Chromosomes 8, 12, and 17 copy number in Astler-Coller stage C colon cancer in relation to proliferative activity and DNA ploidy.

Fluorescence in situ hybridization using centromere-specific DNA probes to chromosomes 8, 12, and 17 was applied to 23 archival paraffin-embedded stage C colonic cancer specimens. Chromosome copy number was related to flow cytometric determinations of S-phase fraction and DNA ploidy. Three to eight copies of chromosomes 8, 12, and 17 were observed at mean frequencies of 28.7%, 37.8%, and 20.9%, respectively. The mean frequency of multiple copies of chromosome 12 was significantly greater than that for chromosome 17 (P < 0.0025). The mean frequency of single copies of chromosome 17 was significantly greater than that for chromosomes 8 and 12 (P < 0.0025 and P < 0.0005, respectively). Regarding the fourth quartile of cases, defined on the basis of the frequency of multiple chromosome copies, the proportion demonstrating moderate to high proliferative activity greatly exceeded the proportion displaying low proliferative activity. The same cases (most chromosomally aberrant) also generally demonstrated DNA aneuploidy. The results indicate a substantial degree of karyotypic instability in advanced colon cancer, particularly in cases with high proliferative activity and DNA aneuploidy.

v-Crk, an effector of the nerve growth factor signaling pathway, delays apoptotic cell death in neurotrophin-deprived PC12 cells.

v-Crk is a member of a class of SH2 and SH3-containing adaptor proteins that have been implicated in regulating the TrkA receptor tyrosine kinase and potentiating Nerve Growth Factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells (Hempstead et al, Mol. Cell Biol. 14: 1964 - 1971). Given the fact that NGF induces both differentiation and survival by binding to TrkA, we examined the rate of apoptotic cell death elicited by NGF-withdrawal in native, v-Crk, and TrkA-expressing PC12 cells. While more than 50% of native PC12 cells underwent apoptosis within 48 h of NGF withdrawal, the v-Crk and TrkA-expressing cells were much more resistant to apoptosis under these conditions, whereby approximately 70 and 95%, respectively, of the cells were alive. The ability of v-Crk to delay apoptosis required prior NGF-dependent differentiation, since naive undifferentiated v-Crk expressing PC12 cells or cells that express v-Crk mutants that are defective in NGF signaling were not protected from apoptosis during growth factor withdrawal. Moreover, addition of 50 ng/ml EGF to serum and NGF deprived v-Crk expressing cells, which also causes neurite outgrowth, promoted complete and long-term survival, although such EGF replacement had no neurotrophic effect on wild-type PC12 cells or PC12 cells overexpressing Human Bcl-2. These experiments suggest that v-Crk potentiation of a receptor tyrosine kinase under conditions of growth factor deprivation is essential for preventing apoptosis. However, unlike native PC12 cells, neither v-Crk or TrkA-expressing PC12 cells exhibited a G1 arrest when incubated for 2 weeks in NGF. Thus, v-Crk and TrkA may protect NGF deprived PC12 by preventing cell cycle arrest and hence an aborted entry into a defective cell cycle. Moreover, during NGF-withdrawal, v-CrkPC12 cells exhibited down regulation in MAP kinase and JNK activities while in native cells, these activities increased within 6 - 8 h after NGF deprivation. Thus, unlike v-Crk-mediated augmentation of differentiation, sustained activation of MAP kinase may not be required for v-Crk-induced cell survival.

Simulation of free radical reactions in biology and medicine: a new two-compartment kinetic model of intracellular lipid peroxidation.

To explore mechanisms of free radical reactions leading to intracellular lipid peroxidation in living systems, we developed a computational model of up to 109 simultaneous enzymatic and free radical reactions thought to be involved in the initiation, propagation, and termination of membrane lipid peroxidation. Rate constants for the various reactions were obtained from the published literature. The simulation model included a lipid membrane compartment and an aqueous cytosolic compartment, between which various chemical species were partitioned. Lipid peroxidation was initiated by the iron-catalyzed, superoxide-driven Fenton reaction. A "C" language computer program implemented numerical solution of the steady-state rate equations for concentrations of nine relevant free radicals. The rate equations were integrated by a modified Euler technique to describe the evolution with time of simulated concentrations of hydrogen peroxide, ferric and ferrous iron, unsaturated lipid, lipid hydroperoxides, superoxide anion, and biological antioxidants, including SOD and catalase. Initial results led to significant insights regarding mechanisms of membrane lipid peroxidation: 1. segregation and concentration of lipids within membrane compartments promotes chain propagation; 2. in the absence of antioxidants computed concentrations of lipid hydroperoxides increase linearly about 40 microM/min during oxidative stress; 3. lipid peroxidation is critically dependent upon oxygen concentration and the modeled dependence is similar to the experimental function; 4. lipid peroxidation is rapidly quenched by the presence of Vitamin E-like antioxidants, SOD, and catalase; 5. only small (1 to 50 microM) amounts of "free" iron are required for initiation of lipid peroxidation; 6. substantial lipid peroxidation occurs only when cellular defense mechanisms have been weakened or overcome by prolonged oxidative stress, hence understanding of the balance between free radical generation and antioxidant defense systems is critical to the understanding and control of free radical reactions in biology and medicine.

Hydroxyl radical generation by postischemic rat kidney slices in vitro.

To quantitate the formation of hydroxyl radicals (HO.) in ischemia and reoxygenation, dimethyl sulfoxide (DMSO) was added to "trap" evolving HO. in normal, in ischemic, and in ischemic and reoxygenated rat kidney slices, incubated in short-term organ culture in vitro. Hydroxyl radical generation was measured as the accumulation of the specific product of DMSO oxidation by HO., methane sulfinic acid (MSA) in the kidney tissue and surrounding medium using a new colorimetric assay. A mean difference of 7 nmol cumulative HO./gram tissue was detected in rat kidney slices subjected to ischemia and reoxygenation. This amount of HO. generation was not significantly greater than that found in nonischemic or in ischemic but not reoxygenated control tissues, and does not appear to represent the highly toxic burst of HO. radicals implied in current theoretical discussions of reperfusion injury. However, the addition of EDTA chelated iron (1:1) to the incubation medium led to marked postischemic HO. generation. We conclude that clearly toxic numbers of HO. radicals are not formed during reoxygenation in rat kidney slices, either because there is insufficient iron, because only a small fraction of cells in the kidney tissue make oxygen radicals, or because cellular defenses against HO. formation are more powerful than currently appreciated.

Detection and quantitation of hydroxyl radical using dimethyl sulfoxide as molecular probe.

Generation of clearly harmful amounts of hydroxyl radicals in biological systems can be studied using DMSO as a molecular probe. DMSO is oxidized by HO. to form the stable, nonradical compound methanesulfinic acid, which is not normally found in living systems and which can be easily extracted from tissue and measured spectrophotometrically. The present method provides a simple, inexpensive assay for methanesulfinic acid in biological materials. As little as 10 nmol of sulfinate can be detected, and interference from diverse biological compounds is minimal. Additionally, there is no interference from a large excess of dimethyl sulfoxide, which is necessary if the assay is to be applied directly to tissues pretreated with DMSO. When straightforward cleanup procedures are utilized, there is minimal interference from glutathione or sulfate, and potentially troublesome interference from detergentlike substances can usually be minimized. Owing to its relative specificity for sulfinic acids at acid pH, the diazonium coupling reaction can thus be exploited to provide an efficient and inexpensive means of detecting methanesulfinic acid in DMSO-pretreated biological materials. The results provide a direct chemical means for measuring cumulative HO. generation.

Highly potent, orally active diester macrocyclic human renin inhibitors.

Replacing one amide bond in macrocyclic renin inhibitors of the general structure 1 and 2 with an ester linkage gave glutamate-derived inhibitors 3 and serine-derived inhibitors 4. While this oxygen-for-nitrogen exchange had little effect on potency in the glutamate series, potency was dramatically increased in the serine series. In this series, the 14-membered ring compounds proved to be more potent than the corresponding 13-membered ring derivatives. Substitution of the ring at the position corresponding to P2' generally increased potency. The absolute configuration at this center was shown to be R for the 4-morpholinomethyl derivative (4o), both by asymmetric synthesis and X-ray crystallography. Replacing the "Boc-Phe" moiety of inhibitor 4o with a variety of substituents led to subnanomolar inhibitors, one of which (the "3(S)-quinuclidinyl-Phe" derivative 33) lowered blood pressure 20 mmHg and completely inhibited plasma renin activity for 6 h in sodium-depleted rhesus monkeys. This compound proved to have limited bioavailability (1% in rats) due to cleavage of the serine ester bond and rapid hepatic extraction.

Characterization of novel cell lines from pleomorphic adenomas of the parotid gland established in a collagen gel system.

The pathobiology of salivary neoplasms can best be studied in a model system that reflects the native state of the tumor. The present study describes the use of a three-dimensional collagen gel (organoid) system in which pleomorphic adenomas of the parotid gland were propagated in vitro. Five pleomorphic adenoma cultures were established as organoid gels and compared with touch-preparations or cryopreserved specimens of native tumor. The organoid cultures demonstrated normal DNA content, the expression of myoepithelial cell proteins, and the production of sulfated acid mucins; these cellular and secretory features mimicked those found in the archival specimens. Further, organoid cultures of pleomorphic adenoma could be initiated after monolayer culture, demonstrating that culture on a plastic support does not alter the nature of the cells. Development of an in vitro culture system that maintains the native state of pleomorphic adenoma is an important tool for studying the pathobiology of these tumors.

Quantitation of the hydroxyl radical by reaction with dimethyl sulfoxide.

This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO.) in aqueous systems. Reaction of HO. with DMSO produces methane sulfinic acid as a primary product, which can be detected by a simple colorimetric assay. To evaluate this method for estimating total HO. production, we studied three model systems, including the Fenton reaction, gamma irradiation of water, and ultraviolet photolysis of hydrogen peroxide, for which the theoretical maximum yield of HO. could be calculated and compared to measured DMSO oxidation. The results confirm that 0.05 to 1 M DMSO may be used to capture nearly all of the expected HO. radicals formed. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO. formation in aqueous systems pretreated with DMSO.

Inhibition of aberrant proliferation and induction of apoptosis in HER-2/neu oncogene transformed human mammary epithelial cells by N-(4-hydroxyphenyl)retinamide.

Epithelial cells from non-cancerous mammary tissue in response to exposure to chemical carcinogens or transfection with oncogenes exhibit hyperproliferation and hyperplasia prior to the development of cancer. Aberrant proliferation may, therefore, represent a modifiable early occurring preneoplastic event that is susceptible to chemoprevention of carcinogenesis. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR), has exhibited preventive efficacy in several in vitro and in vivo breast cancer models, and represents a promising chemopreventive compound for clinical trials. Clinically relevant biochemical and cellular mechanisms responsible for the chemopreventive effects of HPR, however, are not fully understood. Experiments were performed on preneoplastic human mammary epithelial 184-B5/HER cells derived from reduction mammoplasty and initiated for tumorigenic transformation by overexpression of HER-2/neu oncogene, to examine whether HPR inhibits aberrant proliferation of these cells and to identify the possible mechanism(s) responsible for the inhibitory effects of HPR. Continuous 7-day treatment with HPR produced a dose-dependent, reversible growth inhibition. Long-term (21 day) treatment of 184-B5/HER cells with HPR inhibited anchorage-dependent colony formation by approximately 80% (P < 0.01) relative to that observed in the solvent control. A 24 h treatment with cytostatic 400 nM HPR produced a 25% increase (P = 0.01) in G0/G1 phase, and a 36% decrease (P = 0.01) in S phase of the cell cycle. HPR treatment also induced a 10-fold increase (P = 0.02) in the sub-G0 (apoptotic) peak that was down-regulated in the presence of the antioxidant N-acetyl-L-cysteine. Treatment with HPR resulted in a 30% reduction of cellular immunoreactivity to tyrosine kinase, whereas immunoreactivity to p185HER remained essentially unaltered. HPR exposure resulted in time-dependent increase in cellular metabolism of the retinoid as evidenced by increased formation of the inert metabolite N-(4-methoxyphenyl)-retinamide (MPR) and progressive increase in apoptosis. Thus, HPR-induced inhibition of aberrant proliferation may be caused, in part, by its ability to inhibit HER-2/neu-mediated proliferative signal transduction, retard cell cycle progression and upregulate cellular apoptosis.

PAI-1 gene expression is growth state-regulated in cultured human epidermal keratinocytes during progression to confluence and postwounding.

Growth of human keratinocytes (NHKs) in submerged cultures approximates a "wound" response generally considered equivalent to regenerative maturation. Within this context, PAI-1 expression in cultured NHKs appears to be growth state-regulated and is associated with specific NHK subpopulations undergoing migration and proliferation in response to wounding; NHKs transit through specific phases during growth to confluence. Basal layer keratinocytes comprise several classes of nucleated epidermal cells (designated "A," "B," and "C") which are distinguishable on the basis of RNA content, population generation time, and expression of basal cell marker proteins. "A" substrate cells initially give rise to expanding proliferating keratinocyte colonies in vitro, but are rapidly replaced (at the stage of 50-75% culture confluence) by transient amplifying "B" cells and, eventually, the larger "C" subpopulation which subsequently differentiates into suprabasal spinous cells during the postconfluent growth period. Expression of plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor and member of the serpin gene family which is synthesized by "activated" or migrating keratinocytes in vivo, was restricted to the preconfluent stages of cell growth in vitro, a condition equivalent to wound regeneration in situ. PAI-1 mRNA and protein expression was maximal in 50-75% confluent cultures correlating, thereby, with the emergence of the transient amplifying "B" cell population. Flow cytometric analysis revealed that PAI-1 is detected early in expanding NHK colonies, during the initial recruitment of basal cells from the "A" state into the "B" compartment in the absence of significant proliferation, suggesting that PAI-1 may be active in regulating early NHK migratory events, independent of cell proliferation. Thereafter, these PAI-1-expressing "A" cells are recruited into the "B" compartment, where they continue to migrate, proliferate, and enlarge, eventually giving rise to PAI-1-expressing "C" cells. By the time NHK cultures reach exponential growth, virtually no small "A" cells contain immunoreactive PAI-1. PAI-1 expression peaks during preconfluent growth and remains confined to larger basal cell phenotypes. Cellular accumulation of both PAI-1 mRNA and protein appeared to be cell cycle phase-specific and characteristic of progression through an activated G1 growth phase. Induced expression, moreover, was restricted to a "window" in G1 with PAI-1 mRNA evident within 2 h after serum addition to growth-arrested cells and attaining maximal levels (50-fold) at 10 h poststimulation. Induced PAI-1 expression, thus, appears to be a general characteristic of the activated epidermal phenotype in vitro as well as in vivo.

Targeted inhibition of wound-induced PAI-1 expression alters migration and differentiation in human epidermal keratinocytes.

In the adult epidermis, keratinocytes do not normally express the type-1 inhibitor of plasminogen activator (PAI-1). Basal epithelial cell-specific PAI-1 synthesis, however, accompanies epidermal wound repair in vivo in which PAI-1 transcripts and immunoreactive protein are confined to epithelial cells in the migrating tongue and the hyperproliferative zone. A model system using human keratinocytes (HaCaT cells) was developed to assess functional relationships between epithelial growth state transitions and PAI-1 expression. PAI-1 synthesis was maximal in low population density, exponentially growing HaCaT cultures; relative PAI-1 mRNA and protein levels progressively declined as cells attained, and were maintained in, a postconfluent condition. While the fraction of PAI-1(+) keratinocytes remained stable (at approximately 85-90% of the population) throughout the culture period, both PAI-1 mRNA abundance and mean cell-associated PAI-1 protein declined by >90% during prolonged (i.e., 8-day) growth arrest. Similar to epidermal trauma in vivo, scrape wounding of HaCaT monolayers resulted in the rapid and location-specific induction of PAI-1 protein (an increase of 11- to 16-fold relative to unwounded cultures) in cells immediately bordering the injury site. PAI-1 expression was evident in keratinocytes that comprised the opposed migrating fronts and remained elevated until wound closure. Down-regulation of PAI-1 synthesis in HaCaT cells transfected with an inducible LacSwitch-based antisense vector system markedly impaired both the rate and the extent of wound closure. All injuries created in antisense PAI-1 monolayers remained unhealed at day 8 postinjury compared to the 3-day complete repair typical of control cultures. Vector-driven modulation of PAI-1 synthesis was also associated with an increase in the percentage of suprabasal-type keratinocytes within the wound field. PAI-1 expression by migrating HaCaT cells appears necessary to maintain the basal epidermal phenotype and/or appropriate cell-to-substrate adhesion during injury repair.

Carbohydroxamido-oxazolidines: antibacterial agents that target lipid A biosynthesis.

A series of carbohydroxamido-oxazolidine inhibitors of UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase, the enzyme responsible for the second step in lipid A biosynthesis, was identified. The most potent analog L-161,240 showed an IC50 = 30 nM in the DEACET assay and displayed an MIC of 1-3 microg/mL against wild-type E. coli.

DNA analysis of human cholesteatomas.

HYPOTHESIS: The hypothesis tested in this article is that if cholesteatomas are a low-grade squamous cell neoplasm, then evidence of genetic instability, in the form of abnormal or aneuploid amounts of DNA, should be evident. BACKGROUND: Cholesteatoma is a destructive lesion of the middle ear and/or mastoid process that produces complications by erosion of the temporal bone. The clinical hallmarks of cholesteatomas, namely invasion, migration, uncoordinated proliferation, altered differentiation, aggressiveness, and recidivism, are traits typically associated with the neoplastic cell. However, there is little evidence to support or refute the speculation that cholesteatomas are a low-grade squamous cell neoplasm. the existence of defects in the genetic complement of the major cellular constituents comprising a cholesteatoma, fibroblasts and keratinocytes, would support the speculation that cholesteatomas are a neoplasm, since cancers commonly manifest quantitative and qualitative alterations in the normal euploid complement of genetic information, resulting in a cell that has an abnormal or aneuploid amount of DNA. METHODS: DNA content (ploidy) within cholesteatoma tissues was measured by flow cytometry and image analysis. RESULTS: The DNA content of 11 human cholesteatomas and nine postauricular skin specimens was analyzed using flow cytometry, while the DNA content of 10 cholesteatoma specimens was analyzed using image analysis. Interpretable data was obtained from 10 cholesteatoma specimens and six postauricular skin specimens. One cholesteatoma specimen demonstrated an abnormal aneuploid DNA content, whereas the remaining nine cholesteatomas and the six postauricular skin specimens demonstrated a normal euploid DNA content. CONCLUSIONS: We conclude that, due to the lack of overt genetic instability, as evidenced by the presence of a normal euploid DNA content, cholesteatomas are not low-grade neoplasms.