The K-ras, p53, p16 and DPC4 genes are among those most frequently altered in pancreatic ductal carcinoma. We analyzed 22 cell lines for alterations in these genes by direct sequence analysis and methylation-specific polymerase chain reaction. These cell lines showed mutations in K-ras and p53 at frequencies of 91% and 95%, respectively. Alterations in p16INK4a were found in all cases and included nine homozygous deletions, seven mutations and promoter methylation in six cases. Eight cell lines (36%) had an alteration of DPC4, including one mutation and seven homozygous deletions. The most typical mutational profile involved K-ras, p53, and p16INK4a, concurrently aberrated in 20 cases (91%). Eight cell lines had alterations in all four genes. Inactivation of DPC4 was always accompanied by alteration of all of the other three genes. This comprehensive data regarding the cumulative genetic alterations in pancreatic carcinoma cell lines will be of great value for studies involving drug sensitivity or resistance that may be associated with inactivation of a particular gene or molecular pathway.
The ALL) gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to AF4 and AF9. The fused genes encode chimeric proteins. Because the Drosophila homologue ofALL), trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL) transcriptional activity may underlie its action in malignant transformation. To Chromosome 1 1q23 abnormalities are associated with 5-10% of human acute leukemias, in particular in children under the age of 1 year, and in secondary leukemias (1). These leukemias have a lymphoid, myeloid, or lymphoid-myeloid phenotype and uniformly carry a poor prognosis. More than 20 different 11q23 translocations have been identified by now (2, 3). In nearly all of these aberrations, the ALLI gene (also called HRX, MLL, or HTRX) is involved (4-7). ALLI is thought to be the human homologue of the Drosophila trithorax gene, which acts as a positive regulator of the homeotic gene complexes Antennapedia and Bithorax.11q23 chromosome translocations cleave ALL1 within the breakpoint cluster region and fuse it to a partner gene, resulting in production of fused RNAs and presumably of chimeric proteins (4, 5). The chimeric proteins containing the N terminus of ALL1 are thought to be the critical products of the translocations (for review, see ref. 8). Presently, 10 partner genes have been cloned (8-13).The mechanism(s) by which the chimeric ALL1 proteins act to trigger neoplasm is not known. Two models were proposed: the first (5) emphasized gain of function and suggested that the altered protein (specifically ALL1 /ENL) is a hybrid transcription factor in which the DNA binding domain of ALL1The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.localizes the hypothetical transactivation domain of ENL to regulatory sites of target genes. The second (14) suggested that the cleavage of ALL1 protein results in loss of its function and that the partner polypeptide augments this loss. Resolution between the two models would necessitate developing biological and biochemical assays to measure transcriptional regulatory activity of the normal ALL1 protein, of the normal partner proteins, and of the chimeric species. As a first step in this direction, we looked for domains with transcriptional activation or repression potential within ALL1, within the AF4 and AF9 partner proteins (presumed to be nuclear proteins), and within the AF17 partner polypeptide, which contains a dimerization motif. A similar approach to investigate the ALL1 protein was recently reported (15). MATERIALS AND METHODSPlasmids. Effector plasmid vectors were either RSV-GAL4 (16) or pGALM (17). The former contains a GAL4 DNA binding domain (amino acids 1-147) flanked by the Rous sarcoma virus (RSV) promoter and ...
Abstract. To address the question whether leukocyte integrins are able to generate signals activating neutrophil functions, we investigated the capability of mAbs against the common ß chain (CD18), or the distinct a chains of CR3, LFA-1, or gp150/95, to activate neutrophil respiratory burst. These investigations were performed with mAbs bound to protein A immobilized to tissue culture polystyrene . Neutrophils plated in wells coated with the anti-CD18 mAbs IB4 and 60.3 released H202 ; H202 release did not occur when neutrophils were plated in wells coated with an irrelevant, isotype-matched mAb (OKDR), or with mAbs against other molecules (CD16, ß2-microglobulin) expressed on the neutrophil surface at the same density of CD18. Four different mAbs, OKMI, OKM9, OKM10, 60.1, which recognize distinct epitopes of CR3 were unable to trigger H202 or 02 release from neutrophils. However, mAbs against LFA-1 or gpl50/95 triggered both H202 and 02 release from neutrophils . Stimulation of neutrophils respiratory burst by both anti-CD18, and T 13E 02 subfamily of integrins is constituted by three heterodimeric transmembrane molecules that are all expressed by circulating leukocytes (9, 35). Expression of the individual members of the family, which are referred to as LFA-1 (CDlla/CD18), CR3 (CDI lb/CDl8), and gp150/95 (CDllc/CD18), varies in different cell types (35), neutrophils displaying the unique phenotype of expressing all the three leukocyte integrins, and CR3 at a higher density than LFA-1 and gp150/95 (6) .The peculiarity of CR3 is that to recognize a wide array of different molecules, a property that underlies its involvement in adhesion of phagocytes to endothelium (11,17) and fibrinogen (45), uptake of parasites (8, 29-33, 42, 44, 46), and activation of the coagulation cascade (1, 2) . Also, the recognition repertoire of both LFA1 and gp150/95 indicate that these molecules certainly play a role in mediating neutrophil functions in the inflammatory site. LFA-1 has been established as the counter-receptor for ICAM-1 and ICAM-2 expressed by endothelial cells (19,37). Recent data showed
Abstract. Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. £ Cell Biol. 120:777-784). We investigated the dependence of this response on B2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express B2 integrins, and by plating PMN on surface bound and-B2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNE Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNE Surface bound anti-CD18, but not isotype-matched anti-Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether B2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58 f~r) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58f~ in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of B2 integrins such as phorbol-12-myristate 13-acetate (PMA), and formyl methionylleucyl-phenylalanine (FMLP), induced activation of p58~ kinase activity. Activation of p58¢ kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNE Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58 fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN,/~2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between/32 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.
Aimed at developing accurate, reliable and cost-saving analytical techniques for drugs screening we evaluated the potential of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) as a quantitative pre-diagnostic approach for the rapid identification of IR signatures of drugs targeting specific molecular pathways causing Chronic Myeloid Leukemia (CML). To obtain reproducible FTIR absorbance spectra at the necessary spatial resolution we optimized sample preparation and acquisition parameters on a single channel Mercury-Cadmium-Telluride (MCT) detector in the spectral interval of frequencies from 4000 to 800 cm(-1). Single K562 cells were illuminated by Synchrotron Radiation (SR) and a number of ~15 K562 cells spread in monolayer were illuminated by a conventional IR source (Globar), respectively. Combining IR spectral data with the results of complementary biochemical investigations carried out in samples by different analytical methods we identified and cross-validated IR signatures of drugs targeting the oncogenic protein BCR/ABL and its associated abnormal tyrosine kinase activity in K562 cell line. Unsupervised pattern recognition performed by Hierarchical Cluster Analysis (HCA) clustered the spectra of single K562 cells in two distinct groups roughly corresponding to living and to apoptotic cells, respectively. The corresponding IR spectral profiles were assumed to represent drug-resistant and drug-sensitive cells. Significant variations with increasing percentages of apoptotic cells were observed after the treatment of K562 cells with drugs that directly or indirectly target BCR/ABL. In conclusion, we suggest that microFTIR associated with multivariate data analysis may be useful to assess drug compounds in ex vivo cancer cell models and possibly peripheral blast cells from CML patients.
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