p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.     

Cholesterol starvation decreases p34(cdc2) kinase activity and arrests the cell cycle at G2.

As a major component of mammalian cell plasma membranes, cholesterol is essential for cell growth. Accordingly, the restriction of cholesterol provision has been shown to result in cell proliferation inhibition. We explored the potential regulatory role of cholesterol on cell cycle progression. MOLT-4 and HL-60 cell lines were cultured in a cholesterol-deficient medium and simultaneously exposed to SKF 104976, which is a specific inhibitor of lanosterol 14-alpha demethylase. Through HPLC analyses with on-line radioactivity detection, we found that SKF 104976 efficiently blocked the [(14)C]-acetate incorporation into cholesterol, resulting in an accumulation of lanosterol and dihydrolanosterol, without affecting the synthesis of mevalonic acid. The inhibitor also produced a rapid and intense inhibition of cell proliferation (IC(50) = 0.1 microM), as assessed by both [(3)H]-thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or longer resulted in the accumulation of cells specifically at G2 phase, whereas both the G1 traversal and the transition through S were unaffected. The G2 arrest was accompanied by an increase in the hyperphosphorylated form of p34(cdc2) and a reduction of its activity, as determined by assaying the H1 histone phosphorylating activity of p34(cdc2) immunoprecipitates. The persistent deficiency of cholesterol induced apoptosis. However, supplementing the medium with cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, completely abolished these effects, whereas mevalonate was ineffective. Caffeine, which abrogates the G2 checkpoint by preventing p34(cdc2) phosphorylation, reduced the accumulation in G2 when added to cultures containing cells on transit to G2, but was ineffective in cells arrested at G2 by sustained cholesterol starvation. Cells arrested in G2, however, were still viable and responded to cholesterol provision by activating p34(cdc2) and resuming the cell cycle. We conclude that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34(cdc2) activity.     

The crystal structure of p13suc1, a p34cdc2-interacting cell cycle control protein.

p13suc1 binds to p34cdc2 kinase and is essential for cell cycle progression in eukaryotic cells. The crystal structure of S.pombe p13suc1 has been solved to 2.7 A resolution using data collected at the ESRF source, Grenoble, from both native crystals and crystals of a seleno-methionine derivative. The starting point for structure solution was the determination of the six selenium sites by direct methods. The structure is dominated by a four-stranded beta-sheet, with four further alpha-helical regions. p13suc1 crystallizes as a dimer in the asymmetric unit stabilized by the binding of two zinc ions. A third zinc site stabilizes the higher-order crystal packing. The sites are consistent with a requirement for zinc during crystal growth. A likely site for p13suc1-protein interaction is immediately evident on one face of the p13suc1 surface. This region comprises a group of conserved, exposed aromatic and hydrophobic residues below a flexible negatively charged loop. A conserved positively charged area would also present a notable surface feature in the monomer, but is buried at the dimer interface. p13suc1 is larger than its recently solved human homologue p9CKS2, with the extra polypeptide forming a helical N-terminal extension and a surface loop between alpha-helices 3 and 4. Notably, p13suc1 does not show the unusual beta-strand exchange that creates an intimate p9CKS2 dimer. p13suc1 cannot oligomerize to form a stable hexamer as has been proposed for p9CKS2.     

Polyamine metabolism in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620)

The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth--most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.     

Protein kinase B, P34cdc2 kinase, and p21 ras GTP-binding in kidneys of aging rats

Renal nephropathy present in male Wistar rats more than 13 months of age was reported as an indication that the rats were in renal failure. In this study, the renal tissue damage at 14 months of age in male Munich Wistar rats was similar to that reported for Wistar rats, indicating that Munich Wistar rats could be another model for study of kidney function in the aging rat. The usual renal response to injury involves increased cell division and/or reparative processes that involve tyrosine kinase activity (TyrK) and/or guanosine triphosphate-binding (G) protein signal trans-duction pathways. This study reveals the presence of renal tissue damage coinciding with significantly reduced activity of Ras, Akt, and p34cdc2 kinase, the signaling proteins that regulate cell division and/or growth, in renal cortical tissues of aging rats compared to young rats (P < 0.005, P < 0.005, and P< 0.001, respectively). These results suggest that proteins involved in signal transduction pathways associated with cell replication are downregulated in the aging kidney cortex at a time when renal cellular damage is also present.     

Growth inhibition of a colonic adenocarcinoma cell line (HT29) by T cells specific for mutant p21 ras

Interleukin-2 (IL-2)-activated killer cells, also referred to as lymphokine-activated killer (LAK) cells, are stimulated by tumor cells to express cytotoxic activity and to also secrete cytokines such as interferon (IFN) and tumor necrosis factor (TNF ). We previously reported that secretion of cytokines by IL-2-activated T cells (LAK-T cells) is dependent on the initial cross-linking of the T cell receptor (TCR)-CD3-molecular complex, but the cross-linking of accessory molecules, such as LFA-1, CD2, CD44 and CD45, on LAK-T cells can enhance this cytokine production. We have developed an approach involving interspecific gene transfer to define further the contributions of LFA-1 and CD2 to the activation of LAK-T cells. The genes for huICAM-1 (a ligand for LFA-1) and huLFA-3 (a ligand for CD2) were transfected singly and in combination into a null mouse melanoma background, and clonal populations of cells that stably express ICAM-1 and/or LFA-3 were derived. Expression of the introduced ICAM-1 and/or LFA-3 by transfected cells enhanced their ability to bind LAK-T cells; the LFA-1/ICAM-1-mediated binding was not further enhanced by activation with phorbol 12-myristate 13-acetate. ICAM-1- and/or LFA-3-transfected cells, in the presence of immobilized anti-CD3, exhibited a greater ability to stimulate IFN secretion by LAK-T cells compared to the untransfected parental lines. This experimental system, which allows ICAM-1/LFA-1 and CD2/LFA-3 interactions to occur on the LAK-T cell at a site distal from the anti-CD3 signal, extends our understanding of LAK-T cell activation by establishing that both LFA-1/ICAM-1 and CD2/LFA-3 can mediate co-stimulation via adhesion and signaling events.     

Both calcium and ROS as common signals mediate Na(2)SeO(3)-induced apoptosis in SW480 human colonic carcinoma cells

Recent studies have shown that reactive oxygen species (ROS) play a crucial role in Se-induced cell apoptosis. A number of studies have demonstrated that perturbed cellular calcium homeostasis has been implicated in apoptosis. The main objective of this study was to evaluate the role of Ca(2+) in Na(2)SeO(3)-induced apoptosis and the relationship between Ca(2+) and ROS in human colonic carcinoma cells SW480. When SW480 cells were exposed to 25-100 microM Na(2)SeO(3), both cell apoptosis and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Na(2)SeO(3) was able to induce increase of [Ca(2+)](i) and ROS production and disrupt mitochondrial membrane potential (Delta Psi m) in SW480 cells monitored by using a confocal laser scanning microscope. Ca(2+) channel inhibitor CoCl(2) and an intracellular Ca(2+) chelator o-phtalaldehyde, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA) completely inhibited [Ca(2+)](i) increase, but catalase had no effect on Na(2)SeO(3)-induced increase of [Ca(2+)](i). BAPTA-AM, CoCl(2), and mitochondrial Ca(2+) uptake inhibitor ruthenium red blocked Delta Psi m dissipation. The increase of ROS was also suppressed by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine and catalase, respectively. The mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) completely inhibited Na(2)SeO(3)-induced ROS increase. This showed that ROS increase is due to mitochondrial Ca(2+) overload. The Na(2)SeO(3)-induced apoptosis of SW480 cells was also inhibited by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine, and catalase, respectively. The results mentioned above imply that both calcium and Ca(2+)-dependent ROS as a signal molecule mediate apoptosis induced by Na(2)SeO(3) in SW480 cells.     

Influence of the G2 cell cycle block abrogator pentoxifylline on the expression and subcellular location of cyclin B1 and p34cdc2 in HeLa cervical carcinoma cells

The progression of cells from G₂ into mitosis is mainly controlled by formation of the cyclin B1/p34^cdc2 complex. The behaviour of this complex in the irradiation-induced G₂ cell cycle delay is still unclear. A prior study demonstrated that the expression of the cyclin B1 protein is reduced by irradiation, and restored to control levels by the methylxanthine drug pentoxifylline, which is a potent G₂ block abrogator. The present study shows that irradiation, and 2 mM pentoxifylline affect the expression of the cyclin-dependent kinase p34^cdc2 in HeLa cells. Irradiation induces p34^cdc2 levels to increase and cyclin B1 levels to decrease. Addition of pentoxifylline at the G₂ maximum reverses these trends. This is also evident from the cyclin B1/p34^cdc2 ratios which decline after irradiation and are rapidly restored to control levels upon addition of pentoxifylline. It is concluded that cyclin B1 and p34^cdc2 protein expression are important events and act in concert to control the irradiation induced G₂ block. Analysis of cyclin B1 expression in whole cells and in isolated nuclei furthermore show that cyclin B1 is translocated from the nucleus into the cytoplasm when the G₂ block is abrogated by pentoxifylline.     

A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.     

Trichosanthin-induced specific changes of cytoskeleton configuration were associated with the decreased expression level of actin and tubulin genes in apoptotic Hela cells

Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-cancer activities through apoptosis pathway. However, little is known about the effects of TCS on the cytoskeleton configuration and expression of actin and tubulin genes in Hela cell apoptosis. In the present study, apoptotic cytoskeleton structures were observed by confocal immunofluorescence microscopy, absolute amounts of actin and tubulin subunit mRNAs were determined by quantitative real-time PCR assays (QRT-PCR). Our results showed that the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization, depolymerized microfilaments (MFs) accumulated in the coarsened cytoplasm and apoptotic bodies, followed by the formation of a ring microtubule (MT) structure beneath the plasma membrane. Importantly, apoptosis occurred by a suppression of actin and tubulin subunit gene expression. In particular, a rapid decrease in the amounts of gamma-actin mRNA preceded that of beta-actin; alpha- and beta-tubulin mRNAs were subsequently down-regulated in the later stage of Hela cell apoptosis. These results suggested that the execution of Hela cell apoptosis induced by TCS accompanied the specific changes of cytoskeleton configuration and, significantly, decreased the expression level of actin and tubulin subunit genes in different stages.     

Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts

We have examined the effects of elevating the intracellular levels of p34cdc2 kinase by microinjection into living mammalian cells. These studies reveal rapid and dramatic changes in cell shape with cells becoming round and losing the bulk of their cell-substratum contact. Such effects were induced at all times in the cell cycle except at S phase and were fully reversible at S phase or mitosis. Similar results were obtained with the homogeneous catalytic subunit of p34cdc2 kinase or p34cdc2 kinase associated with cyclin B. These alterations were accompanied by a marked reduction in interphase microtubules without the spindle formation, actin microfilament redistribution, and premature chromatin condensation. Although these changes closely mimic the events occurring during early phases of mitosis, p34cdc2 kinase-injected cells were not induced to pass further into division. These data provide detailed evidence that p34cdc2 kinase plays a major prerequisite role in the rearrangement of cellular structures associated with mammalian cell mitosis.     

Regulation of synthesis of p34cdc2 and its homologues and their relationship to p110Rb phosphorylation during cell cycle progression of normal human T cells.

In yeast, the protein kinase p34cdc2 plays a role in regulating both the G2 to M and G1 to S phase transitions. The discovery of multiple homologues of the protein in cells of higher eukaryotic organisms suggests that different cell cycle regulatory events may be performed by different kinases in such cells. Here, the synthesis and metabolism of the human forms of these proteins are described in a normal human cell type, peripheral blood T lymphocytes that have been stimulated to enter the cell cycle in vitro. Using a carboxyl-terminus antiserum specific for true p34cdc2, the protein could first be found in T cells at about 24 to 30 h after stimulation, just before the initiation of DNA synthesis. Three forms of the enzyme could be resolved by denaturing gel electrophoresis: an unphosphorylated form with an apparent molecular mass of 34,500 daltons and two phosphorylated derivatives. In cells synchronized at G2/M phase with nocodazole, p34 was almost entirely in the unphosphorylated form whereas the phosphorylated derivatives were more predominant in cultures arrested at the G1/S border with aphidicolin. The relationship of p34 synthesis to the phosphorylation of p110Rb, an event known to be associated with passage through late G1 and/or the G1/S phase transition, was also investigated. It was noted that p110Rb phosphorylation began before p34 synthesis first became detectable. Furthermore, it appeared that the two events could be largely uncoupled by treating cells with deferoxamine (10 microM), an iron chelating agent that arrests T cells at a point in late G1 phase but substantially before the G1 to S phase transition. Under these conditions, p110Rb phosphorylation was almost completely accomplished in the absence of significant p34 synthesis, a finding that suggests that most or all of p110 phosphorylation is performed by kinases other than p34. Because of this observation, extracts were next examined for p34-like molecules using an antibody against the so-called PSTAIRE domain found in all cdc2 homologues identified to date. A species of protein with a mobility slightly less than true p34 was found, even in resting T cells. Upon stimulation, this protein increased slightly in amount, and a second protein with a mobility greater than p34, a putative p33cdk2, was seen. Not only was the appearance of these proteins not inhibited by deferoxamine but they accumulated in cultures treated with the drug, suggesting that p33, and not p34, may be the G1 phase kinase for p110Rb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of oocyte diameter on meiotic competence, embryo development, p34 (cdc2) expression and MPF activity in prepubertal goat oocytes

The aim of this study was to analyze the relationship between oocyte diameter, meiotic and embryo developmental competence and the expression of the catalytic subunit of MPF, the p34(cdc2), at mRNA, RNA and protein level, as well as its kinase activity, in prepubertal (1-2 months old) goat oocytes. MPF is the main meiotic regulator and a possible regulator of cytoplasmic maturation; therefore, it could be a key factor in understanding the differences between competent and incompetent oocytes. Oocytes were classified according to oocyte diameter in four categories: <110, 110-125, 125-135 and >135 microm and matured, fertilized and cultured in vitro. The p34(cdc2) was analyzed in oocytes at the time of collection (0 h) and after 27 h of IVM (27 h) in each of the oocyte diameter categories. The oocyte diameter was positively related to the percentage of oocytes at MII after IVM (0, 20.7, 58 and 78%, respectively) and the percentage of blastocysts obtained at 8 days postinsemination (0, 0, 1.95 and 12.5%, respectively). The expression of RNA and mRNA p34(cdc2) did not vary between oocyte diameters at 0 and 27h. Protein expression of p34(cdc2) increased in each oocyte category after 27 h of maturation. MPF activity among diameter groups did not vary at 0h but after IVM there was a clear and statistically significant increase of MPF activity in the biggest oocytes.     

Quantification of cyclin B1 and p34(cdc2) in bovine cumulus-oocyte complexes and expression mapping of genes involved in the cell cycle by complementary DNA macroarrays

Although high amounts of cyclin B1 mRNA are present in bovine oocytes arrested at the germinal vesicle (GV) stage, the protein is not detectable. Furthermore, there is a depletion of the stored cyclin B1 mRNA in the oocyte as follicular growth progresses. To assess the effect of follicular growth on the accumulation of M-phase promoting factor (MPF) components, mRNA and protein levels of cyclin B1 and p34(cdc2) were measured in GV oocytes collected from diverse follicle size groups (<2 mm, 3-5 mm, and >6 mm). Because oocytes collected from very small follicles have high levels of cyclin B1 mRNA, the onset of its accumulation in the oocytes was evaluated by in situ hybridization of fetal ovaries. Also, a comparative expression map of cell cycle-related genes expressed in the oocyte and cumulus cells was established using nylon-based cDNA arrays, which allowed the detection of 35 different genes transcribed mostly in oocytes. Both components of the pre-MPF complex were expressed at the mRNA level in GV oocytes, whereas p34(cdc2) was the only pre-MPF protein detected at that stage, thus indicating that meiosis resumption in bovine oocytes is differentially regulated as compared with other mammals, and meiosis resumption seems to be regulated by the translation of cyclin B1 mRNA.     

Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity.

The Vpr accessory gene product of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus is believed to play a role in permitting entry of the viral core into the nucleus of nondividing cells. A second role for Vpr was recently suggested by Rogel et al. (M. E. Rogel, L. I. Wu, and M. Emerman, J. Virol. 69:882-888, 1995), who showed that Vpr prevents the establishment in vitro of chronically infected HIV producer cell lines, apparently by causing infected cells to arrest in the G2/M phase of the cell cycle. In cycling cells, progression from G2 to M phase is driven by activation of the p34cdc2/cyclin B complex, an event caused, in part, by dephosphorylation of two regulatory amino acids of p34cdc2 (Thr-14 and Tyr-15). We show here that Vpr arrests the cell cycle in G2 by preventing the activation of the p34cdc2/cyclin B complex. Vpr expression in cells caused p34cdc2 to remain in the phosphorylated, inactive state, p34cdc2/cyclin B complexes immunoprecipitated from cells expressing Vpr were almost completely inactive in a histone H1 kinase assay. Coexpression of a constitutively active mutant p34cdc2 molecule with Vpr relieved the G2 arrest. These findings strongly suggest that Vpr arrests cells in G2 by preventing the activation of the p34cdc2/cyclin B complex that is required for entry into M phase. In vivo, Vpr might, by preventing p34cdc2 activation, delay or prevent apoptosis of infected cells. This would increase the amount of virus each infected cell produced.     

Effect of dexamethasone on the expression of p34(cdc2) and cyclin B1 in pig oocytes in vitro

The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition. At the G2-M boundary, maturation promoting factor (MPF) activation is usually induced via several ways, including tyrosine dephosphorylation of p34(cdc2) and synthesis of cyclin B according to cell type and species. Previous studies in our laboratory demonstrated that glucocorticoids directly inhibit the meiotic maturation of pig oocytes in vitro. The aim of this study was therefore to investigate the influence of glucocorticoids on the expression of p34(cdc2) and cyclin B1 in resumption of meiosis of pig oocytes. We detected the relative levels and association of p34(cdc2) and cyclin B1. Isolated cumulus-enclosed oocytes were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microgram/ml), LH (0.5 microgram/ml), FSH (0.5 microgram/ml), and estradiol-17beta (1 microgram/ml) in the presence or absence of dexamethasone (DEX) for 24 hr; they then were cultured without hormonal supplements in the presence or absence of DEX for an additional 24 hr. We found that cyclin B1, as well as p34(cdc2), was already present in fully grown G2-arrested pig oocytes when removed from the follicle. In these oocytes, cyclin B1 and p34(cdc2) were already associated in complex. Treatment with DEX at concentrations of 1 microgram/ml or above decreased the level of cyclin B1, but had no effect on the level of p34(cdc2). The exposure of oocytes to DEX also decreased the amount of complexed p34(cdc2)-cyclin B1. These findings suggest that the inhibitory action of DEX on meiotic maturation could be due, at least in part, to the reduced amount of p34(cdc2)-cyclin B1 complex.