Biomedical subjects

E Pisick

Publications and source records attributed to E Pisick.

14 recordsLinked to original sources

PubMed2001

Use of cytokeratins 7 and 20 in determining the origin of metastatic carcinoma of unknown primary, with special emphasis on lung cancer.

Metastatic carcinoma of unknown primary is a common problem, accounting for up to 10-15% of all solid tumours at presentation. Proper identification of the site of origin has prognostic and therapeutic significance. Prior immunohistochemical methods to identify the site of origin have been useful in a limited number of cases. Differential cytokeratin staining may be useful in this setting, particularly in identifying metastases from lung cancer. We have identified 144 cases of metastatic carcinoma of unknown primary to bone, lung or liver at Brigham and Women's Hospital between 1 January 1997 and 1 July 1998. Cytokeratin (CK) 7 and CK20 were used in 75 of these cases to narrow down the possible sites of the primary tumours. All of these cases were ambiguous as to the site of the primary tumour. Forty-five cases were CK7+/CK20-, 15 cases were CK7-/CK20-, 9 cases were CK7-/CK20+ and 6 cases were CK7+/CK20+. Three of the cases were selected for detailed presentation and discussion as well as a discussion of the pertinent literature. Overall, the CK7+/CK20- phenotype favours a lung primary, the CK7+/CK20+ phenotype strongly favours transitional cells (urothelial) carcinoma, the CK7-/CK20+ phenotype favours colorectal carcinoma, while the CK7-/CK20- profile is not helpful.

Biomarkers↗

PubMed2000

Role of the cytoskeletal protein paxillin in oncogenesis.

The focal adhesion is an important cellular structure that is involved in cell signaling, cell motility, and oncogenic transformation. Paxillin is a unique adapter protein that is localized to the focal adhesion and is involved in regulating various functions of the focal adhesion. The predicted amino acid structure for paxillin shows at the amino-terminus five LD motifs, a proline-rich domain, several potential phosphorylation sites and four carboxy-terminal LIM domains. Paxillin interacts with cell surface receptors and the actin cytoskeleton and activates several signal transduction pathways that are known to regulate normal cell physiology. Because paxillin is a central protein within the focal adhesion, it is a common target of many different oncoproteins, such as BCR/ABL, v-Src, and E6. In this review we summarize the current knowledge about the structure/function of paxillin and its family members, its role in integrin, cytokine signaling, and oncogenic transformation.

Amino Acid Motifs↗

PubMed1999

Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility.

Lung cancer can lead to abnormalities of the actin cytoskeleton structure which may be important in transformation. In this study, we have investigated the expression of the cytoskeletal associated protein paxillin in lung cancer. Paxillin is a 68 kDa focal adhesion protein, with four tandem LIM domains at the C-terminus, involved in growth factor receptor, integrin and oncogenic signaling such as v-src, BCR/ABL, and E6 of the papilloma virus. In non-small cell lung cancer (NSCLC) cell lines, paxillin localized to the focal adhesions. The possible role of paxillin in lung cancer cells was assessed by overexpressing green fluorescence protein (GFP)-paxillin construct in two separate NSCLC cell lines (Calu-1 and H661). Over the course of 48 h, GFP-paxillin consistently caused the cells to become round and to decrease cell motility as compared to normal controls, GFP-N-terminus paxillin, or GFP-LIM transfected cells. Because some lung cancers may be quite aggressive and metastasize quickly, which may be related to the cytoskeleton, we determined the expression of paxillin in NSCLC and small cell lung cancer (SCLC) cell lines and patient tumor tissues. Expression of paxillin in NSCLC and SCLC cell lines were determined by Northern blot and Western blot analysis. The expression of paxillin was consistently low in SCLC cell lines, whereas there was paxillin expression in NSCLC cell lines. There was a variability of expression of paxillin in NSCLC tumor tissue as compared to normal lung tissue. In contrast, by immunohistochemistry, we show that there was no detectable expression of paxillin in 5/5 SCLC patients. This data suggests that absence or low level of paxillin protein expression may cause certain lung cancers, such as SCLC, to be more motile and possibly more aggressive.

3T3 Cells↗

PubMed1997

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.

3T3 Cells↗

PubMed1997

Steel factor induces tyrosine phosphorylation of CRKL and binding of CRKL to a complex containing c-kit, phosphatidylinositol 3-kinase, and p120(CBL).

Steel factor (SF) is a growth and survival factor for hematopoietic cells. The receptor for SF, c-Kit, contains intrinsic tyrosine kinase activity, and binding of SF induces rapid tyrosine phosphorylation of several cellular proteins, including c-Kit itself. Activation of c-Kit is shown here to induce tyrosine phosphorylation of CRKL, and CRKL coprecipitated with c-Kit through an interaction that required the CRKL SH3 domains and not the SH2 domain. CRKL associated with c-Kit indirectly as part of a larger complex of proteins. Two proteins in this complex were identified as the p85 regulatory subunit of phosphatidylinositol 3-kinase (p85(PI3K)) and the proto-oncoprotein p120(CBL). Because p85(PI3K) is known to bind to the activated c-Kit receptor, the possibility that CRKL interacted with c-Kit indirectly through p85(PI3K) was investigated. Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85(PI3K )in vitro. However, although a small amount of CRKL was preassociated with p85(PI3K), the interaction was increased after SF stimulation, suggesting that the interactions of these three proteins are complex. We conclude that SF induces the formation of a signaling complex potentially containing CRKL and p120(CBL), both of which bind to c-Kit through p85(PI3K). These data suggest that one function of CRKL in normal cells might be to recruit signaling molecules such as CBL into a complex with PI3K. Such complexes could be important in propagating signals involving PI3K such as gene expression and adhesion.

Adaptor Proteins, Signal Transducing↗

PubMed1997

The BCR/ABL oncogene alters interaction of the adapter proteins CRKL and CRK with cellular proteins.

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary leukemic neutrophils from patients with CML, the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene. In cell lines transformed by BCR/ABL, CRKL was tyrosine phosphorylated, while CRK was not. We looked for changes in CRK- and CRKL-binding proteins in Ba/F3 hematopoietic cell lines which were transformed by BCR/ABL. Anti-CRK II or anti-CRKL immunoprecipitates were probed by far Western blotting with CRK II- or CRKL-GST fusion proteins to display CRK- and CRKL-coprecipitating proteins. There was a striking qualitative difference in the proteins coprecipitating with CRKL and CRK II. In untransformed cells, three major proteins coprecipitated with CRKL, identified as C3G, SOS and c-ABL. Each of these proteins was found to interact with the CRKL-SH3 domains, but not the SH2 domain. After BCR/ABL transformation, the CRKL SH3-domain binding proteins did not change, with the exception that BCR/ABL now coprecipitated with CRKL. Compared to CRKL, very few proteins coprecipitated with CRK II in untransformed, quiescent cells. After BCR/ABL transformation, both the CRKL- and CRK-SH2 domains bound to a new complex of proteins of approximate molecular weight 105-120 kDa. The major protein in this complex was identified as p120CBL. Thus, in these hematopoietic cell lines, CRKL is involved to a greater extent than CRK II in normal signaling pathways that involve c-ABL, C3G and SOS. In BCR/ABL-transformed cells, CRKL but not CRK II, appears to form complexes which potentially link BCR/ABL, c-ABL, C3G, and SOS to the protooncoprotein, p120CBL.

Adaptor Proteins, Signal Transducing↗

PubMed1996

The related adhesion focal tyrosine kinase forms a complex with paxillin in hematopoietic cells.

Related adhesion focal tyrosine kinase (RAFTK), also known as proline-rich tyrosine kinase 2 and cellular adhesion kinase beta, has been recently cloned and characterized as a member of the focal adhesion kinase (FAK) subfamily. RAFTK has an overall 48% amino acid homology to p125(FAK) and contains a kinase domain but lacks a transmembrane region, myristylation sites, and Src homology region 2 and 3 domains. By Northern blot analysis, RAFTK is expressed in myeloid, lymphoid, and megakaryocytic hematopoietic cells. Like p125(FAK), we found that RAFTK interacts with the focal adhesion protein paxillin. In the lymphoid cell line BaF3 and the myeloid cell line 32Dcl3, RAFTK coprecipitates with paxillin. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly, through a segment of paxillin that required amino acids 100-227 and a domain in the C terminus of RAFTK. In vitro, RAFTK could phosphorylate paxillin on tyrosine residues. These results suggest that RAFTK, as well as p125(FAK), may be important in phosphotyrosine-signaling events within the focal adhesion.

Animals↗

PubMed1996

p130CAS forms a signaling complex with the adapter protein CRKL in hematopoietic cells transformed by the BCR/ABL oncogene.

The Philadelphia chromosome (Ph) translocation generates a chimeric tyrosine kinase oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML) and a type of acute lymphoblastic leukemia (ALL). In primary samples from virtually all patients with CML or Ph+ALL, the CRKL adapter protein is tyrosine phosphorylated and physically associated with p210(BCR/ABL). CRKL has one SH2 domain and two SH3 domains and is structurally related to c-CRK-II (CRK) and the v-Crk oncoprotein. We have previously shown that CRKL, but not the related adapter protein c-CRK, is tyrosine phosphorylated in cell lines transformed by BCR/ABL, and that CRKL binds to BCR/ABL through the CRKL-SH3 domains. Furthermore, the CRKL-SH2 domain has been shown to bind one or more cellular proteins, one of which is p120(CBL). Here we demonstrate that another cellular protein linked to BCR/ABL through the CRKL-SH2 domain is p130(CAS). p130(CAS) was found to be tyrosine phosphorylated and associated with CRKL in BCR/ABL expressing cell lines and in samples obtained from CML and ALL patients, but not in samples from controls. In both normal and BCR/ABL transformed cells, p130(CAS) was detected in focal adhesion-like structures, as was BCR/ABL. In normal cells, the focal adhesion proteins tensin, p125(FAK), and paxillin constitutively associated with p130(CAS). However, in BCR/ABL transformed cells, the interaction between p130(CAS) and tensin was disrupted, while the associations between p130(CAS), p125(FAK), and paxillin were unaffected. These results suggest that the BCR/ABL oncogene could alter the function of p130(CAS) in at least three ways: tyrosine phosphorylation, inducing constitutive binding of CRKL to a domain in p130(CAS) containing Tyr-X-X-Pro motifs (substrate domain), and disrupting the normal interaction of p130(CAS) with the focal adhesion protein tensin. These alterations in the structure of signaling proteins in focal adhesion like structures could contribute to the known adhesion abnormalities in CML cells.

Adaptor Proteins, Signal Transducing↗

PubMed1996

The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3' kinase pathway.

Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In an effort to understand the possible contribution of p120CBL to transformation by BCR/ABL, we looked for cellular proteins which associate with p120CBL in hematopoietic cell lines transformed by BCR/ABL. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitated with an 85 kDa phosphoprotein, which was identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK were also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c-CRK bound directly to p120CBL, while the SH3 domains of c-CRK and CRKL bound to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bound directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, could also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins which include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself.

Adaptor Proteins, Signal Transducing↗

PubMed1996

p210BCR/ABL induces formation of complexes containing focal adhesion proteins and the protooncogene product p120c-Cbl.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by the t(9;22) translocation. This translocation creates a unique tyrosine kinase oncogene, bcr/abl, whose product, p210BCR/ABL, is localized to the actin cytoskeleton. One of the major tyrosine phosphoproteins in cells transformed by p210BCR/ABL is the protooncoprotein p120c-Cbl. We have previously shown that p210BCR/ABL induces formation of a multimeric complex of proteins which include p120c-Cbl, phosphotidylinositol-3' kinase, and p210BCR/ABL itself. Here we show that certain focal adhesion proteins are also part of this complex, including paxillin and talin. The sites in paxillin required to bind to p120c-Cbl in this complex have been partially mapped. The interaction of pl20c-Cbl with paxillin is specific, since other focal adhesion proteins, such as p125FAK, vinculin, and alpha-actinin, are not in this complex. The binding of p120c-Cbl to the focal adhesion protein paxillin could contribute to the known adhesive defects of CML cells.

Adaptor Proteins, Signal Transducing↗

PubMed1995

CRKL links p210BCR/ABL with paxillin in chronic myelogenous leukemia cells.

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary neutrophils from patients with CML, the major novel tyrosine-phosphorylated protein is CRKL, an SH2-SH3-SH3 linker protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene product. Anti-CRKL immunoprecipitates from CML cells, but not normal cells, were found to contain p210BCR/ABL and c-ABL. Several other phosphoproteins were also detected in anti-CRKL immunoprecipitates, one of which has been identified as paxillin, a 68-kDa focal adhesion protein which we have previously shown to be phosphorylated by p210BCR/ABL. Using GST-CRKL fusion proteins, the SH3 domains of CRKL were found to bind c-ABL and p210BCR/ABL, while the SH2 domain of CRKL bound to paxillin, suggesting that CRKL could physically link p210BCR/ABL to paxillin. Paxillin contains three tyrosines in Tyr-X-X-Pro (Y-X-X-P) motifs consistent with amino acid sequences predicted to be optimal for binding to the CRKL-SH2 domain (at positions Tyr-31, Tyr-118, and Tyr-181). Each of these tyrosine residues was mutated to a phenylalanine residue, and in vitro binding assays indicated that paxillin tyrosines at positions 31 and 118, but not 181, are likely to be involved in CRKL-SH2 binding. These results suggest that the p210BCR/ABL oncogene may be physically linked to the focal adhesion-associated protein paxillin in hematopoietic cells by CRKL. This interaction could contribute to the known adhesive defects of CML cells.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Increased tyrosine phosphorylation of focal adhesion proteins in myeloid cell lines expressing p210BCR/ABL.

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and induces growth factor independence of hematopoietic cell lines in tissue culture. p210BCR/ABL is localized at least in part to the cytoskeleton, and has been shown to interact directly with actin filaments through an actin binding domain located in the C-terminus of ABL. CML cells have reduced adhesion to some extracellular matrix components but the mechanism of this phenomenon is unknown. In this study we examined tyrosine phosphorylation of focal adhesion proteins in cells expressing p210BCR/ABL. An interleukin-3 (IL-3)-dependent cell line, 32Dc13, was transformed with a BCR/ABL cDNA, and the patterns of localization, expression, and tyrosine phosphorylation of focal adhesion proteins were compared among untransformed 32Dc13 cells with and without IL-3 stimulation and BCR/ABL-transformed 32Dc13 cells. Of the focal adhesion proteins examined, only paxillin exhibited tyrosine phosphorylation in response to IL-3; while in cells transformed by p210BCR/ABL, paxillin, vinculin, p125FAK, talin and tensin were constitutively tyrosine phosphorylated. IL-3 induced a transient association between paxillin and vinculin, while in BCR/ABL-transformed cells, several proteins coimmunoprecipitated with paxillin, including vinculin, p125FAK, talin and tensin. Pseudopodia enriched in focal adhesion proteins were transiently detected in 32Dc13 cells in response to IL-3, but constitutively detected in cells expressing p210BCR/ABL. p210BCR/ABL protein was also found concentrated in punctate structures adjacent to the cell membrane in myeloid cell lines, which often contained vinculin and paxillin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL.

Paxillin is a 68-kDa focal adhesion protein that is phosphorylated on tyrosine residues in fibroblasts in response to transformation by v-src, treatment with platelet-derived growth factor, or cross-linking of integrins. Paxillin has been shown to have binding sites for the SH3 domain of Src and the SH2 domain of Crk in vitro and to coprecipitate with two other focal adhesion proteins, vinculin and focal adhesion kinase (p125fak). After preliminary studies showed that paxillin was a substrate for the hematopoietic oncogene p210BCR/ABL, we investigated the role of this protein in hematopoietic cell transformation and signal transduction. A full-length length cDNA encoding human paxillin was cloned, revealing multiple protein domains, including four tandem LIM domains, a proline-rich domain containing a consensus SH3 binding site, and three potential Crk-SH2 binding sites. The paxillin gene was localized to chromosome 12q24 by fluorescence in situ hybridization analysis. A chicken paxillin cDNA was also cloned and is predicted to encode a protein approximately 90% identical to human paxil-lin. Paxillin coprecipitated with p210BCR/ABL and multiple other cellular proteins in myeloid cell lines, suggesting the formation of multimeric complexes. In normal hematopoietic cells and myeloid cell lines, tyrosine phosphorylation of paxillin and coprecipitation with other cellular proteins was rapidly and transiently induced by interleukin-3 and several other hematopoietic growth factors. The predicted structure of paxillin implicates this molecule in protein-protein interactions involved in signal transduction from growth factor receptors and the BCR/ABL oncogene fusion protein to the cytoskeleton.

3T3 Cells↗

PubMedDate not supplied

Role of serum tumor markers CA 125 and CEA in non-small cell lung cancer.

BACKGROUND: CA 125 and CEA are valuable serum tumor markers that can be used to monitor response to therapy in patients with various solid tumors. Systemic studies of CA125 and CEA have not been evaluated in lung cancer. In this study, we report the serum levels of CA 125 and compared it to CEA in newly diagnosed lung cancer and analyzed the serum levels of these markers pre- and post-therapy. MATERIALS AND METHODS: Two hundred and sixteen patients with newly diagnosed non-small lung cancer were evaluated. CA 125 and CEA levels were correlated with stage and histopathology. RESULTS: CA 125 levels and CEA levels were shown to be lower in patients with early stage disease as compared to patients with unresectable or metastatic disease. CEA levels were significantly higher among patients with adenocarcinoma, while there was no statistically significant relationship between histology and CA 125. There was a statistically significant difference in the CEA and CA 125 levels dependent on tumor size. Thirty-seven patients were analyzed for responses to chemotherapy and responders are more likely to have decreases in CA 125 or CEA. CONCLUSION: When abnormally elevated inpatients witlrlung cancer, CA 125 and CEA are useful indicators of disease extent, a useful clinical therapeutic marker, and may potentially have important prognostic value.

Adenocarcinoma↗