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D G Fernig

Publications and source records attributed to D G Fernig.

At least 55 records · Page 3Linked to original sources
PubMed1998

Stem cells in breast epithelia.

The rodent and human nonpregnant mammary glands contain epithelial, intermediate and myoepithelial cells which have all been isolated as cell lines in vitro. Transforming growth factor-alpha (TGF alpha) and basic fibroblast growth factor (bFGF) are produced by myoepithelial cells and can stimulate the growth of intermediate stem cells in vitro. Epithelial and intermediate cells behave like stem cells in vitro, since they can differentiate into alveolar-like an myoepithelial cells. The myoepithelial differentiation pathway is associated with the early expression of a calcium-binding regulatory protein called p9Ka and the protease, Cathepsin D. Myoepithelial cells are also present in benign lesions but not in malignant mammary carcinomas of rats or humans, whose resultant cell lines fail to differentiate completely along the myoepithelial cell pathway. Loss of the myoepithelial cell in some invasive carcinomas may be compensated, at least in part, by changes in malignant cells. Over-expression of TGF alpha and/or erbB receptors may reduce the requirement for TGF alpha, whilst ectopic production of bFGF and its receptors and p9Ka/Cathespin D may assist in tumorigenesis and in metastasis, respectively. Thus compensation for, or retention of, molecules potentially involved in the differentiation of mammary cells may be a mechanism by which malignancy progresses in some human invasive carcinomas.

Animals↗
PubMed1998

Growth and differentiation of the normal mammary gland and its tumours.

The mammary glands of non-pregnant rodents and humans consist of epithelial, intermediate stem and myoepithelial cells, and these have been isolated as cell lines in vitro. Growth factors produced by the myoepithelial cells, e.g. transforming growth factor alpha (TGF alpha) and basic fibroblast growth factor (bFGF), can stimulate the growth of the intermediate stem cells in vitro. One protein, p9Ka, a calcium binding regulatory protein, arises at an early stage of the differentiation of epithelial into myoepithelial cells in vitro and is associated with the cytoskeleton; another, cathepsin D, is a protease associated with this pathway in vivo. Unlike normal glands and benign lesions, malignant mammary carcinomas of rats and humans do not contain fully differentiated myoepithelial cells, and the resultant cell lines fail to differentiate completely into myoepithelial cells. Loss of the myoepithelial cells in some human invasive carcinomas may account, in part, for compensatory changes in the malignant cells. For example, overexpression of TGF alpha/ErbB-2 receptors may compensate for a decrease in TGF alpha, whereas ectopic production of bFGF and its receptors, and of p9Ka and cathepsin D, may help in tumorigenesis and in metastasis respectively. Thus compensation for, or retention of, molecules potentially involved in growth and/or differentiation by some human invasive carcinomas may be a mechanism by which a malignancy progresses.

Animals↗
PubMed1997

Stimulation of proliferation in human colon cancer cells by human monoclonal antibodies against the TF antigen (galactose beta1-3 N-acetyl-galactosamine).

In many tissues, the TF (Thomsen-Friedenreich) blood group antigen (Galbeta1-3GalNAc alpha-) behaves as an onco-foetal carbohydrate antigen, showing increased expression in malignancy and hyperplasia. Dietary lectins which bind the TF antigen have marked effects on proliferation of epithelial cells without cytotoxicity. This led us to speculate that anti-TF antibodies, including those that naturally occur in humans, might have similar effects. Five anti-TF antibodies, TF2 (human), TF5 (human), 5A8 (mouse), 8D8 (mouse) and BM22 (mouse), but not TFI (human) or 49H.9 (mouse), showed marked dose-dependent stimulation (95-192%) of [3H]thymidine incorporation by HT29 human colon cancer cells. Similar stimulation of proliferation of HT29 cells by these monoclonal antibodies (MAbs) was found when cell count assessment was used. Antibody-stimulated proliferation was inhibited by co-incubation with glycoproteins expressing Galbeta1-3GalNAc alpha- (asialo glycophorin or [Galbeta1-3GalNAc alpha-O-p-aminophenyl]n-human serum albumin). A proliferative effect of these antibodies was also demonstrated on human colon cancer cell lines LS174T and HT29-MTX but not on Caco-2 cells. Although immunoblotting showed similar binding patterns of all the antibodies on HT29 cell membrane extracts, there was little correlation between cell surface binding assessed by immunofluorescence and proliferative response, and internalization of the biotinylated antibody TF5 was demonstrated by confocal microscopy. Our results provide further evidence that cell surface glycoproteins which express TF antigen may play an important role in the regulation of cell proliferation and also suggest that human anti-TF antibodies may have proliferative effects on cells which express TF antigen.

Antibodies↗
PubMed1997

Invasion of human colorectal carcinoma cells is promoted by endogenous basic fibroblast growth factor.

The growth-stimulatory and invasion-promoting effects of basic fibroblast growth factor (bFGF) were examined in 2 series of related human colon carcinoma cell lines (HCT116A, HCT116B and 20-10-1 as well as and LS180, LS174T and ARK1A) that exhibit different invasive potentials. The invasive cell lines 20-10-1 and ARK1A grew more rapidly than their non-invasive counterparts; exogenously added bFGF stimulated the proliferation of all the cells. When extracts of the cells were fractionated on columns of heparin-Sepharose, bFGF-like activity was found in extracts from each cell line. The amount of bFGF-like growth-stimulatory activity was greater in the more invasive cells: the invasive cells 20-10-1 contained 35-fold more activity than the non-invasive HCT116A cells, and the ARK1A cells contained 15-fold more activity than LS180 cells. Relatively small amounts of bFGF-like activity were recovered from medium conditioned by the invasive cells. The bFGF-like growth-stimulatory activity from the cell extracts was neutralised by an antibody to bFGF, and immunoblotting revealed the presence of an 18 kDa immunoreactive polypeptide, consistent with the presence of bFGF in the cell extracts. Exogenously added bFGF caused the usually non-invasive HCT116A cells to invade collagen gels. The HCT116B and 20-10-1 cells that were naturally invasive in a collagen gel assay also showed increased levels of invasiveness in the presence of bFGF, but an antibody that neutralised the activity of bFGF reduced the constitutive invasiveness of these cells. Our results suggest a causal relationship between the endogenous production of bFGF and the invasive potential of human colon carcinoma cells.

Adenocarcinoma↗
PubMed1997

Programmed cell death in bovine mammary tissue during lactation and involution.

Cessation of milk removal causes mammary tissue involution, which in rodents is characterized by extensive tissue degeneration and loss of the majority of luminal epithelial cells by apoptosis. In contrast, bovine mammary tissue shows little histological evidence of tissue remodelling between lactations. In this study, we combined histology with molecular biology to examine the cellular and molecular changes in bovine mammary tissue on cessation of milking. Oligonucleosomal laddering of genomic DNA extracted from lactating tissue indicated that a proportion of cells were dying by apoptosis. This was confirmed by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labelling of apoptotic cells in lactating tissue sections (TUNEL). One week after cessation of milking, alpha-lactalbumin and alpha S1-casein messenger RNA (mRNA) abundance had decreased by 99 and 85%, respectively, whereas lactoferrin mRNA had increased 20-fold. Drying off was also accompanied by an increase in oligonucleosomal laddering of genomic DNA, and by an increase in the proportion of TUNEL-positive cells, which were localized preferentially in regions where alveolar structure had deteriorated. Therefore, termination of lactation was associated with partial loss of the mammary cell population and dedifferentiation of the remainder.

Animals↗
PubMed1996

Effect on tumorigenicity and metastasis of transfection of a diploid benign rat mammary epithelial cell line with DNA corresponding to the mRNA for basic fibroblast growth factor.

To examine the potential role of fibroblast growth factors (FGF) in tumorigenesis and metastasis, plasmid constructs containing the human basic FGF (bFGF) gene, with or without fusion to a secretory signal peptide (IgbFGF), were transfected into the diploid rat mammary epithelial cell line Rama 37. All transfectants possessed multiple copies of the transfected cDNA, which was expressed as the corresponding mRNA and the protein. The amount of bFGF protein was usually greater than the bFGF growth-stimulatory activity that could be recovered from the transfected cells. Nevertheless, the amount of bFGF growth-stimulatory activity secreted by the IgbFGF transfectants (0.08-0.8 ng/ml/24 hr) was sufficient to induce growth in responsive cells. However, the transfectants themselves were refractory to stimulation by exogenously added bFGF, despite possessing a small number of high-affinity receptors for bFGF. When the bFGF or the IgbFGF transfectants were inoculated into the mammary fat pads of syngeneic rats, the tumour incidence was low (0-50%). However, when cells cultured from these tumours were inoculated into the fat pad of syngeneic rats, the tumour incidence was 100%. Tumours were in all cases benign and no metastases were observed. Our results suggest that the role of bFGF in metastasis is not simply one of autocrine/paracrine stimulation of cell growth and that other events may also be required.

Animals↗
PubMed1996

Late signals are required for the stimulation of DNA synthesis in rat mammary fibroblasts by growth factors.

Maximal stimulation of DNA synthesis in quiescent rat mammary (Rama) 27 fibroblasts is elicited by epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) 18 h after the initial addition of the growth factors-the 'lag' period. At maximally-stimulating concentrations, EGF and bFGF are interchangeable 9 h after their initial addition. When the initial concentration of growth factor is below that required to elicit a maximal response, it is possible to increase the level of DNA synthesis by increasing the concentration of growth factor 9 h after its initial addition. When the initial concentration of growth factor is high, substitution by a lower concentration of growth factor after 9 h allows a greater proportion of cells to synthesize DNA than would be expected from a continuous low dose of growth factor. Similar results are obtained when both the growth factor and its concentration are changed 9 h after the initial addition of growth factor. However, when EGF at a low concentration is substituted for a high concentration of EGF or bFGF the resulting increase in the levels of DNA synthesis is greater when EGF rather than bFGF is added for a second time. The half-life of the growth-stimulatory signals delivered by EGF and by bFGF 9 h after their initial addition is 1-2 h. These results suggest that to stimulate DNA synthesis: (i) EGF or bFGF must deliver a signal(s) continuously; (ii) the initial signals produced by EGF and bFGF are equivalent; (iii) the signals produced between 9-18 h by EGF may be different to those produced by bFGF.

Animals↗
PubMed1995

Growth factors and their receptors in neoplastic mammary glands.

Control of the growth of mammary glands is largely exerted in vivo by systemic hormones and locally-produced growth factors, whereas malignant tumours gradually lose the ability to respond to both types of control in vivo. However, the systemic hormones have little direct effect on stimulating the growth of rat or human mammary cell lines in vitro. Estrogens are thought to work by stimulating locally-produced growth factors and/or their receptors, eg transferrin, TGF alpha and IGF-1, and prolactin by a contaminating pituitary mammary growth factor (PMGF). Mammary stem cells intermediate between epithelial and myoepithelial cells are thought to be retained in malignant carcinomas, whereas the TGF alpha and bFGF-producing myoepithelial cells are lost. Hormonal autonomy of carcinomas may develop by overproduction of the locally-produced growth factors, their receptors (including related receptors, eg c-erbB-2) and/or by stem cells differentiating sufficiently to utilise normal control mechanisms, eg refractivity to PMGF and autocrine/paracrine response to bFGF. The failure of the stem cells to differentiate completely to myoepithelial cells in carcinomas greatly reduces the heparan sulphate proteoglycan sink used to sequester to bFGF in normal glands and also removes the possibility of eliminating cells by terminal differentiation, both processes possibly contributing to the uncontrolled growth of the malignant breast cell.

Animals↗
PubMed1994

Fibroblast growth factors and their receptors: an information network controlling tissue growth, morphogenesis and repair.

The stimulation of cellular metabolism by the nine fibroblast growth factors (FGFs) is mediated by a dual-receptor system. This comprises a family of four receptor tyrosine kinases (FGFR) and heparan sulphate proteoglycans (HSPG). The stimulation of cell division by FGFs has an obligate requirement for both partners of the dual-receptor system. The binding of the nine FGFs to the FGFRs is marked by a pattern of overlapping specificity despite alternative splicing events generating a large number of FGFR proteins. Thus many of the FGFR isoforms bind several FGFs. It is likely that each FGF requires a different pattern of sulphation within the heparan sulphate chains for binding. Therefore, the HSPG receptors may provide additional specificity, allowing a cell to fine tune its response to the FGFs present in the extracellular milieu. The HSPG receptors also control the availability of FGFs and hence regulate the transport of FGFs within a tissue. FGF-stimulated cell division would appear to have a mandatory requirement for the FGFs to be translocated to the nucleus via the cytosol after interacting with the dual-receptor system. The consequences of the potential direct action of FGFs in stimulating cell division are examined in the light of current models of signal transduction.

Animals↗
PubMed1994

Secretion of transforming growth factor alpha and expression of its receptor in human mammary cell lines.

The secretion of transforming growth factor alpha (TGF alpha) and the expression of cell-surface receptors for epidermal growth factor (EGF) were measured in a series of human mammary cell lines. The amount of TGF alpha secreted by the cells did not correlate with the phenotype of the cells (epithelial or myoepithelial), the mechanism of immortalization of the cells (SV40 or spontaneous) or the source of the cells (normal mammary gland, benign hyperplastic lesion, malignant tumour). The level of expression of cell-surface receptors for EGF was markedly increased as a consequence of SV40-immortalization of mammary cells, but otherwise did not correlate with the phenotype of the cells or the source of the cells. Much of the increase was accounted for by the appearance of a large number of low-affinity receptors for EGF in the SV40-immortalized cells. It is suggested that one of the mechanisms whereby SV40-immortalization suppresses the senescence of primary cultures of human mammary epithelial cells involves increasing the level of expression of receptors for EGF. In contrast the level of secretion of TGF alpha by cells in culture is probably a consequence of the mechanisms of adaptation of each cell line to culture conditions, and does not reflect the level of secretion of TGF alpha by cells in vivo.

Breast↗
PubMed1993

Reversible inhibition of proliferation of epithelial cell lines by Agaricus bisporus (edible mushroom) lectin.

Galactosyl beta-1,3-N-acetyl galactosamine (Gal beta-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal beta-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) cells and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal beta-1,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gal beta-1,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal beta-1,3-GalNAc. In contrast to PNA, ABL (25 micrograms/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by 16% (95% confidence limit, 12-20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47-52%), and Rama-27 rat mammary fibroblasts by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum-free medium. When assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 micrograms/ml for 49% inhibition). Ten micrograms/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal growth factor (100 pg/ml) and PNA (25 micrograms/ml) and markedly inhibited the stimulatory effect of insulin (50 ng/ml). ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50 micrograms/ml ABL was reversible after removal of the lectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 +/- 0.29) x 10(-7) M with (3.6 +/- 0.3) x 10(7) binding sites/cell. A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.

Adenocarcinoma↗
PubMed1993

Ectopic production of heparin-binding growth factors and receptors for basic fibroblast growth factor by rat mammary epithelial cell lines derived from malignant metastatic tumours.

A rat mammary (Rama) epithelial cell line, Rama 704, derived from normal rat mammary gland does not possess any detectable cell-surface receptors for basic fibroblast growth factor (bFGF), produces a barely detectable level of bFGF mRNA and does not contain detectable levels of bFGF-like activity. Similar results have been obtained with the Rama 37 epithelial cells derived from benign tumours. However, 4 independently isolated epithelial cell lines derived from malignant rat mammary tumours and their metastases possess receptors for bFGF and contain between 2 ng and 9 ng heparin-binding, growth-stimulatory activity per 10(6) cells. The weakly metastatic Rama 600 cells possess high- and low-affinity receptors for bFGF, (KD 20 pM and 8 nM, respectively), while the moderately metastatic Rama 800 cells possess only high-affinity receptors (KD 40 pM). The moderately metastatic C18PLN and 267LU cells, derived from metastases arising from benign Rama 37 cells which had been transfected with DNA from the malignant Rama 800 cells, also possess only high-affinity receptors (KD 36 pM and 80 pM, respectively). Our results show that within the Rama system there is a correlation between the appearance of heparin-binding growth factors and of high-affinity but not low-affinity receptors for bFGF with the malignant phenotype.

Animals↗
PubMed1993

Immunocytochemical identification of basic fibroblast growth factor in the developing rat mammary gland: variations in location are dependent on glandular structure and differentiation.

We raised antiserum to human recombinant basic fibroblast growth factor (rbFGF) in rabbits. With this affinity-purified antiserum, other antisera to rbFGF, and commercial antiserum to bovine pituitary bFGF, we undertook immunocytochemical detection of bFGF in histological sections of rat mammary glands at different developmental stages. In non-growing ducts, anti-bFGF serum stains the basement membrane/myoepithelial cells, whereas in serial sections most of this stain is observed to be associated with anti-laminin-staining basement membranes rather than with anti-callus-keratin-staining myoepithelial cells. The weak staining of the myoepithelial cells is enhanced when NiCl2 is included in the detection system, but little staining for bFGF is observed in the epithelial cells. In growing neonatal ducts from 1-day-old rats, in growing terminal end buds (TEBs) and, to a lesser extent, in growing alveolar buds (ABs) in prepubescent (21-day) and pubescent (50-day) rats, both their inner and outer cells are stained moderately by anti-bFGF sera. In non-growing ducts from rats aged 6 days, in non-growing ABs of rats aged 60 days and more, and in alveoli from pregnant and lactating rats, only the basement membrane/myoepithelial cell area is stained by anti-bFGF sera; the epithelial cells are unstained. Staining of the myoepithelial cells is enhanced by mixtures of rbFGF and anti-bFGF sera in non-growing ducts, but there is little change in the staining of growing TEBs. All staining by anti-bFGF sera is abolished with heparin in the reactions. We suggest that the immunoreactive bFGF is present mainly bound to heparan sulfate glycosaminoglycans in the basement membrane of resting structures, but that immunoreactive bFGF becomes associated with proliferating cells, particularly those intermediate in characteristics between epithelial and myoepithelial cells in growing structures such as TEBs.

Animals↗
PubMed1993

The expression of basic fibroblast growth factor and its receptor in cell lines derived from normal human mammary gland and a benign mammary lesion.

mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.

Breast↗