Pattern analysis of microtubule-polymerizing and -depolymerizing agent combinations as cancer chemotherapies.

Subcellular distribution of mass can be analyzed by a technique that involves culturing cells on interferometers and digitizing their interference contours. Contour sampling resulted in 102 variables per cell, which were predictors of oncogenic transformation. Cell phenotypes can be deconstructed by use of latent factors, which represent the covariance of the real variables. The reversal of the cancer-type phenotype by a combination of microtubule-stabilizing and -depolymerizing agents was described previously. The implications of these results have been explored by clinicians who treated patients with the combination of docetaxel and vinorelbine (Navelbine). The current study was performed to determine the effects of different combinations on phenotype and in phases of the cell cycle other than mitosis. Combinations of paclitaxel with either colchicine, podophyllotoxin, nocodazole, or vinblastine caused phenotype reversal. Paclitaxel analogue, 7-deoxytaxol, by itself caused reversal. Factors #4, (filopodia), #5 (displacement and/or deep invaginations in the periphery), #8, and #12 took on values typical of normal cells, whereas the values of #7 (p21-activated kinase), and #13 (rounding up) shifted toward the cancer-type. All combinations altered microtubule arrangement at the cell edge. Delivery schedules and drug ratios used in clinical studies were subjected to analysis. Clinical response rates were better when the combination was not interspersed with a single agent (P=0.004). The results support the idea that efficacy depends upon simultaneous exposure to both agents, and suggest a novel mechanism for combination therapies. These therapies appear to restore in transformed cells some of the features of a contact-inhibited cell, and to impede progress through the cell cycle even when provided at nanomolar concentrations.

Pbx1 is required for Hox D3-mediated angiogenesis.

Our previous studies showed that the homeobox (Hox) D3 transcription factor induces expression of alphavbeta3 integrin and promotes endothelial cell (EC) migration and angiogenesis. Since binding of Hox 3 factors to target DNA is enhanced by the co-factor Pbx, we investigated whether Pbx1 is also required for angiogenesis. We observed that EC predominantly express the Pbx1b isoform. Nuclear extracts from angiogenic EC express higher levels of active Pbx1 and more effectively form complexes on Pbx1/Hox consensus DNA oligonucleotides as compared to nuclear extracts from quiescent EC. Introduction of anti-sense against Pbx1 impaired the formation of Pbx1/Hox complexes on target DNA consensus in nuclear extracts from angiogenic EC. Anti-sense against Pbx1 also impaired EC migration and blocked angiogenesis induced by bFGF in vivo. Furthermore, although the levels of Hox D3 were unchanged, expression of its target gene, beta3 integrin was reduced, consistent with impaired transcriptional activation by Hox D3. Together, these studies suggest that Pbx1 is required for pro-angiogenic Hox DNA binding and transcriptional activity in endothelial cells.

Novel p21-activated kinase-dependent protrusions characteristically formed at the edge of transformed cells.

During long-term culture, certain lines become neoplastic while accumulating changes in cell shape. Early and late cell populations have characteristic shape phenotypes that have been quantified by computerized assay. Phenotypes are determined from variables describing three-dimensional aspects of the subcellular distribution of mass. The features of cells can be recognized by use of latent factors, which are theoretical variables based on the covariance of the primary variables. Factor #7 represented a cell edge feature different from filopodia. We studied the morphological characteristics and morphogenesis of the feature. Brief exposure of cells from rat tracheal epithelium to phorbol 12-myristate 13-acetate (PMA) enhanced #7 values. The time to reach maximal #7 values was prolonged if PMA was administered with calcium ionophore or lysophosphatidic acid (LPA). Factor #7 was elevated during periods of ruffling suppression and stress fiber reorganization. Cells showing high #7 values were examined by scanning electron microscopy (SEM) and found to exhibit strap-shaped and cupola-shaped projections. Because RhoA regulates stress fiber formation, we sought to perturb #7 features by introducing dominant-acting negative and positive constructs of RhoA, RhoA-N19, and RhoA-V14. Neither affected #7 values. Although overexpression of the kinase inhibitory domain of p21-activated kinase 1 (PAK) had no effect on #7 values, they were affected by overexpression of a domain binding PAK-interacting guanine nucleotide exchange factor (PIX). Because a PAK-PIX complex is implicated in the remodeling of focal complexes (FCs) and recycling of PAK to the cytoplasm, the results implicate a component of FCs in the formation of #7 features. The data suggested that feature formation is driven by activated Cdc42-binding kinase (ACK) and Rac. Moreover, they suggested that the #7 protrusions are neurite-like structures and that their development involves FC regulation.

Hox D3 expression in normal and impaired wound healing.

BACKGROUND: We have previously shown that Hox D3 and Hox B3 can promote angiogenesis. As angiogenesis is essential for wound healing, we examined expression of these genes in the vasculature following wounding in normal and genetically diabetic adult mice with impaired healing. METHODS: In situ hybridization was performed on tissues taken 0, 1, 4, 7, and 14 days following administration of linear wounds in wild-type and genetically diabetic mice. Expression of Hox D3 and Hox B3, angiogenesis, and synthesis of type I collagen were assessed in the wound. RESULTS: Hox B3 was expressed in endothelial cells (ECs) of both medium and small vessels in unwounded tissue, whereas little Hox D3 was detected in resting ECs. Hox D3 expression was significantly upregulated by 1 day after wounding in ECs of vessels immediately adjacent to the wound site, and expression was maintained for at least 7 days. In the diabetic mice, expression of Hox B3 was similar to that of wild-type mice. In contrast, expression of Hox D3 in ECs was significantly lower and delayed during wound repair in diabetic mice. In cultured microvascular ECs, Hox D3 selectively induced high levels of collagen I mRNA expression. Hox D3-deficient wounds of diabetic animals also displayed a reduction in expression and deposition of type I collagen. CONCLUSIONS: These results suggest that reduced angiogenesis and type I collagen in diabetic mice with impaired wound healing may be related to deficient Hox D3 expression, and restoring Hox D3 expression may enhance angiogenesis and wound repair.

Membrane interactions of a new class of anticancer agents derived from arylchloroethylurea: a FTIR spectroscopic study.

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and different lipids such as dimyristoylphosphatidylcholine (DMPC) in the absence and presence of 30 mol% of cholesterol, dimyristoylphosphatidylglycerol (DMPG) and a mixture made of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and DMPC by Fourier transform infrared (FTIR) spectroscopy. The results indicate that the drugs incorporate in the bilayer and cause a decrease of the phase transition temperature and an increase of the conformational disorder of the lipid acyl chains. These effects are dependent on the nature (degree of branching, length of the alkyl chain and presence of a sulfur atom), as well as on the position of the R substituent and are related to the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky branched substituent and those for which the disordering effect on the lipid bilayer is the greatest. On the other hand, the disordering effect is small for the long chain CEUs, such as 4-n-hexadecyl CEU, which have been shown to have weak cytotoxic activity.

Abnormal balance in the angiopoietin-tie2 system in human brain arteriovenous malformations.

Brain arteriovenous malformations (BAVMs) are congenital vascular lesions that often present as cerebral hemorrhage in young adults. The variable nature of the clinical course, especially with respect to spontaneous hemorrhage, recurrence, growth, and regression, suggests that BAVMs are lesions with active angiogenesis and vascular remodeling. We examined mRNA and protein expression of angiopoietin 1 (Ang1) and Ang2 by semiquantitative reverse transcriptase-polymerase chain reaction, in situ hybridization, and Western blot in BAVMs and control brains obtained from temporal lobectomy for medically intractable seizures. Although Ang1 mRNA levels were similar in BAVMs and controls, Ang1 protein levels were 30% lower in BAVMs than in controls. Ang2 mRNA levels were 40% higher and Ang2 protein levels were 8-fold higher in BAVMs than in controls. In situ hybridization showed that the Ang2 mRNA was localized to the perivascular area in BAVMs. This abnormal balance in the Ang-Tie2 system may, in part, explain the aberrant vascular phenotype in BAVMs.

Association of smoking with serum and dietary levels of antioxidants in adults: NHANES III, 1988-1994.

OBJECTIVES: This study examined the association of smoking with serum levels and dietary intakes of antioxidants in a nationally representative sample. METHODS: This study classified 7873 apparently healthy adults aged 17 to 50 years from National Health and Nutrition Examination Survey III (NHANES III) data as nonsmokers or as smokers if their serum cotinine levels were either lower than 14 ng/mL or 14 ng/mL or greater, respectively. SUDAAN software was used for the statistical analysis. RESULTS: Smokers of both sexes had significantly (P < .001) lower serum levels of vitamin C, alpha-carotene, beta-carotene, beta-cryptoxanthin, and lutein/zeaxanthin. Reduction in the serum vitamin E, lycopene, and selenium levels in smokers was slight. Smokers also had significantly lower dietary intakes of vitamin C and beta-carotene. A significant (P < .001) inverse relation was found between serum vitamin C and beta-carotene levels and cotinine levels independent of diet effect, and a positive relation (P < .001) was found between serum levels and dietary intakes. CONCLUSIONS: Antioxidants appear to have differing declines in serum levels as a result of reduced dietary intakes and the effects of smoking.

Homeobox B3 promotes capillary morphogenesis and angiogenesis.

Endothelial cells (EC) express several members of the Homeobox (Hox) gene family, suggesting a role for these morphoregulatory mediators during angiogenesis. We have previously established that Hox D3 is required for expression of integrin alphavbeta3 and urokinase plasminogen activator (uPA), which contribute to EC adhesion, invasion, and migration during angiogenesis. We now report that the paralogous gene, Hox B3, influences angiogenic behavior in a manner that is distinct from Hox D3. Antisense against Hox B3 impaired capillary morphogenesis of dermal microvascular EC cultured on basement membrane extracellular matrices. Although levels of Hox D3-dependent genes were maintained in these cells, levels of the ephrin A1 ligand were markedly attenuated. Capillary morphogenesis could be restored, however, by addition of recombinant ephrin A1/Fc fusion proteins. To test the impact of Hox B3 on angiogenesis in vivo, we constitutively expressed Hox B3 in the chick chorioallantoic membrane using avian retroviruses that resulted in an increase in vascular density and angiogenesis. Thus, while Hox D3 promotes the invasive or migratory behavior of EC, Hox B3 is required for the subsequent capillary morphogenesis of these new vascular sprouts and, together, these results support the hypothesis that paralogous Hox genes perform complementary functions within a particular tissue type.

A grouping interpretation of the modality effect in immediate probed recognition.

In a series of experiments on immediate probed recognition for eight 3-digit numbers, it was shown that if the target modality involved auditory components and the effect of the similarity of the modality of the probe to that of the targets was controlled, unequivocal evidence was obtained for an auditory superiority effect (modality effect) for hit rates for the final items of the list. Moreover, false-alarm rates were significantly lower following targets with an auditory component than they were following silently seen targets. It is argued that this pattern of hits and false alarms is consistent with the idea that targets that have an auditory component yield memory representations that are better grouped as units than are those for targets that are only silently seen; in particular, if a new probe has a first digit that accidentally matches the first digit of a target item, it is more likely that the subject will mistakenly identify this new probe as old (give a false alarm) if the target has only been partially encoded because it was only silently seen.

Extracellular matrix and integrin signalling: the shape of things to come.

The extracellular matrix (ECM) and integrins collaborate to regulate gene expression associated with cell growth, differentiation and survival. Biochemical and molecular analyses of integrin signalling pathways have uncovered several critical cytoplasmic proteins that link the ECM and integrins to intracellular pathways that may contribute to anchorage-dependent growth. A large body of evidence now indicates that the non-receptor protein kinases focal adhesion kinase (FAK) and specific members of the mitogen-activated protein kinases (MAPKs), including the extracellular-signal-regulated kinases (ERKs), mediate these ECM- and integrin-derived signalling events. However, little is known about how FAK and MAPKs contribute to biological processes other than cell proliferation or migration. In addition, remarkably little is known concerning the signalling events that occur in cells that adhere to complex multivalent extracellular matrices via multiple integrin receptors. Given the stringent requirement for attaining a proper morphology in ECM/integrin-directed cell behaviour, it is still not clear how cell shape and tissue architecture impact upon intracellular signalling programmes involving FAK and MAPKs. However, the recent discovery that members of the Rho family of small GTPases are able to regulate ECM/integrin pathways that modulate both cell shape and intracellular signalling provides new insights into how cell morphology and signal transduction become integrated, especially within three-dimensional differentiated tissues.

Extracellular matrix signaling: integration of form and function in normal and malignant cells.

A growing number of studies have established reciprocal linkages between extracellular matrix (ECM)-integrins, growth factor signaling and cell-cell adhesion molecules. ECM-dependent tissue-specific gene expression has also been linked to chromatin remodeling. With respect to tissue morphogenesis and differentiation, crosstalk has been established between the ECM and the homeobox morphoregulatory genes. Each of these linkages is profoundly influenced by the cell's microenvironment and the resulting tissue form. Thus for a cell to achieve a differentiated phenotype, the ECM molecules and their receptors must integrate both form and function. In contrast, mutated genes and aberrant interactions with the microenvironment conspire to undermine this integration, often resulting in malignant transformation.

Interaction between lipid bilayers and a new class of antineoplastic agents derived from arylchloroethylurea: a 2H solid-state NMR study.

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and model membrane of dimyristoylphosphatidylcholine by deuterium nuclear magnetic resonance spectroscopy. The results indicate that the drug incorporates in the bilayer and causes an increase of the lipid acyl chain order, this effect being greater close to the interfacial region of the lipid bilayer. The increase in ordering is dependent on the nature (degree of ramification, length of the alkyl chain, and presence of a sulfur atom) as well as on the position of the R substituent and is correlated with the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky ramified substituent and those for which the ordering effect on the lipid bilayer is the smallest. On the other hand, the ordering effect is greater and seen all along the lipid acyl chains for the long-chain CEUs, such as n-hexadecyl CEU, which have been shown to have very weak cytotoxic activity. Finally, the results obtained as a function of the drug concentration indicate that the ordering effect is seen for lipid to drug molar ratios as low as 20:1.

Validated high-performance liquid chromatographic methods for quantitation of a novel nonsteroidal antiestrogen.

HPLC assays were developed and validated for the quantitation of the novel orally active nonsteroidal antiestrogen EM-800 ¿(S)-(+)-4-[7-(2,2-dimethyl-l-oxopropoxy)-4-methyl-2-[4-[2-(1-pipe ridinyl)- ethoxy]phenyl]-2H-l-benzopyran-3-yl]-phenyl 2,2-dimethylpropanoate¿. The assay involves reversed-phase C18 or C4 columns using different mobile phases with ammonium acetate buffers and UV detection at lambda = 240 nm. The standard curve was linear over the concentration range of 10-1100 micrograms/ml. The precision (% relative standard deviation) values of these methods were in the range of 0.38-0.52 and 1.89-3.45% with C4 and C18 reversed phases, respectively. The limit of detection was found to be 1 microgram/ml. Enantiomeric separation was also obtained using a chiral method (ChiralPak AD column) using a mixture of hexane-reagent alcohol-diethylamine (94.9:5.0:0.1) as mobile phase. These methods were applied to stability studies, evaluation of pharmaceutical dosage forms and in the framework of toxicological studies. Details of some of these applications will be presented.

Induction of the angiogenic phenotype by Hox D3.

Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin alphavbeta3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and integrin alphavbeta3 expression, yet had no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin alphavbeta3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.

Microtubule-associated protein 1 light chain 3 is a fibronectin mRNA-binding protein linked to mRNA translation in lamb vascular smooth muscle cells.

Intimal cushions form in the fetal ductus arteriosus by fibronectin-dependent smooth muscle cell migration which is associated with greater efficiency of fibronectin mRNA translation. We investigated whether the AU-rich element (ARE), UUAUUUAU, in the 3'-untranslated region (3'UTR) of fibronectin mRNA is involved in this mechanism by transfecting smooth muscle cells with plasmids containing the chloramphenicol acetyltransferase coding region with its 3'UTR replaced by fibronectin 3'UTR bearing intact or mutated ARE. More efficient translation of fusion mRNA with intact versus mutated ARE was observed. This effect was amplified in ductus (10.9-fold) compared with nonmigratory, lower fibronectin-producing aorta cells (6.5-fold). Ductus cells transfected with wild-type but not ARE-mutated plasmid reverted to the stellate phenotype of aorta cells associated with reduced fibronectin production. This suggested that plasmid ARE sequesters RNA-binding factors, thereby reducing endogenous fibronectin mRNA translation. We next purified a 15-kD fibronectin ARE-dependent RNA-binding protein and identified it as microtubule-associated protein 1 light chain 3 (LC3). LC3 is present in greater amounts in ductus compared with aorta cells, and overexpression of LC3 in aortic cells by transfection enhances fibronectin mRNA translation to levels observed in ductus cells.

Interaction of 4-tert-butyl-[3-(2-chloroethyl) ureido] benzene with phosphatidylcholine bilayers: a differential scanning calorimetry and infrared spectroscopy study.

We have investigated the interaction between a new antineoplastic drug, 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (tBCEU), and distearoylphosphatidylcholine bilayers using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and high-pressure infrared spectroscopy. The results obtained with the three different techniques indicate that the drug incorporates in the lipid bilayer. More specifically, the incorporation of the tBCEU results in a decrease in the phase transition temperature of the lipid and in an increase in the amount of gauche conformers in the liquid-crystalline phase. In the gel phase, high-pressure FT-IR results indicate that the incorporation of tBCEU decreases the acyl chain packing. In addition, the results suggest the presence of hydrogen bonding between the lipid carbonyl group and a hydrogen bond donor in the tBCEU molecule. A possible candidate for this donor is the NH group adjacent to the phenyl ring. A model is proposed for the incorporation of tBCEU in lipid bilayers, with the hydrophobic portion of the drug intercalated between the lipid bilayers and the hydrophilic region located close to the interfacial region of the bilayer.

Membrane fluidity response to odorants as seen by 2H-NMR and infrared spectroscopy.

Fourier transform infrared spectroscopy (FTIR) and deuterium nuclear magnetic resonance spectroscopy (2H-NMR) have been used to study the location of two odorants, beta-ionone and menthone, in a model membrane of dimyristoylphosphatidylcholine, as well as the effect of the odorants on the structure and dynamics of the phospholipids. The interaction has been investigated for two lipid-to-odorant molar ratios, 10:1 and 1:1. The two odorants were found to affect the fluidity of the membrane. More specifically, the 2H-NMR results indicate that at a lipid-to-odorant molar ratio of 10:1, both beta-ionone and menthone increase the order of the deuterons in the interfacial and headgroup regions of the lipid while the incorporation of the odorants at a lipid-to-odorant molar ratio of 1:1 decreases the order of both the lipid headgroup and acyl chains. On the other hand, the infrared results show that the incorporation of beta-ionone and menthone decreases the phase transition temperature and cooperativity of the lipid acyl chains. The results suggest that the site of incorporation of beta-ionone and menthone is very similar in DMPC membranes.

Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle.

The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments.

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix.

Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.