Simple, semiautomatic assay of cytostatic and cytotoxic effects of antitumor drugs by laser scanning cytometry: effects of the bis-intercalator WP631 on growth and cell cycle of T-24 cells.

Background: Common assays of drug-induced cytotoxicity on adherent cells rely on cell trypsinization followed by count of live and dead cells. To estimate the cell cycle effects, cellular DNA content is analyzed by flow cytometry. This procedure is laborious and time consuming.

Antiangiogenic therapy and tumor progression.

Angiogenesis is necessary for tumor growth (a rationale for antiangiogenic therapy), but hypoxia caused by such a therapy will, in theory, drive tumor progression and metastasis. To reconcile conflicting notions, we discuss that, first, although a shift from normoxia (21% O2) to hypoxia indeed activates cancer cells for aggressive behavior, this may not occur during therapy, because most cancers are not normoxic to start with. Second, only successful antiangiogenic therapy, which is capable of controlling cancer, will select for resistance and progression.

Cycling without cyclins.

Complex oscillations in the activation and inactivation of cyclin-dependent kinase complexes propel mammalian cells through the cycle. A recent spate of studies seems to indicate that many, if not most, of these seemingly essential molecules may, in some senses, be dispensable. If true, that would mean the essential components of the mammalian cell cycle are not much different from those found in prokaryotes.

DNA damage induced by DNA topoisomerase I- and topoisomerase II-inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis.

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (gammaH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of gammaH2AX with: a.

Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation.

Background: Fluorochrome-labeled inhibitors of caspases (FLICA, e.g., FAM-VAD-FMK, FITC-VAD-FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases.

Targeting cancer cells by exploiting their resistance.

Cancer cells are intrinsically resistant to growth arrest and can further acquire multidrug resistance. Current approaches to this problem are intended to reverse, overcome or prevent the drug resistance. However, the resistance of cancer cells can be exploited to kill resistant cells selectively, while sparing sensitive normal cells.

NF-kappaB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis: difficulties in identification of dead cells in such cultures.

Background: Apoptosis and necrosis ("accidental cell death") are distinct modes of cell death. The feature that often distinguishes apoptotic from necrotic cells is preservation of the plasma membrane integrity, reflected by ability of the former cells to exclude cationic dyes such as propidium iodide (PI) for a certain length of time. During necrosis, the plasma membrane is rapidly ruptured and necrotic cells stain intensely with PI.

Cell cycle effects and caspase-dependent and independent death of HL-60 and Jurkat cells treated with the inhibitor of NF-kappaB parthenolide.

The sesquiterpene parthenolide (PRT) is an active component of Mexican-Indian medicinal plants and also of the common herb of European origin feverfew. PRT is considered to be a specific inhibitor of NF-kappaB. Human leukemic HL-60, Jurkat, and Jurkat IkappaBalphaM cells, the latter expressing a dominant-negative IkappaBalpha and thus having non-functional NF-kappaB, were treated with PRT and activation of caspases, plasma membrane integrity, DNA fragmentation, chromatin condensation (probed by DNA susceptibility to denaturation), and changes in cell morphology were determined.

Cell immortality and hallmarks of cancer.

In growth-limiting conditions, cells that express telomerase and inactivate tumor suppressors have a selective advantage due to resistance to growth arrest. Accidentally such cells become immortal.

Why Iressa failed: toward novel use of kinase inhibitors (outlook).

A phase III failure of Iressa, an inhibitor of the EGF receptor (EGFR), is viewed as a surprise. With a few exceptions, however, inhibitors of EGFR cannot be effective as a monotherapy in cancers with additional oncogenic changes (downstream of EGFR), which cause mitogen-independent proliferation. In other cases, combining these inhibitors with chemotherapy may lead to antagonism in cancer cells and/or aggravated side-effects.

In vitro model of "wound healing" analyzed by laser scanning cytometry: accelerated healing of epithelial cell monolayers in the presence of hyaluronate.

Background: In vitro models of "wound healing" rely on analysis of confluent cell cultures that are mechanically wounded, e.g., by scratching the cell monolayer.

Cell senescence and hypermitogenic arrest.

A diverse range of conditions, from mitogenic stimuli to cytotoxic stress, can induce cell senescence. Here, I propose that simultaneous stimulation of mitogen-activated pathways and downstream inhibition of cyclin-dependent kinases leads, ultimately, to cell senescence. This model distinguishes between two types of growth arrest: first, exit to G0 phase, which is caused by the withdrawal of mitogens and can lead to apoptosis; and second, hypermitogenic arrest, which is stimulated by mitogens and can lead to senescence.

Apoptosis, proliferation, differentiation: in search of the order.

In search of the order, we are tempted to universally link cell death, proliferation, differentiation, and senescence. Current models (classical, conflicting signal and quantitative signal models) are restricted, precisely because they attempt to hardware a plethora of end-points of cellular responses. By defining each cellular process in molecular term, one can disconnect proliferation (CDK activation), apoptosis (caspase activation), and differentiation (tissue function genes expression), even though these responses are linked by upstream signal transduction pathways.

A new science-business paradigm in anticancer drug development.

Studies into the molecular mechanisms of cancer have revealed that, with a few exceptions, the disease lacks a specific drug target. Therefore, new anticancer drugs not only take many years and much money to develop but also might not outperform existing drugs. However, what are assumed to be unfavorable hallmarks of cancer, for example drug resistance, can be exploited for selective anticancer therapy and for protection of normal cells.

Are p27 and p21 cytoplasmic oncoproteins?

By causing cytoplasmic mislocation of p27 and p21, the Akt oncogenic kinase functionally inactivates these nuclear tumor suppressor proteins. Is cytoplasmic localization of p27 and p21 simply equivalent to loss of their function or are new functions acquired in the cytoplasm? Indeed, several lines of evidence suggest that cytoplasmic p27 and p21 may be oncoproteins with antiapoptotic activities.

Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest, and cytotoxicity.

By preventing deacetylation of histones, histone deacetylase inhibitors (HDIs) transcriptionally induce p21. Here we show that the HDIs sodium butyrate (Bu), trichostatin A (TSA) and depsipeptide (FR901228) all induced p21, but only TSA and FR901228 caused mitotic arrest (in addition to arrest in G1 and G2). The ability to cause mitotic arrest correlated with the higher cytotoxicity of these compounds.

Flavopiridol inversely affects p21(WAF1/CIP1) and p53 and protects p21-sensitive cells from paclitaxel.

Resting cells are relatively resistant to microtubule-active drugs including paclitaxel (PTX). By causing p53-mediated arrest, pretreatment with low concentrations of doxorubicin (DOX) protected HCT116 cells from the cytotoxicity caused by PTX. Unlike DOX, flavopiridol (FL) did not protect HCT116 cells.

Enhancement of activation-induced apoptosis of lymphocytes by the cytotoxic ribonuclease onconase (Ranpirnase).

Onconase (Onc) is an amphibian ribonuclease of the pancreatic RNase family that is cytostatic and cytotoxic to several tumor lines. It also shows anti-tumor activity in mouse tumor models and is currently in phase III clinical trials. In animal tests and clinical trials Onc shows lesser toxicity and fewer side effects compared to most chemotherapeutic drugs.