Clonal variation and aging of diploid fibroblasts : Cinematographic studies of cell pedigrees

Time-lapse cinematographic (TLC) analysis of clones of human diploid fibroblasts indicate heterogeneity in clonal division behaviour. Variations are noted in interdivision time, clone size and generations per clone. Correlation coefficients for interdivision times of sister pairs are high in young clones and generally low in aged clones. A consistent division pattern at all population doubling levels is one of low average interdivision time for early and late generations of a clone and high average interdivision time for the middle range of generations of a clone. The clonal division patterns observed experimentally have been duplicated in computer simulated pedigrees. The computer model is based on an oscillating system which allows for flux of regulator substances. The critical concentrations of regulator substances determine the clonal division pattern for a given progenitor cell.     

The Death of an Amoeba1

A geneological study shows that about 4% of the cells in healthy, adequately fed Amoeba proteus cultures are inviable. Two different categories of inviability are distinguished. About 44% of all inviability involves twin sisters formed at a division. Another 39% involves single cells with viable sisters and nieces. The inviable singles usually die more rapidly and show fewer visible abnormalities than the twins. The mothers of inviable twins show an increased interdivision time compared to mothers of inviable singles. Both categories of death are more rapid than starvation. The 17% of the deaths which involve aunt-niece pairs appear to be special cases of twin sister or single cell deaths. There is no evidence for stem line division where a cell forms only one viable daughter for several generations. It is proposed that death is a normal occurrence in amoeba populations. It occurs regardless of culture conditions and may be a measure of accumulated lethal mutations in an asexual polyploid organism.     

Variability of cell generation times in a hepatoma cell pedigree

Time-lapse cinematographic analysis of a clone of HTC rat hepatoma cells showed variations in interdivision time within the clone. A positive correlation was found between the interdivision times of mother and daughter cells. The variability of the differences between interdivision times of cells of sister, cousin, second cousin or second-second cousin relationship was calculated. The proportion of cells with large differences in intermitotic times was found to increase with decreasing relationship. The clonal division pattern observed suggests strongly that 'inherited' factors goven the process leading to cell division but that their effects can be modified.     

VARIABILITY OF CELL GENERATION TIMES IN A HEPATOMA CELL PEDIGREE

Time-lapse cinematographic analysis of a clone of HTC rat hepatoma cells showed variations in interdivision time within the clone. A positive correlation was found between the interdivision times of mother and daughter cells. The variability of the differences between interdivision times of cells of sister, cousin, second cousin or second-second cousin relationship was calculated. The proportion of cells with large differences in intermitotic times was found to increase with decreasing relationship. The clonal division pattern observed suggests strongly that ‘inherited’factors govern the process leading to cell division but that their effects can be modified.     

3-Hydrogenkwadaphnin targets inosine 5'-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines

3-Hydrogenkwadaphnin (3-HK) is a recently characterized daphnane-type compound isolated from Dendrostellera lessertii with high anti-tumor activity in animal models. Herein, we report on time- and dose-dependent effects of this compound on growth, differentiation, IMPDH inhibition, cell cycle and apoptosis of a panel of human leukemia cell lines (HL-60, K562 and Molt4). The drug decreased the growth of leukemia cells in less than 24 h of treatment. However, longer exposure times and/or higher concentrations were required to promote cell apoptosis. Cell cycle analysis revealed the accumulation of cells in their G1 phase as early as 12 h after drug exposure but sub-G1 population was recorded after 24 h. Occurrence of apoptosis was constantly accompanied by morphological (staining with DNA-binding dyes) and biochemical (DNA fragments) variations among drug-treated cells. Despite these observations, non-activated normal human PBL were insensitive to the drug action. In addition, treatment of PHA-activated PBL, K562, Molt4 and HL-60 cells with a single dose of the drug for 24 h led to the inhibition of IMPDH activity by almost 37, 38, 44 and 50%, respectively. In contrast, no difference in IMPDH activities were seen between normal PBL and the drug treated PBL cells. Restoration of the depleted GTP concentration by exogenous addition of guanosine (25-50 microM) reversed the drug effects on cell growth, DNA fragmentation and apoptosis. Furthermore, the drug effects were potentiated by exogenous addition of hypoxanthine to the drug-treated cells. Reduction of the drug potency on the non-proliferative (retinoic acid treated) HL-60 cells by almost 40%, compared to the proliferative cells, clearly shows type II IMPDH as one of the main targets of the drug. These results suggest that 3-HK may be a powerful candidate for treatment of leukemia.     

Effect of inhibitors and activators of tyrosine kinase on insulin imprinting in Tetrahymena.

Primary exposure of Tetrahymena cells to insulin gave rise to hormonal (insulin) imprinting in the offspring generations, as judged from the increase in binding upon reexposure to insulin. Vanadate mimicked the action of insulin, inasmuch as it also induced imprinting for insulin, whereas the other tyrosine kinase activator tested, namely H2O2, had no such effect. However, combined treatment with vanadate+H2O2 + insulin induced a more pronounced imprinting for insulin than either insulin or vanadate on their own. The tyrosine kinase inhibitor genistein, a plant flavonoid, did not change the value for insulin binding significantly relative to the control immediately after exposure, but increased it slightly in the offspring generations after 24 h at high dilution. Upon combination with insulin, 10(-4)M genistein inhibited imprinting by insulin. These experimental observations suggest that there may be a key role for tyrosine kinase activity in the mechanism (development) of imprinting.     

Distribution of Interdivisional Times In Proliferating and Differentiating Friend Murine Erythroleukaemia Cells

Abstract. The interdivisional times of Friend murine erythroleukaemia cells which are growing continuously, or during terminal erythroid differentiation after exposure to dimethyl sulphoxide (DMSO), were determined by time lapse video photography. the median interdivisional times were found to increase from 11.75 hr before exposure to DMSO, to 24.0 hr at 72 hr after exposure. This increase in median interdivisional time was accompanied by an increase in heterogeneity of interdivisional times (% CV = 8-5 → 40.8), by an increase in the similarity of sister interdivisional times (r_yy= 0.622 → 0.925), and by a decrease in the fraction of cells observed to divide ( F = 1. 0 → 0.807). Cells exposed to DMSO for 72 hr can be induced to divide at least once with nearly normal interdivisional times, if they are resuspended at a tenfold higher cell concentration. Computer simulations of cell cycle regulation, based on the opposing reactions model of Murphy, generate interdivisional time distributions which resemble the experimental data better than the single transition probability model of Smith and Martin.     

Distribution of interdivisional times in proliferating and differentiating Friend murine erythroleukaemia cells

The interdivisional times of Friend murine erythroleukaemia cells which are growing continuously, or during terminal erythroid differentiation after exposure to dimethyl sulphoxide (DMSO), were determined by time lapse video photography. The median interdivisional times were found to increase from 11.75 hr before exposure to DMSO, to 24.0 hr at 72 hr after exposure. This increase in median interdivisional time was accompanied by an increase in heterogeneity of interdivisional times (%CV = 8.5----40.8), by an increase in the similarity of sister interdivisional times (ryy = 0.622----0.925), and by a decrease in the fraction of cells observed to divide (F = 1.0----0.807). Cells exposed to DMSO for 72 hr can be induced to divide at least once with nearly normal interdivisional times, if they are resuspended at a tenfold higher cell concentration. Computer simulations of cell cycle regulation, based on the opposing reactions model of Murphy, generate interdivisional time distributions which resemble the experimental data better than the single transition probability model of Smith and Martin.     

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0420-4) contains supplementary material, which is available to authorized users.     

A rationale for the clinical development of the thymidylate synthase inhibitor ZD9331 in ovarian and other solid tumours

ZD9331 is an antifolate drug that potently and specifically inhibits thymidylate synthase (TS). In contrast with TS inhibitors such as raltitrexed, it cannot be polyglutamated, leading to antitumour activity independent of folylpolyglutamyl synthetase (FPGS) activity.The growth inhibition IC50 values for ZD9331 and raltitrexed were determined for a panel of 18 human tumour cell lines, that included six colon and six ovarian. The colon lines largely displayed overlapping sensitivities to both drugs with only one of the six lines being drug resistant. In contrast, the ovarian cell lines displayed non-overlapping sensitivities with four being highly resistant to raltitrexed and only one was cross-resistant to ZD9331. Studies were undertaken to explain these results. The colon and ovarian cell lines were characterised for TS activity, and TS and FPGS mRNA expression. TS activity correlated with sensitivity to ZD9331 (r=0.50; p=0.097) and raltitrexed (r=0.74; p=0.0063). Provided the data from the highly drug-resistant cell lines (BE and 41 M) were omitted, TS mRNA expression levels also correlated with ZD9331 (r=0.77; p=0.013) and raltitrexed IC50 (r=0.84; p=0.0031). FPGS mRNA expression correlated with higher sensitivity to raltitrexed relative to ZD9331 (higher ZD9331/raltitrexed IC50 ratios) (r=0.62; p=0.048). Similarly, cell lines with IC50 ratios>median expressed a 1.8-fold higher median level of FPGS mRNA (p=0.0087) compared with those with ratios相似文献   

Natural and chemotherapy-induced clonal evolution of tumors

Evolution and natural selection of tumoral clones in the process of transformation and the following carcinogenesis can be called natural clonal evolution. Its main driving factors are internal: genetic instability initiated by driver mutations and microenvironment, which enables selective pressure while forming the environment for cell transformation and their survival. We present our overview of contemporary research dealing with mechanisms of carcinogenesis in different localizations from precancerous pathologies to metastasis and relapse. It shows that natural clonal evolution establishes intratumoral heterogeneity and enables tumor progression. Tumors of monoclonal origin are of low-level intratumoral heterogeneity in the initial stages, and this increases with the size of the tumor. Tumors of polyclonal origin are of extremely high-level intratumoral heterogeneity in the initial stages and become more homogeneous when larger due to clonal expansion. In cases of chemotherapy-induced clonal evolution of a tumor, chemotherapy becomes the leading factor in treatment. The latest research shows that the impact of chemotherapy can radically increase the speed of clonal evolution and lead to new malignant and resistant clones that cause tumor metastasis. Another option of chemotherapy-induced clonal evolution is formation of a new dominant clone from a clone that was minor in the initial tumor and obtained free space due to elimination of sensitive clones by chemotherapy. As a result, in ~20% of cases, chemotherapy can stimulate metastasis and relapse of tumors due to clonal evolution. The conclusion of the overview formulates approaches to tumor treatment based on clonal evolution: in particular, precision therapy, prediction of metastasis stimulation in patients treated with chemotherapy, methods of genetic evaluation of chemotherapy efficiency and clonal-oriented treatment, and approaches to manipulating the clonal evolution of tumors are presented.     

Proliferative kinetics of human lymphocytes in culture measured by autoradiography and sister chromatid differential staining

A simple combination of autoradiography, to determine when a cell synthesized DNA, and sister chromatid differential staining, to determine how many times a cell has divided, was used to follow up the proliferating fate of human lymphocytes in culture. Cells were incubated continuously with 5-bromodeoxyuridine (BrdU) and pulse-labelled with 0.1 muCi/ml [3H]thymidine at various times after stimulation with phytohemagglutinin (PHA). The cells were then harvested at 4 h intervals up to 72 h, and the percentage of labelled mitoses was determined separately in first, second, or third division cells. The data showed that the cycling cells, whether they began cycling at earlier or later times after stimulation, had about the same generation times of 12--14 h. This confirms that the heterogeneity of cell generations seen in short-term lymphocyte cultures is in large part due to the difference in the times when cells began cell cycling in response to PHA.     

Clonal Transgenerational Effects Transmit for Multiple Generations in a Floating Plant

Environmental conditions of a parent plant can influence the performance of their clonal offspring, and such clonal transgenerational effects may help offspring adapt to different environments. However, it is still unclear how many vegetative generations clonal transgenerational effects can transmit for and whether it depends on the environmental conditions of the offspring. We grew the ancestor ramets of the floating clonal plant Spirodela polyrhiza under a high and a low nutrient level and obtained the so-called 1^st-generation offspring ramets of two types (from these two environments). Then we grew the 1^st-generation offspring ramets of each type under the high and the low nutrient level and obtained the so-called 2^nd-generation offspring ramets of four types. We repeated this procedure for another five times and analyzed clonal transgenerational effects on growth, morphology and biomass allocation of the 1^st- to the 6^th-generation offspring ramets. We found positive, negative or neutral (no) transgenerational effects of the ancestor nutrient condition on the offspring of S. polyrhiza, depending on the number of vegetative generations, the nutrient condition of the offspring environment and the traits considered. We observed significant clonal transgenerational effects on the 6^th-generation offspring; such effects occurred for all three types of traits (growth, morphology and allocation), but varied depending on the nutrient condition of the offspring environment and the traits considered. Our results suggest that clonal transgenerational effects can transmit for multiple vegetative generations and such impacts can vary depending on the environmental conditions of offspring.     

The cytotoxic and anti-proliferative effects of 3-hydrogenkwadaphnin in K562 and jurkat cells is reduced by guanosine

3-hydrogenwadaphnin (3-HK) is a new daphnane-type diterpene ester isolated from Dendrostellera lessertii with strong anti-tumoral activity in animal models and in cultures. Here, prolonged effects of this new agent on proliferation and viability of several different cancerous cell lines were evaluated. Using [(3)H]thymidine incorporation, it was found that the drug inhibited cell proliferation and induced G1/S cell cycle arrest in leukemic cells 24 h after a single dose treatment. The cell viability of Jurkat cells was also decreased by almost 10 %, 31 % and 40 % after a single dose treatment (7.5 nM) at 24, 48 and 72 h, respectively. The drug-treated cells were stained with acridine orange/ ethidium bromide to document the chromatin condensation and DNA fragmentation. These observations were further confirmed by detection of DNA laddering pattern in the agarose gel electrophoresis of the extracted DNA from the treated cells. Treatment of K562 cells with the drug at 7.5, 15 and 30 nM caused apoptosis in 25 %, 45 % and 65 % of the cells, respectively. Exogenous addition of 25-50 microM guanosine and/or deoxyguanosine to the cell culture of the drug-treated cells restored DNA synthesis, released cell arrest at G1/S checkpoint and decreased the apoptotic cell death caused by the drug. These observations were not made using adenosine. However, the drug effects on K562 cells were potentiated by hypoxanthine. Based on these observations, perturbation of GTP metabolism is considered as one of the main reasons for apoptotic cell death by 3-HK.     

Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.     

Clonal Variation In Proliferation Rate of Cultures of Gpk Cells

Pedigrees of twenty-six clones of a line of keratocytes derived from guinea-pig ear epidermis (GPK cells) were analysed from time-lapse film. the mean interdivision time (IDT) for the culture was 1143 φ 215 (SD) min. the mean generation rates (mean reciprocal interdivision times) of clones varied over a range of 3.93–10.2 × 10^-4/min and the standard deviation of the clonal mean generation rates was 16.8% of the average value. Transient intraclonal variations in IDT due to mitoses in a plane perpendicular to the substratum were observed. the data were also analysed on the basis of cell location in sixteen equal zones (quadrats) of the filmed area. the mean generation rate of quadrats was 8.73 x 10^-4/min (SD = 4.9%). the spatial distribution showed some clustering of cells. the mean local density of the clones (2.25 φ 0.62 cells/10^-4 cm²) was significantly higher than the quadrat density (1.76 φ 0.8 cells/10^-4 cm²). There was no significant correlation between clonal density and mean generation rates, whereas for quadrats a significant negative correlation was found (P= 2.7%). the results support the proposition that cell lineage is the major determinant of the proliferation rate of subconfluent cultures.     

Spatial and Temporal Heterogeneity in High-Grade Serous Ovarian Cancer: A Phylogenetic Analysis

Background

The major clinical challenge in the treatment of high-grade serous ovarian cancer (HGSOC) is the development of progressive resistance to platinum-based chemotherapy. The objective of this study was to determine whether intra-tumour genetic heterogeneity resulting from clonal evolution and the emergence of subclonal tumour populations in HGSOC was associated with the development of resistant disease.

Methods and Findings

Evolutionary inference and phylogenetic quantification of heterogeneity was performed using the MEDICC algorithm on high-resolution whole genome copy number profiles and selected genome-wide sequencing of 135 spatially and temporally separated samples from 14 patients with HGSOC who received platinum-based chemotherapy. Samples were obtained from the clinical CTCR-OV03/04 studies, and patients were enrolled between 20 July 2007 and 22 October 2009. Median follow-up of the cohort was 31 mo (interquartile range 22–46 mo), censored after 26 October 2013. Outcome measures were overall survival (OS) and progression-free survival (PFS). There were marked differences in the degree of clonal expansion (CE) between patients (median 0.74, interquartile range 0.66–1.15), and dichotimization by median CE showed worse survival in CE-high cases (PFS 12.7 versus 10.1 mo, p = 0.009; OS 42.6 versus 23.5 mo, p = 0.003). Bootstrap analysis with resampling showed that the 95% confidence intervals for the hazard ratios for PFS and OS in the CE-high group were greater than 1.0. These data support a relationship between heterogeneity and survival but do not precisely determine its effect size. Relapsed tissue was available for two patients in the CE-high group, and phylogenetic analysis showed that the prevalent clonal population at clinical recurrence arose from early divergence events. A subclonal population marked by a NF1 deletion showed a progressive increase in tumour allele fraction during chemotherapy.

Conclusions

This study demonstrates that quantitative measures of intra-tumour heterogeneity may have predictive value for survival after chemotherapy treatment in HGSOC. Subclonal tumour populations are present in pre-treatment biopsies in HGSOC and can undergo expansion during chemotherapy, causing clinical relapse.     

Clonal integration affects growth and sediment properties of the first ramet generation,but not later ramet generations under severe light stress

克隆整合影响严重光胁迫下第一分株世代的生长和沉积物特征但不影响 后续分株世代的生长和沉积物特征 克隆整合通过缓冲环境压力和提高资源获取效率使克隆植物受益。然而,在一个克隆系统中,受益于克隆整合的连接分株世代的数量很少受到关注。我们进行了一个盆栽实验来评估沉水植物苦草 (Vallisneria natans)克隆系统内的生理整合程度,该克隆系统由一个母株和3个依次连接的后代分株组成。 母株生长在正常光照下,而后代分株被严重遮荫。母株与后代分株间的匍匐茎被切断或保持连接,但3个后代分株之间的连接仍然存在。与遮荫的后代分株连接时,苦草未遮荫的母株的光合能力显著增强,但其生物量积累大大减少。克隆整合显著增加了第一分株世代(相邻分株)的生物量积累和土壤的碳氮可用性、胞外酶活性和微生物生物量,但没有增加后续分株世代的这些特征。我们的结果表明,在严重光胁迫下,来自苦草母株的支持可能仅限于克隆系统中相邻的后代分株,这暗示着一个分株世代的效应。我们的结果有助于更好地理解克隆植物的层次结构和分段化。这些发现表明克隆整合程度在分株种群的生态相互作用中起着至关重要的作用。     

Effects of fragmentation of clones compound over vegetative generations in the floating plant Pistia stratiotes

Background and AimsClonal plants dominate many plant communities, especially in aquatic systems, and clonality appears to promote invasiveness and to affect how diversity changes in response to disturbance and resource availability. Understanding how the special physiological and morphological properties of clonal growth lead to these ecological effects depends upon studying the long-term consequences of clonal growth properties across vegetative generations, but this has rarely been done. This study aimed to show how a key clonal property, physiological integration between connected ramets within clones, affects the response of clones to disturbance and resources in an aquatic, invasive, dominant species across multiple generations.MethodsSingle, parental ramets of the floating stoloniferous plant Pistia stratiotes were grown for 3 weeks, during which they produced two or three generations of offspring; connections between new ramets were cut or left intact. Individual offspring were then used as parents in a second 3-week iteration that crossed fragmentation with previous fragmentation in the first iteration. A third iteration yielded eight treatment combinations, zero to three rounds of fragmentation at different times in the past. The experiment was run once at a high and once at a low level of nutrients.ResultsIn each iteration, fragmentation increased biomass of the parental ramet, decreased biomass of the offspring and increased number of offspring. These effects persisted and compounded from one iteration to another, though more recent fragmentation had stronger effects, and were stronger at the low than at the high nutrient level. Fragmentation did not affect net accumulation of mass by groups after one iteration but increased it after two iterations at low nutrients, and after three iterations at both nutrient levels.ConclusionsBoth the positive and negative effects of fragmentation on clonal performance can compound and persist over time and can be stronger when resource levels are lower. Even when fragmentation has no short-term net effect on clonal performance, it can have a longer-term effect. In some cases, fragmentation may increase total accumulation of mass by a clone. The results provide the first demonstration of how physiological integration in clonal plants can affect fitness across generations and suggest that increased disturbance may promote invasion of introduced clonal species via effects on integration, perhaps especially at lower nutrient levels.     

Characterization and cellular localization of nucleophosmin/B23 in HeLa cells treated with selected cytotoxic agents (studies of B23-translocation mechanism).

Previous studies indicated that nucleophosmin/B23, an abundant nucleolar phosphoprotein, accumulated in the nucleoplasm (B23-translocation) of cells after exposure to selected cytotoxic drugs. Attempts were made to understand the B23-translocation mechanism. This paper reports that: (1) B23-translocation is a reversible process. Upon removal of camptothecin, which induced B23-translocation in HeLa cells, nucleophosmin/B23 relocalized into nucleoli within 2 h. Relocation occurs in the presence of cycloheximide which inhibits new protein synthesis. There is no reduction or degradation of nucleophosmin/B23 detected during drug treatments. Nucleophosmin/B23 has a half-life of 18-20 h. Taken together, these results indicate that B23-translocation is a reversible process. Drug treatment causes redistribution of nucleophosmin/B23 in nucleoplasm. (2) Inhibition of RNA synthesis does not cause the B23-translocation. Over 80% of RNA synthesis was inhibited in HeLa cells by treatment with actinomycin D, camptothecin, and methotrexate. While actinomycin D and camptothecin cause B23-translocation in all cells, 40% of methotrexate-treated cells remain untranslocated. (3) There is no significant change of phosphorylation in nucleophosmin/B23 during drug treatment. An identical oligomeric cross-linkage pattern was obtained in drug-treated cells. (4) HeLa cells treated with B23-translocation effective drugs have small and round nucleoli while control cells have large and irregular-shaped nucleoli.