Design, synthesis, and evaluation of estradiol-linked genotoxicants as anti-cancer agents.

A series of bifunctional compounds was prepared consisting of 17beta estradiol linked to a DNA damaging N,N-bis-(2-chloroethyl)aniline. The objective of our studies was to determine the characteristics of the linker that permitted both reaction with DNA and binding of the resultant covalent adducts to the estrogen receptor. Linker characteristics were pivotal determinants underlying the ability of the compounds to kill selectively breast cancer cells that express the estrogen receptor.

Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF-1/CREB and increased expression of glycolytic enzymes, p21(waf1/cip1), and erythropoietin.

Iron chelators are pluripotent neuronal antiapoptotic agents that have been shown to enhance metabolic recovery in cerebral ischemia models. The precise mechanism(s) by which these agents exert their effects remains unclear. Recent studies have demonstrated that iron chelators activate a hypoxia signal transduction pathway in non-neuronal cells that culminates in the stabilization of the transcriptional activator hypoxia-inducible factor-1 (HIF-1) and increased expression of gene products that mediate hypoxic adaptation. We examined the hypothesis that iron chelators prevent oxidative stress-induced death in cortical neuronal cultures by inducing expression of HIF-1 and its target genes. We report that the structurally distinct iron chelators deferoxamine mesylate and mimosine prevent apoptosis induced by glutathione depletion and oxidative stress in embryonic cortical neuronal cultures. The protective effects of iron chelators are correlated with their ability to enhance DNA binding of HIF-1 and activating transcription factor 1(ATF-1)/cAMP response element-binding protein (CREB) to the hypoxia response element in cortical cultures and the H19-7 hippocampal neuronal cell line. We show that mRNA, protein, and/or activity levels for genes whose expression is known to be regulated by HIF-1, including glycolytic enzymes, p21(waf1/cip1), and erythropoietin, are increased in cortical neuronal cultures in response to iron chelator treatment. Finally, we demonstrate that cobalt chloride, which also activates HIF-1 and ATF-1/CREB in cortical cultures, also prevents oxidative stress-induced death in these cells. Altogether, these results suggest that iron chelators exert their neuroprotective effects, in part, by activating a signal transduction pathway leading to increased expression of genes known to compensate for hypoxic or oxidative stress.

Adaptive resistance to nitric oxide in motor neurons.

Nitric oxide (NO) is a free radical produced actively by mammalian cells, including neurons. Low levels of NO can function in intercellular signaling, but high levels are cytotoxic. This cytotoxic potential suggests that cells at risk for NO damage, such as neurons, might have NO resistance mechanisms to prevent cell death, and adaptive resistance to NO-releasing compounds has been reported for some non-neuronal cell types. Here we show that immortalized mouse motor neurons (NSC34 cells) respond to sub-lethal fluxes of pure NO by activating adaptive resistance mechanisms that counteract cytotoxic NO exposure. This adaptive NO resistance is reversible and is paralleled by the induction of the oxidative stress enzyme heme oxygenase 1 (HO-1). An inhibitor of both HO-1 and heme-dependent guanylate cyclase (tin-protoporphyrin IX) greatly sensitized NO-pretreated NSC34 cells to the NO challenge. However, readdition of cyclic GMP (in the form of the 8-bromo derivative) restored rather little resistance, and a more selective guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxaline-1-one (at 10 microM), did not have the sensitizing effect. Therefore, the inducible HO-1 pathway contributes substantially to adaptive NO resistance, while cyclic GMP seems to play at most a small role. A similar adaptive resistance to NO was observed in primary rat spinal chord motor neurons. The activation of NO resistance in motor neurons may counteract age- or disease-related neurodegeneration.

Complex genetic response of human cells to sublethal levels of pure nitric oxide.

NO is a biologically generated free radical that serves diverse roles in mammalian cell signaling and immune-mediated cell killing. Because mammalian cells might be exposed to varying levels of NO, we tested for possible defense genes and proteins induced upon treatment of cells with sublethal fluxes of pure NO. Two-dimensional gel analysis was performed for human embryonic lung fibroblasts (IMR-90) exposed for 90 min to pure NO at approximately 280 nM/s, which revealed the reproducible induction of at least 12 proteins. Among these, a prominent polypeptide had Mr approximately 32,000, similar to the well-known oxidative stress protein heme oxygenase-1 (HO-1). Northern blot analysis of IMR-90 and HeLa cells demonstrated the NO-mediated induction of HO-1 mRNA up to 70-fold over the levels in untreated cells. HO-1 induction depended on the NO dose and subsequent expression time and was maximal 3-5 h after a 1-h exposure to NO at a constant flux of approximately 280 nM/s. The mRNA encoding a tyrosine/threonine phosphatase (CL100/MKP-1) was also NO inducible (approximately 20 fold), whereas there was no increase in expression of the mRNA encoding manganese-containing superoxide dismutase. Induction of HO-1 mRNA was independent of the guanylate cyclase signaling pathway; addition of the analogue 8-bromo-cyclic GMP did not induce the HO-1 transcript, and the soluble guanylate cyclase inhibitor LY-83583 did not block HO-1 induction by NO in IMR-90 cells. Luciferase reporter constructs containing up to 4.7 kb of DNA upstream of the HO-1 transcription start site showed < or = 2.5-fold induction in IMR-90 or HeLa cells exposed to NO. However, HO-1 mRNA was dramatically stabilized after exposure of IMR-90 cells to NO. Even a transient NO exposure produced elevated levels of HO-1 protein for > or = 10 h, whereas continuous low-level NO treatment (35 nM/s) maintained elevated HO-1 mRNA expression for > or = 8 h. These results reveal a complex mammalian response to NO that involves a new level of posttranscriptional control in response to this radical.

Mammalian DNA repair methyltransferases shield O4MeT from nucleotide excision repair.

O6-Methylguanine (O6MeG) and O4-methylthymine (O4MeT) are potentially mutagenic DNA lesions that cause G:C-->A:T and A:T-->G:C transition mutations by mispairing during DNA replication, and the repair of O6MeG and O4MeT by DNA repair methyltransferases (MTases) is therefore expected to prevent methylation-induced transitions. The efficiency of O6MeG and O4MeT repair by different MTases can vary by several hundred-fold and the aim of this study was to establish the biological consequences of such differences in the efficiency of repair. The ability of three microbial and two mammalian MTases to prevent methylation-induced G:C-->A:T and A:T-->G:C transitions is taken as a measure of their ability to repair O6MeG and O4MeT in vivo respectively. All five MTases give complete protection against G:C-->A:T transitions. However, while the microbial MTases give complete protection against A:T-->G:C transitions, the mammalian MTases actually sensitize cells to A:T-->G:C transitions. We hypothesize that the mammalian MTases bind O4MeT lesions in vivo but that, because they are extremely slow at subsequent methyl transfer, binding shields O4MeT from repair by the nucleotide excision repair pathway. Results are presented to support this hypothesis.

Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast.

Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis.

Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds.

Cartilage implants which could potentially be used to resurface damaged joints were created using rabbit articular chondrocytes and synthetic, biodegradable polymer scaffolds. Cells were serially passaged and then cultured in vitro on fibrous polyglycolic acid (PGA) scaffolds. Cell-PGA constructs were implanted in vivo as allografts to repair 3-mm diameter, full thickness defects in the knee joints of adult rabbits, and cartilage repair was assessed histologically over 6 months. In vitro, chondrocytes proliferated on PGA and regenerated cartilaginous matrix. Collagen and glycosaminoglycan (GAG) represented 20 to 8% of the implant dry weight (dw), respectively, at the time of in vivo implantation; the remainder was PGA and unspecified components. Implants based on passaged chondrocytes had 1.7-times as much GAG and 2.6-times as much collagen as those based on primary chondrocytes. In vivo, cartilaginous repair tissue was observed after implantation of PGA both with and without cultured chondrocytes. Six month repair was qualitatively better for cell-PGA allografts than for PGA alone, with respect to: 1) surface smoothness, 2) columnar alignment of chondrocytes, 3) spatially uniform GAG distribution, 4) reconstitution of the subchondral plate, and 5) bonding of the repair tissue to the underlying bone. These pilot studies demonstrate that it is feasible to use cell-polymer allografts for joint resurfacing in vivo.

Kinetics of chondrocyte growth in cell-polymer implants.

In vitro cultivation of cartilage cells (chondrocytes) on biodegradable polyglycolic acid (PGA) scaffolds resulted in implants which could potentially be used to repair damaged joint cartilage or for reconstructive surgery. Cell growth kinetics were studied to define conditions under which the cellularity of implants made from isolated calf chondrocytes reached that of the parent calf cartilage. In static cultures, condrocyte growth rates decreased as either implant thickness or implant cell density increased. Over 4 weeks of cultivation, implant permeability to glucose decreased to 3% that of the plain polymer scaffold; this effect was attributed to the decrease in effective implant porosity associated with cartilage tissue regeneration.In a well-mixed culture, implants 1 cm in diameter by 0.3 cm thick maintained high cell growth rates over 7 weeks and hard normal cell densities. Regenerated cartilage with these dimensions is large enough to resurface small joints such as the trapezium bone at the base of the human thumb. Such implants could not be grown statically, since cell growth stopped at 3-4 weeks and cell densities remained below normal. Optimization of the tissue culture environment is thus essential in order to cultivate clinically useful cartilage implants in vitro. (c) 1994 John Wiley & Sons, Inc.

Composition of cell-polymer cartilage implants.

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.

Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers.

Cartilaginous implants for potential use in reconstructive or orthopedic surgery were created using chondrocytes grown on synthetic, biodegradable polymer scaffolds. Chondrocytes isolated from bovine or human articular or costal cartilage were cultured on fibrous polyglycolic acid (PGA) and porous poly(L)lactic acid (PLLA) and used in parallel in vitro and in vivo studies. Samples were taken at timed intervals for assessment of cell number and cartilage matrix (sulfated glycosaminoglycan [S-GAG], collagen). The chondrocytes secreted cartilage matrix to fill the void spaces in the polymer scaffolds that were simultaneously biodegrading. In vitro, chondrocytes grown on PGA for 6 weeks reached a cell density of 5.2 x 10(7) cells/g, which was 8.3-fold higher than at day 1, and equalled the cellularity of normal bovine articular cartilage. In vitro, the cell growth rate was approximately twice as high on PGA as it was on PLLA; cells grown on PGA produced S-GAG at a high steady rate, while cells grown on PLLA produced only minimal amounts of S-GAG. These differences could be attributed to polymer geometry and biodegradation rate. In vivo, chondrocytes grown on both PGA and PLLA for 1-6 months maintained the three-dimensional (3-D) shapes of the original polymer scaffolds, appeared glistening white macroscopically, contained S-GAG and type II collagen, and closely resembled cartilage histologically. These studies demonstrate the feasibility of culturing isolated chondrocytes on biodegradable polymer scaffolds to regenerate 3-D neocartilage.