An efficient assay for dolichyl phosphate-mannose: protein O-mannosyltransferase.

A novel method for quantifying the reaction product from dolichyl phosphoryl mannose:polypeptide mannosyltransferase (protein mannosyl transferase; PMT), was developed. The assay quantifies the amount of radioactivity incorporated into the acceptor peptide YNPTSV from dolichyl phosphoryl [3H]mannose (Dol-P-Man). A novel delivery system, large unilamellar vesicles (LUV), composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), is used to keep the poorly soluble donor substrate, Dol-P-Man, in solution. The use of LUV allows generation of truly reproducible data and, as an additional benefit, also results in a more than 10 times increase in transfer efficiency. In contrast to the solvent extraction procedures commonly used in previously described PMT assays, the assay reaction product is separated from the radioactive donor substrate on C(18) cartridges. The use of C(18) cartridges allows generation of reproducible data with a low, consistent background and also produces a significant reduction in the time and labor needed for the product workup. In a reaction mixture consisting of 100 microg POPC LUV, 9 x 10(5)cpm (approximately 15 pmol) Dol-P-Man, 100 nmol YNPTSV, and aproximately 4 microg of crude yeast microsomal extract, time-dependent formation of glycosylated product obeys Michaelis-Menten-type kinetics throughout the course of the reaction-until exhaustion of the donor substrate. The linear initial rates of the reaction allowed calculation of an apparent K(m) of 1mM, for the acceptor peptide YNPTSV. Variations in detergent concentration in the assay influence transfer efficiency, possibly through interference with the LUV-based donor substrate delivery system. Hence detergent concentrations should be kept constant.

Partial characterization of the N-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-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-agarose and P- or E-selectin-agarose chromatography. N-linked oligosaccharides were released from the purified, denatured ligand molecule by peptide: N-glycosidase F treatment and, following separation by Sephacryl S-200 chromatography, partially characterized using lectin, ion-exchange and size-exclusion chromatography in combination with glycosidase digestions. The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated, multiantennary complex type structures with extended, poly-N-acetyllactosamine containing outer chains. A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans, in addition to the O-glycans on PSGL-1, may be involved in selectin binding.

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

P-selectin glycoprotein 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), Galp, 3GalNAc (36%), and Galbeta, 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.

The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins.

P- and E-selectins belong to a family of Ca(2+)-dependent lectins and function as receptors for myeloid leukocytes. We have described a panel of monoclonal antibodies which recognize a sialoglycoprotein from human neutrophils and HL-60 promyelocytic cells and inhibit adhesion of these cells to P-selectin. In this study, we show that the E-selectin receptor-globulin (E-selectin Rg) affinity chromatography can isolate specifically only one glycoprotein from [3H]glucosamine-labeled HL-60 cells in a Ca(2+)-dependent manner. This protein has a molecular mass of approximately 120 kDa under reducing conditions, which appears to be identical with the previously characterized glycoprotein ligand for P-selectin. The molecule can be cross-depleted by and cross-bound to the E- and P-selectin columns. The chromatographic profile of desialylated O-linked carbohydrates from molecules purified by P- and E-selectin affinity chromatography are identical. Both have five structures at 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units. PL5 monoclonal antibody to the P-selectin sialoglycoprotein ligand, E-selectin Rg, and antiserum to P-selectin glycoprotein ligand-1 (PSGL-1) all recognize the purified P-selectin ligand on ligand blots and immunoblots. Furthermore, PL5 monoclonal antibody blocks adhesion of HL-60 cells and human neutrophils to E-selectin Rg. Taken together, our results demonstrate that the P- and E-selectin ligand defined in this study is PSGL-1 and suggest that this molecule is an important leukocyte ligand for both P- and E-selectins.

Evidence for paracrine regulation of experimental metastasis in 13762NF rat mammary adenocarcinoma cell clones.

The ability of tumor cell lines to form experimental pulmonary metastases is determined in part by characteristics which are stable over many cell generations; in part by characteristics that are acquired by adaptation or phenotypic instability; but also in part by characteristics which may change over less than one cell generation. This study was designed to examine the hypothesis that tumor cells secrete and respond to paracrine factors which can reversibly modulate metastasis. The number of experimental lung metastases increased for 13762NF rat mammary adenocarcinoma cell clones MTF7 and MTLn3 as they approach 100% confluence. This observation corresponded to increased attachment to bovine brain capillary and bovine corneal endothelial monolayers and to ability of tumor cells to invade reconstituted basement membrane barriers in the Membrane Invasion Culture System (MICS), but did not correspond to cell cycle distribution, susceptibility to NK or PMN cell killing or average cell size/Coulter volume. While changing confluence did not qualitatively alter metastatic potential, modification of metastasis in a quantitative manner suggested that some properties pertinent to metastasis are transient and manipulatable. Tumor cell-conditioned medium (CM) collected from donor cells grown to defined levels of confluence when placed onto recipient cells reversibly raised or lowered metastatic potential depending upon the medium source and confluence of the recipient cells. CM from 20% confluent donor cultures reduced recipient cell metastatic potential. In contrast CM from 100% confluent cultures increased metastatic potential of subconfluent cells. Replacement with fresh unconditioned medium or leaving the medium unchanged did not alter experimental metastasis. These data suggest that metastasis involves steps which may be influenced by paracrine factors elaborated by tumor cells.

Glycosylation of recombinant prorenin in insect cells: the insect cell line Sf9 does not express the mannose 6-phosphate recognition signal.

Sf9 cells infected with a recombinant baculovirus containing the gene for human prorenin were cultured in the presence of [3H]mannose. In vivo labeled prorenin was isolated by immunoprecipitation from the culture medium and digested with Pronase. The oligosaccharide structures on the resulting glycopeptides were analyzed by a combination of lectin, ion-exchange, paper, and high-pressure liquid chromatography. Of the N-linked oligosaccharides isolated from the Sf9-produced prorenin, 98% were of a truncated (trimannosyl) high-mannose type, approximately two-thirds of which contained a fucose residue linked to the reducing N-acetylglucosamine. The remaining 2% constituted a mixture of high-mannose-type structures containing six, seven, or eight mannose residues; none of these structures were core-fucosylated. None of the oligosaccharide structures recovered from recombinant prorenin synthesized by Sf9 cells were phosphorylated or contained any other form of charge. Furthermore, assays for UDP-GlcNAc-lysosomal-enzyme N-acetylglucosamine phosphotransferase demonstrated no activity above background in lysates prepared from Sf9 cells. Blotting of Sf9 cell lysates with an 125I-labeled, soluble form of the cation-independent mannose 6-phosphate receptor failed to detect any proteins carrying the mannose 6-phosphate recognition signal. Taken together, the data suggest that Sf9 cells do not synthesize high-mannose-type oligosaccharides containing mannose 6-phosphate, and consequently it appears unlikely that these cells utilize the mannose 6-phosphate receptor mediated pathway for targeting of lysosomal enzymes.

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%).

Influence of different B-complex recombinants on the outcome of Rous sarcomas in chickens.

Seven major histocompatibility (B) complex recombinants were evaluated for anti-Rous sarcoma response. In experiment 1, the BR5(F21-G19) recombinant haplotype both homozygous and in heterozygous combinations with B19 and B21 haplotypes were compared to B19/B19 and B21/B21 chickens to determine the relative influence of the BF versus BG chromosomal segments on regression of Rous sarcoma virus-induced tumours. In experiment 2, six recombinant haplotypes BR1(F24-G23), BR2(F2-G23), BR3(F2-G23), BR4(F2-G23), BR6(F21-G23) and BR8(F2-G2a,23) present in chickens heterozygous for normal haplotypes B19, B23 or B26 were compared for anti-sarcoma response. A total of 1328 chickens were blood typed for B alloantigens at 17 days of age, inoculated in the wingweb with Rous sarcoma virus at 6 weeks and monitored for anti-tumour immune response over a 10-week period. Genotypes which shared the same BF haplotype, but differed in their BG regions, had similar anti-tumour responses, implicating the BF but not the BG region in tumour regression. Chickens carrying BF2 or BF21 had a strong anti-tumour response, while BF24 conferred a weaker response, regardless of the accompanying normal haplotype.

Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines.

Human parainfluenza virus type 3 (PIV-3) is one of the leading causes of paediatric viral respiratory disease. The PIV-3 genome encodes two envelope glycoproteins, F and HN, which are the major targets for the host antibody response. We have expressed secreted forms of the F and HN proteins and a novel chimeric FHN glycoprotein in insect cells using recombinant baculovirus vectors and secreted forms of the F and FHN glycoproteins in stably transformed Chinese hamster ovary (CHO) cells. Comparison of the mammalian cell- and insect cell-expressed F and FHN proteins by SDS-PAGE showed that the CHO cell-expressed proteins are several kilodaltons larger in size than the baculovirus-produced proteins. A partial characterization of the oligosaccharide structures of the F and FHN proteins revealed that the size difference is due to the different oligosaccharide structures added to these proteins by the two cell lines. The F, HN and FHN proteins were immunoaffinity-purified from the culture medium of baculovirus-infected Sf9 cells and the F and FHN proteins were immunoaffinity-purified from the culture medium of CHO cells. A comparison of the immunogenicity and efficacy of the mammalian cell- and insect cell-produced FHN proteins was tested in cotton rats. The CHO cell- and baculovirus-produced FHN proteins were found to induce similar levels of PIV-3-specific ELISA-positive and neutralizing antibodies and both proteins provided near complete protection when animals were vaccinated with low doses of the FHN protein.

Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells.

Prorenin was isolated by immunoprecipitation from the culture medium of Chinese hamster ovary cells transfected with a human prorenin cDNA. The N-linked oligosaccharide structures on the in vivo [3H]mannose-labeled, purified protein were characterized using a combination of serial lectin affinity chromatography, high-pressure liquid chromatography, ion-exchange chromatography, and size-exclusion chromatography and treatment with specific glycosidases and methylation analysis. Approximately 61% of the oligosaccharides on the molecule are complex type, in the form of tetraantennary (2%), 2,6-branched triantennary (13%), 2,4-branched triantennary (3%), and biantennary (43%) structures. The majority of all complex type structures are core-fucosylated. Sialic acids are linked at the C-3 position of terminal galactose, and the degree of sialylation of the bi- and triantennary structures varies between nonsialylated and fully sialylated; no tetraatennary structure contains more than three sialic acid residues. Recombinant prorenin contains 4% hybrid-type structures, all of which carry a terminal sialic acid residue. The remaining 35% of the structures on the molecule are high mannose type, composed of 5, 6, or 7 mannose residues. Approximately 6% of the high mannose type structures and 10% of the hybrid structures are phosphorylated, as judged by their susceptibility to treatment with alkaline phosphatase. Compositional analysis of an unlabeled preparation of the protein suggested the presence of approximately 1.4 oligosaccharide units per molecule.

Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9.

The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [3H]glucosamine and infected with a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal beta 1-3GalNAc constituting 17 and 66% of the total number of structures, respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked alpha 1-6 to the asparagine-linked N-acetylglucosamine.

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 approximately 50,900, pI approximately 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 approximately 93,000), was present in smaller amounts as cells were passaged in vivo and re-established as in vitro cultures [MTF7 greater than 'primary' tumor-derived lines (sc1, sc3) much greater than local recurrence-derived lines (LR1, LR1a, 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 approximately 110,000), 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.

Tumor-elicited polymorphonuclear cells, in contrast to "normal" circulating polymorphonuclear cells, stimulate invasive and metastatic potentials of rat mammary adenocarcinoma cells.

Circulating polymorphonuclear cell (PMN) levels rise in proportion to the metastatic potential of the tumor in 13762NF mammary adenocarcinoma tumor-bearing rats. These tumor-elicited PMNs (tcPMNs) secrete high levels of the basement-membrane-degrading enzymes, type IV collagenase and heparanase, suggesting that metastatic tumor cells stimulate neutrophilia so that the tcPMNs might assist tumor cell extravasation during metastasis. To test this hypothesis, purified proteose peptone-elicited PMNs from peritoneal exudate, circulating normal PMNs, and tcPMNs were evaluated for their effects on in vitro invasive and in vivo metastatic potentials of syngeneic 13762NF mammary adenocarcinoma tumor cells. tcPMNs caused a dose-dependent increase in invasion through a reconstituted basement membrane barrier in an in vitro invasion assay. At PMN:tumor cell ratios of 30:1, invasion potential significantly (P less than 0.05) rose to 26-fold, 40-fold, and 37-fold for poorly metastatic MTLn2 cells, highly metastatic MTLn3 cells, and moderately metastatic MTF7 cells, respectively. In contrast, purified proteose peptone-elicited PMNs and circulating normal PMNs did not significantly alter invasive potential. Intravenous coinjections of purified proteose peptone-elicited PMNs did not change the number of experimental lung metastases, but tcPMNs at ratios to 50:1 significantly raised the mean number of metastases 23-fold for MTLn2, 3- to 4-fold for MTLn3, and 1.6- to 1.8-fold for MTF7. These results demonstrate that tcPMNs contribute to the metastatic propensity of mammary adenocarcinoma clones by increasing efficiency of invasion through basement membrane.

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 10(6) 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.

Use of the Membrane Invasion Culture System (MICS) as a screen for anti-invasive agents.

The Membrane Invasion Culture System (MICS) assay was adapted for relatively rapid screening of compounds and used to identify anti-invasive drugs that inhibit human and murine tumor cell migration through a reconstituted basement membrane in vitro. Cell lines demonstrating low and high invasive and metastatic potentials were tested with all compounds for tumoricidal effects prior to evaluation in MICS at non-cytotoxic doses. The effect on invasive potential in the MICS assay was determined in 3 categories: (1) 48 hr drug pre-treatment prior to seeding in the MICS (exceptions: 90 min pre-treatment with pertussis toxin and, for some studies, continuous exposure for 2-7 days); (2) peptide or prostaglandins 2 hr after seeding and attachment to the membranes in MICS followed by continuous exposure; and (3) cells receiving neither drug nor peptide treatment and serving as controls in each MICS chamber. Since invasion involves cellular motility and deformability, some cytoskeleton disrupting agents were selected. Of these, vincristine, colcemid and colchicine inhibited invasion but taxol did not. Pre-treatment with cAMP agonists produced conflicting results: dibutyryl cAMP and 8-(4-chloro-phenylthio) cAMP resulted in 50% and 38% reduction in invasion, respectively, whereas 8-bromo cAMP stimulated invasive potential by 30%. Forskolin and cholera toxin both significantly reduced invasiveness. Pre-treatment with 5-azacytidine and araC, to consider the role of methylation and proliferations decreased invasive ability. Anti-metastatic drugs such as gamma-interferon and razoxane inhibited invasive potential but to varying degrees. Treatment of cells with prostaglandins E2, F2 alpha, A2, and D2 were ineffectual; however, indomethacin mildly inhibits invasion (less than 30%).(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity of locally recurrent rat mammary tumour cell lines to syngeneic polymorphonuclear cell, macrophage and natural killer cell cytolysis.

Using a recently developed model for studying the biology of locally recurrent (LR) mammary tumours in the 13762NF rat mammary adenocarcinoma system, we examined the sensitivity to polymorphonuclear cell, macrophage and natural killer cell cytolysis. The parental MTF7(T20) cell line; the 'primary' tumours which arose following subcutaneous inoculation into the mammary fat pad, sc1 and sc3; and the local recurrences (following surgical excision) LR1 and LR1a from sc1, and LR3 from sc3 were all cells generally resistant to specific PMN cytolysis. LPS-activated macrophages caused 25.1%, 38.7% and 58.8% specific cytolysis in MTF7, sc1 and LR1 cells, respectively at E:T of 20:1 and 72 h co-incubation. LR1a, sc3 and LR3 lysis ranged from 0-4.4% under the same conditions. Non-activated macrophages did not lyse any of the cell lines. Locally recurrent and 'primary' tumour cell lines were also not lysed by naive NK cells (range 0.5-4.0% cytolysis). NK cells activated with bropirimine, a potent immunomodulator currently being studied in clinical trials, and/or interleukin-2 were mildly more effective at killing LR cells. Our results show that locally recurrent tumours exhibit heterogeneous sensitivities and are different from 'primary' tumour cells in sensitivities to immune cell killing, but they are not necessarily more or less sensitive. Results with bropirimine-activated or IL-2-activated NK cells emphasize that nonspecific activation is insufficient to eliminate all tumour subpopulations.

The role of polymorphonuclear leukocytes (PMN) on the growth and metastatic potential of 13762NF mammary adenocarcinoma cells.

Circulating neutrophil (PMN) levels can increase in rats bearing subcutaneously growing clones of the 13762NF mammary adenocarcinoma and the level of increase correlates with the metastatic potential of the clone. In rats with poorly metastatic MTC tumors, numbers of circulating PMN did not rise, whereas PMN levels rose 50-fold in rats bearing highly metastatic MTLn3, 12-fold in rats with weakly metastatic MTLn2, and 14-fold in those with moderately metastatic MTF7 tumors. Neutrophilia was caused partly by tumor size, but metastatic potential was a stronger determinant, suggesting that PMNs may play a role in the metastatic process. To determine whether circulating PMNs indeed contribute to cellular metastatic potential, we examined effects of PMN on various aspects of the metastatic process. Experimental metastasis assays involving i.v. co-injections of PMNs yielded a dose-dependent increase in extrapulmonary metastases for MTLn3, but no change in lung colonization potential for any of the clones examined. The change in the metastatic profile was not due to any modification in in vivo distribution of i.v. injected tumor cells or in adhesion to endothelial monolayers in vitro. PMNs also had no effect on in vitro DNA, RNA or protein synthesis and were not cytolytic (E:T 100:1). However, PMNs collected from high-passage MTLn3 tumor-bearing rats had a 50% increase in heparanase and type-IV collagenolytic activity as compared to unstimulated PMNs isolated from normal rats. These results indicate that polymorphonuclear cells may contribute to the metastatic potential of highly metastatic clones from the 13762NF mammary adenocarcinoma cells by assisting in the degradation of basement membrane during extravasation.

Development and characterization of a rat model for locally recurring mammary tumors: sensitivities to 5-fluoro-2'-deoxyuridine, adriamycin, and X-irradiation.

Local recurrence occurs in 4-47% of breast cancer patients and is often associated with development of metastatic foci and resistant cell populations. Thus, recurrent breast cancer indicates a poor prognosis for the patient. Local tumor-derived 13762NF rat mammary adenocarcinoma cell clone MTF7(T20) was injected into the inguinal mammary fat pad and allowed to grow before surgical excision. Individual locally growing (primary) tumors were removed and established in short-term tissue culture. Corresponding local recurrences were excised after regrowth and established in short-term tissue culture. All sublines were tested for in vitro sensitivities to 5-fluoro-2'-deoxyuridine, Adriamycin, and ionizing X-irradiation. Using a clonogenic colony formation assay, responses of individual sublines ranged from 85 to 1500 ng/ml for Adriamycin and 65 to 10,000 nM for FdUrd. Some recurrences were significantly more resistant while others were more sensitive than the corresponding primary tumor lines. All recurrences had smaller 90% lethal dose values than the corresponding parent or primary tumor in response to Adriamycin; whereas, to 5-fluoro-2'-deoxyuridine, 90% lethal dose values revealed that most lines were quite resistant. Statistically significant differences in radiation survival were observed only for lines LR1a and LR5 (more sensitive). There was no apparent correlation between sensitivities to chemotherapy agents or X-irradiation and experimental metastatic potential in LR sublines. These dose-response data indicate that locally recurrent tumors are frequently, but not always, different from the original primary tumor in response to chemotherapy agents and ionizing X-irradiation. Although an exact mechanism is unknown, it is likely that "selective" pressures which eliminate large numbers of cells, in this case surgery, change tumor composition so that recurrent tumors may no longer be equivalent to the tumor mass that was originally excised. This suggests that treatment strategies should be planned accordingly.

Bile and serum immunoglobulin levels during primary and secondary infections with Eimeria tenella in chickens.

Using a radial immunodiffusion assay, total bile and serum IgG, IgM and IgA were measured following primary and secondary exposures to Eimeria tenella. Neither IgG nor IgM could be detected consistently in bile. Biliary IgA peaked at Days 6 and 10 following a primary infection of either 5000 or 10,000 oocysts and remained elevated following a subsequent 10,000-oocyst challenge at Day 10. Serum IgG and IgM levels were not influenced by parasitism and measurable concentrations of serum IgA were not detected.

Delayed wattle reactions in Eimeria tenella infected chickens.

Wattle reactions to an Eimeria tenella antigen and phytohemagglutinin (PHA) were studied in chickens infected with E. tenella. Two trials were conducted using a total of 224 chickens. Four days after infection with one dose of 10,000 sporulated oocysts, the increase in wattle thickness in response to E. tenella antigen was significantly greater than that of uninfected controls. This significant response persisted through day 10 post infection. Wattle response to PHA 3 days after infection were significantly greater than for uninfected controls. Significant differences in response to PHA were maintained throughout the experiment except on day 6. An increased response to PHA from days 7 to 13 post infection was attributed to the lower parasite burden at that time.