To grow mouse mammary epithelial cells in culture.

Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures.

Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells.

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.

Ouabain-sensitive 86Rb(K) influx is linked to transepithelial Na transport in pig kidney cell line.

The pig kidney cell line, LLC-PK1, exhibits rheogenic D-glucose coupled transepithelial Na+ transport that is inhibited by phlorizin. By measuring the difference in initial rates of influx of 86Rb+ with and without coupled Na+ transport, we can demonstrate an 86Rb+ uptake linked to Na+ transport, The simultaneous determination of phlorizin-inhibited Na coupled D-[3H] glucose uptake and 86Rb+ influx allows calculation of an Na+/Rb+ stoichiometry that is consistent with an electrogenic Na+ for Rb+ exchange.

Cell density determines epithelial migration in culture.

The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet.

CO2/bicarbonate stimulates growth independently of PH in mouse mammary epithelial cells.

Mouse mammary cells of the NMuMG line proliferated faster and formed colonies more efficiently when the air above the cells contained 5% CO2. An increase in colony forming efficiency also occurred if the bicarbonate concentration in the medium was higher (44 versus 13 mM). These growth increases induced by the CO2 or bicarbonate occurred even when the control cultures were maintained at the same pH, and they occurred at every pH tested. Both the growth rate and colony forming efficiency of the NMuMG cells were highest at pH 7.0 to 7.3.

Transepithelial transport in cell culture: stoichiometry of Na/phlorizin binding and Na/D-glucose cotransport. A two-step, two sodium model of binding and translocation.

The renal cell line LLC-PK1 cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependent D-glucose coupled transport. The short-circuit current (Isc) was accounted for by net apical-to-basolateral D-glucose coupled Na flux, which was 0.53 +/- 0.09(8) mueq cm-2hr-1, and Isc, 0.50 +/- 0.50(8) mueq cm-2hr-1. A linear plot of concurrent net Na vs. net D-glucose apical-to-basolateral fluxes a gave a regression coefficient of 2.08. As support for a 2:1 transepithelial stoichiometry, sodium was added in the presence of D-glucose and the response of Isc analyzed by a Hill plot. A slope of 2.08 +/- 0.06(5) was obtained confirming a requirement of 2 Na for 1 D-glucose coupled transport. A Hill plot of Isc increase to added D-glucose in the presence of Na gave a slope of 1.02 +/- 0.02(5). A direct determination of the initial rates of Na and D-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2. A coupling ratio of 2 for Na, D-glucose uptake, doubles the potential energy available for Na-gradient coupled D-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependent-phlorizin binding was 1.1 +/- 0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na].(ABSTRACT TRUNCATED AT 250 WORDS)

Furosemide-sensitive Cl transport in embryonic chicken retinal pigment epithelium.

Retinal pigment epithelium- (RPE) choroid-sclera preparations from embryonic chickens were mounted in an Ussing chamber. A spontaneous transepithelial voltage (Ve) of 5.1 mV (retinal side positive) and a resistance of 114 omega . cm2 can be attributed to the RPE. Furosemide and ouabain inhibited the Ve without affecting the resistance when applied to the retinal surface of the preparation but had no effect when applied to the scleral surface. Acetazolamide and amiloride were without effect when applied to either surface. The Ve fell by 100% in Na-free medium, by 70% in Cl-free medium, and was unchanged in HCO3-free medium. When Na and Cl were returned to the medium, the Ve recovered. Dilution potentials and unidirectional flux studies indicated that the RPE is more permeable to Na than to Cl. Isotope flux studies performed under open-circuit conditions showed a net retina-to-choroid flux for both Na and Cl with the net Cl flux abolished by furosemide. Analysis of the Na+ and Cl- electrochemical gradients and the Na permeability across the RPE suggests that the net retina-to-choroid Na+ flux is largely passive, whereas the net retina-to-choroid Cl- flux results from active transport. These results indicate that embryonic chicken RPE possesses furosemide-sensitive Cl transport function. A model of embryonic chicken RPE transepithelial transport is presented.

Mouse mammary cells in D-valine medium.

Cells of a mouse mammary epithelial cell line as well as fibroblasts from a mouse mammary explant were severely inhibited from proliferating in a medium in which D-valine was substituted for L-valine. After the first few days in D-valine medium, the number of epithelial cells did not increase despite the fact that a few percent continued to synthesize DNA. The cells did recognize the presence of the D-valine in the medium because cells in D-valine increased in volume and their numbers remained stationary, whereas cells without valine shrank and the cell numbers decreased with time.

Transepithelial glucose transport in cell culture.

Pig kidney cell line LLC-PK1 cultured on a collagen-coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by circumferential occluding junctions. In the presence of 5.5 mM D-glucose, a potential difference (PD) of 2.8 mV, apical bath negative, short-circuit current Isc of 13.2 microA . cm-2, and transepithelial resistance of 211 omega . cm2 were recorded. Isc and PD were reduced by phlorizin added to the apical bath but were unaffected when phlorizin was placed in the basolateral bath. Ouabain or the replacement of Na by tris-(hydroxymethyl)aminomethane or choline abolished the Isc. The sugar concentrations required to produce the half-maximal Isc were 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methylglucose. When [Na] was reduced, the D-glucose required for half-maximal SCC increased. Isotopically 3H- and 14C-labeled D-glucose were used to determine simultaneous bidirectional fluxes; a resultant net apical-to-basolateral flux was present and could be abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism. The cell culture model provides advantages for investigation of mechanisms of transepithelial glucose transport.

Transepithelial transport in cell culture: D-glucose transport by a pig kidney cell line (LLC-PK1).

The pig kidney cell line LLC-PK1 cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5 mM D-glucose, a PD of 2.8 mV. apical surface negative a SCC of 13 microA cm-2 and transepithelial resistance of 211 ohm.cm2 were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methyl-glucose. When [Na] was reduced, the concentration of D-glucose required for half-maximal SCC increase. Isotopically labeled 3H and 14C D-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK1, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.

Transepithelial transport in cell culture.

In cell culture a kidney epithelial cell line MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia. The cell layer can be readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms-cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCl, of 1.7. Net water flux, apical to basolateral, was 7.3 mul cm-2 hr-1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potential use of a homogeneous cell population in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels.

Biophysics of domes formed by the renal cell line Madin-Darby canine kidney.

When cultured on an impermeable substratum, the renal epithelial cell line Madin-Darby canine kidney (MDCK) forms domes. From a knowledge of dome shape, radius, and hydrostatic pressure difference delta P, from within to outside the domes, biophysical properties of the epithelium can be determined. By microscopy, circular based domes were found to be section of spheres with a constant height-to-radius ratio h/r of 0.684. By using the Laplace equation derived for this geometry and measurements of delta P and r, the values of the tension of cell dish adhesion Ta and dome wall tension Tw were found to be constants of 6.60 and 7.08 torr, respectively. By combining constants, Ta, and h/r, and because domes are section of spheres, delta P and dome volume were shown to be known functions of radius alone. The modulus of elasticity of the epithelium was calculated to be 4.82 X 10(3) dyn/cm2.

Transepithelial transport in cell culture. A theoretical and experimental analysis of the biophysical properties of domes.

Dissociated cells of transporting epithelia, when cultured on an impermeant substratum, form polarized monolayers frequently characterized by the presence of domes. If the assumption is made that the monolayer exhibits a uniform stretch modulus of elasticity and tension of cell-dish adhesion, Ta, then biophysical properties of the epithelium can be predicted. We have shown that for such epithelia, domes should (a) have circular bases, (b) be sections of spheres with a constant height to radius, h/r, ratio, (c) have a dome-wall tension, Tw, that is constant, and (d) have a dome volume that is a function of radius alone. Additionally, a Laplace equation derived for this geometry predicted the hydrostatic pressure from within to outside domes as a decreasing function of radius alone. By microscopy, domes had predominantly circular bases and were found to be sections of spheres with a constant height, h, to radius, r, ratio of 0.684. Using the Laplace equation derived for this geometry and measurements of delta P and r, the tension of cell-dish adhesion, Ta, and dome-wall tension, Tw, were found to be constants of 6.60 and 7.08 torr, respectively. Combining the constants for Ta and h/r ratio, and the fact that domes are sections of spheres, delta P and dome volume were shown to be known functions of radius alone. In addition, the modulus of elasticity of the epithelium was calculated to be 4.82 X 10(3) dyn/cm2.

Transepithelial transport in cell culture: bioenergetics of Na-, D-glucose-coupled transport.

The renal cell line LLC-PK1 cotransports Na and D-glucose from the apical to the basolateral side of the cell monolayer, and the short-circuit current (Isc) measures the net amount of Na transported. Under conditions of maximal cotransport, the addition of phlorizin or removal of Na reversibly decreased oxygen consumption by one-half. In the absence of glycolytic substrates, alpha-methyl-D-glucoside stimulated Isc and oxygen consumption, although the Isc came to a steady state 50% less than when glycolytic substrates were present. The addition of other aerobic substrates did not increase Isc; however, when non-cotransported glycolytic substrates were introduced the Isc returned to a maximum with an associated fall in oxygen consumption and increased lactate production. Thus, in the absence of glycolytic substrates aerobic ATP formation may be rate-limiting for Na, D-glucose cotransport. For this epithelium glycolysis makes an important contribution to the provision of energy for transport. Oxygen consumption does not correlate well with Isc and is not a good measure of the energy used in transport.

Morphologic and clinical determinants of response to therapy in small cell carcinoma of the lung.

The authors reviewed a series of 29 cases of small cell carcinoma of the lung in order to determine the influence of histologic type and other prognostic factors on response to combination chemotherapy and survival. Comparing classical (lymphocyte-like and fusiform) versus nonclassical histologic types (polygonal and other), no significant difference could be found in response to therapy (CR + PR 11/16 versus 8/11), median survival (7 versus 6 months) or median survival of responders (9 versus 10 months). Furthermore, tumors with atypical histology ("other") behaved similar to the overall series. Of clinical factors investigated, only decreased performance status and presence of metastatic disease were significant prognostic indicators. The results suggest that tumor histologic type is not a useful prognostic indicator in small cell carcinoma, and that atypical tumors resembling small cell carcinoma appear to respond similarly to classical small cell carcinoma and should probably be treated as such.

Transepithelial transport by pulmonary alveolar type II cells in primary culture.

Fluid and electrolyte transport by epithelial cells in vitro can be recognized by the ability of cultured cells to form domes and by the electrical properties of monolayer cultures. Pulmonary alveolar epithelial cells are thought to be partially responsible for fluid movement in the fetal lung, but their role in electrolyte transport in the adult lung is not known. We isolated alveolar type II cells from adult rat lung and maintained them on plastic culture dishes alone, on plastic culture dishes coated with an extracellular matrix, and on collagen-coated Millipore filters. Numerous large domes were formed on culture dishes coated with the extracellular matrix; smaller domes were formed on uncoated plastic culture dishes. Sodium butyrate (3 mM) stimulated dome formation. Transmission electron microscopy showed that the epithelial cells had flattened but still retained lamellar inclusions and that the cells were polarized with microvilli on the apical surface facing the culture medium. The electrical properties of the monolayers maintained on collagen-coated Millipore filters were tested in two laboratories. The transepithelial potential differences were 0.7 +/- 0.1 mV (24 filters, seven experiments) and 1.3 +/- 0.1 mV (13 filters, two experiments) apical side negative, and the corresponding resistances were 217 +/- 11 ohm X cm2 and 233 +/- 12 ohm X cm2. Terbutaline (10 microM) produced a biphasic response with a transient decrease and then a sustained increase in potential difference. Amiloride (0.1 mM) completely abolished the potential difference when it was added to the apical side but not when it was added to the basal side, whereas 1 mM ouabain inhibited the potential difference more effectively from the basal side. Thus, type II cells form a polarized epithelium in culture, and these cells actively transport electrolytes in vitro.