Showing papers by "Carlo M. Croce published in 2000"

Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells

Abstract: Bcl-2 and related proteins are key regulators of apoptosis or programmed cell death implicated in human disease including cancer. We recently showed that cell-permeable Bcl-2 binding peptides could induce apoptosis of human myeloid leukemia in vitro and suppress its growth in severe combined immunodeficient mice. Here we report the discovery of HA14-1, a small molecule (molecular weight = 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl-2 protein. In vitro binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological function. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the decrease in mitochondrial membrane potential and activation of caspase-9 followed by caspase-3. Cytokine response modifier A, a potent inhibitor of Fas-mediated apoptosis, did not block apoptosis induced by HA14-1. Whereas HA14-1 strongly induced the death of NIH 3T3 (Apaf-1+/+) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1−/−) mouse embryonic fibroblast cells. These data are consistent with a mechanism by which HA14-1 induces the activation of Apaf-1 and caspases, possibly by binding to Bcl-2 protein and inhibiting its function. The discovery of this cell-permeable molecule provides a chemical probe to study Bcl-2-regulated apoptotic pathways in vivo and could lead to the development of new therapeutic agents.

Identification of a gene at 11q23 encoding a guanine nucleotide exchange factor: evidence for its fusion with MLL in acute myeloid leukemia.

Abstract: We have identified a gene at 11q23, telomeric to MLL, that encodes a guanine nucleotide exchange factor (GEF). This gene is transcribed into a 9.5-kb mRNA containing a 4.6-kb ORF. By Northern analysis, it was found to be expressed in all human tissues examined including peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine, colon, and minimally in thymus. Analysis of the predicted protein sequence indicates that it has strong homology to several members of the family of Rho GEFs that includes such oncogenes as Dbl, Vav, Tiam, and Bcr. A patient with primary acute myeloid leukemia (AML) and a karyotype of 51,XY,+8,+19,+3mar was found to have the 5′ end of MLL at exon 6 fused in-frame with the 3′ end of almost the entire ORF of this gene, which we named LARG for leukemia-associated Rho GEF. Transcriptional orientation of both genes at 11q23 is from centromere to telomere, consistent with other data that suggest the MLL-LARG fusion resulted from an interstitial deletion rather than a balanced translocation. LARG does not appear to have any homology with other MLL partner genes reported thus far. Thus, LARG represents an additional member of the GEF family and a novel MLL fusion partner in acute myeloid leukemia.

Low frequency of alterations of the α (PPP2R1A) and β (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms

Abstract: The phosphatase 2A (PP2A) is one of the major cellular serine-threonine phosphatases. It was recently shown that the gene encoding for the β isoform of its subunit A, PPP2R1B, is altered in human lung and colorectal carcinomas, suggesting a role in human tumorigenesis. Here, we report the detection of mutations in breast, lung carcinomas and melanomas in the genes of both α (PPP2R1A) and β isoforms. Mutations affecting PPP2R1B were found in four breast carcinomas, while mutations in PPP2R1A were found in carcinomas of the breast and of the lung and in one melanoma. Most of the mutations affecting PPP2R1B were exons deletions, suggesting abnormal splicing. These splicing abnormalities were detected in tumor samples in the absence of the normal splicing product, and were not found in several normal controls. In one case, a homozygous deletion present in tumor DNA, and not in the matched normal control was demonstrated. Mutations affecting the PPP2R1A gene were nucleotide substitutions changing highly conserved amino acids and one frame-shift. Although the frequency of alterations is low, the inclusion of both isoforms of subunit A in the genes mutated in human cancer and the addition of breast cancer to the list of neoplasms in which PPP2R1B is altered, strengthen the potential role of PP2A in human tumorogenesis.

Cell Permeable Bcl-2 Binding Peptides: A Chemical Approach to Apoptosis Induction in Tumor Cells

Abstract: Bcl-2 is a potent suppressor of apoptosis, and its overexpression contributes to tumorigenesis in many types of human cancers. To test the possibility of modulating Bcl-2 function as an anticancer strategy, a cell permeable Bcl-2 binding peptide, cell permeable moiety (cpm)-1285, was designed by chemically attaching a fatty acid to a peptide derived from the proapoptotic protein Bad. cpm-1285 entered HL-60 tumor cells, bound Bcl-2 protein, and induced apoptosis in vitro. In contrast, cpm-1285 had little effect on normal human peripheral blood lymphocytes. Furthermore, cpm-1285 had in vivo activity in slowing human myeloid leukemia growth in severe combined immunodeficient mice. These results demonstrate a novel approach for therapeutic intervention of tumor growth in vivo with small molecule inhibitors of Bcl-2.

A novel zinc finger gene is fused to EWS in small round cell tumor

Abstract: Ewing sarcoma family of tumors share recurrent translocations that fuse EWS from 22q12 to five different members of transcription factors namely FLI-1, ERG, ETV1, E1AF and FEV. Different classes of DNA binding proteins, ATF1, WT1 and CHOP are fused to EWS generating distinct tumor phenotypes: clear cell sarcoma, desmoplastic small round cell tumor, and myxoid liposarcoma, respectively. We have cloned a novel gene located at 22q12 fused to EWS by a submicroscopic inversion of 22q in a small round cell sarcoma showing a translocation (t(1;22)(p36.1;q12). The gene, designated ZSG (Zinc finger Sarcoma Gene), is a putative Cys2-His2 zinc finger protein which contains a POZ transcriptional repressor-like domain at the N-terminus. The rearrangement involves intron 8 of EWS and exon 1 of ZSG creating a chimeric sequence containing the transactivation domain of EWS fused to zinc finger domain of ZSG. This product lacks the transcriptional repressor domain at the N-terminus of ZSG. A rearrangement of the second ZSG allele was also found in tumor cells. This is the first example of an intra-chromosomal rearrangement of chromosome 22, undetectable by cytogenetics, activating EWS in soft tissue sarcoma.

Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus.

Abstract: The FHIT gene, located at chromosome 3p14.2, is a tumor suppressor gene often involved in tumors resulting from exposure to environmental carcinogens. We studied 46 pairs of esophageal primary tumors and corresponding normal squamous mucosa specimens by molecular genetic and immunohistochemical methods to investigate the role of the FHIT gene in esophageal carcinoma. In addition, we studied several different types of lesions, such as carcinoma in situ or dysplasia by immunohistochemistry. Loss of heterozygosity at or around the FHIT gene was observed in 35 (76%) primary tumors. Immunohistochemical detection of Fhit protein in the primary tumors demonstrated that 14 (30%) were positive and 32 (70%) were negative. We observed concordance between loss of Fhit protein and loss of heterozygosity and between loss of Fhit protein and RNA abnormalities. Because the FHIT/FRA3B locus is susceptible to damage by environmental carcinogens, we investigated the correlation between Fhit expression and smoking or alcohol habits. In this relatively small study, the patients who were both heavy users of tobacco and alcohol showed a significantly higher frequency of loss of Fhit expression than those who were light users. Noncarcinomatous squamous epithelium showed positive Fhit reactivity in most cases; however, five showed negative Fhit reactivity. Interestingly, all of these five patients had habits of heavy use of tobacco and alcohol. Eight of 12 carcinomas in situ, 2 of 4 severe dysplasias, 4 of 8 moderate dysplasias, and 3 of 9 mild dysplastic lesions showed negative Fhit reactivity. These findings indicated that loss of Fhit expression may be an early event in the development of human esophageal carcinoma and may occur even in normal-appearing squamous epithelium in some patients heavily exposed to environmental carcinogens.

huASH1 protein, a putative transcription factor encoded by a human homologue of the Drosophila ash1 gene, localizes to both nuclei and cell–cell tight junctions

Abstract: During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax (trx)and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homologue of trx, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an ≈10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling.

Regulation of TCL1 Expression in B- and T-Cell Lymphomas and Reactive Lymphoid Tissues

Abstract: Chromosomal rearrangements observed in T-cell prolymphocytic leukemia involve the translocation of one T-cell receptor gene to either chromosome 14q32 or Xq28, deregulating the expression of cellular protooncogenes of unknown function, such as TCL1 or its homologue, MTCP1. In the human hematopoietic system, TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells causes mature T-cell proliferation in transgenic mice. In this study, using a newly generated monoclonal antibody against recombinant TCL1 protein, we extended our analysis mainly by immunohistochemistry and also by fluorescence-activated cell sorting and Western blot to a large tumor lymphoma data bank including 194 cases of lymphoproliferative disorders of B- and T-cell origin as well as reactive lymphoid tissues. The results obtained show that in reactive lymphoid tissues, TCL1 is strongly expressed by a subset of mantle zone B lymphocytes and is expressed to a lesser extent by follicle center cells and by scattered interfollicular small lymphocytes. In B-cell neoplasia, TCL1 was expressed in the majority of the cases, including lymphoblastic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma (60%), and primary cutaneous B cell lymphoma (55%). TCL1 expression was observed in both the cytoplasmic and nuclear compartments, as confirmed by Western blot analysis. Conversely, TCL1 was not expressed in Hodgkin/Reed-Sternberg cells, multiple myelomas, marginal zone B-cell lymphomas, CD30+ anaplastic large cell lymphoma, lymphoblastic T-cell lymphoma, peripheral T-cell lymphoma, and mycosis fungoides. These data indicate that TCL1 is expressed in more differentiated B cells, under both reactive and neoplastic conditions, from antigen committed B cells and in germinal center B cells and is down-regulated in the latest stage of B-cell differentiation.

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas

Abstract: Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

A pentamer transcriptional complex including tal-1 and retinoblastoma protein downmodulates c-kit expression in normal erythroblasts.

Abstract: Human proerythroblasts and early erythroblasts, generated in vitro by normal adult progenitors, contain a pentamer protein complex comprising the tal-1 transcription factor heterodimerized with the ubiquitous E2A protein and linked to Lmo2, Ldb1, and retinoblastoma protein (pRb). The pentamer can assemble on a consensus tal-1 binding site. In the pRb− SAOS-2 cell line transiently transfected with a reporter plasmid containing six tal-1 binding site, pRb enhances the transcriptional activity of tal-1–E12–Lmo2 and tal-1–E12–Lmo2–Ldb1 complexes but not that of a tal-1–E12 heterodimer. We explored the functional significance of the pentamer in erythropoiesis, specifically, its transcriptional effect on the c-kit receptor, a tal-1 target gene stimulating early hematopoietic proliferation downmodulated in erythroblasts. In TF1 cells, the pentamer decreased the activity of the reporter plasmid containing the c-kit proximal promoter with two inverted E box-2 type motifs. In SAOS-2 cells the pentamer negatively regulates (i) the activity of the reporter plasmid containing the proximal human c-kit promoter and (ii) endogenous c-kit expression. In both cases pRb significantly potentiates the inhibitory effect of the tal-1–E12–Lmo2–Ldb1 tetramer. These data indicate that this pentameric complex assembled in maturing erythroblasts plays an important regulatory role in c-kit downmodulation; hypothetically, the complex may regulate the expression of other critical erythroid genes.

Loss of FHIT expression in transitional cell carcinoma of the urinary bladder.

Abstract: Cytogenetic and loss of heterozygosity (LOH) studies demonstrated chromosome 3p deletions in transitional cell carcinoma (TCC). We recently cloned the tumor suppressor gene FHIT (fragile histidine triad) at 3p14.2, one of the most frequently deleted chromosomal regions in TCC of the bladder, and showed that it is the target of environmental carcinogens. Abnormalities at the FHIT locus have been found in tumors of the lung, breast, cervix, head and neck, stomach, pancreas, and clear cell carcinoma of the kidney. We examined six TCC derived cell lines (SW780, T24, Hs228T, CRL7930, CRL7833, and HTB9) and 30 primary TCC of the bladder for the integrity of the FHIT transcript, using reverse transcriptase–polymerase chain reaction (RT-PCR) to investigate a potential role of the FHIT gene in TCC of the bladder. In addition, we tested expression of the Fhit protein in the six TCC-derived cell lines by Western blot analysis and in 85 specimens of primary TCCs by immunohistochemistry. Three of the six cell lines (50%) did not show the wild-type FHIT transcript, and Fhit protein was not detected in four of the six cell lines (67%) tested. Fhit expression also was correlated with pathological and clinical status. A significant correlation was observed between reduced Fhit expression and advanced stage of the tumors. Overall, 26 of 30 (87%) primary TCCs showed abnormal transcripts. Fhit protein was absent or greatly reduced in 61% of the TCCs analyzed by immunohistochemistry. These results suggested that loss of Fhit expression may be as important in the development of bladder cancer as it is for other neoplasms caused by environmental carcinogens.

Definition and Refinement of Chromosome 8p Regions of Loss of Heterozygosity in Gastric Cancer

Abstract: Loss of heterozygosity at several chromosomal loci is a common feature of the malignant progression of human tumors. These regions are thought to harbor one or more putative tumor suppressor gene(s) playing a role in tumor development. Allelic losses on the short arm of chromosome 8 (8p) have been reported as frequent events in several cancers, and three commonly deleted regions have been defined at 8p11.2-12, 8p21-22, and 8p23.1. To evaluate the possible involvement of these regions in gastric cancer, we used eight microsatellite markers to perform an extensive analysis of allele loss at 8p21-22 in 52 cases of primary gastric adenocarcinoma. We found that 44% of tumors showed allelic loss for at least one marker at 8p21-22. The critical region of loss was found to be between markers LPL and D8S258, which displayed loss of heterozygosity in 39% and 33% of cases, respectively. This region is centromeric to the LPL locus and centered on the D8S258 locus. We conclude that 8p22 deletion is a frequent event in gastric cancer and suggest the presence of a putative tumor suppressor gene near the D8S258 locus. Initial steps were taken toward the identification of this gene, which is likely to play an important role in the pathogenesis of gastric cancer and of other tumors as well.

Compositions, kits, and methods relating to the human fez1 gene, a novel tumor suppressor gene

Abstract: The invention relates to isolated polynucleotides homologous with a portion of one strand of the human tumor suppressor gene, FEZ1, and to the tumor suppressor protein encoded thereby, Fez1. The polynucleotides are useful, for example, as probes, primers, portions of expression vectors, and the like. The invention also includes diagnostic, therapeutic, cell proliferation enhancement, and screening methods which involve these polynucleotides and protein. The invention further includes kits useful for performing the methods of the invention.

Modified intranuclear organization of regulatory factors in human acute leukemias: Reversal after treatment

Abstract: Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias.

Expression of Fhit protein during mouse development.

Abstract: Fhit protein has a putative tumor suppressor function in several types of human and experimental cancers. To assess whether Fhit is involved in fetal development we have examined the distribution of Fhit protein in the 12- through 16-day postcoitum mouse fetus and in postnatal day 0 mouse pups by immunocytochemistry. High levels of Fhit protein were observed in the endodermal derivatives, namely, bronchi, trachea, esophagus, stomach, and intestine, in the 12- to 16-day postcoitum mouse fetus and in the postnatal day 0 pup. Other tissues showed a more restricted pattern of Fhit protein expression. These results suggest that Fhit may play a role in the development of specific tissues during mouse development.

Compositions, kits et procedes concernant le gene fez1 de l'homme, nouveau gene suppresseur des tumeurs

Abstract: L'invention concerne des homologues de polynucleotides isoles avec une partie d'un brin du gene suppresseur de tumeur de l'homme, le gene FEZ1. L'invention traite egalement de la proteine suppresseur de tumeur ainsi codee, Fezl. Ces polynucleotides presentent un grand interet, par exemple, comme sondes, comme amorces, comme parties de vecteurs d'expression et similaires. L'invention traite egalement de procedes de diagnostique, de therapie, de proliferation cellulaire et de dosage utilisant ces polynucleotides et ces proteines. L'invention a egalement pour objet des kits permettant de mettre en oeuvre les procedes selon l'invention.