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PubMedDate not supplied

Establishment and characterization of neonatal mouse sertoli cell lines.

Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone. This strategy produced 2 cell lines, which exhibited mixed phenotypes. We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A. The results showed that both cell lines expressed LTAg when the inducer was present in the culture media. When ponasterone A was removed, the majority of the cells died. After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells. One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo. All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1. Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF). Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation.

Alkaline Phosphatase↗
PubMed1985

The system specifying body position in the early development of Xenopus, and its response to early perturbations.

Evidence is presented that the system setting up preliminary specifications for contributions to the axial body plan, across vegetal regions of the Xenopus embryo, acts in a widespread way at early stages. Mechanisms that regulate the spatial profile of this primary positional variable, and thus ensure the constancy and harmony of the body plans normally achieved, have lost this integrative ability by the 4-cell stage one hour after the plasm shifts that precede first cleavage and symmetrize the egg. Abnormal, partial or distorted profiles of the positional system across whole eggs or isolates, recorded by these times, are retained to give correspondingly partial or imbalanced mes/endodermal pattern at tailbud larval stages. There is evidence that subsequent 'back-up' positional interactions, which can heal gross positional discontinuities in isolated presumptive lateral half-eggs and so restore bilateral symmetry, also do this at the price of loss of complete pattern specification. This is probably because of an asymmetrical principle whereby relatively activated (dorsoanterior specified) material can raise the level of originally posterior material on contact, whereas the reverse interaction cannot occur. The observations are discussed in relation to apparently different behaviour in certain other amphibian embryos, and to our knowledge of other positional interactions, normal and also experimentally provoked, such as those that set up the germ layers.

Animals↗
PubMed1995

Prenatal craniofacial development: new insights on normal and abnormal mechanisms.

Technical advances are radically altering our concepts of normal prenatal craniofacial development. These include concepts of germ layer formation, the establishment of the initial head plan in the neural plate, and the manner in which head segmentation is controlled by regulatory (homeobox) gene activity in neuromeres and their derived neural crest cells. There is also a much better appreciation of ways in which new cell associations are established. For example, the associations are achieved by neural crest cells primarily through cell migration and subsequent cell interactions that regulate induction, growth, programmed cell death, etc. These interactions are mediated primarily by two groups of regulatory molecules: "growth factors" (e.g., FGF and TGFalpha) and the so-called steroid/thyroid/retinoic acid superfamily. Considerable advances have been made with respect to our understanding of mechanisms involved in primary and secondary palate formation, such as growth, morphogenetic movements, and the fusion/merging phenomenon. Much progress has been made on the mechanisms involved in the final differentiation of skeletal tissues. Molecular genetics and animal models for human malformations are providing many insights into abnormal development. A mouse model for the fetal alcohol syndrome(FAS), a mild form of holoprosencephaly, demonstrates a mid-line anterior neural plate deficiency which leads to olfactory placodes being positioned too close to the mid-line, and other secondary changes. Work on animal models for the retinoic acid syndrome (RAS) shows that there is major involvement of neural crest cells. There is also major crest cell involvement in similar syndromes, apparently including hemifacial microsomia. Later administration of retinoic acid prematurely and excessively kills ganglionic placodal cells and leads to a malformation complex virtually identical to the Treacher Collins syndrome. Most clefts of the lip and/or palate appear to have a multifactorial etiology. Genetic variations in TGF alpha s, RAR alpha s, NADH dehydrogenase, an enzyme involved in oxidative metabolism, and cytochrome P-450, a detoxifying enzyme, have been implicated as contributing genetic factors. Cigarette smoking, with the attendant hypoxia, is a probable contributing environmental factor. It seems likely that few clefts involve single major genes. In most cases, the pathogenesis appears to involve inadequate contact and/or fusion of the facial prominences or palatal shelves. Specific mutations in genes for different FGF receptor molecules have been identified for achondroplasia and Crouzon's syndrome, and in a regulatory gene (Msx2) for one type of craniosynostosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat.

This is a study of the chemoanatomical organization of the projection from the raphe nuclei to the main olfactory bulb in the rat. A heavy projection from the dorsal and median raphe nuclei to the main olfactory bulb was shown by both retrograde and anterograde tracing techniques. Following injections of 1% wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the main olfactory bulb, up to 1300 neurons were retrogradely labeled in the dorsal and median raphe nuclei, a much larger number than has been suggested by previous studies. By combining 5-HT immunofluorescence with True blue retrograde fluorescent labeling, it was determined that the vast majority of raphe neurons that project to the olfactory bulb contain serotonin. Injections of WGA-HRP into dorsal and/or median raphe produced dense anterograde labeling in the glomeruli, while fewer labeled fibers were observed in the external plexiform layer, internal plexiform layer, and granule cell layer. In contrast to the number of other centrifugal afferents to the bulb, a significant contingent of fibers from the raphe nuclei enters the main olfactory bulb (MOB) from outside in, i.e., via the olfactory nerve layer. Serotonergic fibers in MOB were visualized by immunocytochemistry, and the distribution of specific 5-HT fibers closely matched the distribution of anterograde terminal label resulting from injections of WGA-HRP in the raphe nuclei. The serotonergic fibers have a specific laminar distribution and morphology in MOB. Thus, the density of the serotonergic innervation to the glomerular layer is 2-3 times that of any other layer in MOB. In addition to their preferential innervation of the glomeruli, glomerular and infraglomerular serotonergic fibers are morphologically different. Serotonergic fibers located in the glomerular layer are generally thick (0.25-0.60 micron) compared to the thinner (0.25-0.35 micron) fibers that predominate in infraglomerular layers. Another difference is that the glomerular fibers often contain varicosities that are greater than 1 micron in diameter, while varicosities along infraglomerular fibers are usually barely larger than the axonal diameter. Finally, glomerular fibers are much more intensely stained than infraglomerular fibers. Electrolytic lesions of the dorsal and median raphe caused a total depletion of serotonin fiber staining in the bulb, demonstrating that the raphe nuclei are the sole source of the serotonergic input to the main olfactory bulb. Thus, it has been demonstrated that serotonergic neurons in dorsal and median raphe project very heavily to glomeruli and less heavily to other layers in the main olfactory bulb.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Variable efficiency of retroviral-mediated gene transfer into early-passage cultures of fetal lamb epithelial, mesenchymal, and neuroectodermal tissues.

The relative efficiency of retroviral-mediated gene transfer into early-passage cultures of different tissues of fetal lamb was investigated. Monolayer cultures prepared by plating 1 x 10(6) cells were infected with the Moloney murine leukemia (MoMLV)-based vector pZIP Neo at a multiplicity of infection (moi) of approximately 1 pfu per 2 x 10(2) recipient cells prior to selection for neomycin resistance. At the low moi used, cells from different tissues showed marked differences in efficiency of colony formation in the descending order: brain > kidney > muscle, lung > skin. Brain cells were transduced at least an order of magnitude more efficiently than other cell types, despite the doubling time of brain cell cultures being five times as long. Cultures were analyzed by morphological and immunocytological criteria to determine whether any particular cell types were transduced. A wide variety of morphologically distinct neuron-like and glial-like brain cells were neomycin resistant. The majority of muscle cell colonies were myogenic. Approximately half of the large kidney colonies were epithelial-like. The majority of lung colonies consisted of fibroblasts. The results suggest that cells originating from the surface embryonic germ layer (ectoderm) and/or occupying positions near the fetal external surface have a markedly lower susceptibility to retroviral-mediated gene transduction.

Animals↗
PubMed1992

A novel lamina lucida component of epithelial and endothelial basement membranes detected by LH39 monoclonal antibody.

The murine monoclonal antibody, LH39 was characterized in this study and appeared to bind to a novel basement membrane epitope. This antigen was expressed in the epithelial basement membrane of human tissue derived from all three germ cell layers and in basement membranes surrounding small blood vessels within the stroma of all organs examined. LH39 antigen could be first detected in fetal skin at the dermo-epidermal junction at 7 weeks estimated gestational age but was not present in the dermal vasculature until 16 weeks. When tested against tissue from a range of lower mammalian species, LH39 antigen appeared to be primate-specific. The epithelial basement membrane zone in organotypical cultures, where there is de novo synthesis of basement membrane components, contained abundant LH39 antigen in contrast to other basement membrane components, type IV collagen, laminin, and type VII collagen. Ultrastructural localization of LH39 epitope, using immunogold electron microscopy on unfixed freshly frozen tissue, was to the lamina lucida. No cross-reactivity could be detected between LH39 and laminin, fibronectin, and collagens I, III, IV, and V using the ELISA assay. In vitro studies with a range of proteolytic enzymes suggested that the antigen was non-collagenous in nature. LH39 precipitated a polypeptide with a molecular weight of 185 kD from extracts of metabolically labelled cultured keratinocytes, and polypeptides of 185 and 200 kD from the culture medium. The tissue distribution of LH39 antigen suggested that it may be an epitope within anchoring filaments. Potential applications of this antibody include the study of benign and malignant human vascular disorders, diseases and tumours associated with angiogenesis, epithelial neoplasms, and conditions of tissue regeneration and repair, such as wound healing.

Adult↗
PubMed1995

Neurogenic cells inhibit the differentiation of cardiogenic cells.

In this study, we present evidence that neurogenic cells inhibit the differentiation of cardiogenic cells. When cells of the entire area pellucida at stage 5 were dissociated and reaggregated, the aggregates differentiated into neural tissues and other structures of any germ layer origin, except for heart tissues, despite the fact that the cardiogenic cells are already committed to differentiate. The phenomenon also occurs from stages 6 to 8, during which cells are in a higher state of commitment. Using combinations of different regions of the area pellucida, it was confirmed that neurogenic cells are responsible for the inhibition of cardiogenic cell differentiation. The inhibition is not species-specific because quail and chick neurogenic cells inhibit each other's cardiogenic cell differentiation. Direct contact between cardiogenic and neurogenic cells seems to be necessary for inhibition because cardiogenic cell differentiation takes place if cardiogenic and neurogenic cells are separated by a porous membrane. Dissociated neural tubes from early stages, but not dissociated telencephalon from 2-day-old chicks, also inhibit cardiogenic cell differentiation. These results suggest that neurogenic cells may sequester molecules necessary for the differentiation of cardiogenic cells rather than produce inhibitory molecules.

Animals↗
PubMed1988

Immunohistochemical characterization of pulmonary blastoma.

The immunohistochemical staining patterns of three pulmonary blastomas (PBs) were studied for markers of mesenchymal (vimentin, muscle-specific actin) and epithelial (cytokeratin, epithelial membrane antigen) differentiation to clarify their phenotypic nature and, possibly, their histogenesis. Epithelial markers were confined to the tubular component of the tumors, and the mesenchymal markers were confined to the stromal component, demonstrating that PBs are true mixed tumors. These findings are consistent with origin from a primitive pluripotential stem cell but do not rule out derivation from a single germ layer with subsequent metaplastic transformation.

Aged↗
PubMed1999

The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo.

ActRIA (or ALK2), one of the type I receptors of the transforming growth factor-beta (TGF-beta) superfamily, can bind both activin and bone morphogenetic proteins (BMPs) in conjunction with the activin and BMP type II receptors, respectively. In mice, ActRIA is expressed primarily in the extraembryonic visceral endoderm before gastrulation and later in both embryonic and extraembryonic cells during gastrulation. To elucidate its function in mouse development, we disrupted the transmembrane domain of ActRIA by gene targeting. We showed that embryos homozygous for the mutation were arrested at the early gastrulation stage, displaying abnormal visceral endoderm morphology and severe disruption of mesoderm formation. To determine in which germ layer ActRIA functions during gastrulation, we performed reciprocal chimera analyses. (1) Homozygous mutant ES cells injected into wild-type blastocysts were able to contribute to all three definitive germ layers in chimeric embryos. However, a high contribution of mutant ES cells in chimeras disrupted normal development at the early somite stage. (2) Consistent with ActRIA expression in the extraembryonic cells, wild-type ES cells failed to rescue the gastrulation defect in chimeras in which the extraembryonic ectoderm and visceral endoderm were derived from homozygous mutant blastocysts. Furthermore, expression of HNF4, a key visceral endoderm-specific transcription regulatory factor, was significantly reduced in the mutant embryos. Together, our results indicate that ActRIA in extraembryonic cells plays a major role in early gastrulation, whereas ActRIA function is also required in embryonic tissues during later development in mice.

Activin Receptors, Type I↗
PubMed2006

Bovine embryonic stem cells.

Bovine embryonic stem (bES) cell lines reported to date vary in morphology and marker expression, such as alkaline phosphatase (ALPL), stage-specific embryonic antigen 4 (SSEA4), and octamer-binding transcription factor-4 (OCT4), that normally are associated with the undifferentiated, pluripotent state. This chapter introduces the methods of isolating and maintaining bovine ES cells. These bovine ES cells grow in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells, as well as human EG cells. Throughout the culture period, most of the cells within the colonies stain positive for ALPL and cell surface markers SSEA4 and OCT4. The staining patterns of the bES cells are identical to those of the blastocysts fertilized in vitro (IVF), yet different from most previously reported bovine ES cell lines, which are either negative or not detected. After undifferentiated culture for more than 1 year, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. In addition to bES from IVF, this chapter introduces two methods of generating blastocyst stage embryos other than in vitro fertilization, which are parthenogenetic activation and somatic cell nuclear transfer for the potential application of generation "patient-specific" ES cells.

Animals↗
PubMed1991

Changes in temporal and spatial patterns of Gi protein expression in postimplantation mouse embryos.

We previously demonstrated the presence of GTP-binding proteins, G proteins, in the preimplantation mouse embryo (Jones and Schultz, 1990. Dev. Biol. 139, 250-262). These studies have been extended to the Day 6.5, 7.5, and 8.5 gestation embryo by employing PT-catalyzed ADP-ribosylation and immunoblotting techniques. We report here that the amount of embryonic alpha i increases from Day 6.5 to Day 7.5 of gestation, and remains at about the same level at Day 8.5. In contrast, the extent of PT-catalyzed ADP-ribosylation of Gi alpha protein(s) decreases between Days 6.5 and 7.5--this decrease is global and not restricted to a particular germ layer of the Day 7.5 embryo--and then dramatically increases by Day 8.5 of gestation. In the Day 8.5 gestation embryo, the extent of PT-catalyzed ADP-ribosylation of Gi alpha proteins increases along the anterior-posterior axis, whereas the amount of immunoreactive alpha i subunit decreases along this axis. By using a combination of PT-catalyzed ADP-ribosylation and immunoprecipitation with antisera specific for alpha i1, alpha i2, or alpha i3, we report that all three alpha i subtypes are present in the Day 8.5 gestation mouse embryo. Results of these experiments suggest that an activation of Gi proteins occurs between Days 6.5 and 7.5 of gestation in the postimplantation embryo, a time during which the embryo is gastrulating, and that a decreasing gradient of activation exists along the anterior to posterior axis in the Day 8.5 gestation embryo. Last, we report that oocytes, eggs, and preimplantation embryos possess all three subtypes of alpha i.

Amino Acid Sequence↗
PubMed1994

Sialylation of terminal saccharides of glycoconjugates expressed by murine molar tooth germs developing in vitro and in vivo.

During development of the mammalian tooth germ the pattern of terminal saccharides of glycoconjugates changes, with many structures losing lectin reactivity in a consistent pattern. This study investigated whether the epitopes are lost or become masked by terminal sialylation, using a combination of neuraminidase treatment of sections and sialic acid-reactive lectins. The results suggested that most of the terminal galactosamine and fucose sites in the epithelial enamel organ were removed during morphogenesis. Conversely, during condensation of the dental mesenchyme, masked peanut agglutinin (PNA)-reactive galactose epitopes appeared. During differentiation and organisation of the mesenchyme into odontoblasts and a subodontoblastic layer the PNA-reactive sites became masked again. These regions also specifically expressed sialylated glucosamine. However, at the proliferating epithelial cervical loop galactose sites appeared to be masked. This was more pronounced during in vitro development when abnormal expression of PNA-reactive sites was found at the cervical loop. Additionally, fucosylated sites persisted in the enamel organ, further indicating that the expression of terminal saccharides was disrupted during development in the organ culture system. These data suggest that loss of terminal galactose and galactosamine is related to differentiation of the cells. However, whether this loss occurs by removal or sialic acid masking is not dependent either on the origin of the cells or the epitope being lost.

Animals↗
PubMed1991

The somatic envelopes around the germ-line cells of polytrophic insect follicles: structural and functional aspects.

The polytrophic ovarioles of three insect species, the fruit fly Drosophila melanogaster, the fungus gnat Bradysia tritici, and the honeybee Apis mellifera, were compared morphologically and with respect to the cytological organization of the peripheral somatic layers. Staining with rhodaminyl-phalloidin revealed differences in the organization of the muscle strands of the epithelial sheath and the microfilament pattern in the basal part of the follicular epithelium (mid-vitellogenic stages). Also, the size of the intercellular space between the follicle cells differed considerably in the three analyzed species. The basement membrane of Drosophila and Bradysia follicles was partially digested using purified collagenase. The observed morphological changes indicated that in both species the basement membrane of the follicular epithelium plays an important role in shaping the follicles. The possible functional significance of the species-specific structural differences is discussed.

Actin Cytoskeleton↗
PubMed2000

Patterning the endoderm: the importance of neighbours.

The endodermal germ layer gives rise to the inner epithelial lining of the gastrointestinal tract, while that of the mesoderm gives rise to the outer smooth muscle layer. Much of the work in chick shows that the mesoderm plays an important role in endodermal differentiation, and recent results in Xenopus have begun to elucidate the factors involved in establishing endodermal cell fate. However, little is know about the signals responsible for the initial specification and pattern of the endoderm. In a recent paper, Wells and Melton have investigated the importance of early mesectodermal-endodermal interactions in the initial specification of the early mouse endoderm.(1) They demonstrate that the initial specification and differentiation of the endoderm does not occur cell-autonomously, but requires signals released from the mesectoderm.

Animals↗
PubMed2005

In vitro neuronal differentiation of cultured human embryonic germ cells.

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2microM all-trans-retinoic acid (RA) or 0.1mM dibutyryl cyclic AMP (dbcAMP)/1mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, beta-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.

Biomarkers↗
PubMed2006

In vitro differentiation of human placenta-derived multipotent cells into hepatocyte-like cells.

Multipotent cells isolated from human term placenta (placenta-derived multipotent cells [PDMCs]) have been known to be able to differentiate into mesodermal lineage cells, including adipocytes and osteoclasts. The low infection rate and young age of placenta compared with other tissue origins of adult stem cells make theses cells attractive target for cell-based therapy. However, the differentiation potential of PDMCs toward hepatic cells has not been evaluated yet. In this study, we cultivated PDMCs with hepatic differentiation medium to evaluate the ability of these cells in differentiating toward hepatic cells. After treatment, the morphologies of differentiated PDMCs changed to polygonal epithelial cell-like. The differentiated cells not only show the hepatocyte-like morphologies but also express hepatocyte-specific markers, including albumin and cytokeratin 18. The bioactivity assays revealed that these hepatocyte-like cells could uptake lipoprotein and store glycogen. Furthermore, the addition of rifampicin increased the gene expression of CYP3A4, which is similar with the activities of human liver cells. According to our previous results, PDMCs were capable of differentiating into mesodermal and ectodermal lineage cells. Our results indicate that PDMCs can differentiate into three germ layer cells, which is similar to embryonic stem cells. In conclusion, placenta might be an easily accessible source for progenitor cells that are capable of differentiating toward hepatocyte-like cells in vitro.

Albumins↗
PubMed1991

A unique fibrillar coat on the surface of migrating human primordial germ cells.

Primordial germ cells (PGCs) were studied by electron microscopy in human embryos at 27 and 30 days of gestation. PGCs were mainly found in the hindgut epithelium, some at the stage of separation from the endoderm, and others in migration through the mesenchyme of the dorsal mesentery between the primitive intestinal wall and the coelomic epithelium, including the coelomic angle. A few PGCs were still located in the endodermal epithelium. Before the process of separation and migration, PGCs appeared clearly different from neighboring somatic cells (endodermal, mesenchymal and epithelial cells of the hindgut and the coelomic layer). PGCs located in the endodermal epithelium were large and showed an irregular cell body often provided with filopodia-like processes that contacted somatic cells to form small areas of focal junctions. As a rule, PGCs possessed a large, round nucleus containing one or two conspicuous nucleoli. Lipid droplets, abundant glycogen particles, ribosomes and mitochondria were often observed in their cytoplasm. A detailed ultrastructural analysis revealed a delicate fibrillar coat frequently present on the free surface of PGCs during their migratory phase through the mesenchyme of the dorsal mesentery. This surface coat, likely corresponding to a glycocalyx, appeared as a particular, conspicuous filamentous layer of 30 nm thickness, mainly in the leading pseudopodial projections of PGCs migrating toward their target. The surface coat of PGCs can be associated with the binding sites of specific macromolecular components of the extracellular matrix, including fibronectin, and hence plays a role in PGCs recognition and migration during this special embryonic phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1992

Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines.

Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth-inhibitory effect of exogenous transforming growth factor-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.

Adenocarcinoma↗