Levels Of Cyclin-dependent Kinase Research Articles

Degradation of Cdt1 during S Phase Is Skp2-independent and Is Required for Efficient Progression of Mammalian Cells through S Phase

Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854-30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807-30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase.

Saucernetin-7 isolated from Saururus chinensis inhibits proliferation of human promyelocytic HL-60 leukemia cells via G0/G1 phase arrest and induction of differentiation.

In the present study, we investigated the in vitro effect of saucernetin-7, which is a dineolignan isolated from Saururus chinensis, on the proliferation, cell cycle-regulation and differentiation of HL-60 human promyelocytic leukemia cells. Saucernetin-7 potently inhibited the proliferation of HL-60 cells in both a dose- and time-dependent manner with an IC50, approximately 5 microM. DNA flow-cytometry indicated that saucernetin-7 markedly induced a G1 phase arrest of HL-60 cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent kinase (CDK)6 and cyclin D1 were reduced by saucernetin-7, whereas the steady-state levels of CDK2, CDK4, cyclin D2, cyclin D3 and cyclin E were unaffected. The protein and mRNA levels of a CDK inhibitor p21CIP1/WAF1, but not p27KIP1, were markedly increased by saucernetin-7 and p21CIP1/WAF1 induction is likely to occur at the transcriptional level because actinomycin D blocked this induction. In addition, saucernetin-7 markedly enhanced the binding of p21CIP1/WAF1 with CDK2 and CDK6, resulting in the reduced activity of both kinases and the hypophosphorylation of Rb protein. We furthermore suggest that saucernetin-7 is a potent inducer of the differentiation of HL-60 cells, based on observations such as a reduction of the nitroblue tetrazolium level, an increase in the esterase activities and phagocytic activity, morphology changes, and the expression of CD14 and CD66b surface antigens. In conclusion, the onset of saucernetin-7-induced the G0/G1 arrest of HL-60 cells prior to the differentiation is linked to a sharp up-regulation of the p21CIP1/WAF1 level and a decrease in the CDK2 and CDK6 activities. This is the first report demonstrating that saucernetin-7 potently inhibits the proliferation of human promyelocytic HL-60 cells via the G1 phase cell cycle arrest and differentiation induction.

Differential expression of cell cycle regulatory molecules and evidence for a “cyclin switch” during progression of prostate cancer

Deregulation of the cell cycle can be viewed as both cause and consequence of cancer. Cyclin expression regulates progression through the cell cycle and although some cyclins have been examined in prostate cancer, the spatial and temporal changes in expression of these molecules during progression of autochthonous disease has not been fully explored. Expression patterns of cyclins and cyclin dependent kinases during the different stages of progression in the spontaneous autochthonous TRAMP model were examined by RNAse protection assay, Western blot analysis, and immunohistochemistry. Differential expression of cell cycle regulatory molecules was observed during prostate cancer progression. Levels of the D-type cyclins decreased during progression while expression of cyclin E increased both at the mRNA and protein levels. The level of cyclin A and cyclin B expression increased beginning in early stage tumors and continued to increase throughout progression. The levels of cyclin dependent kinases did not change substantially during progression of the TRAMP model. The spatial and temporal pattern of mitotic cyclin expression during prostate cancer progression suggests that these molecules represent potential therapeutic targets. The differential expression of D-type cyclins may have implications with respect to androgen receptor mediated gene expression.

Accelerated in Vivo Growth of Prostate Tumors that Up-Regulate Interleukin-6 Is Associated with Reduced Retinoblastoma Protein Expression and Activation of the Mitogen-Activated Protein Kinase Pathway

Interleukin-6 (IL-6) is a multifunctional cytokine that activates the signaling pathways of Janus kinases-signal transducers and activators of transcription (STAT) and/or mitogen-activated protein kinases (MAPK) in various tumors. Thus, it modulates cell growth and apoptosis. IL-6 levels are elevated in tissues and sera from prostate cancer patients and IL-6 receptor expression has been detected in prostate cancer cell lines and clinical specimens. Continuous exposure of prostate cancer cells to IL-6 might alter their responsiveness to this cytokine. To gain more insight into the function of IL-6 in prostate carcinoma, we have inoculated LNCaP-IL-6+ cells, generated after prolonged treatment with IL-6, into nude mice (total n = 16, two independent experiments). Controls included animals bearing LNCaP-IL-6- cells, passaged at the same time as LNCaP-IL-6+ cells without supplementation of IL-6. LNCaP-IL-6+ tumor volumes were larger than those of their counterparts at all time points. There were no signs of cachexia in any of the experimental animals and all mice were free of metastases. To better understand the mechanisms responsible for accelerated growth of LNCaP-IL-6+ tumors, we have investigated the expression of cell-cycle regulatory molecules by Western blot analysis. The levels of cyclin-dependent kinase 2 were elevated in LNCaP-IL-6+ cells. There was a strong down-regulation of cyclins D1 and E in the LNCaP-IL-6+ subline. The cell-cycle inhibitor p27 was expressed at a low level in LNCaP-IL-6+ cells and could not be up-regulated by addition of IL-6. Most notably, LNCaP-IL-6+ cells exhibited a reduced expression of the hypophosphorylated form of the retinoblastoma protein (pRb). Accelerated tumor growth in our model system was also associated with alterations in IL-6-signaling pathways. The ability of IL-6 to induce tyrosine phosphorylation of STAT3 was abolished in the LNCaP-IL-6+ subline. In contrast, the levels of the MAPK extracellular signal-regulated kinases 1/2 increased in cells generated after long-term IL-6 treatment. The inhibitor of MAPK kinase PD 98059 retarded the proliferation of LNCaP-IL-6+ but not that of control cells. In summary, we show in the present study that chronic exposure of prostate cancer cells to IL-6 facilitates tumor growth in vivo by abolishment of the growth control by pRb and activation of the MAPK signaling pathway. These findings could be relevant to understand the role of IL-6 in prostate cancer progression.

Decreased proliferation and cell cycle arrest in neoplastic rat pituitary cells is associated with transforming growth factor-β1-induced expression of p15/INK4B

Transforming growth factor beta (TGF-β) is a member of a family of cytokines that regulate differentiation and proliferation in a wide variety of tissues including the pituitary gland. In both the normal pituitary and tumorous cell lines TGF-β1 has anti-proliferative activity, however the intracellular mechanisms responsible have not been defined. In the pituitary derived cell line GH 3, p27 Kip1, a key regulator of G 1/S transition is not expressed, suggesting that this protein is not an effector of the anti-proliferative response following TGF-β1 treatment. Among other TGF-β responsive cell cycle regulators p15 Ink4b has been shown to have anti-proliferative effects associated with cell cycle arrest in other cell types. We therefore examined p15 Ink4b expression in response to TGFβ-1 to determine if this cyclin dependent kinase inhibitor was responsible for anti-proliferative activity in GH 3 cells. Treatment of GH 3 cells with TGF-β1 (0.5–30 ng/ml) showed significant dose dependent growth inhibition ( P<0.001) as assessed by viable cell counts. Maximum growth inhibition (66%) was observed following treatment with 2 ng/ml TGF-β1. FACS analysis carried out in parallel with the growth studies showed treatment was associated with a decrease in the proportion of cells in S-phase (22–9%) and a significant increase in the G 1 fraction from 58 to 75% relative to controls ( P<0.001). The absence of a sub G 1 fraction and reversibility of the G 1 arrest over three cycles showed that these changes were not due to either an apoptotic response or cytoxicity, respectively. Semi-quantitative RT-PCR and Western blot analysis showed no change in the expression level of cyclin dependent kinase 4 (CDK4), p16 Ink4a or p21 Cip1. However, p15 Ink4b mRNA and protein levels showed a 10 and 8 -fold induction, respectively. Increased levels of p15 Ink4b were accompanied by a shift in the phosphorylation status of pRb toward its active hypophosphorylated form. Furthermore, studies of the kinetics of p15 Ink4b induction showed that arrest of cells in G 1 is preceded by induction of p15 Ink4b mRNA and protein. These investigations would suggest that p15 Ink4b is a functional effector of TGF-β1 mediated cell cycle arrest in GH 3 cells. However, our present studies cannot determine if it is the sole mediator. Identification of intracellular target(s) that mediate responses to anti-proliferative signals will increase our understanding of these pathways and aberrations responsible for their dysfunction in tumorigenesis.

Expression of cyclins D1, D2 and E correlates with proliferation of rat stellate cells in culture

Background/Aims: Regulation of cell cycle progression of cultured rat stellate cells was studied. Methods: DNA synthesis was determined by the uptake of [ 3H]thymidine or 5-bromo-2′-deoxyuridine. Cell cycle distribution was analyzed using FACScan of cellular DNA stained with propidium iodide. Expression of cyclins and cyclin-dependent kinase 4 was evaluated by Western and Northern blotting. Results: DNA synthesis of primary-cultured stellate cells was found to accelerate 48 h after plating. Cell cycle analysis revealed that more than 93% of the cells were in G0/G1 phase during the first 48 h after plating. The cell population in S phase abruptly increased to about 16% 72 h after culture and shifted to G2/M phase thereafter. The level of proteins and mRNAs for cyclins D1, D2 and E started to increase 48 h after culture with a concomitant expression of platelet-derived growth factor receptor β, while the level of cyclin-dependent kinase 4 and its mRNA remained unchanged. On the other hand, stellate cells remained in G1 phase when they were cultured in the presence of 3-isobutyl-1-methylxanthine or dibutyryl cAMP after plating. Attenuation of the expression of cyclins D1, D2 and E and platelet-derived growth factor receptor β, but not cyclin-dependent kinase 4 was found in stellate cells cultured with these agents. Further analysis revealed that LY294002, a selective inhibitor of phosphatidylinositol 3-kinase, suppressed DNA synthesis and cyclin D1 expression in a dosedependent manner without affecting platelet-derived growth factor receptor β expression. Conclusions: Induction of G1 cyclins may play crucial roles in cell cycle transition of cultured stellate cells from G1 to S. Expression of platelet-derived growth factor receptor β and activation of phosphatidylinositol 3-kinase may be involved in the process.

Induced Differentiation of U937 Cells by 1,25-dihydroxyvitamin D3 Involves Cell Cycle Arrest in G1 That Is Preceded by a Transient Proliferative Burst and an Increase in Cyclin Expression

AbstractThe hormonal form of vitamin D, 1,25-dihydroxyvitamin D3[1,25(OH)2D3], is a potent inhibitor of cellular proliferation as well as an inducer of differentiation of myeloid leukemic cells to macrophages. We have previously reported that a number of genes are upregulated by 1,25(OH)2D3 during myeloid differentiation, including the cyclin-dependent kinase (CDK) inhibitors p21, p27, 15, and p18, suggesting that cell cycle arrest and differentiation are tightly linked processes. We further explore here the relationship between growth inhibition and differentiation. We report that, upon 1,25(OH)2D3 treatment, U937 cells exhibited an early proliferative burst followed by growth inhibition and subsequent differentiation. Although CDK levels remain constant throughout, this transient increase in proliferation was accompanied by increases in cyclin A, D1, and E protein levels. p21 and p27 levels were also elevated during both the proliferative burst and subsequent inhibition of cell growth. Ectopic overexpression of p21 and/or p27 in U937 cells, in the absence of hormone, resulted in an induction of the expression of monocyte/macrophage-specific markers, whereas overexpression of p15 and p18 had no effect, suggesting that a subset of CDK inhibitors are important for both growth arrest and differentiation and that an early increase in proliferation is somehow a prerequisite for subsequent differentiation. However, no such biphasic behavior was detected in cells that are growth inhibited by 1,25(OH)2D3but do not differentiate, such as MCF-7 cells. Taken together, these results indicate that both growth stimulation and subsequent inhibition precede differentiation and involve induction of both cyclins and p21 and p27, whereas cell cycle arrest of differentiated cells can be achieved simply by elevations in CDK inhibitors.

Induced Differentiation of U937 Cells by 1,25-dihydroxyvitamin D3 Involves Cell Cycle Arrest in G1 That Is Preceded by a Transient Proliferative Burst and an Increase in Cyclin Expression

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3[1,25(OH)2D3], is a potent inhibitor of cellular proliferation as well as an inducer of differentiation of myeloid leukemic cells to macrophages. We have previously reported that a number of genes are upregulated by 1,25(OH)2D3 during myeloid differentiation, including the cyclin-dependent kinase (CDK) inhibitors p21, p27, 15, and p18, suggesting that cell cycle arrest and differentiation are tightly linked processes. We further explore here the relationship between growth inhibition and differentiation. We report that, upon 1,25(OH)2D3 treatment, U937 cells exhibited an early proliferative burst followed by growth inhibition and subsequent differentiation. Although CDK levels remain constant throughout, this transient increase in proliferation was accompanied by increases in cyclin A, D1, and E protein levels. p21 and p27 levels were also elevated during both the proliferative burst and subsequent inhibition of cell growth. Ectopic overexpression of p21 and/or p27 in U937 cells, in the absence of hormone, resulted in an induction of the expression of monocyte/macrophage-specific markers, whereas overexpression of p15 and p18 had no effect, suggesting that a subset of CDK inhibitors are important for both growth arrest and differentiation and that an early increase in proliferation is somehow a prerequisite for subsequent differentiation. However, no such biphasic behavior was detected in cells that are growth inhibited by 1,25(OH)2D3but do not differentiate, such as MCF-7 cells. Taken together, these results indicate that both growth stimulation and subsequent inhibition precede differentiation and involve induction of both cyclins and p21 and p27, whereas cell cycle arrest of differentiated cells can be achieved simply by elevations in CDK inhibitors.

P27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry.

In this study the direct involvement of cadherins in adhesion-mediated growth inhibition was investigated. It is shown here that overexpression of N-cadherin in CHO cells significantly suppresses their growth rate. Interaction of these cells and two additional fibroblastic lines with synthetic beads coated with N-cadherin ligands (recombinant N-cadherin ectodomain or specific antibodies) leads to growth arrest at the G1 phase of the cell cycle. The cadherin-reactive beads inhibit the entry into S phase and the reduction in the levels of cyclin-dependent kinase (cdk) inhibitors p21 and p27, following serum-stimulation of starved cells. In exponentially growing cells these beads induce G1 arrest accompanied by elevation in p27 only. We propose that cadherin-mediated signaling is involved in contact inhibition of growth by inducing cell cycle arrest at the G1 phase and elevation of p27 levels.

Growth promotion by homocysteine but not by homocysteic acid: a role for excessive growth in homocystinuria or proliferation in hyperhomocysteinemia?

Excessive growth of long bones in patients with homocystinuria is still unexplained and previous work incriminating homocysteic acid could not be confirmed by others. In vitro studies from our laboratory showed that homocysteine stimulated growth in a clonogenic assay. This observation made us study plasma cyclin dependent kinase (CDK), homocyst(e)ine and homocysteic acid in 10 patients with homocystinuria and 20 controls. In addition, homocysteine and homocysteic acid were tested in a clonogenic assay to correlate the growth promoting activity with CDK. Plasma CDK (protein) correlated strongly with homocysteine ( r=0.84) but not with homocysteic acid. Supernatants of the clonogenic assay samples showed up to three times higher CDK levels in the presence of homocyst(e)ine but not homocysteic acid. In vitro data and the strong correlation between homocysteine and CDK suggest a role for homocysteine stimulating CDK, the starter of mitosis, with subsequent stimulation of growth.

Select alterations in protein kinases and phosphatases during apoptosis of differentiated PC12 cells.

The involvement of cell cycle-regulatory proteins in apoptosis of neuronally differentiated PC12 cells induced by the removal of nerve growth factor and serum was examined. Three major findings are presented. (1) Cdc2 kinase protein levels increased fivefold in apoptotic PC12 cells by day 3 of serum and nerve growth factor deprivation. Histone H1 kinase activity was increased significantly in p13(suc1) precipitates of apoptotic PC12 cells, which was due to increased activation and/or expression of cdc2 kinase. (2) The protein levels of cyclin-dependent kinase 4, cyclin D, and proliferating cell nuclear antigen that are normally expressed in the cell cycle were increased during neuronal PC12 cell apoptosis. (3) The levels of the catalytic subunit, but not the regulatory subunit of the calcium/calmodulin-dependent protein phosphatase 2B, decreased significantly concomitant with a significant decrease in protein phosphatase 2B activity early in the apoptotic process. Protein phosphatase 2A activity decreased slightly but significantly after 3 days of serum and nerve growth factor deprivation, and no alterations in protein phosphatase 1 were observed during the apoptotic process. These data demonstrate that certain cell cycle-regulatory proteins are inappropriately expressed and that alterations in specific phosphorylation events, as indicated by the increase in histone H1 kinase activity and the decrease in protein phosphatase 2B activity, are most likely occurring during apoptosis of PC12 cells. These observations support the hypothesis that apoptosis may be due in part to a nondividing cell's uncoordinated attempt to reenter and progress through the cell cycle.

Changes in Cyclin-dependent Kinase 2 and p27kip1 Accompany Glial Cell Differentiation of Central Glia-4 Cells

The generation of different glial cell types in the central nervous system depends upon a wide variety of proliferative and differentiative signals. Here we report that changes in the levels of cyclin-dependent kinase 2 (CDK2) and the cell cycle inhibitor p27kip1 accompany the differentiation of central glia-4 (CG-4) progenitor cells to an astrocytic cell phenotype in the presence of fetal calf serum. Although a decrease in CDK2 levels was observed in both oligodendrocyte and astrocyte cells derived from CG-4 cells, a striking increase in the levels of p27 was observed during the differentiation of astrocyte cells. In astrocyte cell extracts, inhibition of CDK2 activity could be overcome with exogenously added cyclin E. Furthermore, depletion of p27 from astrocyte extracts lowered the amount of cyclin E required for CDK2 activation. Taken together, these results suggest that the inhibitory action of p27 upon cyclin E-CDK2 may prevent entry of cells into the S phase and regulate the progression of CG-4 cells toward an astrocytic lineage.

Decreased cyclin dependent kinase in brain of patients with Down Syndrome

In order to study whether phosphokinases might be involved in the neuropathology of Down Syndrome (DS) and Alzheimer disease (AD), cyclin dependent kinase (CDK) activity and protein, phosphokinase C (PKC) and phosphokinase A (PKA) activities have been determined in frontal lobes of DS, AD and control brains. An enzyme linked immunosorbent assay (ELISA) technique for CDK protein, and commercially available enzyme assays for CDK, PKC and PKA activities have been used. The major finding of our study was the remarkable and significant decrease of CDK protein and activity in DS brains in comparison to AD and controls. PKC and PKA were unaffected in both, AD and DS. As CDK controls cell division and differentiation, lowered CDK levels could reflect impaired proliferation and differentiation in DS.

Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells.

Differentiation of murine erythroleukemia cells induced by hexamethylene bisacetamide (HMBA) is associated with accumulation of underphosphorylated retinoblastoma protein (pRB) and an increase in retinoblastoma (RB) gene expression. Here we show that HMBA causes a rapid decrease in the level of cyclin-dependent kinase 4 (cdk4) protein. This decrease results from decreased stability of the protein, while the rate of synthesis of the protein is not affected by HMBA. The decrease in the level of cdk4 protein is followed by suppression of the pRB kinase activity associated with cdk4. Cyclin D3, which can bind and activated cdk4, is increased in HMBA-induced cells and is found in complex with pRB and the transcription factor E2F. In uninduced cells cyclin D3 complexes with pRB and E2F are barely detected. At the later stages of differentiation, MEL cells become arrested in G1 and cdk2 kinase activity is suppressed; this is accompanied by a decrease in the level of cyclin A and cdk2 proteins. Cells transfected with cdk4, which continue to overexpress cdk4 protein during culture with HMBA, are resistant to HMBA-induced differentiation. In contrast, overexpression of cdk2 protein does not inhibit induced differentiation. These findings suggest that suppression of cdk4 is a critical event in the pathway leading to terminal differentiation of erythroleukemia cells.

Suppression of Cyclin-Dependent Kinase 4 during Induced Differentiation of Erythroleukemia Cells

Differentiation of murine erythroleukemia cells induced by hexamethylene bisacetamide (HMBA) is associated with accumulation of underphosphorylated retinoblastoma protein (pRB) and an increase in retinoblastoma (RB) gene expression. Here we show that HMBA causes a rapid decrease in the level of cyclin-dependent kinase 4 (cdk4) protein. This decrease results from decreased stability of the protein, while the rate of synthesis of the protein is not affected by HMBA. The decrease in the level of cdk4 protein is followed by suppression of the pRB kinase activity associated with cdk4. Cyclin D3, which can bind and activated cdk4, is increased in HMBA-induced cells and is found in complex with pRB and the transcription factor E2F. In uninduced cells cyclin D3 complexes with pRB and E2F are barely detected. At the later stages of differentiation, MEL cells become arrested in G1 and cdk2 kinase activity is suppressed; this is accompanied by a decrease in the level of cyclin A and cdk2 proteins. Cells transfected with cdk4, which continue to overexpress cdk4 protein during culture with HMBA, are resistant to HMBA-induced differentiation. In contrast, overexpression of cdk2 protein does not inhibit induced differentiation. These findings suggest that suppression of cdk4 is a critical event in the pathway leading to terminal differentiation of erythroleukemia cells.

Immune dysregulation in TGF-beta 1-deficient mice.

Approximately 2 wk after birth, mice having a TGF-beta 1 null mutation (TGF-beta 1(-/-)) exhibit a progressive wasting syndrome and death. Associated with this phenotype is a multifocal infiltration of lymphocytes and macrophages into target organs, especially the heart, lungs, and salivary glands. To explore the consequences of TGF-beta 1 deficiency on the immune system, lymphocyte phenotype and function were analyzed. Initially, lymphoid organ architecture seemed to be normal and, as symptoms developed, the thymus decreased in size, whereas lymph nodes were enlarged. Phenotypically, the TGF-beta 1(-/-) lymphoid cells seemed to be more differentiated in the thymus and activated in the lymph nodes, but remarkably unaffected in the spleen. Moreover, TGF-beta 1(-/-) spleen and lymph nodes displayed enhanced numbers of proliferating cells, as measured by proliferating cell nuclear Ag and/or cyclin-dependent kinase levels. Consistent with this hyperproliferative response, constitutive levels of IL-2 mRNA were elevated in the thymus and both IL-2 and IL-2R mRNA were increased in the lymph nodes. In contrast with the activation profile of TGF-beta 1(-/-) lymphoid cells in vivo, mitogen challenge of these cells in vitro revealed suppressed proliferation that was associated with a defect in inducible IL-2 mRNA expression and IL-2 secretion. Moreover, the addition of rIL-2 restored the deficient mitogen-induced proliferation. The mechanism leading to T cell anergy remains unclear; however, these data confirm the essential role for TGF-beta 1 in maintaining normal immune function.

Cell cycle-specific induction of Cdk2 expression in B lymphocytes following antigen receptor cross-linking

The ligation of membrane Ig (mIg) on quiescent primary B lymphocytes by mitogenic concentrations of anti-IgM antibodies leads to cell cycle progression. The level of cyclin-dependent kinase 2 (Cdk2) expression was found to be restricted to specific phases of the cell cycle in primary cultures of murine B lymphocytes. Resting G 0 phase, G 1 phase, or B cells arrested near the G 1/S boundary by hydroxyurea contained no detectable Cdk2 protein or associated histone H1 kinase activity. In contrast, B cell entry into S phase was accompanied by an induction in the expression of cellular Cdk2 as judged by immunoblotting of B cell lysates with anti-Cdk2 antibodies. Concomitant with S phase entry was the detection of anti-Cdk2-specific immunoprecipitable histone H1 kinase activity. Further analysis revealed that the amount of cyclin A protein also oscillated during cell cycle, appearing initially in G 1 phase B cells. Cyclin A was found to be associated with Cdk2 in B cells during S phase progression. These results indicate that cross-linking of mIg on primary B lymphocytes results in the “downstream” catalytic activation of Cdk2. The timing of Cdk2 expression and its association with cyclin A suggests that Cdk2 may not be involved in the decision to enter S phase, but rather may provide a role in the maintenance of S phase progression or in preparing B cells to enter M phase.