Late Passage WI-38 Research Articles

Proteasome inhibition increases HuR level, restores heat-inducible HSP72 expression and thermotolerance in WI-38 senescent human fibroblasts

At the end of their replicative potential in vitro, late passage WI-38 human diploid fibroblasts (HDF) have a low basal expression of heat shock protein 72 (HSP72) and an attenuated ability to induce it in response to heat shock. The transient exposure to the specific and reversible proteasome inhibitor MG132 during a mild heat shock induced late passage HDF to synthesize and accumulate high levels of HSP72. This HSP72 expression was long-lasting and appeared to result from both increased cytoplasmic levels and enhanced translation of HSP72 mRNA. The level of HuR, a stabilizing mRNA-binding protein, increased following the MG132 treatment. This result is consistent with the proposed role of HuR in assisting mRNA export to the cytoplasm and in antagonizing its degradation. Furthermore, the previous exposure of late passage HDF to a mild heat shock in the presence of MG132 protected these cells against the otherwise lethal effect of a subsequent severe heat shock. This acquisition of thermotolerance appeared to be correlated with the level of HSP72.

Expression of Transcriptional Repressor Proteins mSin3A and 3B during Aging and Replicative Senescence

Sin3 proteins have a key role in transcriptional repression mediated by histone deacetylation. Mammalian Sin3 proteins, mSin3A and 3B, act as adapter molecules which bind both to repressive transcription factors and to the methyl-CpG-binding proteins (MeCPs) and recruit histone deacetylases to assemble a multiprotein repressor complex. We have recently observed (Biochem. Biophys. Res. Commun. 252, 274–277, 1998) that the expression of mSin3A but not mSin3B protein is induced during neuronal apoptosis. The purpose of this study was to find out whether aging and replicative senescence affect the expression levels of mSin3A and 3B repressor proteins. We studied the expression levels of mSin3A and 3B mRNAs and proteins both in replicative senescence model of WI-38 fibroblasts and in liver and brain tissues of young (4–6 months) and old (26–30 months) male Wistar rats. Replicative senescence of human WI-38 fibroblasts did not affect the expression levels of mSin3A and 3B mRNAs. However, the late passage WI-38 fibroblasts showed a significant decline in the expression level of mSin3A protein. Immortalization of WI-38 fibroblasts with SV-40 transformation increased the expression level of 6.0 kb mSin3A mRNA. Aging of Wistar rats did not affect the expression levels of either mSin3A or mSin3B mRNAs in the liver and frontal cortex. Similarly, the protein levels of mSin3A and 3B were unaffected in the hippocampus, cerebellum and liver tissues during aging. These results show that aging in vivo, in contrast to replicative senescence, does not affect the expression levels of mSin3A and 3B repressor proteins. However, this does not exclude the possible age-related functional changes mediated by mSin3-histone deacetylase complexes.

Attenuation of NF-κB Signaling Response to UVB Light during Cellular Senescence

The ability of cells to adapt to environmental stresses undergoes a progressive reduction during aging. NF-κB-mediated signaling is a major defensive system against various environmental challenges. The aim of this study was to find out whether replicative senescence affects the response of the NF-κB signaling pathway to UVB light in human WI-38 and IMR-90 fibroblasts. The exposure of early passage fibroblasts to UVB light inhibited the proliferation and induced a flat phenotype similar to that observed in replicatively senescent fibroblasts not exposed to UVB light. The UVB radiation dose used (153 mJ/cm2) did not induce apoptosis in either early or late passage WI-38 fibroblasts. UVB exposure induced a prominent activation of the NF-κB signaling pathway both in early and in late passage WI-38 and IMR-90 fibroblasts. Interestingly, the response to UVB light was significantly attenuated in late passage fibroblasts. This attenuation was most prominent in DNA binding activities of nuclear NF-κB complexes. Similar senescence-related attenuation was also observed in the DNA binding activities of nuclear AP-1 and Sp-1 factors after UVB treatment. Immunoblotting and -cytochemistry showed an increase in nuclear localization of p50 and p65 components of NF-κB complexes. Supershift experiments showed that the specific NF-κB complexes contain p50 and p65 protein components but not p52 and c-Rel proteins. Cytoplasmic IκBα showed a marked decrease at protein level but an increase in phosphorylation after UVB treatment. Transient transfection assays with TK5-CAT and TK10-CAT plasmids carrying NF-κB-responsive sites of the TNFα promoter were used to analyze the functional activity of the NF-κB complexes. Results showed that UVB exposure induced an increase in NF-κB-driven CAT expression both in early and in late passage fibroblasts though the response was significantly stronger in early passage fibroblasts. Our results show that the induction of NF-κB-mediated signaling by UVB light is highly attenuated in senescent fibroblasts. This attenuation may reduce the stress resistance during cellular senescence.

Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts

EPC-1 (early population doubling level cDNA-1) is a quiescence-specific gene expressed at high levels by early passage WI-38 fibroblasts under conditions of either density-dependent growth arrest or serum deprivation. Late passage WI-38 cells lose the ability to express EPC-1 under all conditions tested. The decline in EPC-1 mRNA is gradual during the replicative life span and correlates inversely with the population doubling level (PDL) of the cells. The objective of this study was to determine whether the decline in EPC-7 mRNA abundance observed during proliferative senescence also occurs in cultures derived from donors of different ages. To address this question, we examined the abundance of EPC-1 mRNA in 28 skin fibroblast lines established from healthy donors of different ages ranging from 12 fetal weeks to 94 years. EPC-1 expression was measured, under conditions of growth arrest, prior to the end of the replicative life span of the cultures. Despite some variability in steady-state transcript levels among the cell lines, EPC-1 expression was significantly lower in cells derived from the fetal donor group (12-20 gestational weeks) than in cells derived from adult donors. An in vitro age-dependent decline in EPC-1 expression was observed in all the skin lines examined, independent of donor age; however, no significant difference was observed between the young adult donor group (17-33 years) and the old adult donor group (78-94 years). Thus, expression of EPC-1 is linked to the replicative age of the cells and whether the cells are derived from fetal skin or adult skin. In adults, EPC-1 expression is independent of donor age.

Identification of a highly abundant cDNA isolated from senescent WI-38 cells

A cDNA library was constructed from poly(A) + RNA derived from late passage WI-38 cells and differentially screened with cDNA probes from early and late passage cells. From a number of clones which exhibited differences in intensity of hybridization to the early or late passage probes, one was chosen for further analysis because of its large increase in hybridization to the late passage probe. This clone accounted for approximately 1% of the recombinants in the library. The partial cDNA clone shows complete sequence homology to elongation factor I α (EF-I α). Northern analysis of poly(A) + RNA from cells at various population doublings suggested that a 2.2-kb transcript, homologous to EF-I α accumulates as cells near the end of their replicative life span (phase III). When early and late passage cells were reexposed to serum after serum starvation, this transcript decreased in abundance. Additionally, a lower molecular weight transcript (1.6 kb) was detected 18 h following serum stimulation in early passage cells and 9 h after stimulation in late passage cells.

Increase in abundance of a transcript hybridizing to elongation factor I alpha during cellular senescence and quiescence

We have isolated a senescence-specific clone (pSEN) from a cDNA library constructed from late passage WI-38 human diploid fibroblast that accounts for approximately 1% of the recombinants. Nucleotide sequence analysis of the partial cDNA clone has led to the identification of pSEN as elongation factor I alpha. Northern analysis of poly(A)+ RNA from various intermediate population doubling levels shows that a 2.2 kb transcript hybridizes to pSEN but is expressed prior to PDL-40 at very low levels. This transcript begins to accumulate at PDL-40 and is induced approximately 50-fold just prior to senescence. Furthermore, this transcript was shown to be specific to G o of the cell cycle whereas a second, lower molecular weight transcript (1.6 kb) was observed during S phase (Giordano and Foster, unpublished data). The 2.2 kb transcript is also detected in neonatal foreskin cells but very little increase in abundance is observed between early and late passage cells. Sucrose gradient fractionation of RNA from late passage WI-38 cells suggests that the lower molecular weight transcript is associated with the polysome fraction while the 2.2 kb transcript sediments with the nonpolysomal fraction. Thus, the possibility exists that the 1.6 kb transcript is derived from the 2.2 kb transcript.

Cholesterol and cholesteryl ester concentration in different size classes of cultured human fibroblasts: Effect of in vitro aging

Early and late passage WI-38 fibroblasts were fractionated on the basis of cell size by gravity sedimentation, and free and esterified cholesterol concentrations were determined in each fraction. The cholesteryl ester concentration in all size classes of late passage cells was greater than that of all size classes of early passage cells; the average of all fractions of late passage cells was 2.5 times greater (pg/μm 3) and 1.7 times greater (μg/mg protein) than that of all fractions of early passage cells ( p< 0.001). The average free cholesterol concentration (μg/mg protein) in late passage cell fractions exhibited a consistent, but not statistically significant, increase over that in early passage cells. These results indicate that the increase in cholesteryl ester concentration in late passage WI-38 fibroblasts is not solely attributable to the large, non-dividing cells which accumulate in senescing cultures.

Hydrocortisone effects on nucleic acid and nuclear protein content and protein metabolism in aging WI-38 cells

Confluent late passage WI-38 human diploid fibroblast cultures grown in the presence or absence of hydrocortisone for fifteen population doublings were measured cyto-chemically for DNA, nuclear RNA, basic nuclear protein and acidic nuclear protein content during a 12-hour interval following a medium-change stimulation. Radioisotope tracer studies were utilized to assess the effects of prolonged hydrocortisone treatment on protein synthesis and degradation in aging confluent and logarithmically growing cultures. Hydrocortisone-treated cultures exhibited an increased Feulgen—DNA stainability, no difference in basic nuclear protein content, a significantly larger initial decrease followed by an increase in acidic nuclear protein content, and elevated levels of nuclear RNA as compared to untreated controls. Hydrocortisone treatment also resulted in increased incorporation of leucine into protein and decreased breakdown of pre-labeled protein in both confluent and logarithmically growing cultures. These results indicate that many of the metabolic changes associated with proliferation are stimulated by prolonged hydrocortisone treatment of late passage WI-38 fibroblast cells.

Increased prostaglandin F 2α and E 2 production in late passage WI38 diploid fibroblasts

The prostaglandin (PG) F 2α and E 2 concentrations in the culture medium of WI38 cells were determined at various passage levels. It was found that late passage cells produce significantly higher amounts of both PG F 2α and PG E 2.

Cellular senescence and the analysis of generation time distributions

Generation time analysis by time lapse cinematography is an important method for investigating cellular senescence in culture, but its interpretation is complicated by several types of bias and artifact, including small sample size, cut-off bias, changes in global growth rate, and phase of the population growth cycle. When these factors are considered, interpretation of the data base used by previous investigators changes considerably, and does not reveal any differences in growth behavior between middle and late passage WI-38 cells. Nor does it support the transition probability theory either of cell cycle transit or of culture senescence.

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.

The relationship between in vitro cellular aging and in vivo human age.

Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging.

Gene activation in human diploid cells: Age-dependent modifications in the stability of messenger RNAs for nonhistone chromosomal proteins

Serum stimulation of early as well as late passages of nondividing WI-38 human diploid fibroblasts to proliferate, results in DNA synthesis beginning at 12 hours and mitosis at 20 hours. A 2-fold increase in the transcriptional activity of chromatin isolated from early and late passage WI-38 cells is evident one hour following serum stimulation. An increased synthesis and association with the genome of two defined molecular weight classes of nonhistone chromosomal proteins one hour following serum stimulation of early and late passage cells is also observed. The increased chromatin template activity and nonhistone chromosomal protein synthesis occur in early passage cells stimulated to proliferate in the presence of actinomycin D. However, when late passage WI-38 cells are stimulated in the presence of actinomycin D, increases in chromatin template activity and nonhistone chromosomal protein synthesis are not observed one hour following serum stimulation. The possibility that nonhistone chromosomal protein synthesis and activation of transcription early during the prereplicative phase of the cell cycle in early passage human diploid fibroblasts may be regulated at the translational level is discussed. Consideration is also given to the possibility that there may be an age-dependent modification in such a regulatory mechanism.

Genealogies of clones of diploid fibroblasts: Cinemicrophotographic observations of cell division patterns in relation to population age

Direct assessment of cell division patterns of human diploid fibroblasts (WI-38) has been achieved using techniques of time-lapse cinemicrophotography. Pedigrees of progeny cells in clones from middle and late passage WI-38 cultures are presented. The passage 28 clone exhibited a division pattern that was highly synchronous through four generations and had an average interdivision time that increased linearly from the second through sixth generations. Division was essentially logarithmic through the fourth generation. The passage 53 clone was much more variable in interdivision time, less synchronous, and less motile than the passage 28 clone. While there is considerable overlap in the interdivision times of cells of the passage 28 and 53 clones, the late passage cells generally exhibit more variation in interdivision times. The average population doubling time was 16.8 h in thepassage 28 clone and 32.0 h in the passage 53 clone.