Recombinant alpha2(IV)NC1 domain inhibits tumor cell-extracellular matrix interactions, induces cellular senescence, and inhibits tumor growth in vivo

Cellular interaction with the extracellular matrix is thought to be a critical event in controlling angiogenesis and tumor growth. In our previous studies, genetically distinct noncollagenous (NC) domains of type-IV collagen were shown to interact with integrin receptors expressed on the surface of endothelial cells. Moreover, these NC1 domains were shown to inhibit angiogenesis in vivo. Here, we provide evidence that a recombinant form of the alpha2(IV)NC1 domain of type-IV collagen could bind integrins alpha1beta1 and alphavbeta3 expressed on melanoma cells and inhibit tumor cell adhesion in a ligand-specific manner. Systemic administration of recombinant alpha2(IV)NC1 domain potently inhibited M21 melanoma tumor growth within full thickness human skin and exhibited a dose-dependent inhibition of tumor growth in nude mice. Interestingly, alpha2(IV)NC1 domain enhanced cellular senescence in tumor cells in vitro and in vivo. Taken together, these results suggest that recombinant alpha2(IV)NC1 domain is not only a potent anti-angiogenic reagent, but it also directly impacts tumor cell behavior. Thus, alpha2(IV)NC1 domain represents a potent inhibitor of tumor growth by impacting both endothelial and tumor cell compartments.     

Lipopolysaccharide-binding proteins of Limulus amebocyte lysate.

Limulus amebocyte lysate, obtained from horseshoe crab (Limulus polyphemus) blood cells, contains a coagulation system which is activated by bacterial lipopolysaccharide (LPS). A chromatographic fraction of Limulus lysate, containing the endotoxin-sensitive factor(s) which initiates the coagulation cascade, was studied. We utilized a photoreactive, cleavable, radiolabeled derivative of Salmonella minnesota LPS, LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide (LPS-ASD), to identify LPS-binding proteins. The lysate fraction was incubated with LPS-ASD, and LPS-binding proteins were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. An 82-kDa protein, a major protein component of this fraction from Limulus lysate, was identified as a LPS-binding protein in a majority of lysates. Incubation of whole Limulus lysate with antiserum to this protein resulted in enhanced sensitivity of the lysate to LPS, suggesting that this 82-kDa protein is a negative regulator of coagulation. A minor 50-kDa protein component of lysate also was identified as a LPS-binding protein and is a candidate for the LPS-sensitive coagulation protein in L. polyphemus.     

Malignant schwannoma with melanocytic and neuroepithelial differentiation in an infant with congenital giant melanocytic nevus: A complex neurocristopathy

We describe an infant girl, born with a pigmented giant nevus, who developed a malignant schwannoma in the retroperitoneum at 16 months of age. At birth the nevus covered over 50% of her body and histologically was a compound nevus with extension into the deep dermis surrounding dermal appendages. The malignant schwannoma was biphasic with areas composed of spindle and round cells. Ultrastructurally, the majority of the tumor cells exhibited a Schwann cell phenotype, but neuroepithelial and melanocytic cells were identified as well. We believe that this constellation of findings represents a form of neurocristopathy. Neurocristopathy, as defined by Bolande (Hum Pathol 5:409–429, 1974), is a disease that results from aberrations in the migration, growth, or cytodifferentiation of neural crest tissues. These diseases may be simple (a singular pathologic process, usually localized) or complex (multiple neuroectodermal lesions). We report this case because the occurrence of retroperitoneal malignant schwannoma arising in a 16-month-old infant born with a pigmented giant nevus is unique, and may represent a previously undescribed form of a complex neurocristopathy.     

Synthesis and characterization of poly[2,3,9,10,16,17,23,24-octakis(dodecyloxycarbonyl)phthalocyaninatogermoxane]

In this article we describe the preparation of poly[2,3,9,10,16,17,23,24-octakis(dodecyloxycarbonyl)phthalocyaninatogermoxane] by thermal polycondensation of 2,3,9,10,16,17,23,24-octakis(dodecyloxycarbonyl)phthalocyaninatogermanium dihydroxide, and its characterization. The average molar mass of the resulting polymer was determined. High molar masses are assessible, e.g., M?_w = 360000 g/mol, M?_n = 140000 g/mol, at moderate temperature for the polycondensation (200°C). The polymer shows a liquid-crystalline phase in an extremely large temperature range. Furthermore, Langmuir-Blodgett films (LB films) of the polymer and of the monomer were prepared and characterized. In LB films the phthalocyanine rings are preferentially oriented normal to the dipping direction.     

Improved accuracy in diagnostic immunohistochemistry, lectin histochemistry and in situ hybridization using a gold-labeled horseradish peroxidase antibody and silver intensification.

BACKGROUND: Improvements in the use of the avidin-biotin peroxidase complex technique and direct as well as indirect labeled avidin-biotin methods for application in diagnostic immunohistochemistry, lectin histochemistry and in situ hybridization are reported. The new technology combines the advantages of immunoenzyme and immunogold silver staining techniques and can be performed on routinely fixed and paraffin-embedded tissues. EXPERIMENTAL DESIGN: The basic modification of the labeling procedures was introduced at the final revealing step. The histochemical visualization of catalytic activity of horseradish peroxidase by the diaminobenzidine reaction was replaced by the detection of horseradish peroxidase immunoreactivity using anti-horseradish peroxidase-gold complexes and their intensification with silver acetate which is relatively light insensitive. RESULTS: The use of gold-labeled anti-horseradish peroxidase antibodies eliminates the need for quenching of endogenous peroxidase activity. Furthermore, the immunogold silver staining provides improved lateral resolution, higher contrast, and lower background staining as compared with the diaminobenzidine reaction. The new technology has been applied for the localization of different polypeptides in endocrine cells, cytoskeletal elements, cell surface receptors, basal lamina type IV collagen, endothelial cell marker, lectin binding sites, and DNA of various viruses. CONCLUSIONS: We concluded that the anti-horseradish peroxidase-gold complex is of general use in a variety of techniques applying horseradish peroxidase as a marker and should be a valuable alternative to existing enzyme substrate techniques.     

NCAM in developing mouse gonads and ducts

Summary The expression of theNeuralCell AdhesionMolecule, NCAM, in mouse gonads and ducts was studied from fetal life to maturity. The methods used were immunocytochemical staining and Western blotting. The immunocytochemical studies showed that the only structures that remain NCAM-positive throughout life were the mesonephric-derived rete ovarii and rete testis. Also in the fetal gonads some somatic cell lining the groups of differentiating germ cells were stained. In the immature as well as in the mature ovary the granulosa cells and oocytes of growing and large follicles — but not of small follicles — were stained. A particularly strong staining of the cytoplasm of the oocyte, healthy as well as atretic, was seen. All cells of the testis remained negative except for weakly stained residual bodies and late spermatids. At all ages the male ducts showed only weak staining, whereas in the female Müllerian duct the epithelium became strongly positive at puberty. The stroma of the Müllerian duct was positive during a transitory period around day 16 of fetal life in both sexes. One-dimensional gel immunoblotting of total protein from gonads, rete and ducts from immature and mature mice showed that only the two largest isoforms of NCAM (NCAM-A and NCAM-B) were present. The gonads and the rete of both sexes and the adult uterus expressed only NCAM-B, whereas NCAM-A was also detected in the adult epididymis. The present findings suggest that NCAM may be involved in the normal development and formation of both the gonads and ducts. In particular, NCAM may play a part in sustaining the integrity of the rete testis, thus ensuring the pathway for spermatozoa from the testis to the epididymis. Furthermore this cell adhesion molecule may also be important for follicular growth and differentiation.     

Sequestration of tau by granulovacuolar degeneration in Alzheimer''s disease.

Antibodies directed against three regions of tau have been used in a histologic study of granulovacuolar degeneration (GVD) in Alzheimer's disease (AD). Granulovascular degeneration complexes, consisting of a dense granule in a less-dense vacuole, were found in hippocampal pyramidal neurons in all patients studied. Anti-tau antibodies directed against the N-and C-termini, and the repeat region of tau, were found to immunolabel the granule of the GVD complex. Intracellular neurofibrillary tangles also were labeled by these antibodies. In particular, MAb6.423, which recognizes tau protein sequestered in paired helical filaments (PHF) in AD, but not the normal tau proteins so far described in human brain, labeled GVD granules. Contrarily, a generic tau marker (MAb7.51), which immunolabels all known isoforms of isolated and expressed tau protein, including PHF-tau, did not label the GVD granule. These findings demonstrate that the entire tau molecule is sequestered within the GVD granule, and that the tau protein found in GVD complexes is antigenically related to that found in PHFs. There is, however, a difference in the way in which the repeat region of tau is incorporated into the two structures, making the MAb7.51 epitope unavailable in the GVD complex. These findings suggest that the formation of GVD complexes in hippocampal pyramidal neurons vulnerable to neurofibrillary degeneration may represent an alternative pathway for dealing with an aberrant molecular complex, which contributes to the formation of GVD granules and neurofibrillary tangles in AD.     

Hormonally Functional Ovarian Neoplasms

Hormonally functional ovarian neoplasms are those tumors that secrete one or more hormones that are clinically manifested in the patient. The hormone production may have implications for the diagnosis, management, or treatment of the patient. Hormonally functional ovarian neoplasms include tumors that belong to various histologic categories and produce a variety of hormonal effects. Functional ovarian tumors most commonly produce steroid hormones, and such tumors frequently belong in the sec cordstromal and steroid cell categories. In addition, a wide variety of peptide hormones may be produced by ovarian tumors. Although in most instances the neoplastic cells themselves produce the hormones, a wide variety of tumors may induce their stroma to produce steroid hormones. The stroma of ovarian tumors is derived from the ovarian stroma and may, on occasion, resemble specialized ovarian stroma and its derivatives. Cells resembling luteinized stromal cells or luteinized theca cells may be present and appear to be responsible for the resultant hormone secretion.     

Identification of novel regions of allelic loss from a genomewide scan of esophageal squamous-cell carcinoma in a high-risk Chinese population

Esophageal cancer is one of the most common fatal cancers worldwide. Deletions of genomic regions are thought to be important in esophageal carcinogenesis. We conducted a genomewide scan for regions of allelic loss using microdissected DNA from 11 esophageal squamous-cell carcinoma patients with a family history of upper gastrointestinal tract cancer from a high-risk region in north central China. Allelic patterns of 366 fluorescently labeled microsatellite markers distributed at 10-cM intervals over the 22 autosomal chromosomes were examined. We identified 14 regions with very high frequency (>/= 75%) loss of heterozygosity (LOH), including broad regions encompassing whole chromosome arms (on 3p, 5q, 9p, 9q, and 13q), regions of intermediate size (on 2q, 4p, 11p, and 15q), and more discrete regions identified by very high frequency LOH for a single marker (on 4q, 6q, 8p, 14q, and 17p). Among these 14 regions were 7 not previously described in esophageal squamous-cell carcinoma as having very high frequency LOH (on 2q, 4p, 4q, 6q, 8p, 14q, and 15q). The very high frequency LOH regions identified here may point to major susceptibility genes, including potential tumor suppressor genes and inherited gene loci, which will assist in understanding the molecular events involved in esophageal carcinogenesis and may help in the development of markers for genetic susceptibility testing and screening for the early detection of this cancer. Genes Chromosomes Cancer 27:217-228, 2000. Published 2000 Wiley-Liss, Inc.     

Mutations and allelic deletions of the MEN1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms

Endocrine pancreatic tumors (EPT) and neuroendocrine tumors (NET) occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). We analyzed the frequency of allelic deletions and mutations of the recently identified MEN1 gene in 53 sporadic tumors including 30 EPT and 23 NET (carcinoids) of different locations and types. Allelic deletion of the MEN1 locus was identified in 18/49 (36.7%) tumors (13/30, 43.3% in EPT and 5/19, 26.3% in NET) and mutations of the MEN1 gene were present in 8/52 (15.3%) tumors (4/30 (13.3%) EPT and 4/22 (18.1%) NET). The somatic mutations were clustered in the 5' region of the coding sequence and most frequently encompassed missense mutations. All tumors with mutations exhibited a loss of the other allele and a wild-type sequence of the MEN1 gene in nontumorous DNA. In one additional patient with a NET of the lung and no clinical signs or history of MEN1, a 5178-9G-->A splice donor site mutation in intron 4 was identified in both the tumor and blood DNA, indicating the presence of a thus far unknown MEN1 syndrome. In most tumor groups the frequency of allelic deletions at 11q13 was 2 to 3 times higher than the frequency of identified MEN1 gene mutations. Some tumor types, including rare forms of EPT and NET of the duodenum and small intestine, exhibited mutations more frequently than other types. Furthermore, somatic mutations were not restricted to foregut tumors but were also detectable in a midgut tumor (15.2% versus 16.6%). Our data indicate that somatic MEN1 gene mutations contribute to a subset of sporadic EPT and NET, including midgut tumors. Because the frequency of mutations varies significantly among the investigated tumor subgroups and allelic deletions are 2 to 3 times more frequently observed, factors other than MEN1 gene inactivation, including other tumor-suppressor genes on 11q13, may also be involved in the tumorigenesis of these neoplasms.