Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis

Utilization of molecular imaging of apoptosis for clinical monitoring of tumor response to anti-cancer treatments in vivo is highly desirable. To address this need, we now present ML-9 (butyl-2-methyl-malonic acid; MW = 173), a rationally designed small-molecule detector of apoptosis, based on a novel alkyl-malonate motif. In proof-of-concept studies, induction of apoptosis in tumor cells by various triggers both in vitro and in vivo was associated with marked uptake of ³H-ML-9 administered in vivo, in correlation with the apoptotic hallmarks of DNA fragmentation, caspase-3 activation and membrane phospholipid scrambling, and with correlative tumor regression. ML-9 uptake following chemotherapy was tumor-specific, with rapid clearance of the tracer from the blood and other non-target organs. Excess of non-labeled “cold” compound competitively blocked ML-9 tumor uptake, thus demonstrating the specificity of ML-9 binding. ML-9 may therefore serve as a platform for a novel class of small-molecule imaging agents for apoptosis, useful for assessment of tumor responsiveness to treatment. H. Grimberg, G. Levin and A. Shirvan contributed equally to this article.     

Steroid concentrations in plasma, whole blood and brain: effects of saline perfusion to remove blood contamination from brain

The brain and other organs locally synthesize steroids. Local synthesis is suggested when steroid levels are higher in tissue than in the circulation. However, measurement of both circulating and tissue steroid levels are subject to methodological considerations. For example, plasma samples are commonly used to estimate circulating steroid levels in whole blood, but steroid levels in plasma and whole blood could differ. In addition, tissue steroid measurements might be affected by blood contamination, which can be addressed experimentally by using saline perfusion to remove blood. In Study 1, we measured corticosterone and testosterone (T) levels in zebra finch (Taeniopygia guttata) plasma, whole blood, and red blood cells (RBC). We also compared corticosterone in plasma, whole blood, and RBC at baseline and after 60 min restraint stress. In Study 2, we quantified corticosterone, dehydroepiandrosterone (DHEA), T, and 17β-estradiol (E₂) levels in the brains of sham-perfused or saline-perfused subjects. In Study 1, corticosterone and T concentrations were highest in plasma, significantly lower in whole blood, and lowest in RBC. In Study 2, saline perfusion unexpectedly increased corticosterone levels in the rostral telencephalon but not other regions. In contrast, saline perfusion decreased DHEA levels in caudal telencephalon and diencephalon. Saline perfusion also increased E₂ levels in caudal telencephalon. In summary, when comparing local and systemic steroid levels, the inclusion of whole blood samples should prove useful. Moreover, blood contamination has little or no effect on measurement of brain steroid levels, suggesting that saline perfusion is not necessary prior to brain collection. Indeed, saline perfusion itself may elevate and lower steroid concentrations in a rapid, region-specific manner.     

Infusion of a Lipid Emulsion Modulates AMPK and Related Proteins in Rat Liver,Muscle, and Adipose Tissues

The primary objective of this study was to investigate the impact of lipid oversupply on the AMPK pathway in skeletal muscle, liver, and adipose tissue. Male Wistar rats were infused with lipid emulsion (LE) or phosphate‐buffered saline for 5 h/day for 6 days. Muscles exposed to LE for 6 days exhibited increased AMPK and acetyl‐CoA carboxylase (ACC) phosphorylation, along with a greater association between AMPK and Ca²⁺/calmodulin‐dependent protein kinase kinase (CaMKK). No differences in muscle protein phosphatase 2C (PP2C) activity, LKB1 phosphorylation or AMPK and LKB1 association were observed. Muscle ACCβ, and adiponectin receptor 1 (AdipoR1) mRNA levels and PPARγ‐co‐activator 1α (PGC1α) protein levels were also increased in LE‐treated rats. In contrast, AMPK and ACC phosphorylation decreased and PP2C activity increased in rat livers exposed to LE. Hepatic mRNA levels of ACCα, PPARα, AdipoR1, AdipoR2, and sterol regulatory element–binding protein‐1c (SREBP1c) were also reduced after LE infusion. In adipose tissue, there was no significant alteration in AMPK or ACC phosphorylation. These results demonstrate that following lipid oversupply the AMPK pathway was enhanced in rat skeletal muscle while diminished in the liver and was unchanged in adipose tissue. CaMKK in skeletal muscle and PP2C in the liver, at least in part, appear to mediate these alterations. Alterations in AMPK pathway in the liver induced metabolic defects associated with lipid oversupply.     

The turnover kinetics of major histocompatibility complex peptides of human cancer cells

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. Thus, the repertoire of these peptides (the MHC peptidome) should correlate better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. To test the validity of this statement and to determine whether the majority of MHC peptides are derived from short lived proteins, from defective ribosome products, or from regular long lived cellular proteins we analyzed in parallel the turnover kinetics of both MHC peptides and cellular proteins in the same cancer cells. The analysis was performed by pulse-chase experiments based on stable isotope labeling in tissue culture followed by capillary chromatography and tandem mass spectrometry. Indeed only a limited correlation was observed between the proteome and the MHC peptidome observed in the same cells. Moreover a detailed analysis of the turnover kinetics of the MHC peptides helped to assign their origin to normal, to short lived or long lived proteins, or to the defective ribosome products. Furthermore the analysis of the MHC peptides turnover kinetics helped to direct attention to abnormalities in the degradation schemes of their source proteins. These observations can be extended to search for cancer-related abnormalities in protein degradation, including those that lead to loss of tumor suppressors and cell cycle regulatory proteins.     

Intercellular adhesion strengthening as studied through simulated stress by organic acid molecules in potato (Solanum tuberosum L.) tuber parenchyma

Intercellular adhesion in some parenchyma becomes strengthened in response to stress. The present study provides an approach to investigate this phenomenon (usually attributed to pectin methyl esterase and binding of Ca(2+) and/or rhamnogalacturonan-II-borate) through reliable stress simulation by probing organic acid molecules in potato tuber parenchyma. Short-chain monocarboxylic acids induce consistent intercellular adhesion strengthening (3.8-5.3 newton) at pH >or= 3 < pK(a), where pectin methyl esterase activity and Ca(2+) or borate binding are limited, and vice versa at pH > pK(a) with a strength of 1.4-2.0 newton as compared to 0.3-0.4 newton for the nonincubated control. Strengthening of intercellular adhesion is characterized by prominent staining of pectin and protein and immunogold labeling of pectin in the cell wall and the middle lamellar complex, particularly after boiling. Pectin confers strengthening to the primary cell wall, as reflected by: (i) prominent immunogold labeling following boiling; and (ii) puncturing macerated cells by starch gelatinization pressure after enzymatic pectin removal.     

New paradigms of signaling in the vasculature: ephrins and metalloproteases

As our understanding of the control of vasculogenesis and angiogenesis continues to grow, we will be confronted with an increasing number of interacting and intersecting receptor-mediated signaling pathways. If we are to be successful in developing new and novel effective therapeutic reagents that can function as stimulators or inhibitors of these critically important processes, we will have to develop a sophisticated, full understanding of the complex interactions associated with ephrin-based and metalloprotease-based signaling pathways.     

Heparanase induces endothelial cell migration via protein kinase B/Akt activation

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intra-chain sites. Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules. The degranulated heparanase is implicated in extravasation of metastatic tumor cells and activated cells of the immune system. Degranulation and heparanase release in response to an inflammatory stimulus or platelet activation would facilitate cellular extravasation directly, by altering the composition and structural integrity of the extracellular matrix, or indirectly, by releasing HS-bound proinflammatory cytokines and chemokines. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. Here, we provide evidence that addition of the 65-kDa latent heparanase to endothelial cells enhances Akt signaling. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of cell membrane HS proteoglycans and was augmented by heparin. Moreover, addition of heparanase stimulated phosphatidylinositol 3-kinase-dependent endothelial cell migration and invasion. These results suggest, for the first time, that heparanase activates endothelial cells and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor.     

Rho GTPases and phosphoinositide 3-kinase organize formation of branched dendrites

Neurons receive information from other neurons via their dendritic tree. Dendrites and their branches result from alternating outgrowth and retraction. The Rho GTPases Rac and Cdc42 (cell division cycle 42) facilitate the outgrowth of branches, whereas Rho attenuates it. The mechanism of neurite retraction is unknown. Because the adenylyl cyclase activator forskolin causes numerous branched extensions in NG108-15 cells, we have investigated the underlying mechanism in this cell line. In additional studies, we used cultured hippocampal neurons in which forskolin induces branched dendrites. In both cell types, forskolin enhanced the activity of Cdc42, but not that of Rac, although both GTPases were necessary for the formation of branched extensions. Time lapse microscopy showed that forskolin did not increase the rate of addition of new extensions or branches, but it reduced the rate of the retraction so that more branched extensions persisted. Inhibition of phosphoinositide 3-kinase activity by wortmannin or LY294002 also reduced the rate of retraction and thus facilitated dendritic arborization. Forskolin diminished the activity of phosphoinositide 3-kinases. Inhibitors of phosphoinositide 3-kinases not only reduced the retraction but also the addition of new dendrites and branches. This reduction was no longer present when Rho kinase was simultaneously inactivated, suggesting an interaction of phosphoinositide 3-kinases and Rho kinase. The present results show a central role of phosphoinositide 3-kinases in dendrite formation. In neuronal cells, increased levels of cAMP can support dendritic arborization by modulating the activity of the lipid kinase.     

Identification and characterization of phytoplasmal genes, employing a novel method of isolating phytoplasmal genomic DNA

Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes. To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant. Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA. Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized. We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins. Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed.