Expression of c-myc oncogene in human fibroblasts during in vitro senescence

Expression of the c-myc oncogene was determined in pre-confluent early and late passage human (IMR-90) fibroblast by dot blot analysis of cellular mRNA. Significant decreases in c-myc levels were found in late passage when compared to levels found in early passage cells. Cells restimulated with serum after serum restriction also showed reduced levels of c-myc in late passage. Confluent cells expressed levels of c-myc similar to that of pre-confluent cells, when serum stimulation was the same in both cases. Southern blots of Eco R1 digested DNA showed 2 fragments of similar size hybridizing to c-myc sequences in both early and late passage cells.     

Augmented nuclease activity during cellular senescence in vitro

The molecular correlates of the limited proliferative potential of normal human diploid fibroblasts and extensive single-strand breaks in the genomic DNA of these cells were examined by transfection analyses in which DNA replication could be uncoupled from DNA damage and repair. Both supercoiled (fmI), and restriction endonuclease-cleaved, linear (fmIII) molecules of a well-defined bacterial plasmid DNA, pBR322, were transfected into, and subsequently recovered from, early and late passage fibroblasts. Southern blot analysis revealed that fmI DNA was converted by random nicks into fmII DNA slightly more rapidly in late passage cells compared with cells at early passage. Similarly, fmII and fmIII DNAs also sustained multiple random nicks and no appreciable net religation of free ends of fmIII DNA could be detected at either passage. In addition, the efficiency of in vitro ligation of fmIII DNA recovered from late passage cells was also reduced, compared with that from early passage cells, as determined by Southern blotting. These data suggest that in the absence of DNA replication, a putative nuclease activity may contribute to DNA damage observed in senescent cells, which, in turn, may be causally related to their limited replicative potential.     

Effect of low dose rate irradiation on the division potential of cells in vitro. IV. Embryonic and adult human lung fibroblast-like cells

Early and late passage human embryonic lung fibroblasts were compared with early passage adult lung fibroblasts with regards to their survival (number of population doublings), after low dose rate ionizing radiation. It was found that early passage embryonic cells are quite resistant to this type of radiation. Late passage embryonic and early passage adult fibroblasts are more sensitive to ionizing radiations. The results suggest that cell aging is accompanied by an increased sensitivity to low dose rate ionizing radiation and favor the idea that aging in vitro, expressed as a function of the fibroblast division potential, is correlated with aging in vivo.     

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.     

Telomerase expression restores dermal integrity to in vitro-aged fibroblasts in a reconstituted skin model

The lifespan of human fibroblasts and other primary cell strains can be extended by expression of the telomerase catalytic subunit (hTERT). Since replicative senescence is accompanied by substantial alterations in gene expression, we evaluated characteristics of in vitro-aged dermal fibroblast populations before and after immortalization with telomerase. The biological behavior of these populations was assessed by incorporation into reconstituted human skin. Reminiscent of skin in the elderly, we observed increased fragility and subepidermal blistering with increased passage number of dermal fibroblasts, but the expression of telomerase in late passage populations restored the normal nonblistering phenotype. DNA microarray analysis showed that senescent fibroblasts express reduced levels of collagen I and III, as well as increased levels of a series of markers associated with the destruction of dermal matrix and inflammatory processes, and that the expression of telomerase results in mRNA expression patterns that are substantially similar to early passage cells. Thus, telomerase activity not only confers replicative immortality to skin fibroblasts, but can also prevent or reverse the loss of biological function seen in senescent cell populations.     

Functional changes in cellular fibronectin from late passage fibroblasts in vitro

Analysis of fibronectin synthesized by human fibroblasts, at different times during serial subcultivation, reveals functional differences. Fibronectin isolated from late passage cells is defective in promoting cell adhesion, cell spreading, and the formation of focal contacts. These changes are not the result of an inability of late passage cells to interact with fibronectin, since late passage cells become adhesive and form focal contacts in the presence of fibronectin isolated from early passage cells. Therefore, we conclude that late passage cellular fibronectin derived from late passage cells cannot support the cell substrate interactions.     

Histone gene expression remains coupled to DNA synthesis during in vitro cellular senescence

Despite a decrease in the extent to which confluent monolayers of late compared to early passage CF3 human diploid fibroblasts can be stimulated to proliferate, the time course of DNA synthesis onset is similar regardless of the in vitro age of the cells. A parallel and stoichiometric relationship is maintained between the rate of DNA synthesis and the cellular levels of histone mRNA independent of the age of the cell cultures. Furthermore, DNA synthesis and cellular histone mRNA levels decline in a coordinate manner after inhibition of DNA replication by hydroxyurea treatment. These results indicate that while the proliferative activity of human diploid fibroblasts decreases with passage in culture, those cells that retain the ability to proliferate continue to exhibit a tight coupling of DNA replication and histone gene expression.     

Enhanced synthesis of a Mr = 55,000 dalton peptide by senescent human fibroblasts

The secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40-50% lifespan completed), late passage (greater than 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55-60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.     

Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF β-receptors were found to be enhanced in old donor cells strains, whereas the PDGF α-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts. © 1994 Wiley-Liss, Inc.     

Phenylalanyl synthetase function in cultured fibroblasts from subjects with progeria.

Since progeria cells contain a diversity of altered proteins, some aspects of phenylalanyl synthetase function were examined in semipurified extracts of cultured skin fibroblasts using mixed rabbit tRNA as acceptor. No significant differences were found in the Km and Vmax for phenylalanine or ATP in progeria cells compared with controls. Initial velocities of both progeria and control synthetases were lower at late passage owing to either reduced enzyme content or reduced catalytic efficiency. Reverse phase 5 chromatography of tRNAs acylated by progeria and control synthetases gave a single peak of labeled phenylalanine tRNA in all cases with no secondary peaks evident. Total activity of phenylalanyl synthetase in progeria cells was similar to that of control cells at early passage while late-passage control cells had lower specific activities of these synthetases per unit protein.     

Attenuation of NF-kappaB signaling response to UVB light during cellular senescence

The ability of cells to adapt to environmental stresses undergoes a progressive reduction during aging. NF-kappaB-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-kappaB 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-kappaB 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-kappaB 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-kappaB complexes. Supershift experiments showed that the specific NF-kappaB complexes contain p50 and p65 protein components but not p52 and c-Rel proteins. Cytoplasmic IkappaBalpha 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-kappaB-responsive sites of the TNFalpha promoter were used to analyze the functional activity of the NF-kappaB complexes. Results showed that UVB exposure induced an increase in NF-kappaB-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-kappaB-mediated signaling by UVB light is highly attenuated in senescent fibroblasts. This attenuation may reduce the stress resistance during cellular senescence.     

Dupuytren's disease: physiologic changes in nodule and cord fibroblasts through aging in vitro

The pathogenesis of the fibrotic disease Dupuytren's contracture remains unclear. The disease process includes two structurally distinct fibrotic elements, the nodule and the cord. It has been proposed that as the disease progresses, nodules develop into cords. To corroborate that hypothesis, the authors took advantage of cultured fibroblast differences found between gap junction intercellular communication and fibroblast-populated collagen lattice contraction. Paired fibroblast cell lines of nodules and cords derived from four patients with Dupuytren's disease were maintained in culture for at least eight passages. The presence of gap junction intercellular communication in nodule- and cord-derived fibroblasts was documented and reported as a coupling index. The contraction of free-floating nodule- or cord-derived collagen lattices was also documented and reported. Early passage (passage 4) cord-derived fibroblasts showed a significant increase in coupling index compared with passage 4 nodule-derived fibroblasts (4.0 +/- 0.4 versus 2.5 +/- 0.3, respectively), where p < or = 0.01. However, late passage (passage 8) nodule- and cord-derived fibroblasts were equivalent in their coupling index (4.1 +/- 0.4 versus 4.4 +/- 0.4, respectively). Early passage nodule-derived fibroblast-populated collagen lattices contracted by 64 percent, whereas late passage nodule-derived lattices showed less contraction, at only 40 percent. Early and late passage cord-derived lattices contracted 46 and 37 percent, respectively. All nodule- and cord-derived cell lines were statistically equivalent at lattice contraction by passage 8. These in vitro studies support the hypothesis that fibroblasts derived from Dupuytren's contracture nodules change their phenotype after undergoing repeated cell passage, acquiring a cord-like fibroblast phenotype. Dupuytren's nodules represent the early, active form of fibrosis in which cells are more proliferative, better at fibroblast-populated collagen lattice contraction, and display less gap junction intercellular communication. The speculation is that alterations in gap junction intercellular communication may be involved in the progression of Dupuytren's nodules to cords as the disease progresses.     

Autofluorescent structures in cultured WI-38 cells

Human fibroblasts (WI-38) were monitored for intracellular fluorescence at several passage levels. It was found that late-passage cells contained marked increments in particulate intracellular fluorescence when compared with early-passage cells. Intracellular hydrolysis of α-glycerophosphate at pH 5 was used to localize acid phosphatase activity. Most fluorescent structures contained acid phosphatase, and it was concluded that the fluorescence was associated with lysosomes. Autoradiographic analysis of labeled-thymidine uptake into nuclei was employed to distinguish dividing and nondividing cells in cultures of varying passage levels, and these results were correlated with intracellular fluorescence. It was found that cells containing high levels were unable to incorporate thymidine into nuclei. These results suggest a correlation between fluorescence accumulation in lysosomes during aging and inhibition of cell cycling.     

Calcium-mediated cell surface changes in Chinese hamster cells.

Scanning electron microscopy (SEM) revealed quantitative differences in both surface characteristics and spreading behavior of young and senescent WI38 human diploid fibroblasts. Measurement of spreading behavior involved the determination of the rate of cell elongation beyond an axial ratio of 1.5 after time intervals of 1.5, 3, 6, 9, and 12 h following subculture. Early passage cells exhibited a 44–52% increased spreading rate compared with later passage cells, both obeying zero-order kinetics. Surface microvilli and blebs were also found to vary in number and size after those same time intervals following subculture. In young cells, microvilli density was found initially to be very low but then increased steadily with time. Later passage cells exhibited an initial high density of microvilli, followed by a gradual disappearance with time. The density of cellular blebs did not, however, vary significantly with time following subculture. After approx. 24 h, both early and late passage cells resembled each other. These studies do show that physical membrane characteristics of young and senescent cells differ under certain conditions, and suggest the importance of that structure in the phenomena of senescence.     

Presence and possible role of C-ras and nuclear (c-fos and c-jun) proto-oncogene products in preimplantation embryonic development in mice

The presence and possible role of products of nuclear (c-fos and c-jun) and c-ras proto-oncogenes were investigated in preimplantation embryonic development in mice. Polyclonal antibodies to c-fos or c-jun proto-oncogene products did not affect development of in vitro-cultured embryos from two-cell to morula or from morula to late blastocyst stages. However, v-H-ras monoclonal antibody (mAb) to c-ras protein (p²¹), although it did not inhibit the development of in vitro-cultured embryos from two-cell to morula stages, it significantly (P < .001–.005) inhibited the development of morula to late blastocyst stages in a dose-dependent manner. The effects of v-H-ras mAb were specific, since immunoabsorption with synthetic ras peptide completely blocked inhibitory effects of v-H-ras mAb. Neither c-fos nor c-jun antibodies reacted with specific proteins corresponding to c-fos (62 kDa) and c-jun (39 kDa) products on the Western blots of various murine ova/embryos extracts. However, the c-fos and c-jun antibodies reacted with 62 and 39 kDa protein bands, respectively, on the blot of NIH 3T3 cells extract. The v-H-ras mAb specifically identified 21 ± 3 kDa protein corresponding to c-ras p²¹ on the blots of early as well as late blastocyst extracts. The rat control ascites IgG₁ did not react with any protein band on the blots of various ova/embryo extracts. The reactions of v-H-ras mAb on the Western blots of blastocyst extracts were specific, since immunoabsorbed antibody was unable to react with any specific band on blots of early or late blastocyst extract. These results were further confirmed by immunoprecipitation procedure utilizing v-H-ras mAb. Again, the v-H-ras mAb immunoprecipitated a 21 kDa band from early as well as late blastocyst extracts. The rat control ascites IgG₁ did not react with any band corresponding to p ²¹ in the immunoprecipitation procedure. These results suggest that the specific products of nuclear proto-oncogenes, the c-fos and c-jun, are not detected in murine ova and preimplantation embryos, and the respective antibodies do not inhibit embryogenesis, indicating that they may not play a major role in early embryonic development. On the other hand, the product of c-ras proto-oncogene is specifically expressed in the blastocyst-stage embryos and may have a possible role in preimplantation embryonic development in mice. © 1993 Wiley-Liss, Inc.     

Regulation of dihydrofolate reductase gene expression and E2F components in human diploid fibroblasts during growth and senescence

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531–538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (−381 to −17) indicated that nuclear proteins bind to a cluster of cis-elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis-element in the regulation of DHFR gene expression. We found that (1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; (2) Yi binding activity was undetectable in both early passage and senescent cells; and (3) E2F binding activity was serum-inducible, senescence-dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F-1, E2F-4, and E2F-5 mRNA was growth- and senescence-dependent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and senescence-independent. In contrast, E2F-2 mRNA was not detectable in IMR-90 or WI-38 human fibroblasts. Western blot analysis showed that among the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nuclear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells. © 1996 Wiley-Liss, Inc.     

Fibronectin from aged fibroblasts is defective in promoting cellular adhesion

Late passage fibroblasts show decreased cell-substrate adhesion. We provide evidence that the reduced adhesion is due to a defect in the adhesive glycoprotein fibronectin. Late passage cells become more adhesive in culture media that has been conditioned by the growth of early passage cells. Analysis of fibronectins purified from early and late passage cell conditioned media indicates that there are striking differences in their abilities to promote cell adhesion. Young cell fibronectin supports the maximal adhesion of both young and old cells. However, old cells require quantitatively more fibronectin. In contrast, old cell fibronectin is less effective in supporting the adhesion of either cell type. In addition, neither cell type achieves a normal morphology in the presence of old cell fibronectin. The results support the conclusion that the fibronectin released by late passage cells is defective and does not support normal cell-substrate interactions.