The proportion of cells labeled with tritiated thymidine as a function of population doubling level in cultures of fetal, adult, mutant, and tumor origin

112

133

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. overlapping-pathway screen ͉ terminal cell differentiation ͉ senescent phenotype ͉ interferon-inducible gene ͉ evolutionary conserved gene P lasticity of the transformed phenotype is suggested by the ability of differentiation-inducing agents to revert the cancerous properties of specific tumors (1-3). This attribute of tumor cells provides the basis for a potentially less toxic form of therapy, ''differentiation therapy.'' In metastatic human melanoma, a combination of IFN-␤ and the protein kinase C activator mezerein (MEZ) produces irreversible growth arrest, a loss of tumorigenic competence, and terminal differentiation (1, 4). To define gene-expression changes associated with induction of terminal differentiation, a subtracted cDNA library enriched for genes associated with terminal differentiation was constructed (5). This construction was accomplished by subtracting control HO-1 human melanoma mRNAs from IFN-␤ ϩ MEZ-treated HO-1 mRNAs, which were temporally collected over a 24-h period (5). This subtracted cDNA library then was screened by random isolation of phage colonies and Northern blotting, high-density cDNA microarray analysis, and reverse Northern screening followed by Northern blotting (5-7). These approaches have identified both unknown and known genes associated with tumor and normal growth control, cell-cycle regulation, IFN response, differentiation, and apoptosis (5-12). Four classes of melanoma differentiation-associated (mda) genes have been identified (5, 10).Terminal cell differentiation and cellular senescence are characterized by changes in cell morphology, lack of responsiveness to mitogenic stimulation, and irreversible growth arrest (1, 4, 13-18).Normal cells cultured in vitro lose their proliferative potential after a finite number of doublings in a process described as cellular senescence (13). Experiments in human diploid fibroblasts and additional cell types document an inverse correlation between replicative senescence and donor age and a direct relationship between replicative senescence and donor-species life span (13,19,20). In agreement with this relationship, cells from patients with premature aging syndromes such as Werner's syndrome and progeria achieve a quiescent state more rapidly than normal human fibroblasts (21). Although senescence is a time-dependent process, terminal differentiation can be induced in a variety of cell types by appropriate treatme...

Section: Opsmentioning

confidence: 99%

Identification and cloning of human polynucleotide phosphorylase, hPNPase ^old-35 , in the context of terminal differentiation and cellular senescence

Leszczyniecka

Kang

Sarkar

et al. 2002

112

133

“…[3H]thymidine and culture age (13). Theoretical studies have predicted that the distribution of CSs obtained from human fibroblast cultures would be a sensitive index of the number of in vitro PDR (in vitro cell culture "age") (14).…”

mentioning

confidence: 99%

Colony size distributions as a measure of in vivo and in vitro aging.

Smith¹,

Pereira‐Smith²,

Schneider³

1978

142

Recently it has been demonstrated that cell cultures from older human subjects, even during their first few in vitro passages, exhibit diminished replicative ability when compared to parallel cultures derived from young subjects (5). However, these studies have examined the replicative behavior of cell populations. Because human diploid cell cultures are heterogeneous in nature, it is also vital to examine the proliferative behavior of human cells as a function of aging at the level of the individual cell Recent studies on human fetal lung cells (WI-38) have indicated that the distribution of sizes of colonies derived from single cells may be an excellent indicator of the total proliferative capacity (in vitro life-span) of human diploid cell cultures (8).The studies described in this report compare the behavior of cloned human adult skin fibroblasts and fetal lung fibroblasts. In addition, the relationship between colony size distribution (CSD) and in vivo donor age is evaluated. MATERIALS AND METHODSCell cultures were established from 2-mm skin biopsies performed on nonhospitalized volunteer members of the Baltimore Longitudinal Study as described (5). Cell cultures from old (>65 years) and young (20-5 years) donors were examined after 5-15 population doublings (PDs) in vitro.The in vitro life-spans of these cell lines were determined at the Gerontology Research Center by described techniques (5). At low in vitro population doubling level (PDL), frozen stocks of these cultures were prepared by resuspending trypsinized cell suspensions in Eagle's minimal essential medium containing 10% fetal bovine serum and 5% dimethyl sulfoxide and cooled to the temperature of liquid nitrogen at a rate of '14/min.These frozen stocks of adult human skin fibroblasts were rapidly thawed in a 370 water bath, returned to tissue culture, and sent to the W. Alton Jones Cell Science Center where the colony size distributions were determined. Mass cultures were maintained in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate-buffered Eagle's basal medium with 10% fetal bovine serum (Rehatuin, Reheis Chemical Co., Kankakee, IL).All clonal growth experiments were performed in MCDB 102 which was prepared according to the procedures of Ham et al. (9). Each freshly prepared batch of MCDB 102 was tested for its ability to support clonal growth of human diploid fibroblasts at between the 20th and 30th PD. Only those batches that gave results essentially equivalent to those reported previously (9) were used. The lot of fetal bovine serum used was prescreened for its ability to promote clonal growth of WI-38 cells in Eagle's basal medium.Rapidly proliferating cultures were trypsinized prior to confluency. The cultures growing in 25-cm2 flasks were rinsed once with 5 ml of buffered medium without serum and washed with 5 ml of 0.25% crude trypsin in Eagle's basal medium. All except 0. The clonal plates were incubated undisturbed for 14 days at 370 in a 5% C02/95% air atmosphere at 98-100% relative humidity.A 2-week incubation period was ch...

“…Studies based upon clone size analysis indicate an increased number of nondividing cells with advancing population doubling level (PDL) (11,12); however, this result has been criticized as possibly related to cloning conditions not present in mass culture (13). Autoradiographic analysis of HDFC cultures has yielded results that have been interpreted as showing no noncycling population (14), noncycling cells arising only in the last 10 population doubling levels (15), or proportions of noncycling cells progressively increasing with age (16). The inconsistencies are presumably related to problems with method (13) and may be explained in part by the use of [3H]thymine pulse periods that are too short to label slowly dividing cells, or the proliferation of labeled cells during the labeling interval.…”

mentioning

confidence: 99%

Regulation of human fibroblast growth rate by both noncycling cell fraction transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry.

Rabinovitch¹

1983

110

Growth of human diploid fibroblasts in the presence of 5-bromodeoxyuridine, followed by flow cytometric analysis of DNA-specific fluorescence with Hoechst 33258 dye, allows quantitation of the proportion of cells that have not cycled, as well as those in G1 and G2 of two subsequent cell cycles. This technique allows rapid and accurate quantitation of the growth fraction and G1/S transition rate of these cells. The cell cycle kinetics of human diploid fibroblasts at all population doubling levels reveal two components: cycling cells showing a probabilistic rate of G1/S transition, and a variable proportion of noncycling cells. Both the transition probability (rate of exit from G1) and the noncycling proportion of cells change systematically as a function of serum concentration and as a function of population doubling level. The data suggest the existence of an underlying heterogeneity in the population of human diploid fibroblasts with respect to the capacity to divide in the presence of a given concentration of mitogen. Models of cell cycle kinetics must be modified to include regulation of growth by changes in the fraction of cycling cells, as well as by changes in the rate of emit from G1.Heterogeneity in interdivisional times is a common and welldocumented feature of the proliferative behavior of cultured mammalian cells, and whereas S, G2, and M phases are of relatively constant duration, the G1 phase length is highly variable. Of the proposals advanced to explain this variability, one that has been shown to closely fit most experimental data is the transition probability model of Smith and Martin (1). In this model cells remain in a subset of G1 (the "A phase") for a variable length of time, leaving this state with a constant probability of transition per unit time (P), to then complete G1 (the "B phase"). (17), who compensated for cell proliferation by counting cell numbers at the start and end of the experiment. They concluded that noncycling cells are indeed present and progressively increase with culture age. With the objective of performing a more detailed study of these cellular kinetics, we have adapted a technique of flow cytometric assay of growth kinetics based upon Hoechst dye staining of cells grown in 5-bromodeoxyuridine (BrdUrd) (18)(19)(20). The increased ease and accuracy of this technique compared to conventional methods allows an analysis demonstrating that changes in the proliferative rate of HDFC cultures are a result of alterations in both the fraction of noncycling cells and the transition probability of the remaining cycling cells. MATERIALS AND METHODSCell Strains and Culture. HDFC strain 79-81 was explanted from a skin biopsy sample of a normal 27-year-old male, and strain 78-18 was derived from a skin biopsy sample of a 20-week gestational age, karyotypically normal female abortus. Cells were grown as described (21) 2951The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accord...