In vitro and in vivo pharmacological characterizations of the antitumor properties of two new olivacine derivatives, S16020-2 and S30972-1.

S16020-2, a new olivacine derivative and a topoisomerase II inhibitor, has recently entered clinical trials. New analogues and derivatives have been synthesized from the S16020-2 compound. Preliminary data indicate that S30972-1, one of these S16020-2 derivatives, may exhibit a comparatively higher level of antitumor potency associated with an improved therapeutic index than does S16020-2. The antitumor activities of S16020-2 and S30972-1 were therefore characterized both in vitro and in vivo, with Adriamycin and etoposide chosen as reference compounds. The in vitro data show that S30972-1 is a topoisomerase II inhibitor, mediating its activity through an ATP-dependent mechanism such as S16020-2. The two olivacine derivatives exhibited similar activities in vitro at the levels of the global growth of six human cancer cell lines, of the induction of apoptosis, and of the G2 cell cycle phase arrest. The in vivo antitumor activity characterization included the use of two murine leukemia types (P388-LEU and L1210-LEU), two murine lymphoma-like models (P388-LYM and L1210-LYM), two mammary adenocarcinomas (MXT-HI and MXT-HS), and one melanoma (B16). The data show that S30972-1 is actually more efficient in vivo than S16020-2, a feature that may relate to the fact that S30972-1 is less toxic than S16020-2. The S30972-1 compound exhibited in vivo a level of antitumor activity that was also actually higher than that exhibited by Adriamycin and similar to that exhibited by etoposide.

Cellular resistance to the antitumor DNA topoisomerase II inhibitor S16020-2: importance of the N-[2(Dimethylamino)ethyl]carbamoyl side chain.

The new olivacine derivative S16020-2 (NSC-659687) is a DNA topoisomerase II inhibitor endowed with a remarkable antitumor activity against various experimental tumors. In vitro physicochemical properties of this compound, in particular its interaction with DNA and DNA topoisomerase II, were very similar to those of ellipticine derivatives, except for a strictly ATP-dependent mechanism of cleavable complex induction. From the Chinese hamster lung fibroblast cell line DC-3F, a subline resistant to S16020-2, named DC-3F/S16, was selected by adding stepwise increasing concentrations of the drug to the cell growth medium. Whereas DC-3F/9-OH-E cells, a DC-3F subline resistant to 9-hydroxy-ellipticine, are cross-resistant to S16020-2, DC-3F/S16 cells are only very weakly cross-resistant to ellipticine derivatives, indicating that, despite their structural similarity, these compounds may differ in their mechanisms of action. Uptake and efflux rates of S16020-2 were identical in the resistant and the sensitive cells. Topoisomerase IIalpha was expressed at the same level in both sensitive and resistant cells, whereas expression of the beta-enzyme was approximately 50% lower in the resistant cells. Sequencing of both alpha- and beta-isoform cDNAs revealed a point mutation that converts Arg(486) to a Gly in the alpha cDNA, whereas the beta cDNA was not modified. This amino acid substitution in a highly conserved sequence of the enzyme appears to be responsible for the resistance to S16020-2. Comparative analysis of the properties of the ellipticine and S16020-2-resistant cells suggests that S16020-2, which is a DNA intercalator, might also interact with this enzyme amino acid sequence through its side chain.

Mutations in three subdomains of the carboxy-terminal region of collagen type X account for most of the Schmid metaphyseal dysplasias.

We have used the polymerase chain reaction and single strand conformation polymorphism (SSCP) methods to analyse the COL10A1 gene, which encodes collagen type X, in DNA samples from patients with metaphyseal dysplasia type Schmid (SMCD) and other related forms of metaphyseal dysplasia. Five cases of SMCD were sporadic and three others were familial. Abnormal SSCP profiles were observed in six instances. In two families, the altered pattern segregated with the phenotype. The heterozygous mutations corresponded to a glycine substitution by glutamic acid at position 595 and to an asparagine substitution by lysine at position 617. In one sporadic case, the sequence studies demonstrated that the individual was heterozygous for a single base deletion (del T 1908) that produced a premature stop codon. Three additional mutations were single base substitutions that affected highly conserved residues at positions 597, 644 and 648. In two additional individuals with SMCD, in two patients with unclassifiable forms of metaphyseal dysplasia, and in one family with epiphyso-metaphyseal dysplasia, SSCP analysis detected neutral polymorphisms in the entire coding sequence of the gene but no mutations. Our results demonstrate that mutations in the carboxy-terminal region of collagen X are specific for the SMCD phenotype. Mutations appear to be clustered into three small subdomains: one of them is rich an aromatic residues, the second includes the putative N-linked oligosaccharide attachment site and the third contains mostly hydrophilic residues. The absence of clinical variability between patients carrying heterozygous single base substitutions or small deletions suggests that, in both instances, the mutant collagen chains either fail to be incorporated into stable trimers or disturb type X collagen assembly.

Non-collagenous protein screening in the human chondrodysplasias: link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin.

A gel-electrophoretic screening for link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin abnormalities was performed in fetuses, newborn infants, and children with various types of chondrodysplasia. Microdissected freeze-dried sections of upper tibial growth cartilage were extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea, the extracts were submitted to stepwise ion-exchange chromatography to separate the large proteoglycans (aggrecans) from the other components. The latter were analyzed by gel electrophoresis, electrotransferred onto nitrocellulose membranes, and reacted with specific antibodies. Control samples from individuals with apparently normal growth were analyzed in the same runs. Two link protein bands with abnormal electrophoretic migration were found in a sporadic case of spondylometaphyseal dysplasia, Kozlowski type. Three link protein bands with the same migration as in the control samples were found in thanatophoric dysplasia, homozygous achondroplasia, achondrogenesis type II, hypochondrogenesis, Goldblatt syndrome, Desbuquois dysplasia, pseudoachondroplasia, and diastrophic dysplasia. In several pathologic cases with normal electrophoretic pattern of the link proteins, small link protein fragments appeared after reduction. The gel electrophoretic pattern of COMP was studied in thanatophoric dysplasia, diastrophic dysplasia, homozygous achondroplasia, fibrochondrogenesis, hypochondrogenesis, Goldblatt syndrome, and Kniest dysplasia. In all these cases the pattern was the same as in the control samples. The main band of fibromodulin had a normal migration rate in fibrochondrogenesis, Desbuquois dysplasia, Kniest dysplasia, and pseudoachondroplasia. It was delayed in diastrophic dysplasia.

The epidermal growth factor-like domain of the large proteoglycans from articular cartilage (aggrecans). Estimate of content at different ages and in osteoarthritis.

Sequencing of cDNA clones has shown that the carboxy terminal domain of the core protein of large proteoglycans (aggrecans) from human cartilage contains an epidermal growth factor-like (EGF-like) domain which is alternatively spliced. In a previous study it was found that the domain of the translated protein can be recognized by polyclonal antibodies to mouse EGF. A competitive enzyme-linked immunoabsorbent (ELISA) assay has been developed to evaluate the EGF-like domain content of aggrecans at various ages and in osteoarthritis. Fetal aggrecans digested with protease free chondroitinase ABC were adsorbed on polyvinyl chloride microtiter plates followed by blocking with bovine serum albumin and goat serum. Mixtures of known amounts of protein of digested aggrecans and constant amounts of anti-mouse EGF antibodies were incubated and added to plates. The second antibody was peroxidase-conjugate F(ab')2. Fetal, newborn and child aggrecan proteins have a higher content of EGF-like domain than aggrecan proteins from cartilage of older humans. Three areas of cartilages from osteoarthritic joints were separated: cartilages with normal macroscopic appearance, erosion border cartilage and osteophytic cartilage. Values derived from these samples were compared with values derived from nonosteoarthritic aged humans. The content of aggrecans from osteoarthritic cartilage with normal macroscopic appearance was similar to or slightly lower than the latter. The aggrecans from osteophytes had a higher EGF-like domain content. The aggrecans from the erosion border had a variable content, close to noneroded cartilages, to osteophytes or in between the values obtained for noneroded cartilages and for osteophytes. Variations in the amount of newly synthesized aggrecans, in the proteolysis of the carboxy terminal domain of aggrecans and in the alternating splicing of the EGF-like domain might explain the results shown here.

Immunological detection of the EGF-like domain of the core proteins of large proteoglycans from human and baboon cartilage.

Recent data from the literature have shown that cDNA clones for the carboxyterminal domain of the core protein of large proteoglycan monomers from human cartilage contain an EGF-like domain, which appears to undergo alternative splicing. In the present study we have found that articular proteoglycans from human and baboon separated on agarose flat-bed gels and blotted onto nitrocellulose react with a rabbit antiserum to mouse EGF. In addition both forms of the proteoglycans (band I and band II) seen on these gels are reactive. Reactivity is seen with proteoglycans extracted from human articular cartilage of various ages (fetal, newborn, young and aged) and with proteoglycans extracted from cartilage of thanatophoric dysplasia and homozygous achondroplasia. Reactivity is dependent on prior digestion of the nitrocellulose blot with Chase ABC, suggesting masking of epitope by chondroitin sulfate. Reactivity of the EGF antiserum with cartilage proteoglycan core protein was also demonstrated in an ELISA system with core protein as coating antigen. The reactivity appears to reside in a tryptic peptide generated from Chase/keratanase digested core protein. The immunoreactive species migrates as a 68 KDa species on gradient gels. Immunological detection and quantitative analysis of the EGF-like domain could be useful for analysis of various proteoglycan samples.

Age-related changes in small proteoglycans of low buoyant density of human articular cartilage.

Proteoglycans extracted from articular cartilage of large joints of humans aged 4, 11, 70 and 75, were fractionated on associative density gradients. The top fraction (A3) was purified by ion-exchange chromatography and subsequent gel filtration on Sepharose CL 4B in 4 M GuCl, 0.5% Triton x 100. Proteoglycans from young cartilages yielded a narrow rapid migrating band on gel electrophoresis, had a Kav of 0.43 and 0.44 on Sepharose CL 4B, a glucosamine/galactosamine ratio of 0.11 and 0.12 and a glycoprotein core rich in aspartic acid and leucine with a Mr of about 47,000. Proteoglycans from old cartilages gave a wider and slower migrating band on gel electrophoresis, had a wide peak with a Kav of 0.38 and 0.40 on Sepharose CL 4B, a glucosamine/galactosamine ratio of 5.1 and 3.2, a glycoprotein core rich in glutamic acid and glycine, and with a Mr of about 170,000-180,000. Analysis using monoclonal antibodies detected epitopes of keratarn sulfate and of hyaluronic acid binding region in the fractions from old but not in those from young cartilages. Small proteoglycans not derived from the large monomers are the major component of low-buoyant-density fractions of proteoglycans from young cartilages. Fragments of large monomers containing keratan sulfate and hyaluronic acid binding region are the major component of similar fractions from old cartilage.

Proteoglycan electrophoresis on horizontal submerged polyacrylamide-agarose gels.

A method of proteoglycan electrophoresis on submerged horizontal polyacrylamide-agarose gels is described. Several preparations of purified proteoglycans extracted from fetal and young baboon articular cartilage and from mandibular dog-fish cartilage were analyzed. Discrete bands corresponding to proteoglycan monomers of different size were obtained. The results were similar to those obtained using the more tedious electrophoretic separation on cylindrical gel rods.

The protein core of the largest proteoglycan monomer of articular cartilage. Gel electrophoretic pattern and tryptophanyl peptide bond cleavage.

The largest proteoglycan monomer of baboon (Papio papio) articular cartilage was isolated and the protein rich core was obtained after chondroitinase AC II and keratanase digestions. On SDS-PAGE the core yielded a single band with apparent Mr of 290,000. Tryptophanyl peptide bond cleavage of the core with N-chlorosuccinimide/urea gave 4 peptides with apparent Mr of 105,000, 66,000, 62,000 and 56,000

Noncollagenous proteins in cartilage of normal subjects and patients with degenerative joint disease. A gel electrophoretic study.

Normal articular cartilage from subjects of various ages and cartilage from patients with degenerative joint disease were extracted with 4M guanidinium chloride. After dialysis against 8M urea pH 6.8, a 0.2M NaCl fraction was obtained by ion exchange chromatography on DE-52 in 8M urea. This fraction was concentrated, reduced, and analyzed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (7% gels). Six major noncollagenous protein bands (P1-P6) were found; 2 were identified as the link proteins. The approximate molecular weights of P1-P6 were: 87,000, 64,000, 56,000, 46,000, 41,000, and 27,000. A similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of P1-P6 was found in young baboons, in normal young and aged humans, and in patients with degenerative joint disease. Peaks corresponding to extracted collagen were decreased in older patients and increased in patients with degenerative joint disease, even those of advanced age.

Link-proteins and non-collagenous proteins from normal and chondrodysplastic cartilages.

Baboon and human articular and growth cartilage was extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea pH 6.8 the proteins were separated from proteoglycans by ion-exchange chromatography. The concentrated and reduced protein fractions was analyzed by SDS-PAGE. Bands corresponding to collagen and to 6 major non-collegenous proteins were found. Two of the latter were identified with the link-proteins. By using small columns and microconcentration procedures, a gel-electrophoretic analysis of link-proteins extracted from small pieces of cartilage was performed and ten cases of osteochondrodysplasias were studied. No abnormalities were detected in the following syndromes: achondroplasia, diastrophic dwarfism, thanatophoric dwarfism, Jeune disease, spondyloepiphyseal dysplasia congenita, Kozlowski syndrome, osteogenesis imperfecta, polyepiphyseal dysplasia with diabetes mellitus.