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.

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.

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.

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.

Response of B complex haplotypes B22, B24, and B26 to Rous sarcomas.

Five individual male matings of line UNH 105 New Hampshires, in which all males and most females were either B22/B24 or B22/B26, produced 462 progeny that fell into six B complex genotypes: B22/B22, B24/B24, B26/B26, B22/B24, B22/B26, and B24/B26. The genotypes of parents and offspring were determined by blood typing for B alloantigens using a panel of antisera. Six-week-old chickens were inoculated with Rous sarcoma virus (RSV). Resulting tumors were scored for size six times over a 10-week period; based upon these scores, a tumor profile index (TPI) was assigned to each chicken as a criterion of immunological response. The B22/B26 hosts showed the greatest mean response (TPI 3.3) and B24/B24 chickens the lowest response (TPI 4.4), the difference being statistically significant. Dominance in the response to sarcoma was observed when either the B22 or B26 haplotype combined with the B24 haplotype and compared with the appropriate corresponding homozygotes, and when the B22 or B26 heterozygote was compared with B22/B22 and B26/B26 homozygotes.

Estimates of heritability of response to Rous sarcomas of chickens.

The stocks used for this investigation consisted of 1039 F3 generation progeny from the cross of two highly inbred lines and 355 and 462 offspring from subpopulations UNH 105A and UNH 105B, respectively, of a noninbred line of New Hampshires. Matings were such that B complex alleles were segregated in the three experimental populations with minor exceptions. Each chicken was inoculated at 6 weeks of age with a subgroup of Rous sarcoma virus (RSV). Resulting tumors were subjectively scored on a scale from 0 (no tumor) to 6 (massive tumor) six times during a 10-week experimental period. Based upon the six tumor scores, each chicken was then assigned a tumor profile index (TPI), a criterion of antitumor response. The TPI were analyzed by least squares analysis of variance and corrected for hatch, sex, and virus prior to obtaining components of variance and estimates of heritability from a nested analysis of variance. Estimates of heritability from the sire component ranged from 0 to .41 +/- .27 and from the dam component .18 +/- .09 to .26 +/- .14, which are rather low estimates in general.