Aging in vitro and D-glucose uptake kinetics of diploid human fibroblasts

By use of a rapid technique, initial rates of D-glucose transport were obtained during the lifespan in vitro of a commercially available strain of human embryo lung fibroblasts (Flow 2000). The apparent Km of the D-glucose carrier did not change during senescence in vitro: x̄ = 1.8 mM (range 1.3–2.3) in phase II, x̄ = 1.8 mM (range 1.5–2.2) in phase III. Transport rates remained constant in stationary phase II cultures, which had completed between 30% and 80% of their replicative lifespan. A wide variation, however, was observed in terminally differentiated cells (phase III), which showed a two- to threefold increase in average cell size and protein content. In some senescent cultures, glucose transport calculated on a per cell basis was also two-to threefold increased, while it was strongly decreased (-75%) in others. When calculated per unit of cell water, protein, and surface area, respectively, transport rates in phase III cultures ranged from values established for stationary phase II cultures down to very low values. Detaching cells flushed off from senescent cultures did not show measurable rates of glucose transport into the inulin impermeable cell space. Present evidence argues against the idea that an impairment of D-glucose transport might precede loss of replicative potential in aging human fibroblasts. Instead our data indicate that the transport capacity of cell membrane finally decreases during postreplicative senescence in terminally differentiated cells.     

Aging of chick embryo fibroblasts in vitro : III. Polyploid cell accumulation

The accumulation of polyploid cells during the lifespan of chick embryo fibroblasts was examined. The nuclear area of each stained nucleus in the chick cells is virtually proportional to its relative DNA content. Changes in mean nuclear area of the cells with advancing aging were observed. The mean nuclear area increases at the latest culture stage when growth rate notably declines, and their increase follows an increase in multinuclear cells. In the senescent cells, there are the ploidy classes of 2C, 4C, 8C, 16C, 32C and 64C, being defined as 2ⁿC. The mechanism of age-dependent polyploidization is also discussed.     

Aluminum ions induce DNA synthesis but not cell proliferation in human fibroblasts in vitro

The effect in vitro of aluminum (Al) ions on DNA synthesis and human dermal fibroblast proliferation using [Al] concentrations from 1.85 to 74 μM and incubation periods of 1, 2, 3, 4, and 5 d was assessed. The lowest concentration of Al that exerted a slight positive, although not significant, effect on DNA synthesis was 1.85 μM, after d 3 or 5 of incubation. The stimulating action of Al was more evident and statistically significant from concentrations of 3.7 μM and 2 d exposure onward. This Al-induced effect on [³H] thymidine incorporation into DNA increased in a time-dependent manner as [Al] in the culture medium rose, provoking increments of up to 322% above the control at [Al] 74 μM and 5 d incubation. In contrast, Al salts moderately increased fibroblast division in a continuous manner only from 7.4 to 74 μM after 3 d of incubation. Although significant overall, the minimal and inconstant mitogenic activity of Al differs greatly from and is not parallel to DNA synthesis, which is not clearly related to exposure times or Al concentrations. Abnormalities in Al-induced cellular metabolic processes described herein and their influence on the cell cycle may constitute a toxicity mechanism for human tissues, leading to disease development. Further studies are required to determine whether these findings can be extrapolated to in vivo situations.     

Interactions between human fibroblasts and HeLa cells in vitro

Affinity toward each other was demonstrated in co-cultures between HeLa cells and fibroblasts originating from human tumor stromal or normal tissues. Both cell types in the mixed cultures (ratio 1:1, 1:2, 2:1) proliferated normally as shown by 3H-thymidine labeling index estimation for up to 48 hr of co-culture. At ratios of fibroblasts: HeLa lower than 1:10, fibroblasts were eventually eliminated after serial passaging. It was shown that 3H-nucleotides could be transferred between heterologous cells in either direction. Contact of cells was essential for this phenomenon. Transfer of the label from HeLa to fibroblasts required a longer interaction time and was evidently lower than the transfer from fibroblasts to HeLa. 3H-thymidine incorporated into the DNA of either cell type could not be transferred from one cell to another. The model provides a means for studying neoplastic X normal (or tumour stromal) cell interactions in vitro.     

The relationship between pyrimidine dimers and replicating DNA in UV-irradiated human fibroblasts.

The relationship between pyrimidine dimers (measured as endonuclease-sensitive sites) and newly-synthesized DNA has been examined in several different ways, with the following results:- 1. After UV-irradiation of normal human fibroblasts the frequency of pyrimidine dimer sites in sections of DNA which have been synthesized immediately before the UV-irradiation is similar to that in the bulk DNA. 2. The frequency of pyrimidine dimer sites in the parental strands of replicating DNA in UV-irradiated normal human fibroblasts is similar to that in the bulk DNA. 3. In UV-irradiated XP variant cells the size of DNA synthesized in the presence of caffeine immediately after UV irradiation accurately corresponds with the average interdimer distance in the parental DNA. This suggests that in this experimental situation each pyrimidine dimer gives rise to a disocntinuity or a termination site in the daughter strand.     

Correlations between the coding and non-coding regions in DNA.

In this paper various aspects of codon usage and k-tuple correlations in the DNA are compared. It is shown that the correlation structures of the coding and the non-coding regions are very similar and that codon usage is reasonably specific for large groups of organisms. These results suggest that the origin of codon usage is related to the origin and structure of the DNA.     

Proliferative potential of human fibroblasts: an inverse dependence on cell size

Human foreskin fibroblast-like cells were separated on the basis of DNA content and cell size by fluorescence-activated cell sorting. Subpopulations of "large" or "small" cells with the same (G1) DNA content were clonally expanded and found to contain predominantly nondividing or highly proliferative cells, respectively. From the rate of clonal growth, we deduce that small cells divide faster than large cells. Intermediate-sized cells were found to yield primarily smaller ("attenuated") clones. The clonal data can be incorporated into a previously reported kinetic model of clonal attenuation. This version of the model postulates that small "stem" cells yield larger daughters which have only a limited proliferative potential. We also postulate that a progressive increase in cell size can account for the decreasing concentration of DNA polymerase alpha, which has been reported in older cultures.     

Aging of mesenchymal stem cell in vitro

Background  

A hot new topic in medical treatment is the use of mesenchymal stem cells (MSC) in therapy. The low frequency of this subpopulation of stem cells in bone marrow (BM) necessitates their in vitro expansion prior to clinical use. We evaluated the effect of long term culture on the senescence of these cells.     

Stimulation of DNA synthesis in human fibroblasts by thrombin.

Earlier work showed that thrombin stimulates proliferation of human fibroblasts in serumfree medium. This work demonstrates (1) that thrombin has to be prensent during most or all of the G1 period to ensure maximal DNA synthesis, (2) that DNA synthesis increases about three hours later after thrombin than after serum treatment, (3) that both thrombin and serum activate transport of uridine, D--2-deoxy-glucose and putrescine, (4) that thrombin is able to increase 3H-thymidine incorporation also in SV40 transformed human fibroblasts, in HeLa cells and in two continuous monkey cell lines.     

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro

The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [35S]methionine-labeled polypeptide pattern.     

Angiogenesis-inducing ability of human bladder epithelium cell lines and "spontaneously" transformed murine fibroblasts

Ten human bladder epithelium cell lines were tested for their ability to induce blood vessel formation after intradermal injection into irradiated ST/a mice. Cell lines that were shown to be tumorigenic in nude mice, were able to evoke angiogenesis of a higher intensity than nontumorigenic cell lines. No difference was observed between the angiogeneic ability of tumorigenic cells originating from tumors and from in vitro transformed urothelium of nontumor origin. Similarly the origin of nontumorigenic urothelial cell lines did not show any influence on their angiogeneic abilities, but nontumorigenic cell lines which had undergone "infinite growth transformation" exhibited a higher angiogeneic activity than nontumorigenic cell lines with a finite life. The angiogeneic reaction evoked by human bladder epithelium cell lines showed cell dose- and time-dependence; but it was unrelated to the growth potential of the cultured cells. Two "spontaneously" altered sarcoma-producing murine cell lines showed a higher angiogeneic activity than tumorigenic human bladder epithelial cells. The angiogeneic response to these two murine cell lines was unrelated to morphological signs of transformation and to differences in growth rate, serum requirement, saturation density, anchorage dependence, and isoimmunizing properties.     

Stimulation by thyroid hormone analogues of red blood cell Ca2+-ATPase activity in vitro. Correlations between hormone structure and biological activity in a human cell system

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations (10(-10) M) of L-thyroxine (L-T4) and 3,5,3'-triiodo-L-thyronine (L-T3). This human cell system has been utilized to examine a series of iodothyronine and iodotyrosine analogues for structure-activity relationships. Analogue purity was verified by high pressure liquid chromatography. Analogues were studied at a concentration of 10(-10) M and the stimulatory effect of each analogue was compared with that of L-T4 in this system. Essential to Ca2+-ATPase stimulation were occupation of the 3 and 5 phenyl positions by iodide, bromide, or methyl groups, the L-configuration of the alanine side chain, side chain length equal to that of alanine, and a perpendicular (skewed) conformation of the two rings. The 4'-hydroxyl group is not essential to Ca2+-ATPase stimulation in this model system. T3 was 76% as active as T4 in stimulating Ca2+-ATPase activity. The stimulatory effect of 3,5-dimethyl-3'-isopropyl-L-thyronine and 3,5,3',5'-tetrabromo-L-thyronine approximated that of L-T4. Selected tyrosine analogues also stimulated the enzyme. The bioactivities of hormone analogues in this human model of extra-nuclear thyroid hormone action differ in several ways from results obtained previously in other animal model systems in vitro and in vivo.     

Measurement of DNA content and cell volume in senescent human fibroblasts utilizing flow multiparameter single cell analysis

Cell cycle analysis of senescent cultured human fibroblasts by flow cytofluorometry reveals an increased proportion of cells in the G 1 period. An increased variation as well as a slight decrease in cellular DNA contents were observed in both G 1 and G 2 + M senescent cells. Utilizing gated single parameter analysis, the increased cell volumes observed in these senescent fibroblasts were demonstrated to be present in G 1 as well as G2 + M cells.     

Aging of chick embryo fibroblasts in vitro. V. Time course studies on polyploid nucleus accumulation

Age-dependent polyploidization of cultured chick embryo fibroblasts was quantitated using flow microfluorometry. The results confirm the previous observation that ploidy classes developing as a function of fibroblast population doubling are defined as 2ⁿC. Immediately after isolation from embryos, the proportion of 2C nuclei was 95.2–35.7%, decreasing with advancing in vitro age. The proportion of 4C nuclei was only 3.8% at the onset of culture, increasing to 34.5% in senescent cells. The proportion of nuclei 8C and greater increased during the last stage of culture, the highest ploidy class being 128C. On the basis of the polyploidization index, which indicates relative DNA content/cell, chick cells were shown to be considerably polyploidized when they stopped growing.     

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.