Factors controlling smooth-muscle cell proliferation.

Injury to the arterial wall normally elicits a rapid and significant increase in smooth-muscle cell (SMC) replication with the subsequent development of intimal lesions. A variety of factors have been proposed to control SMC replication, but recent work has highlighted the role of basic fibroblast growth factor (bFGF) and platelet-derived growth factor in this process. In the carotid artery of the uninjured rat, we have shown that the SMCs express mRNA for bFGF and that bFGF can be readily extracted from these arteries. Following mechanical injury to the artery, ie, after balloon injury, we suggested that bFGF is released from damaged cells and then stimulates adjacent SMCs. In support of this concept, the infusion of a blocking antibody to bFGF was found to significantly inhibit the early SMC replication induced by use of a balloon catheter. The addition of the antibody at the time of injury, however, did not inhibit the development of intimal lesions. In contrast, studies by us and other investigators have shown that platelet-derived growth factor is not directly important for SMC replication after balloon injury, but that it plays a key role in stimulating the migration of SMCs into the intima. Intimal SMC replication was not inhibited with antibodies to either bFGF or platelet-derived growth factor. Therefore, while significant inroads have been made in understanding the initial events, we still do not fully understand all the processes involved in the proliferation of arterial intimal lesions.     

Drosophila Minus is required for cell proliferation and influences Cyclin E turnover

Turnover of cyclins plays a major role in oscillatory cyclin-dependent kinase (Cdk) activity and control of cell cycle progression. Here we present a novel cell cycle regulator, called minus, which influences Cyclin E turnover in Drosophila. minus mutants produce defects in cell proliferation, some of which are attributable to persistence of Cyclin E. Minus protein can interact physically with Cyclin E and the SCF Archipelago/Fbw7/Cdc4 ubiquitin–ligase complex. Minus does not affect dMyc, another known SCF^Ago substrate in Drosophila. We propose that Minus contributes to cell cycle regulation in part by selectively controlling turnover of Cyclin E.     

Factors controlling the turnover of T memory cells

Summary: Most of the T cells participating in the primary immune response are rapidly eliminated, but small numbers of these cells survive and differentiate into long-lived memory cells. Information on the life history of memory cells can be obtained by studying the component of memory-phenotype T cells found in normal animals; these cells are presumed to represent memory cells specific for various environmental antigens. For CD8⁺ cells, in vivo exposure to viruses and certain other infectious agents causes a large proportion of memory-phenotype (CD44^hi) cells to enter the cell cycle. In this situation, stimulation of CD44^hi CD8⁺ cells does not seem to require T-cell receptor ligation and appears to reflect release of various cytokines, especially type I interferon. The capacity of infectious agents to induce non-antigen-specific stimulation of T cells may play a role in boosting the survival of memory cells and perhaps also in providing an adjuvant function during the primary response.     

The assessment of cell proliferation during 9,10-dimethyl-1,2-benzanthracene-induced hamster tongue carcinogenesis by means of histone H3 mRNA in situ hybridization

The aim of this study was to investigate cell kinetics and ultrastructural changes during carcinogenesis using a hamster 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tongue cancer model. Five squamous cell carcinomas, five dysplastic epithelia, seven hyperplastic epithelia, and four normal epithelia were obtained from 21 hamster tongues by applying 1.0% acetone solution of DMBA on the left lingual mucosa after scratching with a root canal broach. Ultrastructural examination revealed that the number of microvilli increased, whereas that of desmosomes decreased during carcinogenesis. Cell proliferation was analyzed by means of 5-bromodeoxyuridine (BrdU) immunohistochemistry and in situ hybridization (ISH) for histone H3 mRNA. The BrdU and histone H3 mRNA labeling indices (LIs) were lowest for normal epithelium, higher for hyperplastic and dysplastic epithelia, and highest for squamous cell carcinoma. Cytoplasmic histone H3 mRNA and nuclear BrdU were localized in virtually identical areas of serial sections. The correlation coefficient for the relationship between these two LIs was 0.97 (P 0.001). These results suggest that the assessment of cell proliferation using H3 mRNA ISH will be a useful technique for investigating biological behavior during carcinogenesis.     

A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells

The unique property of stem cells to self-renew suggests specific mechanisms that regulate their cell-cycle progression. In the present study, we identify a novel protein, nucleostemin, found in the nucleoli of CNS stem cells, embryonic stem cells, and several cancer cell lines and preferentially expressed by other stem cell-enriched populations. It contains an N-terminal basic domain and two GTP-binding motifs. When stem cells differentiate, nucleostemin expression decreases rapidly prior to cell-cycle exit both in vitro and in vivo. Depletion or overexpression of nucleostemin reduces cell proliferation in CNS stem cells and transformed cells. Mutation analysis indicates that excessive nucleostemin, particularly mutants that lack the GTP-regulatory domain, prevents cells from entering mitosis and causes apoptosis in a p53-dependent manner. The N-terminal basic domain specifies nucleolar localization, the p53 interaction, and is required for the cell death caused by overexpression. This work describes a novel nucleolar mechanism that controls the cell-cycle progression in CNS stem cells and cancer cells.     

RXRalpha mRNA expression is associated with cell proliferation and cell cycle regulation in Hep3B cell

Retinoids are well-characterized differentiation and anti-proliferation agents. The functional role of retinoid x receptor alpha (RXRalpha) in cell proliferation and cell cycle regulation is not well understood. Using human Hep3B cell line, we showed that the mRNA level of RXRalpha was closely associated with cell growth. RXRalpha mRNA expression elevated along with the proliferation of Hep3B cells. This association was most evident in RXRalpha and was also noted with retinoic acid (RA) receptor alpha (RARalpha), but not found in other RARs and RXRs. The expression of RXRalpha and cyclin A mRNA was co-regulated when Hep3B cells were cultured in serum-free medium. The mRNA levels of RXRalpha and cyclin A appeared to be highest in G1/S phase in Hep3B cells treated by aphidicolin. Taken together, our data suggest that RXRalpha may be actively involved in cell proliferation and cell cycle regulation in Hep3B cells.     

Endometrial endothelial cell proliferation during the menstrual cycle

Adult endometrium is a tissue in which physiological angiogenesisoccurs regularly and provides an accessible source of materialfor study. Endometrial biopsies and immunohistochemistry wereused to test two hypotheses: firstly, that there are peaks ofendothelial cell proliferation in human endometrium during themenstrual cycle, and secondly that in-vitro endothelial cellmigratory signal production by human endometrium (measured ina previous study) accompanies endothelial cell proliferativeactivity in vivo. Proliferating cells were identified usinganti-proliferating cell nuclear antigen, and endothelial cellswere identified using anti-CD34. The method was validated bya smaller, separate study using bromodeoxyuridine incorporationand detection with formalin-fixed biopsies, and comparison withproliferating cell nuclear antigen staining. The main study(n=50) showed that significant peaks of endometrial endothelialcell proliferation could not be identified, due to the largevariability in endothelial cell proliferative activity betweenindividuals at the same stage of the menstrual cycle. Endometrialendothelial cell migratory signal production did not correlatewith endothelial cell proliferation (n = 27). Results suggestthat blood vessel growth in the endometrium may occur by a processthat differs from the traditional concept of angiogenesis, whichhas been derived mainly from in-vitro or in-vivo experimentalstudies     

Differential requirement of caspases during naive T cell proliferation

The involvement of Fas and caspase activation during naive T cell proliferation is still controversial. To explore this paradox, we used antigenic variation of PCC 88-104 peptide to dissect pathways of TCR signaling leading to cell proliferation. We demonstrated that strong TCR stimulation but not weak TCR stimulation induced T cell proliferation and was dependent on IETD- but independent of DEVD-specific caspases. In addition, altered TCR ligand induced T cell proliferation in the absence of IETD-, DEVD-specific caspase activities and Fas ligand expression. However, AND-TCR-transgenic mice in lpr/lpr background generated recently have no defect T cell proliferation after stimulation by the agonist peptide, demonstrating that antigen-induced T cell proliferation is independent of Fas-activation pathways. Thus, Fas-independent caspase activation is tightly regulated by the strength of antigenic stimulation during T cell proliferation. These data reveal a novel facet of antigenic caspase regulation during naive CD4 T cell proliferation and provide insights into the function of caspases during T cell homeostasis.     

The importance of the innate immune system in controlling HIV infection and disease

The innate immune system is the first line of defense against invading pathogens and is particularly important in warding off bacterial and viral infections presenting at the mucosal cell surface. From this primitive immune response, the more sophisticated adaptive immune system was derived. Despite nearly two decades of research directed at inducing adaptive immune responses to HIV, no successful immunological therapy or vaccine has been developed. On the basis of recent observations, it is suggested that instead emphasis should now be placed on the alternative arm of the immune system, the innate immune response. Novel approaches should be developed to elicit this rapidly responding immune activity in HIV infection.     

不同增殖能力结肠癌细胞株iNOSmRNA表达的比较研究

目的:探讨iNOSmRNA在结肠癌不同增殖能力细胞株中的表达和作用,研究ATRA对于结肠癌不同增殖能力细胞株iNOSmRNA表达的影响。方法:采用MTT方法确定结肠癌细胞株CW-2和LS174T的生长增殖状况,用RT-PCR和Northernblot方法检测结肠癌中iNOSmRNA的表达量。结果:MTT生长曲线显示结肠癌细胞株LS174T的生长增殖比CW-2快;RT-PCR显示CW-2细胞株有较强的iNOSmRNA表达,而LS174T细胞株iNOSmRNA的表达较弱;Northernblot检测在CW-2中有明显的iNOSmRNA表达,但在细胞株LS174T中表达相对较弱;ATRA对结肠癌CW-2和LS174T细胞株iNOSmRNA的表达量无明显影响。结论:iNOSmRNA对结肠癌细胞株生长有双重作用,即在低增殖结肠癌CW-2呈高表达,可以通过细胞毒或诱导细胞凋亡等作用发挥抗肿瘤效应;在高增殖结肠癌LS174T呈低表达,产生NO作为信号转导的重要分子,增加血供和血管生成,促进肿瘤生长、侵袭和转移。ATRA可以抑制结肠癌细胞株的生长。     

Freeform fabricated scaffolds with roughened struts that enhance both stem cell proliferation and differentiation by controlling cell shape

We demonstrate that freeform fabricated (FFF) scaffolds with a roughened surface topography can support hBMSC proliferation, while also inducing osteogenic differentiation, for maximized generation of calcified, bone-like tissue. Previously, hBMSCs rapidly proliferated, without osteogenic differentiation, during culture in FFF scaffolds. In contrast, hBMSCs underwent osteogenic differentiation, with slow proliferation, during culture in nanofiber scaffolds. Analysis of cell morphology showed that the topography presented by the nanofiber scaffolds drove hBMSC differentiation by guiding them into a morphology that induced osteogenic differentiation. Herein, we hypothesized that using the high-surface area architecture of FFF scaffolds to present a surface roughness that drives hBMSCs into a morphology that induces osteogenic differentiation would yield a maximum amount differentiated hBMSCs and bone-like tissue. Thus, a solvent etching method was developed that imparted a 5-fold increase in roughness to the surface of the struts of poly(ε-caprolactone) (PCL) FFF scaffolds. The etched scaffolds induced osteogenic differentiation of the hBMSCs while un-etched scaffolds did not. The etched scaffolds also supported the same high levels of hBMSC proliferation that un-etched scaffolds supported. Finally, hBMSCs on un-etched scaffolds had a large spread area, while hBMSCs on etched scaffolds has a smaller area and were more rounded, indicating that the surface roughness from the etched scaffolds dictated the morphology of the hBMSCs. The results demonstrate that FFF scaffolds with surface roughness can support hBMSC proliferation, while also inducing osteogenic differentiation, to maximize generation of calcified tissue. This work validates a rational approach to scaffold fabrication where the structure of the scaffold was designed to optimize stem cell function by controlling cell morphology.     

Measurement of daughter cell accumulation during lymphocyte proliferation in vivo

We describe a method to characterize the effects that immunological adjuvants have on in vivo lymphocyte proliferation at the level of daughter cell accumulation. We used standard 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling techniques to follow T cells through multiple rounds of division in experimentally treated mice and measured both the fold-increase in cell number and average number of divisions undergone by each cell. These data were then incorporated into a calculation to determine the average number of daughter cells that accumulated from each round of mitosis, termed the daughter cell accumulation index. In vivo proliferation of T cells that had been stimulated by antigen in the absence of adjuvant was associated with an index value of 1.2, far below the theoretical maximum of 2.0. Low index values indicate poor daughter cell accumulation during proliferation, either because the newly produced cells died or persisted without dividing again. Inclusion of the natural adjuvant lipopolysaccharide (LPS) led to average accumulation of 1.75 daughter cells per cell cycle and an exponential increase in peak clonal expansion. Adjuvant-induced increases in average daughter cell accumulation appeared to account for more of the enhancement in clonal expansion than did adjuvant-induced increases in the number of cell divisions undergone by each cell. Therefore measurement of changes in daughter cell accumulation can be important to understanding how adjuvants influence the yield of proliferating lymphocytes. Measurement of average daughter cell accumulation is likely to be helpful in any cellular context in which it is useful to characterize strictly mitogenic versus accumulative effects.     

The importance of 5-hydroxytryptamine turnover for the analgesic effect of morphine in the chicken

A new method for testing analgesia in young chicks is described. It consists of shining a light on the chick's head till the chick senses the heat and moves. The report in the literature that chickens require very large doses of morphine to produce analgesia was only partly confirmed. Three strains (called A, B &; C) were tested. Only 57% of the chicks showed analgesia after morphine 100mg/kg in strain C, whereas all chicks responded to 30mg/kg in strain B and to 15mg/kg in strain A. The subcutaneous ED₅₀ was 80 mg/kg in C, 17.5 mg in B and 5.6 mg in A. Except for the first days of life, the analgesic responses are not age-dependent (tested up to 48 days). The relationship between 5-hydroxytryptamine turnover in the brain and analgesic effect of morphine was examined in the three strains. Only strain A showed an increase in 5-hydroxyindole acetic acid (of 31%, P < 0.01, in the cerebral hemispheres, and of 23%, P < 0.05, in the optic lobes) and in this respect resembled the mammals.It can be concluded that the phenomenon of the interaction of morphine with 5-hydroxytryptamine-containing neurones, which enhances the analgesic effect of morphine in the mammalian brain, occurs in some strains of chicks but is absent in others. The strain in which it was found had a sensitivity to morphine which approached that seen in mammals.