Differential effects of coverslip-induced hypoxia and cobalt chloride mimetic hypoxia on cellular stress, metabolism, and nuclear structure.

Hypoxia has profound effects on cell physiology, both in normal or pathological settings like cancer. In this study, we asked whether a variant of coverslip-induced hypoxia that recapitulates the conditions found in the tumor microenvironment would elicit similar cellular responses compared to the well established model of cobalt chloride-induced hypoxia. Comparable levels of nuclear HIF-1α were observed after 24 h of coverslip-induced hypoxia or cobalt chloride treatment in CAL-27 oral squamous carcinoma cells. However, cellular stress levels assessed by reactive oxygen species production and lipid droplet accumulation were markedly increased in coverslip-induced hypoxia compared to cobalt chloride treatment. Conversely, mitochondrial ATP production sharply decreased after coverslip-induced hypoxia but was preserved in the presence of cobalt chloride. Coverslip-induced hypoxia also had profound effects in nuclear organization, assessed by changes in nuclear dry mass distribution, whereas these effects were much less marked after cobalt chloride treatment. Taken together, our results show that coverslip-induced hypoxia effects on cell physiology and structure are more pronounced than mimetic hypoxia induced by cobalt chloride treatment. Considering also the simplicity of coverslip-induced hypoxia, our results therefore underscore the usefulness of this method to recapitulate in vitro the effects of hypoxic microenvironments encountered by cells in vivo.

Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes.

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the development of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.

Mechanical profile of human keratinocytes expressing HPV-18 oncogenes.

During tumorigenesis, the mechanical properties of cancer cells change markedly, with decreased stiffness often accompanying a more invasive phenotype. Less is known about the changes in mechanical parameters at intermediate stages in the process of malignant transformation. We have recently developed a pre-tumoral cell model by stably transducing the immortalized but non-tumorigenic human keratinocyte cell line HaCaT with the E5, E6 and E7 oncogenes from HPV-18, one of the leading causes of cervical cancer and other types of cancer worldwide. We have used atomic force microscopy (AFM) to measure cell stiffness and to obtain mechanical maps of parental HaCaT and HaCaT E5/E6/E7-18 cell lines. We observed a significant decrease in Young's modulus in HaCaT E5/E6/E7-18 cells measured by nanoindentation in the central region, as well as decreased cell rigidity in regions of cell-cell contact measured by Peakforce Quantitative Nanomechanical Mapping (PF-QNM). As a morphological correlate, HaCaT E5/E6/E7-18 cells displayed a significantly rounder cell shape than parental HaCaT cells. Our results therefore show that decreased stiffness with concomitant perturbations in cell shape are early mechanical and morphological changes during the process of malignant transformation.

Hypoxia, acidification and oxidative stress in cells cultured at large distances from an oxygen source.

Hypoxia is a condition frequently encountered by cells in tissues, whether as a normal feature of their microenvironment or subsequent to deregulated growth. Hypoxia can lead to acidification and increased oxidative stress, with profound consequences for cell physiology and tumorigenesis. Therefore, the interplay between hypoxia and oxidative stress is an important aspect for understanding the effects of hypoxic microenvironments on cells. We have used a previously developed variant of the method of coverslip-induced hypoxia to study the process of acidification in a hypoxic microenvironment and to simultaneously visualize intracellular levels of hypoxia and oxidative stress. We observed high accumulation of CO2 in hypoxic conditions, which we show is the main contributor to acidification in our model. Also, increased levels of oxidative stress were observed in moderately hypoxic cells close to the oxygen source, where the mitochondrial membrane potential was preserved. Conversely, cells at large distances from the oxygen source showed higher levels of hypoxia, milder oxidative stress and reduced mitochondrial membrane potential. Our results contribute to characterize the interplay between reduced oxygen levels, acidification and oxidative stress in a simple in vitro setting, which can be used to model cell responses to an altered environment, such as the early tumor microenvironment.

Estudio de los efectos de un microambiente hipóxico en queratinocitos humanos in vitro y su correlato con alteraciones del microambiente en la patología de liquen plano oral

La hipoxia es un factor fundamental en el proceso de génesis tumoral, así como en patologías precursoras de cáncer, como es el Liquen Plano Oral (LPO). Objetivo: Determinar si es posible establecer una correlación entre las alteraciones que sufren queratinocitos normales en un microambiente hipóxico in vitro y alteraciones que aparecen en los queratinocitos en el epitelio de la mucosa oral en el contexto de la patología LPO. Métodos: Se estudiaron los cambios morfológicos mediante microscopía de contraste de fases, y la detección de marcadores asociados a hipoxia de queratinocitos humanos (HaCaT), como modelo celular oral, en un microambiente hipóxico generado por la variante del método "Hipoxia inducida por cubreobjetos". Resultados: Mediante microscopía confocal se observó la presencia de los marcadores de hipoxia GLUT-1 y aductos de pimonidazol (Hipoxyprobe) en los cultivos celulares de HaCaT expuestos a un microambiente hipóxico. Además, se observó la presencia del marcador GLUT-1 mediante inmunohistoquímica en tejido epitelial humano derivado de biopsias de la patología LPO. Conclusiones: Se estableció una correlación entre las alteraciones detectadas en queratinocitos humanos inducidos a un microambiente hipóxico in vitro y las alteraciones detectadas in vivo en tejido epitelial de la mucosa oral.

A hipóxia é um fator fundamental no processo de gênese tumoral, bem como em patologias precursoras do câncer, como o Líquen Plano Oral (LPO). Objetivo: Determinar se é possível estabelecer uma correlação entre as alterações que os queratinócitos normais sofrem em um microambiente hipóxico in vitro e as alterações que aparecem nos queratinócitos no epitélio da mucosa oral no contexto da patologia do LPO. Métodos: As alterações morfológicas foram estudadas por microscopia de contraste de fase e a detecção de marcadores associados à hipóxia de queratinócitos humanos (HaCaT), como modelo de célula oral, em um microambiente hipóxico gerado pela variante do método "Hipóxia induzida por lamínulas". Resultados: Por microscopia confocal, observou-se a presença dos marcadores de hipóxia GLUT-1 e Hipoxyprobe em culturas de células HaCaT expostas a um microambiente hipóxico. Além disso, a presença do marcador GLUT-1 foi observada por imuno-histoquímica em tecido epitelial humano derivado de biópsias de patologia de LPO. Conclusões: Foi estabelecida uma correlação entre as alterações detectadas em queratinócitos humanos induzidas em um microambiente hipóxico in vitro e as alterações detectadas in vivo no tecido epitelial da mucosa oral.

Hypoxia is a fundamental factor in the process of tumor genesis, as well as in precursor pathologies of cancer, such as Oral Lichen Planus (OLP). Objective: To determine if it is possible to establish a correlation between the alterations that normal keratinocytes suffer in a hypoxic microenvironment in vitro and alterations that appear in the keratinocytes in the epithelium of the oral mucosa in the context of OLP pathology. Methods: Morphological changes were studied by phase contrast microscopy, and the detection of markers associated with hypoxia of human keratinocytes (HaCaT), as an oral cell model, in a hypoxic microenvironment generated by the variant of the method "Hypoxia induced by coverslips". Results: Using confocal microscopy, the presence of hypoxia markers GLUT-1 and Hipoxyprobe was observed in HaCaT cell cultures exposed to a hypoxic microenvironment. In addition, the presence of the GLUT-1 marker was observed by immunohistochemistry in human epithelial tissue derived from biopsies of OLP pathology. Conclusions: A correlation was established between the alterations detected in human keratinocytes induced in a hypoxic microenvironment in vitro and the alterations detected in vivo in epithelial tissue of the oral mucosa.

Ep-CAM (MOC-31) expression in tooth germ and ameloblastoma

Background: Ep-CAM, a transmembrane glycoprotein expressed in most epithelium in normal conditions, hasdiverse roles in these tissues, including in cell adhesion, proliferation, differentiation, cell cycle regu-lation, migration and intracellular signaling. It is also over-expressed in most malignant neoplasia, partic-ipating in theinitiation, progression, and metastatic dissemination of the tumor. The expression and roles of this protein in oralneoplasia, particularly in odontogenic tumors, remain unestablished. The objective of this study consisted in analyzing the expression of this protein in ameloblastoma and tooth germ.Material and Methods: Ep-CAM (MOC-31) expression was evaluated by immunohistochemistry in tooth germs(TG) (n = 16) ameloblastomas (AM) (n = 60) and 2 ameloblastic carcinomas. Sections were visualized in theirtotality with an optical microscope, and positivity observed in cell membrane and cytoplasm was graded according to the following semi-quantitative scale: Neg, "essentially unstained", for negative sections or staining <5% ofcells; + for staining of 5-50% of cells; ++ for staining >50% of cells.Results: Most tooth germs expressed MOC-31 (81.3%), strong staining was observed both in the inner epitheliumof the enamel organ and in the adjacent stellate reticulum. 16.7% of the AM cases showed MOC-31 expression,the immunoexpression expression was diffuse at the cytoplasmic and membrane level. The only two cases ofameloblastic carcinoma included were strong positive to MOC-31. No correlation was observed between proteinexpression and gender, age, clinical variants, or histological subtypes.Conclusions: Overexpression was found in TG and ameloblastic carcinoma compared to AM; further studies withdifferent experimental strategies are suggested to clarify the biological significance of this finding. (AU)

Computational multifocus fluorescence microscopy for three-dimensional visualization of multicellular tumor spheroids.

SIGNIFICANCE: Three-dimensional (3D) visualization of multicellular tumor spheroids (MCTS) in fluorescence microscopy can rapidly provide qualitative morphological information about the architecture of these cellular aggregates, which can recapitulate key aspects of their in vivo counterpart. AIM: The present work is aimed at overcoming the shallow depth-of-field (DoF) limitation in fluorescence microscopy while achieving 3D visualization of thick biological samples under study. APPROACH: A custom-built fluorescence microscope with an electrically focus-tunable lens was developed to optically sweep in-depth the structure of MCTS. Acquired multifocus stacks were combined by means of postprocessing algorithms performed in the Fourier domain. RESULTS: Images with relevant characteristics as extended DoF, stereoscopic pairs as well as reconstructed viewpoints of MCTS were obtained without segmentation of the focused regions or estimation of the depth map. The reconstructed images allowed us to observe the 3D morphology of cell aggregates. CONCLUSIONS: Computational multifocus fluorescence microscopy can provide 3D visualization in MCTS. This tool is a promising development in assessing the morphological structure of different cellular aggregates while preserving a robust yet simple optical setup.

The Tissue Architecture of Oral Squamous Cell Carcinoma Visualized by Staining Patterns of Wheat Germ Agglutinin and Structural Proteins Using Confocal Microscopy.

OBJECTIVES: Tissue architecture and cell morphology suffer profound alterations during oral cancer and are important markers for its progression and outcome. For precise visualization of tissue architecture in oral cancer, we used confocal microscopy to examine the staining pattern of wheat germ agglutinin, a lectin that binds membrane glycoproteins, and the staining patterns of structural proteins. MATERIALS AND METHODS: Paraffin sections of oral squamous cell carcinoma were stained with fluorescently labeled wheat germ agglutinin and with antibodies against structural proteins, which were revealed by immunohistochemistry with tyramide signal amplification. RESULTS: Membrane localization of wheat germ agglutinin was markedly decreased in the basal layers and in regions of tumor invasion, accompanied by cytoplasmic redistribution of E-cadherin, ß-actin and syndecan-1. Wheat germ agglutinin staining clearly identified tumor clusters within the surrounding stroma, and tumor cells with elongated morphology. CONCLUSIONS: Our results suggest that the wheat germ agglutinin staining pattern is indicative of the degree of cell cohesion in oral squamous cell carcinoma, which decreases in basal layers and invasive tumor clusters with more migratory morphologies. Wheat germ agglutinin staining in combination with confocal microscopy could constitute, therefore, a valuable tool for the study of tissue architecture in oral cancer.

E-Cadherin gene expression in oral cancer: Clinical and prospective data

Background: Low protein expression of E-cadherin in oral squamous cell carcinoma (OSCC) has been associated with clinical and histopathological traits such as metastases, recurrence, low survival and poor tumor differentiation, and it is considered a high-risk marker of malignancy. However, it is still unknown whether low expression of E-cadherin is also present at the mRNA level in OSCC cases. Objective: The aim of this study was to compare E-cadherin mRNA expression in OSCC patients and controls and to correlate the expression with clinical and prospective characteristics. Material and Methods: Forty patients and 40 controls were enrolled. E-cadherin mRNA expression was evaluated by quantitative real-time polymerase chain reaction using TaqMan probes. Results: E-cadherin mRNA expression was significantly decreased in OSCC patients compared to that of controls (p < 0.001). Whereas no significant association between clinical parameters and E-cadherin expression levels was observed, we noted lower E-cadherin expression levels in patients with positive lymph node metastasis. Conclusions: E-cadherin mRNA expression was markedly diminished in OSCC, in agreement with previous re-sults that examined E-cadherin expression at the protein level. E-cadherin is downregulated in the early clinical stages of OSCC, and its mRNA levels do not change significantly in the advanced stages, suggesting that there is limited usefulness of this parameter for predicting disease progression

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Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source.

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid-containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development.

Lamellipodial wrinkles in fish keratocytes as markers of imperfect coordination between extension and retraction during cell migration.

Cell migration involves the precise coordination between extension at the front of the cell and retraction at the rear. This coordination is particularly evident in fast moving cells such as fish keratocytes, where it leads to highly stable gliding motion, propelled at the front by broad, 0.1-0-2 µm thick lamellipodia. Transient uncoupling between extension and retraction can occur if the rear is temporarily stuck, which might eventually lead to cell shape instabilities. We have frequently observed in fish keratocytes the presence of lamellipodial radial wrinkles, detected by confocal, scanning electron and side-view microscopy as folds in the lamellipodium up to 2 µm in height. Using a linear finite elements analysis, we simulated the displacement of cells either with perfect coordination between extension and retraction or with the rear transiently stuck while the front continues extending, and we observed that in this last condition compression stresses arise in the lamellipodium which predict the formation of the observed pattern of lamellipodial wrinkles. In support of the numerical modeling findings, we observed that the transient halting of retraction at the rear using micromanipulation induced the formation of lamellipodial wrinkles in previously flat lamellipodia. The obtained results suggest that the conspicuous lamellipodial wrinkles observed in migrating fish keratocytes are the product of transient imbalances between front and rear displacements, and are therefore useful markers of the short scale dynamics of extension and retraction coordination during cell migration.

Requirement of Pax6 for the integration of guidance cues in cell migration.

The intricate patterns of cell migration that are found throughout development are generated through a vast array of guidance cues. Responding integratively to distinct, often conflicting, migratory signals is probably crucial for cells to reach their correct destination. Pax6 is a master transcription factor with key roles in neural development that include the control of cell migration. In this study, we have investigated the ability of cells derived from cortical neurospheres from wild-type (WT) and Pax6-/- mouse embryos to integrate diverging guidance cues. We used two different cues, either separately or in combination: substratum nanogrooves to induce contact guidance, and electric fields (EFs) to induce electrotaxis. In the absence of an EF, both WT and Pax6-/- cells aligned and migrated parallel to grooves, and on a flat substrate both showed marked electrotaxis towards the cathode. When an EF was applied in a perpendicular orientation to grooves, WT cells responded significantly to both cues, migrating in highly oblique trajectories in the general direction of the cathode. However, Pax6-/- cells had an impaired response to both cues simultaneously. Our results demonstrate that these neurosphere derived cells have the capacity to integrate diverging guidance cues, which requires Pax6 function.

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation.

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome.

A role for PP1/NIPP1 in steering migration of human cancer cells.

Electrical gradients are present in many developing and regenerating tissues and around tumours. Mimicking endogenous electric fields in vitro has profound effects on the behaviour of many cell types. Intriguingly, specific cell types migrate cathodally, others anodally and some polarise with their long axis perpendicular to the electric vector. These striking phenomena are likely to have in vivo relevance since one of the determining factors during cancer metastasis is the ability to switch between attractive and repulsive migration in response to extracellular guidance stimuli. We present evidence that the cervical cancer cell line HeLa migrates cathodally in a direct current electric field of physiological intensity, while the strongly metastatic prostate cancer cell line PC-3-M migrates anodally. Notably, genetic disruption of protein serine/threonine phosphatase-1 (PP1) and its regulator NIPP1 decrease directional migration in these cell lines. Conversely, the inducible expression of NIPP1 switched the directional response of HeLa cells from cathodal to slightly anodal in a PP1-dependent manner. Remarkably, induction of a hyperactive PP1/NIPP1 holoenzyme, further shifted directional migration towards the anode. We show that PP1 association with NIPP1 upregulates signalling by the GTPase Cdc42 and demonstrate that pharmacological inhibition of Cdc42 in cells overexpressing NIPP1 recovered cathodal migration. Taken together, we provide the first evidence for regulation of directional cell migration by NIPP1. In addition, we identify PP1/NIPP1 as a novel molecular compass that controls directed cell migration via upregulation of Cdc42 signalling and suggest a way by which PP1/NIPP1 may contribute to the migratory properties of cancer cells.

PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.

Correct guidance of the migration of neural progenitor cells (NPCs) is essential for the development and repair of the central nervous system (CNS). Electric field (EF)-guided migration, electrotaxis, has been observed in many cell types. We report here that, in applied EFs of physiological magnitude, embryonic and adult NPCs show marked electrotaxis, which is dependent on the PI3K/Akt pathway. The electrotaxis was also evidenced by ex vivo investigation that transplanted NPCs migrated directionally towards cathode in organotypic spinal cord slice model when treated with EFs. Genetic disruption or pharmacological inhibition of phosphoinositide 3-kinase (PI3K) impaired electrotaxis, whereas EF exposure increased Akt phosphorylation in a growth factor-dependent manner and increased phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels. EF treatments also induced asymmetric redistribution of PIP3, growth factor receptors, and actin cytoskeleton. Electrotaxis in both embryonic and adult NPCs requires epidermal growth factor (EGF) and fibroblast growth factor (FGF). Our results demonstrate the importance of the PI3K/Akt pathway in directed migration of NPCs driven by EFs and growth factors and highlight the potential of EFs to enhance the guidance of various NPC populations in CNS repair therapies.

A time-lapse and quantitative modelling analysis of neural stem cell motion in the absence of directional cues and in electric fields.

Neural stem cell (NSC) migration is an important component of their developmental function and therapeutic potential. Understanding their mode of migration and their response to guidance cues can contribute to improved therapies for CNS repair, in which appropriate homing to sites of injury is essential. Using time-lapse imaging, we have analyzed the NSC mode of migration in vitro, both in the absence of directional cues and in the presence of applied electric fields (EFs), previously shown to constitute a strong directional signal for these cells. Without EFs, NSCs displayed an amoeboid motion, characterized by small lamellipodial-like protrusions with changing orientations, leading to highly tortuous migration. In EFs, tortuosity diminished as electrotaxis toward the cathode occurred. EFs suppressed the formation of protrusions oriented toward the anode, suggesting that restriction of protrusions with opposing orientation could underlie the change from tortuous motion to directed migration. Treatment with LY294002, a phosphatidylinositol-3-OH kinase (Pi3K) inhibitor, reduced the cathodal bias of protrusions in EFs and the frequency of changes in direction. We generated a model of NSC migration with only two key parameters, which could accurately reproduce experimental migration patterns, and we used it to show that both effects of LY294002 contribute to impair electrotaxis, although decreased protrusion bias is the most important. Our results show that control of protrusion orientation by EFs is an important component of the electrotactic response. A simple modelling approach might be useful in understanding how diverse pharmacological treatments or genetic deletions affect different kinds of directional cell migration.

Effect of acrylamide on the cytoskeleton and apoptosis of bovine lens epithelial cells.

Acrylamide, a known disrupter of intermediate filaments, has been used to produce the collapse of vimentin filaments in bovine lens epithelial (BEL) cells, and its potential modulation of staurosporine-induced apoptosis has been investigated. In BEL cells, short treatments with acrylamide caused the collapse of vimentin filaments and microtubules and the almost complete disappearance of stress fibers, with thick f-actin bundles remaining in the cell periphery. Actin organization was less affected in cells pretreated with colchicine and in spreading cells, suggesting that extended microtubules and vimentin filaments are required for acrylamide to produce its maximal effects. Acrylamide alone slightly increased apoptosis compared to controls. However, simultaneous exposure to acrylamide and staurosporine for 8h produced significantly less apoptosis than staurosporine alone, and preincubation with acrylamide followed by staurosporine markedly reduced apoptosis at 8 and 24h of treatment. Acrylamide seems therefore to have a dual effect on BEL cell survival.

Necessary conditions for a minimal model of receptor to show adaptive response over a wide range of levels of stimulus.

Sensory systems respond to temporal changes in the stimulus and adapt to the new level when it persists, this pattern of response being maintained in a wide range of levels of stimulus. Here we use a simple model of adaptation developed by Segel et al. (J. Theor. Biol. 120 (1986) 151-179) and extended by Hauri and Ross (Biophys. J. 68 (1995) 708-722) to study the conditions in which it shows wide range of response. The model consists of a receptor that switches between a variable number of states, either by mass action law or by covalent modification. Using a global optimization procedure, we have optimized the adaptive response of the alternatives of the model with different number of states. We find that it is impossible to obtain a wide range of response if the receptor switches between states following mass-action laws, irrespective of the number of states. Instead, a wide range (of five orders of magnitude of ligand concentration) can be obtained if the receptor switches between several states by irreversible covalent modification, in agreement with previous models. Therefore, in this model, expenditure of energy to maintain a large number of covalent modification cycles operating outside equilibrium is necessary to achieve a wide range of response. The optimal values of the parameters present similar patterns to those reported for specific receptors, but there is no quantitative agreement. For instance, ligand affinity varies several orders of magnitude between the different states of the receptor, what is unlikely to be fulfilled by real systems. To see if the minimal model can show adaptive response and range with quantitatively plausible parameter values a sub-optimal receptor was studied, finding that adaptive response of high intensity can still be obtained in at least three orders of magnitude.