IMR-90 Human Diploid Fibroblasts Research Articles

Mitogen-activated Protein Kinase Signaling Mediates Phosphorylation of Polycomb Ortholog Cbx7

Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.

Plasma membrane proteomics identifies Notch1 as a potential regulator of ras-induced senescence

BackgroundOncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism leading to stable cell-cycle arrest in response to oncogene activation. OIS is a heterogeneous phenotype of multiple effector mechanisms; understanding of in-vivo OIS is lacking because of the difficulty in identifying senescence. We wished to establish a cell-surface phenotype of OIS. MethodsWe used ER:Ras IMR90 human diploid fibroblasts, which undergo OIS after 6 days with 4-hydroxytamoxifen (4-OHT). We used SILAC (stable isotope labelling with amino acids in cell culture)-based proteomics and three labelling conditions (light, IMR90 plus 4-OHT; medium [lysine, K+4Da, arginine, R+6Da] ER:ras IMR90; heavy [K+8Da, R+10Da] ER:Ras IMR90 plus 4-OHT), combined with cell surface aminooxybiotinylation before streptavidin pulldown. Tryptic peptides were fractionated by high pH reversed-phase high performance liquid chromatogrpahy and then subjected to liquid chromatograph mass spectrometry. Data were processed by the analytical packages MaxQuant and MASCOT. Hits were validated by fluorescence-activated cell sorting (FACS), quantitative PCR, and immunoblotting. shRNA-mediated knockdown of protein expression used the murine stem cell virus-miR30 system. Findings899 proteins were identified, of which 73% were present at the cell surface by Gene Ontology annotation. Notch1 was significantly upregulated in OIS compared with both control conditions (3·1–3·4 fold). Upregulation was confirmed by both FACS and immunoblotting. Downstream Notch target-genes were also upregulated in OIS. Treatment with the γ-secretase inhibitor DAPT increased both senescence-associated heterochromatin foci (SAHF) and senescence-associated β galactosidase (SA-β-gal) activity, features of OIS. However, Notch1 knockdown reduced both SAHF and SA-β-gal. The effect of DAPT was not mediated through canonical Notch1 signalling since RBP-j knockdown had no effect upon DAPT-mediated SAHF and SA β-gal upregulation. Numb, a canonical Notch pathway inhibitor, is upregulated in OIS. Numb may underpin a transition switch from canonical to non-canonical Notch signaling in OIS. InterpretationPlasma membrane proteomics has identified a cell-surface phenotype of OIS. Notch 1 cell surface expression and downstream target-genes are upregulated in OIS. The transition to OIS is correlated with a transition from canonical to non-canonical Notch1 signalling that may be driven by Numb expression. FundingCancer Research UK.

Aberrant regulation and modification of heat shock factor 1 in senescent human diploid fibroblasts

Induction of the heat shock response (HSR), determined by hsp70-luciferase reporter and HSP70 protein expression, is attenuated as a function of age of the IMR-90 human diploid fibroblasts. To better understand the underlying mechanism, we evaluated changes in the regulation and function of the HSF1 transcription factor. We show that the activation of HSF1 both in vivo and in vitro was decreased as a function of age, and this was attributable to a change in the regulation of HSF1 as the abundance of HSF1 protein and mRNA was unaffected. HSF1 was primarily cytosolic in young cells maintained at 37 degrees C, and heat shock promoted its quantitative nuclear translocation and trimerization. In old cells, some HSF1 was nuclear sequestered at 37 degrees C, and heat shock failed to promote the quantitative trimerization of HSF1. These changes in HSF1 could be reproduced by treating young cells with H2O2 to stunt them into premature senescence. Flow cytometry measurement of peroxide content showed higher levels in old cells and H2O2-induced premature senescent cells as compared to young cells. Experiments using isoelectric focusing and Western blot showed age-dependent changes in the mobility of HSF1 in a pattern consistent with its S-glutathiolation and S-nitrosylation; these changes could be mimicked by treating young cells with H2O2. Our results demonstrated dynamic age-dependent changes in the regulation but not the amount of HSF1. These changes are likely mediated by oxidative events that promote reversible and irreversible modification of HSF1 including S-glutathiolation and S-nitrosylation.

Increased abundance of cytoplasmic and nuclear caveolin 1 in human diploid fibroblasts in H 2O 2-induced premature senescence and interplay with p38α MAPK

Treatment of IMR-90 human diploid fibroblasts with a sublethal concentration of H 2O 2 induces premature senescence. We investigated the protein abundance, subcellular localization and involvement of caveolin 1 in premature senescence. Caveolin 1 is a scaffolding protein able to concentrate and organize signaling molecules within the caveolae membrane domains. We report the first evidence of increased nuclear and cytoplasmic localization of caveolin 1 during establishment of H 2O 2-induced premature senescence. Moreover, we demonstrate that phosphorylation of caveolin 1 during treatment with H 2O 2 is dependent on p38α mitogen-activated protein kinase.

Knocking Down p53 with siRNA Does Not Affect the Overexpression of p21WAF‐1 after Exposure of IMR‐90 hTERT Fibroblasts to a Sublethal Concentration of H2O2 Leading to Premature Senescence

Premature senescence of IMR-90 human diploid fibroblasts (HDFs) expressing telomerase was induced by exposure to sublethal concentration of H(2)O(2), with appearance of several biomarkers of cellular senescence like enlarged cell shape, senescence-associated beta-galactosidase (SA ss-gal) activity, and cell cycle arrest. The induction of stress-induced premature senescence (SIPS) was associated with a transient increase in DNA-binding activity of p53 and an increased expression of p21(WAF-1). p53 small interferent RNA (siRNA) affected the basal level of p21(WAF-1) mRNA but did not affect the overexpression of p21(WAF-1) after stress. This siRNA approach confirms previous results obtained with other methods.

Identification of p38 MAPK-dependent genes with changed transcript abundance in H 2O 2-induced premature senescence of IMR-90 hTERT human fibroblasts

Premature senescence of IMR-90 human diploid fibroblasts expressing telomerase (hTERT) establishes after exposure to an acute sublethal concentration of H 2O 2. We showed herein that p38 MAPK was phosphorylated after exposure of IMR-90 hTERT cells to H 2O 2. Selective inhibition of p38 MAPK activity attenuated the increase in the proportion of cells positive for senescence associated β-galactosidase activity. We generated a low density DNA array to study gene expression profiles of 240 senescence-related genes. Using this array, p38 MAPK inhibitor and p38 MAPK small interferent RNA, we identified several p38 MAPK-target genes differentially expressed in H 2O 2-stressed IMR-90 hTERT fibroblasts.

Increased transcription and modified growth state-dependent expression of the plasminogen activator inhibitor type-1 gene characterize the senescent phenotype in human diploid fibroblasts.

The type-1 inhibitor of plasminogen activator (PAI-1) is a major physiologic regulator of pericellular proteolytic activity and, as such, influences matrix integrity, cell-to-substrate adhesion, and cellular proliferation. Excessive accumulation of both PAI-1 mRNA and protein correlates with the progressive acquisition of morphological and growth traits characteristic of the senescent phenotype (Mu and Higgins, 1995, J. Cell. Physiol., 165:647-657). Compared to early-passage IMR-90 human diploid fibroblasts, a late-passage senescence-associated 11-fold elevation in steady-state PAI-1 mRNA content reflected a 15-fold increase in constitutive PAI-1 gene transcription. Differential mRNA stability was not a factor in age-associated PAI-1 overexpression in IMR-90 cells. Upon removal of serum, early-passage human fibroblasts enter into a state of growth arrest with marked down-regulation of PAI-1 synthesis. Rapid induction of both the 3.0- and 2.2-kb PAI-1 mRNA species was evident upon serum-induced "activation" of quiescent early-passage fibroblasts; induced PAI-1 transcripts were maximal at 2 hr post-serum stimulation and declined in late G1 prior to entry into S phase. In contrast, late-passage (p32) fibroblasts maintained a significant level of PAI-1 expression under serum-free culture conditions. Although the PAI-1 gene was further responsive to serum in senescent cells, transcript abundance remained elevated and actually increased over the 12 to 16 hr post-serum addition period (a time when early-passage fibroblasts down-regulate PAI-1 mRNA content). Development of the senescent phenotype in human fibroblasts is associated, therefore, with significant changes in PAI-1 gene regulation. Such reprogramming involves predominantly transcriptional events and results in a marked increase in steady-state PAI-1 transcript abundance involving both the 3.0- and 2.2-kb mRNA species.

Dramatic attenuation of hypusine formation on eukaryotic initiation factor 5A during senescence of IMR-90 human diploid fibroblasts.

Deoxyhypusine synthase catalyzes the conversion of lysine to deoxyhypusine residue on the eukaryotic initiation factor 5A (elF-5A) precursor using spermidine as the substrate. Subsequent hydroxylation of the deoxyhypusine residue completes hypusine formation on elF-5A. Hypusine formation is one of the most specific polyamine-dependent biochemical events in eukaryotic cells. Although changes in polyamine metabolism have been demonstrated in human diploid fibroblasts during senescence (Chen and Chang, 1986, J. Cell. Physiol., 128:27-32.), it is unclear whether or not polyamine-dependent hypusine formation itself is an age-dependent biochemical event. In the present study, hypusine-forming activity was measured by a radiolabeling assay in cells whose polyamines have been depleted by prior treatment of alpha-difluoromethyl ornithine (DFMO). In addition, an in vitro cross-labeling assay was developed for simultaneous measurement of the deoxyhypusine synthase activity and protein substrate (elF-5A precursor) amount. We showed that the hypusine-forming activity in low-passage presenescent IMR-90 cells [population doubling level (PDL) = 15-23, termed young cells] was prominently induced by serum whereas little or no hypusine-forming activity could be detected in late-passage senescent cells (PDL = 46-54, termed old cells). The striking difference in hypusine-forming activity between young and old cells was due to changes in both deoxyhypusine synthase activity and elF-5A precursor amount in IMR-90 cells during senescence. However, Northern blot analysis showed no significant difference in the elF-5A messenger RNA (mRNA) between young and old cells, suggesting that the age-dependent attenuation of elF-5A precursor protein may be regulated at either translational or post-translational level.

SV40 T antigen increases the expression and activities of p34cdc2, cyclin A, and cyclin B prior to immortalization of human diploid fibroblasts

SV40 T antigen induces karyotype instability soon after it is expressed in human diploid fibroblasts and ultimately promotes cell immortalization and tumorigenesis. Protein levels and activities of mitotic cell cycle proteins have been shown to be elevated in several immortal cell lines relative to their normal parental cells, suggesting a possible role for the aberrant regulation of mitosis in karyotype instability. We show here that IMR-90 human diploid lung fibroblasts expressing the SV40 tumor antigens display increased protein levels and associated enzymatic activities of cyclin A, cyclin B, and p34cdc2 long before crisis and immortalization. These elevations cannot be explained by faster cell growth or altered cell cycle distributions. Increased protein levels were not totally accounted for by elevated levels of the corresponding mRNA, indicating that T antigen modulates expression at least partially by posttranscriptional mechanisms. These results indicate that perturbation of mitotic regulatory proteins precedes crisis, and imply that altered mitotic control is a direct consequence of T antigen expression rather than an outcome of secondary events associated with immortalization.

Global change of gene expression at late G1/S boundary may occur in human IMR‐90 diploid fibroblasts during senescence

The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR-90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263:11431-11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR-90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR-90 cells but not in old IMR-90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age-dependent attenuation in activities. In contrast, expression of growth-related genes such as eIF-5A, c-Ha-ras, and beta-actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp-1 binding site as the most common cis-element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled.

Transient cold shock induces the heat shock response upon recovery at 37 degrees C in human cells.

We evaluated the effects of a transient cold shock followed by recovery and incubation at 37 degrees C on the regulation of heat shock gene expression in the IMR-90 human diploid fibroblasts and HeLa cells in tissue culture. We showed that preincubation of cells at 4 degrees C induced the synthesis and accumulation of the heat shock proteins (HSPs) upon recovery at 37 degrees C, and the degree of this induction was directly related to the time that the cells spent at 4 degrees C. Assays on the abundance of the hsp 70 transcript, the hsp 70 gene promoter activity, and the trimerization and activation of heat shock factor (HSF) to bind to its consensus heat shock element (HSE) provided evidence that this induction of the heat shock response in cells recovering from a transient cold shock is attributable to a transcriptional event mediated by the activation of HSF. Further, the induction was a response to the temperature upshift from 4 to 37 degrees C as opposed to the 4 degrees C treatment itself; quantitation of the HSE-binding activity of cells incubated at 4 degrees C without recovery and incubation at 37 degrees C gave no evidence of an activated response. Analysis of the effects of protein synthesis inhibitors demonstrated that neither cycloheximide nor puromycin was effective in blocking the induction of HSE-binding activity in cells recovering from a transient cold shock. Experiments on the time course and temperature dependence of this induction of HSE-binding activity showed that the onset, magnitude, and duration of this induction were directly proportional to the severity of the cold stress (measured by time and temperature). We discuss the possible mechanism(s) involved in this induction of the heat shock genes at 37 degrees C by a transient cold shock and the biological implications of this observation.

Decline of Protein Kinase C Activation in Response to Growth Stimulation during Senescence of IMR-90 Human Diploid Fibroblasts

We have studied the activation of protein kinase C (PKC) during senescence of human IMR-90 fibroblasts by analyzing the phosphorylation of its in vivo substrate MARCKS (myristoylated alanine-rich C kinase substrate). It was found that the extent of TPA-induced phosphorylation of MARCKS was not significantly different between young and old IMR-90 fibroblasts. In contrast, the increase of MARCKS phosphorylation after serum stimulation was 4.5-fold in young fibroblasts as compared to 1.8-fold in old fibroblasts. Analysis of PKC by Western blotting showed that the levels of PKC were not changed during senescence of IMR-90 fibroblasts. However, the generation of diacylglycerol in response to serum stimulation declined in old fibroblasts. These results suggest that the efficiency of signal transduction mediated by diacylglycerol generation and PKC activation during the mitogenic response is age-dependent in human IMR-90 fibroblasts.

A specific CCAAT-binding protein, CBP/tk, may be involved in the regulation of thymidine kinase gene expression in human IMR-90 diploid fibroblasts during senescence.

The aging of IMR-90 human diploid fibroblasts in culture is accompanied by a 5-7 fold decrease in the level of thymidine kinase (TK) mRNA and TK activity (Chang, Z. F., and Chen, K. Y. (1988) J. Biol. Chem. 263, 11431-11435). We have employed a gel mobility shift analysis to investigate the molecular basis of the age-dependent attenuation of TK gene expression. Several cis-elements including two inverted CCAAT boxes, located at base pairs (bp) -36 and -67, and GC-rich Sp1 binding sites have been identified in the TK promoter. A 28-bp (-91 to -64) fragment containing the distal inverted CCAAT element was excised from the TK promoter to examine possible differences in nuclear protein binding between young and old IMR-90 cells. A prominent DNA-protein complex was identified in serum-stimulated young cells by a gel mobility shift assay. Competition analysis indicated that the binding was highly specific. The nuclear protein responsible for the complex formation was named CBP/tk (CCAAT Binding Protein for TK gene) since methylation interference assay showed that the inverted CCAAT box was involved in binding. The appearance of the CBP/tk-28-bp complex in IMR-90 cells was (i) serum-dependent, becoming prominent 12-24 h after serum stimulation, and (ii) age-dependent, prominent only in young but not in old IMR-90 cells. Similar serum- and age-dependent complex formations were also observed using a 67-bp fragment (-63 to +4) containing the proximal CCAAT element and a TATA box. In contrast, the binding activities for the Sp1 sequence were the same in young and old cells and appeared to be serum-independent. CBP/tk binding activity in nuclear extracts was abolished by heat (60 degrees C, 5 min) or treatment with proteinase K (0.1 microgram/ml) and sodium dodecyl sulfate (0.005%), but not by Nonidet P-40 or Triton X-100. Treatment of nuclear extracts with alkaline phosphatase or lectins (concanavalin A and wheat germ agglutinin) did not affect the binding activity. Metal chelators such as 1,10-ortho-phenanthroline (0.5 mM) inhibited the CBP/tk binding activity. Cycloheximide added to the serum-stimulated cultures at an early or mid-G1 phase inhibited the CBP/tk binding activity. The half-life of the serum-induced CBP/tk binding activity was estimated to be less than 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Tributyltin is a potent inducer of the heat shock response in human diploid fibroblasts.

Submicromolar concentrations of tributyltin (TBT), a commercially used organotin compound, were found to induce the expression of several stress proteins, most notably HSP89 and HSP70, in IMR-90 human diploid fibroblasts in a time- and dose-dependent manner. This induction can be demonstrated by quantitation of 1) synthesis of the heat shock proteins (HSPs), 2) relative abundance of mRNA of hsp70, and 3) transient expression of a human hsp70 promoter driven reporter gene. TBT also increased the abundance of mRNA of heme oxygenase, whereas heat shock was without effect. Analysis of protein binding to a consensus heat shock element (HSE) by electrophoretic mobility shift assay suggests that the induction of the heat shock response by TBT was attributable to activation of the heat shock transcription factor (HSTF).

The heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases: evidence from the effects of okadaic acid.

We examined the effects of okadaic acid (OA), a potent and specific inhibitor of serine phosphatases 2A and 1, on the transient expression of an hsp 70 promoter-reporter gene construct in IMR-90 human diploid lung fibroblasts. We showed that OA markedly potentiated the heat-induced but not the basal expression of pHBCAT, a full-length human hsp-70-promoter-driven CAT gene construct. This effect of OA was dose and time dependent and promoter specific. Importantly, the potentiating effects of OA appeared to be independent of the binding of the activated heat shock transcription factor (HSTF) to its consensus DNA sequence, the heat shock element (HSE). Thus, OA had no effect on the HSTF DNA-binding activity as measured by mobility shift assay, and mutation of the HSE sequence did not obliterate the stimulatory effects of OA on reporter gene expression under a heat shock condition, although heat shock by itself was without effect. Analysis of the status of phosphorylation of the largest subunit of RNA polymerase II provided evidence that this effect of OA is attributable, at least in part, to the increased phosphorylation of RNA polymerase II. These results provided evidence that the heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases. We propose that the heat-induced transcriptional activation of hsps is associated with phosphorylation of component(s) of the transcription complex; one of the likely candidates being the transcriptionally engaged RNA polymerase II. OA, by inhibiting phosphatase 2A and 1 activity, enhanced this phosphorylation and potentiated the transcriptional activation of hsps.

5-Azacytidine-induced demethyelation of DNA to senescent level does not block proliferation of human fibroblasts

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.

Molecular events involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid fibroblasts

We examined the induction, by heat shock, of heat shock transcription factor (HSTF) DNA-binding and hsp 70 gene promoter activities during aging of the IMR-90 human diploid fibroblasts. Cells with population doubling level (PDL) ranging from 15-48 were heat shocked at temperatures of 39, 42, and 45 degrees C for various time periods; the binding of HSTF to its consensus DNA was determined by gel retardation assay and the promoter activity of the human hsp 70 gene was analyzed by transient expression of reporter gene activity. We observed that the induction of HSE-binding activity was inversely related to the PDL of the cells used. Importantly, as cells progress through their life span, a higher temperature and a longer period of heat shock were needed to evoke an optimal increase in HSE-binding activity. A substantial and rapid (within 30 min) increase in HSE-binding activity was observed when PDL 20 cells were heat shocked at 39, 42, or 45 degrees C. However, PDL 35 cells did not respond to 39 degrees C, and PDL 48 cells responded slowly to heat shock at 45 degrees C, but not 39 or 42 degrees C. Experiments on the heat induced increase in hsp 70 promoter driven reporter gene expression provided similar information on the age-dependent decrease in transcriptional activation of hsps. These results were further corroborated by quantitation of the abundance of mRNA of hsp 70. Analysis of the cAMP induced expression of the rat somatostatin promoter driven CAT gene provided evidence that the decrease in transcriptional activation of hsps in aging diploid cells was not a reflection of a generalized dysfunction of signal transduction. We conclude that functional changes in the heat shock response occur before cells lose their capacity to replicate, and we suggest that these changes are likely to have a central role in the expression of the aging phenotype.

Heat shock induction of HSP 89 is regulated in cellular aging

Induction of heat shock proteins (HSPs) was evaluated as a function of age of the IMR-90 human diploid fibroblasts. Heat shock (42°C) markedly increased the synthesis of proteins with apparent molecular weights of 98, 89, 72, 50, 42 and 25 KDa, with HSP 89 and 72 being most prominent. This heat shock induction of HSPs was inversely correlated to the population doubling level (PDL) of the cell cultures used. For example, the synthesis rate of HSP 89 increased from a basal level of 1% of total protein synthesis to a maximum of 8–10% at 6–8 hrs after heat shock of young cells (PDL 18); in the old cells (PDL 51), the increase was from 1% to a maximum of 4% at 10–12 hrs after initiation of the heat shock. Western blot analysis showed that HSP 89 constituted approximately 2 and 10% of total cellular proteins in control and heat shocked (42°C, 12–24 hrs) young cells; corresponding values for the old cells were 2 and 4.5%, respectively. Northern blot quantitation of the amount of mRNA hybridizable to cDNA probes of HSP 89 provided evidence that this age-dependent decrease in induction of HSP 89 in IMR-90 cells was attributable to a transcriptional/pre-translational mechanism.

Attenuated induction of heat shock gene expression in aging diploid fibroblasts

We examined the effects of cellular aging on the regulation of heat shock gene expression in IMR-90 human diploid fibroblasts. Heat shock (42-43 degrees C) and canavanine (200-400 micrograms/ml) were used to evoke the heat shock response in these cells. We showed that heat shock induced the synthesis of proteins with apparent molecular weights of 98,000, 89,000, 78,000, 72,000, 64,000, 50,000 and 25,000, with heat shock protein (HSP) 89 and 72 being most prominent. Canavanine induced the synthesis of the four high molecular weight HSPs, particularly HSP 89 and HSP 78, without noticeably enhancing synthesis of the low molecular weight HSPs. We found that, while a similar series of HSPs were induced in the young and old cells, there was a marked decrease in the magnitude of this induction in the old cells. Using cells with defined population doubling levels, we observed a direct correlation of the inducibility of HSP synthesis and the replicative potential of the cells used. Analysis of the amount of translatable and hybridizable mRNA, by the methods of in vitro translation and Northern blot hybridization, demonstrated that the induction of HSPs synthesis can be accounted for by increases in their mRNA. Nuclear runoff transcription provided evidence that the decrease in inducible expression of the HSPs in aging IMR-90 cells was attributable to a transcriptional mechanism. This conclusion was substantiated by analysis of the hsp 70 promoter activity in transient expression assay of the hsp 70 promoter-chloramphenicol acetyltransferase construct. We propose that there is an age-associated dysfunction in the signaling mechanism of the heat shock response.

Altered intracellular protein degradation in aging: A possible cause of proliferative arrest

Many proteins that control cell-cycle progression are short-lived. Therefore, alterations in protein degradation are as likely as changes in transcription and/or translation in causing the proliferation arrest of senescent cells. Several different pathways of intracellular protein degradation have been identified, and both cytosolic and lysosomal pathways operate in most cells. We have used red cell-mediated microinjection to study degradation of radiolabelled proteins introduced into IMR-90 human diploid fibroblasts at early and late population doubling levels. Lysosomal pathways of protein degradation are reduced in senescent cells, and this defect may account for many characteristics of aging, including the accumulation of posttranslationally altered proteins. These abnormal proteins may then stimulate cytosolic, ubiquitin-dependent proteolytic pathways that are also responsible for the degradation of crucial regulatory proteins. Unknown short-lived proteins are also required for some step in lysosomal proteolysis, and this connection between the two degradative systems may cause the age-related changes in protein degradation to be progressive. Several experimental approaches are available to test whether altered protein degradation significantly contributes to proliferative arrest of senescent cells.