Cell Cycle In Human Fibroblasts Research Articles

Cyclin C Regulates Human Hematopoietic Stem/Progenitor Cell Quiescence

Hematopoietic stem cells (HSCs) can remain quiescent or they can enter the cell cycle, and either self-renew or differentiate. Although cyclin C and cyclin dependent kinase (cdk3) are essential for the transition from the G(0) to the G(1) phase of the cell cycle in human fibroblasts, the role of cyclin C in hematopoietic stem/progenitor cells (HSPCs) is not clear. We have identified an important role of cyclin C (CCNC) in regulating human HSPC quiescence, as knocking down CCNC expression in human cord blood CD34(+) cells resulted in a significant increase in quiescent cells that maintain CD34 expression. CCNC knockdown also promotes in vitro HSPC expansion and enhances their engraftment potential in sublethally irradiated immunodeficient mice. Our studies establish cyclin C as a critical regulator of the G(0)/G(1) transition of human HSPCs and suggest that modulating cyclin C levels may be useful for HSC expansion and more efficient engraftment.

Polo-like Kinase-2 Is Required for Centriole Duplication in Mammalian Cells

Centriole duplication initiates at the G1-to-S transition in mammalian cells and is completed during the S and G2 phases. The localization of a number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling the centriole duplication cycle. Here we show that the human Polo-like kinase 2 (Plk2) is activated near the G1-to-S transition of the cell cycle. Endogenous and overexpressed HA-Plk2 localize with centrosomes, and this interaction is independent of Plk2 kinase activity. In contrast, the kinase activity of Plk2 is required for centriole duplication. Overexpression of a kinase-deficient mutant under S-phase arrest blocks centriole duplication. Downregulation of endogenous Plk2 with small hairpin RNAs interferes with the ability to reduplicate centrioles. Furthermore, centrioles failed to duplicate during the cell cycle of human fibroblasts and U2OS cells after overexpression of a Plk2 dominant-negative mutant. These results show that Plk2 is a physiological centrosomal protein and that its kinase activity is likely to be required for centriole duplication near the G1-to-S phase transition.

Intracellular free Ca2+ in the cell cycle in human fibroblasts: transitions between G1 and G0 and progression into S phase.

Intracellular free calcium ([Ca2+]i) has been proposed to play an important part in the regulation of the cell cycle. Although a number of studies have shown that stimulation of quiescent cells with growth factors causes an immediate rise in [Ca2+]i (Rabinovitch et al., 1986; Vincentini and Villereal, 1986; Hesketh et al., 1988; Tucker et al., 1989, Wahl et al., 1990), a causal relationship between the [Ca2+]i transient and the ability of the cells to reenter the cell cycle has not been firmly established. We have found that blocking the mitogen-induced elevation of [Ca2+]i with the cytoplasmic [Ca2+]i buffer dimethyl BAPTA (dmBAPTA) also blocks subsequent entry of cells into S phase. The dose response curves for inhibition of serum stimulation of [Ca2+]i and DNA synthesis by dmBAPTA are virtually identical including an anomalous stimulation observed at low levels of dmBAPTA. Reversal of the [Ca2+]i buffering effect of dmBAPTA by transient exposure of the cells to the Ca2+ ionophore ionomycin also reverses the inhibition of DNA synthesis 20-24 h later. Ionomycin by itself does not stimulate DNA synthesis. These data are consistent with the conclusion that a transient increase in [Ca2+]i occurring shortly after serum stimulation of quiescent fibroblasts is necessary but not sufficient for subsequent entry of the cells into S phase. This study is the first to show a direct relationship between early serum stimulated Cai2+ increase and subsequent DNA synthesis in human cells. It also goes beyond recent studies on BALB/3T3 cells by providing dose response data and demonstrating reversibility, which are strong indications of a cause and effect relationship.

Dolichol delays G1-arrest for one cell cycle in human fibroblasts subjected to depletion of serum or mevalonate.

It is well-established that some product(s) or metabolite(s) of mevalonate is (are) critical for growth of mammalian cells. In the search for this (these) compound(s) it seems meaningful to distinguish between compounds needed for cell cycle progression in proliferating cells and compounds needed for growth activation of arrested cells. By using time-lapse video recording we have studied the possible regulatory role of cholesterol, dolichol and mevalonate in the cell cycle of human diploid fibroblasts (HDF). HDF, which are serum-dependent, were rapidly growth-arrested in the first part of G1 upon removal of serum factors. They also responded to mevinolin (an HMG CoA reductase inhibitor) by a similar G1-block, indicating that a mevalonate-derived product is involved in the G1-located cell cycle control of HDF. Interestingly, dolichol counteracted the G1-block caused by both types of treatment. Hence, the early G1-cells could traverse the remainder of the cell cycle and divide despite depletion of serum or mevalonate. We also demonstrated that addition of dolichol resulted in a significant decrease in the rate of protein degradation. This protein stabilizing effect may constitute the mechanism by which dolichol delays the G1-arrest of HDF.

Replicative potential and the duration of the cell cycle in human fibroblasts: Coordinate stimulation by epidermal growth factor

The in vitro replicative potential of human diploid fibroblasts can be increased by polypeptide growth factors such as epidermal growth factor (EGF). Also, the cycle time of EGF-stimulated cells is, on average, decreased and their mitotic cell volume is reduced. Therefore, the regulation of cell size by the duration of the cell cycle may be one process which determines replicative potential. The growth response to a continuous presence of EGF, however, appears to be limited by eventual desensitization to the growth factor.

Production of interferon-β upon induction with polyinosinic acid:polycytidylic acid during the cell cycle of human fibroblasts

The production of interferon-β was examined at various stages of the cell cycle in synchronized and unsynchronized cell populations induced by poly(I):poly(C). Human fibroblasts were synchronized with mitotic detachment and, at different stages of the cell cycle, poly(I):poly(C) was added for induction of interferon-β. One hour after induction, cell-free medium was collected and assayed for secreted interferon-β. The cells were then fixed and stained with a DNA-specific fluorochrome, 4′,6-diamidino-2-phenylindole (DAPI), for cell cycle analysis by microfluorometry. The data indicated that interferon-β was produced in every stage examined of the cell cycle. In addition, the level of intracellular interferon-β was quantitatively measured in single cells of an unsynchronized cell population using a specific antibody. In the same individual cell, DAPI fluorescence intensity was measured for determination of the cell cycle position. The results show that interferon-β protein can be detected throughout the cell cycle.

Influence of native collagen on the cell cycle

The influence of native collagen-solutions on the cell cycle of human-fibroblasts was investigated. The results point out that the phase of cell-specific function is distinctly lengthened to the debit of a reduced rate of mitosis. That seems to be desirable, because the cells of the implantation-layer would produce more rapidly and augmentedly their cell-specific product--usually collagen--and incorporation of the implanted material would occur quite earlier. This effect seems also to be responsible for the promotion of granulation of "Dermodress", a fleece of collagen I for temporary covering of debrided areas (5).