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C M Croce

Publications and source records attributed to C M Croce.

517 records · Page 29Linked to original sources
PubMedDate not supplied

Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers.

BACKGROUND: The nucleotide-binding protein Fhit, among the earliest and most frequently inactivated proteins in lung cancer, suppresses tumor formation by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion protein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression profiles. According to the Rosetta Stone hypothesis, if the separate Nit and Fhit genes could be shown to occur in the same subset of genomes (that is, to share a phylogenetic profile), then the existence of a fusion protein in invertebrates and the coordinated expression of separate mRNAs in mouse suggest that Nit and Fhit function in the same pathway and that the structure of invertebrate NitFhit may reflect the nature of Nit-Fhit interactions. RESULTS: To satisfy the phylogenetic profile criterion for functional significance of protein fusion events, we cloned additional Nit homologs from organisms with Fhit homologs. We used fluorescent nucleotide analogs of ApppA to follow the purification and to characterize the nucleotide specificity of NitFhit from Caenorhabditis elegans, crystallized the 200 kDa tetrameric complex, and solved the structure of NitFhit from a single mercury derivative phased by two-wavelength anomalous diffraction. CONCLUSIONS: Nit monomers possess a new alpha-beta-beta-alpha sandwich fold with a presumptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stranded beta box that binds Fhit dimers at opposite poles and displays Nit active sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit. Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit.

Acid Anhydride Hydrolases↗
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Nucleotide sequence of cloned cDNA of human c-myc oncogene.

Like other transforming genes of retroviruses, the v-myc gene of the avian virus, MC29, has a homologue in the genome of normal eukaryotic cells. The human cellular homologue, c-myc, located on human chromosome 8, region q24 leads to qter (refs 1, 2), is translocated into the immunoglobulin heavy-chain locus on human chromosome 14 (ref. 3) in Burkitt's lymphoma, suggesting that c-myc has a primary role in transformation of some human haematopoietic cells. In addition, c-myc is amplified in the human promyelocytic leukaemia cell line, HL60 (refs 6, 7) which also contains high levels of c-myc mRNA. Recently, Colby et al. reported the nucleotide sequence of the human c-myc DNA isolated from a genomic recombinant DNA library derived from human fetal liver. This 4,053-base pair (bp) sequence includes two exons and one intron of the myc gene, and the authors have suggested the existence of a human c-myc mRNA of 2,291 nucleotides that has a coding capacity for a protein of molecular weight (Mr) 48,812. We have approached the problem of accurately defining the characteristics of the human c-myc mRNA and c-myc protein by determining the sequence of the c-myc cDNA isolated from a cDNA library prepared from mRNA of a clone of the K562 human leukaemic cell line. K562 cells are known to contain c-myc mRNA which is similar in size to the c-myc mRNA of other human cell types. We report here the sequence of 2,121 nucleotides of a human c-myc mRNA and demonstrate that its 5' noncoding sequence does not correspond to the sequence of the reported genomic human sequence. However, our data confirm that the intact human c-myc mRNA can encode a 48,812-Mr protein with a sequence identical to that reported by Colby et al.

Amino Acid Sequence↗
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Association of amplified oncogene c-myc with an abnormally banded chromosome 8 in a human leukaemia cell line.

Several unusual chromosome structures have been described in drug-resistant cell lines and in certain tumours. These structures include elongated homogeneously staining regions (HSRs), small extrachromosomal paired chromatin bodies (double minutes, DMs) and abnormally banded regions (ABRs) with strong but anomalous band patterns. There is evidence that these are alternative forms of gene amplification, with HSRs breaking down to form DMs, and DMs integrating into the chromosome to generate HSRs and ABRs. Recently, it was demonstrated that, compared with several normal and leukaemia human cells, DNA sequences representing the human homologue of the onc gene of the avian myelocytomatosis virus (MC29), the so-called c-myc gene, were amplified in HL-60 cells. This is a human promyelocytic leukaemia cell line established in the laboratory of one of us (R.C.G.) at the National Cancer Institute (Bethesda, Maryland) in 1977, and widely used for studies on myeloid and monocytic differentiation. Amplification of the gene was present in primary leukaemic cells of the patient, and DMs were noted in some of these cells as well as in early passages of the HL-60 line. No structure resembling HSRs or ABRs were noted in karyotypic studies at this early stage and there were no alterations involving the long arm of chromosome 8 (8q), to which the c-myc gene has recently been mapped. We have now re-examined the karyotype of the HL-60 line, using cells frozen at various times during its continuous passage at the Wistar Institute (Philadelphia, Pennsylvania) to look for chromosomal abnormalities that might be associated with the amplification of c-myc. We find that, beginning in 1979, HL-60 cells at the Wistar Institute no longer had DMs, but did show an abnormal 8q+ chromosome, replacing a normal chromosome 8, and representing an ABR reflecting the site of myc gene amplification.

Cell Line↗
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Suppression of the normal mouse c-myc oncogene in human lymphoma cells.

In Burkitt's lymphoma, which carries the t(8;14) chromosome translocation, the c-myc oncogene normally located on band q24 of human chromosome 8 (refs 1-3) translocates to the heavy-chain locus on chromosome 14 (refs 1, 4, 5); this results in transcriptional deregulation of the translocated c-myc oncogene, which is transcribed constitutively at elevated levels, while the normal c-myc oncogene on the uninvolved chromosome 8 is either silent or expressed at very low levels (A.ar-R. and C.M.C., unpublished results). We have now introduced the active c-myc oncogene of proliferating mouse spleen cells into human lymphoma cells carrying the t(8;14) chromosome translocation by hydridization, and have examined the hybrids for expression of the human and murine c-myc oncogene. The results of this analysis, reported here, indicate that the active mouse myc gene is shut off at the transcriptional level in the human lymphoma cells, implying that human B cells at the stage of differentiation of lymphoma cells used in this study are nonpermissive for normal c-myc transcription.

Animals↗
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Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) chromosome translocation.

The t(11;14) (q13;q32) chromosome translocation has been reported in diffuse small and large cell lymphomas and in chronic lymphocytic leukaemia (B-CLL) and multiple myeloma. Because chromosome band 14q32 is involved in this translocation, as well as in the t(8;14) (q24;q32) translocation of the Burkitt tumour, interruption of the immunoglobulin heavy-chain locus was postulated for this rearrangement. We have cloned the chromosomal joinings between chromosomes 11 and 14 and also between chromosomes 14 and 18, in B-cell tumours carrying translocations involving these chromosomes, and suggested the existence of two translocated loci, bcl-1 and bcl-2, normally located on chromosomes 11 (band q13) and 18 (band q21) respectively, involved in the pathogenesis of human B-cell neoplasms. The results indicate that in the leukaemic cells from two different cases of CLL, the breakpoints on chromosome 11 are within 8 nucleotides of each other and on chromosome 14 involve the J4-DNA segment. Because we detected a 7mer-9mer signal-like sequence with a 12-base-long spacer on the normal chromosome 11, close to the breakpoint, we speculate that the t(11;14) chromosome translocation in CLL may be sequence specific and may involve the recombination system for immunoglobulin gene segment (V-D-J) joining.

B-Lymphocytes↗
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Expression of a translocated c-abl gene in hybrids of mouse fibroblasts and chronic myelogenous leukaemia cells.

Chronic myelogenous leukaemia (CML) is a clonal disease arising from malignant transformation of pluripotent hematopoietic stem cells. In most cases, it is characterized by the presence of the Philadelphia (Ph1) chromosome (22q-) which results from a reciprocal translocation between chromosomes 9 and 22 (refs 1-3). In this translocation, the human homologue of the Abelson virus oncogene, c-abl, normally on chromosome 9, is moved to chromosome 22, while c-sis, the cellular homologue of the simian sarcoma virus oncogene, is moved from chromosome 22 to chromosome 9 (refs 4-6). CML cells carrying the t(9;22) chromosomal translocation are known to produce an 8-kilobase (kb) c-abl transcript in addition to the normal 6- and 7-kb transcripts and to express the normal p145 abl protein and a p210 c-abl protein possessing a tyrosine kinase activity not detected in the p145 species. Results of our analyses using somatic cell hybrids between a mouse fibroblast line and two human CML-derived cell lines which carry the Ph1 chromosome and are phenotypically identical to the fibroblast parent indicate that only the hybrid cells containing Ph1 chromosome express both the 8-kb c-abl RNA and the p210 protein. Thus, expression of the altered c-abl transcripts and protein depends on the presence of the Ph1 chromosome and is not myeloid-specific.

Animals↗
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Localization of gene for human p53 tumour antigen to band 17p13.

Recently the gene for the cellular tumour antigen p53, a phosphoprotein found in increased concentration in a variety of human cells, had been mapped to region 17q22 by in situ hybridization techniques and has been shown to translocate to the chromosome carrying the translocation [t(15; 17)] associated with acute promyelocytic leukaemia (APL). Based on this finding it has been postulated that this gene has a role in the pathogenesis of APL. Here we present evidence that the gene for p53 is not located on the long arm of chromosome 17, but maps to band 17p13. We therefore suggest that this gene is not directly involved in the chromosome translocation observed in APL.

Antigens, Neoplasm↗
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The t(8; 14) chromosomal translocation occurring in B-cell malignancies results from mistakes in V-D-J joining.

The reciprocal chromosome translocation, t(8;14), involving the heavy chain locus on chromosome 14 and the c-myc oncogene on chromosome 8 is a characteristic of the B-cell malignancies Burkitt's lymphoma and acute lymphoblastic leukaemia (ALL). We have cloned and sequenced the t(8; 14) breakpoints of an African Burkitt's lymphoma cell line, P3HR-1, and a pre-B cell ALL cell line, 380. In each case the region of chromosome 8 involved has recombined with a JH region on chromosome 14. The two sites of breakage on chromosome 8 lie within 70 base pairs (bp) of one another. At each joining site, sequences homologous to the signal sequences thought to be recognized by the V-D-J recombinase were identified, as were N regions. In B-cell chronic lymphocytic leukaemias (B-CLL) carrying the t(11; 14) chromosome translocation and in follicular lymphomas carrying the t(14; 18) translocation, the V-D-J recombinase is implicated in the mechanism of chromosomal translocations. We speculate that the same enzymatic mechanism is responsible for the t(8; 14) translocations in African Burkitt's lymphoma and pre-B cell ALL.

Base Sequence↗
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A new fused transcript in Philadelphia chromosome positive acute lymphocytic leukaemia.

The leukaemic cells of more than 90% of chronic myelogenous leukaemia (CML) patients and of 10% of acute lymphocytic leukaemia (ALL) patients carry the t(9:22) (q34:q11) translocation which generates the Philadelphia chromosome (Ph1). In CML the abl gene is translocated from chromosome 9 to the centre of the bcr gene on chromosome 22 and this results in production of chimaeric bcr-abl RNA translated into a protein of relative molecular mass (Mr) 210,000 (210K). Our data indicate that in ALL abl is translocated into the 5' region of the bcr gene. The consequence of this is the expression of a fused transcript in which the first exon of bcr is linked to the second abl exon. This transcript encodes a 190K protein kinase.

Amino Acid Sequence↗
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Antiapoptosis potential of bcl-2 oncogene by dephosphorylation.

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.

Apoptosis↗
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Fragile histidine triad gene and skin cancer.

Five years ago the fragile histidine triad (FHIT) gene including the most common fragile site locus of the human genome, FRA3B, was identified. The gene is altered in many types of cancer and several data support the idea that FHIT has to be considered a tumor suppressor. FHIT abnormalities were investigated in some skin tumors. Fifty-seven per cent of Merkel cell carcinomas displayed abnormal FHIT products but the involvement of FHIT in human non-melanoma skin cancer is still unclear. Because the murine Fhit locus is similar to its human homologue and is altered in cancer cell lines, we have established a strain of Fhit-deficient mice. After N-nitrosomethylbenzylamine treatment, the spectrum of tumors developed by the Fhit-deficient mice was similar to those observed in a familial skin cancer condition, the Muir-Torre syndrome, although there is no clear evidence yet for a relationship of FHIT and the human syndrome. Because cancer cells lacking in FHIT are defective in apoptosis, we propose the Fhit-deficient mouse as a model to understand a possible proapoptotic mechanism deficiency in the human syndrome.

Acid Anhydride Hydrolases↗