Chromosome arm painting probes.
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Chromosome Aberrations↗
Biomedical subjects
P Meltzer
Publications and source records attributed to P Meltzer.
Microdissection and microcloning of chromosomal alterations in human breast cancer.
The recognition of recurring sites of chromosome changes in malignancies has greatly facilitated the identification of genes implicated in the pathogenesis of human cancers. Based especially upon recent studies [1-4], it appears increasingly likely that a subset of recurring chromosome alterations will be recognized in human breast cancer. Currently recognized chromosome changes characterizing breast carcinoma include the recognition of cytologic features of gene amplification (e.g. double minutes [dmins] and homogeneously staining regions [HSRs]) [5-8]. As these and other chromosome regions are implicated in recurring abnormalities in breast cancer, it will become increasingly important to have band- or region-specific genomic libraries and probes in order to facilitate high resolution physical mapping and ultimately to clone breast cancer related genes [9]. Toward this end an important recent development in physical mapping has been the establishment of chromosome microdissection as a rapid and reproducible approach to rapidly isolate and characterize chromosome region-specific DNA, greatly facilitating the initial steps in positional cloning of disease-related genes [10-13]. In this brief report, we will highlight the application of chromosome microdissection to the generation of region-specific probes for both fluorescent in situ hybridization (FISH) and the generation of genomic microclone libraries. Additionally, efforts using this methodology to generate a microclone library encompassing the early onset breast/ovarian cancer (BRCA1) gene will be presented.
Breast Neoplasms↗
Application of chromosome microdissection probes for elucidation of BCR-ABL fusion and variant Philadelphia chromosome translocations in chronic myelogenous leukemia.
Fluorescence in situ hybridization (FISH) has become an increasingly important method for assessing chromosome rearrangement. The reciprocal translocation constituting the Philadelphia (Ph) chromosome [t(9;22)(q34;q11)] characterizes more than 90% of patients with chronic myelogenous leukemia (CML). However, in the remaining cases the Ph chromosome (genetically characterized by the fusion of the BCR-ABL genes) is thought to arise through complex translocations that are often not readily apparent using routine chromosome-banding analysis. For this reason we have developed unique band-specific probes for two-color FISH that detect unequivocally the Ph chromosome, and its derivatives. Results of the application of these probes are illustrated by analysis of 11 cases of CML (9 of which contain "variant" translocations). The probes were generated by chromosome microdissection and in vitro amplification of the bands involved in the Ph translocation, leading to an extremely fast and sensitive approach to identify this alteration in leukemic cell populations.
Base Sequence↗
p53 Mutation and MDM2 amplification in human soft tissue sarcomas.
The p53 and MDM2 genes were analyzed in 24 human soft tissue sarcomas (11 malignant fibrous histiocytomas and 13 liposarcomas). Alterations of p53, consisting of point mutations, deletions, or overexpression, were detected in one-third (8 of 24) of the sarcomas. MDM2 gene amplification was detected in another 8 tumors, but no tumor contained an alteration of both genes. Monoclonal antibodies reactive with the human MDM2 gene product were developed, and immunohistochemical analysis revealed nuclear localization and overexpression of MDM2 in those tumors with amplified MDM2 genes. These data support the hypothesis that p53 and MDM2 genetic alterations are alternative mechanisms for inactivating the same regulatory pathway for suppressing cell growth.
Animals↗
Molecular cytogenetic analysis of i(12p)-negative human male germ cell tumors.
The i(12p) chromosome has been shown to characterize more than 80% of male germ cell tumors (GCTs) and is an important diagnostic marker. Although recent cytogenetic analyses of GCTs have defined nonrandom chromosome abnormalities in these tumors, no attempt has so far been made to compare i(12p)-positive and -negative tumors in terms of their cytogenetic, histologic, and clinical features. During a 5-year period, we have ascertained 202 GCTs, of which 117 had clonally abnormal karyotypes. Among the latter, 91 had one or more copies of i(12p), whereas 26 lacked an i(12p). We report here the karyotypic analysis of these 26 i(12p)-negative GCTs. In this group, nonrandom sites of chromosomal rearrangements included 12p13 (9/26) and 1p11-q11 (5/26). Comparison of the cytogenetic features of i(12p)-negative tumors with i(12p)-positive tumors revealed the only significant difference to be rearrangements affecting 12p13 in the former (35%) as compared to their absence in the latter (3%). Hybridization of metaphase preparations of 9 i(12p)-negative tumors with a chromosome 12 painting probe and with a microdissected 12p painting probe revealed extra copies of chromosome 12 segments incorporated into marker chromosomes whose composition could not otherwise be resolved by banding analysis; all were shown to be derived from 12p. These data demonstrate that both i(12p)-negative and -positive groups are characterized by an increased copy number of 12p, which is consistent with a lack of significant clinical or biological difference between them. An increased 12p copy number thus is a specific aberration of significance to the development of germ cell tumors.
Chromosome Aberrations↗
Related subunits of NF-kappa B map to two distinct loci associated with translocations in leukemia, NFKB1 and NFKB2.
The chromosomal locations of the human genes NFKB1 and NFKB2, which encode two alternative DNA binding subunits of the NF-kappa B complex, p105 and p49/p100, respectively, have been determined. p105 was assigned to 4q21.1-q24 and p49/p100 to chromosome 10 by Southern blot analysis of panels of human/Chinese hamster cell hybrids. The locations were confirmed by fluorescence in situ hybridization and mapped with greater resolution to 4q23 and 10q24, respectively. These results demonstrate that these members of the NF-kappa B family are unlinked. Interestingly, p49/p100 as well as p105 maps to regions associated with certain types of acute lymphoblastic leukemia.
Animals↗
Establishment of two new cell lines derived from human breast carcinomas with HER-2/neu amplification.
Two human cell lines (UACC-812 and 893), both containing significant amplification of the HER-2/neu gene, were established from biopsy specimens of breast carcinomas. One patient had Stage II breast carcinoma; the other had metastatic disease. Characterisation of these lines has revealed that both are highly aneuploid containing multiple clonal chromosome alterations, have doubling times near 100 h, and are oestrogen and progesterone receptor negative. Electron microscopy demonstrates that both lines contain numerous microvilli, cytoplasmic filaments, multivesicular bodies, and desmosomes. Immunoblot analysis for P-glycoprotein using the monoclonal antibody C219 was negative for both patient cell lines. These relatively rare cell lines may represent a useful model to investigate human breast carcinomas.
Adult↗
Epidermal growth factor receptor overexpression and trisomy 7 in a case of Barrett's esophagus.
Increased expression of epidermal growth factor receptor linked with abnormalities of chromosome 7 has been described in a number of malignancies, but not in premalignant lesions. We report on one patient of nine studied with Barrett's esophagus who had trisomy 7 and showed increased expression of epidermal growth factor receptor. Southern blot analysis demonstrated that the protooncogene c-erb-B, which encodes the epidermal growth factor receptor, was not amplified. However, increased expression of the corresponding mRNA was evident on dot blot analysis. Epidermal growth factor (EGF) binding studies were consistent with increased expression of the receptor with approximately 2.3 x 10(5) molecules of EGF bound per cell. These data suggest that chromosome 7 abnormalities can occur in Barrett's esophagus.
Barrett Esophagus↗
Optimization of immunocytochemical P-glycoprotein assessment in multidrug-resistant plasma cell myeloma using three antibodies.
Seeking to optimize the immunocytochemical assay of P-glycoprotein, a 170-kilodalton (P-170) molecule associated with multidrug resistance, we experimented with a variety of antibodies (JSB-1, C219, and MRK-16), fixation conditions, and titers using both human myeloma cell lines and clinical myeloma specimens. Under optimized conditions, using all three antibodies and the cell lines as standards and controls, the ICC method proved sensitive, specific, reliable, rapid, and within the realm of everyday hospital laboratory expertise. The 3 anti-P-glycoprotein antibodies revealed different reactivities with P-170. Both C219 and JSB1 were optimized by fixation in cold acetone. With MRK-16 optimal results were obtained on unfixed or formalin fixed specimens. Under optimal fixation and titering conditions, low level (DOX 4) detection was possible. Given that the three antibodies differ in reactivity and recognize different P-170 epitopes, it follows that using the antibodies in a small panel is a useful strategy in increasing the likelihood of detecting true P-glycoprotein expression by the immunocytochemical method. In dilution experiments, the immunocytochemical method was as sensitive as RNase protection assay and more sensitive than Western blot detection. Immunocytochemistry coupled to computer-assisted single-cell densitometry, showed a strong correlation (R = 0.98) between cellular P-170 density and in vitro resistance to doxorubicin. Multidrug-resistant specific probes for RNA expression and Western blot assays confirmed the specificity of P-170 expression in both cell lines and clinical samples. Thus, a small panel of antibodies, under optimized immunocytochemical conditions, appears to have potential as a rapid, sensitive, clinically useful assay for multidrug resistance in myeloma.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Role of glutathione and its associated enzymes in multidrug-resistant human myeloma cells.
Multidrug resistance (MDR) is a phenomenon associated with the emergence of simultaneous cross-resistance to the cytotoxic action of a wide variety of structurally and functionally unrelated antineoplastic agents. The present study was undertaken to determine if 8226 human myeloma cells possessing the MDR phenotype had an increased ability to resist the intercalating drug doxorubicin (DOX) via glutathione-based detoxification systems. Glutathione S-transferase (GST) was isolated by affinity chromatography, and the enzyme activity was assessed using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. There was no difference in overall GST activity between the sensitive and resistant cells. Using a cDNA probe (pGTSS1-2) for the human placental, anionic GST isoenzyme, no overexpression of mRNA for this isoenzyme was noted in the resistant line. When glutathione peroxidase activity (GSH-px) was assessed using either H2O2 or cumene hydroperoxide as substrate, again there was no difference in enzyme activity. Non-protein sulfhydryl (NPSH) levels were found to be elevated significantly in the resistant 8226/DOX40 subline (19.2 +/- 0.1 nmol NPSH/10(6) cells) as compared to the drug-sensitive parental subline 8226/S (11.6 +/- 1.9 nmol NPSH/10(6) cells) (P less than 0.001). In addition, when the 8226/DOX40 cells were cultured in medium without doxorubicin, there was a consistent decline in NPSH values reaching a steady state identical to that of the 8226/S cells. However, the decrease in NPSH level was not accompanied by a change in the level of doxorubicin resistance as assessed by colony-forming assays. Depletion of glutathione by D,L-buthionine-S,R-sulfoximine had no effect on doxorubicin sensitivity in either subline. Thus, it appears that GSH-based detoxification systems are not causally involved in maintaining the MDR phenotype in 8226 human myeloma cells; rather they appear to comprise an epiphenomenon associated with the resistance selection procedure.
Buthionine Sulfoximine↗
Alpha-globulins suppress human leukocyte tumor necrosis factor secretion.
The secretion of tumor necrosis factor (TNF) by human peripheral blood mononuclear cells was suppressed by either whole human plasma alpha-globulins or purified alpha 1-acid-glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin in a concentration-dependent manner. alpha 1-Antitrypsin was found to be the most suppressive of the purified proteins tested and completely blocked TNF release at concentrations above 1.25 mg/ml. Both alpha 1-acid glycoprotein and alpha 1-antitrypsin blocked TNF secretion by leukocytes which were simultaneously stimulated with either recombinant human interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS). IFN-gamma- and LPS-activated cells were also susceptible to suppression mediated by these two alpha-globulins and the inhibition produced by 5 mg/ml alpha 1-antitrypsin was greater than that caused by either 1 microM prostaglandin E2 or 10 ng/ml transforming growth factor-beta 1. The level of TNF mRNA in TNF-secreting and alpha-globulin-suppressed cells was examined and found to be equal in both groups. The suppressive effect of whole alpha-globulins was confined to the inhibition of TNF secretion and these plasma proteins had no effect on the cytolytic activity of the recombinant cytokine as measured on murine L-929 target cells. Thus the alpha-globulins, which are a major fraction of the circulating plasma proteins, may function in TNF homeostasis by controlling TNF secretion without inhibiting the biological activity of the released cytokine.
Adult↗
Application of natural partial digests to pulsed-field gel analysis of the amplified MDR locus.
The analysis by pulsed-field gel electrophoresis of partial digestion products visualized by probes for the human multidrug resistance (MDR) locus has been used to further establish the restriction map of this region. Results place the MDR1 and MDR2 genes on a single SfiI fragment, with partial digestion products establishing the distance between these genes to be 230-250 kb. The feasibility and potential advantages of using "natural" partials to generate detailed restriction maps within an amplified DNA domain are discussed.
Blotting, Southern↗
Effects of tumor necrosis factor on sensitive and multidrug resistant human leukemia and myeloma cell lines.
Two human tumor cell lines exhibiting acquired multidrug resistance (MDR) with increased expression of a cell surface glycoprotein (GP-170) were tested for their sensitivity to human recombinant tumor necrosis factor (rTNF). The drug resistant mutant lines (CEM/V, a T-cell leukemia line resistant to vinblastine, and 8226/D, a multiple myeloma line resistant to doxorubicin), were markedly more sensitive to rTNF in clonogenic assay than were their drug-sensitive parental lines (CEM, 8226). As determined by radioreceptor assay, the number of cell surface receptors for rTNF did not differ on the parental and drug-resistant lines. During the first 24 hours after addition of rTNF, there was a decrease in intracellular ATP content in the CEM/V line but not in the CEM line. No differential effect of rTNF on ATP content was observed between 8226 and 8226/D. As determined by RNA dot-blot analysis, total cellular RNA for GP-170 was increased in the 8226/D cells. After rTNF exposure, expression of total cellular RNA for GP-170 was not altered. Accumulation of radiolabeled doxorubicin by 8226/D cells was not altered by previous or coincubation with rTNF. These findings suggest that the effects of rTNF on MDR cells is not related to TNF receptor number and is mediated at a step subsequent to rTNF binding and not by either inhibition of synthesis of GP-170 or by alteration in the function of the GP-170 efflux pump.
Cell Line↗
Antifertility, spermicidal and ultrastructural effects of gossypol and derivatives administered orally and by intratesticular injections.
Because there are problems, in men, associated with the use of gossypol related to reversibility and, infrequently, hypokalemia, several laboratories around the world have resorted to the synthesis and evaluation of experimental analogs and optical isomers of gossypol in an attempt to find a compound which retains its pharmacologically desirable antifertility effects while eliminating its suboptimal ones. The present study documents: (a) the effects of fourteen new, orally-administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters, (b) the in vitro effects of these compounds as well as of the optical isomers of gossypol against hamster and human sperm, and (c) the morphological and antifertility effects of intratesticular injections of gossypol-PVP and its optimal isomers in the rat. The results of the study demonstrate that these new analogs are not effective as male antifertility agents and that their in vitro activity is unrelated to their in vivo contraceptive potential. In addition, this report establishes the validity of the intratesticular injection model for the analysis of the mechanism of action of gossypol and its analogs by making these compounds directly available at the testicular site. The significance of these findings is discussed.
Administration, Oral↗
Wasting disease associated with Epstein-Barr virus infection.
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Child↗
Ultrastructural, fertility, and spermicidal studies with isomers and derivatives of gossypol in male hamsters.
The effects of fourteen new, orally administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters and the spermicidal properties of these compounds, as well as of the optical isomers of gossypol against hamster and human sperm in vitro, are reported in this study. Test compounds were administered to adult male hamsters by daily gavage for 9 weeks at doses ranging from 15 to 50 mg/kg. The results of this study have demonstrated that the fourteen new gossypol analogs evaluated herein are not effective as male antifertility agents and their in vitro activity or lack of activity as spermicides is unrelated to their in vivo contraceptive potential. In addition, the results of the study suggest that (1) the isopropyl moiety of the gossypol molecule, like the aldehyde group, is essential for its mechanism of action and (2) the pathognomonic defect in the mitochondrial sheath induced by gossypol appears to be related to its unique activity as a male antifertility agent. The significance of these findings is discussed.
Animals↗
Gene amplification in cancer: a molecular cytogenetic approach.
Many human tumors have been shown to exhibit cytologic evidence of gene amplification. In several instances the amplified genes have proven to be cellular oncogenes. We present an approach for combining cytogenetic and molecular biological techniques to systematically investigate this phenomenon.
Base Sequence↗
Enhanced expression of epidermal growth factor receptor correlates with alterations of chromosome 7 in human pancreatic cancer.
Recently, the gene for the epidermal growth factor (EGF) receptor has been mapped to chromosome 7p, the short arm of chromosome 7 [Shimizu, N., Kondo, I., Gamou, M. A., Behzadian, A. & Shimizu, Y. (1984) Somatic Cell Mol. Genet. 10, 45-53]. Utilizing EGF binding in saturation studies, karyology, and cDNA hybridization experiments, we have sought to determine whether there is a correlation between dosage or alteration of chromosome 7 and enhanced expression of EGF receptor in cultured human pancreatic carcinoma cells. Saturation binding studies with 125I-labeled EGF were performed at 4 degrees C with four established human pancreatic cancer cell lines: T3M4, PANC-1, COLO 357, and UACC-462. Analysis of binding data revealed enhanced numbers of EGF receptors in all four cell lines. Chromosome banding analysis revealed clonal structural alterations of chromosome 7p in the cell lines T3M4, PANC-1, and COLO 357, whereas UACC-462 displayed multiple copies of chromosome 7. Hybridization studies using a radiolabeled EGF receptor cDNA probe failed to demonstrate DNA sequence amplification in any cell line but confirmed the presence of EGF receptor mRNA in these cells in approximate proportion to EGF receptor number. Our results suggest that enhanced expression of EGF receptor in human pancreatic cancer can be associated with either structural or numerical alterations of chromosome 7.
Cell Line↗