Correlation of (Na---K)-ATPase activity with growth of normal and transformed cells

The (Na⁺---K⁺)-stimulated Mg²⁺-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na⁺---K⁺)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na⁺---K⁺)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.     

Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.     

Synthesis and first in vitro cytotoxicity studies of bis(2-chloroethyl) amino group containing polymers. Pharmacologically active polymers: 22

Monomers containing the cytotoxic bis(2-chloroethyl)amino group (nor-nitrogen mustard), linked in deactivated form via urethane and O-acylated hydroxamic acid bonds to polymerizable methacrylic acid derivatives, have been prepared. Besides the homopolymerization, these monomers were copolymerized with hydrophilic comonomers to obtain water-soluble polymers. The linkages used are expected to undergo intracellular enzymatic or hydrolytic cleavage, releasing the cytotoxic bis(2-chloroethyl)amino moiety from the polymeric carrier. Preliminary in vitro cytotoxicity data for the polymers against three rat and mouse experimental tumour lines (Walker 256 carcinosarcoma, L1210 murine leukaemia and ADJ/PC6A plasma cell tumour) are reported. An approximately 10² fold difference in cytotoxic potency was found, depending on the type of the cleavable spacer group.     

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.     

Characterization of antibody-mediated inhibition of natural killer (NK) cytotoxicity: Evidence for blocking of both recognition and lethal hit stages of cytolysis

Rat antisera prepared against murine, periodate-activated alloimmune cytotoxic lymphocytes (termed RAT¹) have previously been shown to effectively block T-cell-mediated cytotoxicity (CMC) at the “lethal hit” stage of cytolysis (J. C. Hiserodt and B. Bonavida, J. Immunol.126, 256, 1981). Both natural killer (NK) and cytotoxic T lymphocytes (CTL) have been shown to mediate lysis by the same pathway, namely binding of effector to target cells, programming for lysis, and killer cell-independent target cell lysis. This result suggested that the molecular mechanism of NKCMC and CTLCMC may also be similar. In this context, RAT¹-mediated blocking of CTL was examined for its ability to block NKCMC. The results show that (1) addition of RAT¹ serum or IgG fractions blocked NKCMC in the absence of complement in a 4-hr ⁵¹Cr-release assay, and blocking was directed at the effector cell; (2) at the single-cell level, RAT¹ serum blocked the formation of conjugates between effector and target cells; (3) in a Ca²⁺-pulse experiment, in which the effectors and targets were first allowed to bind in the absence of Ca²⁺ for 1 hr at 37 °C, followed by the addition of Ca²⁺ to initiate the lytic event, RAT¹ was capable of blocking cytotoxicity after conjugate formation at the Ca²⁺-dependent lethal hit stage of cytolysis. The similarity of results in RAT¹ blocking experiments of both the CTL and NK systems suggests a common molecular mechanism of cytolysis.     

The roles of the opioid peptides in controlling thyroid stimulating hormone release

We have studied the role of the opioid peptides in controlling TSH secretion. Morphine sulfate significantly decreased, while naloxone had no effect on, basal plasma TSH levels of female rats. In contrast, naloxone blocked the stress-induced fall in plasma TSH. Microinjection of β-endorphin into the third ventricle resulted in a fall in TSH while such injection of naloxone into the posterior hypothalamus increased TSH. Microinjection of β-endorphin directly into the pituitary caused a rise in plasma TSH. It is concluded that opioid peptides probably play no role in basal TSH secretion, but are involved in the stress-induced fall in TSH. Furthermore, it appears that opioid peptides have a site of action in the hypothalamus to decrease TSH and a direct pituitary action to increase TSH.     

DNA repair in UV-irradiated heteroploid cells at different phases of the cell cycle

The variation of DNA repair activity during the cell cycle was studied by analysing the UV-stimulated DNA synthesis in cells synchronized in mitosis. This activity was detected both by autoradiography and by directly measuring the incorporation of tritiated thymidine in cells irradiated and incubated in the presence of hydroxyurea. Cells in all phases were found to be able to perform repair. However the activity appeared to be considerably lower in mitotic cells than in cell in other phases. Increasing values of repair capacity were observed in G1 cells, in mixed G2, S and M cells and in asynchronous cells. The relationship between these findings and data on survival rates in the same synchronized cells is discussed.     

Regulation of DNA synthesis in cytochalasin B (CB)-induced binucleate HeLa cells

The effects of timing and duration of cytochalasin B (CB) treatment on the kinetics of the initiation of DNA synthesis in mono- and binucleate HeLa cells, synchronized in the G1 phase of the cell cycle by the reversal of a mitotic block (N₂O at 80 PSI), were studied. In the control, bi-, tri- and tetranucleate cells entered S phase slightly earlier than the mononucleate cells at a rate proportional to the number of their nuclei. The difference between any two adjacent sub-populations was less than 0.5 h. However, the binucleate cells produced by a 90 min CB treatment immediately after the reversal of the mitotic block exhibited a considerably shorter G1 period as compared to mononucleate cells (a difference of 1.5 h). This exaggerated difference in the duration of G1 period between mono- and binucleate cells disappeared when the CB treatment was delayed by 75 or 90 min indicating that it was an experimental artifact. From this study, we conclude that there is naturally some degree of nuclear cooperation in the multinucleate systems, particularly with regard to the initiation of DNA synthesis, which is not influenced by CB treatment.     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.     

Evidence for the identification of lymphocyte activating factor as the adherent cell-derived mediator responsible for enhanced antibody synthesis by nude mouse spleen cells

Human adherent peripheral blood leukocytes spontaneously elaborate both a thymocyte proliferative factor and a factor which augments the in vitro anti-sheep erythrocyte (SRC) plaque-forming cell (PFC) response of nu/nu mouse spleen cells. Nonadherent leukocytes do not spontaneously elaborate either factor. The adherent cell-derived factors appear to have an identical molecular weight (approximately 14,500 Daltons) as determined by Sephadex gel filtration. The data support the hypothesis that the molecule(s) mediating both enhancing activities is identical to the previously described adherent leukocyte product, LAF.     

Interactions of a 5-nitroxide-labeled poly(uridylic acid) with poly(adenylic acid)

The physicochemical properties of a high-molecular-weight spin-labeled nucleic acid, (RUGT,U)_n, synthesized by enzymatic copolymerization, were evaluated by uv and ESR spectroscopy. It was shown earlier that spin labeling of nucleic acids by chemical modification to an extent which gives a nitroxide-to-nucleotide ratio greater than 0.002 can cause noticeable lattice perturbations (A. M. Bobst, A. Hakam, P. W. Langemeier, and S. Kouidou (1979), Arch. Biochem. Biophys. 187, 339–345). The presence of RUGT, a 5-nitroxide-labeled uridine residue, in a (U)_n lattice at a $\text{RUGT}\text{U}$ ratio of 0.01 is shown here not to affect the complexation with (A)_n, since the uv melting temperature (T₀^OD) of the 2 → 1 transition and the hypochromicity changes were the same for (RUGT,U)_n· (A)_n and (U)_n·(A)_n. ESR measurements indicated that the nitroxide radical reflects the transition accurately within the error limit, although a slight destabilization of the spinlabeled segment could not be excluded. Computer simulations showed conclusively that the spin melting temperature (T_m^sp) corresponds to the temperature at which half of the spin-labeled segments are no longer complexed, for the ESR spectrum at T_m^spcan be simulated with equal contributions from the line shapes of ESR spectra taken before and after the transition. Arrhenius plots obtained by using two different approaches for computing correlation times were qualitatively the same. Computer analysis also revealed that the formation of a (RUGT,U)_n·(A)_n complex can be described by a two-state model, in contrast to results obtained with chemically spin-labeled (U)_n. Thus, using (RUGT,U)_n over chemically spin-labeled (U)_n can offer distinct advantages.     

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.     

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.     

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.     

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.     

A glycoprotein involved in aggregation of D. discoideum is distributed on the cell surface in a nonrandom fashion favoring cell junctions

We studied the distribution on the cell surface of a glycoprotein (gp150) involved in the aggregation process of Dictyostelium discoideum. Using immunohemocyanin labeling of intact aggregates and visualization by scanning electron microscopy (SEM), we found a distribution gradient of gp150 wherein the concentration was enriched at or near sites of cell contact. When the distribution of gp150 on the cell surface was examined with immunoferritin and transmission electron microscopy (TEM), we found that gp150 was closely associated with the plasma membrane.     

Regulation of 1,25(OH)2 vitamin D3 receptor content in cultured LLC-PK1 kidney cells limits hormonal responsiveness

Receptor content in cultured kidney (LLC-PK1) cells was found to be modulated following the introduction of a culture medium change, declining to 40% of control values at 18 h. Scatchard analysis indicated that the reduced 1,25(OH)2-[3H]D3 nuclear binding we detected was due to decreased abundance of receptors (3811 vs 1619 sites/cell) with no change in the Kd (0.4-0.5 nM). Cells with reduced receptors exhibited diminished ability to respond to 1,25(OH)2D3 as measured by induction of 25(OH)vitamin D-24-hydroxylase activity. There was a close coupling between decreased receptor levels and diminished hormone responsiveness. The data suggest the absence of "spare" receptors and that receptor abundance is a limiting factor in cell responsiveness to 1,25(OH)2D3.     

δ-Aminolevulinic acid synthetase: Regulation of activity in various tissues of the aging rat

The basal and ethanol-induced activities of the rate-limiting enzyme of heme biosynthesis, δ-aminolevulinic acid (ALA) synthetase were measured in the liver, heart, kidney, and brain of young, adult, and aged Sprague-Dawley rats. When assayed in whole mitochondria derived from either fed or 24-h fasted animals, the basal levels of hepatic ALA synthetase activity decreased dramatically as a function of age. An equivalent decrease was seen in the ethanol-induced activity although the ratio of induced to basal activities did not change with age. In the heart, ALA synthetase activity also decreased significantly during aging. The activity was not induced by ethanol and was decreased markedly by fasting. By contrast, kidney ALA synthetase activity showed no age-related changes. The activity was unaffected by fasting and showed a variable induction response to ethanol. Brain ALA synthetase activity displayed a significant age-dependent decrease in its activity which was neither affected by fasting nor sensitive to induction by ethanol. The data presented are consistent with the hypothesis that ALA synthetase activity is subject to metabolic regulation. Further, they indicate that while the enzyme activity is regulated in a tissuespecific manner, a time-dependent decrease is a general feature of the aging animal.     

The relationship between cell growth, macromolecular synthesis and poly ADP-ribose polymerase in lymphoid cells

Homogeneous populations of different chloroplast classes (organelles with morphologically intact, swollen and broken external envelopes) have been separated by combinations of differential and silica sol density gradient centrifugation. Organelles with intact envelopes purified in silica (Ludox) gradients are rather stable against osmotic rupture. The ultrastructural analysis of isolated organelles revealed colloid precipitates predominantly at the organelle membranes. Evidence is presented that Mg²⁺ and strong cationic membrane groups participate in the colloid/membrane interaction. Physiological activities of silica gradient-purified unbroken chloroplasts were compared with those of organelles isolated in silica sol-free media (chloroplastic NAD-dependent malic dehydrogenase, Mg²⁺-independent; NAD-dependent phosphoglycerate kinase/triosephosphate dehydrogenase, Mg²⁺-dependent; light-dependent phosphoglycerate and CO₂ reduction; incorporation of deoxyribonucleotide triphosphates in an acid-insoluble, DNase-sensitive form). Altered or no activities were recovered with silica-treated plastids. Care should therefore be taken if silica gradient-purified organelles are used for investigations of physiological processes. However, due to the high resolving power of the gradient and the isotonicity of the isopycnic steps the method is recommended and has successfully been applied for the isolation of pure high mol wt substances from cell organelles, e.g. for higher plant chloroplast and nuclear DNA.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.