Induction of heat shock proteins in young and senescent human diploid fibroblasts

When human diploid fibroblasts, TIG-1, reached the end of their proliferative lifespan in vitro, they still could synthesize proteins with molecular weight of about 70,000, 85,000 and 116,000 following the heat shock at 46 degrees C for 10 min as well as young cells did so.     

Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus.

Four DNA-negative temperature-sensitive (ts) mutants of human cytomegalovirus, belonging to different complementation groups, were studied for their ability to induce cell DNA synthesis and DNA polymerase in permissive human embryo lung (HEL) and nonpermissive rabbit lung (RL) cells. These is mutants stimulated host cell DNA synthesis in HEL and RL cells and DNA polymerase activity in HEL and RL cells at permissive (33.5°) and nonpermissive temperatures (39.5°). Salt stimulation of induced DNA polymerase activity was used to distinguish between virus and cell DNA polymerase from HEL cells. DNA polymerase activity was stimulated by 100 mM (NH₄)₂SO₄ at either 33.5 or 39.5° in cultures infected with three of the mutants (ts 9, is 153, and ts 155). However, DNA polymerase activity was not stimulated by 100 mM (NH₄)₂SO₄, in cultures infected with one of the ts mutants (ts 13). These data suggest that at least four cistrons control the synthesis of virus DNA and that virus DNA synthesis is not required for the induction of cell DNA synthesis.     

Lack of recovery from oxygen-induced damage to colony formation and DNA synthesis in senescent human diploid fibroblasts

The colony formation and DNA synthesis of cultured human fibroblasts were inhibited under hyperbaric oxygen. When maintained in a growth-arrested state for a few days after oxygen exposure, early passage diploid and SV-40-transformed cells repaired the oxygen-induced damage, while late passage diploid cells did not. In early passage cells to which cycloheximide, a protein synthesis inhibitor, was administered, no recovery from the damage to colony formation was found, indicating that de novo protein synthesis is necessary to the repair process. The repair function of the late passage cells against oxidative damage seems to be impaired. Oxidative stress may be involved in cellular ageing.     

Induction of DNA strand breaks by ethylene oxide in human diploid fibroblasts

In vitro exposure of normal human diploid fibro-blasts (strain VH-10) to ethylene oxide (EtO) induced DNA strand breaks in the dose range of 2.5–30 mMh of EtO. Alkaline DNA unwinding (ADU), neutral filter elution (NFE), pulsed field gel electrophoresis (PFGE), and the comet assay were used to measure DNA single (SSBs) and double strand breaks (DSBs). Different induction rates of SSBs and DSBs, depending on applied method and also on treatment conditions (cells in monolayer or in suspension were used), were found. A dose-dependent increase of DNA strand breaks was found by the ADU method in the dose range of 2.5–20 mMh of EtO when treatment was performed in monolayer and in suspension. DSBs were detected by NFE only when the cells were treated with EtO in suspension (doses 10–30 mMh). The highest induction rate of DSBs (about 4 DSBs per 100 Mbp per 1 mMh of EtO) was detected in suspension with PFGE applied. We have shown that heat-labile sites are formed by EtO. Presumably, the different DSB levels detected by PFGE and NFE result from the conversion of these sites to DSBs during cell lysis at elevated temperature in the PFGE method. The results of the comet assay confirmed that apoptotic processes are not involved in the formation of DSBs in our experimental conditions (less than 1% of apoptotic cells were observed at all doses studied). Possible mechanisms for the induction of DNA strand breaks by EtO-treatment are discussed. The capacity to repair DSBs in EtO-exposed (5–7.5 mMh) cells was studied, and it was found that a considerable part of the damage (about 50%) could be repaired during 18 hr of incubation. © 1994 Wiley-Liss, Inc.     

Increased unscheduled DNA synthesis in aged human diploid fibroblasts

Using Hayflick's model, ultraviolet (UV) induced unscheduled DNA synthesis was compared in cells at a low, middle and high population doubling level (PDL). Concomitant DNA replication was prevented by arresting all cultures in the G₁ phase by lowering the serum concentration. After UV-irradiation cells at a high PDL incorporated 1.5–2 times more [³H] thymidine into DNA than cells at a low and middle PDL. These findings seem to indicate that the repair capacity of cells at a high PDL is more than those of cells at low and middle PDL. Alternatively, the higher incorporation might be explained by a difference in the pool sizes of precursors of cells at different PDL. These possibilities were examined by adding fluorodeoxyuridine to the system to reduce de novo synthesis of deoxythymidine triphosphate (dTTP), and measuring the pool size of dTTP and the specific activity of [³H] dTTP in cells at different PDL. The results indicated that the increased incorporation in fact reflects increased unscheduled DNA synthesis in cells at a high PDL.     

Histone acetylation and deacetylation in senescent human diploid fibroblasts

We measured the kinetics of histone H4 hyperacetylation and kinetics of deacetylation for all core histones in young and senescent human diploid fibroblast-like cells. Both cell populations contained a fraction of histone H4 characterized by very rapid acetylation and deacetylation. The kinetics of these reactions were similar in young and senescent cells. The distribution of acetylated species of core histones was also similar in young and senescent human diploid fibroblast-like cells. These results indicated that previously-reported alterations in chromatin template activity in senescent cells were due to a mechanism other than histone acetylation.     

DNA synthesis in chromatin preparations from human fibroblasts infected by cytomegalovirus

Summary Chromatin prepared from (¹⁴C)-thymidine pulse labelled cytomegalovirus-infected human fibroblasts 72 hours postinfection exhibited under appropriate conditions endogenous activity of (³H)-thymidine triphosphate incorporation which was relatively salt-resistant and phosphonoacetic acid-sensitive. Isopycnic centrifugation of the doubly labelled DNA in CsCl revealed that cell-free incorporation occurred into viral as well as into host cell DNA. Density labelling experiments with bromodeoxyuridine triphosphate suggested the incorporation into viral DNA to be due to replicative DNA synthesis. Chromatin from infected cells contained, in addition to cellular, viral DNA polymerase activity.With 5 Figures     

Abortive infection of human diploid cells by murine cytomegalovirus

Inoculation of human diploid cells (WI-38) with murine cytomegalovirus (MCMV) did not result in the synthesis of any infectious virions. However, morphological changes typical of the cytopathic effects (CPE) of MCMV were detectable within 12 hr of infection. The CPE included rounding, swelling, and detachment of cells. The nuclei of infected cells were enlarged, and intranuclear inclusions were visible by May Grunwald-Giemsa staining and by the indirect fluorescent-antibody test. All cells infected at a multiplicity of infection of 3 showed CPE, and these cells could not be passaged successfully. Cell lysates and exhausted media from infected WI-38 cultures did not produce any CPE in WI-38 cells. The virus absorbed to WI-38 cells with the same efficiency as to mouse embryo fibroblast cells (MEF). Samples of MCMV in which virus infectivity for MEF cells had been inactivated by ultraviolet irradiation or by exposure to 56 C failed to produce any of the above signs. MCMV-specific CPE did not occur in the presence of actinomycin D (1 mug/ml) or puromycin (20 mug/ml), but 5'-fluoro-2'-deoxyuridine at 1 x 10(-4)m did not prevent CPE or the development of intranuclear inclusions.     

Glycosphingolipid synthesis in human fibroblasts infected by cytomegalovirus

Infection of serum-deprived human foreskin fibroblasts (HFF-cells) by cytomegalovirus (HCMV) resulted in enhanced precursor incorporation into glycosphingolipids (GSL). Analysis of the component patterns revealed a unique biphasic effect on the rate labeling of neutral GSL. Early during infection or in the presence of phosphonoacetic acid, radiolabel was found predominantly in ceramide tri- and tetrahexoside, whereas late after infection label in ceramide monohexoside exceeded that in the other components. Determination of the chemical amounts of neutral GSL components from infected cultures supported the view of increased biosynthesis of ceramide tri- and tetrahexoside early, and of ceramide monohexoside late postinfection. Changes, comparable to those observed under the influence of "early" viral functions were observed also during S-phase of serum-stimulated HFF cells. With respect to acidic GSL a decrease of metabolic labeling occurred late during infection. Relatively little alteration was found in the component pattern.     

Unimpaired histone synthesis in human fibroblasts infected by human cytomegalovirus

Serum-starved human foreskin fibroblasts were infected by human cytomegalovirus (Towne strain) that is thought to induce DNA replication in host cells during lytic infection. At various times postinfection, the cultures were pulse labeled with either³H-thymidine or¹⁴C-thymidine and³H-lysine to examine DNA synthesis and histone synthesis, respectively. Isopycnic centrifugation of labeled DNA in CsCl revealed that precursor incorporation into host-cell DNA was enhanced over the control around 24 h postinfection and decreased after onset of viral DNA synthesis which reached a peak around 72 h postinfection. For analysis of histones³H-lysine-labeled proteins of lysates of unfractionated cells and of chromatin preparations were subjected to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and subsequent fluorography. Comparison of the fluorograms from the various pulses postinfection suggested that³H-lysine incorporation into histones exhibited no major variations concurrent with the changes of host-cell DNA synthesis. In contrast, herpes simplex virus type 1 was found progressively to extinguish histone synthesis in the course of the cellular infection. Furthermore, histone synthesis in phosphonoacetic acid-treated cytomegalovirus-infected cultures was not enhanced over that in mock-infected controls. These observations do not support the view that human cytomegalovirus induces host-cell DNA replication under the conditions used.Dedicated to Professor Dr. R. Siegert on the occasion of his 65th birthday     

Relationship between cell replication and volume in senescent human diploid fibroblasts.

Since alterations in cell replication rate and cell volume distribution are two of the earliest changes seen in the culture of human diploid cells, it was decided to examine the relationship between these parameters. After standardization of the conditions for cell volume measurements (enzyme treatment, temperature and stage of cell growth), a close correlation was observed between cell population doubling time and cell volume in WI-38 cells at various levels of in vitro passage. Cell populations which replicate more slowly (trisomic-21 fibroblasts, fetal skin fibroblasts) also demonstrated a shift to larger cell volumes when compared with control rapidly replicating cell populations at the same level of in vitro passage. Similar shifts to larger cell volumes were produced by reducing serum concentration, decreasing incubation temperature and inhibiting DNA synthesis. Separation of senescent WI-38 cells on the basis of cell volume revealed that the cell fractions with the largest modal cell volume contained the highest percentage of slow or nonreplicating cells. Therefore, an inverse relationship appears to exist between growth rate and cell volume in cultured human diploid fibroblasts.     

Appearance of the terminal senescent cell population in human diploid fibroblasts analyzed by flow cytometry

We studied changes in the distribution pattern of relative RNA content during the in vitro aging of TIG-3 cells by flow cytometry (FACS III). Propidium iodide (PI) does not stain total cellular RNA, but it intercalates specifically into double-helical regions of both DNA and RNA. In applying this principle to RNA, we stained double-stranded RNA (dsRNA) in whole cells with PI after DNA digestion with DNase. The results showed that dsRNA distribution patterns were relatively constant at 7-75 population doublings (PD) but were significantly altered after 77 PD. The distribution patterns were similar as those for cell volume measured with a Coulter Counter. The total cellular dsRNA contents increased linearly at the senescent phase of their in vitro life span. In contrast, the mean dsRNA contents (50% dsRNA contents) rapidly increased to 77-79 PD, but decreased somewhat at 81-83 PD. Two-dimensional histograms of the dsRNA contents versus cell size were little altered from 25 PD to 75 PD. However, a population with relatively larger cell volume and weaker fluorescence intensity appeared and increased after 79 PD. This cell population group may be categorized as "terminal senescent cells" that no more divide in respect that the dsRNA content decreases in spite of the increase of total RNA content.     

The early effects of human cytomegalovirus infection on macromolecular synthesis in human embryonic fibroblasts

Summary Cytomegalovirus inhibits overall protein and RNA synthesis within 3 hours of infection of human embryonic fibroblasts. This effect is similar to that seen in herpes infected cells.With 2 Figures     

Time-dependent differential gene expression in lysophosphatidic acid-treated young and senescent human diploid fibroblasts

The gene expression profiles of lysophosphatidic acid (LPA)-treated young and senescent human diploid fibroblasts (HDFs) were examined using cDNA microarray analysis. The expression of some genes, including EGR 1/3 and MRRF, was controlled by LPA similarly in young and senescent cells, showing a typical time-dependent up-and-down expression profile. In contrast, some other genes, including DUSP6, CYR61, and F3, showed sustained upregulation in senescent HDFs later after LPA treatment. These genes might be involved in altered LPA responsiveness during the aging process.     

Actin distribution and synthesis in human fibroblasts infected by cytomegalovirus

Summary Early functions of human cytomegalovirus were found to induce a loss of microfilaments as revealed by indirect immunofluorescence and by the use of fluorescent phalloidin. Actin synthesis in infected cultures, on the other hand, appeared to be largely unchanged as estimated from the specific radioactivity of cytoplasmic actin.With 2 Figures     

Failure in S6 protein phosphorylation by serum stimulation of senescent human diploid fibroblasts, TIG-1

When quiescent young or senescent human diploid cells, TIG-1, were metabolically labeled with 32Pi and stimulated with 10% fetal bovine serum, the phosphorylation of ribosomal S6 protein was enhanced in young cells but not in senescent cells while that of some other proteins were increased in both cells. Inability to stimulate the phosphorylation of S6 protein in senescent cells after serum addition may be the primary cause of the failure of enhancement in protein synthesis followed by the block of prereplicative events dependent on protein synthesis and thus of the failure of cells to enter S phase. However, when the cell-free preparation from serum-stimulated senescent cells was incubated with [gamma-32P]ATP, S6-kinase activity was stimulated and S6 in ribosomal fraction was susceptible to phosphorylation as observed in young cells. Differences in S6 phosphorylation of senescent cells between in vivo and in vitro was discussed.     

Evidence for gene silencing by DNA methylation in normal human diploid fibroblasts

Human diploid fibroblasts, strain MRC-5, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) in the S phase of the cell cycle. The frequency of TG^RHPRT^– cells was increased up to 20-fold in comparison to control untreated cultures. Representative TG^R clones were unable to grow in HAT, and these were treated with 5-azacytidine (5-aza-CR). In many cases subsequent growth in HAT medium was observed, but in others it is likely that the cells had run out of growth potential. The results provide the first evidence of the silencing and reactivation of a gene in normal diploid mammalian cells.     

Stimulation of host DNA synthesis and induction of early antigens by ultraviolet light irradiated human cytomegalovirus

Summary The ultraviolet (UV-)sensitivity of the human cytomegalovirus (HCMV) genes coding for very early complement fixing and early antigens in human embryonic fibroblasts (HEF) and mouse embryonic fibroblasts (MEF) and the relation of these genes to the ability of the virus to stimulate host cell DNA synthesis were investigated.After 14 minutes of UV-irradiation of the virus inoculum only the very early complement fixing nuclear antigen (CMNA) developed in the HEF cells and only the early cytoplasmic antigen(s) was present in the MEF. In both HEF and MEF, host cell DNA synthesis was stimulated. We conclude that the ability of HCMV to stimulate host DNA synthesis is an early function of the viral genome and shows a high resistance to UV-irradiation.There is no direct correlation, however, between the ability of the virus to stimulate host cell DNA synthesis and the genes which code for the CMNA or for early cytoplasmic antigens.With 4 FiguresThis research was supported by a grant from the Hungarian Ministry of Health (Contract No. 2-11-0801-02-1/VG).     

Mitogenic activity in human embryonic fibroblasts early after infection by human cytomegalovirus.

We have characterized the nonspecific lymphocyte stimulation by extracts of human cytomegalovirus-infected human embryonic fibroblasts. Cell extracts prepared at 5 h postinfection (early extract) and 72 h postinfection (late extract) were both highly mitogenic in lymphocyte preparations from adult blood, cord blood, and rabbit blood. Maximum stimulation of the lymphocytes was observed on day 3 after the addition of early or late extract under optimal conditions. Early extract stimulated both the E-rosetting and the EAC-rosetting subpopulations of human lymphocytes. The mitogenic activity appeared before 5 h postinfection and was fairly stable at 30 degrees C for 5 h.     

Ultrastructural study on the sequence of human cytomegalovirus infection in human diploid cells

Summary The sequence of human CMV infection in WI 38 cells at high input multiplicity was studied by electron microscopy.The majority of entering virus particles were degraded within the cytoplasmic vacuoles but few particles released their capsids into the cytoplasm. Unenveloped particles with a distinct core were observed to persist over a period of 24 hours post-infection. Virus particles in the nucleus were only detectable 48 hours post-infection and thereafter. Process of formation and segregation of primary lysosome-like dense bodies was observed in association with the termination of the virus assembly cycle.This investigation was supported, in part, by grants from the John A. Hartford Foundation and Public Health Service Research Grant #NS06859 from the National Institute of Neurological Diseases and Stroke.