Zeenat Gunja-Smith

CD44v3,8–10 is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells

In the present study, we have employed a unique breast cancer cell line (Met‐1, which was derived from a high metastatic potential tumor in transgenic mice expressing polyomavirus middle T oncogene) to study the role of CD44 variant isoform(s) in the regulation of metastatic breast tumor cell behavior. The results of reverse transcriptase–polymerase chain reaction, Southern blot, nucleotide sequencing, immunoprecipitation, and immunoblot analyses indicated that these cells express a major CD44 isoform (molecular weight ≈260 kDa) containing a v3,8–10 exon insertion (designated as CD44v3,8–10). In addition, we have determined that CD44v3,8–10 binds specifically to the cytoskeletal proteins such as ankyrin. Biochemical analyses, using competition binding assays and a synthetic peptide identical to NGGNGTVEDRKPSEL (a sequence located between aa480 and aa494 of CD44v3,8–10) indicate that this 15‐amino acid peptide binds specifically to the cytoskeletal protein ankyrin (but not to fodrin or spectrin). This peptide competes effectively for ankyrin binding to CD44v3,8–10. Therefore, we believe that the sequence 480NGGNGTVEDRKPSE494L, located at the cytoplasmic domain of CD44v3,8–10, is required for the ankyrin binding. We have also detected that CD44v3,8–10‐containing Met‐1 cells are capable of forming membrane spikes or “invadopodia” structures and undergo active migration processes. Treatments of Met‐1 cells with certain agents including anti‐CD44v3 antibody, cytochalasin D (a microfilament inhibitor), and W‐7 (a calmodulin antagonist), but not colchicine (a microtubule disrupting agent) effectively inhibit “invadopodia” formation and subsequent tumor cell migration. Further analyses using zymography assays and double immunofluorescence staining indicated that CD44v3,8–10 is closely associated with the active form of matrix metalloproteinase, MMP‐9, in a complex within “invadopodia” structures. These findings suggest that CD44v3,8–10 plays an important role in linking ankyrin to the membrane‐associated actomyosin contractile system required for “invadopodia” formation (coupled with matrix degradation activities) and tumor cell migration during breast cancer progression. J. Cell. Physiol. 176:206–215, 1998.

A glycogen-debranching enzyme from Cytophaga

Potato R-enzyme [1 ] and bacterial pullulanase [2, 3] hydrolyse the a-1,6-linkages in pullulan and a-limit dextrins and also cleave the a-1,6-branch linkages in amylopectin [4]. The availability of highly purified preparations of pullulanase from Aerobacter aerogenes [5] has made this enzyme invaluable in the analysis of the fine structure of amylopectin [6]. Pullulanase, however, is of limited use in the analysis of glycogen structure because, although it is able to hydrolyse some branch linkages in degraded glycogen, it has little or no action on the undegraded macromolecule [7]. Yeast isoamylase [8] hydrolyses a limited proportion of the interchain linkages of amylopectin and glycogen but, unlike pullulanase, does not act on the 1,6-linkages of pullulan. Recently, an extracellular isoamylase from a new strain o f Pseudomonas was reported to hydrolyse almost all the branch linkages of amylopectin and glycogen [9, 10]. The importance of this type of enzyme for the analysis of glycogen structure prompts us to report the discovery of an isoamylase in a species of Cytophaga. Studies of the partially purified enzyme indicate that its specificity of action is similar to that of the Pseudomonas enzyme and that it has the ability to hydrolyse the branch linkages of amylopectin and glycogen with the complete dismemberment of the branched macromolecules.

The Marfan Syndrome: A Deficiency in Chemically Stable Collagen Cross-Links

THE reduced tensile strength of tissues supporting the ocular lenses, cardiac valves, and aorta in heritable connective-tissue diseases is probably due to a defective organization of collagen. Sinc...

Up-Regulation of Matrix Metalloproteinase-9 in T Lymphocytes of Mammary Tumor Bearers: Role of Vascular Endothelial Growth Factor

Matrix metalloproteinase-9 (MMP-9), a matrix-degrading enzyme, is crucial in tumor invasion and metastasis and is implicated in leukocyte extravasation. In this report, we demonstrate that during growth of the D1–7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is progressive up-regulation of MMP-9 in splenic T cells at both the transcriptional and translational levels. Our previous work has identified several factors produced by this tumor, including PGE2, GM-CSF, and phosphatidyl serine; however, none of these agents induces increased production of MMP-9 by normal splenic T cells. Although not produced by the tumor, TNF-α and IL-6 are up-regulated in both macrophages and B cells in tumor-bearing mice. Exposure of normal T cells to these two cytokines, however, also fails to up-regulate MMP-9 production. Vascular endothelial growth factor (VEGF) is produced by many tumors, and we determined that the mammary tumor used in our studies expresses high levels of this angiogenic growth factor. Importantly, splenic T cells from tumor bearers constitutively produce increased amounts of VEGF, and treatment of normal T cells with VEGF results in up-regulated MMP-9 production. Of crucial importance is the finding that tumor-infiltrating T cells also produce high levels of VEGF and MMP-9. Our studies indicate that VEGF can act directly on T lymphocytes and that elevated VEGF levels may contribute to the aberrant MMP-9 secretion by mammary tumor bearers’ T cells.

An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples

An enzyme-linked immunosorbent assay method has been developed for the determination of desmosine. The method is based on an inhibition immunoassay (under nonequilibrium conditions) and uses rabbit antisera directed against a desmosine-bovine serum albumin conjugate and microtiter plates coated with desmosine-gelatin conjugate. The assay quantitates desmosine in the range 2.5-50 pmol in tissue and urine samples. Important applications of this rapid and sensitive assay are in studying elastin metabolism and in screening for monoclonal antibodies against desmosine. Methods are described for obtaining a constant level of substitution of desmosine per molecule of bovine serum albumin and for preparing a desmosine-gelatin coating antigen. Five different antibody preparations directed against desmosine exhibit 15-20% cross-reactivity toward pyridinoline (3-hydroxypyridinium), a nonreducible collagen crosslinking compound also present in urine and many tissue samples.

Changes in Desmosine and Pyridinoline Crosslinks During Rapid Synthesis and Degradation of Elastin and Collagen in the Rat Uterus

The wet weight of the rat uterus increased 8-fold during pregnancy and fell by 70% within 5 days postpartum. Uterine collagen increased about 5-fold during pregnancy and also fell by 70% within 5 days. The crosslink pyridinoline remained constant at 0.28 mole/mole collagen at every time point, with the possible exception of 11-12 days of pregnancy. The pyridinoline link can therefore form within the short time span of a few days, a feature presumed to be necessary to maintain the full mechanical strength of the uterus during labor. Uterine elastin increased about 8-fold during pregnancy, but the desmosines did not keep pace and fell from a normal value of 1.43 mole/mole elastin to a low of 0.89 at term. Moreover, elastin content reached a maximum several days prior to parturition and then declined continuously to 5 days postpartum. During this decline there was a selective loss of the poorly crosslinked elastin. The desmosines cannot be used as a direct measure of uterine elastin content, because of their continuously changing levels. Desmosines and pyridinoline were measured both by ELISA and by the amino acid analyzer. The two methods gave almost identical results when elastin and collagen were first separated from each other.

Content of the collagen and elastin cross-links pyridinoline and the desmosines in the human uterus in various reproductive states.

During pregnancy the collagen content of the human uterus increases sevenfold and the elastin content increases fourfold to fivefold. The stable pyridinoline cross-link is found in uterine collagen at a level of 0.11 mol per mole of collagen. The same ratio, or a higher one, is found at the end of pregnancy, indicating that pyridinoline synthesis keeps pace with the rapid synthesis of collagen. This cross-link would participate in the maintenance of high mechanical strength of the uterus needed during parturition. Uterine elastin contains 2.4 residues of desmosine plus isodesmosine in 1000 residues of amino acids. This value falls to 0.95 at term, indicating that synthesis of desmosines does not keep pace with the synthesis of elastin. Therefore, desmosine measurements do not provide an accurate index of elastin changes in pregnancy. Collagen and elastin contents in nongravid uteri increase with successive pregnancies; the cross-links remain constant during this change.

Evidence for the periplasmic location of glycogen in Saccharomyces.

Abstract Treatment of yeast cells with hot alkali fails to solubilize a significant amount of glycogen. The insoluble glycogen is readily hydrolyzed by insolubilized α-amylase indicating that the apparent insolubility of this glycogen fraction does not result from its physical entrapment within an insoluble glucan membrane. The alkali-insoluble glycogen fraction of glutaraldehyde treated-cells is rapidly degraded by a mixture of snail gut enzymes during the formation of spheroplasts but the alkali-soluble glycogen fraction is unaffected. These results indicate that a major fraction of yeast glycogen is located outside the cellular membrane.

Activation of cartilage stromelysin-1 at acid pH and its relation to enzyme pH optimum and osteoarthritis

Stromelysin-1 was purified from human articular cartilage and compared to synovial fibroblast enzyme and to recombinant enzyme. If the latent enzyme was incubated at pH 5.5 with substrates such as aggrecan, it spontaneously became active. Incubation of latent zymogen alone at pH 5.5 gave increasing activation over a periodof at least 5 hours. However, this activation process was not accompanied by any shift in molecular weight even after continued incubation for 18 hours. Maximum activity observed was 45–60% of that seen with APMA activation. Stromelysin-1 has a pH optimum of 5.5–6.5 on various macromolecular and peptide substrates. Interaction with TIMP is reduced at pH 5.5 relative to that at 7.5. The hypothesis is presented that osteoarthritis may be initiated by acid activation of stromelysin-1.

Considerations of aortic elastin chemistry in the genesis of the intimal plaque (Broad-Breasted White turkey)

Abstract Fragmentation of internal and external elastic membranes occurs in the abdominal but not in the thoracic aorta of the Broad-Breasted White turkey during the early developmental period. In this early period, isolated elastin from the abdominal aorta contains more polar amino acid residues than elastin of the thoracic aorta. Additionally, the relative amount of a chemically unstable elastin cross-link, dehydrolysinonorleucine, is greater in the abdominal as compared to the thoracic aorta during the early developmental and adult periods. Cells of the abdominal aorta exposed to a higher arterial pressure and a greater rate of arterial pressure rise ( dp dt ) and in a region with a higher collagen:elastin ratio (reduced compliance to pulse-pressure-volume deformations) may synthesize a chemically unstable elastin which breaks down in regions of accentuated wall stress. Though these breaks of the internal elastic membranes, modified smooth muscle cells may migrate, proliferate and ultimately synthesize new elastin in forming the intimal plaque.