Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture.

A coculture method is described for ensheathing glial cells from adult rat olfactory nerve, serving as a substrate for the regrowth of neurites from adult rat retinal ganglion cells. Immunocytochemically identified phenotypes present in primary cultures of olfactory nerve cells are described, and their ability to promote neurite outgrowth is compared with neonatal astrocytes and Schwann cells, with other nonglial cells, and with laminin. Ensheathing cell cultures were more effective than any other substrate tested and also directed the orientation of regrowing neurites. In comparison with cultured Schwann cells, which released neurotrophic factors into the culture medium, there was no evidence of a similar activity in ensheathing cell cultures. Combinations of ensheathing cell-conditioned medium and substrates of laminin, merosin, or 3T3 cells also failed to show the release of factors enhancing either survival or neurite outgrowth from retinal ganglion cells. Evidence is presented for a partial inhibition of neurite outgrowth in the presence of calcium channel antagonists or an intracellular calcium-chelating reagent. This provides evidence for a contribution from an intracellular calcium signaling mechanism, possibly implicating ensheathing cell adhesion molecules in promoting neurite outgrowth.

Spontaneous orientation of transplanted olfactory glia influences axonal regeneration.

Transplanted olfactory ensheathing cells (OECs) have previously been demonstrated to support axonal growth and myelination in the adult rat CNS. Here, the capacity of donor OECs to control the direction of axonal regeneration has been investigated following transplantation, as elongated columns, into the thalamus of adult rats. The OECs formed a 'glial bridge' which extended from the thalamus to the hippocampus. Transplanted OECs rapidly adopted a spindle-shaped morphology which was orientated along the vertical axis of the transplant. Numerous host axons grew into the transplants and followed the highly orientated OEC cell matrix across the choroid fissure. Thus, the spontaneous elongation and orientation of donor OECs may support highly directional host axonal growth across natural barriers within the CNS.

Spontaneous immortalisation of ensheathing cells from adult rat olfactory nerve.

In this report, we describe the isolation of a cell line, Rolf B1.T, from cultures of adult rat olfactory nerve cells. Rolf B1.T cells have an antigenic phenotype which closely resembles that of olfactory ensheathing cells. In routine culture conditions, Rolf B1.T cells constitutively express glial fibrillary acidic protein, S1OO, the low-affinity neurotrophin receptor p75 NGF, laminin, tenascin, and the neural cell adhesion molecule (N-CAM); a variable proportion of the cells also express cadherin, which is regulated by local culture conditions and is associated positively with cell proliferation status. We provide evidence that the association may be indirect and linked to a related parameter such as local cell density. Rolf B1.T cells arose from a population of less well-differentiated cells after a spontaneous immortalisation event. The cells retain many characteristics of normal cells, are dependent on serum growth factors for their proliferation, and fail to grow in semi-solid agar. Rolf B1.T cells support the regrowth of neurites from adult retinal ganglion cells in vitro in a heterologous co-culture system and will have potential value in investigations into the mechanisms of glial support for axonal regeneration from adult mammalian central neurons.

Immunocytochemical analysis of glial cells in the hypomyelinated optic nerve of the BW mutant rat.

The Browman-Wyse (BW) rat is a mutant with structural defects of the visual system, including a failure of the proximal (retinal) end of the optic nerve to myelinate. This latter abnormality is correlated with an absence of CAII+ oligodendrocytes, but we have previously shown that astrocytes are normally distributed, as judged by morphological characteristics of GFAP+ cells in vivo. We have further examined in vitro the immunohistochemical characteristics of macroglia isolated from the BW optic nerve, either as cell suspensions or after 4 days in culture. Cell cultures derived from the hypomyelinated proximal segment of BW optic nerves contained very few 0-2A progenitor cells (from which oligodendrocytes and cells with the GFAP+/A2B5+ phenotype develop), whereas over 90% of the glia were Schwann cells. A proportion of these few 0-2A progenitor cells differentiated normally after 4 days in vitro into both progeny phenotypes in appropriate media. Accordingly, we conclude that the myelination deficiency in the BW optic nerve could be explained as a failure of 0-2A progenitor cells to populate fully the proximal extremity of the nerve during development. Since most glia isolated from adult optic nerves did not adhere to the culture substrate, we analysed the phenotypes of freshly isolated cells in suspension. Comparing optic nerves of normal adult rats with those of BW mutants, a significantly higher fraction of the GFAP+ cells reacted with A2B5 in cell suspensions of the latter. The double-labelled cells which are present in abnormally high numbers may be the differentiated progeny of 0-2A progenitors in the hypomyelinated segment of nerve. One explanation for these findings is that Schwann cells within the BW nerve induce the differentiation of 0-2A progenitor cells to the GFAP+/A2B5+ phenotype. We investigated this possibility using conditioned medium from cultured Schwann cells which increased tenfold the frequency of GFAP+/A2B5+ cells in normal neonatal rat optic nerve cultures. Oligodendrocyte numbers showed a concomitant decline with increasing concentration of Schwann cell conditioned medium. Hypomyelination in the BW rat optic nerve may therefore arise because Schwann cells, present in the proximal segment of the nerve, not only impede the migration of 0-2A progenitor cells but also release a factor which induces those 0-2A progenitor cells which arrive in the proximal segment of the nerve to differentiate into GFAP+ cells at a critical stage in oligodendrocyte development.

Oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells from neonatal and adult rat optic nerve differ in their responsiveness to platelet-derived growth factor.

We have investigated, in vitro, the mitogenic responsiveness to platelet-derived growth factor (PDGF) of oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerve and their differentiation into oligodendrocytes. Progenitor cells from adult optic nerves differentiate into oligodendrocytes in a limiting concentration of foetal calf serum more slowly than in cultures of neonatal cells. Nevertheless, differentiation of oligodendrocytes from progenitors is nearly complete by 6 days in vitro, with 50% expressing galactocerebroside by 4-5 days. In these experiments, adult optic nerve cells were grown in medium containing PDGF, a potent mitogen for neonatal O-2A progenitor cells, and yet the decline in numbers of O-2A progenitor cells matches the rise in oligodendrocyte numbers. We suggest that this is because adult O-2A progenitor cells differ from their neonatal counterparts and do not show the same proliferative response in the presence of exogenous PDGF. We tested this hypothesis by a quantitative autoradiographic analysis of tritiated thymidine-labelled nuclei, comparing percentages of labelled adult and neonatal O-2A lineage glial cells in low-serum medium, in the presence of absence of PDGF, with their response to a monolayer of neonatal rat cortical type 1 astrocytes or astrocyte-conditioned medium. Whereas, adult O-2A progenitors responded to astrocyte monolayers and to conditioned medium from astrocyte cultures, there was no dose-dependent response to PDGF-BB over a wide range of concentrations. Antibodies to human PDGF neutralise the growth-promoting activity of astrocyte-conditioned medium for neonatal O-2A cells but do not neutralise astrocyte-conditioned medium stimulation of adult O-2A progenitor cells. This indicates that the principal astrocyte-derived growth factor(s) for adult O-2A progenitor cells is unlikely to be PDGF.

The conditioning effect of optic nerve injury upon axonal regrowth from adult rat retinal ganglion cells explanted in vitro.

An in vitro assay was used to determine the effects of conditioning nerve lesions on the regeneration of adult rat retinal ganglion cell (RGC) axons from retinal explants. Following the conditioning lesion (CL) of unilateral optic nerve transection, maximal regrowth was seen from RGC explanted from ipsilateral retinae 10 days post-CL. Explants from this group initiated axonal regrowth earlier and a greater percentage regrew axons when compared with explants from normal rats. Axonal regrowth from explants of retinae contralateral to CL was also seen earlier than normal. In further experiments, the effects of both exposure of the optic nerve sheath in the orbit and the incision of the dura without injury to optic nerve axons were studied. The conditioning effect of a dural incision was found to be the same as that of optic nerve transection, whilst exposure of the optic nerve sheath had no conditioning effect on RGC axonal regrowth in vitro.

Regeneration of adult rat retinal ganglion cell processes in monolayer culture: comparisons between cultures of adult and neonatal neurons.

The aim of the present study was to develop a model culture system for the study of factors controlling regeneration of axons from injured adult mammalian central nervous system. We show, for the first that retinal ganglion cells (RGC) dissociated from adult rat retina, regrow processes in vitro over long distances under over appropriate conditions in monolayer culture. Most importantly, adult RGC depend completely upon the presence of a preformed layer of neonatal cortical astrocytes, whereas RGC from neonatal retinae are supported by indigenous retinal glia which spread and proliferate to form a monolayer of cells upon which RGC regrow processes. Adult retinal glia fail to spread on the culture surface and proliferate in the same way and we suggest that this is a major factor in limiting the survival of adult RGC on an acellular substrate such as polylysine. Laminin does not substitute for the presence of a glial monolayer. These findings indicate that at least one type of adult CNS neuron is capable of regenerating its processes in vitro in an environment which includes a cellular component of CNS tissue.

Neither laminin nor prior optic nerve section are essential for the regeneration of adult mammalian retinal ganglion cell axons in vitro.

Retinal explants obtained from normal adult rats and from operated animals in which the optic nerve had been sectioned 10 days previously were cultured in either serum-containing or serum-free medium on poly-L-lysine and laminin substrata. Regenerating ganglion cell axons growing from these explants have been identified using monoclonal antibodies against Thy-1.1 cell surface glycoprotein and the 200-kDa subunit neurofilament protein. Irrespective of substratum or medium composition, axons regenerated from 28-49% of normal rat retinal explants. This percentage increased to 60-84% of explants from operated rats. There were no significant differences in percentages of explants from normal or operated rats showing neurite outgrowth when substrata of either poly-L-lysine or laminin were compared in serum-free medium. In serum-containing medium the results were less easily interpreted due to the presence of an outgrowth of non-neuronal (glia and mesenchymal) 'flat cells', which served as a preferred axonal substratum in many cases. Thus we show that adult rat retinal ganglion cell axons will regrow in vitro, and that a 'priming' optic nerve section will increase this response. In neither case is the response laminin-dependent.

Elevated production of growth factor by human premalignant colon adenomas and a derived epithelial cell line.

Growth factor activity which stimulates anchorage-independent growth (AIG) in a rat fibroblast line, was detected in human premalignant adenoma tissue from familial polyposis coli colectomy specimens and in serum-free culture supernatant from an adenoma cell line PC/AA. The activity extracted from adenoma tissue was compared quantitatively in the AIG bioassay with extracts of normal mucosa from split thickness colorectal tissue. Adenoma tissue yielded three times the amount of acid-extractable protein g-1 wet wt and adenoma extracts consistently had significantly greater specific activity over a wide protein concentration range. Activity extracted from adenoma tissue and from the derived cell line PC/AA were compared qualitatively after fractionation by gel filtration. Both extracts showed almost identical profiles of biological activity after assay of individual fractions for AIG stimulation, suggesting that the factor(s) originates from the epithelial component of the adenoma tissue since PC/AA is a pure epithelial cell line. Activity eluted as two major peaks with apparent mol. wts of 9 kd and 20-25 kd (relative to standards) in both cases. This report demonstrates for the first time that elevated production of a growth factor may be an early change in the evolution of human colorectal cancer from small, premalignant adenomas.

Growth factor production during multistage transformation of epithelium in vitro. I. Partial purification and characterisation of the factor(s) from a fully transformed epithelial cell line.

Transforming growth factor (TGF)-like activity is characterised from one of a series of salivary epithelial cell lines, CSG 211, chemically transformed in vitro. In this transformation system, we can demonstrate multiple stages in the acquisition of a malignant phenotype by normal diploid ductal epithelial cells from male mouse submandibular gland. The fully transformed, tumorigenic cell TGF-like activity in serum-free supernatants resembles no other well-characterised growth factor and has an apparent molecular weight (Mr) of 14 kd. There is also evidence of a higher Mr activity, which is separable by anion exchange chromatography. We show that the premalignant, nontumorigenic progenitor cells of this line do not produce demonstrable TGF-like activity and that this property is therefore acquired as CSG 211 cells become carcinoma producing.

Presence of two cytogenetic forms of amplified DNA: evidence for a role in tumor growth in an intraspecific mouse hybrid cell line.

An intraspecific mouse hybrid epithelial cell line, F5/B, is described in which the homogeneously staining region (HSR)-containing marker chromosome from one parent is absent in about half of the cells. It is replaced in these cells by double minutes (DM), an alternative form of amplified DNA, which is liable to loss because of its instability at mitosis. DM probably arise from the breakdown of the HSR during clonal growth of F5/B. Subclones were derived possessing one or another cytogenetic feature, and their cloning efficiency in vitro and tumorigenicity in syngeneic animals were compared. There were no differences in in vitro tumorigenicity, but in vivo DM-containing subclones were significantly less tumorigenic than HSR-containing subclones or the F5/B parent hybrid. In tumors that developed after long latent periods, cells had increased numbers of DM compared with the inoculated population, demonstrating a selective advantage in vivo for cells with a high DM content. These results indicate a role for the amplified DNA in tumor growth.

The isolation and characterization of colorectal epithelial cell lines at different stages in malignant transformation from familial polyposis coli patients.

The genetic disease familial polyposis coli (hereditary adenomatosis of the colon and rectum) provides an excellent model for the study of tumour progression in the large bowel. We have isolated and characterized four epithelial cell lines from colorectal tumours from polyposis coli patients. These cell lines are grown on collagen-coated Petri dishes in the presence of mouse 3T3 feeder cells in medium containing 20% foetal bovine serum. Of these cell lines three were isolated from premalignant adenomas and one from an adenocarcinoma. All four lines have a characteristic cuboidal epithelial morphology, and their epithelial origin was confirmed by positive staining with a monoclonal antibody which reacts specifically with the keratin filaments of simple epithelia. The adenoma-derived lines display ultrastructural features characteristic of colonic epithelium including desmosomes, microvilli and mucin droplets. One of the adenoma-derived cell lines, designated PC/AA, has retained differentiated functions in culture, namely mucin production, after 21 in vitro passages. PC/AA has a karyotype of 46, XY with no detectable chromosome rearrangements. The adenoma-derived lines could be passaged from clumps of cells but not from single cells even in the presence of 3T3 feeder cells. The carcinoma-derived line, designated PC/JW, could however grow from single cells in the presence of a feeder layer. The one premalignant adenoma-derived line tested so far, PC/AA, did not produce tumours in athymic nude mice. In contrast, the carcinoma-derived line, PC/JW was tumorigenic in athymic nude mice. PC/JW produced moderately well-differentiated tumours which were histologically similar to the adenocarcinoma from which the cell line was isolated. PC/JW has a near-diploid chromosome number with an isochromosome (1q), an isochromosome (14q) and an (Xp; 17q) translocation. Unidentified marker chromosomes were present in a few cells. The features at present which distinguish the carcinoma-derived line from the adenoma-derived lines are tumorigenicity, growth from single cells and chromosomal abnormalities. The isolation and characterization of differentiating human epithelial cell lines at different stages in malignant transformation provide an opportunity to examine the cellular and molecular mechanisms controlling tumour progression in the large intestine, and to obtain an insight into the multistep process of human epithelial carcinogenesis.

TPA affects early and late stages of chemically-induced transformation in mouse submandibular salivary epithelial cells in vitro.

The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), is a potent mitogen in mouse submandibular epithelial cells in vitro. Effects of TPA were investigated on various stages in the transformation of this cell type by the carcinogen benzo[a]pyrene. There was no evidence of enhancement of the frequency of induction of foci of preneoplastic epithelial cells following a high, transforming dose of carcinogen, but TPA promoted this early stage in transformation if given at weekly intervals after a subthreshold, initiating exposure to carcinogen. Foci from all TPA-treated cultures appeared earlier and grew in size more rapidly than those induced by carcinogen alone. There was no evidence from these experiments, however, that foci from TPA-treated cultures were more likely to give rise to permanent epithelial cell lines or that these cell lines became tumorigenic earlier. Preliminary results showed that one out of two permanent, preneoplastic epithelial cell lines examined responded to the promoter at a late stage in transformation by showing enhanced tumorigenicity in syngeneic animals.

Chromosome changes associated with the progression of cell lines from preneoplastic to tumorigenic phenotype during transformation of mouse salivary gland epithelium in vitro.

Two mouse salivary gland epithelial cell lines, CSG 211 and CSG 205/2B1, isolated during carcinogen-induced neoplastic transformation in vitro, were analyzed cytogenetically before and after they acquired the ability to produce carcinomas in syngeneic animals. With the use of Giemsa banding techniques, chromosome changes were identified that were associated with the transition from a preneoplastic to a fully transformed (tumorigenic) phenotype during serial passage in vitro. Results were compared with those from a third cell line of similar origin, CSG 225, which was tumorigenic at the earliest passage tested. These cell lines were found to be subtetraploid, which confirms previous data, and the tumorigenic lines showed consistent losses of copies of chromosomes 1, 4, 7, 9, and 14. Compared with their preneoplastic counterparts, the loss of no single chromosome seems to be sufficient to generate the tumorigenic phenotype, but the loss of a combination of some or all of these chromosomes appears to be important in the phenotypic transition. In CSG 211 the loss of chromosome 7 is probably more important in this respect than loss of the other chromosomes listed. The karyotype of this cell line undergoes major structural rearrangement, which suggests that loss of specific regions of chromosomes 1 and 9 is also important.

Changes in DNA content during in vitro transformation of mouse salivary gland epithelium.

DNA content was measured in single cells in situ during the in vitro development of neoplastic transformation in epithelial cells from mouse salivary glands. The earliest change in DNA content was found in stage III of the transformation process (a preneoplastic stage). In foci of these cells, the percentage of tetraploid (4C) cells was 30--60% compared with the starting tissue and early primary cultures, which were diploid. The stage III foci eventually gave rise to subtetraploid tumorigenic cell lines. The studies suggested that during in vitro development of neoplastic transformation in salivary gland epithelium, tetraploids were generated that then underwent a period of chromosome instability and loss. Evidence was also presented that a similar mechanism may be operating in transformation of salivary gland epithelium in vivo. A possible mechanism for transformation on the basis of chromosome imbalance was discussed.

Cell-mediated mutagenesis in cultured Chinese hamster cells by polycyclic hydrocarbons: mutagenicity and DNA reaction related to carcinogenicity in a series of compounds.

Three polycyclic hydrocarbons, benz(a)anthracene, 3-methylcholanthrene and 7,12-dimethylbenz(a)anthracene, have been studied in a cell-mediated mutagenesis system using BHK 21 cells to metabolize the hydrocarbons and V-79 cells as targets for detecting induced cytotoxicity and mutation. In large-scale experiments, the DNA of V-79 cells was analyzed by column chromatography to determine the nature and true extent of reaction of hydrocarbons with dexoyribonucleosides. Products with DNA formed by the two carcinogenic compounds were qualitatively very similar to those reported to occur in vivo and in primary cell cultures. Binding indices were calculated from the tritium content of DNA-hydrocarbon products, related to overall metabolism, for these two compounds together with benzo(a)pyrene and 7-methylbenz(a)anthracene using data from a previous study. These values reflected differences in carcinogenic potency between the compounds. Induced mutation frequencies were related to the extent of DNA reaction with each compound. At equivalent extents of DNA reaction with hydrocarbon products, levels of induced mutation were not significantly different.

Different tumours induced by benzo(a)pyrene and its 7,8-dihydrodiol injected into adult mouse salivary gland.

A comparison has been made between the carcinogenic activities of benzo(a)pyrene and the proposed proximate carcinogen, benzo(a)pyrene 7,8-dihydrodiol, in the adult C57BL mouse submandibular salivary gland. In preliminary studies using a range of doses, the dihydrodiol was slightly less active than the parent hydrocarbon in this system. There was a difference in the type of tumour induced by the 2 compounds. Benzo(a)pyrene induced tumours of the salivary glands at the site of injection, whereas the dihydrodiol induced malignant lymphosarcomas, particularly of the thymus, which were often metastatic to other orgnas. Possible reasons for the different sites of action of the 2 compounds are discussed.