A potent DNA synthesis inhibitor expressed by the immortal cell line SUSM-1

We have previously reported the production of DNA synthesis inhibitor proteins by both quiescent and senescent human diploid fibroblasts. Young, proliferating fibroblasts do not produce such inhibitors, but are capable of responding to either the quiescent or senescent cell DNA synthesis inhibitors. Recently, we have analyzed the immortal cell line SUSM-1 (derived from normal liver fibroblasts following exposure to carcinogen) for inhibitory activity. We have found that SUSM-1 cells produce a factor capable of inhibiting DNA synthesis in young fibroblasts. Crude extracts prepared from SUSM-1 cells inhibit DNA synthesis in a dose-dependent manner at concentrations 10-fold lower than those of either senescent or quiescent fibroblast cell extracts. SUSM-1 cells are incapable of responding to the inhibitor they produce, as are three other immortal human cell lines tested. One immortal cell line, HeLa, does respond to the SUSM-1 inhibitor, though to a lesser degree than observed with normal young fibroblasts. One hypothesis is that the DNA synthesis inhibitor protein(s) of senescent cells plays a role in determining the finite in vitro life span of normal cells. The results reported here suggest that SUSM-1 cells may have escaped senescence through loss of a receptor or cofactor for the inhibitor protein(s).     

Changes induced by expression of the v-src gene in the regulation of cell proliferation. Hypothesis and preliminary results

Similarities between the mode of action of growth factors and the oncogene product (pp 60 src protein) of Rous Sarcoma virus have been described. However, a major difference is that addition of growth factors does not induce a malignant transformation of cells. The present work proposes a hypothesis concerning this difference. Various data suggest that density-dependent inhibition (DDI) of growth in non-transformed cells is due to the diffusion of growth inhibitory molecules. Inhibitory factors of 45 K (IDF 45) and 12 K have been fractionated. We assume that the stimulation of DNA synthesis induced by growth factor addition to dense quiescent cultures of non-transformed cells leads to an increase in the activity of autocrine inhibitory molecules in such a manner that the growth factor stimulatory effect is only transient, and cells re-enter the Go phase. On the contrary, the stimulation of DNA synthesis by v-src transformation would not be counterbalanced by inhibitory diffusing factors and cells would not enter Go phase. We present preliminary results which support this assumption. Dense quiescent cultures of chick embryo fibroblasts infected by Ny 68 virus (ts mutant for transformation of Rous Sarcoma virus) were stimulated to proliferate either by addition of growth factors in cultures maintained at 41 degrees C or by expression of transformation (by the cell transfer from 41 to 37 degrees C, the permissive temperature for expression of transformation). Stimulation of DNA synthesis by growth factors was totally inhibited by the inhibitory diffusing factors of 45 K (IDF45) whereas the stimulation of DNA synthesis produced by transformation was reproducibly not decreased by IDF45.     

Overexpression of human NOX1 complex induces genome instability in mammalian cells

The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because of their potential to damage macromolecules, including DNA. To investigate possible links between high ROS levels, oxidative DNA damage, and genomic instability in mammalian cells, we established a novel model of chronic oxidative stress by coexpressing the NADPH oxidase human (h) NOX1 gene together with its cofactors NOXO1 and NOXA1. Transfectants of mismatch repair (MMR)-proficient HeLa cells or MMR-defective Msh2(-/-) mouse embryo fibroblasts overexpressing the hNOX1 complex displayed increased intracellular ROS levels. In one HeLa clone in which ROS were particularly elevated, reactive nitrogen species were also increased and nitrated proteins were identified with an anti-3-nitrotyrosine antibody. Overexpression of the hNOX1 complex increased the steady-state levels of DNA 8-oxo-7,8-dihydroguanine and caused a threefold increase in the HPRT mutation rate in HeLa cells. In contrast, additional oxidatively generated damage did not affect the constitutive mutator phenotype of the Msh2(-/-) fibroblasts. Because no significant changes in the expression of several DNA repair enzymes for oxidative DNA damage were identified, we suggest that chronic oxidative stress can saturate the cell's DNA repair capacity and cause significant genomic instability.     

Isolation of a population of transiently transfected quiescent and senescent cells by magnetic affinity cell sorting.

Rous sarcoma virus (RSV) and cytomegalovirus (CMV) promoters were tested for activity in proliferating and nonproliferating (quiescent or senescent) human embryo fibroblasts. These promoters were cloned upstream of the coding sequence for the Tac subunit of the interleukin 2 receptor, and activity was calculated from the fraction of Tac antigen positive cells detected in a coupled transient transfection/magnetic affinity cell sorting assay. Differences in promoter activities are substantial in quiescent cells: the efficiency of the RSV promoter is no greater than background whereas the CMV promoter is equally active in serum concentrations ranging from 0.5 to 20%. While both promoters are functional in growing cells (WI-38 and HeLa), the CMV promoter exhibits twofold greater activity. Surprisingly, in senescent cells both promoters exhibit the same degree of activity.     

Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination.

Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or aging. Studies of eccDNA structure and mode of formation provide insight into mechanisms of instability of the mammalian genome. Previous studies have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA sequences. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate the complementary chromosomal sequences. Comparing the recombination junctions within the eccDNAs with the chromosomal sequences from which they were derived revealed that nonhomologous recombination was involved in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest that rejoining of ends of fragmented DNA is responsible for the generation of a substantial portion of the eccDNAs found in HeLa S3 cells.     

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.     

Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states

DNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. alpha-Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE-cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, alpha- and beta-polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late-passage and postconfluent early passage fibroblasts, amounts of alpha-polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The alpha-polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCI and could be inhibited by a monoclonal IgG fraction prepared against KB cell alpha-polymerase. By several criteria, there was no significant differences in levels of UV-stimulated repair synthesis observed in early or late-passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of alpha-polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for alpha-polymerase from late-passage cells may be attributed more generally to the nondividing nature of these cells.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

Ubiquitin is a heat shock protein in chicken embryo fibroblasts.

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.     

The comet assay: a method to measure DNA damage in individual cells

We present a procedure for the comet assay, a gel electrophoresis-based method that can be used to measure DNA damage in individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell. DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring (e.g., DNA damage in earthworms crawling through toxic waste sites) to measurement of DNA damage in specific genomic sequences. This protocol can be completed in fewer than 24 h.     

Senescent and quiescent cell inhibitors of DNA synthesis : Membrane-associated proteins

Cytoplasts derived from senescent and quiescent human diploid cells inhibit DNA synthesis initiation when fused with cells capable of proliferation. When the cytoplasts were subjected to a variety of conditions (trypsin and cycloheximide treatment and growth on fibronectin), this inhibitory activity was lost, suggesting that the inhibitors involved were proteins associated with the surface membranes of the cells. We have studied the quiescent cell inhibitor in greater detail and determined that surface membrane-enriched preparations isolated from quiescent cells and proteins extracted from these membrane preparations have DNA synthesis-inhibitory activity.     

Quiescent human diploid fibroblasts. Common mechanism for inhibition of DNA replication in density-inhibited and serum-deprived cells

The mechanism for cessation of proliferation in density-inhibited quiescent human diploid fibroblasts (HDF) and serum-deprived quiescent HDF was compared in two ways. Density-inhibited HDF were fused to either replicating HDF or SV40-transformed HDF and DNA synthesis was measured in the resulting heterokaryons. DNA synthesis was inhibited in the replicating HDF nuclei in heterokaryons in a way that suggested that entry into S phase was blocked, but ongoing DNA synthesis was not inhibited. In contrast, DNA synthesis was induced in the quiescent nuclei in heterokaryons formed with SV40-transformed HDF. Previous experiments had shown that serum-deprived HDF also behave in this way in heterokaryons. To test this similarity further, we examined the inhibitory activity of cell membranes prepared from both types of quiescent HDF. We found that both types of quiescent HDF contain DNA synthesis-inhibitory activity that is (1) effective on replicating HDF; (2) ineffective on SV40-transformed HDF; (3) sensitive to heat and trypsin. Thus, these results support the hypothesis that both density-inhibited HDF and serum-deprived HDF share a common mechanism for arrest in G1 phase. They also suggest that a membrane-bound protein plays a role in the inhibition of DNA synthesis in quiescent HDF.     

Human diploid fibroblasts (HDF) can induce DNA synthesis in cycling HDF but not in quiescent HDF or senescent HDF

HeLa cells in S phase induce DNA synthesis in cycling cells, serum-deprived quiescent cells, and non-replicative senescent cells following cell fusion. In contrast normal human diploid fibroblasts (HDF) do not induce DNA synthesis in either quiescent cells or senescent cells. Instead, the replicative HDF nuclei are inhibited from entering S phase in heterokaryons formed with these two types of non-replicative cells. These differences in the inducing capabilities of normal HDF and HeLa cells raise the question whether normal HDF in S phase can induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Thus, the hypothesis that initiation of DNA synthesis in cycling cells is positively controlled by inducer molecules appears to be valid for normal HDF as well as for transformed cells such as HeLa.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk