Stable alterations at the cell membrane of Chinese hamster ovary cells resistant to the cytotoxicity of phytohemagglutinin.

Chinese hamster ovary (CHO) cells selected for resistance to the cytotoxicity of phytohemagglutin (PHA) have been found to exhibit stable alterations at their plasma membranes. The PHA-resistant (PhaR) cells bind markedly less 125I-PHA than do sensitive CHO cells and also exhibit an increased sensitivity to the cytotoxicity of concanavalin A, a lectin of different receptor specificity. Mutagenesis with ethylmethanesulfonate increases the proportion of PhaR cells 20- to 100-fold. PHA-resistant cells maintained for up to 8 months in continuous culture in the absence of the selective agent have retained the PhaR phenotype. These and other characteristics of the experimental system suggest that CHO cells selected for PHA resistance are authentic somatic cell mutants. The Pha marker appears to behave recessively in hybrids formed between PhaR and PhaS cells.     

Quantitative morphological analysis of adriamycin-resistant human K562 leukemic cells

The morphological changes associated with Adriamycin resistance in a human leukemic cell line have been investigated by image analysis. An Adriamycin-resistant subline of the human erythroleukemic K562 cell line has been established. Three sets of cells have been analysed: sensitive cells, resistant cells cultured in the continuous presence of Adriamycin, and resistant cells cultured without the drug. Image analysis shows that Adriamycin-resistant K562 cells display significant morphological changes as compared with sensitive cells, at both the nuclear and cytoplasmic levels. These changes make it possible to separate sensitive and resistant cells automatically and with a classification accuracy of 76% and only four cytological parameters. Image analysis may therefore offer an interesting tool for studying drug resistance in leukemic cells, from both an experimental and a clinical point of view.     

Bcl-2 inhibits apoptosis by increasing the time-to-death and intrinsic cell-to-cell variations in the mitochondrial pathway of cell death

BH3 mimetics have been proposed as new anticancer therapeutics. They target anti-apoptotic Bcl-2 proteins, up-regulation of which has been implicated in the resistance of many cancer cells, particularly leukemia and lymphoma cells, to apoptosis. Using probabilistic computational modeling of the mitochondrial pathway of apoptosis, verified by single-cell experimental observations, we develop a model of Bcl-2 inhibition of apoptosis. Our results clarify how Bcl-2 imparts its anti-apoptotic role by increasing the time-to-death and cell-to-cell variability. We also show that although the commitment to death is highly impacted by differences in protein levels at the time of stimulation, inherent stochastic fluctuations in apoptotic signaling are sufficient to induce cell-to-cell variability and to allow single cells to escape death. This study suggests that intrinsic cell-to-cell stochastic variability in apoptotic signaling is sufficient to cause fractional killing of cancer cells after exposure to BH3 mimetics. This is an unanticipated facet of cancer chemoresistance.     

Does plasmolysis increase the drought tolerance of plant cells?

Summary Iljin's experiments withRhoeo discolor, cabbage and red beet which seemed to demonstrate increased drought resistance of plasmolysed tissue have been repeated, but his results could not be confirmed. The tissue plasmolysed in sucrose solutions, died either during stepwise plasmolysis and deplasmolysis or else later on, during exposure to unsaturated air within one or two days, even at the highest humidities. Iljin's error was apparently produced by a wrong interpretation of his tests of viability: the plasma of his dead cells did not disintegrate and retained anthocyanin by tonoplast plasmolysis.Plasmolysis proved harmful to all three objects investigated.In view of these results and earlier criticism of Iljin's analogous experiments on frost resistance by others, all experimental evidence produced in support of Iljin's mechanical theory of drought resistance should be reexamined.     

Assessing the genetic component of the susceptibility of mice to viral infections.

Laboratory mice often exhibit wide differences in susceptibility when infected experimentally with viruses. Based on such observations, experiments have been designed to investigate the determinism of these differences at the molecular level, and a few genes that play a major role in the innate mechanisms of defence of the species toward viral aggressions have been characterised. For example, the extraordinary resistance of SJL mice to experimental infections with hepatitis virus strain A59 is the consequence of a structural alteration of a cell adhesion molecule which normally binds to the spikes of the virus, allowing its entry into the cells. If the virus cannot bind to the molecule, or if the molecule is absent, epithelial cells of the intestine and liver are not infected and mice are resistant. In the same way, most--not to say all--laboratory strains of mice are susceptible to infections with orthomyxoviruses or flaviviruses because essential molecules, the synthesis of which is normally triggered by interferon, are defective in these mice. Wild mice, by contrast --probably because they are constantly exposed to natural infections--are resistant. Finally, some mouse strains resist experimental infections by the mouse cytomegalovirus 1 (MCMV-1) because, once infected, these mice synthesise a molecule at the surface of infected cells which allows immediate recognition and killing by natural killer (NK) cells. With the exuberant development of mouse genetics and the constant generation of new mutant alleles, it is likely that many more genes with an impact on the phenotype of resistance or susceptibility will be identified in the forthcoming years. These genes are probably numerous, however, and many of them presumably interact with each other and/or have additive effects. This might slow down progress in our understanding of the innate mechanism of defence.     

Measurement of the membrane potential in small cells using patch clamp methods

The resting membrane potential, E(m), of mammalian cells is a fundamental physiological parameter. Even small changes in E(m) can modulate excitability, contractility and rates of cell migration. At present accurate, reproducible measurements of E(m) and determination of its ionic basis remain significant challenges when patch clamp methods are applied to small cells. In this study, a mathematical model has been developed which incorporates many of the main biophysical principles which govern recordings of the resting potential of 'small cells'. Such a prototypical cell (approx. capacitance, 6 pF; input resistance 5 GΩ) is representative of neonatal cardiac myocytes, and other cells in the cardiovascular system (endothelium, fibroblasts) and small cells in other tissues, e.g. bone (osteoclasts) articular joints (chondrocytes) and the pancreas (β cells). Two common experimental conditions have been examined: (1) when the background K(+) conductance is linear; and (2) when this K(+) conductance is highly nonlinear and shows pronounced inward rectification. In the case of a linear K(+) conductance, the presence of a "leakage" current through the seal resistance between the cell membrane and the patch pipette always depolarizes E(m). Our calculations confirm that accurate characterization of E(m) is possible when the seal resistance is at least 5 times larger than the input resistance of the targeted cell. Measurement of E(m) under conditions in which the main background current includes a markedly nonlinear K(+) conductance (due to inward rectification) yields complex and somewhat counter-intuitive findings. In fact, there are at least two possible stable values of resting membrane potential for a cell when the nonlinear, inwardly rectifying K(+) conductance interacts with the seal current. This type of bistable behavior has been reported in a variety of small mammalian cells, including those from the heart, endothelium, smooth muscle and bone. Our theoretical treatment of these two common experimental situations provides useful mechanistic insights, and suggests practical methods by which these significant limitations, and their impact, can be minimized.     

Rejection of tumors of the B cell lineage by idiotype-vaccinated mice

Idiotypic determinants of immunoglobulins of malignant B lymphocytes and plasma cells are tumor-specific antigens and have been used extensively in immunotherapy studies. The mechanisms involved in resistance to tumor challenge following idiotype vaccination are poorly understood. Although a predominant role has been attributed to anti-idiotype antibodies, both humoral and cellular immune responses are probably involved. Cell-mediated responses may be particularly effective against tumor cell variants that do not express the idiotype on the cell surface and are therefore resistant to anti-idiotype antibodies but continue to produce one of the original immunoglobulin polypeptides that may be processed and presented to T cells. In this report we describe two experimental models of idiotype vaccination in which antibodies are unlikely to play a role, and hence tumor immunity is attributed to cell-mediated responses. One model consists of the murine B lymphocyte tumor 38C-13 and its idiotype-negative variant DB2, which has lost the idiotypic specificity of the parental 38C-13 cell line through the production of a different light chain but expresses the original heavy chain. Vaccination of mice with the purified IgM of 38C-13 induced resistance to 38C-13 tumor cells as well as to the variant cells. Although immunized mice produced high levels of anti-idiotype antibodies that bound to 38C-13 cells, no binding of antibodies to DB2 cells occurred. The finding that idiotype-vaccinated mice were resistant to idiotype-negative DB2 cells suggested that cellular mechanisms are involved in mediating resistance. The second model consists of the two plasma cell line JLμs and JLμm, which produce IgM with an identical specificity. Whereas one of them (JLμs) secretes the IgM, the other one(JLμm) can neither secrete nor deposit it on the cell surface. Immunization against JLμs IgM followed by tumor challenge resulted in prolonged survival of both JLμs- and JLμm-challenged mice. Although sera of immunized mice contained high levels of anti-idiotype antibodies, they did not react with the plasmacytoma cells. Similarly to the results obtained in the 38C-13 experimental model, these results suggest that a non-antibody-mediated mechanism was involved in the resistance of mice to tumor growth. Received: 11 June 1998 / Accepted: 26 November 1998     

Modeling the Transfer of Drug Resistance in Solid Tumors

ABC efflux transporters are a key factor leading to multidrug resistance in cancer. Overexpression of these transporters significantly decreases the efficacy of anti-cancer drugs. Along with selection and induction, drug resistance may be transferred between cells, which is the focus of this paper. Specifically, we consider the intercellular transfer of P-glycoprotein (P-gp), a well-known ABC transporter that was shown to confer resistance to many common chemotherapeutic drugs. In a recent paper, Durán et al. (Bull Math Biol 78(6):1218–1237, 2016) studied the dynamics of mixed cultures of resistant and sensitive NCI-H460 (human non-small lung cancer) cell lines. As expected, the experimental data showed a gradual increase in the percentage of resistance cells and a decrease in the percentage of sensitive cells. The experimental work was accompanied with a mathematical model that assumed P-gp transfer from resistant cells to sensitive cells, rendering them temporarily resistant. The mathematical model provided a reasonable fit to the experimental data. In this paper, we develop a new mathematical model for the transfer of drug resistance between cancer cells. Our model is based on incorporating a resistance phenotype into a model of cancer growth (Greene et al. in J Theor Biol 367:262–277, 2015). The resulting model for P-gp transfer, written as a system of integro-differential equations, follows the dynamics of proliferating, quiescent, and apoptotic cells, with a varying resistance phenotype. We show that this model provides a good match to the dynamics of the experimental data of Durán et al. (2016). The mathematical model shows a better fit when resistant cancer cells have a slower division rate than the sensitive cells.     

Characteristics of expression of the tetracycline resistance gene from the plasmid pBR322 in Bacillus subtilis cells

The expression of Tc resistance gene derived from plasmid pBR322 has been studied in Bacillus subtilis cells where this alien gene is not usually expressed. Fragments of Bacillus subtilis chromosome were inserted into the Tc resistance gene promoter region of the hybrid plasmid pGG20 and the expression of this gene was registered. Plasmid pGG20 confers a constitutive mode of Tc resistance in Escherichia coli cells. In contrast, the inducibility of Tc resistance gene expression in Bacillus subtilis cells has been reported. Optimal concentration for the highest inducibility of Tc resistance by the antibiotic has been determined.     

Targeting the Hedgehog Pathway to Mitigate Treatment Resistance

‘Hedgehog’ (HH) molecules are secretory signaling proteins that were first discovered in Drosophila. Three HH homologues have been identified in humans including Sonic hedgehog (SHH), Indian hedgehog (IHH) and Desert hedgehog (DHH). During embryonic development, the Hedgehog (HH) signaling pathway is critical, and it regulates both proliferation and differentiation of various types of stem cells.1This article provides a brief overview of HH signaling, summarizes the correlation between HH signaling and treatment resistance of cancer cells, and discusses the recent advances in targeting this signaling cascade to overcome treatment resistance with supporting experimental results.     

Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.     

Anti-Candida resistance in the mouse brain and effect of intracerebral administration of interleukin 1

The effects of intracerebral and intravenous Candida albicans infection on experimental meningo-encephalitis in mice were compared. Naive mice inoculated with two C. albicans strains of different pathogenicity (highly virulent CA-6 and poorly virulent PCA-2) were more resistant to infection when the yeasts were inoculated by the intracerebral rather than the intravenous route. In immunized mice, in which systemic immunity had been induced by long-term colonization with low-virulence PCA-2 cells, increased intracerebral resistance to challenge with virulent Candida was observed at about two weeks post-infection. In contrast, the inoculation of PCA-2 cells directly into the brain resulted in early, long-lasting activation of local microbicidal mechanisms against intracerebral challenge with CA-6, Staphylococcus aureus or Aspergillus fumigatus. Increased local anti-Candida resistance was also observed upon intracerebral injection of human recombinant interleukin 1. These data suggest that, in addition to the intracerebral expression of systemic antifungal immunity, microbial mechanisms may be locally activated in the brain, possibly through release of endogenous interleukin 1.     

Overcoming barriers to understanding the cellular basis of aluminium resistance

There appears to be an emerging consensus that resistance to aluminium (Al) is mediated at the cellular level. Virtually all current hypotheses which seek to explain the basis of Al resistance have a cellular focus, including those which postulate that external mechanisms limit the rate of Al entry across the membrane and/or protect sensitive extracellular sites, as well as those which postulate that internal mechanisms detoxify Al in the cytoplasm. If Al resistance is a cellular phenomenon, it should be expressed in single cells. Attempts to demonstrate resistance in cell culture systems, however, have not been uniformly satisfying. Considerable uncertainty has arisen from use of experimental conditions which favour formation of insoluble or non-toxic Al species. This problem has plagued research which has attempted to select for Al resistance in cell culture systems, as well as research which has attempted to express existing patterns of differential resistance in cell culture systems. Despite technical problems such as this, work at the cellular level has provided some important contributions. Most importantly, we now know resistance to Al can be expressed at the cellular level. We have discovered also that plant cells accumulate Al much more rapidly in cell culture systems than in intact roots and that isolated cells are more sensitive to Al than complex tissues. While this type of research is still hampered by a number of technical barriers, it would appear that more rapid progress could be achieved if greater emphasis was placed on true experimental work. Furthermore, we need to begin evaluating experimental data in the context of an integrated Al stress response if we are to achieve a full understanding of the cellular basis of Al resistance.     

Organ-associated macrophage precursor activity: isolation of candidacidal and tumoricidal effectors from the spleens of cyclophosphamide-treated mice

We recently reported the modulating effects of a single injection of the anti-neoplastic drug cyclophosphamide (Cy; 150 mg/kg i.p.) on in vivo resistance against the experimental Candida albicans infection. Increased resistance to microbial challenge occurred 12 to 18 days after treatment. We now show that the increased resistance is paralleled by the appearance of potent nonadherent nonphagocytic effectors in the spleen (day 12) that are capable both of candidacidal activity and natural killer (NK) activity against YAC-1 cells. The cells mediating the two reactivities have a low buoyant density, a strong proliferating activity in response to the macrophage colony stimulating factor (CSF-1), and are unable to kill the NK-insensitive lines EL-4 and P815. A clear cut isolation of macrophage precursor cells from this Percoll low density fraction has been performed in an indirect rosette assay on the basis of their positivity for the surface markers recognized by the highly specific rat-anti-mouse macrophages, monoclonal antibodies M143 and F4/80. We obtained an extremely homogeneous population of cells in the early stage of macrophage differentiation that is responsible for all the candidacidal activity and for a major part of the NK activity observed in the spleen of Cy-treated mice, and which is extremely sensitive to CSF-1 induction.     

Cancer stem cells: implications for the progression and treatment of metastatic disease

Metastasis is the major cause of death for cancer patients with solid tumours, due mainly to the ineffectiveness of current therapies once metastases begin to form. Further insight into the biology of metastasis is therefore essential in order to gain a greater understanding of this process and ultimately to develop better cancer therapies. Metastasis is an inefficient process, such that very few cells that leave a tumour successfully form macrometastases in distant sites. This suggests that only a small subset of cells can successfully navigate the metastatic cascade and eventually re-initiate tumour growth to form life-threatening metastases. Recently, there has been growing support for the cancer stem cell (CSC) hypothesis which stipulates that primary tumours are initiated and maintained by a small subpopulation of cancer cells that possess "stem-like" characteristics. Classical properties of normal stem cells are strikingly reminiscent of the observed experimental and clinical behaviour of metastatic cancer cells, including an unlimited capacity for self renewal; the requirement for a specific 'niche' or microenvironment to grow; use of the stromal cell-derived factor 1 (SDF-1)/chemokine receptor 4 (CXCR4) axis for migration; enhanced resistance to apoptosis and an increased capacity for drug resistance. Therefore, in addition to playing a role in primary tumour formation, we believe that CSCs are also key players in the metastatic process. We will review the current evidence supporting this idea and discuss the potential implications of the CSC hypothesis with regards to experimental investigation and treatment of metastatic disease.     

Quantifying the relationship between multiple immunological parameters and host resistance: probing the limits of reductionism

Although reductionist experimental designs are excellent for identifying cells, molecules, or functions involved in resistance to particular microbes or cancer cells, they do not provide an integrated, quantitative view of immune function. In the present study, mice were treated with either dexamethasone (DEX) or cyclosporin A (CyA), and immune function and host resistance were evaluated. Multivariate statistical methods were used to describe the relative importance of a broad range of immunological parameters for host resistance in mice treated with various dosages of DEX. Multiple regression and logistic regression analysis indicated that changes in 24 immunological parameters explained a substantial portion of the changes in resistance to B16F10 tumor cells or streptococcus group B. However, at least 40% of the change in host resistance remained unexplained. DEX at all dosages substantially suppressed numerous relevant immunological parameters, but significantly decreased resistance to Listeria monocytogenes only at the highest dosage. In contrast, CyA substantially decreased resistance to L. monocytogenes at dosages that caused relatively minor suppression of just a few immunological parameters (unfortunately, CyA data and host resistance data for L. monocytogenes were not suitable for multivariate analysis). These results illustrate that mathematical models can be used to explain changes in host resistance on the basis of changes in immune parameters, and that moderate changes in relevant immunological parameters may not produce the types of changes in host resistance expected on the basis of results from reductionist experimental designs.     

Consequences of cell-to-cell P-glycoprotein transfer on acquired multidrug resistance in breast cancer: a cell population dynamics model

Background  

Cancer is a proliferation disease affecting a genetically unstable cell population, in which molecular alterations can be somatically inherited by genetic, epigenetic or extragenetic transmission processes, leading to a cooperation of neoplastic cells within tumoural tissue. The efflux protein P-glycoprotein (P-gp) is overexpressed in many cancer cells and has known capacity to confer multidrug resistance to cytotoxic therapies. Recently, cell-to-cell P-gp transfers have been shown. Herein, we combine experimental evidence and a mathematical model to examine the consequences of an intercellular P-gp trafficking in the extragenetic transfer of multidrug resistance from resistant to sensitive cell subpopulations.     

Detection of recombinant P-glycoprotein in multidrug resistant cultured cells

The MDR1 multidrug resistance gene encodes a high molecular weight membrane-spanning cell surface protein, P-glycoprotein, that confers multidrug resistance by pumping various cytotoxic drugs, including vinblastine, doxorubicin or paclitaxel, out of cells. Overexpression of P-glycoprotein in human tumors has been recognized as a major obstacle for successful chemotherapy of cancer. Thus, P-glycoprotein represents an important drug target for pharmacological chemosensitizers. Initially, cell culture models to study the multidrug resistance phenotype were established by selecting drug-sensitive cells in step-wise increasing, sublethal concentrations of chemotherapy agents. P-glycoprotein was found to be overexpressed in many of these models. Multidrug resistant cells can also be generated by transfection of cultured cells with the MDR1 gene, followed by selection with cytotoxic drug at a concentration that kills all untransfected host cells. Transfectants expressing wild-type or mutant recombinant P-glycoprotein have significantly contributed to our understanding of the structure of P-glycoprotein and its molecular and cellular functions. Additionally, the MDR1 gene has also been used as a selectable marker for the transfer and coexpression of non-selectable genes. This article details means for detection of P-glycoprotein in DNA-transfected or retrovirally transduced, cultured cells. Different experimental approaches are described that make use of specific antibodies for detection of P-glycoprotein. Strategies to visualize P-glycoprotein include metabolic labeling using ³⁵S-methionine, labeling with a radioactive photoaffinity analog, and non-radioactive immunostaining after Western blotting.     

盐胁迫对芦苇细胞超微结构的影响

于2009年3月从辽宁盘锦双台河口湿地挖取芦苇根茎并人工桶栽,待缓苗成功后进行不同浓度的盐胁迫处理,用透射电镜观察芦苇细胞超微结构对不同盐度胁迫的响应,以明确芦苇细胞的耐盐性。结果表明:芦苇细胞可承受4.0%以下浓度的盐胁迫。当盐度介于0%～4.0%时,芦苇细胞膜系统开始遭到破坏,使芦苇细胞受损的膜结构发生局部内陷或萎缩变形,细胞器表面变得凹凸不平,或将功能丧失的细胞器清理出细胞外,出现破裂和解体,以响应结构损伤的膜系统的修复,使细胞功能得到修复;当盐度为4.0%时,芦苇细胞叶绿体、线粒体、细胞核等具有膜结构细胞器及细胞壁遭到破坏,造成芦苇细胞膜系统的不可逆损伤,使细胞正常的物质代谢与能量转换和信息传递无法完成,导致芦苇细胞新陈代谢过程的中断,芦苇细胞生命活动趋于停止;在8.0%浓度盐胁迫下,芦苇细胞膜系统结构完全消失解体,导致芦苇细胞直接死亡。     

Hydrodynamic and mechanical aspects of endothelial permeability

The passive transport of water through the endothelial cell layer junctions is considered from the standpoint of hydrodynamic theories based on ultrastructural information. The local geometry of tight junctions based on molecular level forces and elastic membrane properties has been modeled and leads to estimates of the hydraulic resistance of the clefts. It is shown that the large resistance measured experimentally can be accounted for in this model. The transport of large macromolecules via vesicles which diffuse across the endothelial cell has been developed, but recent experimental data do not appear to support this mechanism as a primary pathway. Fused vesicles forming an open channel appear to be rare. Leaky junctions, such as around dying endothelial cells or produced by cytoskeletal changes within the cells, may be important in control of endothelial permeability. Another kind of model is a fiber matrix model of the endocapillary layer, extending into the intercellular clefts which can also account for the molecular seiving properties of the endothelial cell layer but may produce a large resistance to water flux.