The role of apoptosis-induced proliferation for regeneration and cancer

Genes dedicated to killing cells must have evolved because of their positive effects on organismal survival. Positive functions of apoptotic genes have been well established in a large number of biological contexts, including their role in eliminating damaged and potentially cancerous cells. More recently, evidence has suggested that proapoptotic proteins-mostly caspases-can induce proliferation of neighboring surviving cells to replace dying cells. This process, that we will refer to as "apoptosis-induced proliferation," may be critical for stem cell activity and tissue regeneration. Depending on the caspases involved, at least two distinct types of apoptosis-induced proliferation can be distinguished. One of these types have been studied using a model in which cells have initiated cell death, but are prevented from executing it because of effector caspase inhibition, thereby generating "undead" cells that emit persistent mitogen signaling and overgrowth. Such conditions are likely to contribute to certain forms of cancer. In this review, we summarize the current knowledge of apoptosis-induced proliferation and discuss its relevance for tissue regeneration and cancer.     

Stability conditions of cell populations in vivo

Specific exogenous stimulating and inhibiting substances act on cell proliferation and differentiation rates. An additional internal regulatory mechanism is shown to be necessary to ensure the regulation of cell proliferation in vivo. Various internal regulatory mechanisms have been envisaged. Stability conditions and experimental data led us to reject all of them except those postulating that an inhibitory signal produced by the differentiating cells acts on the rate of cell proliferation. This finding supports the theory concerning the mechanism of action of "chalone-like" inhibiting substances.     

Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria

Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but they have not historically been accessible to extensive genetic manipulation. Here we utilize RNA-mediated genetic interference (RNAi) to introduce large-scale gene inhibition studies to the classic planarian system. 1065 genes were screened. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. We assessed the effects of inhibiting genes with RNAi on tissue homeostasis in intact animals and stem cell (neoblast) proliferation in amputated animals identifying candidate stem cell, regeneration, and homeostasis regulators. Our study demonstrates the great potential of RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.     

Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.     

Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation

Nectins and nectin-like molecules (Necls) are immunoglobulin-like transmembrane cell adhesion molecules that are expressed in various cell types. Homophilic and heterophilic engagements between family members provide cells with molecular tools for intercellular communications. Nectins primarily regulate cell-cell adhesions, whereas Necls are involved in a greater variety of cellular functions. Recent studies have revealed that nectins and NECL-5, in cooperation with integrin alphavbeta3 and platelet-derived growth factor receptor, are crucial for the mechanisms that underlie contact inhibition of cell movement and proliferation; this has important implications for the development and tissue regeneration of multicellular organisms and the phenotypes of cancer cells.     

PCNA、Ki67 在胰腺疾病中的研究进展

PCNA、Ki-67是与细胞增殖有关的核抗原,在增殖的组织细胞中呈阳性表达,反映组织细胞的增殖活性,是细胞增殖的重要标记物。PCNA、Ki-67在正常发育的胚胎组织、糖尿病、胰腺肿瘤、胰岛移植等胰腺疾病及其他疾病中均高表达,同时也与其他系统肿瘤和疾病密切相关。PCNA、Ki-67作为增殖指标可以用于评价胰腺疾病、胰岛细胞移植后细胞再生数量及其他疾病的诊断、治疗及判断预后。目前已将它们视为细胞的标志物,用于细胞增殖的动力学研究,在临床病理上具有很大的应用前景。未来PCNA、Ki67将广泛应用于临床及基础研究,尤其用于研究胰腺疾病的新靶点、探索糖尿病的发病机制,对疾病的预防和治疗及胰岛移植具有一定的应用前景及意义。     

Notch与相关调控信号的相互作用在伤口愈合中的调控

皮肤是哺乳动物最重要的组织之一.当皮肤受损时,受损组织通过系列伤口愈合反应的生理和心理作用被修复,实现组织再生.再生反应主要发生在胚胎发育早期,伤口自愈能力随着机体的成熟而减弱;并且哺乳动物的组织重塑过程较为复杂,在不正确的信号引导下,可能引起并发症而导致创面愈合异常.研究表明,伤口微环境的稳态和信号分子的辅助作用是愈...     

Contribution of growth and cell cycle checkpoints to radiation survival in Drosophila

Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy.     

Maintenance of Functional Stem Cells in Isolated and Cultured Adult Intestinal Epithelium

We have previously described a method for the primary culture of adult large intestinal epithelium, suggesting that stem cells had survived both the isolation and the culture procedures. However, as no markers for such cells exist, confirmation of stem cell survival is difficult-only the functional properties can be used to define them. Unfortunately, many of these (e.g., differentiation, crypt regeneration) do not occur in culture, probably due to suboptimal conditions. To address this problem both freshly isolated and cultured small and large intestinal crypts were grown subcutaneously in an immunocompromized mouse. All initially formed cysts lined by a simple epithelium which gradually became multicellular and formed invaginations containing many mitoses and apoptoses. Epithelial differentiation, as assayed by Goblet cell mucin production, was also apparent. Mucin maturation was also typical of the normal intestine. The lumen was frequently filled with mucin and apoptotic bodies. Interestingly, in grafts displaying pronounced crypt-like morphology the regions of proliferation were situated toward the base of the structure and the Goblet cells toward the lumen, i.e., a typical crypt-like morphology. Hence, functional adult stem cells appear to survive isolation and tissue culture, permitting organotypic regeneration, possibly involving homeobox gene expression. This may now allow direct stem cell characterization and experimental manipulation, such as transfection, and may ultimately permit transplantation and therapeutic gene therapy.     

Antagonistic control of cell fates by JNK and p38-MAPK signaling

During the development and organogenesis of all multicellular organisms, cell fate decisions determine whether cells undergo proliferation, differentiation, or aging. Two independent stress kinase signaling pathways, p38-MAPK, and JNKs, have evolved that relay developmental and environmental cues to determine cell responses. Although multiple stimuli can activate these two stress kinase pathways, the functional interactions and molecular cross-talks between these common second signaling cascades are poorly elucidated. Here we report that JNK and p38-MAPK pathways antagonistically control cellular senescence, oncogenic transformation, and proliferation in primary mouse embryonic fibroblasts (MEFs). Similarly, genetic inactivation of the JNK pathway results in impaired proliferation of fetal hepatoblasts in vitro and defective adult liver regeneration in vivo, which is rescued by inhibition of the p38-MAPK pathway. Thus, the balance between the two stress-signaling pathways, MKK7-JNK and MKK3/6-p38-MAPK, determines cell fate and links environmental and developmental stress to cell cycle arrest, senescence, oncogenic transformation, and adult tissue regeneration.     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.     

Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells

Summary Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-β1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.     

Partial characterization of mosquito larvae extract inducing dna synthesis alterations on human mononuclear cells

A crude mosquito larvae and dialysed extract alters the mitotic rate of several epithelial cell populations in normal young and adult hepatectomized mice. A crude extract also showed a biphasic effect on the proliferation of human mononuclear cells (MNCs), either stimulating or inhibiting them depending on the dose applied. In the present paper, we assayed the effect of the dialysed mosquito larvae extract and two different protein fractions on human MNCs. Analysis of cell viability after culture indicated that the extract did not have toxic effects. Our results show a dual response of the MNCs to the dialysed, as well as to the protein fraction, with the highest molecular weight inhibiting or stimulating proliferation, depending on the dose applied. The protein fraction with the lowest molecular weight (range between 12-80 kDa) showed only an inhibitory effect on cell proliferation.     

Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with transforming growth factor-beta (TGF-beta). Muscle-specific protein synthesis and satellite cell fusion were used as indicators of muscle differentiation; a dose-dependent inhibition of differentiation was observed in response to TGF-beta. In addition, TGF-beta depressed cell proliferation in a dose-dependent manner. Half-maximal inhibition of differentiation was seen with a TGF-beta concentration of approximately 0.1 ng/ml. Although proliferation was not inhibited, it was depressed and half-maximal suppression of proliferation occurred in response to 0.1-0.5 ng TGF-beta/ml. Neonatal rat myoblasts were also subjected to TGF-beta treatment, and similar results were observed. Neonatal cells, however, were more sensitive to TGF-beta than satellite cells, as indicated by the reduced concentrations of TGF-beta required to inhibit differentiation and reduce the rate of proliferation. Under identical culture conditions proliferation of muscle-derived fibroblasts were also depressed. The differentiation inhibiting effect of TGF-beta on satellite cells was reversible. It has been suggested that TGF-beta could be an important regulator of tissue repair, and its in vitro effects on satellite cells suggest a possible role in regulation of muscle regeneration.     

异质性干细胞增殖过程中的熵变化

干细胞在多细胞生物体内广泛存在,其增殖过程在生命体的生长、发育、衰老、组织修复过程中起着重要作用。正常组织中的细胞增殖过程受到严格的控制,干细胞的异常增殖与恶性肿瘤、肥胖症、再生障碍性贫血等疾病有密切关系。生命体内异质性细胞的增殖过程是复杂的动力系统行为,干细胞异常增殖过程伴随细胞的可塑性变化和细胞间相互作用的再平衡过程,如何对这一过程进行定量描述是重要的研究课题。本文构建包含细胞的增殖分化指标和异常增殖性指标异质性的干细胞增殖模型,通过所建立的模型研究由于微环境变化引起的细胞异常增殖过程的熵变化,建立不同增殖条件下的系统熵变化与宏观动力学和系统参数之间的关系。结果表明,在细胞微环境变化引起异常增殖和恢复的过程中,系统的熵与细胞数量之间存在对应关系,而与微环境变化的路径无关。 此外,熵对细胞数量的依赖关系在异常增殖和恢复阶段表现出不同的行为,显示了生物过程的微观不可逆性。本文从物理学的角度对细胞异常增殖过程中熵变化与细胞数量变化的动力学给出定量刻画,为定量描述异质性干细胞增殖过程给出新的研究思路。     

Skin tissue generation by laser cell printing

For the aim of ex vivo engineering of functional tissue substitutes, Laser-assisted BioPrinting (LaBP) is under investigation for the arrangement of living cells in predefined patterns. So far three-dimensional (3D) arrangements of single or two-dimensional (2D) patterning of different cell types have been presented. It has been shown that cells are not harmed by the printing procedure. We now demonstrate for the first time the 3D arrangement of vital cells by LaBP as multicellular grafts analogous to native archetype and the formation of tissue by these cells. For this purpose, fibroblasts and keratinocytes embedded in collagen were printed in 3D as a simple example for skin tissue. To study cell functions and tissue formation process in 3D, different characteristics, such as cell localisation and proliferation were investigated. We further analysed the formation of adhering and gap junctions, which are fundamental for tissue morphogenesis and cohesion. In this study, it was demonstrated that LaBP is an outstanding tool for the generation of multicellular 3D constructs mimicking tissue functions. These findings are promising for the realisation of 3D in vitro models and tissue substitutes for many applications in tissue engineering.