A theory for analysis of cell populations with non-cycling S phase cells

Analyses of cell populations that have been labeled in vivo with analogs of thymidine that are incorporated by cells synthesizing DNA and then monitored over time by bivariate flow cytometry sometimes detect populations of cells that have S phase DNA content but that have not acquired label. Two alternative explanations for the lack of labeling are that either the cells were not exposed to the label or that the cells stopped DNA synthesis and ceased progression through S phase. To help determine which scenario is the more likely, a model has been devised for studying a population of cells that includes the possibility that cells in S phase will cease DNA synthesis. In this model, the initial fraction of unlabeled cells in S phase depends on two rates: the growth rate of the total population and the number of cells that cease progression through S phase per unit time. The model is used to analyze the changing quantities which can be measured by monitoring the population of cells over time and is used to estimate the two rates required to compute the initial fraction of unlabeled S phase cells. Thus, the initial fraction of unlabeled cells can be compared with that predicted by the population dynamics to determine whether one explanation for the failure of some cells to be labeled is preferable to the other, which in turn might offer information about tumor microvascular or cytologic properties.     

On the use of the Von Foerster equation for the solution and generalization of a problem studied by S. A. Tyler and R. Baserga

In a population of cells labeled with a single injection of tritiated thymidine at timet=0, it is assumed that a constant fraction, 1?z, of the cells which are potentially able to divide fail to do so, and that the cells which do divide all have identical generation time,D. Death and emigration of cells are neglected. In mitosis, the partitioning of label among the two daughter cells is supposed to follow the binomial probability law. Using the formalism developed by H. Von Foerster the fraction of labeled cells in the total population is computed as a function oft, the time after injection of label. Ift is an integral multiple ofD the results coincide with those of S. A. Tyler and R. Baserga.     

Transformation and Deoxyribonucleic Acid Size: Extent of Degradation on Entry Varies with Size of Donor

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.     

Time course of selenite metabolism in confluent cultures of mouse mammary epithelial cells

The distribution of 75Se selenite in various macromolecules over time was evaluated in confluent cultures of the LMA mouse mammary epithelial cell line. 75Se label did not distribute equally to all fractions at the same time point. The label initially entered the selenonucleic acid fraction and subsequently increasing quantities were detected in the selenoprotein fraction. The time period of increasing inhibition of DNA synthesis correlated with increasing levels of selenoproteins. The major selenoprotein found in this time frame in confluent phase LMA cells was an acidic, 58K selenoprotein.     

Metabolic relationships of the isolated fractions of the pectic substances of actively growing sycamore cells

1. d-Glucose and l-arabinose serve as precursors of the pectic polysaccharides of sycamore suspension-callus tissue. 2. The rates and characteristics of the incorporation of radioactive sucrose, glucose and mesoinositol by sycamore callus tissue have been compared and shown to be different. 3. The time-course of the incorporation of radioactive glucose into the major fractions within the cells has been determined. Approx. 7-10% of the radioactivity incorporated is present in the whole pectin of the cells. 4. A study of the continuous incorporation of radioactive glucose showed that the neutral arabinan-galactan fraction of the pectin quickly became saturated with the radioactive label. During the incorporation of radioactivity from a pulse of radioactive glucose the neutral fraction became progressively less labelled, with a corresponding increase in the radioactivity of the weakly acidic pectinic acid, which is known to contain neutral sugars. 5. When the cells were exposed to a pulse of radioactive l-arabinose, the label accumulated first in the neutral fraction and then after 4hr. it passed to the weakly acidic pectinic acid with a corresponding decrease in the radioactivity of the neutral fraction. 6. The product that was initially labelled during the first hour of exposure of the cells in the stationary phase to radioactive glucose was identified as an incompletely methylated galacturonan in which the radioactivity was present in the anhydrogalacturonide residues. This polysaccharide probably acts as the precursor of the polyuronide portions of both the strongly acidic and weakly acidic pectinic acids. 7. The observations are discussed in relation to the structure of the pectic substances and their function in cell growth and development. A tentative model for their metabolic relationship is put forward.     

Improved method for computing potential doubling time from flow cytometric data

Relative movement methods use the timed progression of the mean fluorescence of cells which have been labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, TS. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity v is introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to ln(2)Ts/Tpot so that Tpot (equal to ln(2)Ts/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.     

Modeling survival of experimentally virus-infected laboratory animals--exploration of the survival diagram

In a recent paper (Sühnel & Veckenstedt, 1989, J. theor. Biol. 137, 27) we have proposed a new method of plotting survival data from experimentally virus-infected laboratory animals; the survival diagram. In this diagram two experiments, for which the mean number of virions inoculated is kept fixed but other parameters may vary, are compared. The variations in two basic quantities of survival analysis are simultaneously displayed: the standard mean survival time and the relative mean challenge virus dose, which is via a dose-response relation interrelated with the fraction of animals dying. It is analyzed in which manner variations in the kinetic parameters and the critical virus level necessary to produce a particular effect influence the location of the points of comparison in the survival diagram. The analysis presented is a prerequisite for further applications of this diagram and of the underlying mathematical model.     

Degradation of cell constituents during starvation of Salmonella enteritidis

Salmonella enteritidis starved in Fernbach flasks used acid-alcohol-soluble material and RNA as endogenous reserves during starvation. Organisms starved in a fermentor system consumed acid-alcohol-soluble material, RNA and protein to maintain viability. Half-life survival times were 132 h and 118 h for the Fernbach and fermentor-starved cells, respectively. The acid-alcohol-soluble fraction of the cell consisted mainly of peptides or protein. This fraction accounted for most of the loss of label from 14C-labelled cells during the first 5 days of starvation and presumably contains the primary endogenous reserve. Although the residue fraction of fermentor-starved cells provided 35% of the total loss of 14C from the cells by the 5th day, a small net increase in 14C activity of the residue fraction of Fernbach-starved cells was observed. Differences observed appeared to be due to the method of starvation.     

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12. Detection of a λ phage fraction with a retarded mode of DNA injection

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage λ in cells irradiated with ultraviolet prior to infection. We demonstrate that restriction of λ is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of λ. At this time more than 60% of the λ DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of λ DNA from infected cells and a mild detergent treatment removing adsorbed phages from the cellular surface showed that only a small specific fraction of all λ infections is destined to escape restriction due to restriction alleviation. This fraction (10–20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of λ phages explains why the SOS function restriction alleviation could initially be discovered. We show that the retarded mode of DNA injection is not required for another SOS function acting on λ DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).     

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12. Detection of a lambda phage fraction with a retarded mode of DNA injection

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. We demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing absorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. We show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).     

Membrane transformations in aging potato tuber slices

When potato tuber slices (Solanum tuberosum L.) are incubated with radioactive choline, labeled membrane-bound phospholipids are formed. If potato slices are aged for 0 to 24 hours before exposure to radioactive choline, the distribution of the labeled phospholipids undergoes both quantitative and qualitative changes. Quantitatively, there is a marked increase in the total lipoidal radioactivity with aging time. Qualitatively, there is a shift in the kinds of subcellular fractions that are being labeled. Fresh slices incorporate most of the lipoidal radioactivity in the microsomes. Slices aged for 9 hours incorporate most of the label in a fraction consisting of single membrane-bound cisternae, which are presumed to be dictyosomal fragments. Slices aged for 24 hours before incubation with radioactive choline incorporate the greater portion of the label in this same fraction, but a significant portion of the label is found in a heavier, mitochondria-containing fraction.     

A new model of thermal inactivation and its application to clonogenic survival data for WiDr human colonic adenocarcinoma cells

Based on the analysis of clonogenic survival data for human colonic adenocarcinoma cells (WiDr) after a single heating, a new model is proposed to describe cell survival after hyperthermia quantitatively. The effects of heat are explained as heat-induced cell damage assuming a first-order (single-hit) and a second-order (cumulative damage) process. Thus cell survival at a specified temperature can be described by the linear-quadratic (LQ) model. The proposed model is based on an alternative definition of the (single) thermal dose, given as the (normalized) product of heating time and a specified nonlinear function of the increase in temperature (relative to a threshold temperature) to be interpreted as the thermal dose rate. In further analogy to the modeling of the effects of low-dose-rate radiation, an inherent capacity of the cells to repair sublethal damage is assumed, and these effects are quantified by the usual g factor measuring incomplete repair effects. The model defines thermal dose-response and isoeffect dose relationships, enabling a direct (i. e. single-step) analysis of the available thermal response data. Additionally, the analysis of our data based on heating times in the range from 0 to 360 min and temperatures from 41 to 46 degrees C and covering a broad spectrum of different densities of cells seeded for colony formation did not yield any evidence of the existence of a breaking point usually derived from Arrhenius plots based on the single-hit, multitarget model and the Arrhenius equation. The model includes no specific assumptions describing the development of thermotolerance, which can be assumed to be negligible under our experimental conditions. The proposed thermal dose-response model correlates satisfactorily with the in vitro survival data for WiDr adenocarcinoma cells.     

On the validity of time‐dependent AUC estimation in the presence of cure fraction

During the last decades, several approaches have been proposed to estimate the time‐dependent area under the receiver operating characteristic curve (AUC) of risk tools derived from survival data. The validity of these estimators relies on some regularity assumptions among which a survival function being proper. In practice, this assumption is not always satisfied because a fraction of the population may not be susceptible to experience the event of interest even for long follow‐up. Studying the sensitivity of the proposed estimators to the violation of this assumption is of substantial interest. In this paper, we investigate the performance of a nonparametric simple estimator, developed for classical survival data, in the case when the population exhibits a cure fraction. Motivated from the current practice of deriving risk tools in oncology and cardiovascular disease prevention, we also assess the loss, in terms of predictive performance, when deriving risk tools from survival models that do not acknowledge the presence of cure. The simulation results show that the investigated method is valid even under the presence of cure. They also show that risk tools derived from survival models that ignore the presence of cure have smaller AUC compared to those derived from survival models that acknowledge the presence of cure. This was also attested with a real data analysis from a breast cancer study.     

Cholesterol from Degenerating Nerve Myelin Becomes Associated with Lipoproteins Containing Apolipoprotein E

Apolipoprotein E is synthesized and secreted by rat sciatic nerve consequent to several types of injury. It has been proposed that endoneurial apolipoprotein E, in analogy to its role in systemic cholesterol transport, is involved in the salvage and reutilization of myelin cholesterol during degeneration and regeneration. To test this hypothesis, nerve lipids were prelabeled via intraneural injection of [3H]acetate. Four weeks later the nerves were crushed. From 1 to 12 weeks later, crushed nerves were examined for extracellular lipoprotein-bound cholesterol label. By 2 weeks after injury, 10% of the endoneurial lipid label was in a soluble form that was releasable into incubation medium. This released fraction was enriched in labeled cholesterol, and its labeled lipid composition was constant, in contrast to the changing distribution of label in the nerve with time after injury. On a KBr gradient, the released lipid label cofractionated with the released apolipoprotein E at densities similar to that of lipoproteins. These data indicate that at least some myelin cholesterol in injured nerve becomes associated with apolipoprotein E-containing lipoproteins and thus is available for reutilization via the hypothesized model.     

A General Class of Semiparametric Transformation Frailty Models for Nonproportional Hazards Survival Data

Summary We propose a semiparametrically efficient estimation of a broad class of transformation regression models for nonproportional hazards data. Classical transformation models are to be viewed from a frailty model paradigm, and the proposed method provides a unified approach that is valid for both continuous and discrete frailty models. The proposed models are shown to be flexible enough to model long‐term follow‐up survival data when the treatment effect diminishes over time, a case for which the PH or proportional odds assumption is violated, or a situation in which a substantial proportion of patients remains cured after treatment. Estimation of the link parameter in frailty distribution, considered to be unknown and possibly dependent on a time‐independent covariates, is automatically included in the proposed methods. The observed information matrix is computed to evaluate the variances of all the parameter estimates. Our likelihood‐based approach provides a natural way to construct simple statistics for testing the PH and proportional odds assumptions for usual survival data or testing the short‐ and long‐term effects for survival data with a cure fraction. Simulation studies demonstrate that the proposed inference procedures perform well in realistic settings. Applications to two medical studies are provided.     

Starvation and the size structure of zooplankton communities*

The survival time of zooplankton under conditions of total starvation is expressed as a function of weight-specific respiration rate and the fraction of initial (pre-starvation) body weight which may be lost prior to death. Data from the literature on these two components of survival time are used to formulate a general expression of survivorship of zooplankton at 20°C as a function of body weight: t=2·95 w^0·25, where t is in days and w is μg dry weight. Survival time data from the literature on 25 marine and freshwater species are compared to this prediction as are new data on Daphnia pulex Leydig, D. magna Straus and Simocephalus serrulatus (Koch). The effects of age-specific (non-starvation) mortality are considered; in particular, older individuals of each of these species survive for shorter periods of time than predicted and an interaction between age-specific and starvation-induced mortality is proposed. The effects of total and partial starvation on the size structure of zooplankton communities are discussed.     

Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions. Earlier reports suggest that alphaB-crystallin could prevent stress-induced aggregation of actin in vitro. However, the molecular mechanisms by which alphaB-crystallin stabilizes actin filaments in vivo are not known. Using the H9C2 rat cardiomyoblast cell line as a model system, we show that upon heat stress, alphaB-crystallin preferentially partitions from the soluble cytosolic fraction to the insoluble cytoskeletal protein-rich fraction. Confocal microscopic analysis shows that alphaB-crystallin associates with actin filaments during heat stress and the extent of association increases with time. Further, immunoprecipitation experiments show that alphaB-crystallin interacts directly with actin. Treatment of heat-stressed H9C2 cells with the actin depolymerzing agent, cytochalasin B, failed to disorganize actin. We show that this association of alphaB-crystallin with actin is dependent on its phosphorylation status, as treatment of cells with MAPK inhibitors SB202190 or PD98059 results in abrogation of this association. Our results indicate that alphaB-crystallin regulates actin filament dynamics in vivo and protects cells from stress-induced death. Further, our studies suggest that the association of alphaB-crystallin with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Patterns of acetylcholine phosphocholine, and choline phosphoglycerides labeling from (methyl-14C)choline.

The uptake and metabolism of [14C]choline in dissociated rat brain embryo cell cultures was examined as a function of the extracellular choline concentration. Choline uptake did not follow normal Michaelis-Menten kinetics, but rather exhibited two components with apparent Km of 0.016 mM and 0.96 mM. At low choline concentrations (high affinity uptake) most of the [14C]choline label was present in the phosphocholine fraction prior to the appearance of label in phospholipids. At high choline concentrations (low affinity uptake) a large proportion of the radioactivity was converted into acetylcholine. The dissimilarities between the formation of phosphocholine and acetylcholine as a function of choline concentration might be explained by the existence of two mutually independent enzymatic activities with different Km affinities for choline. Kinetic data augmented by double label studies, suggested that formation of choline phosphoglyceride proceeds entirely via a phosphocholine intermediate. Nearly all radioactivity in the lipid fraction is incorporated into choline phosphoglycerides. A higher turnover rate of choline incorporation into choline phosphoglycerides, accompanied by an increase in the levels of glycerophosphocholine, was observed in older cultures as compared to younger cultures. The metabolic implications of these findings in cultured brain cells in comparison with other in vitro systems are discussed.