Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Fine root growth and demographic responses to nutrient patches in four old-field plant species

Proliferation of roots in a nutrient patch can occur either as a result of an increase in root length (morphological response) or by a change in root birth or death rates (demographic responses). In this study we attempted to distinguish between these two mechanisms of response to nutrient patches and to compare the responses of four old-field plant species (two annuals, two perennials). For all four species combined, there were significant increases in root numbers and root length in fertilized patches. Root proliferation in fertilized patches was largely due to increased birth (=branching) rates of new roots. However, there was also a significant increase in root death rates in the fertilized patches which reduced the magnitude of the increase in net root numbers. Plots for individual species suggested they differed in the magnitude and timing of root proliferation in fertilized patches due to differences in root birth and death rates. However, because of the limited sample size in this study, there was only a marginally significant difference among species in root birth rates, and no difference in death rates. Further studies are currently underway to better quantify species differences in the demographic mechanism, as well as magnitude, of response to nutrient patches and if this would affect the ability to exploit small-scale heterogeneity in soil resources.     

In vitro culture of vanhoutte's spirea explants from ‘secondary cultures’ and dormant stems forced in solutions containing plant growth regulators

Explants from new growth of forced dormant stems and secondary cultures of Vanhoutte's spirea were cultured on Linsmaier and Skoog (L.S.) medium containing benzyladenine (BA), indoleacetic acid (IAA), thidiazuron (TDZ), and zeatin. The dormant stems were forced by immersing their basal portions in forcing solutions containing 626 µM 8-hydroxyquinoline citrate (8-HQC) and 2% sucrose. BA and gibberellic acid (GA₃) were also added into the forcing solutions to determine if explants obtained from the new growth will benefit from this treatment when culturedin vitro.L.S. medium supplemented with 5 µM BA alone, 5 µM BA plus 1 or 5 µM IAA, and 0.5 or 0.75 µM TDZ alone produced the best shoot proliferation for both sources of explants. BA and GA₃ appeared to be taken up from the forcing solution by the new softwood growth. BA in the forcing solution stimulatedin vitro shoot proliferation in different degrees depending on the period of treatment, while GA₃ caused lessin vitro shoot proliferation. It is proposed that forcing solutions containing plant growth regulators (P.G.R.) are a useful approach for manipulating responses of plant tissues culturedin vitro.     

Promotion of root emergence in vitro from rhizome buds of Lapageria rosea cv. Nashcourt after proliferation in the presence of paclobutrazol

Medium components that may influence the emergence of adventitious root primordia in rhizome buds of Lapageria rosea (Ruiz et Pav.) cv. Nashcourt in vitro were examined. Sucrose was the only medium component tested that affected root emergence. Increasing the sucrose concentration in the medium from 10 to 30 g 1^-1 stimulated adventitious root emergence from rhizome buds that had been proliferated on a medium containing the gibberellin biosynthesis inhibitor paclobutrazol (5 M). Successful clonal propagation of L. rosea cv. Nashcourt can now be achieved by a rhizome bud proliferation stage(s) in the presence of paclobutrazol followed by an adventitious root emergence stage in the absence of paclobutrazol.     

Tissue Fixation for Immunohistochemical Detection of Proliferating Cell Nuclear Antigen with PC10 Monoclonal Antibody

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3-24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehydecontaining solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.     

Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin

Summary During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.     

Patterns of cellular proliferation and migration in the developing tectum mesencephali of the frog Rana temporaria and the salamander Pleurodeles waltl

The development of the tectum mesencephali was studied in the frog Rana temporaria and the salamander Pleurodeles waltl by means of nuclear staining and by labeling of cells with bromodeoxyuridine (BrdU). The general spatial and temporal pattern of cell proliferation and cell migration is the same in both species, despite drastic differences in overall tectal morphology. However, the salamander species differs from the frog species by (1) a generally lower cell proliferation rate, (2) a reduction in the activity of the lateral proliferation zone, and (3) a reduction in the formation of superficial cellular layers. Because point (3) affects processes that occur late in ontogeny, our experiments provide evidence that the simple morphology of the tectum of Pleurodeles waltl, compared with the multilayered tectum of Rana, is a consequence of a paedomorphic alteration of the ancestral developmental pattern of the amphibian tectum.     

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon,Oncorhynchus keta

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stagespecific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a genetransfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were estblished. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 celllines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue nonspecific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.