N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage.

Bovine articular cartilage was cultured both in the presence and in the absence of human recombinant interleukin-1 alpha (IL-1) (100 units/ml). Addition of this cytokine stimulated matrix degradation approx. 3-fold. This increased degradation permitted characterization of the large chondroitin sulphate proteoglycan (aggrecan) fragments accumulating in the media. When compared with controls, the proteoglycans isolated from the medium of cultures treated with IL-1 exhibited a decrease in the Kav. (control 0.25; IL-1-treated 0.37), determined by Sepharose CL-2B chromatography. This decrease in proteoglycan size was accompanied by a decreased ability of these monomers to associate with hyaluronic acid. Thus only 20% of the proteoglycans isolated from the medium of IL-1-treated cultures, compared with 39% for control cultures, had the capacity to form high-M(r) aggregates with hyaluronic acid. SDS/PAGE analysis of the proteoglycans from the media of IL-1-treated cultures demonstrated several large proteoglycan protein-core bands (M(r) 144,000-380,000). The protein-core bands with M(r) 144,000-266,000 exhibited a significantly decreased reactivity with monoclonal antibody 1-C-6 (specific for domains G1 and G2). The N-terminal amino acid sequence of four of these protein-core bands (M(r) 144,000, 173,000, 214,000 and 266,000) yielded sequences LGQRPPV-Y-PQLF(E), AGEGP(S)GILEL-GAP(S)-AP(D)M, GLG-VEL-LPGE and (A)RGSVIL-AKPDFEV-P-A. A comparison of these N-terminal amino acid sequences with the published proteoglycan sequence for bovine nasal cartilage [Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259], rat chondrosarcoma [Doege, Sasaki, Horigan, Hassell & Yamada (1987) J. Biol. Chem. 262, 17757-17769] and human articular cartilage [Doege, Sasaki, Kimura & Yamada (1991) J. Biol. Chem. 266, 894-902] permitted assignment of their relative positions on the core protein. Furthermore, on the basis of this similarity to published sequence, putative sites of enzymic cleavage were constructed. These theoretical cleavage sites revealed a glutamic acid residue in the P1 position and an uncharged polar or non-polar residue in the P1' position.

Primary structure and function of stromelysin/transin in cartilage matrix turnover.

Stromelysin/Transin is a member of the matrix metalloprotease gene family. This metalloprotease is synthesized as a preproenzyme with a predicted size of 53,977 Da including a 17 amino acid signal peptide. Prostromelysin is secreted from normal and transformed cells in two forms with apparent molecular masses on NaDodSO4 gels of 60 and 58-kDa. The minor 60-kDa species contains N-linked oligosaccharide(s). Stromelysin consists of three domains the amino terminal propeptide(s) domain contains the tribasic amino acid sequence RRK which is important in the proteolytic activation of this zymogen by trypsin-like serine proteases. The second domain consists of the catalytic domain which contains the zinc binding site. The carboxyl-terminal hemopexin domain has no known function and can be removed without a loss of enzymatic activity. Stromelysin has a broad range of substrate specificity including proteoglycans, casein, fibronectin, laminin, native type IV and IX collagen and gelatin but not type I collagen. In the presence of trypsin or plasmin, catalytic amounts of this enzyme can also fully activate interstitial fibroblast collagenase. We have developed a panel of monoclonal antibodies against stromelysin which will be useful for the tissue localization of the various species of this enzyme in tissues. In addition, we have demonstrated that either human rIL-1 (alpha) or rTNF (alpha) can stimulate the expression of this enzyme in cultured bovine articular cartilage at least 10-fold. Based on western blot analysis, the zymogen form of the enzyme was the major enzyme species detected in either the media or cartilage matrix compartments of cytokine treated cultures.

A synthetic mycobacterial heat shock peptide prevents adjuvant arthritis but not proteoglycan-induced synovitis in the rat.

Intradermal immunization of female Lewis rats with 100 micrograms of a nine-amino acid synthetic peptide corresponding to the arthritic T cell-reactive epitope of mycobacterial heat shock protein, three weeks prior to induction of adjuvant arthritis, produced inhibition of day 16 ankle swelling and histologic score. Intraarticular injection of 10 micrograms of bovine articular cartilage proteoglycan monomer emulsified in heavy mineral oil into normal Lewis rat stifle joints produced several hallmarks of chronic synovitis at day 16. Pre-treatment with nonapeptide did not inhibit proteoglycan-induced synovitis. These results indicate that tolerance to the critical epitope of heat shock protein does not abrogate the ability of proteoglycan to induce synovitis in rats.

A heparan sulfate-containing fraction of bone marrow stroma induces maturation of HL-60 cells in vitro.

Constituents of the bone marrow microenvironment have the capacity to influence both normal and malignant hematopoietic cell behavior. For example, HL-60 human promyelocytic leukemia cells in vitro display a more mature phenotype when grown on a bone marrow stroma-derived matrix. To elucidate which component(s) of the stromal matrix is capable of modulating HL-60 cell phenotype, matrices were treated with a variety of chemicals and enzymes prior to being used in the differentiation assay. Treatment of matrices with collagenase, pronase, chondroitinase, or chloroform:methanol:ether could not abolish the differentiation-promoting activity of bone marrow stroma. In contrast, the activity was destroyed by alkali treatment (0.5 M NaOH for 18 h) or heparinase/heparitinase enzymes. Heparin added to cultures increased maturation of HL-60 cells as determined by esterase production, Fc rosette formation, and morphological appearance. Other stromal components such as laminin, fibronectin, collagen I, collagen IV, or chondroitin sulfate did not alter the HL-60 leukemia cell phenotype. Stroma-derived matrix material which labeled with [35S]sulfate and eluted on a DEAE ion-exchange column as a high ionic fraction in 1.5 M LiCl and 7.5% sodium dodecyl sulfate contained the active fraction. A heparan sulfate proteoglycan component isolated by polyacrylamide-agarose gel electrophoresis induced a more mature HL-60 phenotype, and digestion with heparinase/heparitinase in the presence of protease inhibitors abrogated the effects on HL-60 phenotype. We conclude that a heparan sulfate-associated fraction of the bone marrow matrix plays a key role in the regulation of leukemic cell maturation.

Bone marrow matrix modulation of HL-60 phenotype.

The initiation and maintenance of cellular differentiation for a variety of cell types has been shown to be influenced by the microenvironment. To investigate the influence of bone marrow stroma on leukemic cell differentiation, HL-60 human promyelocytic leukemia cells were grown in the presence of Triton-treated extracellular matrix derived from normal human bone marrow stromal cells. This bone marrow matrix microenvironment had a dramatic impact on the phenotypic expression of this malignant line. HL-60 cellular proliferation, morphology, nonspecific esterase activity, formation of Fc rosettes, and sensitivity to induction by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were all influenced by the presence of matrix molecules. In contrast, stromal cell-conditioned media did not alter HL-60 phenotype. Thus, HL-60 cells appear to retain responsiveness to a human bone marrow stromal cell-derived matrix despite their ability to grow autonomously. Studies of the interaction of leukemic cells and marrow stroma in vitro may provide important information concerning the regulation of leukemic cell behavior.

Modulation of cell shedding and glycosaminoglycan synthesis of human malignant keratinocytes by all-trans-retinoic acid and hydrocortisone in vitro.

Physiologic concentrations (5 X 10(-8) M) of all-trans-retinoic acid (RA) caused a 2- to 3-fold increase in the rate of cell desquamation of a malignant keratinocyte line (SqCC/Y1) grown in serum-free medium. Measurement of the incorporation of [35S]sulfate and [3H]glucosamine into cetylpyridinium chloride-precipitable glycosaminoglycans (GAGS) demonstrated that RA treatment did not alter total GAG production. In addition, compartmental distribution was not affected by RA, with 50-70% of GAGS being recovered from the medium, 25% from the pericellular matrix, and the remainder from the cells. Relatively small amounts of GAGS were associated with shed cells in RA-treated cultures, presumably reflecting a relatively short association of these cells with the monolayer before desquamation. Chondroitin sulfate (Ch-S), heparin/heparan sulfate (Hep-S), and hyaluronic acid (HA) were the GAG species identified in SqCC/Y1 cultures by gel-exclusion chromatography. RA reduced the relative amount of HA in the trypsin-sensitive pericellular compartment by 50%. Since the proportions of Ch-S and Hep-S were not affected by RA, the findings suggest that the altered ratio of HA to sulfated GAGS in this fraction may contribute to the increased cell desquamation. Hydrocortisone (10(-6) M) reversed the effect of RA on cell shedding, and increased the proportion of pericellular HA relative to that found in cultures exposed to RA alone. These findings support the concept that the relative proportion of HA to sulfated GAGS may be important in the intercellular cohesion of keratinocytes. In addition, the relative decrease in HA and the predominance of Ch-S over Hep-S in SqCC/Y1 cultures differed from results reported with normal keratinocytes, indicating that this property may be associated with the malignant phenotype.

Interference with tumor cell-induced degradation of endothelial matrix on the antimetastatic action of nafazatrom.

The antithrombotic compound nafazatrom was evaluated in several in vivo and in vitro assays to elucidate the mechanism of its antimetastatic activity. C57BL/6 mice bearing B16 amelanotic subcutaneous tumors treated with 100 mg nafazatrom/kg/day exhibited a sixfold reduction in metastatic pulmonary lesions compared to lesion numbers in controls. The reduction in metastatic lesions was not accompanied by changes in primary tumor growth, and up to 1 microgram nafazatrom/ml did not inhibit tumor cell proliferation in vitro. Treatment of C57BL/6 mice with nafazatrom prior to iv inoculation of tumor cells failed to inhibit lung colony formation. In vitro exposure of exponentially growing B16 amelanotic cells to nafazatrom (1 microgram/ml for 72 hr) in culture did not change their ability to adhere to endothelial cell monolayers. B16 amelanotic cells degraded the matrix material of bovine endothelial cell monolayers; a heparin sulfate proteoglycan appeared to be the predominant matrix component released by these tumor cells, as judged by resistance to chondroitin ABC lyase and sensitivity to heparitinase and pronase degradation. Nafazatrom (1 microgram/ml for 72 hr) inhibited the solubilization of matrix components by approximately 60%. Tumor cell degradation of matrix components is an important event in the pathogenesis of metastasis. Thus the interference with this process appears to provide an explanation for the inhibition of malignant cell dissemination in vivo by nafazatrom.

Glycosaminoglycan production and distribution in cloned B16 murine melanoma cell lines exhibiting different lung colony-forming efficiencies.

Three cloned B16 murine melanoma cell lines were characterized with respect to their ability to a) detach from a plastic tissue culture substratum, b) form experimental pulmonary metastases, and c) produce and process glycosaminoglycans (GAGS). All three of the cell lines formed pulmonary metastases to different extents. Chondroitin sulfate and heparin-heparan sulfate were the major GAGS produced by all of the clones. Although the major compositional analyses of the cell lines were similar, some differences were apparent. The clone never selected for lung colony-forming efficiency and with the lowest metastatic potential (B16YL1) exhibited a reduced cell-associated glycosaminoglycan (GAG) matrix, shed the highest proportion of labeled GAGS into the medium, and was the most easily removed from the tissue culture substratum. In contrast, the other two cell lines (B16YM1 and B16YH1), derived from cultures selected for lung colony-forming efficiency, exhibited GAG-enriched cellular coats and were more resistant to EDTA-induced detachment from the plastic substratum. Both the B16YL1 and B16YM1 clones, which exhibited the lowest lung colony-forming efficiencies, shed an unsulfated chondroitin species into the extracellular fraction that was not evident in the most metastatic clone B16YH1. Despite these differences, cellular GAG matrix development and detachment from the plastic substratum showed only a partial correlation with lung colony-forming efficiency, presumably expressing the complexity of this biological phenomenon. Nonetheless, the findings suggest that GAGS may play an important role at several steps along the cascade of events leading to the successful dissemination of malignant cells.

Reversible effects of retinoic acid on glycosaminoglycan synthesis during differentiation of HL-60 leukemia cells.

Glycosaminoglycans (GAGs) play an important role in cell-cell and cell-substratum interactions, and undergo specific changes during neutrophil development. Previous studies (Luikart, S.D., Maniglia, C. A., and Sartorelli, A. C. Cancer Res., 44: 2907-2912, 1984) have shown that both dimethyl sulfoxide and 4-beta-phorbol-12-beta-myristate-13-alpha-acetate decreased GAG production by a hypoxanthine-guanine phosphoribosyl transferase-deficient clone of HL-60 promyelocytic leukemia cells prior to the appearance of a mature myeloid or monocytoid phenotype. To expand these investigations further, GAGs were analyzed by cetylpyridinium chloride precipitation and DEAE-Sephacel ion-exchange chromatography after labeling of parental HL-60 cultures with [35S]sulfate and D-[3H]glucosamine for 6 h, following treatment with 1 microM all-trans retinoic acid (RA). Chondroitin sulfate represented the major GAG species produced, although endo-beta-galactosidase-sensitive undersulfated macromolecules which possibly might be keratan sulfate, were also identified. GAG production decreased over a time period of 144 h in culture. RA treatment reduced the amount of radiolabeled cell-associated GAGs by 50% after 48, 96, and 144 h of exposure. In contrast, commitment to myelocytic maturation of the majority (i.e., approximately 60%) of the cells occurred between 72 and 96 h of RA treatment. Concurrently with the appearance of mature granulocytic cells, two-thirds of the radiolabeled GAGs were recovered from the medium, compared to one-third in untreated cultures, a phenomenon that resulted in an overall alteration in the distribution of GAGs. When RA was removed by washing after either 48 h (i.e., precommitment to differentiation) or 96 h (i.e., postcommitment to differentiation), a 1.5- to 3.5-fold increase in GAG production was noted 48 h later; this increase was unrelated to the medium change or to alterations in cell cycle distribution. The amounts of endo-beta-galactosidase-sensitive macromolecules were unaltered. Thus, although 1 microM RA inhibited the synthesis of chondroitin sulfate by HL-60 leukemia cells, this inhibition was reversible by removal of the drug and appeared to be unrelated to the commitment to myelocytic maturation.

Glycosaminoglycan synthesis during differentiation of HL60/HGPRT-leukemia cells induced by dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate.

Glycosaminoglycans (GAGs) are polyanionic components of the cell surface that have been shown to play an important role in the cellular differentiation of many embryonic systems, as well as in the maturation of the developing human leukocyte. For this reason, the production of GAGs during the induction of myelocytic and macrophage-like differentiation of the human promyelocytic leukemia cloned cell line HL60/HGPRT- was studied. The major GAG component of HL60/HGPRT- was chondroitin 4-sulfate. This molecule has been reported to be the major GAG constituent of normal granulocytes and myeloid leukemia cells as well. Treatment of HL60/HGPRT- cultures with dimethyl sulfoxide, which initiates myeloid maturation, or 12-O-tetradecanoylphorbol-13- acetate, which induces the formation of macrophage-like cells, resulted in a 43 and 34% reduction, respectively, of the incorporation of [35S]sulfate into total GAGs at a time when greater than 80% of the cells were morphologically immature and were unable to reduce nitroblue tetrazolium dye. This reduction occurred primarily in GAGs associated with the cells, which decreased by 75% after exposure to these agents. Therefore, the distribution of GAGs between the cellular and medium compartments was altered by exposure to inducers. A phorbol ester with no capacity to induce differentiation, 4 alpha-phorbol-12, 13-didecanoate, elicited a reproducible but less dramatic decrease in cell-associated GAGs. The reduction in [35S]-sulfate incorporation into GAGs, therefore, may be an important step in leukocyte differentiation and may provide a useful biochemical probe of the maturation process.

N-(substituted-phenyl)-D-glycopyranosylamines and their O-acetyl derivatives as potential modifiers of the formation of glycosaminoglycans.

D-Arabinosyl, D-ribosyl, D-glucosyl, D-galactosyl, D-mannosyl, and L-rhamnosyl N-glycosides of p-aminobenzoic acid and their O-acetyl derivatives have been synthesized, and their ability to (a) inhibit the replication of cultured B16 melanoma cells and (b) modify the synthesis of glycosaminoglycans by these neoplastic cells have been evaluated. The most cytotoxic compound of the series was N-(p-carboxyphenyl)-2,3,4-tri-O-acetyl-D-arabinopyranosylamine (8), which produced 50% inhibition of cellular proliferation at a concentration of 4 microM; a number of other compounds were relatively cytotoxic, causing 50% inhibition of cell replication at levels of 18 to 49 microM. These effects were not due to modification of glycosaminoglycan biosynthesis, since these compounds were ineffective as inhibitors or initiators of the formation of these macromolecules.

Influence of collagen substrata on glycosaminoglycan production by B16 melanoma cells.

A cloned metastatic murine melanoma cell line exhibited similar growth characteristics when propagated on either type I collagen, type IV collagen, or plastic. However, cells grown on both types of collagen exhibited an altered cellular morphology and on type IV collagen only, an increased substrate adhesiveness, relative to those maintained on a plastic substratum. Incorporation of [3H]glucosamine and [35S]sulfate into glycosaminoglycans (GAGs) of cells grown on collagen substrates was 20% and 40% less, respectively, than cells grown on plastic, whereas degradation of cell-associated [35S]sulfate-labeled GAGs was similar in cells grown on collagen or plastic. Although the composition of GAGs was similar in all cultures, consisting of approximately 60% chondroitin and 40% heparin or heparan sulfate, the degree of sulfation of the heparin or heparan sulfate molecules was markedly decreased in cultures grown on collagen. The results indicate that the composition of the extracellular matrix influences the biological behavior of B16 melanoma cells, in part by altering the amount and nature of the GAG molecules produced.

Synthesis of N-(Substituted-phenyl)-D-xylopyranosylamines as potential modifiers of the formation of glycosaminoglycans.

N-(Substituted-phenyl)-D-xylopyranosylamines and their O-peracetyl derivatives have been synthesized and tested for their ability (a) to inhibit the replication of cultured B16 melanoma cells and (b) to modify the synthesis of glycosaminoglycans by these neoplastic cells. The most cytotoxic compound synthesized was N-(p-methoxyphenyl)-D-xylopyranosylamine (6), which produced 50% inhibition of cellular proliferation at a concentration of 2 microM; a number of other compounds were relatively cytotoxic, causing 50% inhibition of cell replication at levels of 12 to 25 microM. Several of the synthesized xylosides appeared to be capable of serving as artificial initiators of glycosaminoglycan synthesis, with the most active agents causing approximately 2- to 4-fold increases in the incorporation of [35S]sulfate into the glycosaminoglycans of B16 melanoma cells excreted into the culture medium.

The effect of 2,6-bis(diethanolamino)-4-piperidinopyrimido [5,4-d]-pyrimidine (RA233) on growth, metastases and lung colony formation of B16 melanoma.

The dissemination of malignant cells from a primary tumor to distant host sites appears to be influenced by blood platelets of the hemostatic system. Under conditions that did not inhibit primary tumor growth, RA233 decreased both the incidence and frequency of spontaneous lung metastases by 66% and 69%, respectively. Although RA 233 effectively inhibited the formation of spontaneous metastases, oral administration of RA233 prior to and after the intravenous inoculation of 1 X 10(5) B16F10 murine melanoma cells failed to inhibit subsequent lung colony formation. These findings indicate that RA233 has antimetasttic activity, and that inhibition of platelet aggregation is not the sole determinant of this action.

Solubilization of murine melanoma xylosyltransferase and galactosyltransferase activities and their inactivation by dialdehyde nucleosides.

The enzymes involved in the initial steps in the biosynthesis of glycosaminoglycans were examined in the murine B16 melanoma. Approximately 60% of the melanoma xylosyltransferase activity and nearly all of the galactosyltransferase activity were membrane-bound; these enzymatic activities were solubilized by treatment with Nonidet P-40 and potassium chloride, and the Michaelis constants for the substrates and acceptor molecules were determined and found to be comparable to those reported for these enzymes from the chick embryo and a rat chondrosarcoma. Dialdehyde nucleosides, which have been reported to alter the activity of several enzymes involved in nucleic acid synthesis, inhibited both xylosyltransferase and galactosyltransferase activities, with galactosyltransferase being considerably less sensitive than xylosyltransferase. The inhibition of xylosyltransferase by dialdehyde nucleosides was irreversible, with no apparent specificity for the base moiety of the dialdehyde nucleosides.

Two types of adriamycin inhibition of a homologous RNA synthesizing system from L1210 cells.

Purified L1210 RNA polymerase II was utilized to study the effect of adriamycin on the kinetics of RNA synthesis. In this study it was found that the adriamycin induced two types of inhibition of RNA synthesis with native DNA as a template. At low drug/DNA-P molar ratios, adriamycin functions as an uncompetitive inhibitor, whereas at high drug/DNA-P molar ratios adriamycin inhibits RNA synthesis competitively. However, when denatured DNA was utilized as template, only competitive inhibition was observed. It is proposed that two modes of inhibition of native DNA-dependent RNA synthesis represents two types of adriamycin-DNA complexes.