An eye-derived growth factor regulates epithelial cell proliferation in the cultured lens

Lenses in organ culture permit an analysis of factors acting on epithelial cell growth, while keeping the normal steric constraints of the cell population. By employing this technique with radioautography of epithelial whole mounts, we showed that the DNA synthesis found in the epithelia of cultured bovine lenses follows an organized spatial and temporal pattern during culture. Within the first 48 h, active cells were located at the preequatorial region ("germinative zone"), a distribution consistent with the in vivo spatial organization of multiplying cells. Starting at about 48 h, cells from the central region of the epithelium--a nonproliferating population--were triggered to synthesize DNA in the presence of eye-derived growth factor (EDGF). When cultured in serum-free medium, only a small fraction of the cells was labeled, but when a low serum concentration was present, this fraction reached 50% of the cell population. The stimulatory effect of EDGF required a lag period, but its effect reached a maximum exceeding that found for serum. However, the cells from the germinative region, having a cell density three- to four-fold higher than the central region, were not stimulated to proliferate. This occurred irrespective of the presence of EDGF or serum. If this growth-stimulatory activity derived from the retina were the actual factor controlling cell proliferation in the lens in vivo, then the results presented here would point to the presence of a regulatory mechanism similar to that known for some other hormones.     

Appearance of contractile activity in muscular dysgenesis (mdgmdg) mouse myotubes during coculture with normal spinal cord cells

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation expressed in the homozygous mutant as lack of skeletal muscle contraction. To test the ability of normal neurons to form neuromuscular contacts with, and/or possibly induce contractions in $\text{mdg}\text{mdg}$ muscle, dispersed cell cultures of normal and dysgenic muscle from newborn mice were cocultured with normal embryonic rat, mouse, and chick dissociated spinal cord cells. Contraction was induced in $\text{mdg}\text{mdg}$ muscle 1 to 10 days (depending upon the species of the neuronal source) following establishment of the cocultures. Control experiments indicated that the dispersed spinal cord preparations were free of myoblasts capable of fusing with $\text{mdg}\text{mdg}$ muscle. The establishment of neuromuscular contacts in the rat neuron cocultures was monitored by cytochemical staining of acetylcholinesterase (AChE), autoradiography of ¹²⁵I-α-bungarotoxin-bound acetylcholine receptors (AChR), and electrophysiological study of muscle membrane activity. Patches of high AChE activity were similar in size and distribution to high-density clusters of AChR on both control and $\text{mdg}\text{mdg}$ myotubes cocultured with rat neurons. The resting membrane potentials of normal myotubes and those of $\text{mdg}\text{mdg}$ myotubes in the presence of neurons were similar (? ?52 mV). The mepp frequency and the mepp amplitude distribution were the same for both control and mutant cocultured muscle. Thus, normal rat spinal cord neurons were capable of forming normal, functional neuromuscular junctions with $\text{mdg}\text{mdg}$ myotubes, and contractions were induced under coculture conditions, in otherwise noncontracting mutant muscle.     

Persistence of exogenous, inserted ganglioside GM1 on the cell surface of cultured cells

Ganglioside GM₁, which can insert spontaneously into the membrane of intact cells, has been measured after insertion into transformed fibroblasts by cholera toxin (choleragen) binding, for which ganglioside GM₁ is the natural receptor. Choleragen binding is not altered in starved, quiescent cells over a four-day period. Dividing cells show decreased binding in proportion to cell division. Thus, neither dividing nor quiescent cells appear to metabolize or otherwise degrade this membrane component.     

Stimulatory cells in the generation of T-cell-mediated cytotoxicity: Potent stimulating activity of a small radioresistant spleen cell population (RSCs)

The combined effects of irradiation followed by cultivation on a total spleen cell population in order to study the evolution of the stimulating potential in the in vitro generation of allogeneic cytotoxic T lymphocytes (CTLs) were tested. Results revealed that, after 3 days and up to at least 7 days of cultivating irradiated (1000 rad) spleen cells, the remaining living cells (radioresistant spleen cells or RSC) have the same potential to generate CTLs as irradiated noncultivated spleen cells. RSC can resist a 5000-rad irradiation and induce a primary cytotoxic response pattern similar to that of total spleen cells; they act in primary as well as in secondary cultures with optimal responder to RSC ratios of about 100, but are still stimulatory at MLC ratios up to 1000 or 5000. They are lysed by specific allogeneic CTLs and readily inhibit the specific lysis of H-2-identical labeled targets by CTLs. RSCs do not express unusual levels of H-2 or Ia antigens and do stimulate purified T cells. Alloantisera anti-H-2 are able to completely block the RSC-induced generation of CTL. This RSC population may prove to be a good model to study non-H-2- or H-2-associated, nonserologically detectable determinants interacting in the generation of T-cell-mediated cytotoxicity.     

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt₂-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.     

Interaction of far- and near-ultraviolet radiation. The occurrence of photo-augmentation and photo-recovery in cultured mammalian cells

Guided by the phenomena of photo-augmentation and photo-recovery, which have been described with respect to the induction of erythema in human skin, experiments were undertaken with cultured mammalian cells to study whether irradiation with far- and near-ultraviolet radiation results in an interaction at the cellular level with respect to cell survival and induction of mutations. Evidence was found for both photo-augmentation and photo-recovery. Photo-augmentation (more than an additive effect) was observed for cell survival when the long-wave ultraviolet irradiation (UVA) preceded the short-wave ultraviolet irradiation (UVB). Photo-recovery (less than an additive effect) was observed for cell survival if the UVA was given after or simultaneously with the UVB. The latter effect, however, was strongly influenced by dose: doses of UVA higher than 20 000 J/m2 no longer lead to photo-recovery in cell survival. For mutation induction, reduction in mutant frequency appears indicated for both combinations of UVA and UVB and for high and low doses of UVA.     

Exposure to 4-aminopyridine prevents depressant effects of opiates on sensory-evoked dorsal-horn network responses in spinal cord cultures

The depressant effects of morphine (0.1-1 microM) on sensory-evoked dorsal-horn network responses in explants of mouse spinal cord with attached dorsal root ganglia (DRGs) were rapidly restored after addition of 4-aminopyridine (4-AP; 0.1 mM) and major components of these cord responses were stably maintained in the presence of the opiate. Moreover, prior exposure of cord-DRG explants to 0.1 mM 4-AP prevented the depressant effects of 0.1 microM morphine on DRG-evoked dorsal-horn responses, and the effects of 1-10 microM morphine were at least partly antagonized. Increased Ca++ levels (5 microM) attenuated the depression of dorsal horn responses by 1-10 micro M morphine and these effects of Ca++ were greatly enhanced in the presence of 4-AP--in some cultures, concentrations of morphine as high as 100 micro M were strongly antagonized during test periods up to 2 hours. Receptor assays showed that 0.1 mM 4-AP +/- 5 mM Ca++ had no effect on stereospecific opiate binding, indicating that the antagonist actions of these agents in our cultures do not occur at the level of the opiate receptor. The relevance of our in vitro studies of 4-AP antagonism of opiate-depressant effects on sensory-evoked dorsal-horn network responses for analyses of problems in opiate analgesia has been strengthened by a recent report demonstrating that 4-AP does, in fact, reverse morphine analgesia in rats, as determined by tail flick tests.     

The multicomponent nature of teratoma cell adhesion factor

The multicomponent nature of teratoma cell adhesion factor has been demonstrated. Fractionation of crude ascites fluid on a DEAE cellulose ion exchange column shows that two or more components are involved in teratoma adhesion factor (TAF) activity. Glycoproteins (or proteoglycans) in fractionated ascites fluid were localized in polyacrylamide gels. The possible role of these sugar-containing molecules in teratoma cell adhesion and current hypotheses on the mechanism of carbohydrate involvement in intercellular adhesion are discussed.     

Transport of phosphoenolpyruvate through red cell membrane in acidified isotonic sucrose medium.

Organic phosphate compounds added exogenously are impermeable to the red cell membrane in physiological conditions. However, when the cells were incubated in an acidified iso-osmotic sucrose medium(pH 4.2), phosphoenolpyruvate passed freely the membrane, though other glycolytic intermediates and nucleotides failed to permeate the membrane. During incubation of the PEP loaded red cells,PEP was metabolized rapidly and almost one-to-one stoichiometry was observed in the relationship between 2,3DPG production and PEP depletion.     

The effects of dihydropyridines on neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of (3H) norepinephrine and (3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of (3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.     

A serum-free medium supplemented with multiplication-stimulating activity (MSA) supports both proliferation and differentiation of chondrocytes in primary culture

The effect of hormones influencing cartilage metabolism on the growth of chondrocytes isolated from rabbit and rat ribs was investigated in serum-free medium. Insulin supported growth of the cells slightly, whereas calcitonin and parathyroid hormone did not. On the other hand, multiplication-stimulating activity (MSA), a substance partially purified from serum-free medium conditioned by the growth of Buffalo rat liver cells, markedly induced not only proliferation of the chondrocytes but also their synthesis of acid mucopolysaccharides, the characteristic cartilage phenotype, in serum-free medium. These cells maintained this specialized cellular function of differentiated chondrocytes for at least 21 days in serum-free medium. A combination of MSA and other hormones, such as insulin, calcitonin, and parathyroid hormone was even more effective in stimulating sulfation of glycosaminoglycans. These rabbit and rat chondrocytes cultured in completely defined medium seem to be a good experimental system for studies on chondrogenesis and endochondral ossification.     

X-chromosome activity in heterokaryons and hybrids between mouse fibroblasts and teratocarcinoma stem cells

To determine whether 2X-active cells contain factors capable of reactivating the inactive mammalian X chromosome, fibroblast lines, having a cytologically or genetically marked inactive X, were fused with 2X-active mouse embryos or ovarian teratocarcinoma stem cells. Fusions with 2–16 cell embryos were uninformative because no mitosis occurred in heterokaryons. Fusions with 2X-active teratocarcinoma cells, and screening for re-expression of alleles on the inactive X showed that reactivation did not occur with detectable frequency in heterokaryon. Hybridization of HPRT^?M. musculus × M. caroli cells with XO HPRT^? teratocarcinoma cells yielded hybrids with a frequency of >10^?6; these hybrids all expressed the Hpt allele on the inactive M. caroli X, but not the M. caroliGpd or Pgk. Late replication-banding studies of hybrids and 6-thioguanine-resistant revertants showed that the reactivated Hp⁺ allele was still located on the late replicating X. Similar results were obtained with hybridization of this line to 1X-active (male-derived) fibroblast lines, indicating that hybridization per se, rather than a specific factor contributed by the teratocarcinoma cell partner, was reponsible for the frequent localized derepression of the Hpt⁺ allele on the inactive X.     

Cytotoxic effect of a T-strain mycoplasma (Ureaplasma urealyticum) on cultured normal human cells (WI-38)

Normal human embryonic lung fibroblasts (WI-38) were infected with Ureaplasma urealyticum, a urea hydrolysing mycoplasma. It was possible to observe reduced rates of multiplication of infected cells and reduced plating efficiency as well as the morphological changes usually associated with mycoplasma infection of animal cells in vitro. The cytotoxic effect on multiplication was sensitive to aureomycin but not penicillin. It was not related to depletion of amino acids or nucleic acid precursors from the cell culture medium but appeared to require that the host cells be growing. Ureaplasmas could not be recovered from cell culture medium after 4 days post infection and their characteristic urease activity could not be demonstrated either in cell culture medium or associated with the cells after the first cell subcultivation. [³H]TdR was incorporated into the nuclei of infected cells and the percent labelled nuclei was reduced compared with uninfected cells. Nuclear labelling indices of infected cells increased as the cells were subcultivated by trypsinization suggesting that the ureaplasmas were removed from the host cell surface by this treatment. In general, the effects of ureaplasmas on WI-38 cells do not appear to be as pronounced as effects of other mycoplasmas on animal cells in culture. It is clear, nonetheless, that the urea hydrolysing mycoplasmas can infect cells in culture and cause discernible effects on the growth and metabolism of these cells.     

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes.

A procedure for continuous measurement of ¹⁴CO₂ production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [¹⁴C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO₂ and lactic acid production.     

Replacement of serum with a defined medium increases beta-adrenergic receptor number in cultured glioma cells

Lymphocyte chromosomes from a cercopithecoid species, Macaca mulatta, were studied for the occurrence of lateral asymmetry in constitutive heterochromatin. The technique consisted of growing the lymphocytes for one cell cycle in BrdUrd, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 SSC and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin of the nucleolar organizer marker chromosome (no. 13 of the complement). In these chromosomes, the darkly staining region was confined at any given point to a single chromatid, while the corresponding region on the sister chromatid was lightly stained. This pattern of asymmetric staining in the constitutive heterochromatic region was not observed in any other chromosome of Macaca mulatta. The lateral asymmetry of constitutive heterochromatin in this species is presumed to reflect the strand bias in the distribution of thymine in the alphoid DNA fractions.     

Guinea pig T-cell in vitro proliferative responses to tyrosine-azobenzenearsonate-pulsed and azobenzenearsonate-coupled macrophages have different specificities

The cell interactions involved in azobenzenearsonate-N-acetyl-tyrosine (ABA-tyr)-induced delayed hypersensitivity in the guinea pig were studied by in vitro blastogenesis. The ABA-sensitive lymphocyte was demonstrated to be a T lymphocyte and its presence in peritoneal exudate cells was shown to be much higher than spleen or lymph node populations. The secondary response of ABA-sensitized lymphocytes to ABA-tyr in culture is dependent on the presence of an accessory cell, with both splenic and peritoneal macrophages being equally effective. ABA coupled directly to macrophages as an immunogen induced strong responses to itself and not to ABA-tyr-pulsed macrophages or ABA-tyr in solution. The reverse was true in animals, immunized with ABA-tyr. ABA conjugated to thymocytes, L₂C leukemia cells, and guinea pig erythrocytes however, did not elicit significant responses. The results obtained in animals immunized with ABA- or ABA-tyr-modified cells was similar whether or not CFA was used. The difference in specificity shown between ABA-coupled and ABA-tyr-pulsed macrophages favors a single receptor hypothesis for T-cell recognition.     

Selectivity in metabolic cooperation between cultured mammalian cells

Selective communication between cultured mammalian cells was detected as selectivity in metabolic cooperation. Whilst the majority of the cell types examined (human skin fibroblast, PC13, G3, Don, PyY) showed metabolic cooperation at almost all (>95%) of their homotypic cell-to-cell contacts, they did not necessarily show cooperation at such a high proportion of their heterotypic contacts. Less than 10% of G3/human fibroblast contacts, and usually less than 30% of G3/PC13 contacts were observed to be positive for metabolic cooperation. L cells differed from these other cell types in that they formed permeable junctions at a greater proportion of their heterotypic cell-to-cell contacts (contacts between L and PyY cells) than their homotypic contacts. We question why it was that the contacts between any two poorly-compatible cell types were positive for metabolic cooperation in only a small proportion of cases. We could find no indication that this phenomenon was attributable to heterogeneity within the cell stocks. Time course studies upon PC13 and G3 cells showed that the proportion of cooperation-positive contacts was not constant but that it continued to increase over many hours of co-culture. In comparing the homotypic and heterotypic interactions of these cell types, selectivity manifested as a difference in the rate of appearance of permeable junctions. We discuss possible explanations for these findings.     

Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells

Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5-10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause "down regulation" of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a Mr 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system.     

Cell cycle-dependent expression of surface antigens in murine T-lymphoma cells : II. Murine leukemia virus gp70 increases in S phase

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.