Improved intervention of atherosclerosis and cardiac hypertrophy through biodegradable polymer-encapsulated delivery of glycosphingolipid inhibitor.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glycosphingolipid synthesis inhibitor, holds promise for the treatment of atherosclerosis and cardiac hypertrophy but rapid in vivo clearance has severely hindered translation to the clinic. To overcome this impediment, we used a materials-based delivery strategy wherein D-PDMP was encapsulated within a biodegradable polymer composed of poly ethylene glycol (PEG) and sebacic acid (SA). PEG-SA was formulated into nanoparticles that were doped with (125)I-labeled PEG to allow in vivo bio-distribution and release kinetics of D-PDMP to be determined by using γ-scintigraphy and subsequently, by mass spectrometry. Polymer-encapsulation increased the residence time of D-PDMP in the body of a treated mouse from less than one hour to at least four hours (and up to 48 h or longer). This substantially increased in vivo longevity provided by polymer encapsulation resulted in an order of magnitude gain in efficacy for interfering with atherosclerosis and cardiac hypertrophy in apoE-/- mice fed a high fat and high cholesterol (HFHC) diet. These results establish that D-PDMP encapsulated in a biodegradable polymer provides a superior mode of delivery compared to unconjugated D-PDMP by way of increased gastrointestinal absorption and increased residence time thus providing this otherwise rapidly cleared compound with therapeutic relevance in interfering with atherosclerosis, cardiac hypertrophy, and probably other diseases associated with the deleterious effects of abnormally high glycosphingolipid biosynthesis or deficient catabolism.

Glucosamine modulates chondrocyte proliferation, matrix synthesis, and gene expression.

OBJECTIVE: To investigate the effects of glucosamine (GlcN) on chondrocyte proliferation, matrix production, and gene expression for providing insights into the biochemical basis of its reported beneficial effects in osteoarthritis (OA). METHODS: Dose-dependent effect of GlcN on cell morphology, proliferation, cartilage matrix production and gene expression was examined by incubating primary bovine chondrocytes with various amounts of GlcN in monolayers (2D) and in cell-laden hydrogels (3D constructs). Histology, immunofluorescent staining and biochemical analyses were used to determine the effect of GlcN on cartilage matrix production in 3D constructs. The impact of GlcN on gene expression was evaluated with real-time polymerase chain reaction (PCR). RESULTS: GlcN concentration and culture conditions significantly affected the cell behavior. Quantitative detection of matrix production in cell-laden hydrogels indicated a relatively narrow window of GlcN concentration that promotes matrix production (while limiting cellular proliferation, but not cell viability). Notably, GlcN enhanced cartilage specific matrix components, aggrecan and collagen type II, in a dose-dependent manner up to 2 mM but the effect was lost by 15 mM. Additionally, GlcN treatment up-regulated transforming growth factor-beta1 (TGF-beta1) mRNA levels. CONCLUSION: Results indicate that culture conditions play a significant role in determining the effect of GlcN on chondrocytes, explaining both the previously reported beneficial and deleterious effects of this sugar. The ability of GlcN to alter TGF-beta1 signaling provides a biochemical mechanism for GlcN activity on chondrocytes that up to now has remained elusive. The observed anabolic effect of optimal GlcN concentrations on chondrocytes may be useful in formulating effective cartilage repair strategies.

Substrate specificity of the sialic acid biosynthetic pathway.

Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

New directions in carbohydrate engineering: a metabolic substrate-based approach to modify the cell surface display of sialic acids.

This review discusses new directions in the emerging field of carbohydrate engineering. Specifically, it describes substrate-based methodologies that are complementary to the recombinant DNA techniques that now dominate metabolic and cellular engineering endeavors. A substrate-based approach consists of intercepting a biosynthetic pathway with an unnatural analog of a metabolic intermediate. The unnatural compound competes with the endogenous substrate for biosynthetic incorporation into a cellular component by action of the natural enzymes of the cell. The incorporation of the unnatural compound into the cellular architecture can directly modulate cellular properties and biological processes. Alternatively, a molecular handle can be included in the design of the unnatural substrate that allows further elaboration upon reaction with an externally delivered reagent. The sialic acid biosynthetic pathway is presented as a model system to illustrate both the practical aspects and theoretical considerations of a substrate-based cellular engineering approach. Specific applications of carbohydrate-based cell surface engineering include chemical construction of new glycosylation patterns on cells, new approaches to targeting tumor cell with either diagnostic or therapeutic agents, and installation of novel receptors on cells for facilitating viral-mediated gene delivery.

Metabolic selection of glycosylation defects in human cells.

Changes in glycosylation are often associated with disease progression, but the genetic and metabolic basis of these events is rarely understood in detail at a molecular level. We describe a metabolism-based approach to the selection of mutants in glycoconjugate biosynthesis that provides insight into regulatory mechanisms for oligosaccharide expression and metabolic flux. Unnatural intermediates are used to challenge a specific pathway, and cell surface expression of their metabolic products provides a readout of flux in that pathway and a basis for selecting genetic mutants. The approach was applied to the sialic acid metabolic pathway in human cells, yielding novel mutants with phenotypes related to the inborn metabolic defect sialuria and metastatic tumor cells.

Characterizing glycosylation pathways.

Numerous factors that influence cell-surface carbohydrate composition remain to be elucidated. The combination of novel biochemical and metabolism-based approaches with emerging genomic methods promises to accelerate efforts to understand glycosylation.

Metabolic delivery of ketone groups to sialic acid residues. Application To cell surface glycoform engineering.

The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Using a recently described method (Mahal, L. K., Yarema, K. J., and Bertozzi, C. R. (1997) Science 276, 1125-1128), we delivered a uniquely reactive ketone group to endogenous cell surface sialic acid residues by treating cells with the ketone-bearing metabolic precursor N-levulinoylmannosamine (ManLev). The ketone undergoes highly selective condensation reactions with complementary nucleophiles such as aminooxy and hydrazide groups. The detailed quantitative parameters of ManLev metabolism in human and nonhuman-derived cell lines were determined to establish a foundation for the modification of cell surfaces with novel epitopes at defined cell-surface densities. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. The remodeled cells were endowed with novel lectin binding profiles as determined by flow cytometry analysis. The simplicity and generality of this method make it well suited for use in the study of carbohydrate-mediated cell surface interactions.

Chemical approaches to glycobiology and emerging carbohydrate-based therapeutic agents.

The contributions of cell surface oligosaccharides to critical biological processes such as leukocyte-endothelial cell adhesion, bacterial and viral infection, and immunological recognition of tumor cells and foreign tissue are now understood in significant molecular detail. These discoveries at the forefront of biological research have motivated the design of synthetic glycoconjugates as tools for the fundamental study of glycobiology and as candidates for future generations of therapeutic and pharmaceutical reagents. During the past two years, significant progress has been made in the design and synthesis of carbohydrate-based inhibitors of selectins, receptors involved in the attachment of leukocytes to endothelial cells at sites of inflammation. Monomeric and multivalent oligosaccharides that bind to bacterial and viral receptors have been shown to abrogate infection by agents such as Helicobacter pilori, influenza virus and HIV. The identification of certain cell surface oligosaccharides as potent antigens has prompted their use in tumor vaccines, and inspired new approaches to the management of tissue rejection subsequent to xenotransplantation. To better understand how cell surface oligosaccharides function within their native context, novel chemical approaches to modulating cell surface oligosaccharides structures are now being developed. These stratergies for cell surface 'glycoform remodeling' promise to facilitate the investigation of carbohydrate mediated cell-cell interactions.

Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis.

Cell surface oligosaccharides can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharides metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate.

Synthesis and biological activity of DNA damaging agents that form decoy binding sites for the estrogen receptor.

It is a goal of cancer chemotherapy to achieve the selective killing of tumor cells while minimizing toxicity to normal tissues. We describe the design of selective toxins forming DNA adducts that attract the estrogen receptor (ER), a transcription factor that is overexpressed in many human breast and ovarian tumors. The compounds consist of 4-(3-aminopropyl)-N,N-(2-chloroethyl)-aniline linked to 2-(4'-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former moiety is a DNA damaging nitrogen mustard and the latter is a ligand for the ER. The connection between these groups was refined to permit DNA adducts formed by the mustard portion of the molecule to present the ligand domain so that it was able to interact efficiently with the ER. By using 16-mers containing specific DNA adducts, it was determined that monoadducts and putative intrastrand crosslinks were preferred targets for the ER over interstrand crosslinks. A series of structurally related 2-phenylindole mustards was prepared, some of which were selectively toxic to the ER-positive breast cancer cell line MCF-7, as compared with the ER(-) negative line MDA-MB231. The ability both to bind to DNA and to interact significantly with the ER were essential to achieve selective lethality toward ER(+) cells. Compounds forming DNA adducts without the ability to bind receptor showed similar toxicities in the two cell lines. Several models could explain the selective toxicity of the mustard-phenylindole compounds toward ER(+) cells. The favored model suggests that a mustard-DNA adduct is shielded by the ER from DNA repair enzymes and hence cells possessing an abundance of the ER selectively retain the adduct and are killed.

Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-intrastrand d(GpTpG)-cisplatin adduct.

Nucleotide excision repair by mammalian enzymes removes DNA damage as part of approximately 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion. We analyzed this dual incision reaction at a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a closed circular duplex DNA substrate. Incisions were formed in the DNA with human cell extracts in which DNA repair synthesis was inhibited. The nicks were mapped by restriction fragment end labeling and primer extension analysis. Principal sites of cleavage were identified at the 9th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion. The predominant product was found to be a 26-mer platinated oligonucleotide by hybridization to a 32P-labeled complementary DNA probe. Oligonucleotides were formed at the same rate as the 3' cleavage, suggesting that both incisions are made in a near-synchronous manner. There was, however, a low frequency of 5' incisions in the absence of 3' cleavage. The dual incision reaction was reconstituted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF and IF7. All of these components were required in order to observe any cleavage.

Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II).

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.

Effects of DNA adduct structure and distribution on the mutagenicity and genotoxicity of two platinum anticancer drugs.

cis-Diamminedichloroplatinum(II) (cis-DDP) and cis,trans,cis-ammine(cyclohexylamine)-dibutyratodichloroplatinu m(IV) (ACDDP) are anticancer drugs that bind to DNA, forming replication blocking adducts. ACDDP, probably manifests its cytotoxicity through the metabolite cis-ammine(cyclohexylamine)dichloroplatinum(II) (ACDP). The biological effects of ACDP and cis-DDP were compared by studying polymerase inhibition in vitro and mutagenesis and genotoxicity in vivo in the duplex genome of bacteriophage M13mp18 replicated in Escherichia coli. cis-DDP and ACDP adducts were equally genotoxic within the statistical error limits of the analysis. Survival of genomes platinated by either drug, increased by threefold in cells pretreated with u.v. irradiation to induce the SOS functions of the host. In the M13mp18 lacZ' gene fragment, the mutagenicity of ACDP was lower than that of cis-DDP; the difference was approximately twofold at a dose of two adducts per 370 base-pair mutational target. Mutagenesis by both compounds was SOS-dependent. The structural basis for lower mutagenicity of ACDP is proposed to be its reduced reactivity at d(ApG) sites. This effect is attributed to an orientational isomerism that precludes the formation of one of two possible DNA adducts at d(ApG) residues. The types of mutations induced for both drugs were similar, but they occurred with different distributions. Both compounds induced primarily G-->T transversions at d(GpG) sites whereas G-->A transitions and A-->T transversions, many at d(ApG), d(GpNpG), and d(GpG) sites, were also well represented. The mapping of DNA adducts by DNA synthesis inhibition revealed excellent correlation between the location of DNA lesions and the sites of mutations. Analysis of the distribution of mutations and the distribution of potential platination sites revealed no sequence-dependent mutation hotspots; i.e. mutagenesis was random throughout the lacZ' region of the M13mp18 bacteriophage genome. These results offer insights into the molecular mechanism of mutagenicity of platinum anticancer drugs.

Mutagenicity and genotoxicity of the major DNA adduct of the antitumor drug cis-diamminedichloroplatinum(II).

The mutagenicity and genotoxicity of cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] (G*G*), the major DNA adduct of the antitumor drug cisplatin, has been investigated in Escherichia coli. A duplex bacteriophage M13 genome was constructed to contain the G*G* adduct at a specific site in the (-) strand. The singly platinated duplex genome exhibited a survival of 22% relative to that of the unplatinated control genomes, and this value rose to 38% in cells treated with ultraviolet light to induce the SOS response. Singly platinated single-stranded genomes were also produced. Replication of the single- and double-stranded genomes in vivo yielded SOS-dependent, targeted mutations at frequencies of 1.3% and 0.16%, respectively. The mutagenic specificity of G*G* in both single- and double-stranded DNA was striking in that 80-90% of the mutations occurred at the 5'-platinated G. Approximately 80% of the mutations were G-->T transversions at that site. A model of mutagenesis is presented to explain this mutational specificity with respect to current understanding of platinum-DNA adduct structure.