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At least 19 recordsLinked to original sources
PubMed1995

Cyclin D2 is a moderately oscillating nucleoprotein required for G1 phase progression in specific cell types.

To explore regulation and function of cyclin D2, a candidate cell cycle-regulatory proto-oncogene, we examined subcellular localisation, cell type- and cell cycle-dependent expression, and requirement of cyclin D2 protein for G1 progression, in a panel of 40 human normal and cancer cell types. Except for lymphoid cells and sarcoma cell lines, expression of cyclin D2 was considerably more restricted than that of cyclin D1, whereas both D-type cyclin proteins were low or undetectable in cells lacking functional retinoblastoma gene product. In G1 cells, the cyclin D2 protein was more resistant to extraction and localised predominantly to nuclei, whereas it became more soluble and distributed in both nuclei and cytoplasm from G1/S transition onwards. Centrifugal elutriation and multiparameter flow cytometry analyses of several cell types showed moderate cell cycle oscillation with maximum levels of the cyclin D2 protein reached in late G1. Microinjection and/or electroporation of antibodies to cyclin D2 during G1 arrested the cyclin D2-expressing lymphocytes, breast myoepithelium, and U-2-OS sarcoma cells in G1 phase, whereas cyclin D2-negative cell types were unaffected by such treatment. Consistent with the putative proto-oncogenic role of cyclin D2 in specific cell types, our data show that this G1 cyclin has properties closely resembling those of cyclin D1, including the essential positive role in regulation of G1.

Animals↗
PubMed1995

Overexpression of cyclin D2 in chronic B-cell malignancies.

Tumor progression in B-cell chronic lymphocytic leukemia (B-CLL) is thought to result from the gradual accumulation of small resting G0/G1 phase lymphoid cells rather than the proliferation of actively dividing cells. The recent identification of G1 cyclins that are likely to control both the progression through G0 and G1 phase and the G1/S transition prompted us to study the mRNA expression of D-type cyclins in the peripheral blood lymphocytes from 34 patients with B-CLL, 7 patients with lymphoplasmacytic lymphoma (LPL), and 2 patients with mantle cell lymphoma (MCL). Cyclin D2 mRNA was, on average, 5- to 10-fold overexpressed in most of the samples studied (B-CLL, 29/34; LPL, 7/7; MCL, 0/2) as compared with normal resting B lymphocytes, in which cyclin D2 mRNA was barely detectable. In situ hybridization with cyclin D2 digoxigenin-labeled mRNA probe showed that all the cells from a given sample were stained with approximately the same intensity. Cyclin D3 was never detected in any of the samples tested, whereas cyclin D1 was expressed in only the 3 cases (1 LPL and 2 MCL) bearing a t(11;14) translocation. A trisomy 12 was found in 4 of 19 (21%) B-CLL or LPL cases for which cytogenetic analysis was available. Although the cyclin D2 gene has been mapped to chromosome 12p13, there was no apparent correlation between trisomy 12 and the level of cyclin D2 expression. Cell cycle analysis by flow cytometry after staining with propidium iodide consistently showed that more than 96% of the cells were in G0/G1 phase, whatever the importance of cyclin D2 overexpression was, and that cyclin D2 overexpression in B-CLL was not associated with any modifications of the cell cycle repartition. No consistent overexpression of cyclin D2 was found in acute myeloid leukemias. In conclusion, overexpression of cyclin D2 mRNA was found to be an almost constant feature in B-CLL and LPL. Therefore, it led us to hypothesize, with the support of data from some transfection experiments previously reported in murine hematopoietic cell lines, that cyclin D2 might play a role in B-CLL pathogenesis, possibly by preventing cells from programmed cell death.

Cyclin D2↗
PubMed1995

Expression of cyclin D2 in Epstein-Barr virus-positive Burkitt's lymphoma cell lines is related to methylation status of the gene.

The cyclin D2 gene is not expressed in resting primary B lymphocytes or in group I Burkitt's lymphoma (BL) cell lines that retain the characteristics of authentic BL cells. Expression of cyclin D2 is induced in primary B lymphocytes following infection with Epstein-Barr virus (EBV) or transfection of the EBV genes EBNA-LP and EBNA-2. However, attempts to induce cyclin D2 expression in BL cell lines by the enforced expression of EBV genes were unsuccessful. Since the demethylation agent 5-azacytidine has been shown to modulate viral gene expression in BL cells, we explored the possibility that methylation plays a significant role in the control of cyclin D2 expression. We show that 5-azacytidine treatment of the Mutu CI 179 BL cell line led to expression of cyclin D2 RNA and that expression correlated with differences in the methylation status of a CCGG restriction enzyme site near the transcription initiation region of the cyclin D2 gene. Thus, methylation appears to play a direct role in the regulation of the cyclin D2 locus in BL.

Antigens, Viral↗
PubMed1993

Inhibition of granulocyte differentiation by G1 cyclins D2 and D3 but not D1.

Growth factor-induced signals govern the expression of three D-type cyclins, which, in turn, function as regulatory subunits of cyclin-dependent kinases (cdks) to control cell cycle transitions during the late G1 interval. 32D myeloid cells, which self-renew as uncommitted precursors in interleukin 3 (IL-3), express cyclins D2 and D3 (but not D1) in complexes with cdk4 and cdk2. When transferred to granulocyte colony-stimulating factor (G-CSF), 32D cells stop dividing and terminally differentiate to mature neutrophils. Cyclin D and cdk4 expression ceased as cells underwent growth arrest in G-CSF, but cdk2 levels were sustained. 32D cells engineered to ectopically express D-type cyclins exhibited contracted G1 intervals with a compensatory lengthening of S phase but remained IL-3 dependent for cell growth; those overexpressing cyclins D2 and D3 (but not D1) were unable to differentiate and died in G-CSF. Cyclin D2 mutants, which cannot efficiently bind to, or functionally interact with, the retinoblastoma protein (pRb) or its relatives (p107) did not block differentiation. Conversely, the introduction of a catalytically inactive cdk4 mutant into cells overexpressing cyclin D2 restored their G-CSF response. The persistence of cdk2 and its predilection to functionally interact with cyclins D2 and D3 rather than D1 might explain the specificity of the differentiation blockade.

Amino Acid Sequence↗
PubMed1995

Cyclin D2 and Ha-Ras transformed rat embryo fibroblasts exhibit a novel deregulation of cell size control and early S phase arrest in low serum.

The D-type cyclins are growth factor-regulated delayed early functions which peak at the G1/S transition, are thought to regulate entry into S phase and have been implicated in tumorigenesis. Here, we show that cyclin D2 can co-operate with Ha-Ras to impose a novel transformed state on rat embryo fibroblasts (REF). While clonal cyclin D2/Ha-Ras REF transformants exhibit a characteristic transformed phenotype in high serum, in low serum they arrest cell proliferation and display profound morphological and cytological changes indicating loss of control of cell mass and deregulation of the G1/S transition. Notably, in low serum, despite re-establishment of actin cables and arrest of proliferation, cell mass continues to increase, creating giant cells up to 10 x normal size. Also, during low-serum culture the cells make a very gradual but progressive entry into S phase, reaching a 2.4N DNA content after 6 days. PCNA is expressed and 2N and 4N cells are largely absent, and thus the cells undergo a novel S phase arrest. While transfer to low serum induced the retinoblastoma protein to enter its dephosphorylated state, and cyclin A, cyclin B and cdc2 levels to decrease, all as normal, cyclin E, cdk4, cdk2 and the exogenous cyclin D2 persisted at high levels. These results indicate that cyclin D2 and Ha-Ras can transform cells when mitogenic signals from growth factors are provided. However, in low serum, co-operation of cyclin D2 and Ha-Ras provides only a subset of the progression signals and these are sufficient for G1-related cell mass increase and S phase entry, but are insufficient for full cell cycling.

Animals↗
PubMed1995

Differential expression of cyclin D2 and cdc2 genes in proliferating and nonproliferating alveolar epithelial cells.

Alveolar epithelial cells (AEC) proliferate during embryonic and fetal life, while in the adult lung AEC form a highly differentiated population that does not usually divide. Herein, we tested the hypothesis that differential expression of specific cell cycle control genes may occur during AEC development and transformation. We compared normal rat AEC in primary culture with transformed AEC for the expression of D-type G1 cyclins and cyclin-dependent protein kinases (cdc2 and cdk2). Cyclin D1 mRNA and protein were expressed at comparable levels in both normal rat AEC and in transformed AEC. In contrast, high levels of cyclin D2 mRNA and protein expression were only observed in normal 19-day fetal rat AEC and in transformed mink Mv1Lu cells derived from fetal mink lung epithelium. Moreover, treatment either with antisense oligodeoxynucleotides directed against cyclin D2 mRNA or with genistein (a tyrosine kinase inhibitor) caused significant inhibition of [3H]thymidine incorporation into DNA as well as inhibition of cyclin D2 expression in normal 19-day fetal rat AEC. p34cdc2 (but not p33cdk2 or p34cdk4) was expressed at progressively decreasing levels with corresponding histone H1 kinase activities during rat AEC development (19-day fetal > 21-day fetal > 13-day postnatal > adult rat AEC). The levels of p34cdc2 histone H1 kinase activity were significantly up-regulated or amplified in adult rat type 2 AEC following hyperoxic injury and repair and in transformed AEC. Collectively, these data support an important functional role for cyclin D2 and cdc2 genes in determining the proliferative versus nonproliferative phenotype of AEC during lung development, injury and repair, and transformation.

Animals↗
PubMed1994

MN20, a D2 cyclin found in brain, is implicated in neural differentiation.

Cyclins are regulatory proteins that promote the progression of dividing cells through the cell cycle. D-type cyclins are important mediators of the transition from G1 into S phase of the cell cycle and are thought to be widely expressed in mitotically active tissues (Matsushime et al., 1991b; Inaba et al., 1992; Xiong et al., 1992). We report the isolation of a cDNA clone, MN20, which represents a D2 cyclin message form whose expression pattern is highly restricted to brain. MN20 is not ubiquitous, but rather it is expressed only in restricted neuronal precursor populations, for example, in proliferating granule neuroblasts of the cerebellum but not hippocampus. Strikingly, MN20 expression is also found in postmitotic neuronal precursor cells of the embryonic cerebral cortex, but not in the dividing cortical neuroblasts. These observations suggest that the D2 cyclin gene serves regionally specific functions in neuronal differentiation, some of which may be distinct from the promotion of cell cycle progression and which act at the interface between mitosis and the assumption of mature neuronal morphology.

Animals↗
PubMed1995

Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-beta 1-mediated growth inhibition in EBV-positive B cells.

The normal cell cycle is regulated by several molecules, such as the tumor-suppressor protein pRb, the G1 cyclins, the cyclin-dependent kinases, and their inhibitors. These regulators are targeted by negative growth regulatory signals, such as that provided by TGF-beta. Here, we show that the presence of either wild-type EBV or its transforming latent membrane protein-1 (LMP-1) results in the loss of TGF-beta 1-mediated growth inhibition in human B cells. Chemical cross-linking with 125I-labeled TGF-beta 1 showed an essentially normal TGF-beta receptor profile in EBV-positive and EBV-negative Burkitt's lymphoma cell lines, and these receptors were shown to be functional in transducing signals, as evidenced by the TGF-beta 1-mediated modulation of junB gene expression. However, TGF-beta 1 did not induce dephosphorylation of pRb in EBV (or LMP-1)-positive cells as opposed to EBV-negative cells, suggesting a dichotomy in the TGF-beta 1 signaling pathway leading to separable gene regulatory and growth inhibitory responses. Furthermore, LMP-1 was found to induce the expression of cyclin D2; normal B cells or EBV-negative Burkitt's lymphoma cells do not express D-type cyclins. Taken together, these data point to a potential mechanism underlying EBV-mediated B cell transformation whereby constitutive induction of key cell cycle regulators by LMP-1 can lead to pRb hyperphosphorylation and uncontrolled cell proliferation.

B-Lymphocytes↗
PubMed1995

Use of semiquantitative reverse transcription-polymerase chain reaction to study gene expression in normal human skin fibroblasts following low dose-rate irradiation.

One way to study the effect of radiation on gene expression is to monitor changes in the levels of specific messenger RNAs. We describe the use of reverse transcription-polymerase chain reaction (RT-PCR) analysis, a faster and more sensitive procedure than the traditional techniques to monitor RNA levels. Using RT-PCR, we confirmed previous results showing increased levels of GADD45 transcripts after high dose-rate X-irradiation in normal human fibroblasts. No differences were observed in the transcript levels of beta-ACTIN, beta-MICROGLOBULIN, Cu-Zn SUPEROXIDE DISMUTASE (SOD-1) and CATALASE. In cells exposed to 3-6 Gy low dose-rate gamma-irradiation we observed increased levels of the GADD45 transcript and lower transcript levels of the genes TOPOISOMERASE II alpha, FACC, CYCLIN A and CYCLIN B. No differences were detected in the transcript levels of beta-ACTIN, beta-MICROGLOBULIN, SOD-1, URACYL-DNA GLYCOSYLASE, CYCLIN C, CYCLIN E, CYCLIN D1, CYCLIN D2, CYCLIN D3, TOPOISOMERASE I and TOPOISOMERASE II beta.

Actins↗
PubMed1995

Distinct patterns of expression of the D-type cyclins during testicular development in the mouse.

The three D-type cyclins have been shown to be differentially expressed in a number of isolated cell types and cell lines, suggesting distinct roles in cell cycle regulation in particular cell lineages. The testis provides unique opportunities to study genes involved in cell cycle regulation, since it contains cells in both mitosis and meiosis as well as differentiated cells with little proliferation activity. Major transcripts of 4.2 kb, 6.8 kb, and 2.3 kb were detected in the adult mouse testis by Northern hybridization analyses for cyclin D1, cyclin D2, and cyclin D3, respectively. Additional transcripts of 1.8 and 2.7 kb were detected by Northern hybridization for cyclin D3 in the testis, but not in other tissues, and these transcripts were limited to germ cells. Northern and in situ hybridization analyses of normal and germ cell-deficient testes showed the surprising result that cyclin D1 was expressed in a pattern consistent with expression in the non-dividing Sertoli cells. Cyclin D2 levels appeared slightly enriched in germ cell-deficient testes as compared to intact testis, but in situ hybridization analysis did not reveal any distinct cellular localization. Also surprising was the observation that cyclin D3 expression was highest in the non-dividing, haploid, round spermatids. The possible roles of these cyclins in the events of spermatogenesis are discussed.

Animals↗
PubMed1995

Expression of cyclins A, D2 and D3 in individual normal mitogen stimulated lymphocytes and in MOLT-4 leukemic cells analyzed by multiparameter flow cytometry.

Cyclins are regulatory subunits of the cyclin dependent kinases (CDKs), the enzymes that drive the cell through the respective phases and check-points of the cell cycle. The expression of cyclins in non-tumor cells, regulated by timely induction of their synthesis and proteolysis, is scheduled, occurring at discrete periods of the cell cycle. Using multiparameter flow cytometry we have recently observed that expression of cyclins B1 and E in individual normal lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) and lymphocytic leukemic MOLT-4 cells was similar, restricted to particular phases of the cycle: cyclin B1 was detected only in G2+M- and cyclin E in late G1 and early S-phase cells. In the present study we have measured the expression of cyclins A, D2 and D3 in these cells. The presence of cyclin A was restricted to late S and G2 phases, both in the case of lymphocytes and of MOLT-4 cells. Over 95% of the non-stimulated lymphocytes were both cyclin D2 and D3 negative. Mitogenic stimulation with PHA-induced expression of cyclins D2 and D3 in over 50% cells, which corresponds to the percentage of cells that respond to this mitogen in cultures. Expression of these proteins peaked between 8 and 24 h after addition of PHA, and then decreased at the time of cell entrance to S. During exponential growth (48-72 h after stimulation with PHA) expression of the D-type cyclins was diminished: only between 5-10% of the lymphocytes had levels of cyclin D3 as high as G1 cells between 8-24 h after PHA stimulation. Populations of proliferating lymphocytes and MOLT-4 cells were very heterogeneous in terms of expression of D-type cyclins by individual cells. While expression of cyclin D2 in exponentially growing MOLT-4 cells was similar to that of proliferating lymphocytes, the percent of cells expressing cyclin D3 as well as the degree of expression, was higher in MOLT-4 cells, regardless of the phase of the cycle. These results, with our earlier observations of the untimely expression of cyclins B1 and E in several other tumor lines, suggest that altered expression of cyclins may be a frequent feature of malignancy.

Analysis of Variance↗
PubMed1995

Mouse mammary hyperplasias and neoplasias exhibit different patterns of cyclins D1 and D2 binding to cdk4.

Deregulated expression of G1 cyclins D1 and D2 is a feature of some neoplasias. This study examined the altered expression of D1 and D2 cyclins, both the total pool and as associated with cdk4 and cdk2, at different stages of mouse mammary tumorigenesis. Three different mammary hyperplastic outgrowth lines, TM2, TM10 and TM12, and their respective tumors were examined. Increasing levels of the cyclin D1 protein pool, D1 binding to cdk4 and cdk2 and cdk4 kinase activity were closely correlated with tumorigenesis. In constrast, cyclin D2 binding to cdk4 was predominant in hyperplasias and much less in tumors, where cyclin D1 became predominant. However, the cyclin D2 pool showed increases of 15-65 times in hyperplasias compared with normal gland and further increases of 11-15 times in two of three different tumors. The message level for cyclin D1 increased only 2-3 times in tumors compared with normal gland. Cyclin D2 mRNA was highest in normal tissue and decreased only marginally in tumors. These results suggest that cyclin D2 functions uniquely from cyclin D1 in the early stages of mouse mammary tumor development. Cyclin D2 bound to cdk4 may act to guarantee a low level of kinase activity in hyperplasias and may be an attempt to direct the mammary epithelial cells through differentiation rather than proliferation. This interaction may be one of the negative regulatory mechanisms in the early stages in mouse mammary tumor development, until cyclin D1 totally replaces cyclin D2 binding to cdk4, which would activate the high levels of cdk4 kinase activity observed in neoplasias.

Animals↗
PubMed1994

EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein-Barr virus.

Epstein-Barr virus (EBV) is unusual among DNA tumour viruses in that the virus particle is able to infect and immortalize resting cells with very high efficiency. Mutation of the viral genome has indicated that at least six viral genes (LMP-1 and EBNAs 1, 2, 3A, 3C and LP) are essential for immortalization. We demonstrate that the activation of a G1 cyclin, cyclin D2, is an early event following infection with EBV and that cyclin D2 activation is dependent on the expression of viral genes. The different levels of cyclin D2 transcripts in Burkitt's lymphoma cell lines expressing different subsets of EBV immortalizing genes suggest an involvement of EBNA-2 or EBNA-LP in cyclin D2 regulation. By exposing resting primary B cells to a purified preparation of the EBV surface glycoprotein gp340, we have been able to achieve efficient expression of plasmid DNAs introduced by electroporation. Vectors encoding two viral genes, EBNA-2 and EBNA-LP, are sufficient to activate the expression of cyclin D2 in this system. Thus, the progression of resting B lymphocytes into the G1 phase of the cell cycle can be reconstituted in the absence of virus by the cooperation of two of the six viral genes required for immortalization.

Antigens, Viral↗
PubMed1994

Differential requirements for interleukin-2 distinguish the expression and activity of the cyclin-dependent kinases Cdk4 and Cdk2 in human T cells.

We examined the expression and activity of Cdk4 and Cdk2 in resting, competent, and proliferating normal human T cells. Expression of Cdk4 but not of Cdk2 was induced in competent T cells independent of an IL-2 signal. This up-regulation of Cdk4 mRNA and protein was resistant to the immunosuppressant drugs cyclosporin A (CsA) and FK506. A further increase in Cdk4 expression was seen upon stimulation of competent T cells by IL-2, as was de novo expression of Cdk2. Cyclin D2, a Cdk4 partner, showed similar patterns of regulation as Cdk4. The increases in Cdk4 and cyclin D2 expression seen in competent T cells were functionally significant since Cdk4 immunoprecipitates from these cells phosphorylated recombinant RB protein in vitro. Despite the lack of an increase in the expression of Cdk2, a small pool of pre-existing Cdk2 protein detected in resting T cells could be activated upon induction of competence. These data demonstrate that 1) the signals that lead to induction of competence in T cells stimulate an IL-2-independent and CsA-resistant phase of Cdk4 and cyclin D2 expression, Cdk4 kinase activity, and Cdk2 kinase activity, and 2) IL-2 stimulates a second phase of Cdk4 and cyclin D2 expression and de novo expression of Cdk2 in these cells. The data show that the expression and activity of these major cell cycle regulatory proteins are controlled differentially by growth factors and indicate a role for Cdk4 and cyclin D2 in T-cell cycle entry and/or early G1 progression and for Cdk2 in later G1 progression and G1/S transition.

CDC2-CDC28 Kinases↗
PubMed1992

Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.

Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.

Amino Acid Sequence↗
PubMed1994

Signal transduction of steel factor and granulocyte-macrophage colony-stimulating factor: differential regulation of transcription factor and G1 cyclin gene expression, and of proliferation in the human factor-dependent cell line MO7.

Steel factor (SF) synergizes with a variety of hemopoietins to support the growth and differentiation of human progenitor cells. The human factor-dependent cell line MO7 has been used as a model to study the interaction of SF with other growth factors such as GM-CSF, because both factors support the proliferation of this cell line and are synergistic in combination. Previous studies have shown that this effect is not readily explained by the synergistic activation of early, cytosolic signal transduction intermediates such as tyrosine kinases, Raf-1, MAP2 kinase, or phospholipase C gamma. In an attempt to further explore the biological and biochemical mechanisms of the synergy between SF and GM-CSF, we examined the effects of these growth factors on the regulation of nuclear proto-oncogenes, cell cycle control genes, and G1-->S transition of MO7 cells. Individually, GM-CSF was a much more potent growth factor for MO7 cells than SF, particularly under serum-free conditions. Only GM-CSF, but not SF, was able to stimulate G1-->S transition of MO7 cells after factor deprivation for 24 h. Northern blot analyses showed also differential effects of GM-CSF and SF on the expression of some nuclear proto-oncogenes and G1 cyclins. GM-CSF (10 ng/ml), but not SF (20 ng/ml) increased the expression of c-myc and cyclin D2 mRNA, whereas both factors caused transient increases of c-fos and cyclin D3 mRNAs. When added simultaneously, GM-CSF and SF induced an at least additive increase of c-fos mRNA expression; this effect required the presence of fetal calf serum. No additive effects of GM-CSF and SF on c-myc, cyclin D2 or D3 mRNA expression were observed. C-jun and c-myb mRNAs were constitutively expressed in the MO7 cell line, but not further increased after stimulation with GM-CSF or SF for 15 min to 48 h. The inability of SF to induce growth promoting genes such as c-myc and cyclin D2 may explain why this cytokine does not support sustained proliferation of MO7 cells. These observations suggest that SF and GM-CSF exert different effects on the expression of genes involved in regulatory pathways of cell proliferation, but the molecular mechanism of synergy remains to be elucidated.

Cell Division↗
PubMed1995

Regulation of synthesis and activity of the PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), a major cyclin D-associated cdk4 homologue in normal human T lymphocytes.

The PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), which shares extensive sequence homology (approximately 70%) with cdk4, was identified as the earliest inducible member of the cdk family of proteins in human T lymphocytes induced to proliferate in vitro by stimulation either with phorbol 12,13-dibutyrate and ionomycin (PDB/I) or PHA. The p40cdk6 protein was present in resting cells and increased amounts were detected 6 h after stimulation. It increased in amount throughout the first cell cycle but was present in reduced amounts at later times. Activity of the kinase, determined by in vitro phosphorylation of recombinant truncated retinoblastoma tumor suppressor gene (Rb) protein (p60Rb), paralleled p40cdk6 protein amounts. Cyclins D2 and D3 were the major cyclins associated with p40cdk6, with D2 predominating in early G1 phase. Both PDB and ionomycin were required for maximal accumulation of p40cdk6, but either agent alone stimulated some increase in amount and activity of the protein. p40cdk6 also increased in amount in cells activated in the presence of cyclosporin A or FK506, drugs that inhibit production of IL-2 and cell proliferation, suggesting that initial induction occurred independently of IL-2-mediated cell cycle progression. Furthermore, increased accumulation of p40cdk6 protein and activity occurred in cells rendered "competent" (responsive to IL-2) by a brief treatment with PDB/I. Thus, increased accumulation of the protein and its activity begin before IL-2/IL-2 receptor interaction, suggesting that the cdk6-cyclin D2 complex might be involved in acquisition of the competent state in human T lymphocytes.

Cell Cycle↗