A reason for intermittent fasting to suppress the awakening of dormant breast tumors.

For their growth, dormant tumors, which lack angiogenesis may critically depend on gradients of nutrients and oxygen from the nearest blood vessel. Because for oxygen depletion the distance from the nearest blood vessel to depletion will generally be shorter than for glucose depletion, such tumors will contain anoxic living tumor cells. These cells are dangerous, because they are capable of inducing angiogenesis, which will "wake up" the tumor. Anoxic cells are dependent on anaerobic glucose breakdown for ATP generation. The local extracellular glucose concentration gradient is determined by the blood glucose concentration and by consumption by cells closer to the nearest blood vessel. The blood glucose concentration can be lowered by 20-40% during fasting. We calculated that glucose supply to the potentially hazardous anoxic cells can thereby be reduced significantly, resulting in cell death specifically of the anoxic tumor cells. We hypothesize that intermittent fasting will help to reduce the incidence of tumor relapse via reducing the number of anoxic tumor cells and tumor awakening.

Two-dimensional semi-parametric alignment of chromatograms.

We present a comprehensive alignment algorithm that extends the semi-parametric approach to two dimensions. The algorithm is based on modeling shifts with a two-dimensional "warp function" such that the sample chromatogram - its shifts corrected with the warp function - is adjusted to the reference chromatogram by minimizing the squared intensity difference. A warp function approach has the advantage that overlapping peaks are easily dealt with compared to other proposed two-dimensional algorithms. Another advantage is that missing peaks are allowed if the absence of these peaks has little numerical effect on the warp function computation and if these peaks occur between existing peaks. Performance of the algorithm is demonstrated using GC×GC data from three batches of three diesel oil samples and LC-MS data from a mouse breast cancer data set.

A new mathematical pharmacodynamic model of clonogenic cancer cell death by doxorubicin.

Previous models for predicting tumor cell growth are mostly based on measurements of total cell numbers. The purpose of this paper is to provide a new simple mathematical model for calculating tumor cell growth focusing on the fraction of cells that is clonogenic. The non-clonogenic cells are considered to be relatively harmless. We performed a number of different types of experiments: a long-term drug "treatment", several concentrations/fixed time experiments and time-series experiments, in which human MCF-7 breast cancer cells were exposed to doxorubicin and the total number of cells were counted. In the latter two types, at every measurement point a plating efficiency experiment was started. The final number of colonies formed is equal to the number of clonogenic cells at the onset of the experiment. Based on the intracellular drug concentration, our model predicts cell culture effects taking clonogenic ability and growth inhibition by neighboring cells into account. The model fitted well to the experimental data. The estimated damage parameter which represents the chance of an MCF-7 cell to become non-clonogenic per unit time and per unit intracellular doxorubicin concentration was found to be 0.0025 ± 0.0008 (mean ± SD) nM(-1) h(-1). The model could be used to calculate the effect of every doxorubicin concentration versus time (C-t) profile. Although in vivo parameters may well be different from those found in vitro, the model can be used to predict trends, e.g. by comparing effects of different in vivo C-t profiles.

A split microdrive for simultaneous multi-electrode recordings from two brain areas in awake small animals.

Complex cognitive operations such as memory formation and decision-making are thought to be mediated not by single, isolated brain structures but by multiple, connected brain areas. To facilitate studies on the neural communication between connected brain structures, we developed a multi-electrode microdrive for chronically recording ensembles of neurons in two different brain areas simultaneously. The "split drive" contains 14 independently movable microdrivers that were designed to hold tetrodes and to permit day-to-day adjustment of dorsoventral position in the brain. The limited weight of the drive allowed rats to adjust well to the headstage after recovering from surgery and permitted stable recording sessions across at least several weeks. In addition to describing the design and assembly of the split drive, we also discuss some important individual parts of microdrives used for tetrode recordings in general. Furthermore, the split drive was applied to two widely separated and connected brain structures, the hippocampus and ventral striatum. From these two areas, stable ensemble recordings were conducted in rats performing a reward-searching task on a triangular track, yielding group sizes of about 15 and 25 units in the dorsal hippocampus and ventral striatum, respectively.

Epidermal growth factor receptor-induced activator protein 1 activity controls density-dependent growth inhibition in normal rat kidney fibroblasts.

Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these cells irresponsive to EGF. Using an activator protein (AP)-1 sensitive reporter construct, we show that AP-1 activity is strongly decreased in density-arrested NRK cells, but is restored after relaxation of densitydependent growth inhibition by removing neighboring cells. EGF could not induce AP-1 activity or S-phase entry in density-arrested cells, but could do so after pretreatment with retinoic acid, which enhances EGF receptor expression. Our results support a model in which the EGF receptor regulates density-dependent growth control in NRK fibroblasts, which is reflected by EGF-induced mitogenic signaling and consequent AP-1 activity.

Cancer: a Systems Biology disease.

Cancer research has focused on the identification of molecular differences between cancerous and healthy cells. The emerging picture is overwhelmingly complex. Molecules out of many parallel signal transduction pathways are involved. Their activities appear to be controlled by multiple factors. The action of regulatory circuits, cross-talk between pathways and the non-linear reaction kinetics of biochemical processes complicate the understanding and prediction of the outcome of intracellular signaling. In addition, interactions between tumor and other cell types give rise to a complex supra-cellular communication network. If cancer is such a complex system, how can one ever predict the effect of a mutation in a particular gene on a functionality of the entire system? And, how should one go about identifying drug targets? Here, we argue that one aspect is to recognize, where the essence resides, i.e. recognize cancer as a Systems Biology disease. Then, more cancer biologists could become systems biologists aiming to provide answers to some of the above systemic questions. To this aim, they should integrate the available knowledge stemming from quantitative experimental results through mathematical models. Models that have contributed to the understanding of complex biological systems are discussed. We show that the architecture of a signaling network is important for determining the site at which an oncologist should intervene. Finally, we discuss the possibility of applying network-based drug design to cancer treatment and how rationalized therapies, such as the application of kinase inhibitors, may benefit from Systems Biology.

Induction of p53 up-regulated modulator of apoptosis messenger RNA by chemotherapeutic treatment of locally advanced breast cancer.

PURPOSE: In biopsies of patients with locally advanced breast cancer, we investigated the in vivo changes of the gene expression pattern induced by chemotherapy to find genes that are potentially responsible for the efficacy of the drug. EXPERIMENTAL DESIGN: Early cellular responses to chemotherapy-induced damage, both in vivo and in vitro, were investigated by analyzing chemotherapy-induced changes in gene expression profiles. Core biopsies were taken from nine patients with locally advanced breast cancer, before and at 6 hours after initiation of doxorubicin-based chemotherapy. Both samples were cohybridized on the same microarray containing 18,000 cDNA spots. RESULTS: The analysis revealed marked differences in gene expression profile between treated and untreated samples. The gene which was most frequently found to be differentially expressed was p53 up-regulated modulator of apoptosis (PUMA). This gene was up-regulated in eight of nine patients with an average factor of 1.80 (range, 1.36-2.73). In vitro MCF-7 breast cancer cells exposed to clinically achievable doxorubicin concentrations for 6 hours revealed marked induction of PUMA mRNA, as well. CONCLUSIONS: This is the first report describing PUMA mRNA to be up-regulated as a response to chemotherapy in patients. Because PUMA is a known member of the family of BH3-only proapoptotic proteins, this finding suggests PUMA's potential importance for the response to anticancer drugs.

Synergistic activation of signalling to extracellular signal-regulated kinases 1 and 2 by epidermal growth factor and 4 beta-phorbol 12-myristate 13-acetate.

Signal transduction pathways are often embedded in complex networks, which result from interactions between pathways and feedback circuitry. In order to understand such networks, qualitative information on which interactions take place and quantitative data on their strength become essential. Here, we have investigated how the multiple interactions between the mitogen-activated protein kinase cascade and protein kinase C (PKC) affect the time profile of extracellular signal-regulated kinase (ERK) phosphorylation upon epidermal growth factor (EGF) stimulation in normal rat kidney fibroblasts. This profile is a major determinant for the cellular response that is evoked. We found that EGF stimulation leads to a biphasic ERK-PP pattern, consisting of an initial peak and a relaxation to a low quasi-steady state-phase. Costimulation with the EGF and PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA) resulted in a similar pattern, but the ERK-PP concentration in the quasi-steady state-phase was synergistically higher than after stimulation with either EGF or PMA only. This resulted in prolonged signalling to ERK. PMA increased the EGF concentration sufficient to obtain half-maximum ERK phosphorylation. These data suggest that PKC amplifies EGF-induced signalling to ERK, without increasing its sensitivity to low EGF concentrations. Furthermore, PKC inhibition did not affect the ERK-PP time profile upon EGF stimulation and a cellular phospholipase A2 (cPLA(2)) inhibitor did not decrease the synergistic effect of EGF and PMA. This indicates that the positive feedback loop from ERK to Raf via cPLA(2) and PKC does not contribute significantly to signalling from EGF to ERK in normal rat kidney cells. Taken together, we provide a quantitative description of which reported interactions in this network affect the time profile of ERK phosphorylation.

Simulation model of doxorubicin activity in islets of human breast cancer cells.

During cytotoxic chemotherapy, cancer cells are exposed to a dynamic concentration-versus-time curve. Besides the area under this curve, the shape of this curve may determine the cytotoxic effect. This report describes the concept that cell damage is determined by the molar drug accumulation history inside the tumor cells. Cell numbers of large populations of human MCF-7 cells exposed to three different doxorubicin concentration-versus-time profiles were recorded for 31 days. The drug accumulation history in the cells was calculated using cellular drug transport parameters derived from doxorubicin uptake and efflux measurements on MCF-7 cells attached to culture dishes. Recovery of the proliferation rate of a cell population after drug exposure was described using a mathematical model of cell damage. The model fitted well to the proliferation assays. It allowed for comparison of the effects of changes in doxorubicin concentration-versus-time profiles in vitro. The model was then used to predict the effect of the changes in the doxorubicin concentration profile in vivo, in tumor islets, after a bolus injection of doxorubicin. In the model doxorubicin exposure resulted in less cell damage inside the tumor islets than at the rim.

Resistance to cytotoxic and anti-angiogenic anticancer agents: similarities and differences.

Intrinsic resistance to anticancer drugs, or resistance developed during chemotherapy, remains a major obstacle to successful treatment. This is the case both for resistance to cytotoxic agents, directed at malignant cells, and for resistance to anti-angiogenic agents, directed at non-malignant endothelial cells. In this review, we will discuss mechanisms of resistance which have a bearing on both these conceptually different classes of drugs. The complexity of drug resistance, involving drug transporters, such as P-glycoprotein, as well as resistance related to the tissue structure of solid tumors and its consequences for drug delivery is discussed. Possible mechanisms of resistance to endothelial cell-targeted drugs, including inhibitors of the VEGF receptor and EGF receptor family, are reviewed. The resistance of cancer cells as well as endothelial cells related to anti-apoptotic signaling events initiated by cell integrin-matrix interactions is discussed. Current strategies to overcome resistance mechanisms are summarized; they include high-dose chemotherapy, tumor targeting of cytotoxics to improve tumor uptake, low-dose protracted (metronomic) chemotherapy and combinations of classical agents with anti-angiogenic agents. This review discusses primarily literature published in 2001 and 2002.

Tissue transport of anti-cancer drugs.

Blood-borne drug molecules are transported through as well as around cells in tissue. For small molecule drugs with a molar weight <1000, the wall of the capillary blood vessels in tumors usually is not a barrier. Just after a rise in the drug concentration in the blood, the cells closest to the microvessels are exposed to the highest drug concentrations. Short or long lasting concentration gradients away from the capillary vessels will develop. Since in a tumor the distance to the nearest blood vessel can be relatively large, inefficient transport of drugs to some cancer cells may limit drug efficacy. Studies on in vitro drug gradients have given insight into the factors determining this transport. Small intercellular distances, high cellular drug influx and low drug efflux rates, and high intracellular and extracellular drug binding favor the development of drug gradients. In the absence of drug metabolism, gradients "level out" over time and may reverse as the blood concentration drops. Understanding the drug transport process from the microvessels to every cancer cell will be important for optimizing cancer chemotherapy. Cancer cells that can "hide" for the drug may lead to regrowth of the tumor.

Analysis of multiple gene expression array experiments after repetitive hybridizations on nylon membranes.

Nylon membrane-based macroarrays form a widely available alternative to microarrays for the collection of large-scale gene expression data. To carry out repetitive hybridization experiments with nylon cDNA arrays, we used phosphorothioate 33P-cDNA, followed by stripping under relatively mild conditions. We were able to use the same membranes more than 10 times without a measurable reduction in their performance. Thus, our protocol allowsfor more comparative studies of multiple data sets obtained from sequential hybridizations of the same set of membranes. We demonstrate how to analyze repetitive macroarray experiments and to determine the reliability or statistical significance of the gene expression data obtained. Both the averaging of signals per gene and the reversal of nylon membranes had a favorable effect on accuracy. By self-self comparisons, we show that in a duplicate experiment with four membranes, a 2-fold change in the gene expression can be measured reliably.

A doxorubicin-CNGRC-peptide conjugate with prodrug properties.

There is increasing interest in the exploitation of molecular addresses for the targeting of tumor imaging or therapeutic agents. A recent study demonstrated anticancer activity in human xenografts of doxorubicin (DOX)-peptide conjugates targeted to the tumor vascular endothelium, among them DOX coupled to the cyclic pentapeptide CNGRC [Science 279 (1998) 377]. In order to learn more about the mechanism of action of this type of DOX-peptide conjugates, we have studied the interaction of DOX-CNGRC with primary human umbilical cord vein endothelial cells (HUVEC) and tumor cells under defined in vitro conditions. We used a DOX conjugate, in which the cyclic CNGRC peptide, for which an in vivo endothelial address has recently been identified as aminopeptidase N (APN)/CD13, has been coupled via a hydrolysable spacer to the C-14 anthracycline-side chain. First we determined that the t(1/2) of DOX-CNGRC conjugate in human blood was 442 min (at 37 degrees ) allowing sufficient time for endothelial targeting when administered i.v. When cultured cells were exposed for 30 min to DOX-CNGRC a more cytoplasmic localization of fluorescent drug was seen when compared to DOX exposure and intracellular DOX-CNGRC was identified after extraction from the cells. This revealed differences in the cellular uptake process of the conjugate compared to DOX. The antiproliferative effect of DOX-CNGRC was determined by 30 min exposure in medium with a high protein content in order to mimick the in vivo targeting situation. In this medium, the IC(50) was 1.1 microM for highly CD13 expressing HT-1080, 1.45 microM for CD13 negative SK-UT-1 sarcoma cells and 6.5 microM for CD13 positive HUVEC. The IC(50) of DOX for these cells were 1.0, 2.0 and 7.3 microM, respectively. Although DOX-CNGRC inhibited the peptidase activity of CD13 up to 50%, our data do not favor an important role for the enzyme inhibition in the cytotoxic effect of the conjugate. The antitumor activity was tested in nude mice bearing human ovarian cancer xenografts (OVCAR-3). A weekly i.v. administration (3mg/kg DOX-equivalent, 3x) showed a minor (40%) growth delay, which does not indicate efficacy better than that expected for free DOX. In conclusion, this study indicates that the antiproliferative and anti-angiogenic effects of DOX-CNGRC as reported before, are likely caused by the cytostatic effects of intracellularly released parent drug DOX, independent of CD13 expression/activity. More research is needed to identify the optimal specific chemical configuration of DOX-peptide conjugates for in vivo targeting and receptor-mediated cellular uptake.