Regular Growth Medium Research Articles

Inhibition of DNA methyltransferase inhibits DNA replication.

Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.

Altered Regulation of Fibronectin Gene Expression in Werner Syndrome Fibroblasts

Fibronectin (FN) production was quantified in the extracellular medium and in extracts of human diploid fibroblasts (HDF) by enzyme-linked immunosorbent assay and by immunoprecipitation of [35S]methionine biosynthetically labeled proteins. FN output was increased in normal late passage (old) HDF and in prematurely senescent Werner syndrome (WS) fibroblasts, compared to normal early passage (young) HDF. Output was maximal when cells were preconfluent, exceeding the level found at confluence and postconfluence. For all cell types the highest proportion of newly synthesized FN was found in the extracellular compartment. Both young and old HDF exposed to regular growth medium containing 15% fetal bovine serum (FBS) secreted twice as much FN as when exposed to serum-free medium, and cognate mRNA levels were commensurate with protein output. WS fibroblasts secreted 1.6-fold more FN than old HDF when both cell types were exposed to medium containing 15% FBS. Immunofluorescent analysis revealed greater association of FN with WS and old HDF than with young HDF. Furthermore, WS cells exposed to 15% FBS contained 3.4-fold more FN mRNA and produced seven times more FN protein compared to WS cells exposed to serum-free medium. Thus, WS fibroblasts secrete more FN than old normal and young normal HDF in the presence of 15% FBS due to augmentation of FN mRNA levels and enhanced efficiency of FN mRNA translation and/or post-translational processing.

Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells

When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.

A simple method for in situ freezing of anchorage-dependent cells including rat liver parenchymal cells.

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at -80 degrees C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.

HPV-18 immortalization of human keratinocytes

The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin. Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection. Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture. Four cell lines termed FE-A, FEH 18L, FEP185, and FEP18-11 have been maintained in culture for 1 1 2 -2 years and were selected for further analysis. In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames. Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins. However, none of the cell lines tested were tumorigenic. The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 m M calcium chloride. All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally. K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.

Contact Inhibitory Factor Also Restores Anchorage and Serum Dependence to Hamster Melanoma Cells

Conditioned medium (CM) from confluent cultures of the contact-inhibited hamster melanocytic cell line, FF, contains a biologic activity, contact inhibitory factor (CIF), which reversibly restores density-dependent growth to melanoma cells. When a hydrophobic affinity-concentrated extract of CIF-containing CM was incorporated in agarose at a concentration of 1000 micrograms protein/ml, it restored anchorage-dependent growth to RPMI 1846 hamster melanoma cells. Colony-forming efficiency in CIF-treated wells decreased to 5% from levels of 51.5% in controls prepared with regular growth medium. In addition, CIF-containing CM restored serum-dependent growth to RPMI 1846 cells, markedly restricting proliferation in 1% calf serum-containing medium. Control cultures containing 1% calf serum and either complete growth medium or CM from the non-contact-inhibited hamster melanoma line itself, supported proliferation of RPMI 1846 cells to levels 3.9 X and 3.7 X that of CIF-treated cultures, respectively. CIF is the first factor derived from contact-inhibited mammalian cell cultures that has been shown to restore density-, anchorage-, and serum-dependent growth to malignant melanoma cells.

DNA-mediated restoration of phenylalanine hydroxylase gene expression in enzyme-deficient derivatives of enzyme-constitutive mouse cell hybrids.

Phenylalanine hydroxylase (PH) gene expression is not extinguished in hybrids between PH- mouse A9 cells, or its neomycin-resistant derivative A9Neo-3, and PH+ mouse erythroleukemia (MEL) cells, PHC-3A, in contrast to its extinction in hybrids between A9Neo-3 and PH+ rat hepatoma cells, FT-2. Two different types of 6-thioguanine (TG) -resistant derivatives of these A9 X PHC-3A hybrids (LP), are generated in regard to PH gene expression. In regular growth medium supplemented with 10(-4) M TG (Tyr+/TG), TGr derivatives, all of which continue to express PH, occur with high frequency (approximately equal to 10(-3). In contrast, in tyrosine-deficient selective medium, supplemented with 10(-4) M TG (Tyr-/TG), no actively growing colonies are observed. Nevertheless, small colonies containing quiescent cells can be rescued by supplementing the medium with tyrosine. The rescued TGr clones do not express any detectable level of PH. Biochemical, hybridization, and cybridization analyses of one such rescued clone, LPTG-3, showed that these cells lack the regulatory factor capable of activating PH gene in PH- MEL cells. The PH- phenotype of LPTG-3 cells can be converted to the PH+ phenotype by transfection with restriction enzyme-digested or -undigested PHC-3A or mouse liver DNA. Therefore, these cells could be used to clone a fragment of DNA involved in PH gene regulation through DNA-mediated gene transfer methods.

Bacteriophage T4D receptors and the Escherichia coli cell wall structure: role of spherical particles and protein b of the cell wall in bacteriophage infection

The nature of the interaction of bacteriophage T4D and the outer cell wall of its host, Escherichia coli B, has been investigated. Bacteria with altered or modified cell walls have been obtained by two different growth procedures: (i) growth in high osmolarity medium or (ii) growth in broth in the presence of divalent heavy metal ions. When these altered host cells were washed and subsequently added to regular growth medium, they interacted with added phage particles, but successful infection did not occur. Most of the phage particles released from these treated cells were observed to have full heads and an altered tail structure. The altered phage tails had contracted sheaths and unusual pieces of the bacterial cell wall attached to the distal portion of the exposed phage tail tube. Phage released from bacteria grown in the high osmolarity medium had attached cell wall pieces of two major types, these pieces being either 40 or 21 nm in diameter. The smaller-type cell wall pieces (21 nm) were formed by three spheres each measuring 7 nm in diameter. Phage particles released from cells previously exposed to the divalent metal ions had only one 7-nm cell wall sphere attached to the distal end of the tail tube. It was found that these 7-nm spheres (i) are normal components of the cell wall and are morphologically similar to endotoxin, (ii) are held in place on the cell wall by a component of the cell wall called protein b, and (iii) are most likely the site of penetration of the phage tail tube through which the phage DNA enters the host cell.

Inhibition of conjugation and cell division in Tetrahymena pyriformis by tunicamycin: A possible requirement of glycoprotein synthesis for induction of conjugation

Tunicamycin, a glucosamine-containing antibiotic inhibited the conjugation process of Tetrahymena pyriformis. Sexual pairing was prevented completely when 1.5 μg/ml of tunicamycin was added to a mixture of the two mating types. Tunicamycin caused preferential inhibition of glycoprotein synthesis in Tetrahymena pyriformis. At 1.5 μg/ml and 6 μg/ml tunicamycin inhibited by 40% and 60% respectively [ 3H]-glucosamine incorporation into material precipitated by ethanol, while it did not affect [ 14C]-leucine incorporation. Cell division was also inhibited when the drug was added either to the regular growth medium or to the starvation medium.

Pachytene mapping of the male Chinese hamster.

Minced seminiferous tubules of male Chinese hamsters when treated with a mixture of trypsin (one part) and McCoy's 5a growth medium with 20% fetal calf serum (nine parts) at 4 degrees C, washed twice with the regular growth medium, incubated at 37 degrees C in growth medium for 4 h, and harvested without Colcemid and hypotonic pretreatments, gave excellent pachytene morphology for studies on chromomere patterns. The Giemsa banding patterns of all somatic metaphase chromosomes except the sex chromosomes of the hamster cells correspond well to the chromomere patterns of meiotic pachytene bivalents.

Reversible Effects of Lysolecithin on Fusion of Cultured Rat Muscle Cells

Lysolecithin prevented the natural cell fusion in monolayer cultures of rat leg muscle cells when it was added in the range of 120 to 160 μg/ml of complete growth medium. The cells rapidly recovered the ability to fuse after withdrawal of the lysolecithin medium and replacement by the regular growth medium. Lysolecithin prevented myogenic cell fusion within a narrow concentration range in the presence of sufficient concentration of calcium and pH values between 7.4 and 7.8. Comparisons between effects of lysolecithin, high cyclic-AMP and low calcium-containing media on cultures of muscle cells were also made. The elaboration of long, thin processes from the bipolar ends of myogenic cells was a feature particularly noticeable with lysolecithin and cyclic-AMP-containing culture plates. The possible influences of other, non-specified, natural surfactants, reacting with lipoprotein micelles of the plasmalemma and subsequently affecting the orientation and arrangement of cell boundaries during differentiation of various cell types, are discussed.

Early events of cell differentiation in Naegleria gruberi. Synergistic control by electrolytes and a factor from yeast extract

For amoebae of Naegleria gruberi NEG-M growing in an axenic medium, one component of the medium, yeast extract, controls whether phenotypic transformation into flagellates occurs. Fractionation of the yeast extract yielded two components that control transformation: electrolytes and an unidentified Factor. Electrolytes prevent transformation if present above a certain concentration. For NaCl and KCl, for example, the critical concentration is 0.08 M. The effect is not specific to particular ions, nor is it due simply to ionic strength. Osmotic pressure produced by nonpenetrating nonelectrolytes like sucrose can also inhibit transformation, but the effect of electrolytes is not due solely to osmotic pressure. The unidentified Factor, a small molecule, probably aromatic (adsorbs reversibly to charcoal), can prevent transformation at a concentration that does not increase the osmotic pressure of the solution, and that is estimated to be not more than 0.001 M. The action of Factor in controlling transformation is much potentiated by electrolytes, such that a combination of one-eighth the electrolyte concentration needed to prevent transformation with one-fourth the Factor concentration completely prevents transformation. The synergistic action with Factor is not specific to a particular ion, but there are differences. For example, Na + is much more effective than Cs +. Factor and osmotic pressure do not act synergistically. Amoebae grown in the presence of extra Factor adapt to it, and will transform in concentrations of Factor and of electrolyte that prevent transformation of unadapted cells. These adapted cells also can transform in regular growth medium. It is concluded that Factor and electrolytes are involved in the normal control of transformation of these axenic Naegleria. The results provide a starting point for studying early events in this cell differentiation.