Mitogenic Treatment Research Articles

The primary mitogen (TCPOBOP)-induced hepatocyte proliferation is resistant to transforming growth factor- β-1 inhibition

Transforming growth factor (TGF)-β-1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens. In this study, we compared the proliferative response in the liver between wild-type and transgenic mice, overexpressing active TGF-β-1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene). The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real-time polymerase chain reaction analysis of cell cycle-related genes. Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF-β-1 expression following the mitogenic treatment. TGF-β-1 does not inhibit the primary mitogen-induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment.

Protein Kinase C δ Stimulates Apoptosis by Initiating G1 Phase Cell Cycle Progression and S Phase Arrest

Overexpression of protein kinase C delta (PKCdelta) stimulates apoptosis in a wide variety of cell types through a mechanism that is incompletely understood. PKCdelta-deficient cells are impaired in their response to DNA damage-induced apoptosis, suggesting that PKCdelta is required to mount an appropriate apoptotic response under conditions of stress. The mechanism through which it does so remains elusive. In addition to effects on cell survival, PKCdelta elicits pleiotropic effects on cellular proliferation. We now provide the first evidence that the ability of PKCdelta to stimulate apoptosis is intimately linked to its ability to stimulate G(1) phase cell cycle progression. Using an adenoviral-based expression system to express PKCalpha,-delta, and -epsilon in epithelial cells, we demonstrate that a modest increase in PKCdelta activity selectively stimulates quiescent cells to initiate G(1) phase cell cycle progression. Rather than completing the cell cycle, PKCdelta-infected cells arrest in S phase, an event that triggers caspase-dependent apoptotic cell death. Apoptosis was preceded by the activation of cell cycle checkpoints, culminating in the phosphorylation of Chk-1 and p53. Strikingly, blockade of S phase entry using the phosphatidylinositol 3-kinase inhibitor LY294002 prevented checkpoint activation and apoptosis. In contrast, inhibitors of mitogen-activated protein kinase cascades failed to prevent apoptosis. These findings demonstrate that the biological effects of PKCdelta can be extended to include positive regulation of G(1) phase cell cycle progression. Importantly, they reveal the existence of a novel, cell cycle-dependent mechanism through which PKCdelta stimulates cell death.

Functional Characterization of SDF-1 Proximal Promoter

Stromal-cell derived factor 1 (SDF1) is a CXC chemokine that binds and signals through the CXCR4 receptor, playing an essential role in embryonic B lymphopoiesis, myelopoiesis and organogenesis. The CXCR4/SDF1 pathway is associated with several pathologies. CXCR4 serves as a fusion cofactor for lymphotropic strains of human immunodeficiency virus type 1 and SDF1 inhibits viral entry. Moreover, recent works suggest an important role for SDF1 in metastasis progression and autoimmune diseases such as rheumatoid arthritis. To understand the molecular mechanisms that regulate SDF1 expression, we have cloned and functionally analysed its 5' flanking regulatory region. An SDF1-promoter luciferase construct showed high levels of reporter gene activity in transient transfection experiments. DNase I footprinting analysis revealed that the proximal promoter was occupied by six putative Sp1-binding motifs. Binding of Sp1 to the promoter was confirmed by electrophoretic mobility shift assay, and its importance in SDF1 gene expression verified by in vitro mutagenesis. Particularly, mutation of an Sp1 motif located between -57 and -39 upstream of the main transcription start-site resulted in a marked reduction in promoter activity. It has been shown that the SDF1 expression could be induced by mitogenic stimuli, X-ray radiation or treatment with IL1beta, depending on cell environment. We have analysed the effect of these stimuli on SDF1 promoter transactivation in three different cell lines. Phorbol myristated acetate plus ionomycin increased promoter activity in U373 and LC5 but repressed it in MS5 cells. On the contrary, gamma irradiation promoted SDF1 transcription in MS5 cells but not in the other cell lines. Interferon-gamma acted as a transcriptional repressor in U373 and LC5 but not in MS5 cells. Finally, IL1beta functions as mild activator only in U373 cells. The present study demonstrates that these stimuli mediate SDF1 production through promoter activation in a cell-specific manner.

Skp2 increases the number of new hair cells generated by math1 in vivo

Problem: Hair cell loss in mammals is irreversible, leading to permanent hearing impairment. To restore hearing, it is necessary to generate new hair cells to replace the lost cells. Math1, the mouse homolog of the Drosophila gene atonal is necessary for hair cell development. Over-expression of Math1 in the adult guinea pig cochlea can generate new hair cells (Kawamoto et al., 2003); however, the number of new hair cells generated by Math1 over-expression is small. This study was designed to determine whether a mitogenic co-treatment can increase the number of new hair cells generated by Math1 gene therapy. For that purpose, we used a recombinant adenovirus with a Skp2 gene insert. Skp2 is an F-box protein that causes G1 to S transition through ubiquitination of p27Kip1. Methods: Normal adult guinea pigs were inoculated with the Skp2 vector (Ad.Skp2) along with an adenovirus encoding the Math1 gene (Ad.Math1) into the scala media of the second turn in their left cochlea. Control groups were inoculated with Ad.Math1, Ad.empty, or artificial endolymph. Two months later, we evaluated the surface morphology of the organ of Corti and surrounding tissues using scanning electron microscopy (SEM). Results: The numbers of ectopic hair cells and the cells with stereochilia in organ of Corti in the combined Ad.Math1/Ad.Skp2 group were significantly higher compared to the group inoculated with Ad.Math1 alone. Animals inoculated with Ad.empty or artificial endolymph displayed no ectopic hair cells. BrdU incorporation study showed that Skp2 over-expression induced mitosis of the cells in the organ of Corti and surrounding tissues. Conclusion: These findings suggest that increasing the number of nonsensory cells through a mitogenic treatment can enhance the number of new hair cells generated upon Math1 over-expression. Significance: Further development of this approach may provide a powerful tool for treating profound sensorineural hearing loss in human beings. Support: None reported.

Mitosis and differentiation in T-cells under cytotoxic action of Echinococcus granulosus hydatid fluid

In the T-cell line, D10, thymidine uptake was used to measure the proportion of cells in S-phase, and the MTT assay to measure the number of viable cells. The effect of Echinococcus granulosus hydatid fluid (HF) on the lymphocytes was assayed in 3-day cultures of the T-cell line, D10, in increasing concentrations of HF. Apparent cytotoxic effects of HF were recorded as a log-linear decline in S-phase activity, which was reduced by the presence of IL-1, IL-2, or a combination of the two. In the presence of IL-2, however, mitogenic treatment with concanavalin A increased the cytotoxic effect in 3-day cultures, while in day-2 cultures, HF itself showed mitogenic effect. HF-induced decline in S-phase activity was not matched by a parallel decline in viable cells, suggesting that the apparent cytotoxicity of HF could result from cell-cycle arrest. Depending on its origin, HF enhanced membrane expression of CD25 and CD38 on human peripheral blood lymphoblasts, and diminished that of CD28. Taken together, these changes suggest that HF can induce T-cell mitosis and reduce co-stimulation with subsequent T-cell anergy or apoptosis.

Identification and characterization of mRNAs differentially expressed in thyroid cells stimulated by a mitogenic treatment

The aim of our work is to identify new genes and proteins involved in the control of the proliferation of thyroidcells as putative protooncogenes and antioncogenes. Several strategies are discussed. A first study has allowed to identify three new genes. Further search will use the differential display and gene arrays methodology. The role of the identified proteins codedby the genes is studied in vitro by the search of partner proteins by the double hydrid method and in vivo by mice gene knockout technology.

Constitutive and Inducible Levels of egr-1 and c-myc Early Growth Response Gene Expression in Self-Renewing B-1 Lymphocytes

B-1 lymphocytes constitute a mature B cell population that differs from conventional B cells in signaling requirements for cell cycle progression, being unusually responsive to PKC agonism but refractory to antigen receptor stimulation. Further, B-1 cells are self-renewing and derive from surface immunoglobulin-positive precursors. A previous report on clonally derived B-1 cells suggested that increased levels of expression of c-myc might underlie these unusual growth characteristics. This issue has now been reexamined using primary B-1 cells. The possible role of egr-1, in addition to c-myc, in specifying the responsiveness of B-1 cells was evaluated by determining levels of gene expression at baseline and after mitogenic treatment. Expression of the two genes was similar in B-1 and B-2 cells prior to stimulation. Subsequently, B-1 cells responded with higher levels of gene expression than B-2 cells regardless of ultimate cell cycle progression, with the exception of stimulation by anti-Ig. Overall, there was no apparent direct correlation between stimulated levels of expression of egr-1 or c-myc and mitogen-induced S phase entry, nor were B-1 cells characterized by constitutively elevated levels of either proto-oncogene that might explain their capacity for self-renewal.

Differential regulation of mRNAs encoding protein kinase C isoenzymes in activated human B cells

Stimulation of human B cells via HLA class II antigens leads to an increase of PKC activity as a consequence of a transcriptional upregulation of the PKC. Extending previous data, other known B-cell activators, which include anti-IgM, SAC, and TSST1, are shown here to increase the cytosolic PKC activity significantly. Human B cells express significant mRNA levels of the PKCα, β, δ, ϵ, and ζ species while the γ species is consistently absent. The levels of PKCα and ϵ mRNA are increased by exposure to a nonmitogenic anti-IgM antibody in a lymphoblastoid B-cell line while PKCβ and δ mRNA are instead downregulated by this agent. An anti-HLA class II antibody (D1.12) induced an increase of PKCα, β, and δ mRNA. A time study of PKC mRNA levels in anti-IgM-treated cells showed that the accumulation of the PKCα mRNA precedes the increase of PKC enzymatic activity. Moreover, PKCβ mRNA decreased following treatment with SAC while, on the contrary, it increased following TPA, anti-HLA class II (1.35) mAb, or mitogenic anti-IgM treatment. Our results underline the complexity of signal transduction via the PKC pathway by revealing that the PKC isoforms are differentially regulated and are in keeping with the idea that they may have distinct physiologic roles in human B cells

Characterization and Functional Activity of Dendritic Cells from Rat Choroid

Cell preparations from the posterior eye cup of the eye cultured for 2 days exhibited accessory activity for T-cell responses to a mitogenic treatment and stimulatory activity in a mixed leukocyte reaction (MLR), two functions characteristic of dendritic cells (DC). These activities both partitioned with cells having a low buoyant density, another characteristic of DC. Immunomagnetic separations with monoclonal antibodies against lymphoid dendritic cell surface antigens revealed that the accessory activity of the low-density cells was entirely associated with a small population of cells positively selected by these antibodies. Immunofluorescent staining with these same antibodies also revealed a small subpopulation of low-density cells having the morphology of DC. On cryostat sections of eye tissue the positively-stained cells were localized in the choroid and were not observed within the sclera or the retina. Based on these results we conclude that there are functional DC in the choroid, and we speculate that they may have a significant role in the inflammatory process during posterior uveitis.

Oscillating levels of adenylate and guanylate cyclase activities in rat embryo fibroblasts stimulated to divide

Basal activities of membrane-bound adenylate and guanylate cyclase were determined in confluent rat embryo cells stimulated to proliferate by either the renewal of serum-supplemented growth medium or the addition of a mitogen, the 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A transient increase in guanylate cyclase activity was observed within minutes following either treatment while adenylate cyclase activity either abruptly declined in serum-stimulated cells or remained unaffected in TPA-treated cells. In response to both mitogenic treatment, adenylate and guanylate cyclase activities varied reciprocally throughout the pre-replicative phase up to DNA synthesis. The lower levels of guanylate over adenylate activity ratio occurred prior to the onset of the replicative phase whereas the higher levels were coincident with DNA synthesis. A similar pattern of oscillating levels of sodium-fluoride-stimulated adenylate and lubrol-treated guanylate cyclase activities was observed.

Differential effects of concanavalin A and phytohemagglutinin on murine immunity: Suppression and enhancement of humoral immunity

The abilities of concanavalin A (Con A) and phytohemagglutinin P (PHA) to selectively induce different T-cell activities affecting humoral immunity were evaluated. The mitogens were intravenously injected before, with, or after injection of sheep red blood cells (SRBC) into mice, and the 3 to 6-day plaque-forming cell (PFC) responses were assessed. Mitogenic treatment differentially influenced the resultant in vivo PFC responses to SRBC. The in vivo suppressive effects induced by Con A were shown to be temporary; only the Day 4 PFC response was inhibited. Con A given 3 hr before, with, or after the antigenic challenge enhanced the PFC response. In contrast, PHA given at all intervals inhibited both the 4- and 5-day PFC response. Neither mitogen appeared to affect the kinetics of the in vivo PFC response to SRBC. Both mitogens enhanced in vivo DNA synthesis by the splenic cells, and Con A appeared biphasic in its stimulation. Con A-induced effects on the humoral immune response were short-lived and transient, while PHA induced a longer-lasting effect on humoral immunity.

Differential effects of concanavalin A and phytohemagglutinin on murine immunity: Suppression and enhancement of cell-mediated immunity

To assess the effects of the selective T-cell mitogens concanavalin A (Con A) and phytohemagglutinin P (PHA) on cell-mediated immunity (CMI), the mitogens were injected before, with, or after intravenous (iv) challenge of mice with Listeria monocytogenes. Mitogenic treatment differentially influenced the CMI response to Listeria. Con A enhanced listericidal activity when given before or with Listeria challenge, but Con A suppressed the CMI response when given after infection with Listeria. In contrast, PHA enhanced listericidal activity at all intervals. Since Con A, but not PHA, affected the growth of Listeria in the spleens of mice 24 and 48 hr after infection, Con A was shown to have an immediate effect on the development of listericidal activity and PHA was shown to have a delayed effect. In addition, Con A induction of immediate nonspecific listericidal activity was short-lived, while PHA induced a longer-lasting effect on resistance to Listeria. The mitogen-induced effects in the CMI response to Listeria were shown to be dependent upon the activities of activated T cells. The enhancement and suppression of listericidal activity appears to result from the activation of different T-cell subpopulations, known to be stimulated preferentially by Con A or PHA.