Generation of cell types with myoepithelial and mesenchymal phenotypes during the conversion of rat mammary tumor epithelial stem cells into elongated cells.

Epithelial cell lines isolated from benign rat mammary tumors converted to elongated cells that showed some aspects of myoepithelial differentiation. This cellular conversion was blocked in cells isolated from malignant tumors. For investigation of the pathway of the conversion, a rat mammary epithelial cell line (Rama 25) that converted to elongated cells through a series of morphologically distinct intermediates was isolated. These intermediates formed a series in the order: Rama 25, Rama 25-I2, Rama 25-I1, Rama 25-I4, and elongated cells. These cell lines were examined for aspects of myoepithelial or mesenchymal differentiation with the use of a polyclonal antibody to type IV collagen and a keratin monoclonal antibody, LP34 (myoepithelial markers), or a polyclonal antibody to type I procollagen (mesenchymal marker) for cells grown on plastic or as tumors in nude mice. The more epithelial-like cell lines Rama 25 and Rama 25-I2 produced relatively small amounts of type IV collagen and did not stain with LP34 or anti-type I procollagen. The flatter, polygonal cell line Rama 25-I1 stained more strongly with the antibody to type IV collagen but did not stain with anti-type I procollagen. Rama 25-I1 cells, and to a lesser extent Rama 25-I4 cells in tumors, contained a network of cytoplasmic filaments that stained strongly with LP34. The elongated cells, Rama 25-I4 and Rama 25-floaters (Rama 25-FL), did not stain with LP34 in vitro but produced an extracellular matrix that stained with antibodies to both type I procollagen and type IV collagen. The results obtained with these marker proteins suggested that, in this series of morphologic intermediates, the myoepithelial phenotype was best expressed in Rama 25-I1 cells and to a lesser extent in 25-I4 cells. However, this phenotype was relatively unstable, converting to elongated cells, some of which have decreased myoepithelial and increased mesenchymal characteristics. Such a pathway may explain the mixed population of cells seen in some types of benign mammary tumor.

D-periodic assemblies of type I procollagen.

The solubility limit of purified chick type I procollagen, incubated at 37 degrees C in phosphate-buffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness (approximately 8 nm) and up to 1 micron wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.

Inhibitors of mammalian tissue collagenase and metalloproteinases suppress ovulation in the perfused rat ovary.

We have examined the effects of a new synthetic inhibitor of mammalian tissue collagenase, CI-1 (N-[3-N-(benzyloxycarbonyl)amino-1-(R)carboxypropyl]L-leucyl-O-methyl-L- tyrosine N-methylamide; G. D. Searle SC 40827), and a general metalloproteinase inhibitor, 1,10-phenanthroline, on ovulation, as judged by the observation of follicular rupture, and on progesterone production of the perfused rat ovary. Ovaries of PMSG (20 IU)-primed rats were perfused for 21 h, and samples of medium were taken for analysis of progesterone concentration. The number of ovulations was estimated by counting the number of oocytes released into the perfusion chamber. Ovaries were stimulated with LH (0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM), and this treatment resulted in a mean of 17.2 ovulations/treated ovary. 1,10-Phenanthroline dose-dependently inhibited ovulation, with 0, 0.2, and 12.5 ovulations/treated ovary at 1.0, 0.1, and 0.01 mM, respectively. This inhibition of ovulation closely paralleled the inhibition of extracted collagenase from uterus and ovary. However, 1,10-phenanthroline also suppressed progesterone release in a dose-dependent manner. Addition of the collagenase inhibitor (CI-1; 25 microM) 1 h after LH plus IBMX inhibited ovulation (6.3 ovulations/treated ovary). Its relatively inactive stereoisomer (CI-2; 25 microM) did not suppress ovulation (20.0 ovulations/treated ovary). CI-1 inhibited extracted uterine collagenase 50% at a concentration of 2 microM, whereas CI-2 was only 1/15th as effective. There was an 80% loss of CI-1 from the medium during the perfusions. Neither CI-1 nor CI-2 had any effect on LH plus IBMX-stimulated progesterone release. These data demonstrate that the general metalloproteinase inhibitor 1,10-phenanthroline is able to inhibit ovulation, but also inhibits steroidogenesis. The more specific inhibitor of collagenase, CI-1, can inhibit ovulation without affecting steroid production. These data indicate an important role for collagenase in the ovulatory process.

Identification of disulphide-bonded type X procollagen polypeptides in embryonic chick chondrocyte cultures.

A high-Mr (Mr 120,000), disulphide-bonded collagenous polypeptide was observed to co-purify with the prox1(X) chain during isolation of cartilage collagens from culture medium of embryonic chick tibiotarsal chondrocytes. This high Mr polypeptide was subsequently shown by two-dimensions l SDS-PAGE and peptide mapping to represent a dimer of the prox1(X) chain of type X collagen linked by disulphide bonding in the non-collagenous domains.

The effects of inhibitors of cysteine-proteinases and collagenase on the resorptive activity of isolated osteoclasts.

The effects of specific inhibitors of cysteine-proteinases ((Z-Phe-Ala-CHN2: benzyloxycarbonyl-phenyl-alanyl alanyl diazomethane and E-64: trans-epoxy-succinyl-L-leucylamido(4-guanidino)-butane) and collagenase and collagenase ((Cl-1: N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide) have been tested on the osteoclastic resorption of dentine. Chick osteoclasts were cultured in the presence or absence of 12.5 microM Z-Phe-Ala-CHN2, 40 or 60 microM E-64, or 40 or 100 microM Cl-1 for 1 or 2 days. In addition, osteoclasts were cultured on oyster shell calcitostracum with or without 12.5 microM Z-Phe-Ala-CHN2. Specimens were studied by light microscopy to count cells and resorption features and by scanning electron microscopy (SEM) stereophotogrammetry for the measurement of the depths, plan-areas and volumes of resorption pits. The numbers, depths and volumes (but not the plan-areas) of the resorption pits in dentine were significantly reduced by Z-Phe-Ala-CHN2 and E-64. Thus, for a given plan-area, the volumes and the depths of resorption pits were smaller in these experimental groups compared with control dentine specimens. The overall inhibition of resorption was at least 75%. Cl-1 did not have this inhibitory effect on the numbers or sizes of resorption pits in dentine. When the oyster calcitostracum was used as a substrate for the osteoclasts, Z-Phe-Ala-CHN2 did not reduce the numbers or volumes of pits, but increased the plan-areas and prevented the formation of deeper pits.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a congenital defect of factor VII in a colony of beagle dogs: the clinical use of the plasma.

A severe, pure deficiency of factor VII has been defined in haematological and biochemical terms in the Alderley Park colony of beagle dogs. By modification of the conventional factor VII assay to improve its specificity an apparent low activity of between 1 and 2.5% of the normal beagle has been found. When this plasma is used as a substrate for factor VII assays it compares well with human factor VII-deficient plasma in the measurement of both raised and depressed factor VII activity. It also appears to be a valuable reagent in the quality control of tissue thromboplastin extracts.

Studies on the major fucosylated glycoprotein released into the medium by cultured human skin fibroblasts.

Electrophoretic analysis of the proteins synthesized by cultured human skin fibroblasts demonstrated that the major component released into the medium is a high molecular weight fucosylated glycoprotein (MFGP). Gel filtration chromatography under denaturing and reducing conditions indicated that MFGP has a molecular weight of approx. 250,000; but appears to behave as a disulphide-linked dimer when unreduced. MFGP is further distinguished as the major labelled macromolecule in the medium after incubation of fibroblasts with [35S]cysteine. The role of this glycoprotein is unknown but it bears a striking resemblance to a presumptive structural glycoprotein recently shown to be secreted by arterial smooth muscle cells in culture, and may also be related to a group of much studied cell surface glycoproteins.

Studies on the assembly of the rat lens capsule. Biosynthesis and partial characterization of the collagenous components.

1. Isolated rat lens capsules synthesized hydroxy[3H]proline-containing polypeptides when incubated with [3H]proline. 2. The collagenous polypeptides synthesized during a 2 h incubation were analyzed by both gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and shown to have an apparent mol.wt. of approx. 180,000. 3. No evidence was obtained for conversion of these polypeptides into a lower-molecular-weight species in experiments where capsules were labelled for 2 h and chased with non-radioactive proline for up to 22 h. However, a time-dependent incorporation of the 180,000-mol.wt. species into a larger collagenous component was observed and this could be prevented by the inclusion of beta-aminopropionitrile in the incubation medium. 4. The radioactive components synthesized by the capsules correspond to subunits of the intact lens capsule and the direct incorporation of the polypeptide of mol.wt. 180,000 into deoxycholate-insoluble basement membrane was demonstrated.

Production of monoclonal antibodies recognising N-ethylmaleimide during attempted generation of monoclonal antibodies to human type I procollagen.

Mice were immunised for the production of monoclonal antibodies to human procollagen (I) using antigen purified from fibroblast conditioned medium. The procedure for procollagen (I) preparation included the addition of proteinase inhibitors N-ethylmaleimide, phenylmethylsulphonyl fluoride and ethylenediaminetetraacetic acid to prevent damage by proteolytic cleavage. Four of the five monoclonal antibodies subsequently produced were found to recognise the thiol proteinase inhibitor N-ethylmaleimide but not type I procollagen prepared in the absence of N-ethylmaleimide. One of these monoclonal antibodies was examined further and shown to recognise beta-galactosidase after it had been reacted with N-ethylmaleimide. As far as we are aware this is the first time that monoclonal antibodies have been produced which recognise N-ethylmaleimide. Our findings indicate an unexpected reaction between procollagen and N-ethylmaleimide and prompt the suggestion that the use of N-ethylmaleimide in the purification of procollagen and other proteins should be reexamined.

Biosynthesis and release of glycoproteins by human skin fibroblasts in culture.

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-(3)H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated (3)H was present as [(3)H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [(3)H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [(3)H]fucose-labelled material before and after trypsin digestion. 3. The [(3)H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000-250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [(3)H]fucose and (14)C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [(35)S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.

The nature of the microfibrillar glycoproteins of elastic fibres. A biosynthetic study.

1. Cell cultures propagated from foetal bovine ligamentum nuchae synthesized and secreted two glycoproteins, designated MFP I and MFP II, that are closely related to elastic-fibre microfibrils. Glycoproteins MFP I (apparent mol.wt. 150 000) and MFP II (apparent mol.wt. 300 000) were metabolically labelled, separated from other culture-medium components by immunoprecipitation with a specific anti-(microfibrillar protein) serum, and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and sodium dodecyl sulphate/gel-filtration chromatography. 2. Ligament cells also synthesized and secreted fibronectin, but salt-fractionation and immunoprecipitation studies with a specific anti-(cold-insoluble globulin) serum established that neither glycoprotein MFP I nor glycoprotein MFP II was related to fibronectin. 3. The secretion of glycoprotein MFP I, but not that of glycoprotein MFP II, was enhanced by the addition of ascorbate to the culture medium. 4. Ascorbate-supplemented ligament cells incorporated [3H]proline into glycoprotein MFP I, and 36% of the nondiffusible proline residues were hydroxylated, exclusively as 4-hydroxy[3H]proline. Less than 1% of the total proline residues in [3H]proline-labelled glycoprotein MFP II were hydroxylated. 5. Ascorbate-supplemented cells incorporated [14C]lysine into glycoprotein MFP I and 30% of the non-diffusible lysine residues were hydroxylated. 6. Newly secreted glycoprotein MFP I was digested by highly purified bacterial collagenase to yield polypeptide fragments of apparent mol.wts. 50 000 and 30 000. Glycoprotein MFP II was not digested by bacterial collagenase. 7. The results suggest that elastic-fibre microfibrils are composed of a novel collagenous glycoprotein MFP I in association, as yet undefined, with a non-collagenous glycoprotein MFP II.

An ultrastructural study of fibroblasts derived from bovine ligamentum nuchae and their capacity for elastogenesis in culture.

Fibroblast cultures were readily propagated from fetal bovine ligamentum nuchae. The ligament cells were easily cultured by standard techniques and were maintained in culture flasks for up to 57 days. During this time they accumulated an extensive extracellular matrix which contained the main structural elements of the parent tissue, namely collagen and elastic fibres. Elastogenesis was seen to proceed in two phases: the formation of parallel bundles of 10--12 nm wide microfibrils followed by the deposition within these bundles of amorphous elastin-like material. Elastic fibres were not produced in cultures that were supplemented with ascorbic acid either in the absence or presence of the lathyrogen BAPN.

Ehlers-Danlos syndrome in two siblings with deficient lysyl hydroxylase activity in cultured skin fibroblasts but only mild hydroxylysine deficit in skin.

Two siblings suffered from Ehlers-Danlos syndrome characterized by skin fragility, joint laxity and dermal hyperelasticity. The association with microcornea and muscle hypotonia allowed the preliminary classification into type VI according to McKusick. Ultrastructure analysis of skin biopsies revealed poor integration of collagen fibrils into fibres; accordingly, the texture of the connective tissue appeared irregular. Lysyl hydroxylase activity of cultured skin fibroblasts was markedly reduced in the cells of the two patients. Preliminary studies revealed intermediate activity in the cells cultured from the skin of the parents. This finding suggested an autosomal recessive mode of inheritance. Unexpectedly and in contrast to the 3 cases reported in the literature, the hydroxylysine deficit in the patients' skin was, for reasons not yet understood, only mild. Therefore, amino acid analysis of skin is not adequate for the diagnosis of lysyl hydroxylase-deficient Ehlers-Danlos syndrome type VI.

Elastogenesis and microfibrillar glycoprotein synthesis by bovine ligamentum nuchae cells in culture.

Cell cultures propagated from fetal bovine ligamentum nuchae were found to accumulate an extensive extracellular matrix containing collagen and elastic fibers when maintained for periods up to 8 weeks. The synthesis by these cells of two glycoproteins, designated MFP I and MFP II, which are immunologically related to components of the elastic fiber microfibrils was investigated. MFP II, apparent mol. t. 300,000, was shown to be a non-collagenous glycoprotein whereas MFP I, apparent mol. wt. 150,000, was found to be a novel collagenous glycoprotein containing hydroxyproline and glycosylated hydroxylysine residues but exhibiting properties of distinct from the known collagens. Two glycoproteins having characteristics similar to MFP I and MFP II were also identified in an extract of the cell layer and in a "microfibrillar protein preparation" from intact nuchal ligament.

The identification of glycoproteins associated with elastic-tissue microfibrils.

Cell cultures derived from foetal bovine ligamentum nuchae accumulate extracellular fibrils morphologically identical with elastic-tissue microfibrils. Two glycoproteins synthesized by the ligament cells are closely related to the matrix microfibrils as assessed by immunological and chemical extraction techniques.