A model with 'growth retardation' for the kinetic heterogeneity of tumour cell populations

In the present paper we propose a continuous cell population model based on Shackney's idea of growth retardation. Cells are characterized by two state variables: the cell maturity x, 0 < or = x < or = 1, and a state variable T that identifies the rate of maturation along cell cycle. During their life span, cells can change T at random by jump transitions to T values corresponding to slower maturation rates, while at each jump the maturity x is conserved. Both the time evolution of the population and the exponential stationary solution are numerically computed. The distribution of the cell cycle transit time in asynchronous exponential growth is investigated by Monte Carlo simulation. An approximated formula for the distribution of cell cycle time is also provided.     

A mathematical model for analysis of the cell cycle in cell lines derived from human tumors

The growth of human cancers is characterised by long and variable cell cycle times that are controlled by stochastic events prior to DNA replication and cell division. Treatment with radiotherapy or chemotherapy induces a complex chain of events involving reversible cell cycle arrest and cell death. In this paper we have developed a mathematical model that has the potential to describe the growth of human tumour cells and their responses to therapy. We have used the model to predict the response of cells to mitotic arrest, and have compared the results to experimental data using a human melanoma cell line exposed to the anticancer drug paclitaxel. Cells were analysed for DNA content at multiple time points by flow cytometry. An excellent correspondence was obtained between predicted and experimental data. We discuss possible extensions to the model to describe the behaviour of cell populations in vivo.     

Modelling cell population growth with applications to cancer therapy in human tumour cell lines

In this paper we present an overview of the work undertaken to model a population of cells and the effects of cancer therapy. We began with a theoretical one compartment size structured cell population model and investigated its asymptotic steady size distributions (SSDs) (On a cell growth model for plankton, MMB JIMA 21 (2004) 49). However these size distributions are not similar to the DNA (size) distributions obtained experimentally via the flow cytometric analysis of human tumour cell lines (data obtained from the Auckland Cancer Society Research Centre, New Zealand). In our one compartment model, size was a generic term, but in order to obtain realistic steady size distributions we chose size to be DNA content and devised a multi-compartment mathematical model for the cell division cycle where each compartment corresponds to a distinct phase of the cell cycle (J. Math. Biol. 47 (2003) 295). We then incorporated another compartment describing the possible induction of apoptosis (cell death) from mitosis phase (Modelling cell death in human tumour cell lines exposed to anticancer drug paclitaxel, J. Math. Biol. 2004, in press). This enabled us to compare our model to flow cytometric data of a melanoma cell line where the anticancer drug, paclitaxel, had been added. The model gives a dynamic picture of the effects of paclitaxel on the cell cycle. We hope to use the model to describe the effects of other cancer therapies on a number of different cell lines.     

Modelling cell death in human tumour cell lines exposed to the anticancer drug paclitaxel

Most anti-cancer drugs in use today exert their effects by inducing a programmed cell death mechanism. This process, termed apoptosis, is accompanied by degradation of the DNA and produces cells with a range of DNA contents. We have previously developed a phase transition mathematical model to describe the mammalian cell division cycle in terms of cell cycle phases and the transition rates between these phases. We now extend this model here to incorporate a transition to a programmed cell death phase whereby cellular DNA is progressively degraded with time. We have utilised the technique of flow cytometry to analyse the behaviour of a melanoma cell line (NZM13) that was exposed to paclitaxel, a drug used frequently in the treatment of cancer. The flow cytometry profiles included a complex mixture of living cells whose DNA content was increasing with time and dying cells whose DNA content was decreasing with time. Application of the mathematical model enabled estimation of the rate constant for entry of mitotic cells into apoptosis (0.035 per hour) and the duration of the period of DNA degradation (51 hours). These results provide a dynamic model of the action of an anticancer drug that can be extended to improve the clinical outcome in individual cancer patients.Revised version: 9 October 2003     

Linking cell division to cell growth in a spatiotemporal model of the cell cycle

Cell division must be tightly coupled to cell growth in order to maintain cell size, yet the mechanisms linking these two processes are unclear. It is known that almost all proteins involved in cell division shuttle between cytoplasm and nucleus during the cell cycle; however, the implications of this process for cell cycle dynamics and its coupling to cell growth remains to be elucidated. We developed mathematical models of the cell cycle which incorporate protein translocation between cytoplasm and nucleus. We show that protein translocation between cytoplasm and nucleus not only modulates temporal cell cycle dynamics, but also provides a natural mechanism coupling cell division to cell growth. This coupling is mediated by the effect of cytoplasmic-to-nuclear size ratio on the activation threshold of critical cell cycle proteins, leading to the size-sensing checkpoint (sizer) and the size-independent clock (timer) observed in many cell cycle experiments.     

Understanding the relationship between cell death and tissue shrinkage via a stochastic agent-based model

Cell death, a process which can occur both naturally and in response to insult, is both a complex and diverse phenomenon. Under some circumstances, dying cells actively contract and cause their neighbors to rearrange and maintain tissue integrity. Under other circumstances, dying cells leave behind gaps, which results in tissue separation. A better understanding of how the cellular scale features of cell death manifest on the population scale has implications ranging from morphogenesis to tumor response to treatment. However, the mechanistic relationship between cell death and population scale shrinkage is not well understood, and computational methods for studying these relationships are not well established. Here we propose a mechanically robust agent-based cell model designed to capture the implications of cell death on the population scale. In our agent-based model, algorithmic rules applied on the cellular level emerge on the population scale where their effects are quantified. To better quantify model uncertainty and parameter interactions, we implement a recently developed technique for conducting a variance-based sensitivity analysis on the stochastic model. From this analysis and subsequent investigation, we find that cellular scale shrinkage has the largest influence of all model parameters tested, and that by adjusting cellular scale shrinkage population shrinkage varies widely even across simulations which contain the same fraction of dying cells. We anticipate that the methods and results presented here are a starting point for significant future investigation toward quantifying the implications of different mechanisms of cell death on population and tissue scale behavior.     

Multi-type Galton-Watson process as a model for proliferating human tumour cell populations derived from stem cells: estimation of stem cell self-renewal probabilities in human ovarian carcinomas

A mathematical model for proliferation of tumour cell populations is developed. The cell population is assumed to be organized in a hierarchy of decreasing proliferative potential and increasing degree of differentiation. Using some elements of the theory of Multi-type Galton-Watson processes, a method is proposed for the estimation of Psr, the probability of self-renewal of tumour stem cells, from the experimental distribution of clonal unit sizes obtained in cell culture studies. Six data sets from patients with advanced adenocarcinoma of the ovary are used to demonstrate the method. Reasonable estimates are obtained, and the theoretical colony size distributions predicted by the model appear to be in good qualitative agreement with the experimental ones, and lend support to a stem cell model of tumour growth. The possible significance of Psr as a prognostic factor is briefly discussed.     

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial–endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells(hESCs) can self-renew indefinitely and differentiate into all cell types in the human body.Therefore,they are valuable in regenerative medicine,human developmental biology and drug discovery.A number of hESC lines have been derived from the Chinese population, but limited of them are available for research purposes.Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin.These hESCs express alkaline phosphatase and hE...     

Differentiation in vitro of primary cultures and transfected cell lines of epithelial cells derived from the thick ascending limb of Henle''s loop

The use of primary cell cultures derived from defined locations of the kidney has enabled the study of certain kidney cell type-specific characteristics under defined environmental conditions. The use of primary cell cultures, however, has a number of inherent disadvantages, many of which may be overcome by the use of differentiated cell lines of defined origin. In this paper I describe in detail an approach to: (a) the isolation and culture of primary cultures derived from the thick ascending limb of Henle's loop (TALH), and (b) the production of differentiated cell lines by the transfection of these primary cell cultures with early region SV40 virus genes. The characteristics of these cultures and other TALH-derived cell lines are described.     

Structural and functional characteristics of primary cell cultures derived from the adrenal gland tissue of neonatal mallard ducklings (Anas platyrhynchos)

Summary Primary cell cultures were prepared from the adrenal glands of one-day-old mallard ducklings (Anas platyrhynchos). The cells attached equally well to uncoated plastic and glass surfaces and on surfaces that had been coated with collagen. The phase of logarithmic growth occurred between the second and the fourth day, and the cells became confluent between the fifth and the sixth day. Staining with Sudan black B and toluidine blue and viewing fixed preparations by transmission electron microscopy indicated that the cultures consisted mostly of steroidogenic cells. A smaller population of chromaffin cells was also present. Scanning electron microscopy showed that most of the cells had long filopodia, and some cells had numerous surface blebs that were interpreted as exocytotic vesicles. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH the cultured cells released three corticosteroids, namely, corticosterone, aldosterone and deoxycorticosterone. These responses occurred within 15 min of exposure to medium containing 1–24 ACTH and continued throughout a 60-min period of continuous stimulation. The minimally effective concentration of 1–24 ACTH was 0.078 ng per ml (0.0234 nM) and, as the concentration was increased up to 10 ng per ml (2.99 nM), the total output of each hormone during the 60-min incubation period increased significantly according to the following semi-logarithmic relationship: Y=a+b log X, where Y=the total output of hormone, X=the concentration of 1–24 ACTH in the medium, and a=the total output of hormone when the medium contained 1.0 ng of 1–24 ACTH per ml. The total outputs of each hormone in the presence of a maximally effective concentration of 1–24 ACTH, however, were low compared to the responses of similarly stimulated tissue slices taken from the neonatal duckling. It is concluded that most of the cells comprising the confluent cultures were derived from steroidogenic cells in the neonatal adrenal. These cells appeared to retain corticotropin receptors during the course of developing into confluent monolayers, but their diminished steroidogenic capacity to respond when stimulated maximally suggests that some generational changes may have occurred.This work was supported by grants to James Cronshaw and W.N. Holmes from the University of California Committee on Research and the National Science Foundation (DIR-8820923), Washington, DC, USA     

In vitro proliferation and in vivo malignancy of cell lines simultaneously derived from a chemically-induced heterogeneous rat mammary tumor

Summary Identification of clones in primary tumors responsible for proliferation, invasion, and metastasis was carried out. Four different aneuploid established cell lines derived from a ductal infiltrating mammary rat tumor induced by 7,12-dimethylbenz[a]anthracene were studied for proliferative and growth features in vitro and for tumorigenic and metastatic potential in vivo in nude mice. Clones, named RM1, RM2, RM3, and RM4, were characterized by different proliferative activity. Clone RM1 showed the highest proliferative activity by both tritiated thymidine incorporation and S-phase flow cytometry, followed by clone RM4. Conversely, clones RM2 and RM3 showed a lower proliferation rate. Growth-promoting activity, tested on 3T3 Swiss cells, was high in all clones, although RM1 showed significantly lower growth factors—releasing activity. Nude mice tumorigenesis demonstrated a strong tumor induction of line RM1 (100% of the mice after 47±7 d) and a slightly lower tumor induction of line RM4 (70% of the mice after 69±9 d). Line RM3 showed tumor induction in 40% of the mice after 186±16 d. Lines RM2 showed no tumor induction. Metastasis occurred in mice treated with line RM1 only. Therefore, tumorigenesis and metastasis correlate with proliferation but not with the release of growth factors. In conclusion, flow cytometry monitoring of clones from heterogeneous primary tumors proved to be a suitable model for the study of in vivo malignancy and in vitro proliferation.     

Anti-cancer effect of Cassia auriculata leaf extract in vitro through cell cycle arrest and induction of apoptosis in human breast and larynx cancer cell lines

The in vitro anti-cancer effect of Cassia auriculata leaf extract (CALE) was evaluated in human breast adenocarcinoma MCF-7 and human larynx carcinoma Hep-2 cell lines. CALE preferentially inhibited the growth of both the cell lines in a dose-dependent manner with IC₅₀ values of 400 and 500 μg for MCF-7 and Hep-2 cells, respectively. The results showed the anti-cancer action is due to nuclear fragmentation and condensation, associated with the appearance of A₀ peak in cell cycle analysis that is indicative of apoptosis. In addition, CALE treated MCF-7 and Hep-2 cells had decreased expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein, eventually leading a decrease in the Bcl-2/Bax ratio. These results demonstrated that CALE inhibits the proliferation of MCF-7 and Hep-2 cells through induction of apoptosis, making CALE a candidate as new anti-cancer drug.     

大豆低聚糖对人胃癌细胞株BGC-823细胞的细胞凋亡和细胞周期的影响

目的通过观察大豆低聚糖对胃癌癌细胞株BGC-823细胞的细胞周期和细胞凋亡的影响,探索乳酸杆菌发酵滤液对胃癌细胞作用的可能机制。方法用光镜和流式细胞仪分析不同浓度大豆低聚糖对BGC-823细胞的凋亡诱导效果;用流式细胞仪分析不同浓度大豆低聚糖对BGC-823细胞细胞周期的影响。结果大豆低聚糖可以诱导BGC-823细胞的凋亡。形态学观察处理后的BGC-823细胞,可见细胞变形,细胞皱缩,体积变小,细胞间隙增大,细胞核固缩。流式细胞仪分析50 mg/ml和100 mg/ml大豆低聚糖作用48 h和72 h BGC-823细胞的凋亡比例,分别为6.76%和7.93%。50 mg/ml大豆低聚糖作用48 h,引起BGC-823细胞G1期阻滞,100 mg/ml大豆低聚糖作用48 h,引起BGC-823细胞出现S期阻滞。结论大豆低聚糖可诱导部分BGC-823细胞凋亡。大豆低聚糖对BGC-823细胞的生长抑制作用在低浓度时可能通过G1期阻滞实现,在高浓度时可能通过S期阻滞实现。     

The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors

In a senescence study, skin fibroblast cultures grown in the presence of a second batch of fetal calf serum (FCS) revealed delayed onsets of cell culture senescence and prolonged in vitro lifespans when compared to cell cultures grown on the initial batch of serum. These statistically significant differences occurred despite the fact that both sera displayed equal growth promoting abilities as measured by cell culture growth curves performed on parallel cultures with the two sera. When cultures grown in either sera were analyzed separately, the onset of cell culture senescence was earlier and in vitro lifespan was shorter in those cultures derived from the old donor group (ages 63–92) when compared to cultures derived from young donors (ages 21–36).     

First cell cycle of zygotes of the mouse derived from oocytes aged postovulation in vivo and fertilized in vivo

This paper describes an analysis of the first cell cycle of mouse oocytes aged postovulation and fertilized in vivo. For this purpose, we developed a procedure for inducing ovulation in vivo that allows accurate timing of ovulation. The method is based on a luteinizing hormone (LH)-releasing hormone (LHRH) administration at proestrus. This ovulation procedure had no detectable effect on the rate of ovulation or postimplantation embryonic death. We used this method of ovulation induction in an analysis of the separate stages of the first cell cycle of in vivo fertilized postovulation aged oocytes. All stages assessed were shorter in aged oocytes (12 hr postovulation) than in zygotes from unaged oocytes (1 hr postovulation): 1) the time interval between insemination and penetration of the aged oocytes was 1.5 hr shorter than the time interval of the unaged oocytes; 2) pronuclear formation in the fertilized aged oocytes was somewhat quicker than pronuclear formation in fertilized unaged oocytes; 3) in zygotes from aged oocytes, the time between formation of pronuclei and the pronuclear membrane breakdown was 1 hr shorter than in zygotes from unaged oocytes; 4) the first cleavage division was 3 hr advanced in zygotes from aged oocytes compared with the moment of the first cleavage division in zygotes from unaged oocytes. We also determined the glutathione (GSH) content of unaged and aged oocytes to investigate a possible relationship between the rate of pronuclear formation and GSH. The level of GSH was two times lower in oocytes aged postovulation for 12 hr than in unaged oocytes.2+ level of GSH in fertilized, unaged oocytes was half that in