D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins Dl, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone Hi or casein. Virtually all cyclin Dl-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin Dl-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin Dl-cdk4 complexes. Thus, cyclin Dl-associated kinase activity was not detected during the Go-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin Dl alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.D-and E-type cyclins govern the rate of progression of mammalian cells through the first gap phase (Gl) of the cell cycle and enforce the commitment of cells to replicate their chromosomal DNA (reviewed in reference 44). When quiescent cells enter the cell cycle, both classes of cyclins are synthesized during G, phase and associate with cyclin-dependent kinases (cdks) to form holoenzymes whose activities are presumed to facilitate entry into S phase.Cyclin E, first identified by complementation of G, cyclin deficiency in yeasts (22,25), is synthesized in late G, phase and associates with cdk2 to generate a histone Hi kinase that is maximally active during the Gl-to-S phase transition in mammalian cells (10, 23). The enforced overexpression of cyclin E in fibroblasts shortens their G, interval, decreases (but does not eliminate) their dependency on serum growth factors, and leads to a reduction in their size, indicating that cyclin E can be rate limiting for G1 progression (33). Its catalytic partner, cdk2, is also necessary for S-phase entry (34, 48), but it remains unclear whether this is due to its association with cyclin E and/or with cyclin A, which is expressed somewhat later during the cell cycle and steadily accumulates during the S and G2 phases (30,38,39). The activity of the cyclin E-c...

Receptor tyrosine kinases Flt-1 and Flk-1͞ KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1͞KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice ( flt-1 TK؊/؊ ) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1 TK؊/؊ mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.

A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-kappaB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3-TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.

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