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DNA methylation/demethylation constitutes a major consequence in all biological processes involving transcription, differentiation, development, DNA repair, recombination, and chromosome organization. Our earlier studies established that demethylation of CpG rich sequence by human DNA demethylase activity (5-methylcytosine-DNA glycosylase (5MeC-DNA glycosylase)) resembles "base excision DNA repair activity" and creates single-strand breaks on DNA that is associated with proliferating cell nuclear antigen (PCNA). Here in this report, we have identified differential DNA demethylation targets (hemi-methylated vs. fully-methylated) in normal cell lines and cancerous cell lines, and a shortened G(0)/G(1) resting time in cancerous cell lines than the normal cell lines. We have identified that in normal HFL1 fibroblast cell line, DNA demethylase activity targets hemi-methylated CpG specific sites on DNA. This normal cell line DNA demethylase activity associates with PCNA immune complex that is inhibited by CDKI proteins p21(waf1)/Gadd45alpha and Gadd45beta. While in cancerous LnCap and BT20 cell lines DNA demethylase activity targets fully-methylated CpG specific sites on DNA. This cancer cell line DNA demethylase activity is not associated with PCNA immune complex and is not inhibited by CDKI proteins p21(waf1)/Gadd45alpha and Gadd45beta. We have also identified that the fully-methylated CpG specific DNA demethylase activity from cancerous cell lines to associate with p300/CBP protein. These significant observations of variable targets of DNA demethylation and alternate partner proteins for DNA demethylase activity in cancerous cell lines are discussed in terms of double-strand DNA breaks versus single-strand DNA breaks and their role in the exit of G(1)/G(2) cell cycle stages. Also, the inability of cell cycle regulatory proteins like PCNA, p21(waf1), and Gadd45 to control DNA demethylase activity in cancerous cell lines is discussed in terms of accelerated G(1)/G(2) cell cycle stage exit to facilitate unregulated cellular proliferation, loss of control of chromosomal organization, and the development of oncogenesis in cancerous cell lines., (Copyright 2003 Wiley-Liss, Inc.)

beta-Lapachone, a novel anti-neoplastic drug, induces various cancer cells to undergo apoptosis. In a previous report, we showed that beta-lapachone-induced apoptosis of HL-60 cells is mediated by oxidative stress. However, in the present study, we found that beta-lapachone-induced apoptosis of human prostate cancer (HPC) cells may be independent of oxidative stress. In contrast to the 10-fold beta-lapachone-induced increase in H(2)O(2) production seen in HL-60 cells, only a 2- to 4-fold increase was observed in HPC cells. N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibited the apoptosis in DU145 cells after 12 h exposure to beta-lapachone. Nonetheless, NAC, along with other antioxidants, failed to exert similar effect in HPC cells subjected to beta-lapachone treatment for 24 h. Under this premise, we suggest that the oxidative stress may not play a crucial role in beta-lapachone-mediated HPC cell apoptosis. Here we demonstrate that damage to genomic DNA is the trigger for the apoptosis of HPC cells induced by beta-lapachone. According to our results, beta-lapachone stimulates DNA dependent kinase expression and poly(ADP-ribose) polymerase cleavage in advance of significant morphological changes. beta-Lapachone promotes the expression of cyclin-dependent kinase (cdk) inhibitors (p21(WAF1) and p27(Kip1)), induces bak expression, and subsequently stimulates the activation of caspase-7 but not of caspase-3 or caspase-8 during the apoptosis of HPC cells. Taken together, these results suggest that the signaling pathway involving the beta-lapachone-induced apoptosis of HPC cell may be by DNA damage, induction of cdk inhibitors (p21 and p27), and then subsequent stimulation of caspase-7 activation.

Cell cycle checkpoints ensure orderly progression of events during cell division. A microtubule damage (MTD)-induced checkpoint has been described in G(1) phase of the cell cycle (G(1)MTC) for which little is known. The present study shows that the G(1)MTC is intact in activated T lymphocytes from mice with the p21(waf-1) gene deleted. However, p21(waf-1) gene deletion does affect the ratio of cells that arrest at the G(1)MTC and the spindle checkpoint after MTD. The G(1)MTC arrests T lymphocytes in G(1) prior to cdc2 up-regulation and prior to G(1) arrest by p21(waf-1). Once cells have progressed past the G(1)MTC, they are committed to chromosome replication and metaphase progression, even with extreme MTD. The G(1)MTC is also present in a human myeloid cell line deficient in p21(waf-1) gene expression. The p21-independent G(1)MTC may be important in cellular responses to MTD such as those induced by drugs used to treat cancer.

Background: The cyclin-dependent kinase inhibitors (CKIs) have different patterns of expression in vascular diseases. The Kip/Cip CKIs, p27(Kip1) and p21(Cip1), are upregulated during arterial repair and negatively regulate the growth of vascular smooth muscle cells (VSMCs). In contrast, the Ink CKI, p16(Ink4), is not expressed in vascular lesions. We hypothesized that a variation in the inactivation of cdk2 and cdk4 during the G(1) phase of the cell cycle by p27(Kip1), p21(Cip1), and p16(Ink4) leads to different effects on VSMC growth in vitro and in vivo., Methods and Results: The expression of p27(Kip1) and p21(Cip1) in serum-stimulated VSMCs inactivated cdk2 and cdk4, leading to G(1) growth arrest. p16(Ink4) inhibited cdk4, but not cdk2, kinase activity, producing partial inhibition of VSMC growth in vitro. In an in vivo model of vascular injury, overexpression of p27(Kip1) reduced intimal VSMC proliferation by 52% (P<0.01) and the intima/media area ratio by 51% (P<0.005) after vascular injury and gene transfer to pig arteries, when compared with control arteries. p16(Ink4) was a weak inhibitor of intimal VSMC proliferation in injured arteries (P=NS), and it did not significantly reduce intima/media area ratios (P=NS), which is consistent with its minor effects on VSMC growth in vitro., Conclusions: p27(Kip1) and p21(Cip1) are potent inhibitors of VSMC growth compared with p16(Ink4) because of their different molecular mechanisms of cyclin-dependent kinase inhibition in the G(1) phase of the cell cycle. These findings have important implications for our understanding of the pathophysiology of vascular proliferative diseases and for the development of molecular therapies.

The ability of the cardiac myocyte to divide ceases shortly after birth. Thus, following severe injury, e.g., during myocardial infarction, the mature heart is unable to regenerate new tissue to replace the dead or damaged tissue. The identification of the molecules controlling the cessation of myocyte cell division may lead to therapeutic strategies which aim to re-populate the damaged myocardial area. Hence, we have determined the cell cycle profile, expressions and activities of the cyclin-dependent kinase inhibitors (CDKIs), p21CIP1 and p27KIP1, during rat ventricular myocyte development. Fluorescent activated cell sorting (FACS) analyses showed the percentage of S phase myocytes to be decreased significantly throughout development, concomitant with a significant increase in the percentage of G0/G1 and G2/M phase cells. The expression of p21CIP1 and p27KIP1 increased significantly throughout cardiac development and complexed differentially with a number of cyclins and CDKs. Furthermore, an adult myocyte extract reduced neonatal myocyte CDK2 kinase activity significantly (>30%, p<0.05) whereas immunodepletion of p21CIP1 from adult lysates restored CDK2 kinase activity. Thus, p21CIP1 and p27KIP1 may be important for the withdrawal of cardiac myocytes from the cell cycle and for maintaining the G0/G1 and G2/M phase blockades.

The regulation of cyclin D-dependent kinase activity in tissue regeneration in vivo has not been fully described. In young adult rat liver after 70% partial hepatectomy, the association of cyclin D1 with cdk4 was significantly promoted during G1 phase and was maximal at 18 hr, corresponding mainly to late G1. Cyclin D1-dependent kinase activity also strongly increased during G1 phase. The timing of the induction of cyclin D1 / cdk4 complex assembly correlated well with that of cyclin D1-dependent kinase activity. At 18 hr after partial hepatectomy, the amounts of CDK inhibitors p21(CIP1) and p27(KIP1) were also maximal, while only one-tenth of p21(CIP1) and of p27(KIP1) was associated with cyclin D1. These findings suggest that cyclin D1, cdk4 and their association act as promoting factors, and that both p21(CIP1) and p27(KIP1) may have physiological functions as adaptor proteins in additions to their roles as CDK inhibitors in rat liver regeneration., (Copyright 1998 Academic Press.)

Treatment of mammalian cells by DNA-damaging agents leads to various cellular responses. At sufficiently high dosage, cisplatin blocks cell proliferation and finally kills cells; this effect is the basis for its widespread use as an anticancer drug. Cisplatin-treated cells arrest in the G1 phase of the cell cycle, most likely due to a signal generated by the stabilization of p53 and the subsequent induction of p21WAF-1/Cip1. We show here that cisplatin-treated mammalian cells accumulate normal levels of cyclin D1 and cyclin E but fail to produce cyclin A. The block to cyclin A gene expression occurs at the level of transcription and is mediated by an E2F binding site in the cyclin A promoter. It is shown here that, upon cisplatin treatment, transcriptionally active free E2F becomes limiting, coincident with the accumulation of hypophosphorylated species of the retinoblastoma protein family. Immunoprecipitation experiments suggest that the loss of free E2F results, at least in part, from the sequestration of E2F-4/DP-1 heterodimers by p107. A role for the kinase inhibitor p21WAF-1/Cip1 in repression of the cyclin A promoter is supported by our finding that ectopic expression of p21WAF-1/Cip1 is sufficient to inhibit transcription from the cyclin A gene, dependent on the E2F site. The data establish the E2F site in the human cyclin A promoter as a key target for the signaling pathway leading to G1 arrest in response to DNA damage by cisplatin and potentially other genotoxic agents.

The p21MDA6 gene product induces cell cycle arrest in p53-null human leukemic cells exposed to differentiation stimuli. We employed an HL-60 cell line stably transfected with a p21MDA6 antisense construct to compare the effects of p21MDA6 dysregulation on the response of myeloid leukemia cells to differentiating and cytotoxic agents. Antisense-expressing cells (HL-60/AS5) treated with 5 nM PMA for 24 h exhibited attenuated induction of p21MDA6 compared to empty vector controls (HL-60/V2). This phenomenon was accompanied by a reduction in the percentage of cells undergoing G1 arrest (67.6 +/- 4.7 vs 82.9 +/- 1.3; P < or = 0.01) and expressing the monocytic maturation marker cd11b (35.5 +/- 2.8 vs 50.5 +/- 2.4; P < or = 0.005). Although HL-AS5 and HL-60/V2 cells did not exhibit obvious differences in the phosphorylation status of the retinoblastoma protein (pRB), in E2F complex formation, or in p27klp1 induction following PMA exposure, inhibition of activity of cyclin-dependent kinase-2 was attenuated in the antisense-expressing line. A 24-h exposure to 5 nM PMA also reduced the cloning efficiency of HL-60/V2 cells to a significantly greater extent than HL-60/AS5 cells (ie to 30.1 +/- 7.0 vs 57.2 +/- 5.6 of controls; P < or = 0.01). In contrast to the disparate responses to PMA, HL-60/AS5 and HL-60/V2 cells treated with the antimetabolite 1-beta-D-arabinofurano-sylcytosine (Ara-C; 10 microM for 6 h) displayed equal susceptibility to G1 arrest, apoptosis, and inhibition of clonogenicity, phenomena unaccompanied by p21MDA6 and p27klp1 induction, or pRB dephosphorylation. These observations indicate that dysregulation of p21MDA6 in p53-null human myeloid leukemia cells interferes with PMA-related G1 arrest, CDK-2 inhibition, differentiation, and loss of clonogenic survival in the absence of obvious alterations in pRB phosphorylation status or E2F complex formation. They also provide functional evidence that p21MDA6 induction does not appear to be required for Ara-C-induced apoptosis, G1 arrest, or the resulting reduction in the self-renewal capacity of HL-60 cells.

The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors p21(CIP), p27(KIP), and p57(KIP2) all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K(a), respectively, mostly because of a decrease in K(off). At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a G(0) arrest. In agreement with our other data, cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

The experiments described in this report were undertaken to define the parameters that regulate cyclin E/cyclin-dependent kinase 2 (Cdk2) kinase activity in mitotically quiescent, serum-starved fibroblastic cells and in cells that had been stimulated to enter the cell cycle and progress through G1 into S phase. We have analyzed the expression of cyclin E and Cdk2, the extent to which these two proteins form complexes, and the enzymatic activity of cyclin E/cdk2 kinase. Particular attention was focused upon subcellular localization and the effect of compartmentalization on the association between cyclin E and Cdk2. In addition, we have examined the interaction of cyclin E/Cdk2 complexes with two well-characterized inhibitors of Cdk2 kinase activity, Cip1 and Kip1. This represents the first report in which all of these parameters have been measured simultaneously in a single, normal diploid cell line. In G0 cells, there is abundant cyclin E and Cdk2, yet there is little or no detectable Cdk2-dependent histone H1 kinase activity. After serum stimulation, there is a rapid increase in the amount of cyclin E that is bound to Cdk2, although there is no significant change in the abundance of either the cyclin or the Cdk. Immunocytochemical data indicate that cyclin E, Cip1, and Kip1 are located within the nuclei of cell in G0, but very little Cdk2 is observed within the nuclei of serum-starved cells. Cdk2 rapidly enters the nucleus upon serum stimulation. The abundance of the cyclin E/Cdk2 complex increases to the extent that the binding capacity of Cip1 is exceeded about 8-12 h after serum stimulation. The abundance of Kip1 decreases at the same time that the Cip1 threshold is exceeded, so that cyclin E/Kip1-containing complexes decrease by 90% within 8-12 h. Cyclin E/Cdk2 kinase activity begins to increase rapidly thereafter, reaching a maximum level about 16 h after serum stimulation. We have been unable to detect histone H1 kinase activity in complexes that contain cyclin E bound to Kip1 or Cip1. We conclude that compartmentalization is the predominant barrier to activation of cyclin E-dependent kinases in quiescent cells. Cip1 and Kip1 serve to prevent premature activation of cyclin E/Cdk2 complexes that form during G0 or early G1.

p21Sdi1/WAF1/Cip1 inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the phosphorylation of growth-regulatory proteins, including the retinoblastoma tumor suppressor protein (pRb). The ultimate effector(s) of p21 growth inhibition, however, is largely a matter of conjecture. We show that p21 inhibits the activity of E2F, an essential growth-stimulatory transcription factor that is negatively regulated by unphosphorylated pRb. p21 suppressed the activity of E2F-responsive promoters (dihydrofolate reductase and cdc2), but E2F-unresponsive promoters (c-fos and simian virus 40 early) were unaffected. Moreover, the simian virus 40 early promoter was rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 deletion mutants showed good agreement in their abilities to inhibit E2F transactivation and DNA synthesis; and E2F-1 (which binds pRb), but not E2F-4 (which does not), reversed both inhibitory effects of p21. Despite the central role for pRb in regulating E2F, p21 suppressed growth and E2F activity in cells lacking a functional pRb. Moreover, p21 protein (wild type but not mutant) specifically disrupted an E2F-cyclin-dependent protein kinase 2-p107 DNA binding complex in nuclear extracts of proliferating cells, whether or not they expressed normal pRb. Thus, E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for the inhibition of growth or E2F-dependent transcription.

A mutated cyclin-dependent kinase 4 (CDK4) was identified as a tumor-specific antigen recognized by HLA-A2. 1-restricted autologous cytolytic T lymphocytes (CTLs) in a human melanoma. The mutated CDK4 allele was present in autologous cultured melanoma cells and metastasis tissue, but not in the patient's lymphocytes. The mutation, an arginine-to-cysteine exchange at residue 24, was part of the CDK4 peptide recognized by CTLs and prevented binding of the CDK4 inhibitor p16INK4a, but not of p21 or of p27KIP1. The same mutation was found in one additional melanoma among 28 melanomas analyzed. These results suggest that mutation of CDK4 can create a tumor-specific antigen and can disrupt the cell-cycle regulation exerted by the tumor suppressor p16INK4a.

Phosphorylation of cyclin-dependent kinases (CDKs) by the CDK-activating kinase is required for the activation of CDK enzymes. Members of two families of CDK inhibitors, p16/p18 and p21/p27, become physically associated with and inhibit the activity of CDKs in response to a variety of growth-modulating signals. Here, we show that the representative members of both families of CDK inhibitors, p21waf1,cip1, p27kip1, and p18, can prevent the phosphorylation of their CDK partners, CDK2 and CDK6, by CDK-activating kinase. No direct interaction between CDK-activating kinase and the CDK inhibitors could be detected, suggesting that binding of these CDK inhibitors to CDK subunits renders CDK inaccessible to the CDK-activating kinase phosphorylation. These findings suggest that a general mechanism of CDK inhibitor function is to block the phosphorylation of CDK enzymes by CDK-activating kinase.

In the fission yeast Schizosaccharomyces pombe, the cdc10+/SWI family members constitute the cell cycle 'start' genes. res1+ and res2+ are the newly identified members of this family and encode putative association partners of the Cdc10 protein. The Pat1 kinase plays a pivotal role in switching between vegetative growth and sexual development, and its inactivation in haploid cells induces unconditional growth arrest and subsequent meiosis. We have identified as an extragenic suppressor of a temperature sensitive pat1-114 mutant, a new B-type cyclin that negatively regulates conjugation by interacting with these 'start' genes. This cyclin, named Cyc17, is highly homologous with Cdc13, but has no detectable activity as a mitotic cyclin. Deletion of cyc17+ markedly enhances conjugation, despite the presence of nitrogen source, and accelerates growth arrest in G1 upon nitrogen starvation. Conversely, overexpression of the cyc17+ gene strongly inhibits conjugation. The cyc17+ gene is transcribed into 3.2 kb poly(A)+ and 3.0 kb poly(A)- RNAs. Only the poly(A)+ species is expressed during vegetative growth and periodically with a peak in the G1 and S phases of the cell cycle. On the other hand, the poly(A)- transcript is highly induced during conjugation. This induction is lost in res2- cells, whereas the poly(A)+ transcript is significantly reduced in res1- cells. However, the mating inhibition as well as the ability to rescue the pat1 mutation by overexpression of res1+ and res2+ are totally abolished in cyc17- cells. Thus, in S.pombe, a B-type cyclin, regulated by the newly identified cell cycle 'start' genes, plays a crucial role in the control of sexual development.

The transcription factor DRTF1/E2F is believed to play an important role in regulating cellular proliferation because it undergoes a series of periodic interactions with proteins that are known to be important regulators of the cell cycle, including the retinoblastoma gene product (pRb) and cyclin A. Furthermore, certain viral oncogene products, such as adenovirus E1a, disrupt these DRTF1/E2F complexes by sequestering the associated proteins. p107, a protein that is structurally related to pRb, also binds to DRTF1/E2F, and in this study we investigate the functional consequences of this interaction. We show that p107 can repress E2F binding site-dependent transcription and that the adenovirus E1a protein overcomes p107-mediated transcriptional repression. Two distinct but related proteins, pRb and p107, can therefore repress transcription driven by DRTF1/E2F, whereas the E1a protein overrides the repression. We also demonstrate that the transcription repressing properties of p107 and pRb are influenced by the cell type and by differentiation, because neither protein affects transcription in F9 embryonal carcinoma (EC) cells but both do so efficiently in differentiated derivatives. In this respect, the repressing activities of pRb and p107 inversely correlate with the presence of the cellular E1a-like activity previously documented in F9 EC cells. These data suggest that p107 and pRb exert their biological activities in some but not all cell types.