Analysis of correlation between some parameters depending on cell proliferation and life span of "stationary phase aging" cultures and maximum life span of mammals

Earlier we developed a "stationary phase aging" model and introduced a definition of life span of "stationary phase aging" cell cultures. In this model the cells grow after seeding in flasks without subcultivation and medium change. They reach cell saturation density, stop dividing, gradually degrade ("stationary phase aging") and perish. By the term "culture life span" we designate the time from cell seeding until culture death. We designate the culture as dead when the number of living cells is less than 10 per cent of their number at saturation density of cell culture. The life span of transformed Chinese hamster cells was found to be proportional to the duration of their growth from cell seeding up to saturation density, as well as to the number of cell culture doublings and to be inversely proportional to the velocity of cell culture doubling for the same growth period. Maximum life span of mammals is known to be proportional to pregnancy duration and to the age at puberty. We found that maximal life span of mammals was proportional to the number of cell population doublings and inversely proportional to the velocity of cell population doubling during embryonal period or for the time from zygote to growth termination. The dependences for cell cultures and for mammals are analogous to each other.     

镜湖长三肢轮虫的有性生殖

2004年5—11月,对芜湖市镜湖大、小湖区水体中长三肢轮虫(Filinia longiseta)的有性生殖及其与种群密度、水温、水体透明度、水体叶绿素a含量和轮虫总密度等环境因子间的关系进行了研究.结果表明:当水温低于22℃或水体透明度大于一定值(小湖区SD>95cm,大湖区SD>100cm)时,长三肢轮虫不进行有性生殖;而当其本身的种群密度达到一定值(小湖区种群密度>122ind·L-1,大湖区种群密度>113ind·L-1)时才进行有性生殖.长三肢轮虫的混交雌体密度仅与其本身的种群密度以及水体中轮虫总密度之间呈正相关关系(P<0·01),而与水温、叶绿素a含量、水体透明度之间均无明显的相关关系;长三肢轮虫种群的混交率和受精率与水温、水体透明度、叶绿素a含量、轮虫总密度和长三肢轮虫种群密度之间也无明显的相关性.长三肢轮虫的有性生殖发生在种群增长的早期阶段,混交雌体以间断的、多循环的方式产生;最大混交率与最大种群密度同时出现,但最大受精率出现在种群即将消失时.     

Effect of Partial Medium Replacement on Cell Growth and Protein Production for the High-Five™ insect cell line

The potential of spent medium to support the growth and recombinant protein production of High-Five? cells was investigated. Growth in medium consisting of three parts fresh and one part spent medium was comparable to that in fresh medium (maximal specific growth rates of 0.028 and 0.029 h^?1, and maximal cell densities of 4 and 4.5 × 10⁶ cells ml^?1, respectively). Glucose exhaustion coincided with an abrupt decrease of viability. Of 15 amino acids analyzed, not a single one was completely exhausted at the end of the growth phase. Growth in medium consisting of equal parts spent and fresh medium led to lower maximal cell concentration (2.9 × 10⁶ cells ml^?1) with a smoother death phase. Glucose supplementation at the beginning of the culture or at the end of the growth phase did not lead to an increase of either the maximal cell density or the specific growth rate. Infection of High-Five? cells at three different densities (1.4, 2.5 and 4.2 × 10⁶ cells ml^?1) without medium change led to monotonically decreased specific productions for β-galactosidase. Partial (75%) or total medium replacement at the higher infection density restored the specific production at the levels of the intermediate density infection (321, 292 and 389 U.(10⁶ cells)^?1, respectively).     

Lesch-Nyhan mutation: the influence of population density on purine phosphoribosyltransferase activities and exogenous purine utilization in monolayer cultures of skin fibroblasts

In extracts of normal and Lesch-Nyhan (LN) heterozygous skin fibroblast monolayer cultures, hypoxanthine-guanine (H-G) and adenine (A) phosphoribosyltransferase (PRT) activities are correlated. These activities vary concomitantly during the life cycle of a culture: Peak activities occur during exponential growth. The ratio of H-GPRT to APRT activity can manifest heterozygosity at the LN locus when H-GPRT activity, per se, is close to and unreliable different from the normal range. If 2 or 8 × 10⁴ cells are planted per 35 mm petri dish, a strain with clearly heterozygous levels of H-GPRT activity in its cell extract may not reveal its genotype by simultaneous measurement of adenine and hypoxanthine incorporation into its acid-insoluble fraction one day after subculture. In contrast, a 1:1 coculture of normal and mutant cells yields the ratio of adenine to hypoxanthine incorporation expected from the behavior of each cell type separately. In heterozygous cultures at the higher population density, the incorporation of hypoxanthine relative to adenine is 28% greater than in those at the lower population density. A 1:1 mixture of normal and mutant cells exhibited a 20% increase in the relative incorporation of hypoxanthine over the same four fold increase in population density. These observations appear to provide a quantitative expression of the phenomenon of metabolic cooperation between contacting H-GPRT-negative and H-GPRT-positive cells.     

Minimal nutritional requirements for immobilized yeast

The effect of reduced nutritional levels (particularly nitrogen source) for immobilized K. fragilis type yeast were studied using a trickle flow, "differential" plug flow type reactor with cells immobilized by adsorption onto an absorbant packing matrix. Minimizing nutrient levels in a feed stream to an immobilized cell reactor (ICR) might have the benefits of reducing cell growth and clogging problems in the ICR, reducing feed preparation costs, as well as reducing effluent disposal costs. In this study step changes in test feed medium nutrient compositions were introduced to the ICR, followed by a return to a basal medium. Gas evolution rates were monitored and logged on a continuous basis, and effluent cell density was used as an indicator of cell growth rate of the immobilized cell mass. Startup of the reactor using a YEP medium showed a rapid buildup of cells in the reactor during the initial 110 h operation. The population density then stabilized at 1.6 x 10(11) cells/g sponge. A defined medium containing a complex mix of essential nutrients with an inorganic nitrogen source (ammonium sulfate) was able to maintain 90% of the productivity in the ICR as compared to the YEP medium, but proved unable to promote growth of the immobilized cell mass during startup. Experiments on reduced ammonium sulfate in the defined medium, and reduced yeast extract and peptone in YEP medium indicated that stable productivity could be maintained for extended periods (80 h) in the complete absence of any nutrients besides a few salts (potassium phosphate and magnesium sulfate). It was found that productivity rates dropped by 35-65% from maximal values as nitrogenous nutrients were eliminated from the test mediums, while growth rates (as determined by shed cell density from the reactor) dropped by 75-95%. Thus, nutritional deficiencies largely decoupled growth and productivity of the immobilized yeast which suggests productivity is both growth- and non-growth-associated for the immobilized cells. A yeast extract concentration of 0.375 g/L with or without 1 g/L ammonium sulfate was determined to be the minimum level which gave a sustained increase in productivity rates as compared to the nutritionally deficient salt medium. This represents a 94% reduction in complex nitrogenous nutrient levels compared to standard YEP batch medium (3 g/L YE and 3.5 g/L peptone).     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

A viscoelastic model of blood capillary extension and regression: derivation, analysis, and simulation

This work studies a fundamental problem in blood capillary growth: how the cell proliferation or death induces the stress response and the capillary extension or regression. We develop a one-dimensional viscoelastic model of blood capillary extension/regression under nonlinear friction with surroundings, analyze its solution properties, and simulate various growth patterns in angiogenesis. The mathematical model treats the cell density as the growth pressure eliciting a viscoelastic response from the cells, which again induces extension or regression of the capillary. Nonlinear analysis captures two cases when the biologically meaningful solution exists: (1) the cell density decreases from root to tip, which may occur in vessel regression; (2) the cell density is time-independent and is of small variation along the capillary, which may occur in capillary extension without proliferation. The linear analysis with perturbation in cell density due to proliferation or death predicts the global biological solution exists provided the change in cell density is sufficiently slow in time. Examples with blow-ups are captured by numerical approximations and the global solutions are recovered by slow growth processes, which validate the linear analysis theory. Numerical simulations demonstrate this model can reproduce angiogenesis experiments under several biological conditions including blood vessel extension without proliferation and blood vessel regression.     

Regulatory factors produced by lymphocytes. II. Inhibition of cellular DNA synthesis associated with a factor inhibiting DNA polymerase alpha activity.

Supernatants of phytohemagglutinin-stimulated human tonsil cells contain two growth inhibitory factors. These factors, called inhibitors of DNA synthesis (IDS), reduce (3)H-thymidine incorporation into mitogen-stimulated lymphocytes and into growing HeLa cells. By Sephadex chromatography, these factors have volumes of distribution corresponding to about 80,000 and 40,000 daltons. Both factors inhibit the activity of calf thymus DNA polymerase alpha in cell-free assays (termed inhibitor of DNA polymerase, IDP). The larger factor, which is chromatographically separable from alpha-lymphotoxin (alpha-LT), is completely inactivated by heating at 70 degrees C for 15 min. This treatment does not destroy alpha-LT. Using supernatants from PHA-stimulated tonsil cells cultured for 5 days in serum-free medium, we attained a 150-fold purification with a succession of molecular sieving, ion exchange, and adsorption chromatographic procedures. Although not purified to homogeneity, the extensive copurification of IDS and IDP activities and their identical heat inactivation profiles suggest that they are the same entity. IDP separated free of alpha-LT inhibits thymidine incorporation into HeLa cells without causing cell death. alpha-LT purified free of IDS does not inhibit thymidine incorporation into HeLa cells, not even at concentrations 7000 times that necessary to kill 50% of growth-inhibited L cell cultures.     

Stimulation of growth of serum-deprived chick embryo fibroblasts by 3',5'-phosphodiesterase.

In chick embryo fibroblasts (CEF) deprived of serum, DNA synthesis is reduced to a basal level in about 12 h, cell division ceases after 24–36 h and their morphology changes to a rounded, less refringent form. During several days without serum the cAMP content of the cells showed a slow increase or a maintenance of the level found before serum was removed. When CEF deprived of serum for 24 h were treated with beef heart 3′,5′-phosphodiesterase (PHD) the cAMP level fell about 40% after 3 h, ³H-thymidine incorporation into DNA was strongly stimulated with a peak of incorporation at 12 h after the start of PHD treatment, cell morphology returned to that observed before serum deprivation, and at 24 h there was an evident growth in cell population, with a parallel increase in protein content. The growth stimulation by PHD is transitory: after cells had been deprived of serum for 4 days the PHD effect was no longer noticeable on the above parameters. Theophylline (1 mM and 4 mM) inhibited the PHD-mediated stimulation of ³H-TdR incorporation, this could well have been due to its general toxic effect on the cells (see Discussion).     

Epidermal growth factor and insulin-like growth factor-1 preserve cell viability in the absence of protein synthesis

Summary Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide (CHX) terminates in cell death. In the present study we investigated the effect of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin on cell death induced by CHX in the human cancerous cell lines MDA-231 and MCF-7 (breast), KB (oral epidermoid), HEP-2 (larynx epidermoid), and SW-480 (colon), and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHX was shown to induce cell death in a concentration (1 to 60 μg/ml) and time (24 to 72 h)-dependent manner in each of the five cell lines. EGF at physiologic concentrations (2 to 40 ng/ml) reduced cell death close to control level (without CHX) in the cell lines HEP-2, KB, MDA-231, and SW-480, but had almost no effect on cell death in the MCF-7 cells. IGF-1 at physiologic concentrations (2 to 40 ng/ml) reduced cell death nearly to control level in the MCF-7 cells, but had only a partial effect in the other four cell lines. Insulin at supraphysiologic concentration (10 000 ng/ml) mimicked the effect of IGF-1 in each of the cell lines. CHX at concentrations that induced about 60% cell death, inhibited about 90% of protein synthesis as measured by [³H]leucine incorporation. Protein synthesis remained inhibited although cell viability was preserved by EGF or IGF-1. These results indicated that the mechanism by which EGF or IGF-1 preserve cell viability does not require new protein synthesis and may be mediated via a posttranslational modification effect.     

PUMA inactivation protects against oxidative stress through p21/Bcl-XL inhibition of bax death

The tumor suppressor protein p53 activates growth arrest and proapoptotic genes in response to DNA damage. It is known that negative feedback by p21(Cip1/Waf1/Sdi1) represses p53-dependent transactivation of PUMA. The current study investigates PUMA feedback on p53 during oxidative stress from hyperoxia and the subsequent effects on cell survival mediated through p21 and Bcl-X(L). Deletion of PUMA in HCT116 colon carcinoma cells increased levels of p53 and p21, resulting in a larger G(1) population during hyperoxia. P21-dependent increase in Bcl-X(L) levels protected PUMA-deficient cells against hyperoxic cell death. Bax and Bak were both able to promote hyperoxic cell death. Bcl-X(L) protection against hyperoxic death was lost in cells lacking Bax, not PUMA, suggesting that Bcl-X(L) acts to inhibit Bax-dependent death. These results indicate that PUMA exerts a negative feedback on p53 and p21, leading to p21-dependent growth suppressive and survival changes. Enhanced survival was associated with increased Bcl-X(L) to block Bax activated cell death during oxidative stress.     

Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production

Phosphorus depletion was identified in high-cell-concentration fed-batch NS0 myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10(9) and 7 x 10(9) cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10(9) cells/L, which is much greater than ca. 7 x 10(9) cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10(9) cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10(9) cells/L, as compared with 3 x 10(9) cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NS0 cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NS0 cultures.     

Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.     

A possible role of initial cell death due to mechanical stretch in the regulation of subsequent cell proliferation in experimental vein grafts

 The proliferation of vascular cells contributes to the formation of neointima and hypertrophy of the blood vessel wall. Here we show that mechanical stretch possibly regulates the proliferation of vascular cells via the mediation of cell death in a rat vein graft model. The wall of vein grafts is subject to a suddenly increased mechanical stretch due to exposure to arterial blood pressure. Such a stretch induces rapid cell death with a reduction in cell density by ∼60% within the first day after surgery. The initial cell death was followed by an increase in the percentage of proliferating cells, as shown by a BrdU incorporation assay (1.55 ± 1.27%, 8.48 ± 2.27%, 11.93 ± 2.36%, 6.36 ± 1.77%, and 5.60 ± 1.46% at days 1, 5, 10, 20, and 30, respectively). When mechanical stretch was reduced by restraining the vein graft using a polytetrafluoroethylene sheath, the percentage of proliferating cells reduced significantly (0.76 ± 0.76%, 1.70 ± 0.46%, 1.29 ± 0.56%, 0.99 ± 0.83%, and 0.47±0.52% at days 1, 5, 10, 20, and 30, respectively). A further reduction in cell density, induced by local administration of a cell death inducer ceramide to experimental vein grafts (without sheath), enhanced subsequent cell proliferation. In contrast, a prevention of cell death, induced by local administration of a cell death inhibitor tetrapeptide-aldehyde DEVD-CHO to experimental vein grafts (without sheath), significantly reduced subsequent cell proliferation. These results suggest that mechanical stretch induces cell death, which possibly mediates subsequent cell proliferation in the present model. Received: 9 September 2001 / Accepted: 19 November 2001     

Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells

An instrument is described that will maintain a population of mammalian cells at constant cell density while automatically monitoring the growth rate of the culture and the extent of precursor incorporation into a variety of cell products. The apparatus was used in an investigation of cyclic changes in the incorporation of labelled precursors into the DNA, RNA, total protein and myeloma protein synthesized in synchronous cultures of a mouse myeloma line. The incorporation of [(3)H]uridine into trichloroacetic acid-insoluble material reveals a slight periodicity, with maxima and minima corresponding to late S phase and the mitotic phases respectively. The incorporation of [(3)H]lysine into total intracellular protein also shows a slight oscillation, with maxima and minima occurring during the respective G2 and mitotic phases. Cyclical changes in the synthesis of serologically precipitable myeloma protein were found to vary somewhat according to the conditions used to synchronize the cells. In experiments conducted with 4.0mm-thymidine, maximal incorporation of label took place during S phase or early G2 phase. Experiments with 1.0mm-thymidine revealed a significantly less marked periodicity of myeloma protein synthesis.     

Interaction of serum and colony-stimulating factor for survival of a factor-dependent hemopoietic progenitor cell line

The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.