Showing papers by "Carlo M. Croce published in 1994"

Down-Regulation of bcl-2 by p53 in Breast Cancer Cells

Abstract: bcl-2 and p53 gene products have been both linked to programmed cell death pathways. We have analyzed several human breast cancer cell lines for the expression of bcl-2 and p53. We found an inverse correlation between the expression of the two proteins. The result suggested that mutant p53 could substitute for bcl-2 function in breast cancer cells and that could also down-regulate bcl-2 expression. We found, indeed, that overexpression of a mutant p53 (mut 175) in MCF-7 cells could induce down-regulation of bcl-2 both at protein and mRNA level. However, the promoter region of the human bcl-2 gene does not contain the negative regulatory element responsible for the down-regulation. If this mechanism will be proved also for the wild-type p53 allele, it may disclose a possible mechanism for p53-induced apoptosis: down-regulation of bcl-2.

Identification of the TCL1 gene involved in T-cell malignancies.

Abstract: The TCL1 locus on chromosome 14q32.1 is frequently involved in chromosomal translocations and inversions with one of the T-cell receptor loci in human T-cell leukemias and lymphomas. The chromosome 14 region translocated or rearranged involves approximately 350 kb of DNA at chromosome band 14q32.1. Within this region we have identified a gene coding for a 1.3-kb transcript, expressed only in restricted subsets of cells within the lymphoid lineage and expressed at high levels in leukemic cells carrying a t(14;14)(q11;q32) chromosome translocation or a inv(14)(q11;q32) chromosome inversion. The cognate cDNA sequence reveals an open reading frame of 342 nt encoding a protein of 14 kDa. The TCL1 gene sequence, which, to our knowledge, shows no sequence homology with other human genes, is preferentially expressed early in T- and B-lymphocyte differentiation.

ALL-1 partial duplication in acute leukemia

Abstract: The ALL-1 gene, located on chromosome band 11q23, is fused to a variety of other genes by reciprocal chromosomal translocations present in 5-10% of human acute leukemias. We have recently reported the detection by Southern blot of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These include 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11. To characterize the abnormal ALL-1 genes, we cloned the ALL-1 rearrangements from two patients with trisomy 11. Characterization of the clones, together with Southern blot analysis, indicates that the ALL-1 rearrangement in both patients is the result of a direct tandem duplication of a portion of the ALL-1 gene spanning exons 2-6. The partial ALL-1 duplication is also detected by Southern blot analysis in a patient with a normal karyotype. RNA PCR and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Partial duplication of ALL-1, in which a portion of a putative protooncogene is fused with itself, represents an additional genetic mechanism for leukemogenesis. Our findings suggest that the presence of trisomy in malignancy may sometimes indicate the partial duplication of a cellular protooncogene.

Rarity of Somatic and Germline Mutations of the Cyclin-dependent Kinase 4 Inhibitor Gene, CDK4I, in Melanoma

Abstract: Evidence from cytogenetics, multipoint linkage analyses of familial melanoma, and loss of heterozygosity studies of familial and sporadic melanomas support localization of a melanoma susceptibility or tumor suppressor gene at chromosomal region 9p21–23 Recently, the inhibitor of cyclin-dependent kinase 4 ( CDK4I ; also known as 16p INK4 , multiple tumor suppressor 1, or CDKN2 gene) has been mapped to 9p21 and shown to be mutated or deleted in a large fraction of cell lines derived from many tumor types, including melanoma, suggesting that this gene could be a melanoma suppressor gene In order to test for somatic mutations in the CDK4I gene in tumors, DNAs from 30 surgically resected melanomas of both cutaneous and uveal origins were sequenced No mutations were detected in the coding region of the CDK4I gene, while mutations or deletions were detected in 60% (9 of 15) of the cultured melanoma cell line DNAs Among presumptive familial cases, nine of which were members of families with one or two other documented melanoma cases, no germline mutations were detected by sequence analysis A deletion in the second exon of the CDK4I gene was found in one germline allele of a familial melanoma patient from a family with eight affected first degree relatives These results not only support the suggestion that the CDK4I gene is a familial malignant melanoma gene, they also suggest the presence of another suppressor gene locus within 9p21 which is the target of loss of heterozygosity in sporadic melanomas

ALL-1 Tandem Duplication in Acute Myeloid Leukemia with a Normal Karyotype Involves Homologous Recombination between Alu Elements

Abstract: Rearrangements of the ALL-1 gene by reciprocal translocations involving chromosome band 11q23 are frequently associated with human acute leukemia. We have previously reported the detection of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These included 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11 as a sole cytogenetic abnormality. Rearrangement of the ALL-1 genes in two of the patients with trisomy 11 was shown to result from a direct tandem duplication of a portion of the gene spanning exons 2–6. Here we report the characterization of the ALL-1 gene rearrangement in one of the previously reported acute myeloid leukemia patients with a normal karyotype, ALL-1 rearrangement in this patient results from a direct tandem duplication of a portion of the gene spanning exons 2–8. RNA polymerase chain reaction and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Sequence analysis of the genomic fusion region provides evidence for Alu -mediated homologous recombination as a mechanism for partial duplication of the ALL-1 gene.

The c-kit ligand suppresses apoptosis of human natural killer cells through the upregulation of bcl-2

Abstract: The bcl-2 protein plays a central role in the regulation of programmed cell death in a variety of tissues and is pivotal to the survival of lymphocytes in vivo. The growth factors responsible for survival of normal lymphocytes are unknown but are likely to maintain viability in part through the regulation of bcl-2 expression. A subset of human natural killer (NK) cells (CD3-CD56bright) are unique among lymphocytes in their constitutive expression of c-kit, a tyrosine kinase cell surface receptor that binds c-kit ligand (KL). Alone, KL does not promote proliferation or further differentiation of CD56bright NK cells. We now report that, in the absence of serum or additional growth factors, KL prevents apoptosis of cultured CD56bright NK cells, as assessed by DNA fragmentation studies, and maintains viability, as measured by biologic responses (i.e., proliferation and cytotoxicity) to the subsequent addition of other cytokines. Furthermore, we demonstrate that KL induces CD56bright NK cells to express the bcl-2 protein. In the presence of anti-c-kit antibody, the tyrosine kinase inhibitor genistein, or bcl-2 antisense oligonucleotide, the protective effect of KL on the survival of CD56bright NK cells is dramatically reduced. These data demonstrate that the binding of KL to its tyrosine kinase receptor results in the upregulation of bcl-2, thereby preventing apoptosis in this subset of normal human lymphocytes. As soluble KL is plentiful in normal human serum, this survival mechanism may be operative for CD56bright NK cells in vivo.

Suppression of Tumorigenicity of Breast Cancer Cells by Microcell-mediated Chromosome Transfer: Studies on Chromosomes 6 and 11

Abstract: Development of breast cancer has been associated with deletions at multiple chromosomal regions, including 6q, 11p, and 11q. In this study we analyzed the effects of the introduction of chromosomes 6 and 11 on the cell phenotype of the breast cancer cell lines MDA-MB-231 and MCF-7. Chromosome 6 induced alterations of in vitro growth properties and suppressed tumorigenicity of MDA-MB-231 cells. Spontaneous reduction of the transferred chromosome allowed mapping of the tumor suppressor gene(s) to region 6q21-q23 and/or 6q26-q27. Clones MCF-7/H6 underwent a senescence process that lasted five months. Chromosome 11 had no effect on MDA-MB-231 cells, although it suppressed tumorigenicity of MCF-7 cells. A MCF-7/H11 clone lacking the short arm of the transferred chromosome retained tumorigenicity, however, tumor cell growth was significantly reduced. These results suggest that each chromosomal arm may contain genes important for the suppression of MCF-7 tumorigenic properties.

Loss of Heterozygosity at 11q22–q23 in Breast Cancer

Abstract: Studies of loss of heterozygosity (LOH) in breast tumor DNA suggest that several tumor suppressor genes participate in the pathogenesis of breast cancer. Although the short arm of chromosome 11 has been implicated in breast cancer development, no previous LOH studies have indicated the involvement of a suppressor gene on 11q in breast carcinoma. To this end, tumor samples and corresponding normal tissue were collected from 62 unselected patients with primary breast cancer, and the extracted DNA was analyzed by polymerase chain reaction using microsatellite markers on chromosome 11. We found that 39% of the tumors (22 of 57 informative cases) revealed allelic loss in the region 11q22–23, and this loss was independent of LOH found to occur on 11p15. Interestingly, more than 90% of the tumors showed concordant loss of alleles at both 11q and 17p. The marker D11S528 , showing LOH in 39% of informative cases, had the highest frequency of LOH among the markers that were used. The data presented indicate that the common overlapping region of LOH is between the loci D11S35 and D11S29 , suggesting that this area contains a tumor suppressor gene frequently lost in breast cancer.

Molecular Characterization of Prostate-specific Antigen Messenger RNA Expressed in Breast Tumors

Abstract: Prostate-specific antigen (PSA) is considered a highly specific biochemical marker of the prostate gland and is currently used for prostate cancer diagnosis and monitoring of patients with prostate adenocarcinoma. We recently demonstrated, however, that about 30% of female breast tumors produce a M(r) 33,000 protein that has striking similarities to seminal PSA. In this study we characterized the presence of PSA in 6 breast tumors and in the testosterone-stimulated T47D breast cancer cell line at the mRNA level. Using reverse transcriptase-polymerase chain reaction and DNA sequencing techniques we identified PSA mRNA in immunoreactive PSA-positive breast tumors but not in immunoreactive PSA-negative breast tumors. The sequence of the generated polymerase chain reaction products was identical to the sequence of the PSA complementary DNA derived from prostate tissue. The data presented here support the notion that breast tumors produce a M(r) 33,000 protein which is identical to PSA produced by the prostate gland. Our study suggests that the presence of PSA in breast tumors may be used as a new additional biochemical marker for breast cancer prognosis, for the spreading of hematogenous micrometastases, and/or for response to adjuvant treatment.

Nucleophosmin (NPM) Gene Rearrangements in Ki-1-positive Lymphomas

Abstract: The (2;5)(p23;q35) translocation which results in the fusion of the NPM (nucleophosmin) gene on chromosome 5q35 with the novel ALK (anaplastic lymphoma kinase) gene on chromosome 2p23 [S.W. Morris et al., Science (Washington DC), 263: 1281–1284, 1994] is associated with Ki-1 (CD30)-positive anaplastic large cell lymphomas (ALCL); a group of morphologically and immunophenotypically heterogeneous high grade large cell lymphomas (LCL), which share many characteristics with Hodgkin9s disease (HD), including the presence of variable numbers of Reed-Sternberg-like cells and the expression of CD30 antigen. Using a DNA probe immediately 5′ to the NPM coding sequences, we have examined NPM gene rearrangements by Southern blotting in 5 Ki-1-positive lymphoma cell lines carrying a translocation involving the 5q35 breakpoint and in 25 Ki-positive lymphoma tumors, including 9 HD. Using this method, we detected rearrangements in all cell lines with apparent clustering of the breakpoints. Analysis of 25 Ki-1-positive lymphomas indicated that only 4 neoplasms, including two HD, had NPM gene rearrangements. Thus, our findings suggest that only a subset of ALCL has detectable involvement of the NPM gene. In addition, the presence of NPM gene rearrangements in HD indicates the involvement of this gene in a fraction of HD. Thus, NPM gene rearrangements may identify a certain subtype in ALCL and HD which may be closely related.

Characterization and localization of the TCL-1 oncogene product.

Abstract: The TCL-1 gene maps at chromosome 14q32.1 and is activated in T cell leukemias and lymphomas by either chromosome translocations or inversions that juxtapose the TCL-1 gene to the α/δ or the β locus of the T cell receptor. The open reading frame of the TCL-1 gene, coding for a protein of 114 amino acids, was expressed in bacteria and antisera were raised against it. The antibodies recognized the predicted TCL-1 M r 14,000 protein product in cells expressing TCL-1 mRNA. Cell fractionation experiments indicated that the TCL-1 protein is present in the microsomal fraction. These results were confirmed by confocal microscopy. The TCL-1 protein has considerable sequence similarities to the product of the MTCP-1 gene on chromosome Xq28, which is involved in T cell lymphoproliferative diseases. Thus, TCL-1 may represent a member of a novel family of genes involved in lymphoid proliferation and/or survival and in T cell malignancies.

Tumor and Growth Suppression of Breast Cancer Cells by Chromosome 17-associated Functions

Abstract: Losses of functions from chromosome 17 are the most frequent genetic abnormalities in human breast cancer. To assess the biological role of chromosome 17 in the development of breast cancer, we transferred a normal human chromosome 17 to two breast cancer cell lines. No viable clone maintaining an intact chromosome was obtained in either MDA-MB-231 or MCF-7. Only one MDA-231/H17 clone contained the long arm of the transferred chromosome 17. Interestingly, this clone lost the ability to induce tumors in nude mice, indicating that at least one gene mapping to the long arm of chromosome 17 could suppress the tumorigenic phenotype. The p53 protein most likely was responsible for the selective loss of the short arm of the chromosome. Both cell lines have no wild-type p53 activity. MDA-MB-231 carries a single mutant TP53 allele, while MCF-7 carries two wild-type alleles, but p53 protein is excluded from the nucleus. Transfection in both cell lines of vectors expressing wild-type p53 produced only clones with rearrangements of the transfected TP53 complementary DNA. Thus, nonregulated expression of the p53 protein driven by the strong cytomegalovirus promoter may have triggered a rapid process of cell death. Stable expression of a mutant p53 in MCF-7 cells proved that nuclear localization of the protein was possible; however, no progression toward an estrogen-independent tumorigenic phenotype was induced. This work indicates that functional inactivation of the wild-type p53 protein and of the product of a gene located on 17q are essential to the development of breast neoplasms.

Nucleic acid primers for detecting micrometastasis of prostate cancer

Abstract: Oligonucleotides for and a method of diagnosing prostate micrometastasis are provided by the present invention whereby nucleic acids from a tissue sample from a patient are isolated, nucleic acids from the tissue sample specific for prostate cancer are amplified, or a signal generated by hybridization of a probe specific to a prostate cancer specific nucleic acid is amplified; and detection of amplified nucleic acids is indicative of micrometastasis of prostate cancer.

The human eps15 gene, encoding a tyrosine kinase substrate, is conserved in evolution and maps to 1p31-p32.

Abstract: Employing an expression cloning approach for tyrosine kinase substrates, we have previously isolated the coding sequence for a novel putative EGFR substrate, eps15, from NIH3T3 fibroblasts. Eps15 displayed a receptor-specific pattern of tyrosine phosphorylation in vivo and was able to transform NIH3T3 cells upon overexpression. To gain understanding of eps15 function as well as its role in normal and neoplastic proliferation, we cloned the human eps15 coding sequence and studied expression of the human RNA and protein, evolutionary conservation, and chromosomal location. The close structural similarity of human eps15 with the murine homologue is indicated by 89% and 90% identity of nucleotide and predicted amino acid sequences, respectively. Using the human eps15 coding sequence as probe, we demonstrate that eps15 is member of a gene family that is highly conserved during evolution. An essential function of eps15 in cell growth regulation is underscored by our observation of ubiquitous expression at the transcript and the protein level in normal and malignant human cells. The human EPS15 locus maps to chromosome 1p31-p32, a region involved in deletion in neuroblastoma, translocations in acute lymphoblastic leukemia, and exhibiting a fragile site.

Antiapoptosis potential of bcl-2 oncogene by dephosphorylation.

Abstract: The antiapoptosis potential of bcl-2 has now been well established. But the biochemical mechanism of bcl-2 action is still poorly understood. Using the phosphatase inhibitor okadaic acid (OA) or chemotherapeutic agents such as Taxol and 5'-fluorouracil, we found that bcl-2 can be phosphorylated. Since OA or Taxol treatment leads to apoptosis, it seems that phosphorylation of bcl-2 leads to its inactivation. Exposure of several lymphoid cell lines expressing differential amounts of bcl-2 protein to OA resulted in apoptosis of the cells and hyperphosphorylation of bcl-2. Interestingly, the lymphoblastoid cell lines that did not phosphorylate bcl-2 following OA exposure did not undergo apoptosis. Moreover, pro-B cells isolated from patients with acute lymphoblastic leukemias exhibited endogenous phosphorylated forms of bcl-2 and a large number of apoptotic cells, even without OA treatment. Treatment with the phosphatase inhibitor or with chemotherapeutic agents (Taxol, 5'-fluorouracil) led to severe apoptosis of these cells, along with hyperphosphorylation of bcl-2. Phosphoamino acid analysis reveals that bcl-2 is phosphorylated at a serine residue. In summary, our investigation indicates that the phosphorylation pathway involving bcl-2 can be the determinant of cell death in lymphocytes.

Chimeric nucleic acids and proteins resulting from ALL-1 region chromosome abnormalities

Abstract: Methods are provided for the diagnosis and treatment of human leukemias involving breakpoints on chromosome 11 in the ALL-1 locus. The ALL-1 breakpoint region, an approximately 8 kb region on chromosome 11, is also disclosed. The ALL-1 region is involved in translocations in acute lymphocytic, myelomonocytic, monocytic and myelogenous leukemias. Probes which identify chromosome aberrations involving the ALL-1 breakpoint region on chromosome 11 are also provided. cDNA sequences of the ALL-1 gene on chromosome 11, the AF-9 gene on chromosome 9 and the AF-4 gene, and corresponding amino acid sequences are also provided. Probes are provided for detecting chromosome abnormalities involving theses genes. Chimeric genes involved in translocations are disclosed. Monoclonal antibodies for diagnosis and treatment and antisense oligonucleotides for treatment of acute leukemias are also described.

ALL-1 polynucleotides for leukemia detection and treatment

Abstract: Methods are provided for the diagnosis and treatment of human leukemias involving breakpoints on chromosome 11 in the ALL-1 locus. The ALL-1 breakpoint region, an approximately 8 kb region on chromosome 11 is also disclosed. The ALL-1 region is involved in translocations in acute lymphocytic, mylemonocytic, monocytic, and myelogenous leukemias. Probes which identify chromosome aberrations involving the ALL-1 breakpoint region on chromosome 11 are also provided. The cDNA sequence of the ALL-1 gene on chromosome 11 is provided. A partial sequence of the AF-4 gene is also provided in the context of the sequences of the two reciprocal end products of a translocation. Amino acid sequences corresponding to the cDNA sequences of the entire ALL-1 gene and the partial sequence of the AF-4 gene are also provided. Probes are provided for detecting chromosomal abnormalities involving the ALL-1 gene on chromosome 11. Monoclonal antibodies for diagnosis and treatment and antisense oligonucleotides for the treatment of acute leukemias are also described.

Evolutionary conservation of the EPS8 gene and its mapping to human chromosome 12q23-q24.

Abstract: We have previously isolated the coding sequence for a novel substrate for tyrosine kinases, eps8, from NIH3T3 fibroblasts. Eps8 was phosphorylated in vivo by several receptor tyrosine kinases (RTKs) and, upon overexpression, was able to enhance EGFR-mediated mitogenic signaling in NIH3T3 cells. To gain understanding of eps8 function as well as its role in normal and neoplastic proliferation, we cloned the human eps8 coding sequence and studied expression of the human RNA and protein, evolutionary conservation, and chromosomal location. In addition to a previously identified SH3 domain, the predicted amino acid sequence of human eps8 revealed a non-random distribution of prolines, clustered in a way to suggest SH3-binding sites and a putative PH domain. Eps8 was expressed in all epithelial and fibroblastic lines examined and in some, but not all, hematopoietic cells. An essential function of eps8 in cell growth regulation was underscored by its conservation during evolution, where eps8-related sequences were detected as early as in Saccharomyces cerevisiae. Finally, the human EPS8 locus was mapped to chromosome 12q23-q24.

Cellular localization of the bcl-2 protein and response to glucocorticoid stress.

Abstract: We performed immunoelectronmicroscopy, immunofluorescence and subcellular fractionation studies of insect cells (Spodopetra frugiperda or SF9) infected with recombinant baculovirus containing bcl-2 cDNA to determine the cellular localization of the bcl-2 product. Similar studies were also undertaken in pre-B cells carrying a bcl-2 gene activated by t(14;18) chromosomal translocation. By immunogold electron microscopy, bcl-2 was localized at several intracellular sites including the nuclear membrane, endoplasmic reticulum, mitochondria and plasma membrane. Immunofluorescence studies revealed the presence of the bcl-2 product throughout the cytoplasm, whereas biochemical fractionation studies indicated a similar pattern to that observed on electron microscopy. Our investigation clearly indicates that the bcl-2 product is expressed at several intracellular sites. Studies were also undertaken to determine any changes in the subcellular distribution of bcl-2 protein following glucocorticoid exposure of immature B lymphocytes. Although no major changes in the distribution of bcl-2 protein were observed, more aggregated patches of gold labelled bcl-2 particles were found under glucocorticoid stress. Aggregation of bcl-2 molecules might represent dimerization necessary to prevent apoptosis.

The human homologue of the retroviral oncogene qin maps to chromosome 14q13

Abstract: Chromosomal mapping of the human QIN gene (renamed FKH2 by the Human Genome Organization Nomenclature Committee) was initially accomplished by correlation of the presence of the QIN locus with specific chromosome regions in a rodent-human hybrid panel. This analysis revealed that the human QIN gene maps to chromosome region 14q11.2-->14q32, between the TCR and IGH loci. Further analysis by fluorescence in situ hybridization techniques with a human QIN genomic clone refined the human QIN gene localization to 14q13.