Showing papers by "Carlo M. Croce published in 1998"

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

Abstract: The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

The role of the fhit/fra3b locus in cancer

Abstract: Common fragile sites form gaps at characteristic chromosome bands in metaphases from normal cells after aphidicolin induction. The distribution of common fragile sites parallels the positions of neoplasia-associated chromosomal rearrangements, prompting the proposal that fragility disposes to chromosomal rearrangements. Implicit in this hypothesis is that genes at fragile sites are altered by chromosome rearrangement and thus contribute to neoplastic growth. Chromosome band 3p14.2, encompassing the most inducible common fragile region, FRA3B, has been cloned and the FHIT gene, straddling FRA3B, characterized. The gene is inactivated by deletions in cancer-derived cell lines and primary tumors and Fhit protein is absent or reduced in lung, stomach, kidney, and cervical carcinomas, consistent with function as a tumor suppressor. FRA3B thus fulfills the prophecy that fragile site alterations contribute to the neoplastic process through inactivation of a tumor suppressor gene.

Serine-70 Is One of the Critical Sites for Drug-induced Bcl2 Phosphorylation in Cancer Cells

Abstract: Taxoids and other microtubule-damaging drugs are known to induce Bcl2 phosphorylation at the G2-M phase of the cell cycle, with concomitant apoptosis in malignant cells derived from a variety of human malignancies, including leukemia, lymphoma, and breast and prostate cancer. We have investigated the ability of another antineoplastic drug, dolastatin 10, in inducing Bcl2 phosphorylation and apoptosis. We also investigated the effects of a phosphatase inhibitor okadaic acid in the regulation of Bcl2 phosphorylation, cell cycle arrest, and programmed cell death. Moreover, site-directed mutagenesis studies were performed to determine the specific serine residue(s) responsible for drug-induced Bcl2 phosphorylation. Our results indicate that these antimicrotubule agents or okadaic acid can induce posttranslational modification (phosphorylation) of Bcl2 protein at multiple serine residues. Interestingly, mutation of a serine residue at position 70 to alanine can significantly decrease drug-induced posttranslational modification (phosphorylation) of Bcl2 protein. Apparently, Ser70 seems to be a critical site for drug-induced posttranslational modification (phosphorylation) of the Bcl2 protein.

Deregulated expression of TCL1 causes T cell leukemia in mice.

Abstract: The TCL1 oncogene on human chromosome 14q32.1 is involved in the development of T cell leukemia in humans. These leukemias are classified either as T prolymphocytic leukemias, which occur very late in life, or as T chronic lymphocytic leukemias, which often arise in patients with ataxia telangiectasia (AT) at a young age. The TCL1 oncogene is activated in these leukemias by juxtaposition to the α or β locus of the T cell receptor, caused by chromosomal translocations t(14:14)(q11:q32), t(7:14)(q35:q32), or by inversions inv(14)(q11:q32). To show that transcriptional alteration of TCL1 is causally involved in the generation of T cell neoplasia we have generated transgenic mice that carry the TCL1 gene under the transcriptional control of the p56lck promoter element. The lck-TCL1 transgenic mice developed mature T cell leukemias after a long latency period. Younger mice presented preleukemic T cell expansions expressing TCL1, and leukemias developed only at an older age. The phenotype of the murine leukemias is CD4−CD8+, in contrast to human leukemias, which are predominantly CD4+CD8−. These studies demonstrate that transcriptional activation of the TCL1 protooncogene can cause malignant transformation of T lymphocytes, indicating the role of TCL1 in the initiation of malignant transformation in T prolymphocytic leukemias and T chronic lymphocytic leukemias.

Loss of FHIT Expression in Gastric Carcinoma

Abstract: Loss of heterozygosity involving the short arm of chromosome 3 has been reported in gastric and other human tumors. We have cloned and mapped a candidate tumor suppressor gene, FHIT (fragile histidine triad), to this chromosomal region (3p14.2). To investigate the role of FHIT gene alterations in the development of gastric carcinoma, we examined 8 gastric carcinoma-derived cell lines and 32 primary adenocarcinoma samples by Southern blot analysis. We also analyzed the integrity of FHIT transcripts by reverse transcription-PCR. The occurrence of alterations in the FHIT gene and its transcript correlated with the absence of Fhit protein expression by immunoblot analysis in the cancer cell lines. Four of eight cell lines showed deletion or rearrangement within the FHIT gene, together with the absence of the wild-type transcript and the Fhit protein. Among the primary gastric carcinomas, rearrangement of the FHIT gene and/or aberrant reverse transcription-PCR products were detected in 17 of 32 (53%) tumors, and 20 of 30 (67%) samples exhibited an absence of Fhit protein expression. Gastric cancer is thought to develop from carcinogenic exposure, possibly explaining the high frequency of abnormalities in the FHIT gene, a fragile locus exhibiting susceptibility to carcinogen-induced alterations. The consequent absence or reduction of Fhit protein expression is consistent with the proposal that the FHIT gene is a preferential target of environmental carcinogens and that FHIT inactivation plays a role in the development of gastric cancer.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene

Abstract: ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of ≈100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5–10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans

Abstract: The tumor suppressor gene FHIT encompasses the common human chromosomal fragile site at 3p14.2 and numerous cancer cell biallelic deletions. To study Fhit function we cloned and characterized FHIT genes from Drosophila melanogaster and Caenorhabditis elegans. Both genes code for fusion proteins in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases; the D. melanogaster fusion protein exhibited diadenosine triphosphate (ApppA) hydrolase activity expected of an authentic Fhit homolog. In human and mouse, the nitrilase homologs and Fhit are encoded by two different genes: FHIT and NIT1, localized on chromosomes 3 and 1 in human, and 14 and 1 in mouse, respectively. We cloned and characterized human and murine NIT1 genes and determined their exon-intron structure, patterns of expression, and alternative processing of their mRNAs. The tissue specificity of expression of murine Fhit and Nit1 genes was nearly identical. Because fusion proteins with dual or triple enzymatic activities have been found to carry out specific steps in a given biochemical or biosynthetic pathway, we postulate that Fhit and Nit1 likewise collaborate in a biochemical or cellular pathway in mammalian cells.

Transcriptional map of 170-kb region at chromosome 11p15.5: Identification and mutational analysis of the BWR1A gene reveals the presence of mutations in tumor samples

Abstract: Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci D11S601 and D11S679 resulted in the identification of six transcriptional units. Three genes, hNAP2, CDKN1C, and KVLQT1, are well characterized, whereas three genes are novel. The three additional genes were designated BWR1A, BWR1B, and BWR1C. Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress, BWR1C cDNA was described as IPL [Qian, N., Franck, D., O’Keefe, D., Dao, D., Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997) Hum. Mol. Genet. 6, 2021–2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith–Wiedemann syndrome samples that resulted in the identification of genetic alterations in the BWR1A gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that BWR1A may play a role in tumorigenesis.

The role of deletions at the FRA3B/FHIT locus in carcinogenesis.

Abstract: The FHIT gene, which encodes a 1-kb message and a 16.8-kDa protein that hydrolyses diadenosine triphosphate (ApppA) to ADP and AMP in vitro, covers a megabase genomic region at chromosome band 3p14.2. The gene encompasses the most active of the common human chromosomal fragile regions, FRA3B. Over the years, it has been suggested that fragile sites might be especially susceptible to carcinogen damage and that chromosomal regions of nonrandom alterations in cancer cells may coincide with defined fragile sites. Within the FRA3B region, the characteristic induced chromosome gaps can occur across the entire region, but 60% of the gaps are centered on a 300-kb region flanking FHIT exon 5, the first protein-coding exon. Numerous hemizygous and homozygous deletions, translocations and DNA insertions occur within FHIT in cancer cell lines, uncultured tumors, and even in preneoplastic lesions, especially in tissues such as lung that are targets of carcinogens. This supports the proposed cancer-fragile site connection and suggests that the FHIT gene, expression of which is frequently altered in cells showing FHIT locus damage, is a tumor suppressor gene whose inactivation may drive clonal expansion of preneoplastic and neoplastic cells. Replacement of Fhit expression in Fhit-negative cancer cells abrogates their tumorigenicity in nude mice.

Crystal structure of MTCP-1: Implications for role of TCL-1 and MTCP-1 in T cell malignancies

Abstract: Chromosomal rearrangements in the T cell malignancies observed in T cell prolymphocytic leukemias, and in patients with Ataxia telangiectasia involve the translocation of one T cell receptor gene to either chromosome 14q32 or Xq28 (1–3). These genetic aberrations juxtapose cellular protooncogenes to enhancer elements leading to deregulation of oncogene expression (4). The two oncogenes involved in these translocations are MTCP-1 and TCL-1, which constitute a family of genes involved in lymphoid proliferation and T cell malignancies (2, 5, 6). The MTCP-1 (mature T cell proliferation-1) gene in the human X chromosome was the first candidate gene involved in the leukemogenesis of mature T cells (7). Aberrant or overexpressed MTCP-1 transcripts are found in the rare, but recurrent, chromosomal translocation t(X;14) in T cell proliferative diseases (8). About 10% of patients with the genetic disease Ataxia telangiectasia have clonal T cell proliferations with this cytogenetic aberration (1–3). MTCP-1 has two short ORFs that express A1 and B1 transcripts. The shorter A1 transcript is widely expressed in both tumor and nontumor cells. Expression of the B1 transcript is restricted to mature T cell proliferations with t(X;14) translocations (9), including T cell prolymphocytic leukemia tumors from patients with ataxia telangiectasia (10). The function of the B1 transcript of MTCP-1 is not known, but it encodes for a protein of 107 amino acids. The TCL-1 oncogene maps at chromosome 14q32.1 (5). Chromosomal rearrangements at the TCL-1 locus are associated with T cell leukemia of the mature phenotype such as T cell prolymphocytic leukemia, adult T cell leukemia, and chronic T cell leukemias in patients with immunodeficiency syndrome Ataxia telangiectasia. TCL-1 is activated in T cell leukemias and lymphomas by chromosome translocations or inversions that juxtapose the α/δ or the β locus of the T cell receptor to the TCL-1 oncogene. TCL-1 is expressed at high levels in pre-B cells and immature thymocytes, but not in mature B or T cells (11). No expression of TCL-1 was observed in cell lines derived from normal human tissues. Ataxia telangiectasia patients with chromosome 14 rearrangements show expression of TCL-1 in leukemic T cells (10, 12). Similarly, the murine homolog of TCL-1 is expressed early in embryonic development in immature T and B cells (13). The TCL-1 gene codes for a protein of 114 amino acid residues and molecular weight of 14,000 daltons (6). TCL-1 and MTCP-1 proteins share 40% identical amino acid residues and 61% similar amino acids, which strongly suggested that their tertiary structures are similar. However, no similarity was found with other human genes. Therefore, TCL-1 and MTCP-1 represent members of a unique family of genes involved in lymphoid proliferation and T cell malignancies. Human recombinant TCL-1 and MTCP-1 proteins have been expressed in Escherichia coli and purified for structural analysis to investigate their role in development of T cell malignancies (14). CD spectra at neutral pH are similar for both proteins and suggest β-sheet secondary structure. The crystal structure of MTCP-1 has been determined and refined to an R factor of 0.21 at 2.0 Å resolution. A model has been built of the structure of TCL-1. Analysis of these structures and the implications for the cellular function of these oncogene products are discussed.

The Murine Fhit Locus: Isolation, Characterization, and Expression in Normal and Tumor Cells

Abstract: The murine Fhit locus maps near the centromere v proximal Ptprg locus on mouse chromosome 14. The cDNA sequence and structure are similar to those of the human gene, with exons 5–9 encoding the protein. The predominant mRNA in the tissues and cell lines tested was an alternatively spliced form missing exon 3. Most murine cell lines tested, including lines established from normal mouse embryos and tumors, expressed very low or undetectable levels of Fhit mRNA. Most normal mouse tissues expressed wild-type Fhit mRNA, whereas ∼40% of murine lung carcinomas expressed wild-type and aberrant Fhit RT-PCR products that lacked various exons. Several tumorigenic mouse cell lines exhibited homozygous deletions of Fhit exons. We conclude that the murine Fhit gene, like its human counterpart, is a target of alterations involved in murine carcinogenesis.

Alternative splicing, genomic structure, and fine chromosome localization of REV3L.

Abstract: We have localized a human homolog, REV3L, of the Saccharomyces cerevisiae REV3 gene on chromosome region 6q21. The full-length cDNA consists of 10,919 nucleotides, with a putative open reading frame of 9,159 bp for a predicted protein of 3,053 amino acids. The gene contains 33 exons in about 200 kb of genomic DNA. In contrast to the previously reported sequence, an additional exon and an alternative splicing site are demonstrated.

The Biology of Tumors

Abstract: 1.The Role for ink4a in Melanoma Pathogenesis: One Gene, Two Products, Multiple Pathways J. Pomerantz, et al. 2. Identification and Characterization of collaborating Oncogenes in Compound Mutant Mice A. Berns, et al. 3. The Transcription Factor B-Myb Is Phosphorylated and Activated by Cyclin A/Cdk2 O. Bartsch, et al. 4. Distinct Dynamics and Regulatory Signal Transduction of Cell Migration: Lessons from Dendritic Cells, Tumor Cells and T Lymphocytes F. Entschladen, et al. 5. Genomic Instability in Sporadic Colorectal Cancer: A Destabilized Genome Producing Accelerated Cellular Evolution as the Fundamental Nature of Cancer G.R. Anderson, et al. 6. APC and the Early Events of Colon Cancer R.L. White. 7. Genome Scanning and Gene Discovery in Breast and Ovarian Cancer J.W. Gray, et al. 8. Failure of Tumor Immunity Resulting from Inaccessibility of Activated Lymphocytes to Solid Tumors: The Possible Role of the Endothelium R. Ganss, D. Hanahan. 9. Deciphering Molecular Circuitry Using High-Density DNA Arrays D.H. Mack, et al. 10. Use of cDNA Microarrays to Assess DNA Gene Expression Patterns in Cancer P.S. Meltzer, et al. 11. Eh, a Novel Protein: Protein Interaction Domain M. Doria, et al. 12 Additional Chapters. Index.

A novel surface marker (B203.13) of human haemopoietic progenitors, preferentially expressed along the B and myeloid lineages

Abstract: We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi-phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21+ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34+ cells, B203.13 was expressed on early committed myeloid (CD33+) and erythroid (CD71dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.