Calcium-dependent heparin-like ligands for L-selectin in nonlymphoid endothelial cells.

L-Selectin is a calcium-dependent mammalian lectin that mediates lymphocyte trafficking by recognizing sialylated ligands on high endothelial venules in lymph nodes. Although L-selectin probably mediates neutrophil extravasation into nonlymphoid tissues, no corresponding ligand has been characterized. Staining of cultured endothelial cells with an L-selectin chimera (LS-Rg) showed an internal pool of ligands. Metabolic labeling with sulfur-35-labeled sulfate revealed heparin lyase-sensitive ligands that bound LS-Rg in a calcium-dependent, sialic acid-independent manner. A fraction of commercial heparin bound to LS-Rg and LS-Rg bound to heparin-agarose, both in a calcium-dependent manner. Thus, L-selectin recognizes endothelial heparin-like chains, which could be physiological ligands mediating leucocyte trafficking.

The plasminogen receptor, Plg-RKT, is essential for mammary lobuloalveolar development and lactation.

Essentials Plg-RKT-/- female mice give birth, but no offspring of Plg-RKT-/- female mice survive to weaning. Causal mechanisms of potential lactational failure in Plg-RKT-/- mice are unknown. Plg-RKT regulates extracellular matrix remodeling, cell proliferation, apoptosis, fibrin surveillance. Plg-RKT is essential for lactogenesis and mammary lobuloalveolar development. SUMMARY: Background Lactational competence requires plasminogen, the zymogen of the serine protease, plasmin. Plg-RKT is a unique transmembrane plasminogen receptor that promotes plasminogen activation to plasmin on cell surfaces. Plg-RKT-/- mice are viable, but no offspring of Plg-RKT-/- female mice survive to weaning. Objectives We investigated potential lactational failure in Plg-RKT-/- mice and addressed causal mechanisms. Methods Fibrin accumulation, macrophage infiltration, processing of extracellular matrix components, effects of genetic deletion of fibrinogen, expression of fibrosis genes, and proliferation and apoptosis of epithelial cells were examined in lactating mammary glands of Plg-RKT-/- and Plg-RKT+/+ mice. Results Milk was not present in the stomachs of offspring of Plg-RKT-/- female mice and the pups were rescued by foster mothers. Although the mammary ductal tree developed normally in Plg-RKT-/- glands, lobuloalveolar development was blocked by a hypertrophic fibrotic stroma and infiltrating macrophages were present. A massive accumulation of fibrin was also present in Plg-RKT-/- alveoli and ducts. Although this accumulation was decreased when Plg-RKT-/- mice were made genetically heterozygous for fibrinogen, defects in lobuloalveolar development were not rescued by fibrinogen heterozygosity. Transcriptional profiling revealed that EGF was downregulated 12-fold in Plg-RKT-/- glands. Furthermore, proliferation of epithelial cells was not detectable. In addition, the pro-survival protein, Mcl-1, was markedly downregulated and apoptosis was observed in Plg-RKT-/- but not Plg-RKT+/+ glands. Conclusions Plg-RKT is essential for lactogenesis and functions to maintain the appropriate stromal extracellular matrix environment, regulate epithelial cell proliferation and apoptosis, and, by regulating fibrinolysis, preserve alveolar and ductal patency.

Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy.

A major goal of tumor immunotherapy is the induction of tumor-specific T cell responses that are effective in eradicating disseminated tumor, as well as mounting a persistent tumor-protective immunity. We demonstrate here that a genetically engineered fusion protein consisting of human/mouse chimeric anti-ganglioside GD2 antibody and human interleukin-2 is able to induce eradication of established B78-D14 melanoma metastases in immunocompetent syngeneic C57BL/6J mice. This therapeutic effect is mediated by host immune cells, particularly CD8+ T cells and is associated with the induction of a long-lived immunity preventing tumor growth in the majority of animals when challenged up to four months later with B78-D14 cells. This effect was tumor-specific, since no cross-protection against syngeneic, ganglioside GD2+ EL-4 thymoma cells was observed. Furthermore, this tumor-specific protection can be transmitted horizontally to naive, syngeneic SCID mice by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of cytokines provides a means to elicit effective immune responses against established tumors in the immunotherapy of neoplastic disease.

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

Deficiency of plasminogen receptor, Plg-RKT , causes defects in plasminogen binding and inflammatory macrophage recruitment in vivo.

Essentials Plg-RKT is a novel integral membrane plasminogen receptor. The functions of Plg-RKT in vivo are not known. Plg-RKT is a key player in macrophage recruitment in the inflammatory response in vivo. Plg-RKT deficiency is not compatible with survival of the species. SUMMARY: Background Plg-RKT is a novel integral membrane plasminogen receptor that binds plasminogen via a C-terminal lysine exposed on the cell surface and promotes plasminogen activation on the cell surface by both tissue plasminogen activator and urokinase plasminogen activator. Objectives To evaluate the role of Plg-RKT in vivo we generated Plg-RKT-/- mice using a homologous recombination technique. Methods We characterized the effect of Plg-RKT deletion on reproduction, viability, health and spontaneous thrombosis and inflammation. Results Plg-RKT-/- mice were viable and fertile. Survival of Plg-RKT-/- mice and Plg-RKT+/+ littermates was not significantly different. However, quite strikingly, all pups of Plg-RKT-/- females died within 2 days of birth, consistent with a lactation defect in Plg-RKT-/- mothers. Additionally, there was a significant effect of Plg-RKT deficiency on the growth rates of female, but not male, mice. In experimental peritonitis studies, Plg-RKT-/- mice exhibited a marked defect in macrophage recruitment. As a contributing mechanism, the capacity of Plg-RKT-/- macrophages for plasminogen binding was markedly decreased. Conclusions These studies demonstrate that Plg-RKT is required for plasminogen binding and macrophage migration in vivo. In addition, Plg-RKT deficiency is not compatible with survival of the species, due to the death of all offspring of Plg-RKT-/- females. This new mouse model will be important for future studies aimed at delineating the role of cell surface plasminogen activation in challenge and disease models in vivo.

Targeted interleukin-2 therapy for spontaneous neuroblastoma metastases to bone marrow.

BACKGROUND: Advanced (stage 4) cases of neuroblastoma, a childhood cancer of the nervous system, are associated with high relapse rates, even after intensive chemotherapy, radiotherapy, and autologous bone marrow transplantation. Therefore, the use of monoclonal antibodies directed against the neuroblastoma tumor marker disialoganglioside GD2 (GD2), in combination with recombinant human interleukin 2 (rhIL-2), is under clinical investigation. We hypothesize that targeted cytokine immunotherapy with a recombinant anti-GD2 antibody-interleukin 2 fusion protein (ch14.18-IL-2) is superior to a combination of ch14.18 and rhIL-2. Our purpose was as follows: 1) to develop a syngeneic model for murine neuroblastoma that expresses GD2 and features both experimental and spontaneous metastases to bone marrow and liver, and 2) to assess anti-GD2-targeted IL-2 therapy in this mode. METHODS: A hybrid neuroblastoma cell line was used to generate the GD2-positive NXS2 cell line. Bone marrow and liver metastases were quantified by reverse transcription-polymerase chain reaction for tyrosine hydroxylase and by organ weight or counts of macroscopic tumor foci, respectively. All P values reported are two-sided. RESULTS: Injection of NXS2 cells resulted in disseminated bone marrow and liver metastases exhibiting stable, but heterogeneous expression of GD2. Treatment with fusion protein (10 microg/day for 6 days) effectively suppressed growth of both experimental and spontaneous metastases to bone marrow and liver (P<.001). In contrast, a mixture of rhIL-2 and ch14.18 at equivalent dose levels was inefficient. Only mice treated with ch14.18-IL-2 showed a twofold prolongation in life span (P<.001). CONCLUSION: Targeted IL-2 therapy with a ch14.18-IL-2 fusion protein elicits an effective antitumor response. Our data suggest that a study of ch14.18-IL-2 as an adjuvant treatment in patients with minimal residual disease may be of value.

Antigens associated with a human lung adenocarcinoma defined by monoclonal antibodies.

Monoclonal antibodies KS1/4, KS1/9, and KS1/17 were developed in this laboratory from a fusion of the murine myeloma cell line P3X63Ag8 with spleens of BALB/c mice previously primed with UCLA P3 cells derived from a human adenocarcinoma of the lung. Monoclonal antibodies KS1/4 and KS1/17 seemed to recognize similar glycoprotein antigens on the lung carcinoma cells by indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. However, mapping of [3H]lysine- and [3H]arginine-labeled tryptic peptides of antigens in specific immunoprecipitates of lung carcinoma cells by high-pressure liquid chromatography revealed a one peptide difference. Antibody KS1/9 did not immunoprecipitate any identifiable protein from detergent extracts of the immunizing cell line by routine methods and appears to detect a glycolipid antigen. Immunocytochemical analysis of tissue sections showed this monoclonal antibody to be reactive with adenocarcinomas of the lung and not with the other histological types of lung carcinoma or normal tissue. Monoclonal antibodies KS1/4 and KS1/17, however, reacted with 3 major histological types of lung cancer and minimally with the proximal tubules of normal kidney and the epithelium of bronchioles.

Involvement of B lymphocytes in the growth inhibition of human pulmonary melanoma metastases in athymic nu/nu mice by an antibody-lymphotoxin fusion protein.

Antibody-cytokine fusion proteins can target biologically active cytokines to various tumor sites, achieving local concentrations sufficient to induce host immune responses leading to tumor elimination. Here, we demonstrate the therapeutic efficacy of a tumor-specific antibody-lymphotoxin fusion protein (ch225-LT) on xenografted pulmonary metastases of human melanoma. In vitro studies indicated a direct cytotoxic effect of such construacts on melanoma cells via the induction of apoptosis, as demonstrated by cell cycle analysis and DNA fragmentation. However, ch225-LT lacked any therapeutic effect in immune deficient C.B17 scid/beige and scid/scid mice, indicating the insufficiency of this direct mechanism in vivo. In contrast, in athymic nu/nu mice, ch225-LT completely inhibited outgrowth of the xenografted tumor. This therapeutic effect was accompanied by infiltrations of CD45+, Mac-1+, and asialo-GM1+ cells into the tumor; B220+ cells were present in the surrounding tissue and the periphery of the tumor. The functional role of asialo-GM2+ cells was confirmed by in vivo depletion studies. Our data indicate that an antibody-lymphotoxin fusion protein effectively inhibits the growth of disseminated melanoma metastases by mechanisms that function in the absence of mature T cells, but require B, NK, and other asialo-GM1+ cells.

Detection of ganglioside GD2 in tumor tissues and sera of neuroblastoma patients.

A murine monoclonal antibody (monoclonal antibody 126) produced against cultured human neuroblastoma cells (LAN-1) was found to be specifically directed to a disialoganglioside (GD2) antigen preferentially expressed on both cell lines and tissues derived from melanoma and neuroblastoma. In enzyme-linked immunosorbent assays, monoclonal antibody 126 failed to react with leukemic and lymphoblastoid cells as well as with a variety of carcinoma and sarcoma cell lines. Immunohistological analysis by the immunoperoxidase technique revealed strong reactivity of monoclonal antibody 126 with frozen and formaldehyde-fixed neuroblastoma and melanoma tissues. Tissues from patients with glioma or with small cell cancer of the lung showed faint staining, whereas those from individuals with sarcoma, lymphoma, and a variety of other neoplasms proved to be negative. Sera of neuroblastoma patients showed significantly elevated GD2 levels compared to normal children (p less than 0.001) and children with other tumors (p less than 0.001) as determined by a quantitative competitive enzyme-linked immunosorbent assay. Furthermore, the GD2 serum level of one neuroblastoma patient, when followed serially, was found to correlate with progression of disease, suggesting the potential usefulness of this assay for the diagnosis and monitoring of neuroblastoma.

Mutational and nonmutational activation of p21ras in rat colonic azoxymethane-induced tumors: effects on mitogen-activated protein kinase, cyclooxygenase-2, and cyclin D1.

Azoxymethane (AOM)-induced colonic carcinogenesis involves a number of mutations, including those in the K-ras gene and CTNNB1, that codes for beta-catenin. Prior in vitro studies have also demonstrated that wild type p21(K-ras) can be activated by epigenetic events. We identified 15 K-ras mutations in 14 of 84 AOM-induced colonic tumors by three independent methods. By single strand conformational polymorphism, we also observed mutations in 22 of 68 tumors in exon 3 of CTNNB1. A highly sensitive method was then used to measure p21ras activation levels. All tumors assayed possessing K-ras mutations had significantly higher p21ras activation levels (8.8 +/- 1.5%; n = 13) compared with that of control colon (3.7 +/- 0.4; n = 6; P < 0.05) or tumors without such mutations (4.2 +/- 0.4%; n = 70; P < 0.05). Among tumors with wild-type K-ras, there was a subset of tumors (18 of 70) that had significantly higher p21ras activation levels (8.0 +/- 0.9%; n = 18) compared with control colons. In three of four tumors examined with activated wild-type p21ras, we observed increased c-erbB-2 receptor expression and decreased Ras-GAP expression. In contrast, only one of eight tumors examined with wild-type ras and nonactivated p21ras demonstrated these alterations. Mitogen-activated protein kinase (MAPK) activation and cyclooxygenase-2 (COX-2) expression were increased in tumors with mutated or activated wild-type p21ras, compared with their nonactivated counterparts. Although beta-catenin mutations did not alter COX-2 expression or MAPK activity, mutations in either K-ras or beta-catenin significantly increased cyclin D1 expression. In contrast, in tumors with wild-type but activated p21-ras, cyclin D1 expression was not enhanced. Thus, the spectrum of changes in MAPK, COX-2, and cyclin D1 is distinct among tumors with ras or beta-catenin mutations or nonmutational activation of p21ras.

De-N-acetyl-gangliosides in humans: unusual subcellular distribution of a novel tumor antigen.

The disialoganglioside GD3 is a major antigen in human melanomas that can undergo 9-O-acetylation of the outer sialic acid (giving 9-OAc-GD3). Monoclonal antibody SGR37 detects a different modification of the GD3, de-N-acetylation of the 5-N-acetyl group (giving de-N-Ac-GD3). We found that conventional immunohistochemistry of the SGR37 antigen is limited by a reduction in reactivity upon fixation with aldehydes (which presumably react with the free amino group) or with organic reagents (which can extract glycolipids). We optimized conditions for detection of this antigen in unfixed frozen tissue sections and studied its distribution in human tissues and tumors. It is expressed at low levels in a few blood vessels, infiltrating mononuclear cells in the skin and colon, and at moderate levels in skin melanocytes. In contrast, the antigen accumulates at high levels in many melanomas and in some lymphomas but not in carcinomas. In positive melanomas, expression is sometimes more intense and widespread than that of GD3. Both 9-O-acetylation and de-N-acetylation of GD3 seem to occur after its initial biosynthesis. Isotype-matched antibodies against GD3, 9-O-acetyl-GD3 and de-N-acetyl-GD3 were used to compare their subcellular localization and trafficking. 9-O-acetyl-GD3 colocalizes with GD3 predominantly on the cell surface and partly in lysosomal compartments. In contrast, de-N-acetyl-GD3 has a diffuse intracellular location. Adsorptive endocytosis of antibodies indicates that whereas GD3 remains predominantly on the cell surface, de-N-acetyl-GD3 is efficiently internalized into a compartment that is distinct from lysosomes. Rounding up of melanoma cells occurring during growth in culture is associated with relocation of the internal pool of de-N-acetyl-GD3 to the cell surface. Thus, a minor modification of the polar head group of a tumor-associated glycosphingolipid can markedly affect the subcellular localization and trafficking of the whole molecule. The high levels of the SGR37 antigen in melanomas and lymphomas, its selective endocytosis from the cell surface, and its relocation to the cell surface of rounded up cells suggest potential uses in diagnostic or therapeutic approaches to these diseases.

Elimination of established murine colon carcinoma metastases by antibody-interleukin 2 fusion protein therapy.

A recombinant humanized antibody-interleukin 2 fusion protein (huKS1/4-IL-2) was used to direct IL-2 to the tumor microenvironment and elicit a T cell-mediated eradication of established pulmonary and hepatic CT26-KSA colon carcinoma metastases in syngeneic BALB/c mice. This antitumor effect was specific because a fusion protein, which was nonreactive with these tumor cells, failed to exert any such effect. The efficacy of the huKS1/4-IL-2 fusion protein in eliminating metastases was documented because mixtures of monoclonal antibody huKS1/4 with recombinant human IL-2 were ineffective and, at best, only partially reduced tumor load. Two lines of evidence indicated the eradication of metastases and the absence of minimal residual disease in animals treated with the fusion protein: first, the lack of detection of CT26-KSA cells by reverse transcription-PCR, which can detect one tumor cell in 10(6) liver cells; and second, the tripling of life span. The effector mechanism involved in this tumor eradication is dependent on T cells because the IL-2-directed therapy is ineffective in T cell-deficient SCID mice. The essential effector cells were further characterized as CD8+ T cells by in vivo depletion studies. Such T cells, isolated from tumor-bearing mice after fusion protein therapy, elicited MHC class I-restricted cytotoxicity in vitro against colon carcinoma target cells. Taken together, these data indicate that fusion protein-directed IL-2 therapy induces a T cell-dependent host immune response capable of eradicating established colon cancer metastases in an animal tumor model.

Analysis of MLH1 and MSH2 expression in ovarian cancer before and after platinum drug-based chemotherapy.

Preclinical studies have demonstrated a relationship between DNA mismatch repair (MMR) status and sensitivity to cisplatin and carboplatin. MMR-deficient cells are resistant to both drugs, and selection for cisplatin resistance in vitro is sometimes accompanied by loss of MMR protein expression. We used immunohistochemical staining techniques to investigate hMLH1 and hMSH2 expression in paired ovarian tumor sections from 54 ovarian cancer patients before and after platinum-based therapy. We sought associations between hMLH1 and hMSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. hMLH1 and hMSH2 staining decreased significantly after platinum-based therapy. The percent of malignant cells that stained positive correlated with the intensity of nuclear staining for both proteins; staining for hMLH1 correlated well with staining for hMSH2. Unexpectedly, expression of nuclear hMLH1 correlated negatively with response to treatment. Expression of nuclear hMLH1 and hMSH2 was positively correlated with pretreatment CA125 level, and expression of nuclear hMSH2 was positively correlated with change in CA125 level after treatment. Tumor stage was associated with expression of nuclear hMSH2, and tumor histological subtype was associated with both hMLH1 and hMSH2 staining. No association was found between expression of either protein and overall survival. These results indicate that the tumor is biologically altered after chemotherapy consistent with treatment-induced selection for cells expressing lower hMLH1 and hMSH2 levels. However, immunohistochemical staining for either hMLH1 or hMSH2 was not highly predictive of drug sensitivity as measured by response or survival.

Marked antiangiogenic and antitumor efficacy of AG3340 in chemoresistant human non-small cell lung cancer tumors: single agent and combination chemotherapy studies.

Effective therapy is needed to improve the survival of patients with advanced lung cancers. We studied the effects of a selective metalloprotease inhibitor, AG3340, on chemoresistant human non-small cell lung cancer tumors (line MV522) in vivo. Mice bearing s.c. tumors were given twice-daily oral doses of AG3340. As a single agent, AG3340 inhibited angiogenesis (up to 77%) and tumor growth (up to 65%) in a dose-dependent manner at well-tolerated daily doses up to 400 mg/kg/day and induced significant tumor necrosis. In contrast, tumors were relatively insensitive to carboplatin with approximately 25% growth inhibition observed at a maximum tolerated dose of approximately 30 mg/kg/week (given i.p., twice weekly). Carboplatin inhibited tumor growth markedly only at toxic doses, demonstrating a superior therapeutic index of AG3340 to carboplatin in this tumor model. A suboptimal dose of AG3340, when used in combination with an ineffective maximum tolerated dose of carboplatin, resulted in greater tumor growth inhibitions than those produced by either agent alone. Similarly, growth inhibition was enhanced when AG3340 was used in combination with paclitaxel. Cotreatment with carboplatin did not alter AG3340 plasma concentrations achieved acutely after oral dosing. These data demonstrate an antiangiogenic and antitumor effect of AG3340 when used as a single agent and enhanced growth inhibitions when AG3340 is used in combination with cytotoxic agents. These data suggest that treatment with this novel matrix metalloprotease inhibitor may be beneficial in advanced lung cancers and other chemoresistant malignancies.

A tyrosine sulfated human glycoprotein with an unusual cell distribution.

We describe a human integral cell surface glycoprotein (Mr 142 kD), recognized by monoclonal antibody M2B3. This glycoprotein is absent from resting peripheral blood lymphocytes, but becomes expressed in significant levels with mitogen activation. The M2B3 glycoprotein is present on epithelial cells in the basal layer of epidermal and esophageal tissue as well as in several fresh tumors examined. In addition, it is present on smooth muscle tissue throughout the gastrointestinal tract, but is absent from smooth muscle from several other tissue sources, skeletal muscle and cardiac muscle. The M2B3 glycoprotein is similar, but not identical, in apparent Mr to a transformation-associated glycoprotein, Q14. Further, the M2B3 and Q14 species are related antigenically. Nonetheless, M2B3 and Q14 are distinct glycoproteins based on clear differences in cell distribution and in partial peptide mapping. The M2B3 antigen described herein is sulfated on tyrosine, and represents one of the few cell surface proteins described to date that is sulfated on tyrosine residues. Our studies suggest the function of the M2B3 glycoprotein is likely to be associated with cell proliferation of cell adhesion.

Lymphocyte-mediated alopecia in C57BL/6 mice following successful immunotherapy for melanoma.

Successful immunotherapy of established B16 melanoma metastases in C57BL/6 mice can be achieved by antibody-targeted interleukin-2 administration. This therapeutic effect is accompanied in approximately 20% of the animals by induction of a population of lymphocytes that migrates to and substantially disrupts the cytoarchitecture of the skin, which results in progressive alopecia. The histologic changes associated with the hair loss, i.e., peri-, and intrafollicular inflammatory infiltrates consisting of both activated CD4+ and CD8+ T cells, as well as expression of major histocompatibility complex class I antigens on subinfundibular follicle epithelium, are similar to those observed in human alopecia areata. Furthermore, the alopecic phenotype can be transmitted horizontally by passive transfer of lymphocytes from treated animals to naïve mice. Since lymphocytes from treated animals either lacking or displaying signs of alopecia are able to transmit these phenotypic changes to a similar percentage of naïve animals, the initiation of alopecia seems to be dependent on the coincidence of at least two different events: the presence of specific lymphocyte populations as well as specific features of the skin disclosing a target for these cells.

Immunohistochemical analysis of the distribution of the human ATPase (hASNA-I) in normal tissues and its overexpression in breast adenomas and carcinomas.

Human ATPase (hASNA-I) is a novel human gene recently cloned on the basis of homology to the arsA gene of bacteria. Its protein product is an ATPase that is free in the cytoplasm and bound in the perinuclear area and nucleolus in human cells. We prepared the hASNA-I-specific 5G8 monoclonal antibody and used it to investigate the expression of hASNA-I in normal human tissues and breast cancers. hASNA-I was detected immunohistochemically only in the epithelial cells of the liver, kidney, and stomach wall, in the adrenal medulla, in the islet cells of the pancreas, in the red pulp of the spleen, and in cardiac and skeletal muscle. No staining was observed in the uterus, testis, lung, thyroid, cerebellum, and large intestine. Although no staining was also observed in normal breast tissue, all four cases of breast fibroadenomas and all 15 cases of either primary or metastatic breast carcinoma demonstrated increased staining. No embryological or functional common denominator is readily apparent. However, the increased expression in malignant breast cells is of particular interest with respect to the use of this antibody for screening of cytological specimens.

Endothelial cells enhance spontaneous metastasis of human lung carcinoma cells in athymic mice.

The process of adhesion to endothelial cells is an important step in the progression to metastatic disease. The use of human neoplastic cell lines (now increasingly used in the study of the mechanisms of metastasis) in studying interactions with normal endothelial cells is therefore pertinent. In this report, the enhanced ability of a metastatic variant, MV522, of a human lung carcinoma cell line to adhere to endothelial cell monolayers is demonstrated. The ability to spontaneously metastasize from subcutaneous sites in athymic mice, of in vitro selected endothelial adhesive subpopulations of MV522 is also shown.

Broad antitumor and antiangiogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clinical trials.

We studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP-2 and -9), MT-MMP-1 (MMP-14), and collagenase-3 (MMP-13) in many tumor models. AG3340 produced dose-dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose-response relationship was explored in three models: COLO-320DM colon, MV522 lung, and MDA-MB-435 breast. Dose-dependent inhibitions of tumor growth (over 12.5-200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen-independent PC-3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD-31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16-F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front-line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.

Chromogranin A epitopes: clues from synthetic peptides and peptide mapping.

Chromogranin A (CgA) is a 48 kDa acidic protein in neuroendocrine secretory vesicles whose primary structure is now known. We used synthetic peptides, synthetic peptide antisera, intact molecule antisera, chymotryptic peptide mapping, microsequencing, immunoblotting, and immunoprecipitation to probe the location of immunodominant domains within the CgA molecule. Polyclonal anti mid-molecule, anti N-terminal and anti C-terminal antibodies specifically visualized CgA (both bovine and human) in one and two dimensional immunoblots of adrenal chromaffin vesicles, and the stain CgA fragments further suggested bidirectional (both N- and C-terminal) cleavage or processing of CgA. Anti intact CgA immunoblotting of HPLC-separated peptides from chymotrypsin-digested bovine CgA revealed several strongly immunoreactive internal peptides, two of which were positioned by N-terminal amino acid sequencing: CgA91ff. and CgA197ff.. A single synthetic peptide (CgA79-113) was recognized by three antibodies developed against the intact CgA molecule: two polyclonal rabbit antisera as well as a monoclonal mouse antibody. Not all antigenicity algorithm-predicted domains were immunogenic, suggesting that some of these predicted domains may not be accessible. Polyclonal anti mid-molecule, anti N- and anti C-terminal synthetic peptide antisera specifically immunoprecipitated 125I-labeled bovine CgA from aqueous solution; mid-molecule antisera precipitated substantially greater amounts than terminal antisera. The immunoprecipitation results suggested exposed terminal as well as interior hydrophilic epitopes in the molecule in its intact, native conformation. 125I-human CgA was best precipitated by anti N-terminal antisera, consistent with greatest interspecies sequence conservation at the N-terminus of CgA. The terminal antisera reacted immunohistochemically in a granular pattern with adrenal medullary chromaffin cells (but not adrenal cortical cells) and pancreatic islet cells (but not pancreatic exocrine acini). Thus, synthetic and chymotryptic peptides yielded novel and specific insights into the structure, conformation, vesicular processing and interior immunodominant domains of CgA.