Quiescent Human Diploid Fibroblasts Research Articles

Resistance of Quiescent Human Diploid Fibroblasts to High Dose of External Oxidative Stress and Induction of Senescence

In response to external oxidative stress/DNA damaging agents, mammalian cells may choose one of the following pathways to avoid propagation of the damaged cells: repair the DNA and proceed with the normal cell cycle; trigger apoptosis; or undergo senescence to block cell division. If these safeguard mechanisms fail, cells containing damaged/mutated DNA will continue to propagate leading to cancer. Working with Human Diploid Fibroblasts, we have observed that young quiescent fibroblasts, unlike dividing fibroblasts, do not undergo apoptosis when subjected to high dose of external oxidative stress. Interestingly, when those quiescent fibroblasts are sub-cultured following H2O2 treatment, they display all the features of the senescent cell phenotype. Our results have indicated that p21 and MnSOD over-expression in quiescent cells is highly correlated to resistance to oxidative stress and may induce senescence. Moreover, there was no observable DNA damage in quiescent fibroblasts after 500 μM H2O2 treatment even though oxidative damage to lipids and proteins was detected both before and after treatment. Most importantly, the mitochondrial membrane potential in quiescent cells remained unchanged even after exposure to a high dose of external oxidative stress. In dividing cells, Bcl-2 expression was down-regulated whereas Bax expression was up-regulated following oxidative stress. On the other hand, Bcl-2 levels remained high and Bax was down-regulated in quiescent cells under identical treatment. Our results reveal that the over-expression of p21 and Mn-SOD and the down-regulation of Bax in quiescent cells could be responsible for their resistance against external oxidative stress and onset of senescence.

Oxidative stress-induced apoptosis in dividing fibroblasts involves activation of p38 MAP kinase and over-expression of Bax: resistance of quiescent cells to oxidative stress.

Oxidative stress has been postulated to be involved in aging and age-related degenerative diseases. Cell death as a result of oxidative stress plays an important role in the age related diseases. Using human diploid fibroblasts (HDF) as model to study the mechanism of cell death induced by oxidative stress, a condition was standardized to induce apoptosis in the early passage sub-confluent HDFs by a brief exposure of cells to 250 microM hydrogen peroxide. It was observed that p38 MAP kinase (MAPK) was activated soon after the treatment followed by over-expression of Bax protein in cells undergoing apoptosis. An interesting finding of the present study is that the confluent, quiescent HDFs were resistant to cell death under identical condition of oxidative stress. The contact-inhibited quiescent HDFs exhibited increased glutathione level following H(2)O(2)-treatment, did not activate p38 MAP kinase, or over-express Bax, and were resistant to cell death. These findings indicated that there was a correlation between the cell cycle and sensitivity to oxidative stress. This is the first report to our knowledge that describes a relationship between the quiescence state and anti-oxidative defense. Furthermore, our results also suggest that the p38MAPK activation-Bax expression pathway might be involved in apoptosis induced by oxidative stress.

Basal and induced amounts of interleukin-6 mRNA decline progressively with age in human fibroblasts.

Interleukin-6 (IL-6) is a multi-functional cytokine that plays a role in the body's response to injury and infection. IL-6 expression is induced in young human diploid fibroblasts (HDF) in response to a number of agents including fetal bovine serum, 12-O-tetradecanoylphorbol-13-acetate, double-stranded RNA, and forskolin. In contrast, we find that senescent HDF are markedly deficient in their ability to express IL-6 in response to serum, double-stranded RNA, and 12-O-tetradecanoyl-phorbol-13-acetate, whereas forskolin is still an effective inducer for senescent cells. Thus, specific pathways for stimulating IL-6 expression appear to be blocked in senescent HDF. The basal amount of IL-6 mRNA in unstimulated senescent HDF is also much lower than in unstimulated young quiescent HDF. Likewise, the amount of IL-6 protein produced by senescent HDF is decreased at least 10-fold. Both the basal and induced levels of IL-6 declined progressively with aging of the HDF in culture, e.g. IL-6 cannot be induced above the young basal level by approximately 65% of life-span completed. If a similar decrease in IL-6 expression takes place in HDF in vivo, it could contribute to the decline in wound healing and the increase in the number and severity of infections experienced by aged individuals.

Serine/threonine protein kinases and calcium-dependent protease in senescent IMR-90 fibroblasts

Three enzymes relevant to signal transduction were compared in replicating, quiescent and senescent human diploid fibroblasts (HDF). These were Ca 2+-dependent thiol protease (calpain), cAMP-dependent protein kinase (Pk-A), and calcium/phospholipid-dependent protein kinase C (Pk-C). The amounts of these enzymes in quiescent HDF were slightly greater or the same as in replicating HDF. In contrast, senescent HDF exhibited higher Pk-C, Pk-A and proteolytic activities than did either replicating or quiescent cells. While the elevated protein kinase activities could be accounted for by the larger size of senescent cells relative to younger cells, the increased calpain activity exceeded this size differential. Immunoblotting studies with antisera to both Pk-C and calpain demonstrated increased enzyme concentrations in parallel with the increased activities. Photolabeling cell extracts with an analog of cAMP, 8-N 3-[ 32P]cAMP provide an estimates of Pk-A concentration. By this criterion, senescent HDF have more Pk-A molecules than do young cells that are either replicating or quiescent. Only the type I isozyme of Pk-A (Pk-A-I) was observe in any of these cells. Photolabeling with 8-N 3[ 32P]cAMP demonstrated more degradative fragments of the Pk-A regulatory subunit (RI) in senescent cells also. This is a logical consequence of the increased calpain activity in senescent cells, since RI is a substance for calpain. These results imply that senescent cells do not fail to enter S phase owing to inadequate concentrations of Pk-A or Pk-C. Rather, the increased quantities of these enzymes in senescent cells may reflect aberrations elsewhere along signal transduction pathways that coordinate cell size with cell proliferation.

Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation.

Senescent human diploid fibroblasts (HDF) contain no detectable cdc2 mRNA or p34cdc2 protein. Similarly, young quiescent HDF have only low levels of cdc2 mRNA and protein. After serum stimulation, quiescent HDF accumulate increasing amounts of cdc2 mRNA and protein and go through DNA synthesis and mitosis. In contrast, serum-stimulated senescent HDF fail to accumulate detectable amounts of cdc2 mRNA and protein and fail to enter S phase. Mitosis is likewise deficient in senescent cells even when they have been induced to synthesize DNA by simian virus 40 large tumor antigen. Since p34cdc2 or its homologues appear to be required for DNA synthesis and mitosis in eukaryotes, a lack of these molecules in serum-stimulated senescent HDF could be an important reason for their inability to enter S phase or mitosis. Nuclear microinjection of cdc2 DNA into senescent HDF causes rounding up of the cells but no induction of DNA synthesis. Since cyclins A and B are important cofactors of the protein kinase activity of p34cdc2 or its homologues, we analyzed expression of these genes in serum-stimulated senescent HDF and determined that they contain little or no cycA or cycB mRNA. These deficiencies may be relevant to the lack of DNA synthesis and mitosis in senescent HDF.

Failure to Phosphorylate the Retinoblastoma Gene Product in Senescent Human Fibroblasts

Heterokaryon studies suggest that senescent and quiescent human diploid fibroblasts (HDF) contain a common inhibitor of entry into S phase. DNA synthesis can be induced in senescent and quiescent HDF by fusing them with cells containing DNA viral oncogenes such as SV40 T antigen, adenovirus E1A, or human papillomavirus E7. Both senescent and quiescent HDF contained the unphosphorylated form (p110Rb) of the retinoblastoma protein, a putative inhibitor of proliferation. After serum stimulation, senescent HDF did not phosphorylate p110Rb and did not enter S phase, whereas quiescent HDF phosphorylated p110Rb and entered S phase. These findings, combined with the observations that T antigen, E1A, and E7 form complexes with, and presumably inactivate, unphosphorylated p110Rb, suggest that failure to phosphorylate p110Rb may be an immediate cause of failure to enter S phase in senescent HDF.

Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe.

We have used the technique of reverse transcription coupled to the polymerase chain reaction to detect mRNA precursors [heterogeneous nuclear RNA (hnRNA)] transcribed from the thymidine kinase (TK) gene of human diploid fibroblasts. With this method, the amplification products of both hnRNA (containing the introns) and mature mRNA can be detected on Southern blots with appropriate hybridization probes. With the experimental conditions used, the sensitivity of the technique is such that TK mRNA can be detected in as few as 20 S-phase cells. TK hnRNA is maximally expressed early in the S phase of the cell cycle after quiescent human fibroblasts are stimulated to proliferate. At this point, the ratio of TK hnRNA to TK mRNA is 1:155. A small amount of TK hnRNA can be detected in populations of cells that appear to be quiescent. However, the presence of the precursor in these populations correlates with the number of cells still cycling. No TK hnRNA can be detected in truly quiescent human diploid fibroblasts, suggesting that in these cells, the TK gene is not transcribed in G0.

Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: characterization and comparison to quiescent cell inhibitor.

Cell membranes prepared from senescent human diploid fibroblasts (HDF) inhibited entry into S phase by 35% when added to the medium of replicating young HDF. This membrane-associated inhibitory activity was (i) sensitive to trypsin, heat, and periodate, which suggests that the inhibitor is a glycoprotein, and (ii) not able to inhibit DNA synthesis in simian virus 40-transformed HDF, which indicates that not all types of cells are sensitive to this inhibitor. Quiescent young HDF also have a surface membrane-associated inhibitor of DNA synthesis. A comparison of the senescent HDF and quiescent HDF inhibitor activities indicates that they may have the same chemical and physical nature and the same specific activity, but their regulation is different. The inhibitory activity of quiescent young HDF is abolished within 20 hr after refeeding with fresh serum-containing medium, whereas that of senescent HDF remains unchanged. Quiescent old HDF (two or three population doublings remaining) exhibit an intermediate response to serum with approximately two-thirds of the inhibitory activity abolished. The fraction of cells in S phase at 20-24 hr post-stimulation (37% in young HDF, 24% in old HDF, and 0% in senescent HDF) is inversely proportional to inhibitor levels. This suggests that inability to neutralize the inhibitory activity in response to serum stimulation could be involved in the inability of senescent HDF to enter S phase. Disappearance of the inhibitory activity from quiescent young HDF occurs late in G1 phase. Thus, the inhibitor may play a role in determining the length of the G0 to S phase transition in these cells.

Quiescent human diploid fibroblasts: Common mechanism for inhibition of DNA replication in density-inhibited and serum-deprived cells

The mechanism for cessation of proliferation in density-inhibited quiescent human diploid fibroblasts (HDF) and serum-deprived quiescent HDF was compared in two ways. Density-inhibited HDF were fused to either replicating HDF or SV40-transformed HDF and DNA synthesis was measured in the resulting heterokaryons. DNA synthesis was inhibited in the replicating HDF nuclei in heterokaryons in a way that suggested that entry into S phase was blocked, but ongoing DNA synthesis was not inhibited. In contrast, DNA synthesis was induced in the quiescent nuclei in heterokaryons formed with SV40-transformed HDF. Previous experiments had shown that serum-deprived HDF also behave in this way in heterokaryons. To test this similarity further, we examined the inhibitory activity of cell membranes prepared from both types of quiescent HDF. We found that both types of quiescent HDF contain DNA synthesis-inhibitory activity that is (1) effective on replicating HDF; (2) ineffective on SV40-transformed HDF; (3) sensitive to heat and trypsin. Thus, these results support the hypothesis that both density-inhibited HDF and serum-deprived HDF share a common mechanism for arrest in G1 phase. They also suggest that a membrane-bound protein plays a role in the inhibition of DNA synthesis in quiescent HDF.

Human diploid fibroblasts (HDF) can induce DNA synthesis in cycling HDF but not in quiescent HDF or senescent HDF

HeLa cells in S phase induce DNA synthesis in cycling cells, serum-deprived quiescent cells, and non-replicative senescent cells following cell fusion. In contrast normal human diploid fibroblasts (HDF) do not induce DNA synthesis in either quiescent cells or senescent cells. Instead, the replicative HDF nuclei are inhibited from entering S phase in heterokaryons formed with these two types of non-replicative cells. These differences in the inducing capabilities of normal HDF and HeLa cells raise the question whether normal HDF in S phase can induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Thus, the hypothesis that initiation of DNA synthesis in cycling cells is positively controlled by inducer molecules appears to be valid for normal HDF as well as for transformed cells such as HeLa.

Inducers of DNA synthesis: levels higher in transformed cells than in normal cells.

The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.

Human platelet-derived growth factor stimulates amino acid transport and protein synthesis by human diploid fibroblasts in plasma-free media.

Purified human platelet-derived growth factor (PDGF) induces an increase in amino acid uptake via system A in quiescent human diploid fibroblasts. Cells must be exposed to PDGF for 45 min to obtain maximum transport stimulation. Transport stimulation requires protein synthesis. Transient exposure to PDGF, alone, in the absence of plasma components can stimulate transport. Acid-insoluble [3H]leucine incorporation is also stimulated by PDGF treatment, and this event also does not require the presence of plasma components. Finally, antiserum to PDGF that blocks PDGF-stimulated DNA synthesis in these cells also blocks PDGF-stimulated amino acid uptake and protein synthesis. Increased amino acid uptake and protein synthesis that occur soon after addition of fresh serum to quiescent cells can be attributed to the action of PDGF acting alone and should be useful as markers for the investigation of early cellular events caused by PDGF.