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The retinoblastoma protein as a fundamental mediator of growth and differentiation signals.

The retinoblastoma gene (RB) is the prototype of the tumor suppressor genes, which play critical roles in the genesis of cancer in humans. Mouse models created through gene knock-out and transgenic methods were established for exploring and manipulating RB in vivo. These models and several other pieces of evidence have shown that the retinoblastoma protein (Rb) plays dual roles in gating cell cycle progression and promoting cellular differentiation. The molecular mechanisms involved in these roles are becoming more obvious in some biological systems: Rb sequesters the transcription factor of E2F to regulate entry of cell cycle but enhances the activities of another class of the transcription factors, exemplified by NF-IL6, to initiate terminal cellular differentiation. Thus, the Rb protein can serve as a mediator for extracellular signals of growth or differentiation. The fundamental question of why only limited cell types are susceptible to tumor formation when Rb expression is lost, however, remains unanswered at present.

Animals↗

Retinoic acid: biochemistry and metabolism.

Retinoic acid, unlike the naturally occurring vitamin A (retinol), is a minor component of the human diet. It is formed in vivo from retinol and has many metabolites. The biological activity of the metabolites is not higher than that of retinoic acid itself, indicating that the metabolites must be products of retinoic acid catabolism. Little is known about the enzymatic systems responsible for forming retinoic acid or about how it enters the cell. Discovering the molecular mechanism(s) of retinoic acid activity in cellular metabolism is important to understanding its physiologic role. The pharmacologic effects of high doses of retinoic acid may be caused by its action on cellular membranes. Conversely, low concentrations appear to produce physiologic effects. Results of experiments with animals and with cell cultures indicate that the primary physiologic role of retinoic acid is in cellular differentiation. Retinoic acid influences genomic expression, inducing the appearance of some proteins while suppressing the expression of others. The existence of an intracellular retinoic acid-binding protein suggests that it may mediate the physiologic effects of retinoic acid on cellular differentiation.

Animals↗

The extracellular matrix components laminin, fibronectin, and collagen IV are present among the epithelial cells forming Rathke's pouch.

Cell-matrix interactions probably play a cooperative role with cell-hormone interactions to ensure normal differentiation of the adenohypophysis. The extracellular matrix (ECM) surrounding adult adenohypophysial cells contains laminin but its embryonic development has not been described. This study was carried out to test the hypothesis that adenohypophysial cells are associated with components in the ECM prior to cellular differentiation in the adenohypophysis. Fetuses were removed from Golden Syrian hamsters every 12 h from embryonic days 8.5-14. Coronal and sagittal 5-microns-thick sections of paraplast-embedded embryos were stained for fibronectin, laminin, or collagen IV using avidin-biotin immunoperoxidase staining. The adenohypophysial anlage was observed initially as a group of epithelial cells (Rathke's pouch) in the roof of the stomatodeum in contact with a basement membrane. The basement membrane stained positively for laminin, collagen IV and fibronectin. Lighter staining for laminin, fibronectin and collagen IV was observed between the developing adenohypophysial cells in Rathke's pouch. Proliferative activity was apparent in the antero-inferior region of Rathke's pouch and resulted in the formation of the bulk of the adenohypophysis. Mesenchyme infiltrates the region between the base of Rathke's pouch and the oral epithelium, thus separating the two. The basement membrane surrounding the pouch appears to become discontinuous in the regions of high proliferative activity. These results show that ECM components appear early during the development of adenohypophysial cells prior to their cellular differentiation into hormone-containing cells. This association between ECM components and developing adenohypophysial cells provides the anatomical basis for cell-ECM interactions to influence adenohypophysial development and differentiation.

Animals↗

Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines, IFN-inducible protein 10, monokine induced by IFN, and IFN-inducible T cell alpha chemoattractant in human cardiac allografts: association with cardiac allograft vasculopathy and acute rejection.

CXCR3 chemokines exert potent biological effects on both immune and vascular cells. The dual targets suggest their important roles in cardiac allograft vasculopathy (CAV) and rejection. Therefore, we investigated expression of IFN-inducible protein 10 (IP-10), IFN-inducible T cell alpha chemoattractant (I-TAC), monokine induced by IFN (Mig), and their receptor CXCR3 in consecutive endomyocardial biopsies (n = 133) from human cardiac allografts and corresponding normal donor hearts (n = 11) before transplantation. Allografts, but not normal hearts, contained IP-10, Mig, and I-TAC mRNA. Persistent elevation of IP-10 and I-TAC was associated with CAV. Allografts with CAV had an IP-10-GAPDH ratio 3.7 +/- 0.8 compared with 0.8 +/- 0.2 in those without CAV (p = 0.004). Similarly, I-TAC mRNA levels were persistently elevated in allografts with CAV (6.7 +/- 1.9 in allografts with vs 1.5 +/- 0.3 in those without CAV, p = 0.01). In contrast, Mig mRNA was induced only during rejection (2.4 +/- 0.9 with vs 0.6 +/- 0.2 without rejection, p = 0.015). In addition, IP-10 mRNA increased above baseline during rejection (4.1 +/- 2.3 in rejecting vs 1.8 +/- 1.2 in nonrejecting biopsies, p = 0.038). I-TAC did not defer significantly with rejection. CXCR3 mRNA persistently elevated after cardiac transplantation. Double immunohistochemistry revealed differential cellular distribution of CXCR3 chemokines. Intragraft vascular cells expressed high levels of IP-10 and I-TAC, while Mig localized predominantly in infiltrating macrophages. CXCR3 was localized in vascular and infiltrating cells. CXCR3 chemokines are induced in cardiac allografts and differentially associated with CAV and rejection. Differential cellular distribution of these chemokines in allografts indicates their central roles in multiple pathways involving CAV and rejection. This chemokine pathway may serve as a monitor and target for novel therapies to prevent CAV and rejection.

Acute Disease↗

IL-5, IL-8 and GM-CSF immunostaining of sputum cells in bronchial asthma and chronic bronchitis.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-5 or IL-8 have been suggested to play an important role in the pathogenesis of eosinophilic airway inflammation in bronchial asthma or neutrophilic airway inflammation in chronic bronchitis, respectively, However, GM-CSF and IL-8 have biological activities to either eosinophils or neutrophils. OBJECTIVE: To investigate the contribution of these cytokines to airway inflammation, we compared the cellular differential and immunolocalization of GM-CSF, IL-5 and IL-8 in sputum cells from patients with bronchial asthma and chronic bronchitis. METHODS: Cytospins of sputum cells from 12 patients with bronchial asthma and 12 with chronic bronchitis were subjected to cellular differential counting and immuno-cytochemistry with anti-human GM-CSF, IL-5 and IL-8 antibody. RESULTS: The predominant cells in bronchial asthma were eosinophils and lymphocytes, while those in chronic bronchitis were neutrophils. All cytokines examined were detected in either bronchial asthma or chronic bronchitis, although the percentage of GM-CSF and Il-5 positive cells in bronchial asthma (53.4 +/- 6.0 [mean +/- SEM]% and 9.7 +/- 2.8%, respectively) was significantly higher than that in chronic bronchitis (11.4 +/- 2.5%; P < 0.001 and 1.7 +/- 0.3%; P < 0.007, respectively). In contrast, the percentage of IL-8 positive cells in chronic bronchitis (23.8 +/- 7.0%) was significantly higher than that in bronchial asthma (7.& +/- 1.9%; P < 0.04). The cells positive for IL-5 were lymphocytes in bronchial asthma and chronic bronchitis. The cells positive for GM-CSF in bronchial asthma were predominantly eosinophils, while those in chronic bronchitis were monocytes/macrophages and neutrophils. In contrast, neutrophils are mainly positive for IL-8 in chronic bronchitis, while monocytes/macrophages and bronchial epithelial cells are positive for IL-8 in bronchial asthma. CONCLUSION: The immunochemical comparison of GM-CSF and IL-8 localization in sputum cells between bronchial asthma/chronic bronchitis suggests the differential regulation and roles of these cytokines in eosinophilic vs neutrophilic airway inflammation, resulting in the development of different types of airway inflammation.

Adult↗

Establishment and characterization of the growth and pulmonary metastasis of a highly lung metastasizing cell line from canine osteosarcoma in nude mice.

Highly lung metastasizing model of canine osteosarcoma in nude mice was established from five subcutaneous implantation cycles of lung tumor deposits. The selection of cells with increased metastatic properties from the parent POS canine osteosarcoma cell line recovered medium sized and polygonal Highly Metastasizing POS cells (HMPOS). The doubling time of HMPOS and POS in culture averaged 30 +/- 1.2 hr and 32 +/- 1.3 hr respectively, and their cell growth patterns in vitro were comparable to their in vivo growth patterns. HMPOS cells produced more tumor deposits (> 20 nodules, > 1 -mm in diameter) of various sizes with replacement of lung tissues at 12 weeks after implantation. POS cells produced fewer and smaller lung deposits (< 10 nodules, 1-mm in diameter). Tumor size and number of metastatic tumor deposits showed a regular association. HMPOS cells developed an osteoblastic type of cellular differentiation subcutaneously and in the lungs. HMPOS micrometastasis along the alveolar walls and blood vessels at 4 weeks averaged 6-7 small tumor locus. Each micrometastatic locus contained an average of 5-7 tumor cells, and developed a pleomorphic osteoblastic type of cellular differentiation. An average of 4 macrometastatic nodules could be seen at 6 weeks, composed of an average of 23 tumor cells, 10 nodules at 8 weeks, 12 nodules at 10 weeks and 20 nodules at 12 weeks. These model provides an opportunity for the evaluation of new treatments against canine lung metastatic osteosarcoma in a nude mice model.

Animals↗

Influence of diabetic metabolic state on fracture healing in spontaneously diabetic rats.

BACKGROUND: Type 1 diabetes mellitus (DM) has been shown to alter the properties of bone and impair fracture healing in both humans and animals. The objective of this study was to examine changes in the histomorphometrical, histological and mechanical parameters of bone and remodeling during fracture healing, depending on the diabetic metabolic state in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats, a rat strain that represents a close homology to DM in man. METHODS: On the basis of blood glucose values at the time of surgery, postoperative blood glucose course and postoperative insulin requirements, 90 spontaneously diabetic BB/OK rats were divided into groups with well-compensated or poorly compensated metabolic state. Forty-five LEW.1A rats served as the normoglycemic controls. The femurs were fractured in a standardized procedure and then allowed to heal for 2, 4 and 6 weeks. RESULTS: Our study showed that, in terms of bone histomorphometry, within the first 4 weeks after fracture, severe mineralization disorders occurred exclusively in the rats with poorly compensated diabetic metabolic states with a significant decrease of all fluorochrome-based parameters of mineralization, apposition, formation and timing of mineralization, as well as a delay in cellular differentiation and significantly decreased values of biomechanical properties in comparison to the spontaneously diabetic rats with well-compensated metabolic states and to the control rats. CONCLUSIONS: With a controlled insulin therapy and a resulting well-compensated diabetic metabolic state, mineralization and cellular differentiation disorders, as well as the decreased values of biomechanical properties in the fracture repair in the poorly compensated diabetic metabolic state with very severe hyperglycemia can be avoided.

Animals↗

Effects of radiation therapy on chondrocytes in vitro.

The negative irradiation complications of growth loss leading to limb length asymmetry and pathological fracture incurred following radiation therapy in pediatric patients has led to a renewed interest in understanding the specific effects of irradiation on the growth plate and the surrounding bone. In the present report, we examined the radiation therapy effects on primary rat growth cartilage chondrocytes in order to determine the chondrocyte radiosensitivity relative to other bone cell constituents and tumor cells, the postirradiation temporal progression of radiation-induced alterations in chondrocyte function, and the time course for the functional restoration of chondrocyte pathways that drive the eventual recovery in growth function. We employed an in vitro primary rat costochondral growth cartilage cell culture model system to evaluate the radiation therapy effects on proliferative chondrocytes using serial radiation doses (0-20 Gy) that are well within the clinically relevant range. Following irradiation, all of the following occurred in a dose-dependent manner: proliferation decreased, cytotoxicity increased, several markers of apoptosis increased, markers of radiation-induced cellular differentiation increased, and cell synthetic activity was disturbed. Alterations in proliferation, cell death, and induction of apoptosis are likely due to a transient radiation-induced derangement of the parathyroid hormone-related protein-Indian hedgehog proliferation-maturation pathway. Alterations in cellular differentiation and cell synthetic activity are novel observations for chondrocytes. Further, these results correspond very well to our previous work in an in vivo Sprague-Dawley rat model, making this model particularly relevant to researching the radiation therapy effects on longitudinal growth.

Alkaline Phosphatase↗

Chicken fetal antigen: example of an antigenically complex oncodevelopmental membrane glycoprotein.

CFA is a complex onco-developmental membrane marker system that is being investigated at the cellular, molecular, genetic and functional levels. CFA appears to be a family of genetically-determined carbohydrate side chains attached to a membrane-bound polypeptide chain. Unique combinations of the thirteen serologically detectable CFA determinants are expressed on erythroid and lymphoid cells of all avian species tested. Whether or not CFA determinants are hematopoietic specific markers remains to be established. The CFA determinants are proving to be useful membrane markers for the study of normal as well as neoplastic hematopoietic cellular differentiation. The probability that CFA determinants are differentiation-stage-specific markers for cells of both the erythroid and lymphoid series coupled with the observation that one of the CFA determinants acts as a cell membrane binding site for an arbovirus makes this antigen system a valuable tool for studying the cellular differentiation blockage hypothesis of neoplasia.

Aging↗

Abnormalities in the differentiation and cellular properties of hyperplastic lens epithelium from strains of chickens selected for high growth rate.

Similar morphological abnormalities of the lens of the eye of two unrelated strains of chicks, both of which had been selected for high growth rate, were found to be associated with epithelial cells showing marked deviations from the normal in cell surface properties, mitotic rate, capacity for differentiation, and in DNA, RNA and protein metabolism. The relationship between these modified properties and the observed morphology is discussed.

Animals↗

Notch signalling in the regulation of peripheral T-cell function.

The Notch signalling pathway plays a highly-conserved role in regulating the cellular differentiation and proliferation events that characterise pattern formation in the embryo. As cells in the embryo respond to environmental signals, similarly T-cells in the peripheral immune system must monitor their environment for antigens and respond accordingly by entering one of several potential differentiation pathways. Recent studies have identified a role for the Notch pathway in regulating the responses of T-cells in the periphery. In this review, we discuss these findings in the context of the Notch signalling pathway's role as an orchestrator of cellular differentiation, and propose a central role for Notch as a regulator of immune system function.

Animals↗

Prenatal diethylstilbestrol exposure alters murine uterine responses to prepubertal estrogen stimulation.

Prepubertal estrogen stimulation was used to investigate the effects of prenatal diethylstilbestrol (DES) exposure on subsequent growth, secretory activity, and cellular differentiation of the mouse uterus in vivo. Secretory activity was examined using sensitive silver staining of two-dimensional gel electrophoresis of uterine luminal fluid (ULF). Decreased uterine growth response, decreases in ULF quantity and protein concentration, alterations in specific ULF proteins, and altered cellular differentiation were found. This system provides a method for evaluation of the effects of prenatal exposure to DES or other compounds on the estrogen-induced secretory activity of the uterus. The alterations found in this study may be partially responsible for the decreased fertility in this mouse model and may have implication for DES-exposed women.

Animals↗

The epithelial-specific Ets factors occupy a unique position in defining epithelial proliferation, differentiation and carcinogenesis.

The Ets family of transcription factors regulates many biological processes. Within the Ets family are a subset of proteins that have epithelial restricted expression patterns, both in tissues and cell lines. These Epithelial-specific Ets (Ese) factors, cluster into two groups based on the sequence of the Ets DNA binding domain: (i) Ese1, Ese2 and Ese3, and (ii) the more divergent, Pdef. Gene targeting and cell culture studies have demonstrated that Ese factors are important for cellular differentiation. Furthermore, the ability of Ese factors to regulate expression of genes that are markers of differentiation supports this model. Significantly, the expression profile of these Ese factors is altered in epithelial cancers relative to the respective normal tissues. It is likely that this variation in expression affects downstream target genes important for carcinogenesis such as oncogenes and tumor suppressor genes. Understanding the role of these Ese factors in normal cellular differentiation may elucidate specific mechanisms by which cancer cells can become undifferentiated.

Animals↗

Ultrastructural and quantitative dynamics of the granulosa of ovarian follicles of the lizard Gerrhonotus coeruleus (family Anguidae).

The progression of ovarian follicular development in the Northern Alligator Lizard has been documented ultrastructurally and by enumeration of cells, with a focus on changes in the granulosa component of the follicle. The pattern of cellular differentiation of the granulosa entails, as in other lizards, the transformation of a simple, cuboidal epithelium in small follicles into a complex layer consisting of three types of cells. Marked differences in size and ultrastructure of the cell types indicate different functional states: the smallest cells are little differentiated and serve primarily as stem cells to other granulosa cells throughout follicular growth, whereas the larger "intermediate" and "pyriform" cells do not divide and show ultrastructural features indicative of synthetic activity. Contrary to some views that this latter cell type is the final step in cellular differentiation and provides organelles and cytoplasm to the oocyte through an intercellular bridge, the results of this study suggest that only relatively small molecules such as ribosomal RNA might pass between cells. Further, these observations support the interpretation that a heterogeneous granulosa results from the fusion in early follicular stages of some cells that are in surface contact with the oocyte. Several of the cytological features of the larger granulosa cell types are seen in the oocyte and in germ-line cells generally, such as highly dispersed chromatin, large nucleoli, abundant nuclear pores, mitochondrial "rosettes," annulate lamellae, "ribosome bodies," and surface microvilli. This strongly suggests that the cytology of large granulosa cells is induced by the oocyte. The heterogeneous granulosa persists only through previtellogenesis and at the onset of exogenous yolk uptake by the oocyte it becomes a secondarily homogeneous layer. The appearance of the granulosa at this stage is similar to that of reptiles whose granulosa remains a single-cell layer throughout folliculogenesis (e.g., turtles and crocodilians). Thus, although follicular development has been scrutinized in only a few representative genera of reptiles to date, the course of follicular development among lizards is similar in detail and involves the transitory development of a heterogeneous population of cells. This feature appears to be exclusive to the squamate reptiles.

Animals↗

Fatty acid composition of ganglioside GM3 of renal glomerular epithelial SGE1 cells during spontaneous dome formation in vitro.

Ganglioside GM3, one of acidic components of membrane glycosphingolipids (GSL), has been known to change its content quantitatively during growth and differentiation of various cells in vitro. Detailed analysis of lipid portion of GM3 of rat renal glomerular SGE1 cells revealed that fatty acids with long carbon chains, especially that of C24:0 and C24:1 increased, while that of short C18:0 and C20:0 decreased after spontaneous dome formation. Since not only fatty acid composition of neutral GSL, sulfatide and phospholipid but also composition of long-chain bases (LCB) did not change, it was suggested that only C24 fatty acid of GM3 specifically increased in relation to dome formation. The spontaneous dome formation has been reported to be related with induction of cellular differentiation in many transporting epithelial cells. We thus assume that the change of fatty acid composition of GM3 is involved in cellular differentiation of SGE1 cells.

Animals↗

Differential expression of p63 isoforms in normal tissues and neoplastic cells.

The p63 gene encodes at least six different proteins with homology to the tumour suppressor protein p53 and the related p53 family member p73. So far, there have been limited data concerning the expression patterns of individual p63 proteins, due to a lack of reagents that distinguish between the different isoforms. Three antibodies have been produced specifically directed against the two N-terminal isoforms (TAp63 and DeltaNp63) and the C-terminal region of the p63alpha proteins. TAp63 proteins are located suprabasally in stratified epithelia compared with the N-terminal truncated forms, which are more abundantly expressed in the basal cell layer, indicating a switch in expression of p63 isoforms during normal cellular differentiation. Analysis of squamous cell carcinomas shows DeltaNp63alpha to be the most widely expressed isoform, compatible with a role for this protein in promoting neoplastic cell growth in these tissues. DeltaNp63 protein expression is also restricted to basal cells in breast and prostate, whilst TAp63 isoforms are more widely expressed in these tissues as well as in tumours at these sites. TAp63, but not DeltaNp63 or p63alpha, is detected in normal colon and in colon carcinoma. TAp63 proteins are also expressed in the nuclei of a sub-population of lymphoid cells and in most malignant lymphomas, whereas DeltaNp63 proteins are not expressed. Taken together, a hitherto unrecognized regulation of p63 isoform expression in vivo has been uncovered, with different p63 proteins expressed during differentiation and in different cell types. The data indicate roles for specific p63 isoforms not only in maintaining epithelial stem cell populations, but also in cellular differentiation and neoplasia.

Adenocarcinoma↗

In situ hybridization detection of human papillomavirus DNAs and messenger RNAs in genital condylomas and a cervical carcinoma.

Routinely processed formalin-fixed paraffin-embedded sections from anogenital condyloma acuminatum and an invasive squamous cell carcinoma of the cervix were examined by in situ hybridization for the detection of human papillomavirus (HPV) DNAs and messenger RNAs. Asymmetric, single-stranded, tritium-labeled RNA probes for both the coding and the nonsense strands of HPVs 6, 11, 16, 18, and 31 were hybridized and washed under stringent conditions and detected by autoradiography. Type-specific HPV DNAs were detected with specific nuclear localization, while HPV messenger RNAs gave much higher signals and had clear-cut cytoplasmic localization. Cross-hybridization was observed only with closely related viruses. The level of signal obtained seemed to be linked to the degree of cellular differentiation, with koilocytotic cells labeling the most heavily. However, messenger RNA could be detected in even relatively undifferentiated cells within areas of dysplasia and invasive carcinoma. In situ hybridization is a sensitive and specific method for investigation of the dynamic interplay of papillomavirus replication and gene expression, cellular differentiation, and neoplastic transformation.

Carcinoma, Squamous Cell↗

Induction of process outgrowth in vertebrate and invertebrate cell lines by a 2-pyridinyl thiosemicarbazone.

Regulation of differentiation in cells of disparate origin is often mediated by widely differing molecular signals and receptor mechanisms. For example, two neuron-like cell lines used extensively as models for molecular control of differentiation, the steroid-sensitive Kc line from Drosophila and the polypeptide- and cyclic nucleotide-sensitive PC12 line from rat, share no obvious growth factor or hormone receptors. However, we have found that a thiosemicarbazone, 1-pyrrolidinecarbothioic acid [1-(2-pyridinyl)ethylidene] hydrazide, one of a class of synthetic antineoplastic agents, induces process outgrowth - a marker of cellular differentiation - in cells of both of these lines. Moreover, the thiosemicarbazone induces process outgrowth in cells of mutant clones of these lines that are refractory to treatment with growth factors or hormones. Activity of the thiosemicarbazone is dependent upon the alpha-(N)-heterocyclic ring. These findings show that the 2-pyridinyl thiosemicarbazone mimics the effects of diverse epigenetic factors in inducing process outgrowth similar to that seen in cellular differentiation of these cell lines induced by natural regulators. Regulation may be by a mechanism, common to both invertebrate and vertebrate cells, which occurs downstream from the receptors that have been previously shown to mediate epigenetically induced differentiation.

Animals↗