Cyclin B2-null mice develop normally and are fertile whereas cyclin B1-null mice die in utero

Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34cdc2. To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34cdc2 kinase to the essential substrates of cyclin B2.

Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells

The activation of cyclin-dependent kinases (cdks) has been implicated in apoptosis induced by various stimuli. We find that the Fas-induced activation of cdc2 and cdk2 in Jurkat cells is not dependent on protein synthesis, which is shut down very early during apoptosis before caspase-3 activation. Instead, activation of these kinases seems to result from both a rapid cleavage of Wee1 (an inhibitory kinase of cdc2 and cdk2) and inactivation of anaphase-promoting complex (the specific system for cyclin degradation), in which CDC27 homolog is cleaved during apoptosis. Both Wee1 and CDC27 are shown to be substrates of the caspase-3-like protease. Although cdk activities are elevated during Fas-induced apoptosis in Jurkat cells, general activation of the mitotic processes does not occur. Our results do not support the idea that apoptosis is simply an aberrant mitosis but, instead, suggest that a subset of mitotic mechanisms plays an important role in apoptosis through elevated cdk activities.

Prominence of the dopamine D2 short isoform in dopaminergic pathways

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.

Activation of ventrolateral preoptic neurons by the somnogen prostaglandin D2

Prostaglandin D2 (PGD2) is an extensively studied sleep-promoting substance, but the neuroanatomical basis of PGD2-induced sleep is only partially understood. To determine potential regions involved in this response, we used Fos immunohistochemistry to identify neurons activated by infusion of PGD2 into the subarachnoid space below the rostral basal forebrain. PGD2 increased nonrapid eye movement sleep and induced striking expression of Fos in the ventrolateral preoptic area (VLPO), a cluster of neurons that may promote sleep by inhibiting the tuberomammillary nucleus, the source of the ascending histaminergic arousal system. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus. PGD2 also increased Fos immunoreactivity in the basal leptomeninges and several regions implicated in autonomic regulation. These observations suggest that PGD2 may induce sleep via leptomeningeal PGD2 receptors with subsequent activation of the VLPO.

Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27KIP1, or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.

Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15INK4b by transforming growth factor β treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15INK4b-independent transforming growth factor β-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16INK4a protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.

Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15INK4B blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27kip-1 blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of β transforming growth factor (TGFβ) in serum-free medium decreased levels of p15INK4B and increased colony formation and retroviral-mediated transduction of primary human CD34+ cells. Although TGFβ neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27kip-1 coupled with TGFβ-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFβ, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.

Posttranslational regulation of cyclin D1 by retinoic acid: A chemoprevention mechanism

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G1 in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G1-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G1 active in the prevention of human bronchial epithelial cell transformation.

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19Ink4d and p27Kip1, in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G2 and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19Ink4d and p27Kip1 cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.