Cumulative Population Doublings Research Articles

Clonal Variation in Phenotype and Life Span of Human Embryonic Fibroblasts (MRC-5) Transduced with the Catalytic Component of Telomerase (hTERT)

Expression of telomerase (hTERT) in certain cell types has been shown to extend cellular life span without malignant transformation. We studied the phenotype of 26 telomerase-transduced fibroblast clones (TTFC) generated from a mass culture of hTERT retrovirally transduced MRC-5 cells. About two-thirds of the transduced clones senesced at the expected time or shortly thereafter, despite high levels of expression of telomerase and telomere length maintenance. The remaining one-third of the clones were “immortalized” (followed for over 200 cumulative population doublings ). All clones maintained a nontransformed phenotype: contact inhibition, anchorage dependency, lack of tumor formation in nude mice, dose dependency to serum and growth factors, low expression of a matrix metalloproteinase associated with metastatic invasion (MMP-9) and high expression of its inhibitor TIMP-1, and no cytogenetic abnormalities by G-banding. In addition, fibroblast-specific biological parameters, such as colony size, production of collagenase, and response to MMC and gamma radiation were tightly regulated at the clonal and subclonal levels.

Energetic stress induces premature aging of diploid human fibroblasts (Wi-38) in vitro

Oxidative phosphorylation is the main endogenous source for the generation of reactive oxygen species (ROS). In order to investigate the influence of enhanced ROS production on the in vitro senescence of Wi-38 fibroblasts, cells were cultivated in medium with elevated (hypertonic) NaCl concentrations. The number of active Na +/K +-ATPase molecules per cell was found to be increased. A rise in both respiration and glycolysis as evidenced by the increases in oxygen and glucose consumption and lactate production was revealed. Cells stayed alive in medium with NaCl concentrations of up to 0.30 M and could be adapted to growth under these hypertonic conditions (high-NaCl tolerant cells). These cells exhibited an increased cell size and protein content. A growing number of cells showed stress fibers and granulation. The proliferation rate and the maximum number of cumulative population doublings of these high-NaCl tolerant cultures were reduced and saturation density was decreased. Thus, these cells under energetic stress due to increased energy requirements for active ion transport expressed features typical for aging in vitro. We conclude therefore that energetic stress induces premature aging in human diploid fibroblasts.

Does MMP-2 expression and secretion change with increasing serial passage of keratocytes in culture?

The effects of ageing on matrix metalloprotease degradation of the extracellular matrix during corneal wound healing are largely unknown. The following study used an in vitro model of ageing to assess changes in MMP-2 RNA expression and protein secretion. Early passage (EP) EK1.BR keratocyte cultures from 14 to 18 cumulative population doublings (cpds) and late passage (LP) cultures from 40 to 47 cpds were used to isolate protein and mRNA samples. Total protein from EP and LP cultures was measured using the Bradford protein assay. Zymographic analysis of EP and LP samples was carried out to compare MMP-2 activity. Northern blot analysis was used to assess changes in MMP-2 mRNA expression by EP and LP cultures, using a digoxigenin (DIG) based chemiluminescent detection system. LP cultures secreted more total protein per cell. MMP-2 but not MMP-9 activity was detected in keratocyte cultures. Densitometric analysis of zymograms and calculation of MMP-2 activity indicated a significant increase in MMP-2 activity per cell (P<0.05, n=11). No difference was observed in the levels of MMP-2 mRNA expressed by EP and LP cultures. An increase in MMP-2 activity per cell by LP cultures suggests that senescent keratocytes increase their degradative capacity. Similar changes in the keratocyte phenotype within the ageing cornea may alter the balanced response necessary for adequate wound healing and may have implications for the therapeutic use of MMP inhibitors in the eye.

MRNA levels of the linker histone variant, H1o, in mitotically active human diploid fibroblasts as a function of the phases of the cell cycle and cumulative population doublings

Senescence and differentiation have many similarities with respect to certain aspects of gene expression and cell cycle related events. One linker histone variant tightly associated with differentiation is the H1 variant, H1o. The work of this investigation has focused on the expression of H1o during the phases of the cell cycle and as a function of increasing cumulative population doublings (CPD) in an in vitro model ageing cell system, namely, human diploid fibroblasts. Increased H1o mRNA levels were found during the S phase of the cell cycle contrary to H1o protein relative synthesis rates, which were found to be increased during the Go phase of the cell cycle. These results were obtained in actively proliferating cell populations. However when the proliferative rate of the overall population begins to drop (CPD 50), H1o mRNA levels tend to remain stable throughout the Go, G1 and S phases. On the other hand, no changes in the H1o relative synthesis rates were found as a function of increasing CPD. Uncoupling of H1o protein and mRNA levels has been observed in numerous differentiating systems. The analogous mode in which H1o gene expression is regulated in both these two systems reinforces the opinion that senescence and differentiation may have similarities at the level of chromatin remodelling.

A serum-free medium formulation supporting growth of human umbilical cord vein endothelial cells in long-term cultivation.

A serum-free medium formulation - TUD-1 - was developed supporting growth of HUVEC in tissue culture. Special features of the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences in final cell density compared to controls cultivated with serum.

A human breast epithelial cell type with stem cell characteristics as target cells for carcinogenesis.

Two types of human breast epithelial cells (HBEC) have been characterized. In contrast to Type II HBEC, which express basal epithelial cell phenotypes, Type I HBEC are deficient in gap junctional intercellular communication and are capable of anchorage-independent growth and of expressing luminal epithelial cell markers, estrogen receptors, and stem cell characteristics (i.e. the ability to differentiate into other cell types and to form budding/ductal organoids on Matrigel). A comparative study of these two types of cells has revealed a high susceptibility of Type I HBEC to immortalization by SV40 large T antigen, although both types of cells are equally capable of acquiring an extended life span (bypassing senescence) after transfection with SV40. The immortalization was accompanied by elevation of a low level of telomerase activity in the parental cells after mid-passage ( approximately 60 cumulative population doubling levels). Thus HBEC do have a low level of telomerase activity, and Type I HBEC with stem cell characteristics are more susceptible to telomerase activation and immortalization, a mechanism which might qualify them as target cells for breast carcinogenesis. The immortalized Type I HBEC can be converted to highly tumorigenic cells by further treatment with X rays (2 Gy x 2) and transfection with a mutated ERBB2 (also known as NEU) oncogene, resulting in the expression of p185(ERBB2) which is tyrosine phosphorylated.

Effects of ageing on proliferative ability, and the expressions of secreted protein, acidic and rich in cysteine (SPARC) and osteoprotegerin (osteoclastogenesis inhibitory factor) in cultures of human periodontal ligament cells

Secreted protein, acidic and rich in cysteine (SPARC) has been suggested to play an important role in wound repair and mineralization. Osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) is a secreted protein that inhibits the maturation, activity and survival of osteoclasts. An examination of the changes in expression of these proteins as well as their proliferative ability according to ageing in cultured cells may elucidate characteristic changes in periodontal tissues induced by ageing. In the present study, proliferative ability and the expression of SPARC and OPG/OCIF were compared between cultures of human periodontal ligament (HPL) cells obtained from young and senior donors (in vivo cellular ageing) as well as in cultures of HPL cells at early and late passages (in vitro cellular ageing). Cumulative population doubling levels and cell population doubling time of HPL cells from young donors were greater and shorter, respectively, than those of HPL cells from senior donors. Levels of SPARC mRNA in HPL cells increased with cellular ageing in vivo. No change in the levels of OPG/OCIF mRNA in HPL cells with cellular ageing in vivo was observed. The changes in proliferative ability and the mRNA levels of SPARC and OPG/OCIF with cellular ageing in vitro were similar to those with ageing in vivo. This study demonstrated for the first time a relationship between in vivo and in vitro cellular ageing in the functional changes in HPL cells. These findings suggest that the impairment of periodontal ligament repair with ageing is due to the decrease in proliferative ability in HPL cells with ageing. Furthermore, the increase in SPARC with ageing may be related to changes in metabolism of periodontal ligament that occur with ageing.

Accumulation of Short Telomeres in Human Fibroblasts Prior to Replicative Senescence

The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1–2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5–1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence.

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 μM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated β-galactosidase activity, overexpression of p21 Waf-1/SDI-1/Cip1, and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 μM t-BHP or a single stress under 450 μM H 2O 2. These corresponding genes include fibronectin, osteonectin, α1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-α binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Histone variants of H2A and H3 families are regulated during in vitro aging in the same manner as during differentiation.

In a previous communication, we showed that the H2A.1/H2A.2 histone variant ratio decreases in a linear manner during the in vitro aging of human diploid fibroblasts. This ratio is known to decrease in the same manner in progressive stages of development and in the process of differentiation, and is thus considered to be a biochemical marker for differentiation. A detailed analysis of the synthesis of H2A and H3 histone variants as a function of cumulative population doublings in the same in vitro cell system is presented in this study. Quantitative analysis of these variants in the G0 phase, synchronized fibroblasts has shown that their relative amount in chromatin, as well as their biosynthesis rate, change during in vitro aging of human diploid fibroblasts, revealing both up-and down-regulation of certain variants as a function of cumulative population doublings. Furthermore, we show by morphometric studies employing the seven distinct fibroblast morphotypes, as described by the Bayreuther classification, that this regulation is attributable to the replicative sub-populations. These results reveal that histone variants of the H2A and H3 families are regulated during in vitro aging in the same manner as that during differentiation.

Long‐term effects of repetitive exposure to a static magnetic field (1.5 T) on proliferation of human fetal lung fibroblasts

The aim of the study was to assess the effects of repetitive exposures to a static magnetic field (1.5 T) on human fetal lung fibroblast (HFL) proliferation. HFL were exposed three times a week for 1 hr to a static magnetic field for 3 weeks. Cells were subcultured every week. Population doublings (PD) and cumulative population doublings (CPD) were calculated weekly. Colony formation assays, bromodeoxyuridine enzyme-linked immunosorbent assay, and cell cycle analysis were performed weekly. After the third week, proliferation kinetics were assessed. Over a period of 3 weeks no statistically significant differences between the PD and CPD of exposed and control cells could be detected. Clonogenic activity, DNA synthesis, cell cycle, and proliferation kinetics were not altered by magnetic field exposure. The data do not provide evidence that repetitive exposures to a static magnetic field (1.5 T) exert effects on HFL proliferation. Magn Reson Med 41:464–468, 1999. © 1999 Wiley-Liss, Inc.

OSTEOGENESIS IN VITRO IN RAT TIBIA-DERIVED OSTEOBLASTS IS PROMOTED BY THE HOMEOPATHIC PREPARATION, FMS*CALCIUMFLUOR

We studied the effects of in vitro treatment of differentiating osteogenic cells with FMS*Calciumfluor, to determine whether it caused changes in proliferative or differentiation potential of osteoblasts. FMS*Calciumfluor was developed for the therapy of post-menopausal and age-related osteoporosis on the basis of the principles of resonance homeopathy and VTR Vega test. Its daily prescribed therapeutical usage is about 30,000-fold less in fluoride concentration than that recommended for NaF associated with calcium monophosphate. Rat tibial osteoblast (ROB) primary cultures represent populations of early osteoblasts and their derivative cultures of more than 60 cumulative population doubling (CPD) represent more mature osteogenic cells. Both these populations were shown to undergo in vitro differentiation, as monitored by the sequential expression of markers that define the stages of the osteogenic progression. Here we report that continual treatment of ROB during osteogenesis with FMS*Calciumfluor modulated the expression of critical osteogenic markers: alkaline phosphatase (AP), an indicator of osteoblast maturation, and45Ca incorporation into the matrix and nodule formation, events of the last phase of osteogenesis and a measure of osteoid mineralization. Treatment did not affect proliferation, or expression and activation of metalloproteinases (MMP). AP activity and levels of AP mRNA were increased by treatment with FMS*Calciumfluor; the incorporation of radiolabelled Ca into the matrix was also increased and the formation of nodules occurred in a shorter time and with higher frequency than in untreated control cultures. The effects of FMS*Calciumfluor were concentration dependent and specific for its modalities of preparation, and were observed at a concentration about three orders of magnitude lower than similar effects reported in the literature by treatment of osteoblast cultures in vitro with NaF.

T-lymphocyte long-term cultures have a constant histone variant pattern during aging.

Human peripheral long-term T-lymphocyte cell cultures show some characteristics similar to those of fibroblast cell lines, the latter of which have been used as in vitro systems for cellular aging studies for many years. Both show a limited in vitro life span, as well as a progressive prolongation of their cell cycle with increasing age. However, whereas T-cell cultures die from apoptosis at the end of their proliferative capacity, fibroblasts can be maintained for long periods of time in stationary cultures as postmitotic senescent cells. Previous studies analyzing the histone variant pattern of a human lung embryonic fibroblast cell line have shown that this pattern changes as a function of cumulative population doublings in a manner not unlike that found in terminally differentiating systems. In the present study the histone variant composition of long-term T-cell cultures was analyzed as a function of population doublings and compared to a human diploid fibroblast system. The results from this study provide a distinction at the molecular level among these two in vitro aging model systems, because it was found that long-term T-cell cultures show a constant histone variant constitution throughout their in vitro life, dissimilar to previous findings using the fibroblast cell system.

DNA Topoisomerase Inhibitors Induce Reversible Senescence in Normal Human Fibroblasts

Inhibitors of DNA topoisomerases I and II induced arrest in cell division in normal human fibroblasts depending on cell divisions. Arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated β-galactosidase. In these cells, p16ink4awas upregulated, whereas p21waf1or p53 was not altered. Upon removal of the inhibitors, the cells resumed growth but their cumulative population doublings were reduced dose dependently. Accelerated telomere shortening was not observed in the arrested cells. These results suggest that DNA topoisomerase inhibitors are efficient and reversible inducers of premature senescence in normal human cells.

Amino acid depletion modulates vascular endothelial growth factor production during the life span of human vascular smooth muscle cells.

The role of nutrient supply in the replicative capacity and secretory phenotype of cultured human diploid cells is unclear. We examined the relationship between amino acid privation, the secretion of vascular endothelial growth factor (VEGF) and growth phenotype of vascular smooth muscle cells (VSMC), and endothelial cells. Cultures of VSMCs, but not endothelial cells, were growth inhibited by exposure to medium that was 75% deficient in leucine, methionine, arginine, and cysteine over two passages. Exposed VSMC cultures exhibited an increased vulnerability to apoptosis. The maximal cumulative population doubling of the exposed cells was reduced significantly compared with the control cells (25.7 +/- 2.0 doublings vs. 27.9 +/- 2.1 doublings; P < 0.03). Constitutive VEGF production first became evident in the later passages of the exposed and nonexposed cell cultures. However, production of VEGF was 17-fold greater in the exposed cultures at the tenth passage (P < 0.001). The replicative capacity and constitutive production of VEGF in VSMCs in culture may be programmed by transient privation of amino acids. These observations are relevant to new concepts concerning the pathogenesis of vascular disease.

The regenerative capacity of adult human hepatocytes: clonal expansion of hepatocytes producing albumin in culture

Upon injury, the liver has the capability to replace lost hepatocytes by at least two processes. In the unipotential model hepatocytes exit quiescence after injury and replicate, while in the multipotential model non-parenchymal epithelial cells replicate and differentiate into mature hepatocytes. To test the former process, we cloned normal adult human hepatocytes expressing differentiated function, and monitored the continued expression of these differentiated functions as they proliferated and expanded in cell culture. Fifty-eight cell clones, derived from two different normal adult human liver primary cell lines, expressing albumin and cytokeratin 18, but not cytokeratin 19, as markers of hepatocyte differentiation, were isolated. These cell clones were serially subpassaged, five to eight times, and the expression of hepatocyte markers was determined using immunocytochemistry. Of the 58 clones isolated, 56 (96%) were positive for both albumin and cytokeratin 18, and two were negative for albumin but positive for cytokeratin 18. Upon subpassage of the clones, the cells continued to expand before senescence at a cumulative population doubling of 27.1±0.8. All of the cell strains, originating from clones producing albumin and cytokeratin 18, continued to express these two markers through senescence. The data indicate that mature normal human hepatocytes are capable of producing progeny hepatocytes, supporting the unipotential model for replacement of hepatocytes in the human liver.

Repeated mild heat shock delays ageing in cultured human skin fibroblasts.

The effects of repetitive mild heat shock (30 min, 41 degrees C) on growth and various cellular and biochemical characteristics of human skin fibroblasts undergoing ageing in vitro were analysed. Human skin cells not only tolerated more than 30 repeated heat shocks throughout their replicative lifespan, but also maintained several characteristics of young cells until late in life. Whereas the growth rates, population doubling rates, and cumulative population doubling levels achieved in vitro remained unaffected, age-related changes in cellular morphology, cell size, cytoskeletal organisation, autofluorescence and neutral beta-galactosidase activity were significantly slowed down by repeated mild heat shock. These hormesis-like effects of stress-induced defence processes can be useful to elucidate the role of maintenance and repair mechanisms in ageing.

Cytochemical Detection of a Senescence-Associated β-Galactosidase in Endothelial and Smooth Muscle Cells from Human and Rabbit Blood Vessels

A β-galactosidase activity has recently been used as a histochemical marker of replicative senescence in human fibroblasts and keratinocytes. To establish whether this marker could be used to detect senescence of vascular cells, we have investigated its presence in cultures of serially passaged human umbilical vein endothelial cells and rabbit aortic smooth muscle cells. β-Galactosidase activity was detected by light microscopy using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside. In endothelial cell cultures, lysosomal β-galactosidase activity, which is detected at pH 4.0, was present in all cells regardless of their replicative age. In contrast, senescence-associated β-galactosidase activity, which is detected at pH 6.0, was absent in the majority of cells in early passage cultures (<15 cumulative population doublings), but was present in a large proportion of cells (up to 62%) in late passage cultures (>30 cumulative population doublings); in intermediate passage cultures (15–30 cumulative population doublings) it was found in fewer than 15% of the cells. The increase in the percentage of senescence-associated β-galactosidase-positive cells correlated with a decrease in the cell density at confluence and with a marked increase in cell size. Counterstaining with an antibody directed against the endothelial cell marker CD31 showed that senescent cells retained the expression of this antigen. Senescence-associated β-galactosidase was also detected in serially passaged, but not in primary explant cultures of rabbit aortic vascular smooth muscle cells. The presence of senescence-associated β-galactosidase in cultured vascular smooth muscle cells and endothelial cells suggests that this marker could be used to study the role of cellular senescence in vascular disease.

Accelerated Telomere Shortening in Fibroblasts After Extended Periods of Confluency

Telomere length in MRC-5 fibroblasts remains constant if the cells are proliferation-inhibited for up to 3 months by confluency. However, the apparent frequency of single-stranded sites in telomeres, measured as sensitivity to degradation by S1 nuclease, increases about fourfold during this extended inhibition of proliferation. After release of the cells, the frequency of telomeric single-stranded sites decreases to control values, and the telomere shortening rate increases about threefold as compared to controls proliferating without inhibition. This acceleration is transitory, the telomere shortening rate decreases to control values after about two population doublings after release. Finally, temporarily arrested fibroblast populations senesce at a lower cumulative population doubling level, but at about the same telomere length, as continuously proliferating controls. The data suggest that metabolic time-dependent single-strand degradation is a major cause of telomere shortening. They support the idea that telomere shortening plays an important role in triggering cellular senescence.

Accelerated Proliferative Senescence of Rat Embryo Fibroblasts after Stable Transfection of Multiple Copies of the c-Myc DNA-Binding Sequence

The protooncogene c-myc positively regulates cellular proliferation whereas it exhibits negative effects on both cellular senescence and differentiation. Ectopic overexpression of c-myc in transfection experiments or titration of the c-myc mRNA by antisense oligonucleotides has demonstrated that small changes of the concentration of cellular c-myc mRNA or protein levels can be crucial for these processes. In view of the role of c-Myc as a transcription factor, most of these effects may be mediated via its binding to specific DNA sequences. Here we studied the cellular reactions after manipulating the cellular concentration of c-Myc DNA-binding sites. Multiple copies of the c-Myc-binding sequence GACCACGTGGTC or, alternatively, the control sequence GACCAGCTGGTC that displays only a poor affinity for c-Myc were stably introduced into the genome of rat embryo fibroblasts. Transfection with the c-Myc-binding sequence yielded much lower clone numbers and sizes than transfection with the control sequence. After polyclonal selection and further subcultivation cells transfected with c-Myc-binding sequence exhibited a reduced growth rate and achieved less than two-thirds of the cumulative population doublings before becoming senescent and irreversibly growth arrested compared to the controls. Southern blot analysis demonstrated that 30 binding sequences on average were integrated into the cellular genome. Our results can be interpreted as competition of the ectopically introduced c-Myc-binding sequences with the functional genomic ones and assume that a fairly low number of the latter exist in the normal cellular genome. Hence, only a low copy number of introduced c-Myc-binding sequences is sufficient to cause signs of accelerated proliferative senescence.