Culture substrate dependence of mouse fibroblasts survival at 4° C

Summary When L-929 mouse fibroblasts grown in Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) were stored in a monodisperse suspension at 4° C, the viability decreased rapidly from the beginning of storage. The viability in this study was determined by counting electronically the number of cells with the capacity to attach to glass substrate and with the membrane boundary resistant to a proteolytic digestion. When, however, the dissociated cells were preincubated briefly at 37° C, and subsequently stored at 4° C as they were attaching on a glass substrate, the rapid loss of viability could be reduced effectively. A biphasic survival profile consisting of an initial phase of slowly decreasing viability and the subsequent phase of rapidly decreasing viability were then observed. The rapid viability loss occurred not only when the cell suspension was prepared by mechanical dislodging but also after trypsinization or dispase treatment. Such viability loss was also observed when the dissociated cells were not stored at 4° C directly but preincubated in a monodisperse suspension at 37° C in a siliconized plate and then stored at 4° C. The above results show that the rapid loss of viability is associated closely with the fact that the cells were not attached to the substrate but in suspension. This work was supported in part by grants-in-aids from the Institute of Physical and Chemical Research and from the Mitsubishi Foundation.     

Early events during substrate adhesion of normal and virus-transformed mouse fibroblasts.

The relationship between attachment of Balb/c3T3 cells and their SV40 transformants to glass or plastic substrates and deposition of substrate-attached material (SAM-proteoglycans implicated in substrate adhesion) has been examined very early after inoculation of cells subcultured with ethylenebis (oxyethylenenitrilo) tetra-acetic acid (EGTA). The metabolic inhibitors cycloheximide and colchicine minimally affected the kinetics or short-term stability of attachment of cells or deposition of SAM. SAM deposition on to the substrate began immediately after inoculation of cells and was maximal prior to the highest cell attachment level (30-40 min after inoculation). At 4 degrees C, there was no attachment of cells to the substrate and no deposition of leucine- or glucosamine-radiolabelled SAM on to the substrate. 3T3 cells deposited SAM to a maximal level earlier during the attachment process than SV40-transformed cells. SVT2 cells deposited much smaller amounts of SAM (measured on a per-cell basis) to 3T3 SAM-coated substrates during attachment processes, whereas 3T3 cells and concanavalin A (con A) revertant variants of SVT2 cells, which have regained density-dependent inhibition of growth, deposited identical amounts of SAM (per-cell) on untreated or SAM-coated substrates. Serial attachment experiments with SVT2 cells indicated that all SVT2 cells reduced their deposition amounts on SAM-coated substrates, rather than there being an ability of a small proportion of cells to attach preferentially to SAM-coated substrates while being unable to deposit SAM themselves. The data are consistent with the presence of a sizeable pool of SAM-like proteoglycans being present on the surface of EGTA-removed cells whose deposition may be a requirement for, but may not necessarily be sufficient for, stable adhesion of cells to the substrate.     

Spreading of normal and transformed fibroblasts in dense cultures

Cell spreading in dense cultures of normal mouse embryo fibroblasts and of the two lines of mouse transformed fibroblasts was examined by electron microscopy. The mean number of cell layers in culture and cell population density per unit area of the substrate were detetmined; the mean area of the cell projection on the substratum was found from these data.Normal fibroblasts formed multilayefed sheet in dense culture. The cells in this sheet were well-spread. These cells formed thin lamellae (lamellar cytoplasm) over the surface of other cells and over the intercellular substance. The mean cell area in dense culture was not smaller than that of the cell spread on the substratum in sparse culture.Dense cultures of two transformed lines (M 22 and L) had differing morphologies: cultures of one line (M 22) were multilayered, those of the other line (L) were monolayered. Decreased spreading and almost complete (M 22) or complete (L) absence of lamellar cytoplasm were characteristic of both transformed lines. The mean area of the cell in dense cultures of both lines was several times smaller than that of their normal progenitors.It is concluded that similar reactions leading to the spreading accompanied by the formation of lamellar cytoplasm can be induced by the contact of fibroblast with various surfaces: that of the substratum in sparse culture, that of other cells and of intercellular structures in dense culture. Deficiency of these reactions characteristic for transformed fibroblasts may be responsible for abnormal morphology of their cultures.     

Long-term growth of chicken fibroblasts on a collagen substrate

In 3 separate experiments, cells derived from chick embryo muscle explants have been grown in either Waymouth's medium or Ling's (AN.54) medium with 20% human placental cord serum. Continuous transfer and new outgrowth from a succession of 2 × 2 mm fragments has continued for periods of 24 to 44 months. Continuous growth was achieved only on a collagen substrate, and no continuous growth was obtained when cells were transferred to glass. When incubator temperature was raised to 43 °C over a period of 1 month, new cell types developed and had the capacity to both survive and grow directly on glass for several months.     

Surface glycosyltransferases on cultured mouse fibroblasts

Previous work has suggested the presence of galactosyltransferases on the outer surface of the plasma membrane of a malignant and a nonmalignant cell line. This paper summarizes data indicating that three other classes of glycosyltransfeases are similarly located on cell surfaces. In addition to the original two cell lines examined, BALB/c 3T3 and BALB/c 3T12, two other lines of BALB/c origin have been investigated. These are the SV40–transformed 3T3 line and one of the revertants of the virally infected cells that is no longer malignant but retains a viral genome.     

Formation of bundles of microfilaments during spreading of fibroblasts on the substrate

Electron microscopy was used to study the sites of formation of bundles of parallel microfilaments in the early stages of spreading of normal mouse embryo fibroblasts on the substrate. Bundles of microfilaments were not found in suspended cells. Contact of the surface of spherical cells with the substrate was not sufficient for the formation of bundles: these bundles were not seen near the under surface of cells that were already attached to the substrate but had not yet developed cytoplasmic outgrowths at their periphery. Peripheral cytoplasmic outgrowths (microspikes and lamellar processes) attached to the substrate were found to be the only sites of localization of the first bundles of microfilaments seen in the spreading cells. It is suggested that surface and/or cytoplasm of the newly-formed peripheral cytoplasmic outgrowth may have some special properties necessary for the initiation of the development of microfilament bundles.     

Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts.

We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to the lipophilic antifolates trimetrexate and piritrexim. These data suggested that R50 cells may possess additional mechanism(s) of antifolate resistance, such as MTX transport alteration. Flow cytometric analysis using fluorescein methotrexate revealed comparable levels of fluorescein MTX displacement with lipophilic antifolates in viable R50 and B11 0.5 cells, but marked insensitivity of R50 cells to MTX competition, thus suggesting a poor uptake of MTX into R50 cells. Analysis of the kinetic parameters of dihydrofolate reductase from R50 cells neither showed alterations in enzyme affinities for various antifolates nor in the Michaelis constant for folic acid and NADPH nor a change in the pH activity optimum. R50 cell-free extracts contained wild-type levels of folylpoly-gamma-glutamyl synthetase activity. However, following metabolic labeling with [3H]MTX, no MTX polyglutamates could be detected in R50 cells. We conclude that the high level of MTX resistance in R50 cells is multifactorial, including overexpression of dihydrofolate reductase, reduced MTX transport, and possibly altered formation of MTX polyglutamates. The potential interactions between the different modalities of MTX resistance in R50 cells are being discussed.     

High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate

We have developed a new approach to detect mechanical forces exerted by locomoting fibroblasts on the substrate. Cells were cultured on elastic, collagen-coated polyacrylamide sheets embedded with 0. 2-micrometer fluorescent beads. Forces exerted by the cell cause deformation of the substrate and displacement of the beads. By recording the position of beads during cell locomotion and after cell removal, we discovered that most forces were radially distributed, switching direction in the anterior region. Deformations near the leading edge were strong, transient, and variable in magnitude, consistent with active local contractions, whereas those in the posterior region were weaker, more stable, and more uniform, consistent with passive resistance. Treatment of cells with cytochalasin D or myosin II inhibitors caused relaxation of the forces, suggesting that they are generated primarily via actin-myosin II interactions; treatment with nocodazole caused no immediate effect on forces. Immunofluorescence indicated that the frontal region of strong deformation contained many vinculin plaques but no apparent concentration of actin or myosin II filaments. Strong mechanical forces in the anterior region, generated by locally activated myosin II and transmitted through vinculin-rich structures, likely play a major role in cell locomotion and in mechanical signaling with the surrounding environment.     

Defining the identity of mouse embryonic dermal fibroblasts

Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase‐seq and histone modification ChiP‐seq data on various cell‐types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell‐types. We found a subset of the signature genes whose expression is dependent on Wnt/β‐catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415–430, 2016. © 2016 Wiley Periodicals, Inc.     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.     

ATP-resistant variants of transformed mouse fibroblasts

Addition of ATP to cultures of transformed mouse fibroblasts, 3T6 cells, resulted in cell growth inhibition, whereas the growth of the non-transformed counterparts, 3T3 cells, was only slightly affected. The inhibition was found to be specific for adenine nucleotides, and concentration dependent. At relatively low concentrations (e.g., 1.0 mM) the effect of ATP was cytostatic, whereas at higher concentrations (e.g., 1.0 mM) a cytotoxic effect was exerted. ATP-resistant variants of 3T6 cells were selected by exposure of cultures to gradually elevated concentrations of ATP. The variants were found to resemble the non-transformed counterparts, 3T3 cells, more than the 3T6 parent cells, by the following criteria: ATP-induced alterations in the membrane potential, changes in membrane permeability, cell growth inhibition, and colony formation on soft agar. The data indicate that long exposure of the transformed cells to external ATP results in redifferentiation and reduction in their tumorigenicity.     

Defining the substrate specificity of mouse cathepsin P

Cathepsin P is a recently discovered placental cysteine protease that is structurally related to the more ubiquitously expressed, broad-specificity enzyme, cathepsin L. We studied the substrate specificity requirements of recombinant mouse cathepsin P using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp (Abz, ortho-aminobenzoic acid and EDDnp, N-[2,4-dinitrophenyl]ethylenediamine). Systematic modifications were introduced resulting in five series of peptides to map the S(3) to S(2)(') subsites of the enzyme. The results indicate that the subsites S(1), S(2), S(1)('), and S(2)('), present a clear preference for hydrophobic residues. The specificity requirements of the S(2) subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. The S(3) subsite of the enzyme presents a broad specificity, accepting negatively charged (Glu), positively charged (Lys, Arg), and hydrophobic aliphatic or aromatic residues (Val, Phe). For several substrates, the activity of cathepsin P was markedly regulated by kosmotropic salts, particularly Na(2)SO(4). No significant effect on secondary or tertiary structure could be detected by either circular dichroism or size exclusion chromatography, indicating that the salts most probably disrupt unfavorable ionic interactions between the substrate and enzyme active site. A substrate based upon the preferred P(3) to P(2)(') defined by the screening study, ortho-aminobenzoic-Glu-Ile-Phe-Val-Phe-Lys-Gln-N-(2,4-dinitrophenyl)ethylenediamine (cleaved at the Phe-Val bond) was efficiently hydrolyzed in the absence of high salt. The k(cat)/K(m) for this substrate was almost two orders of magnitude higher than that of the original parent compound. These results show that cathepsin P, in contrast to other mammalian cathepsins, has a restricted catalytic specificity.