Growth slow-down and growth arrest of human colon carcinoma cells HCT-8 in vitro after exposure to 5-fluoro-2'-deoxyuridine

Abstract. Cellular heterogeneity in drug response denotes a mixed response among individual cells in a drug treated population. Individual cell responses may be more complex than 'cell kill'and 'no response'. In this study we employed a colony formation assay and high-resolution image analysis to detect the various responses such as immediate and delayed cessation of growth, growth delay and growth slow-down, at the level of the individual colony. The evaluation was carried out using a human ileocaecal adenocarcinoma cell line (HCT-8) and the anti-tumour agent 5-fluoro-2'-deoxyuridine (FdUrd). In the presence of a drug concentration which, in standard monolayer assays, inhibits the growth to about 50% (IC₅₀) only about 20% of the colonies ceased to grow and the remaining colonies grew at a growth rate of about 70% of control. At an FdUrd concentration which, in standard monolayer assays, reduced growth by >90% (>IC₉₀), about 50% of the cells grew, with growth rates of about 30% of control. The slowing of growth, most prominent at lower drug concentrations, should be considered in determining mechanisms of drug action at the individual cell level. In clinical situations in which high drug doses are precluded by toxicity to normal tissues, growth slow-down may play a significant role in tumour response.     

Enhanced transformation and chemosensitivity of NIH3T3 cells transduced with hepatoma up-regulated protein

Hepatoma up-regulated protein (HURP) is a recently identified novel cell-cycle-regulated gene. The HURP gene is overexpressed in human hepatocellular carcinoma and transitional cell carcinoma. The cellular function of HURP is not fully understood. In this study, the NIH3T3 cells transduced with the exogenous HURP gene manifested the general characteristics of tumor cells, which had higher growth rate in low-serum media and advanced ability of colony formation on agarose-based plates. Transduced HURP was capable of specifically enhancing the chemosensitivity of deoxycytosine analogs, such as gemcitabine, ARA-C, and 5-AZA-CdR, but neither had an effect on the response of DNA intercalating agents, such as cisplatin, carboplatin, and doxorubicin, nor on the response of microtubule stabilizers, such as paclitaxel, docetaxel, and vinblastine. These results indicate that the HURP gene might be a potential oncogenic gene and capable of enhancing the chemosensitivity of deoxycytosine analogs in NIH3T3 cells.     

Epidermal growth factor (EGF)- and scatter factor/hepatocyte growth factor (SF/HGF)-mediated keratinocyte migration is coincident with induction of matrix metalloproteinase (MMP)-9

Receptor tyrosine kinases are key regulators of cellular function including cell growth, differentiation, migration, and morphogenesis. Disruptions of receptor tyrosine kinase signaling pathways are often associated with changes in cellular proliferative capacity and tumorigenesis. Both receptor-specific and cell type-specific factors may contribute to the ultimate cellular responses observed after receptor activation. In this regard, we find that both normal keratinocytes and their tumorigenic counterparts display differential responses to activation of receptor tyrosine kinases. Multiple ligands were mitogenic for keratinocytes, but only epidermal growth factor (EGF), transforming growth factor α (TGFα), and scatter factor/hepatocyte growth factor (SF/HGF) promoted cell motility as assessed by colony dispersion (scattering) and in vitro reepithelialization. Interestingly, growth factor specificity for motility coincided with ligand-mediated cell invasion through a reconstituted basement membrane and induction of the 92-kDa metalloproteinase (MMP-9) activity as determined by gelatin zymogram analysis. Inhibitors of MMP activity or addition of an MMP-9 neutralizing antibody resulted in the loss of growth factor-induced colony dispersion, suggesting a functional role for MMP-9 induction during this response. Coordinate regulation of MMP-9 induction and the migratory response are likely to contribute to the enhanced invasive potential observed in response to EGF and SF/HGF. Our findings suggest that alternate receptor-mediated signaling pathways leading to differences in gene expression may be involved in complex cellular responses such as colony dispersion or invasion. J. Cell. Physiol. 176:255–265, 1998. © 1998 Wiley-Liss, Inc.     

A computer model for the growth and differentiation of a fungal colony on solid substrate

Mold growth and differentiation are closely related to the formation of secondary products. In solid-substrate fermentations this interrelationship is often more completely realized than in submerged cultures. Solid substrate reactions are used commercially in a limited manner in the western world, but are relatively common in Asia. Basic studies in solid-substrate fermentation should yield results applicable to all types of commercial mold fermentations for the production of a secondary product. This paper presents a relatively simple model for the growth of a mold colony on a solid surface with a defined medium utilizing glucose. Unlike submerged cultures the model must account for both cellular differentiation and the spatial heterogeneity in the system. Model parameters were estimated independently using literature values. The results of the simulation studies suggest that mass transfer limitations are at least partially responsible for the proliferation of differentiated structures on solid substrates as compared to liquid cultures. Since the concentration profile depends on the depth of the substratum, conditions that enhance conidia production can be achieved by controlling the depth of the solid medium.     

Unbalanced cell growth and increased protein synthesis induced by chemotherapeutic agents

Unbalanced cell growth as manifested by an increase in cellular volume and in cellular dry mass following exposure to a variety of chemotherapeutic agents has been shown for neoplastic cells in vitro and human leukemic cells in vivo. The purpose of the present investigation was to test the hypothesis that unbalanced cell growth results from a disassociation of cell growth and cell division due to the blocking effect of chemotherapeutic agents. Monolayer cultures of CHO fibroblasts were studied in terms of their response to two chemotherapeutic agents that differ significantly in their mode of action, adriamycin and chlorambucil. Following exposure to these drugs, cell volume increased at a rate of from 1% to 4% per h; the total cell protein increased at a rate of from 4% to 7% per h. These changes were observed in both log and stationary phase cultures. Thus exposure to adriamycin and chlorambucil was followed by a more rapid rate of protein synthesis relative to the rate of degradation, resulting in larger cells with more protein whether or not the cells were actively in the division cycle. This is inconsistent with the hypothesis that unbalanced growth results simply from a disassociation of the cell division cycle from cell growth. These observations suggest that a final common pathway in the mode of action of chemotherapeutic agents may be the induction of unscheduled protein synthesis resulting in unbalanced cell growth.     

Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous γ-irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after busulphan than after vinblastine even though their proliferation rate is increased. The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan. These results suggest that following cytotoxic agents the agar colony forming cells (“committed” stem cells) are not self-maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.     

The Effect of Spaceflight on Growth of Ulocladium chartarum Colonies on the International Space Station

The objectives of this 14 days experiment were to investigate the effect of spaceflight on the growth of Ulocladium chartarum, to study the viability of the aerial and submerged mycelium and to put in evidence changes at the cellular level. U. chartarum was chosen for the spaceflight experiment because it is well known to be involved in biodeterioration of organic and inorganic substrates covered with organic deposits and expected to be a possible contaminant in Spaceships. Colonies grown on the International Space Station (ISS) and on Earth were analysed post-flight. This study clearly indicates that U. chartarum is able to grow under spaceflight conditions developing, as a response, a complex colony morphotype never mentioned previously. We observed that spaceflight reduced the rate of growth of aerial mycelium, but stimulated the growth of submerged mycelium and of new microcolonies. In Spaceships and Space Stations U. chartarum and other fungal species could find a favourable environment to grow invasively unnoticed in the depth of surfaces containing very small amount of substrate, posing a risk factor for biodegradation of structural components, as well as a direct threat for crew health. The colony growth cycle of U. chartarum provides a useful eukaryotic system for the study of fungal growth under spaceflight conditions.     

Cellular automata simulations of fungal growth on solid substrates

Growth of filamentous fungi on the surface of cereal grains is a critical aspect of solid substrate fermentation (SSF). Numerous mathematical models have been developed to describe various aspects of fungal growth in SSF. These models consider hyphal geometry and nutrient availability as determinants of colony morphology and fungal physiological state. This work describes the use of cellular automata (CA) as an alternative method of modeling fungal growth. CA models reliant on a very limited set of rules or "knowledge base" display a rich array of behaviors that mimic fungal growth. By incorporating probablistic growth rules into CA models, colony characteristics such as biomass accumulation rate, colony radial growth rate, mycelial density and fungal differentiation are readily generated.     

Kinetics of circadian band development in Neurospora crassa

The circadian rhythm of Neurospora crassa can be seen as a conidiation rhythm that produces concentric rings of bands (conidiating regions) alternating with interbands (non-conidiating regions) on the surface of an agar medium. To follow quantitatively this rhythm, densitometric analysis, gravimetric procedures, and video microscopy were employed. The circadian behavior of N. crassa is commonly monitored by cultivation in race tubes; in this work we report different growth kinetics during cultivation in conventional Petri dish cultures. Two different growth parameters were measured: total colony mass (true growth rate) and distance (colony radial expansion or hyphal elongation). Determinations of cellular mass revealed a dramatic circadian oscillation with a marked drop in growth rate during new interband formation followed by a sharp increase during the development of a new conidiation band. On the other hand, we found that the radial expansion of the colony previously reported to decrease periodically seemed unaffected by the circadian clock. Densitometric analysis showed no initial difference in the expanding margin of the colony, independent of whether that area was destined to be a band or an interband. The band areas increased rapidly in density for about 15 h whereas the interband areas maintained an equally rapid rate of increase for only 6h. The density of band areas kept increasing slowly for almost 40 h, along with an increase in the amount of conidia. Video microscopy showed the importance of cytoplasmic flow in colony development with continuous forward flow to support hyphal morphogenesis and reverse flow to support an extended period of conidiogenesis. Our results indicate that the circadian system of Neurospora can be expressed at the level of cellular mass formation, not just as the developmental conidiation rhythm.     

Tumor heterogeneity: morphological, molecular and clinical implications

Malignant tumors are characterized by their great heterogeneity and variability. There are hundreds of different types of malignant tumors that harbour many oncogenic alterations. The tumor heterogeneity has important morphological, molecular and clinical implications. Except for some hematopoietic and lymphoproliferative processes and small cell infant tumors, there are not specific molecular alterations for most human tumors. In this review we summarize the most important aspects of carcinogenesis and chemoradiosensitivity of malignant cells. In this regard, some oncogenes such as neu, ras and bcl-2 have been associated with cellular resistance to treatment with anticancer agents. The knowledge of oncogenic alterations involved in each tumor can be important to correlate the morphological features, the genetic background, the prognosis and the clinical response to treatment with anticancer agents. Based on the molecular background of the tumor there are new cancer gene therapy protocols. For example using adenovirus Ela in tumors with overexpression of neu oncogene, inhibitors of tyrosine kinase specific for the PDGF receptor in glioma, inhibitors of farnesil transferase to prevent ras activity in tumors with mutations in the ras gene.     

T cell regulation of myelopoiesis: analysis at a clonal level

Colony-stimulating factor (CSF) production by a series of cloned human T lymphocyte cell lines was examined by substituting cloned T cells for peripheral blood mononuclear cells in the feeder layer of a double-layer agar CFU-C assay system. Of 12 T cell lines tested, all produced CSF when stimulated by specific antigen, whereas CSF production in the absence of stimulation was generally negligible. In the case of soluble antigen-specific (ragweed or tetanus toxoid) clones, this required both nominal antigen and the appropriate MHC gene product on autologous antigen-presenting cells, whereas in the case of clones specific for EBV-transformed B cell lines (allogeneic or autologous), surface-bound EBV-related antigen and MHC was necessary. When tested in this manner, CSF production by different cloned T cells was heterogeneous in both amount and subclass. Thus, although most clones stimulated growth of granulocyte, monocyte, and eosinophil colonies, certain clones were identified which preferentially stimulated some colony types but not others. This heterogeneity was particularly evident with respect to eosinophil colony production. In addition, a soluble inhibitor of granulocyte colony growth was produced by one clone. These findings provide further support for the notion that antigen-specific T cells may, on activation, regulate myelopoiesis in a precise way, and provide a possible cellular basis for selective eosinophilia, monocytosis, or neutrophilia seen in certain disease states.     

1,25-Dihydroxycholecalciferol-induced differentiation of myelomonocytic leukemic cells unresponsive to colony stimulating factors and phorbol esters

The murine myelomonocytic leukemia cell line WEHI-3B D+, which differentiates in response to granulocyte colony stimulating factor (G-CSF), can also be induced to differentiate into monocyte-macrophages by phorbol myristate acetate (PMA) treatment, whereas the WEHI-3B D- subline, which is unresponsive to G-CSF and PMA, can be induced to differentiate to granulocytes as well as monocytes by 1,25-dihydroxycholecalciferol [1,25-(OH)2 D3], the biologically active metabolite of vitamin D3. A newly developed variant of the WEHI-3B D+ line, named WEHI-3B D+ G, which was responsive to G-CSF but not to PMA, was also differentiated to granulocytes by 1,25-(OH)2 D3. Although vitamin D3 has been reported to induce macrophage differentiation in responsive tumor cells, this is the first demonstration that 1,25-(OH)2 D3 can induce granulocyte differentiation. In both differentiation pathways, cessation of cellular proliferation accompanies changes in morphologic and cytochemical properties of the cells. This suggests that leukemic cell lines unresponsive to differentiation agents acting at the cell surface retain their ability to differentiate in response to agents that do not act via the plasma membrane such as 1,25-(OH)2 D3, which has cytosolic/nuclear receptors. Vitamin D3 could act through different cellular pathways inducing differentiation or by bypassing only the first step of a common differentiation cascade used by agents with cell surface receptors such as CSF. These results suggest that low doses of 1,25-(OH)2 D3 may be useful in combination with hemopoietic growth factors (CSFs) as therapeutic agent to induce leukemic cell differentiation in vivo.     

Mathematical deconvolution uncovers the genetic regulatory signal of cancer cellular heterogeneity on resistance to paclitaxel

Drug resistance remains a major problem in combating malignancies, resulting critical the resistance to paclitaxel used in the treatment of many different cancers. Elucidating the cellular heterogeneity composition of tumours may be relevant to designing more effective treatment strategies on drug resistance. In particular, such heterogeneity correlates with the measurement of gene expression below the population level. However, experimental assays capturing differential response are limited and cannot discern the variation in gene expression specific to different cellular types in tumour populations. These limitations led us to consider a mathematical modelling approach, in which the gene expression of cellular subpopulations is recovered by deconvolution. Mathematically, the deconvolution is a multi-linear regression-based problem. We combined herein data on cellular subpopulation frequency composition with gene expression values from 16 tumour lines (8 resistant and 8 sensitive to paclitaxel treatment) to find genes that are differentially expressed between paclitaxel resistant and paclitaxel sensitive tumour lines in different cellular subpopulations. The results indicate that many genes differentially expressed between paclitaxel resistant and sensitive cancer lines are only detected when considering their heterogeneous cellular composition. Overall, our methodology is thought to keep in mind phenotypic heterogeneity improving our resolution in the identification of biomarkers on resistance to chemo-therapeutic agents.     

Recovery of exponentially growing cultures of Klebsiella pneumoniae NCIB 418 after heat shocks

Exponentially growing cultures of Klebsiella pneumoniae were subjected to heat shocks in the superoptimal and supermaximal temperature ranges for growth on glucose in a defined mineral salts medium. Transitory changes in the specific growth rate constant during recovery were evident. The response was heat shock temperature and exposure time dependent. Cell viability determinations, based on colony counts, indicated complete recovery from heat treatments at superoptimal temperatures. In contrast, at supermaximal temperatures, discrepancies in colony counts on different agars were observed. The kinetic response of the specific growth rate constant after a heat shock at supermaximal temperatures is explained by segregation within the bacterial population     

Quantitative Characterization of Cellular Membrane-Receptor Heterogeneity through Statistical and Computational Modeling

Cell population heterogeneity can affect cellular response and is a major factor in drug resistance. However, there are few techniques available to represent and explore how heterogeneity is linked to population response. Recent high-throughput genomic, proteomic, and cellomic approaches offer opportunities for profiling heterogeneity on several scales. We have recently examined heterogeneity in vascular endothelial growth factor receptor (VEGFR) membrane localization in endothelial cells. We and others processed the heterogeneous data through ensemble averaging and integrated the data into computational models of anti-angiogenic drug effects in breast cancer. Here we show that additional modeling insight can be gained when cellular heterogeneity is considered. We present comprehensive statistical and computational methods for analyzing cellomic data sets and integrating them into deterministic models. We present a novel method for optimizing the fit of statistical distributions to heterogeneous data sets to preserve important data and exclude outliers. We compare methods of representing heterogeneous data and show methodology can affect model predictions up to 3.9-fold. We find that VEGF levels, a target for tuning angiogenesis, are more sensitive to VEGFR1 cell surface levels than VEGFR2; updating VEGFR1 levels in the tumor model gave a 64% change in free VEGF levels in the blood compartment, whereas updating VEGFR2 levels gave a 17% change. Furthermore, we find that subpopulations of tumor cells and tumor endothelial cells (tEC) expressing high levels of VEGFR (>35,000 VEGFR/cell) negate anti-VEGF treatments. We show that lowering the VEGFR membrane insertion rate for these subpopulations recovers the anti-angiogenic effect of anti-VEGF treatment, revealing new treatment targets for specific tumor cell subpopulations. This novel method of characterizing heterogeneous distributions shows for the first time how different representations of the same data set lead to different predictions of drug efficacy.     

Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype

Escherichia coli DEAD-box protein A (DbpA) is an ATP-dependent RNA helicase with specificity for 23S ribosomal RNA. Although DbpA has been extensively characterized biochemically, its biological function remains unknown. Previous work has shown that a DbpA deletion strain is viable with little or no effect on growth rate. In attempt to elucidate a phenotype for DbpA, point mutations were made at eleven conserved residues in the ATPase active site, which have exhibited dominant-negative phenotypes in other DExD/H proteins. Biochemical analysis of these DbpA mutants shows the expected decrease in RNA-dependent ATPase activity and helix unwinding activity. Only the least biochemically active mutation, R331A, produces small colony phenotype and a reduced growth rate. This dominant slow growth mutant will be valuable to determine the cellular function of DbpA.     

A genetically engineered cell-based biosensor for functional classification of agents.

Cell-based biosensors (CBBs) utilize whole cells to detect biologically active agents. Although CBBs have shown success in detecting the presence of biological agents, efforts to classify the type of agent based on functional activity have proven difficult because multiple biochemical pathways can lead to the same cellular response. However, a new approach using a genetically-engineered cell-based biosensor (GECBB) described in this paper translates this cross-talk noise into common-mode noise that can be rejected. The GECBB operates by assaying for an agent's ability to differentially activate two populations of cells, wild-type (WT) cells and cells genetically engineered to lack a specific receptor, knockout (KO) cells. Any biological agent that targets the knocked out receptor will evoke a response in the WT but not in the KO. Thus, the GECBB is exquisitely sensitive to agents that effect the engineered pathway. This approach provides the benefits of an assay for specific functional activity while simplifying signal analysis. The GECBB implemented was designed to be sensitive to agents that activate the beta 1-adrenergic receptor (beta 1-AR). This was achieved by using mouse cardiomyocytes in which the beta 1-AR had been knocked out. The cellular signal used in the GECBB was the spontaneous beat rate of the two cardiomyocyte syncitia as measured with microelectrode arrays. The GECBB was able to detect the beta-AR agonist isoproterenol (ISO) at a concentration of 10 microM (P<0.005).     

A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model--a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis.     

Testing the grain-size model for the evolution of phenotypic plasticity

Phenotypic plasticity is the ability of a genotype to modify its phenotypic characteristics in response to different environments. Theory predicts that adaptive plasticity should primarily evolve in organisms that experience heterogeneous environments. An organism's dispersal rate is a key component in these models, because the degree of dispersal partly determines the extent of environmental heterogeneity. Here, I provide the first large-scale test of the theoretical prediction that phenotypic plasticity evolves in association with dispersal rate using meta-analysis of data from 258 experiments from the literature on plasticity in marine invertebrates. In line with predictions, phenotypic plasticity is generally greater in species with higher dispersal rates, suggesting that dispersal and environmental heterogeneity are important selective agents for evolution of plasticity in marine habitats.     

Modelling the growth of Trichoderma virens with limited sampling of digital images

AIMS: Using limited digital image sampling, a model of fungal growth in soil that considers both hyphal production and lysis was constructed for two strains of Trichoderma virens over a range of four temperatures. MATERIALS AND METHODS: A growth model was developed by fitting the radial cross sectional data with a modified form of the Ratkowsky equation to determine maximum growth rate and a modified Arrhenius equation to determine maximal rate of decrease in area covered by mycelia. The parameters obtained from a combined equation were then verified by using the data obtained from the whole colony to determine the appropriateness of the model. CONCLUSIONS: Using a limited data set and a combination of the Ratkowsky and Arrhenius equations, the mycelial coverage of the T. virens colony was determined, relating microscopic hyphal growth to macroscopic colony growth. This model was sufficiently robust to predict growth across four temperatures for a genetically modified and wild-type strain of T. virens. SIGNIFICANCE AND IMPACT OF STUDY: By using simple assumptions for the increase and eventual decline in fungal growth on a resource-limited medium, this model constructs an initial framework onto which additional parameters such as nutrient consumption could be incorporated for prediction of fungal growth.