Senescent human diploid cells in culture: survival, DNA synthesis and morphology.

Senescent human diploid cell cultures (WI-38 and WI-26) were studied during Phase III for survival time, ability to synthesize DNA, and nuclear morphology. Periodic transfers made during a 6-month period of Phase III showed that cultures were maintained with only slight variations in cell number. No sign of spontaneous acquisition of infinite proliferative potential was observed. The increase in the number of multinucleated cells found during the period of observation showed that progressive changes occur in Phase III. A certain proportion of cells maintained the ability to incorporate tritiated thymidine throughout the period of observation.     

Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts.

We studied the expression of 11 cell cycle-dependent genes in senescent WI-38 fibroblasts and compared the results to those obtained in WI-38 cells from early passages (young cells). Every gene we examined is expressed in the senescent cells at levels similar to those in the young cells, including two genes maximally expressed at the G1/S phase boundary--genes for thymidine kinase and histone H3. The results clearly show that senescent, noncycling WI-38 cells are not similar to quiescent cells. Rather, such senescent WI-38 cells may be blocked just prior to the onset of DNA synthesis.     

The type II insulin-like growth factor receptor does not mediate deoxyribonucleic acid synthesis in human fibroblasts

Two insulin-like growth factor (IGF) receptors, the type I and type II IGF receptors, have been described. While substantial evidence indicates that the type I receptor is involved in the regulation of cell division, it is uncertain if the type II receptor also mediates this response. Similarly, the role of the insulin receptor in mediating DNA synthesis remains controversial. To address these questions, we used a monoclonal antibody (alpha IR-3) to specifically inhibit type I IGF receptor activity and examined the effects of this inhibition on IGF- and insulin-stimulated DNA synthesis in human fibroblasts. WI-38 human embryonic lung fibroblasts have both type I and type II IGF receptors, as determined by cross-linking [125I] IGF-I and [125I]IGF-II to monolayers of these cells. In serum-free medium both IGF-I and IGF-II stimulate DNA synthesis in WI-38 fibroblasts, with half-maximal effects occurring at 1.5 +/- 0.3 (+/- SD) and 3.4 +/- 1.4 nM, respectively. At maximally effective concentrations, however, both hormones stimulate DNA synthesis to equal levels. alpha IR-3 binds to the type I, but not the type II, IGF receptor on WI-38 cells. It also inhibits IGF binding to the type I receptor on these cells. alpha IR-3 competitively inhibited both IGF-I- and IGF-II-stimulated DNA synthesis in WI-38 cells, but had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated DNA synthesis. These results indicate that in WI-38 fibroblasts the mitogenic effects of both IGF-I and IGF-II are mediated through the type I receptor and that the type II IGF receptor is not directly involved in this response. To define the role of the insulin receptor in mediating DNA synthesis we compared the effects of alpha IR-3 on insulin-stimulated DNA synthesis in a variety of human cell lines under identical experimental conditions. With WI-38 and HEL, another human embryonic lung fibroblast cell line, alpha IR-3 competitively inhibited the mitogenic effect of insulin. However, in two other fibroblast cell lines (GM498 and HES) and an osteogenic sarcoma cell line (MG63), alpha IR-3 inhibited IGF, but not insulin-stimulated DNA synthesis. These results indicate that human cell lines differ in the receptor type through which insulin stimulates DNA synthesis and that these differences are intrinsic properties of the cell lines and are not artifacts resulting from differences in experimental conditions.     

Thymidine labelling index as a criterion of aging in vitro.

For logarithmically growing cultures of the human diploid cell lines WI-38 and WI-26, there is an exponential increase in the fraction of cells not incorporating [3H]dT under conditions of continuous labelling. Thymidine uptake, phosphorylation and incorporation into DNA can be correlated with cell proliferation. In addition, determination of the labelling index is reproducible within relatively broad limits of thymidine concentrations and specific activity. The plateauing phenomenon of the curve expressing percent-labelled nuclei versus time, which occurs in populations with less than 100% cycling cells, is largely due to radiation damage to the cells. The results of these studies provide insight into the population dynamics of aging fibroblast-like cell cultures. More importantly, however, the measurement of labelling index as described can be used as a reproducible and quantitative measure of the age of the diploid cell culture.     

Increased RNA Synthesis in Nuclear Monolayers of WI-38 Cells Stimulated to Proliferate

Nuclear monolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ. In nuclear monolayers, incorporation of [(3)H]UTP into RNA is dependent on the presence of the other three nucleotide triphosphate and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium.RNA synthesis increases 15 min after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase.     

Genetics of the connective tissue proteins: Assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [(3)H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17.     

Release of somatomedin-like activity by cultured WI-38 human fibroblasts

Confluent cultures of normal diploid WI-38 human embryonic lung fibroblasts released somatomedin (SM)-like activity into their incubation medium during culture in serum-free medium. This postculture medium (conditioned medium) stimulated cell division in these same cultured WI-38 fibroblasts and 35SO4 uptake by hypophysectomized rat cartilage in vitro. The conditioned medium also contained immunoreactive SM (IRSM) activity which yielded parallel dose-response curves to human serum in a RIA for SM. The IRSM activity measured in conditioned medium was not the artifactual result of effects of possible SM-binding proteins or proteolytic enzymes in conditioned medium. These studies suggest that cultured WI-38 fibroblasts produce and release SM-like activity which has SM-like biological activity and is immunoreactive with a basic SM purified from human plasma Cohn fraction and having similarity with SM-C and insulin-like growth factor-I. Human GH appears to stimulate production and release of IRSM activity by these cells.     

Effects of human Na(+)/dicarboxylate cotransporter 3 on the replicative senescence of human embryonic lung diploid fibroblasts

To investigate the role of human Na(+)/dicarboxylate cotransporter 3 (hNaDC3) in the replicative senescence of normal human embryonic lung diploid fibroblasts (WI-38), a retroviral vector containing hNaDC3 was constructed. hNaDC3 was introduced into normal WI-38 cells through infection with the retroviral virus. Monoclones were selected with G418. The integration and expression of exotic genes were confirmed by Northern blot and Western blot. When compared with the control cells, WI-38 cells transfected with hNaDC3 cDNA showed significant suppression of growth rate (by 40%), increase of positive rate of SA-beta-gal staining, decrease of mitochondrial membrane potential, shortening of telomere length, and increase of P16 and P21 expression. The morphology characteristics of senescent fibroblasts appeared earlier. Our results have, for the first time, demonstrated that high expression of hNaDC3 may be able to, at least partly, promote the cellular senescence of human diploid fibroblasts.     

Inhibition of phytohemagglutinin-induced DNA synthesis and of adenosine deamination by 5-azacytidine and 5-aza-2'-deoxycytidine in intact human cells

5-Azacytosine nucleosides block phytohemagglutinin-induced thymidine incorporation into DNA in intact human blood lymphocytes in a dose-dependent manner at 1-100 micrograms/ml. Maximal inhibition was observed when mitogen and 5-azacytidine or 5-aza-2'-deoxycytidine were added to the culture in admixture. Both analogues also inhibit deamination of exogenous adenosine in stimulated cells without affecting the synthesis of nucleotides from adenosine, adenine or hypoxanthine. Similar inhibitory effect of 5-aza-2'-deoxycytidine on the metabolism of adenosine was observed in normal (WI-38) and SV40 virus-transformed (VA-13) human fibroblasts.     

Synthesis of nuclear proteins during DNA repair synthesis in human diploid fibroblasts damaged with ultraviolet radiation of N-acetoxy-2-acetylaminofluroene.

We have examined the accumulation of newly synthesized nuclear proteins into nuclei during DNA repair synthesis in confluent WI-38 human diploid fibroblasts damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluroene. In contrast to a marked stimulation of DNA repair synthesis, stimulation of amino acid incorporation into histone polypeptides or into the various molecular weight classes of nonhistone nuclear proteins was not observed. These results suggest that detectable stimulation of newly synthesized nuclear protein incorporation into nuclei does not accompany DNA repair synthesis induced by ultraviolet radiation or a direct acting chemical carcinogen. At least for the special case of repair, DNA synthesis may be uncoupled from histone synthesis.     

Gene Activation in WI-38 Fibroblasts Stimulated to Proliferate: Requirement for Protein Synthesis

Confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by fresh medium containing 30% fetal-calf serum. Up to 80% of the cells are stimulated to divide, with a peak of DNA synthesis between 15 and 21 hr. 1 hr after the change of medium there is a 70% rise in chromatin template activity. Cycloheximide inhibited the increase in chromatin template activity. A requirement for RNA synthesis was investigated by incubating stimulated and unstimulated cells with 10 μg/ml of actinomycin D. In spite of a 95% inhibition of RNA synthesis in whole cells, purified chromatin from stimulated cells showed the usual increase in template activity. These experiments implicate a requirement for protein synthesis in template activation, and imply that the synthesis of this (or these) protein(s) is independent of RNA synthesis and regulated by a purely translational mechanism.     

Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization.

We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs.     

Ginsenoside Rg1 delays tert-butyl hydroperoxide-induced premature senescence in human WI-38 diploid fibroblast cells

Tert-butyl hydroperoxide (t-BHP), an analog of hydroperoxide, induced characteristic changes of senescence in human diploid fibroblasts WI-38 cells. It was reported that ginsenoside Rg1, an active ingredient of ginseng, ameliorated learning deficits in aged rats. The present study was aimed to investigate whether ginsenoside Rg1 can delay the premature senescence of WI-38 cells induced by t-BHP and to explore the underlying molecular mechanisms. First, Rg1 pretreatment markedly reversed senescent morphological changes in WI-38 cells induced by t-BHP. Second, t-BHP treatment alone resulted in an increase in the protein levels of P16 and P21, and a decline in intracellular adenosine 5'-triphosphate (ATP) level and mitochondrial complex IV activity. Ginsenoside Rg1 pretreatment had significant effects of attenuating these changes. These data indicate that ginsenoside Rg1 has an anti-aging effect on t-BHP-induced premature senescence in WI-38 cells. This effect may be mediated by regulating cell cycle proteins and enhancing mitochondrial functioning.     

Cell cycle regulation of human histone H1 mRNA.

A cloned genomic DNA fragment containing a human histone H1 gene has been used to analyze histone H1 gene expression in two human cell lines (HeLa S3 and WI-38). The cellular abundance of histone H1 mRNA was compared with that of core (H2A, H2B, H3, and H4) histone mRNAs as a function of the cell cycle: core and H1 histone mRNA levels are related both to each other and to the apparent rate of DNA synthesis and are rapidly destabilized after DNA synthesis inhibition. The use of three synchronization protocols, and of transformed and normal diploid cells in culture, suggests that the detected core and H1 histone mRNA levels are regulated by similar mechanisms in continuously dividing human cell lines and nondividing cells stimulated to proliferate.     

Biosynthesis of the third and fifth complement components by isolated human lung cells

Increasing research efforts have been directed at determining the contribution of locally synthesized (cell-derived) complement in host defense and inflammation. In the studies presented here, we determined the ability of a continuous cell line of type II pneumocytes (A549) and a cell line of human lung fibroblasts (WI-38) to produce complement components in vitro. Complement biosynthesis by A549 pneumocytes and WI-38 fibroblasts was demonstrated by incorporation of [35S]methionine into immunoprecipitable complement proteins. Using this technique, A549 pneumocytes were demonstrated to synthesize Clr, Cls, C4, C3, C5, C6, C7, C8, C9, Factor B, Factor H, Factor I, and C1s inactivator. In comparison, WI-38 fibroblasts were shown to synthesize Cls, C4, C3, C5, C6, C8, and C9. Because previous work has demonstrated the central role of C3, C5, and their activation products in regulating lung inflammation and tissue injury, we further investigated the production of C3 and C5 by both lung pneumocytes and fibroblasts using enzyme-linked immunospecific assays. A549 cells cultured in the presence of 15% fetal bovine serum (FBS) produced antigenic C3 (135 ng C3/ml/24 h) at a greater rate than did identical cells maintained in serum-free culture conditions (70 ng C3/ml/24 h). Similarly, antigenic C5 production by A549 pneumocytes was greatest in the presence of FBS when compared with cells maintained in serum-free culture conditions (245 ng C5/ml/24 h versus 155 ng C5/ml/24 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term treatment with cortisol: influence on proliferation, aging and glycosaminoglycan synthesis of cultured human diploid fibroblasts (WI-38)

Long-term treatment (several weeks and months) of cultured human diploid fibroblasts (WI-38) with cortisol (1.4 x 10(-7) M) stimulated proliferative activity and cellular glycosaminoglycan synthesis, thus counteracting the normal in vitro aging process. Characterization of the individual glycosaminoglycan types revealed an increased portion of cellular hyaluronic acid in cells treated with cortisol. Elevated synthesis of total glycosaminoglycans and, especially, of hyaluronic acid was found in the percellular pool (as determined by the amount liberated from the cells by trypsin treatment).     

Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts.

The trace element selenium is essential for clonal growth of diploid fibroblasts from human fetal lung (WI-38) in media containing small amounts of serum protein. Maximum growth stimulation is obtained when 30 nM neutralized selenious acid is added to a synthetic medium containing 1.5 mg/ml of dialyzed fetal bovine serum protein (equivalent to a 3% serum concentration). Serum appears to be a source of selenium in most culture media, since higher concentrations of serum protein or whole serum mask the selenium requirement of WI-38 cells. Selenium is also required by a Chinese hamster cell line that can be grown in a protein-free synthetic culture medium.     

Growth factors as probes of cell aging

We present examples of four types of alterations which contribute to the senescence phenotype of WI-38 cells: a) in senescent cells there is an increased lability of the tyrosine autophosphorylation capacity of detergent isolated EGF receptor; b) following serum stimulation, the calmodulin protein level fails to increase in senescent cells, although the calmodulin mRNA level increases as expected; c) following heat shock at 43°C, senescent cells produce both less RNA and less protein for the HSP70 and HSP90 genes; d) we find that membranes isolated in basic buffer from senescent or young cells increase the EGF proliferative response of senescing cells, in contrast to the finding by others that membranes isolated in neutral buffer inhibit cell proliferation (Pereira-Smith et al., Senescent and quiescent cell inhibitors of DNA synthesis Exp. Cell Res. 160, 297–306, 1985; Stein and Atkins, Membrane-associated inhibition of DNA synthesis in senescent human diploid fibroblasts: Characterization and comparison to quiescent cell inhibitor. Proc. Natl. Acad. Sci. USA 83 9030–9034, 1986). We conclude that senescence alterations are complex and occur at many levels, and that senescence changes are not identical to quiescence features.     

A New Synthetic RNA-Dependent DNA Polymerase from Human Tissue Culture Cells

Two DNA polymerases that can copy synthetic RNA polymers are present in human tissue culture cells. These enzymes which have each been purified about 500-fold, are present in both HeLa cells, which are derived from a cervical carcinoma, and in WI-38 cells, a normal diploid strain originating from human embryonic lung tissue. These synthetic RNA-dependent DNA polymerases are identified by their ability to copy efficiently the ribo strand of synthetic oligonucleotide-homopolymer complexes, and differ in this respect from the known DNA-dependent DNA polymerases found in HeLa cells. The template requirements of these new DNA polymerases resemble that of the RNA-dependent DNA polymerases of the RNA tumor-viruses.