with induction of p27(Kip1) and inhibition of cyclin-dependent kinase 3 in human lung squamous carcinoma CH27 cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27(Kip1) and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21(CIP1/Waf1) proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor beta (RARbeta) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16(Ink4A), p15(Ink4B), p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin-Cdk complexes showed that RA increases p27(Kip1) expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27(Kip1). These results suggest that increases in the levels of p27(Kip1) and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

Protein kinase Cdelta inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1).

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCalpha has little effect on proliferation, whereas PKCdelta slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCalpha (PKCalphaEC) or PKCdelta (PKCdeltaEC) to determine the role of specific cell cycle regulatory proteins in the PKCdelta-induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKCdeltaEC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKCdeltaEC. In the PKCdeltaEC line, serum induced p27(Kip1) but not p16(Ink4a) or p21(Cip1). Serum did not affect p27(Kip1) levels in the control vascular endothelial cell line. Immunoprecipitation-Western blotting analysis of p27(Kip1) showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27(Kip1). In the PKCdeltaEC line, serum did not increase the amount of cyclin D1 bound to p27(Kip1). Transfection of full-length p27(Kip1) antisense into the PCKdeltaEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27(Kip1) in the PKCdelta-mediated S-phase arrest.     

Efficient down-regulation of cyclin A-associated activity and expression in suspended primary keratinocytes requires p21(Cip1)

When suspended in methylcellulose, primary mouse keratinocytes cease proliferation and differentiate. Suspension also reduces the activity of the cyclin-dependent kinase cdk2, an important cell cycle regulatory enzyme. To determine how suspension modulates these events, we examined its effects on wild-type keratinocytes and keratinocytes nullizygous for the cdk2 inhibitor p21(Cip1). After suspension of cycling cells, amounts of cyclin A (a cdk2 partner), cyclin A mRNA, and cyclin A-associated activity decreased much more rapidly in the presence than in the absence of p21(Cip1). Neither suspension nor p21(Cip1) status affected the stability of cyclin A mRNA. Loss of p21(Cip1) reduced the capacity of suspended cells to growth arrest, differentiate, and accumulate p27(Kip1) (a second cdk2 inhibitor) and affected the composition of E2F DNA binding complexes. Cyclin A-cdk2 complexes in suspended p21(+/+) cells contained p21(Cip1) or p27(Kip1), whereas most of the cyclin A-cdk2 complexes in p21(-/-) cells lacked p27(Kip1). Ectopic expression of p21(Cip1) allowed p21(-/-) keratinocytes to efficiently down-regulate cyclin A and differentiate when placed in suspension. These findings show that p21(Cip1) mediates the effects of suspension on numerous processes in primary keratinocytes including cdk2 activity, cyclin A expression, cell cycle progression, and differentiation.     

p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells

The cyclin-dependent kinase (Cdk) inhibitors p21(Cip1) and p27(Kip1) have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21(Cip1) and p27(Kip1) in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21(Cip1) or p27(Kip1) arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21(Cip1) arrested cells in both G(1) and G(2) cell cycle phases, p27(Kip1) blocked the G(1)/S-phase transition. Furthermore, although both p21(Cip1) and p27(Kip1) associated with Cdk6, only p27(Kip1) significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21(Cip1) triggered megakaryocytic maturation, whereas p27(Kip1) resulted in the expression of erythroid markers. Consistently, p21(Cip1) and p27(Kip1) were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21(Cip1) and p27(Kip1) in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable.     

Retinoic Acid-Mediated G1 Arrest Is Associated with Induction of p27 and Inhibition of Cyclin-Dependent Kinase 3 in Human Lung Squamous Carcinoma CH27 Cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27^Kip1 and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21^CIP1/Waf1 proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor β (RARβ) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16^Ink4A, p15^Ink4B, p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin–Cdk complexes showed that RA increases p27^Kip1 expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27^Kip1. These results suggest that increases in the levels of p27^Kip1 and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.     

Changes in the activity of cyclin-kinase complexes governing cell transition from G1 phase to DNA replication phase in E1A + c-Ha-ras transformants transfected with the bcl-2 gene

The antiproliferative effect of human bcl-2 gene transferred to E1A + c-Ha-ras-transformed rat embryo fibroblasts, which are characterized by the absence of cell cycle checkpoints after damage and by a high proapoptotic sensitivity was studied. Ionizing irradiation, adriamycin treatment, and serum starvation were shown to induce G1/S arrest in E1A + c-Ha-ras-transformants. Bcl-2 antiproliferative effect in E1A + c-Ha-ras-transformants was not associated with alterations in Cdk2, cyclin E and A contents. G1/S arrest following irradiation or serum starvation was accompanied by a decrease in kinase activity associated with cyclin E-cdk2, whereas G1/S arrest in tetraploid subpopulation after adriamycin treatment did not correlate with a decrease in cyclin E-associated kinase activity. Cyclin A-associated kinase activity did not decrease after any used treatment. Transfection of bcl-2 in E1A + c-Ha-ras-transformants resulted in elevated expression of cyclin-cdk complexes inhibitor p21/Waf-1, but not p27/Kip. Damaging agents caused p21/Waf-1 and p27/Kip accumulation, but bcl-2 overexpression did not restore functions of these inhibitors, since p21/Waf-1 and p27/Kip were unable to suppress cyclin-cdk complexes activity after damage. These results suggest that bcl-2 transfection in E1A + c-Ha-ras-transformants is likely to result in irradiation- or serum starvation-induced G1/S arrest accomplished by a selective decrease in cyclin E-associated kinase activity. Adriamycin-induced G1/S arrest seems to be realized via cyclin-cdk complexes activity-independent way involving antiproliferative targets downstream of cyclin E-cdk2 and cyclin A-cdk2 complexes.     

E7 abolishes raf-induced arrest via mislocalization of p21(Cip1)

The cellular response to oncogenic Ras depends upon the presence or absence of cooperating mutations. In the absence of immortalizing oncogenes or genetic lesions, activation of the Ras/Raf pathway results in a p21(Cip1)-dependent cellular arrest. The human papillomavirus oncoprotein E7 transforms primary cells in cooperation with Ras and abolishes p21(Cip1)-mediated growth arrest in the presence of various antimitogenic signals. Here we have utilized a conditional Raf molecule to investigate the effects of E7 on p21(Cip1) function in the context of Raf-induced cellular arrest. E7 bypassed Raf-induced arrest and alleviated inhibition of cyclin E-CDK2 without suppressing Raf-specific synthesis of p21(Cip1) or derepressing p21(Cip1)-associated CDK2 complexes. Activation of Raf led to nuclear accumulation of p21(Cip1), and we provide evidence that this effect is mediated by inhibition of Akt, a regulator of p21(Cip1) localization. Loss of Akt activity appears to be an important event in the cellular arrest associated with Raf-induction, since maintenance of Akt activity was necessary and sufficient to bypass Raf-induced arrest. In agreement, expression of E7 sustained Akt activity and reduced nuclear accumulation of p21(Cip1), resulting in decreased association between p21(Cip1) and cyclin E-CDK2. Taken together, these data suggest that E7 inhibits p21(Cip1) function in the context of Raf signaling by altering Raf-Akt antagonism and preventing the proper subcellular localization of p21(Cip1). We propose that E7 elicits a proliferative response to Raf signaling by targeting p21(Cip1) function via a novel mechanism.     

The protein SET regulates the inhibitory effect of p21(Cip1) on cyclin E-cyclin-dependent kinase 2 activity.

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1) has a dual role in the regulation of the cell cycle; it is an activator of cyclin D1-CDK4 complexes and an inhibitor of cyclins E/A-CDK2 activity. By affinity chromatography with p21(Cip1)-Sepharose 4B columns, we purified a 39-kDa protein, which was identified by microsequence analysis as the oncoprotein SET. Complexes containing SET and p21(Cip1) were detected in vivo by immunoprecipitation of Namalwa cell extracts using specific anti-p21(Cip1) antibodies. We found that SET bound directly to p21(Cip1) in vitro by the carboxyl-terminal region of p21(Cip1). SET had no direct effect on cyclin E/A-CDK2 activity, although it reversed the inhibition of cyclin E-CDK2, but not of cyclin A-CDK2, induced by p21(Cip1). This result is specific for p21(Cip1), since SET neither bound to p27(Kip1) nor reversed its inhibitory effect on cyclin E-CDK2 or cyclin A-CDK2. Thus, SET appears to be a modulator of p21(Cip1) inhibitory function. These results suggest that SET can regulate G(1)/S transition by modulating the activity of cyclin E-CDK2.     

Functions of p21Waf1 in Norm and in Stress

Cyclin-dependent kinase inhibitor p21^Waf1/Cip1 plays the key part in cell cycle arrest at the G1/S checkpoint in response to DNA damage, and is involved in the assembly of active cyclin–kinase complexes, in particular, cyclin D–Cdk4/6. Recent studies extended the range of known p21^Waf1/Cip1 functions. In addition to the cell-cycle control, p21^Waf1/Cip1 participates in important cell processes such as differentiation, senescence, and apoptosis. The balance of p21^Waf1/Cip1 functional activity appears to shift depending on the cell state (senescence, exposure to stress, expression of viral oncogenes). This is due to direct or indirect interaction with various modulators or to modification (phosphorylation, partial proteolysis) of p21^Waf1/Cip1. The review considers the structure of p21^Waf1/Cip1, its posttranslational modification, interactions with various cell or viral proteins, and their effects on the p21^Waf1/Cip1 function and on the cell.     

Insulin/insulin-like growth factor-I and estrogen cooperate to stimulate cyclin E-Cdk2 activation and cell Cycle progression in MCF-7 breast cancer cells through differential regulation of cyclin E and p21(WAF1/Cip1)

Estrogens and insulin/insulin-like growth factor-I (IGF-I) are potent mitogens for breast epithelial cells and, when co-administered, induce synergistic stimulation of cell proliferation. To investigate the molecular basis of this effect, a MCF-7 breast cancer cell model was established where serum deprivation and concurrent treatment with the pure estrogen antagonist, ICI 182780, inhibited growth factor and estrogen action and arrested cells in G(0)/G(1) phase. Subsequent stimulation with insulin or IGF-I alone failed to induce significant S-phase entry. However, these treatments increased cyclin D1, cyclin E, and p21 gene expression and induced the formation of active Cdk4 complexes but resulted in only minor increases in cyclin E-Cdk2 activity, likely due to recruitment of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/Cip1) into these complexes. Treatment with estradiol alone resulted in a greater increase in cyclin D1 gene expression but markedly decreased p21 expression, with a concurrent increase in Cdk4 and Cdk2 activity and subsequent synchronous entry of cells into S phase. Co-administration of insulin/IGF-I and estrogen induced synergistic stimulation of S-phase entry coincident with synergistic activation of high molecular mass (approximately 350 kDa) cyclin E-Cdk2 complexes lacking p21. To determine if the ability of estrogen to deplete p21 was central to these effects, cells stimulated with insulin and estradiol were infected with an adenovirus expressing p21. Induction of p21 to levels equivalent to those following treatment with insulin alone markedly inhibited the synergism between estradiol and insulin on S-phase entry. Thus the ability of estradiol to antagonize the insulin-induced increase in p21 gene expression, with consequent activation of cyclin E-Cdk2, is a central component of the synergistic stimulation of breast epithelial cell proliferation induced by simultaneous activation of the estrogen and insulin/IGF-I signaling pathways.     

sst2 somatostatin receptor mediates cell cycle arrest and induction of p27(Kip1). Evidence for the role of SHP-1.

Activation of the somatostatin receptor sst2 inhibits cell proliferation by a mechanism involving the stimulation of the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory events leading to sst2-mediated growth arrest are not known. Here, we report that treatment of Chinese hamster ovary cells expressing sst2 with the somatostatin analogue, RC-160, led to G1 cell cycle arrest and inhibition of insulin-induced S-phase entry through induction of the cyclin-dependent kinase inhibitor p27(Kip1). Consequently, a decrease of p27(Kip1)-cdk2 association, an inhibition of insulin-induced cyclin E-cdk2 kinase activity, and an accumulation of hypophosphorylated retinoblastoma gene product (Rb) were observed. However, RC-160 had no effect on the p21(Waf1/Cip1). When sst2 was coexpressed with a catalytically inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1 induced entry into cell cycle and down-regulation of p27(Kip1) and prevented modulation by insulin and RC-160 of p27(Kip1) expression, p27(Kip1)-cdk2 association, cyclin E-cdk2 kinase activity, and the phosphorylation state of Rb. In mouse pancreatic acini, RC-160 reverted down-regulation of p27(Kip1) induced by a mitogen, and this effect did not occur in acini from viable motheaten (mev/mev) mice expressing a mutant SHP-1 with markedly deficient enzymes. These findings provide the first evidence that sst2 induces cell cycle arrest through the up-regulation of p27(Kip1) and demonstrate that SHP-1 is required for maintaining high inhibitory levels of p27(Kip1) and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27(Kip1).     

Dual and opposing roles of ERK in regulating G(1) and S-G(2)/M delays in A549 cells caused by hyperoxia

This study explores the role of ERK activation in regulating G(1) and S-G(2)/M delays during hyperoxia. We demonstrate here that exposing A549 human alveolar type 2 adenocarcinoma cells to hyperoxia (95% O(2)) for 0.5-24 h time-dependently increases phospho-ERK, phospho-p53(Ser15), p53, and p21(CIP1) protein levels. Decreasing phospho-ERK with the pharmacological inhibitors, PD98059 and U0126, markedly suppresses hyperoxia-stimulated phospho-p53(Ser15), p53, and p21(CIP1), and also restores the hyperoxia-reduced kinase activities of cyclin D1/E1-Cdks. Our results suggest that ERK activation during hyperoxia contributes to the p53/p21-mediated G(1) checkpoint. However, inhibition of ERK signaling during hyperoxia further delays S-phase entry and progression. Hyperoxia induces significant expression of cyclin A/B1 and translocation of cyclin A into nuclei while marginally decreasing cyclin A/B1-Cdks kinase activities, which may be related to nuclear association with p21. Interestingly, inhibition of ERK signaling markedly suppresses the elevation of cyclin A/B1 proteins and cyclin A/B1-Cdks kinase activities during hyperoxia. Taken together, the results presented here suggest that hyperoxia-activated ERK acts upstream of p53 and p21 to suppress G(1)-Cdk activities; however, it is also required for induction of cyclin A/B1 and maintenance of cyclin A/B1-Cdk activities that oppose delays in S-phase entry and progression.