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M M Zutter

Publications and source records attributed to M M Zutter.

At least 37 records · Page 2Linked to original sources
PubMed1997

NAG-2, a novel transmembrane-4 superfamily (TM4SF) protein that complexes with integrins and other TM4SF proteins.

Transmembrane-4 superfamily (TM4SF) proteins form complexes with integrins and other cell-surface proteins. To further characterize the major proteins present in a typical TM4SF protein complex, we raised monoclonal antibodies against proteins co-immunoprecipitated with CD81 from MDA-MB-435 breast cancer cells. Only two types of cell-surface proteins were recognized by our 35 selected antibodies. These included an integrin (alpha6beta1) and three different TM4SF proteins (CD9, CD63, and NAG-2). The protein NAG-2 (novel antigen-2) is a previously unknown 30-kDa cell-surface protein. Using an expression cloning protocol, cDNA encoding NAG-2 was isolated. When aligned with other TM4SF proteins, the deduced amino acid sequence of NAG-2 showed most identity (34%) to CD53. Flow cytometry, Northern blotting, and immunohistochemistry showed that NAG-2 is widely present in multiple tissues and cell types but is absent from brain, lymphoid cells, and platelets. Within various tissues, strongest staining was seen on fibroblasts, endothelial cells, follicular dendritic cells, and mesothelial cells. In nonstringent detergent, NAG-2 protein was co-immunoprecipitated with other TM4SF members (CD9 and CD81) and integrins (alpha3beta1 and alpha6beta1). Also, two-color immunofluorescence showed that NAG-2 was co-localized with CD81 on the surface of spread HT1080 cells. These results confirm the presence of NAG-2 in specific TM4SF.TM4SF and TM4SF-integrin complexes.

Amino Acid Sequence↗
PubMed1997

Binding of phosphorylated Sp1 protein to tandem Sp1 binding sites regulates alpha2 integrin gene core promoter activity.

The alpha2beta1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the alpha2beta1, integrin in cells with megakaryocytic differentiation, we characterized the 5' flanking region of the alpha2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5' flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the alpha2 integrin gene located between bp -30 and -92 that is required for transcriptional activity of the alpha2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp -30 to bp -92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5', the 3', or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5' Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5' and the 3' Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

Antigens, CD↗
PubMed1996

PDGF induction of alpha 2 integrin gene expression is mediated by protein kinase C-zeta.

Platelet-derived growth factor (PDGF) stimulates fibroblasts to move over collagen and contract three-dimensional collagen gels, processes important in wound repair and fibrocontractive diseases. These processes depend on alpha 2 beta 1 integrin ligation of collagen and PDGF induces the expression of this integrin. Several lines of evidence presented here suggest that PKC-zeta plays a role in alpha 2 integrin gene expression. The induction was blocked by chemical inhibitors for protein tyrosine kinases (PTK), genistein, and protein kinase C (PKC), chelerythrine, and bisindolylmaleimide GF 109203X. Cells depleted of phorbol 12-myristate 13-acetate (PMA)-inducible PKCs by chronic treatment with PMA still demonstrated an alpha 2 response to PDGF indicating that a non-PMA-sensitive PKC isoform was required. PDGF induced kinase activity in PKC-zeta immunoprecipitates. Antisense oligonucleotides complementary to 5' end of PKC-zeta mRNA sequences blocked the PDGF-induced increase of alpha 2 mRNA levels up to 70%, indicating PKC-zeta, a non-PMA-sensitive PKC isoform, is a component of the PDGF stimulatory pathway for alpha 2 mRNA synthesis. A 961-base pair (bp) upstream region of alpha 2 gene/CAT construct transfected into human dermal fibroblasts was positively regulated by PDGF as judged by CAT enzymatic levels. Both PTK and PKC inhibitors blocked PDGF-stimulation of the alpha 2 promoter fragment/CAT construct, indicating that the phosphorylation requirement occurred at alpha 2 promoter-directed transcription level. Therefore, we propose that PDGF-stimulatory pathway of alpha 2 integrin gene expression involves multiple cellular protein kinases, one of which is PKC-zeta.

Adult↗
PubMed1996

Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins).

Here we identified several new integrin/TM4 protein complexes on the cell surface. By immunoprecipitation using nonstringent conditions, and by reciprocal immunoprecipitation, we found that alpha 3 beta 1 and alpha 6 beta 1 integrins but not alpha 2 beta 1, alpha 5 beta 1, or alpha 6 beta 4 integrins associated with CD9 and CD81 in alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, alpha 6 beta 1/CD81, and alpha 6 beta 1/CD9 complexes. Also, cross-linking experiments established that alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, and alpha 3 beta 1/CD63 associations occur on the surface of intact cells and suggested that a critical interaction site is located within extracellular domains. Cross-linking in conjunction with reimmunoprecipitation indicated that larger multi-component alpha 3 beta 1/TM4/TM4 complexes (alpha 3 beta 1/CD9/CD63, alpha 3 beta 1/CD81/CD63, and alpha 3 beta 1/CD9/CD81) also could be detected on the cell surface. Immunofluorescent staining showed redistribution of alpha 3 beta 1/TM4 complexes toward the periphery of cells plated on various extracellular matrix substrates and also showed that these complexes were localized in cell footprints. Staining of human tissues yielded additional results consistent with co-localization of alpha 3 beta 1 and CD9, CD63, and CD81 proteins. In conclusion we suggest that the prevalence of integrin/TM4 complexes in diverse cellular environments is indicative of their general physiological importance.

Antigens, CD↗
PubMed1996

Juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is an aggressive myeloproliferative disorder of childhood that differs both clinically and pathologically from adult type, Philadelphia chromosome positive chronic myelogenous leukemia, and from the other myeloproliferative disorders that are more common in adulthood. The disease can have widely varying clinical presentations and shares many features with the monosomy 7 syndrome and chronic myelomonocytic leukemia. With no specific marker chromosome, establishing the diagnosis can be difficult, and relies on a constellation of clinical, pathologic, and laboratory findings. This article discusses the differential diagnosis of JCML with an emphasis on the pathologic findings and laboratory data that are particularly important for confirming the diagnosis. The sensitivity, specificity, and clinical utility of cell culture colony assays are reviewed. Finally, current knowledge of the biology of JCML and some of the controversies regarding this disease are discussed.

Biopsy, Needle↗
PubMed1996

Immunophenotypic analysis of hematologic malignancy by laser scanning cytometry.

The authors tested a newly-developed computerized laser scanning cytometer (LSC) as a means of performing immunophenotypic analysis of hematologic specimens within their community hospital. Results were compared on a case-by-case basis with parallel flow cytometric and immunohistochemical data. A total of 71 specimens analyzed include 22 excised lymph nodes or other tissue biopsies, 18 peripheral bloods, 17 bone marrow aspirates, 7 body fluids, and 7 fine-needle aspiration biopsies of lymphoid tissue. The LSC proved to be a useful instrument capable of generating simultaneous two-color immunofluorescent data directly analogous to that obtained via conventional flow cytometry. However, laser scanning cytometric analysis provides advantages over flow cytometric analysis, because the LSC measures cells on a slide rather than in a fluid stream. Specifically, cells can be microscopically examined at any time--before, during, or after automated immunofluorescent analysis. In addition, specimen preparation techniques are less restricted and more cost efficient. Lastly, even extremely small and/or hypocellular specimens (such as body fluids and fine-needle aspiration biopsies) can be successfully analyzed.

Diagnosis, Computer-Assisted↗
PubMed1995

Regulation of alpha 2 integrin gene expression in cells with megakaryocytic features: a common theme of three necessary elements.

The alpha 2 beta 1 integrin mediates interactions between cells and the extracellular matrix molecules, collagen and/or laminin. The alpha 2 beta 1 integrin is expressed in a variety of cell types, but in cells of hematopoietic lineage, expression is restricted to megakaryocytes and platelets. Increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of transcriptional activation of the alpha 2 gene. We have begun to characterize the role of the 5' flanking region of the alpha 2 integrin gene in regulating expression during megakaryocyte differentiation. A 5-kb fragment of the 5' region directs both cell type and differentiation-dependent expression of a reporter gene in the pluripotent hematopoietic K562 cells upon megakaryocytic differentiation and in the megakaryocytic cell line, Dami. Analysis of a series of 5' deletion mutants indicates that expression of the alpha 2 integrin gene in cells with megakaryocytic features requires a core promoter region, a silencer region, and megakaryocytic enhancers in the distal 5' end. The organization of these three distinct regulatory regions of the alpha 2 promoter/enhancer suggests a common theme for megakaryocytic gene regulation shared with other megakaryocyte-specific proteins, including alpha IIb integrin subunit and platelet factor 4.

Antigens, CD↗
PubMed1995

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells.

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.

Animals↗
PubMed1995

Alteration of collagen-dependent adhesion, motility, and morphogenesis by the expression of antisense alpha 2 integrin mRNA in mammary cells.

Although integrins are known to mediate adhesive binding of cells to the extracellular matrix, their role in mediating cellular growth, morphology, and differentiation is less clear. To determine more directly the role of the alpha 2 beta 1 integrin, a collagen and laminin receptor, in mediating the collagen-dependent differentiation of mammary cells, we reduced expression of the integrin by the well differentiated human breast carcinoma cell line, T47D, by stably expressing alpha 2 integrin antisense mRNA. Flow cytometry demonstrated that the antisense-expressing clones had levels of alpha 2 beta 1 integrin on their surfaces that were decreased by 30-70%. Adhesion of antisense-expressing clones to both collagens I and IV was decreased relative to controls in a manner that correlated with the level of cell surface alpha 2 beta 1 integrin expression. Adhesion to fibronectin and laminin were not affected. Motility across collagen-coated filters in haptotaxis assays was increased for only those clones that exhibited intermediate levels of adhesion to collagen, suggesting that an intermediate density of cell-surface alpha 2 beta 1 integrin optimally supports cell motility. When cultured in three-dimensional collagen gels, T47D cells organized in a manner suggestive of a glandular epithelium. In contrast, antisense-expressing clones with decreased alpha 2 beta 1 integrin were not able to organize in three-dimensional collagen gels. The growth rate of T47D cells was reduced when the cells were cultured in three-dimensional collagen gels. Unlike adhesion, motility, and morphogenesis, growth rates were unaffected by reduction of alpha 2 beta 1 integrin expression. Our results suggest that adhesive interactions mediated by a critical level of surface alpha 2 beta 1 integrin expression are key determinants of the collagen-dependent morphogenetic capacity of mammary epithelial cells.

Animals↗
PubMed1994

The human alpha 2 integrin gene promoter. Identification of positive and negative regulatory elements important for cell-type and developmentally restricted gene expression.

The alpha 2 beta 1 serves as a collagen receptor or a collagen/laminin receptor, depending upon cell type. Expression of the integrin is regulated during normal cellular differentiation and is altered during carcinogenesis. We have previously demonstrated that increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of increased alpha 2 mRNA due to transcriptional activation of the alpha 2 integrin gene and that the decreased expression of the integrin in breast adenocarcinoma is due to decreased steady-state levels of alpha 2 mRNA. We now report the identification and characterization of the 5'-flanking region of the alpha 2 integrin gene. The 5'-untranslated region of the alpha 2 mRNA extends 129 base pairs 5' to the site of translation initiation. The promoter region lacks TATA and CAAT boxes but contains an abbreviated initiator sequence and six Sp1 binding sites. Consensus binding sites for AP-1 and AP-2 complexes, a GATA box, a Pu.1 box, and two palindromic motifs with potential to bind the estrogen receptor are also present. A 961-base pair fragment of the 5'-flanking region directs both cell type- and differentiation-specific expression of a reporter gene in T47-D epithelial cells and in pluripotent hematopoietic K562 cells upon megakaryocytic differentiation.

Amino Acid Sequence↗
PubMed1994

Dysplastic haemopoiesis following orthotopic liver transplantation: comparison with similar changes in HIV infection and primary myelodysplasia.

To validate scientifically our prior empiric observations that patients develop significant haemopoietic dysplasia following solid organ transplantation, we developed a quantitative lineage-specific scoring system to evaluate dysplastic features of bone marrow aspirates and core biopsies. We used this scoring system to compare retrospectively randomly selected bone marrow aspirates and core biopsies from 19 patients undergoing orthotopic liver transplantation (OLT), 21 with a known history of human immunodeficiency virus (HIV) infection, and 18 with primary or chemotherapy-related myelodysplastic syndromes (MDS). Our results show that the OLT patient group developed significant but milder haemopoietic dysplastic changes than the HIV or MDS groups, and that the MDS group developed more severe dysplasia of the myeloid lineage than the other groups. The possible roles for drugs and infectious agents in the pathophysiology of dysplastic changes are discussed.

Biopsy↗
PubMed1993

Recurrent neuroendocrine (Merkel cell) carcinoma of the skin presenting as marrow failure in a man with systemic lupus erythematosus.

Neuroendocrine carcinoma of the skin, Merkel cell carcinoma, is an aggressive malignancy with a high incidence of local recurrence and metastasis. We describe a case of neuroendocrine carcinoma of the skin with unusual metastatic spread to the bone marrow in a 55-year-old man with systemic lupus erythematosus. Following resection of a left temporal subcutaneous nodule and complete left parotidectomy and radical lymph node dissection, the patient presented with pancytopenia. Although focal bony metastatic disease was not identified, his bone marrow was diffusely replaced by metastatic carcinoma. High-dose chemotherapy allowed a brief remission; however, the patient subsequently died with central nervous system metastases.

Bone Marrow Diseases↗
PubMed1993

Megakaryoblastic leukemia presenting as a temporal bone granulocytic sarcoma. Case report.

The unusual presentation of acute megakaryoblastic leukemia as a temporal bone granulocytic sarcoma in an infant without systemic manifestations of leukemia is reviewed. Leukemia should be considered in the differential diagnosis of skull and skull-based lesions since the appearance on neuroradiological imaging is not unique in this diagnosis. Surgical treatment, as in this case, is limited to obtaining tissue for diagnosis and draining the infection.

Child, Preschool↗
PubMed1993

Altered integrin expression in adenocarcinoma of the breast. Analysis by in situ hybridization.

The integrin superfamily of adhesion receptors mediates interactions between cells and the extracellular matrix. Our earlier immunohistochemical analysis showed that normal mammary epithelium expressed high levels of the alpha 2 beta 1 collagen/laminin receptor and intermediate levels of the alpha 5 beta 1 fibronectin receptor. In contrast, malignant cells of adenocarcinoma of the breast exhibited marked diminution or loss of the alpha 2 beta 1 and alpha 5 beta 1 integrins. We have now evaluated the level of alpha 2, alpha 5, and beta 1 integrin subunit messenger (m)RNA by in situ hybridization in adenocarcinoma of the breast. Normal breast ducts and ductules expressed high levels of all three integrin subunit mRNAs. Poorly differentiated lesions expressed low to undetectable levels of alpha 2, alpha 5, and beta 1 mRNA. Well- and moderately differentiated lesions expressed all three subunits at intermediate levels. Thus, decreased expression of the alpha 2 beta 1 and alpha 5 beta 1 integrins in mammary carcinoma is the result of decreased steady-state integrin subunit mRNA levels due to altered expression of the integrin genes.

Adenocarcinoma↗
PubMed1992

Differential regulation of the alpha 2 beta 1 and alpha IIb beta 3 integrin genes during megakaryocytic differentiation of pluripotential K562 cells.

Expression of the alpha 2 beta 1 and alpha IIb beta 3 integrin genes is differentially regulated during megakaryocytic differentiation of pluripotent K562 cells induced with phorbol 12,13-dibutyrate. Upon megakaryocytic differentiation, steady-state alpha 2 mRNA increased markedly from the undetectable level present in the uninduced cells. The level of beta 1 mRNA did not change. Expression of alpha IIb beta 3 is regulated differently. beta 3 mRNA was undetectable in uninduced cells but increased significantly following induction. alpha IIb mRNA was detectable at a low level prior to induction, but at an increased level following differentiation. Altered mRNA stability did not contribute to changes in mRNA levels. Nuclear run-off experiments revealed a 20-fold increase in alpha 2 gene transcription upon megakaryocytic differentiation, but no change in transcription of the beta 1 gene. Transcription of both the alpha IIb and beta 3 genes increased 10- and 5-fold, respectively. Thus, the increase in alpha 2 beta 1 protein which accompanies the megakaryocytic differentiation of K562 cells is a consequence of the increased steady-state level of alpha 2 mRNA due to transcriptional activation of the alpha 2 gene. The long-lived beta 1 mRNA is not altered during differentiation. In contrast, increased alpha IIb beta 3 protein appears due to increased steady-state levels of both alpha IIb and beta 3 mRNAs that result from transcriptional activation of both integrin genes.

Cell Differentiation↗
PubMed1992

Induced cell surface expression of functional alpha 2 beta 1 integrin during megakaryocytic differentiation of K562 leukemic cells.

The pluripotential hematopoietic cell line K562 was studied as a model of inducible integrin expression accompanying differentiation. Differentiation along the megakaryocytic pathway was induced with phorbol 12,13-dibutyrate and differentiation along the erythroid pathway with hemin. Induction of megakaryocytic differentiation was associated with changes in cell morphology and with increased cell-cell and cell-substrate adhesion and spreading. Erythroid differentiation was not associated with changes in morphology or adhesion. Cell surface expression of the IIb-IIIa and alpha 2 beta 1 integrins increased markedly with phorbol treatment but decreased with hemin treatment. Phorbol-treated K562 cells, but not control cells or hemin-treated cells, adhered to collagen substrates in a Mg(2+)-dependent manner which was specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1 integrin. Northern blot analysis revealed that megakaryocytic differentiation of K562 cells was accompanied by de novo expression of the alpha 2 integrin mRNA with no change in the level of mRNA for the beta 1 subunit. K562 cells provide a model of differentiation-dependent, regulated integrin expression in which expression is up- or down-regulated depending upon the differentiation pathway selected.

Antibodies↗