Paul A. Aeed

Characterization of the O-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)

P-selectin glypoprotein ligand-1, PSGL-1, a specific ligand for P-, E-, and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin and platelet P-selectin- or E-selectin receptor globulin-agarose chromatography. The O-linked oligosaccharides on the ligand were released by mild alkaline sodium borohydride treatment and analyzed by a combination of ion-exchange, size exclusion, lectin, and paper chromatography, together with specific exoglycosidase treatments and chemical modifications. Approximately 91% of the radioactivity released from PSGL-1 was recovered in five O-linked glycans: GalNAc (approximately 4% of the total structures), Galβ, 3GalNAc (36%), and Galβ, 3GalNAc substituted with one (45%), two (6 %), or three (3%) N-acetyllactosamine repeat units. None of these structures contained fucose, and the majority were substituted with at least one sialic acid. The N-acetyllactosmine-containing structures appeared to be core 2. The remaining 9% of the radioactivity recovered in O-linked oligosaccharides from PSGL-1, eluted in two peaks at 11.8 and 10.2 glucose units, on size-exclusion chromatography. Results from lectin chromatography and chemical and enzymatic degradation experiments suggest that the major portion of the radioactivity in these peaks is associated with sialylated N-acetyllactosamine-type oligosaccharides, substituted with fucose at the penultimate residue in the nonreducing end. Since both sialic acid and fucose reportedly are crucial requirements for selectin binding, these results suggest that only a minor portion, approximately 4.5%, of the O-linked oligosaccharides on PSGL-1 are involved in the interaction with the selectins.

Characterization of the oligosaccharide structures on bee venom phospholipase A2

The N-linked oligosaccharide structures on bee venom phospholipase A2 were investigated. The oligosaccharides on purified phospholipase A2 were released by hydrazinolysis and labeled in vitro by reduction with NaB3H4. Following purification, the labeled oligosaccharides were characterized by size exclusion chromatography in combination with digestion with specific glycosidases. Linkage positions were determined by methylation analysis. Four types of structures were identified on the molecule, all of which were of truncated high-mannose type and none of which contained any alpha-(1-->2)-linked mannose residues. The majority of the structures were Man3 oligosaccharides with (43%) or without (38%) a fucose residue linked alpha-(1-->6) to the reducing N-acetylglucosamine. The remaining 19% of the oligosaccharides on the molecule were identified as a Man5 oligosaccharide without core fucose (9.6%) and a core-fucosylated Man4 structure (9.2%).

Tumor progression- and metastasis-associated proteins identified using a model of locally recurrent rat mammary adenocarcinomas

A recently established model for local breast cancer recurrence using the 13762NF rat mammary adenocarcinoma was used to evaluate biologic and biochemical properties related to clinical outcome for this class of tumors. Sublines isolated from local tumor regrowths following surgical resection differed from each other and from the ‘parental’ cell lines for multiple phenotypes, including metastatic propensity. Local recurrence- and primary tumor-derived sublines were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), lectin binding to electrophoretically separated proteins, and lactoperoxidase-catalyzed cell surface iodination; and differential protein patterns were compared to tumor progression and metastatic potential. 2D-PAGE revealed several quantitatively different spots which correlated with lung colonization potential. In particular, quantities of an apparently unique, non-cell-surface protein, P50.9 (Mr ∼ 50 900, pI ∼ 7·3) correlated inversely with metastatic propensity, suggesting that it may be associated with, among other possibilities, the negative regulation of the metastatic phenotype. P50.9 was unrelated to four similarly sized metastasis-associated proteins—tumor autocrine motility factor; the rat analog of tumor suppressor, p53; rat cytokeratin 14 or procathepsin D—as determined by amino acid analysis. A major wheat germ agglutinin binding sialoglycoprotein, gp93 (Mr ∼ 93 000), was present in smaller amounts as cells were passaged in vivo and re-established as in vitro cultures [MTF7 > ‘primary’ tumor-derived lines (scl, sc3) ≫ local recurrence-derived lines (LR1, LRla, LR3, LR4, LR5, LR6)]. Besides cell surface glycoprotein losses, two of six local recurrence-derived sublines expressed a wheat germ agglutinin-binding sialoglycoprotein, gp110 (Mr ∼ 110000), previously undetected on any of the other cell lines including the parental populations. gp110 was found in LR3 and LR6 which were relatively highly metastatic; however, correlation with metastatic potential failed because gp110 was not present on the metastatic parental cell line, MTF7. These results demonstrate specific quantitative and qualitative protein differences associated with the selection of locally recurrent mammary tumors.

Neutrophil-mediated transfer of polar substances from liposomes to mammary tumor cells in vitro

Abstract Little accumulation of fluid-phase markers, lucifer yellow CH (LY) and [ 14 C] sucrose, was observed in 13762NF rat mammary adenocarcinoma clone MTF7, when a cell monolayer was incubated at 37°C for 60 min with reverse-phase evaporation vesicles (REVs) containing the markers. However, time-dependent accumulation was observed when the REVs were preincubated with polymorphonuclear neutrophils (PMNs) and added to the tumor cell layer. In contrast, accumulation of liposomal lipid marker, [ 3 H]dipalmitoylphosphatidylcholine (DPPC), was independent of PMNs and approximately 10 times faster than those of the fluid-phase markers. The PMN-mediated transfer of LY and sucrose from REVs to MTF7 cells is attributed to PMN binding to the tumor cells and possibly high concentrations of the marker molecules in the unstirred layer of the tumor cells. The latter could have resulted from continuous phagocytosis and exocytosis of REV contents by PMNs. DPPC transfer must be spontaneous and could be simply collision-mediated. Significance of these in vitro observations is related to the role of PMNs in metastasis in vivo and the potential for drug delivery targeted to metastatic cells.

Experimental model for locally recurring mammary tumors. Development, morphology, karyotype, growth kinetics, and experimental metastatic potential

A rat model was established for evaluating the biology of locally recurring mammary tumors after surgical resection of the primary tumor. Eight distinct cell lines were independently derived from primary tumors and local recurrences after surgical removal of 13762NF rat mammary adenocarcinoma clone MTF7(T20). In vivo tumor doubling times between the “parental” MTF7(T20) cell line, primary tumor‐derived cell lines sc1 and sc3, and the local recurrence (LR) sublines varied after the inoculation of 106 tumor cells into the mammary fat pad of female Fischer 344 rats. Doubling times were shorter for LR3, and LR4, LR5, and LR6 than their primaries sc3 and MTF7(T20), respectively, and longer for LR1 and LR1a than their primary tumor sc1. The LR sublines varied considerably for their experimental metastatic potentials. Both increases and decreases in metastatic potential were seen compared to MTF7(T20), sc1, and sc3. Karyotype analysis by G‐banding revealed the presence in the LR sublines of several marker chromosomes, previously identified in MTF7 at tissue cultures 11 and 35. Two new chromosome markers were identified: M54, shared by MTF7(T20), sc1, LR4, LR5 and LR6, and M55, shared by MTF7(T20), sc1, LR1, sc3, LR3, LR4, and LR6. These data indicate that local tumor regrowth after surgical excision of the primary tumor in this model most likely selects the growth of tumor cell subpopulations already present within the primary tumor. Differences in growth kinetics, karyotype, and metastatic potential between the parental MTF7(T20), primary tumors sc1 and sc3, and their LR sublines may reflect in vivo influences on the phenotypic diversity generated during the development of local mammary tumor recurrences after surgical treatment of the primary tumor.