Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Subtraction hybridization identifies a transformation progression-associated gene PEG-3 with sequence homology to a growth arrest and DNA damage-inducible gene

Cancer is a progressive multigenic disorder characterized by defined changes in the transformed phenotype that culminates in metastatic disease. Determining the molecular basis of progression should lead to new opportunities for improved diagnostic and therapeutic modalities. Through the use of subtraction hybridization, a gene associated with transformation progression in virus- and oncogene-transformed rat embryo cells, progression elevated gene-3 (PEG-3), has been cloned. PEG-3 shares significant nucleotide and amino acid sequence homology with the hamster growth arrest and DNA damage-inducible gene gadd34 and a homologous murine gene, MyD116, that is induced during induction of terminal differentiation by interleukin-6 in murine myeloid leukemia cells. PEG-3 expression is elevated in rodent cells displaying a progressed-transformed phenotype and in rodent cells transformed by various oncogenes, including Ha-ras, v-src, mutant type 5 adenovirus (Ad5), and human papilloma virus type 18. The PEG-3 gene is transcriptionally activated in rodent cells, as is gadd34 and MyD116, after treatment with DNA damaging agents, including methyl methanesulfonate and γ-irradiation. In contrast, only PEG-3 is transcriptionally active in rodent cells displaying a progressed phenotype. Although transfection of PEG-3 into normal and Ad5-transformed cells only marginally suppresses colony formation, stable overexpression of PEG-3 in Ad5-transformed rat embryo cells elicits the progression phenotype. These results indicate that PEG-3 is a new member of the gadd and MyD gene family with similar yet distinct properties and this gene may directly contribute to the transformation progression phenotype. Moreover, these studies support the hypothesis that constitutive expression of a DNA damage response may mediate cancer progression.

The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.

Cancer is a disease characterized by defects in growth control, and tumor cells often display abnormal patterns of cellular differentiation. The combination of recombinant human fibroblast interferon and the antileukemic agent mezerein corrects these abnormalities in cultured human melanoma cells resulting in irreversible growth arrest and terminal differentiation. Subtraction hybridization identifies a melanoma differentiation associated gene (mda-7) with elevated expression in growth arrested and terminally differentiated human melanoma cells. Colony formation decreases when mda-7 is transfected into human tumor cells of diverse origin and with multiple genetic defects. In contrast, the effects of mda-7 on growth and colony formation in transient transfection assays with normal cells, including human mammary epithelial, human skin fibroblast, and rat embryo fibroblast, is quantitatively less than that found with cancer cells. Tumor cells expressing elevated mda-7 display suppression in monolayer growth and anchorage independence. Infection with a recombinant type 5 adenovirus expressing antisense mda-7 eliminates mda-7 suppression of the in vitro growth and transformed phenotype. The ability of mda-7 to suppress growth in cancer cells not expressing or containing defects in both the retinoblastoma (RB) and p53 genes indicates a lack of involvement of these critical tumor suppressor elements in mediating mda-7-induced growth inhibition. The lack of protein homology of mda-7 with previously described growth suppressing genes and the differential effect of this gene on normal versus cancer cells suggests that mda-7 may represent a new class of cancer growth suppressing genes with antitumor activity.

The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (mda-7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda-7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda-7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda-7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda-7, Ad.mda-7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.mda-7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda-7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda-7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda-7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.

Localization of the Wilson’s disease protein product to mitochondria

Wilson’s disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.

Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing sarcoma.

Olfactory neuroblastoma (ONB) is a malignant tumor of the nasal mucosa whose histogenesis is unclear. A relationship to neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is based on morphologic similarities and the expression of similar neural antigens. However, the clinical presentation of ONB differs from that of NB, and MYCN amplification characteristic of NB is not observed. We have therefore examined the relationship of this malignancy to other classes of neural tumors. In previous studies, two ONB cell lines demonstrated cytogenetic features and patterns of protooncogene expression suggestive of a relationship to the Ewing sarcoma family of childhood peripheral primitive neuroectodermal tumors (pPNETs). The pPNETs show t(11;22)(q24;q12) or t(21;22)(q22;q12) chromosomal translocations fusing the EWS gene from 22q12 with either the FL11 gene on 11q24 or the ERG gene on 21q22. We therefore analyzed ONBs for the presence of pPNET-associated gene fusions. Both cell lines showed rearrangement of the EWS gene, and fluorescence in situ hybridization (FISH) of each case demonstrated fusion of EWS and FL11 genomic sequences. Moreover, both lines expressed EWS/FL11 fusion transcripts with in-frame junctions between exon 7 of EWS and exon 6 of FL11 as described for pPNETs. We identified similar gene fusions in four of six primary ONB cases. None of the cases expressed tyrosine hydroxylase, a catecholamine biosynthetic enzyme widely expressed in NB. Our studies indicate that ONB is not a NB but is a member of the pPNET family.

Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression

Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Myb-related protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression.

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

Unique, small sequences (sequence tag sites) have been identified at the 3′ ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3′ unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3′ hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3′ end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.

Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the XPD gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults

Reduction of 5,10-methylenetetrahydrofolate (methyleneTHF), a donor for methylating dUMP to dTMP in DNA synthesis, to 5-methyltetrahydrofolate (methylTHF), the primary methyl donor for methionine synthesis, is catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR). A common 677 C → T polymorphism in the MTHFR gene results in thermolability and reduced MTHFR activity that decreases the pool of methylTHF and increases the pool of methyleneTHF. Recently, another polymorphism in MTHFR (1298 A → C) has been identified that also results in diminished enzyme activity. We tested whether carriers of these variant alleles are protected from adult acute leukemia. We analyzed DNA from a case–control study in the United Kingdom of 308 adult acute leukemia patients and 491 age- and sex-matched controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. The MTHFR 677TT genotype was lower among 71 acute lymphocytic leukemia (ALL) cases compared with 114 controls, conferring a 4.3-fold decrease in risk of ALL [odds ratio (OR = 0.23; 95% CI = 0.06–0.81]. We observed a 3-fold reduction in risk of ALL in individuals with the MTHFR 1298AC polymorphism (OR = 0.33; 95% CI = 0.15–0.73) and a 14-fold decreased risk of ALL in those with the MTHFR 1298CC variant allele (OR = 0.07; 95% CI = 0.00–1.77). In acute myeloid leukemia, no significant difference in MTHFR 677 and 1298 genotype frequencies was observed between 237 cases and 377 controls. Individuals with the MTHFR 677TT, 1298AC, and 1298CC genotypes have a decreased risk of adult ALL, but not acute myeloid leukemia, which suggests that folate inadequacy may play a key role in the development of ALL.

A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells

A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) (5-methyltetrahydrofolate:(acceptor) oxidoreductase, EC 1.7.99.5), a key regulatory enzyme in one-carbon metabolism, results in a thermolabile variant of the MTHFR enzyme with reduced activity in vitro. In the present study we used a chromatographic method for folate analysis to test the hypothesis that this mutation would be associated with altered distribution of red blood cell (RBC) folates. An alteration was found as manifested by the presence of formylated tetrahydrofolate polyglutamates in addition to methylated derivatives in the RBCs from homozygous mutant individuals. 5-Methyltetrahydrofolate polyglutamates were the only folate form found in RBCs from individuals with the wild-type genotype. Existence of formylated folates in RBCs only from individuals with the thermolabile MTHFR is consistent with the hypothesis that there is in vivo impairment in the activity of the thermolabile variant of MTHFR and that this impairment results in an altered distribution of RBC folates.

Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast

Mutant forms of the BRCA2 gene contribute significantly to hereditary breast cancer. Isolation of the normal and mutant forms of the BRCA2 gene with its natural promoter would greatly facilitate analysis of the gene and its contribution to breast cancer. We have accomplished the direct isolation of the 90-kb gene from total human DNA by transformation-associated recombination in yeast using a small amount of 5′ and 3′ BRCA2 sequence information. Because the entire isolation procedure of a single chromosomal gene could be accomplished in approximately 2 weeks, the transformation-associated recombination cloning approach is readily applicable to studies of chromosome alterations and human genetic diseases.

Cloning of a gene (RIG-G) associated with retinoic acid-induced differentiation of acute promyelocytic leukemia cells and representing a new member of a family of interferon-stimulated genes

In a cell line (NB4) derived from a patient with acute promyelocytic leukemia, all-trans-retinoic acid (ATRA) and interferon (IFN) induce the expression of a novel gene we call RIG-G (for retinoic acid-induced gene G). This gene codes for a 58-kDa protein containing 490 amino acids with several potential sites for post-translational modification. In untreated NB4 cells, the expression of RIG-G is undetectable. ATRA treatment induces the transcriptional expression of RIG-G relatively late (12–24 hr) in a protein synthesis-dependent manner, whereas IFN-α induces its expression early (30 min to 3 hr). Database search has revealed a high-level homology between RIG-G and several IFN-stimulated genes in human (ISG54K, ISG56K, and IFN-inducible and retinoic acid-inducible 58K gene) and some other species, defining a well conserved gene family. The gene is composed of two exons and has been mapped by fluorescence in situ hybridization to chromosome 10q24, where two other human IFN-stimulated gene members are localized. A synergistic induction of RIG-G expression in NB4 cells by combined treatment with ATRA and IFNs suggests that a collaboration exists between their respective signaling pathways.

Altered regulation of platelet-derived growth factor receptor-α gene-transcription in vitro by spina bifida-associated mutant Pax1 proteins

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-α gene (PDGFRα). Mice heterozygous for the PDGFRα-mutation Patch, and at the same time homozygous for the undulated mutation in the Pax1 gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRα and Pax1. Using the human PDGFRα promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the PDGFRα gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the undulated-mutation or the Gln → His mutation previously identified by us in the PAX1 gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRα-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRα expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein–DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the PDGFRα gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRα expression may be causally related to NTDs.

Dissociation of Oct-1 from the Nuclear Peripheral Structure Induces the Cellular Aging-associated Collagenase Gene Expression

The cellular aging-associated transcriptional repressor that we previously named as Orpheus was identical to Oct-1, a member of the POU domain family. Oct-1 represses the collagenase gene, one of the cellular aging-associated genes, by interacting with an AT-rich cis-element in the upstream of the gene in preimmortalized cells at earlier population-doubling levels and in immortalized cells. In these stages of cells, considerable fractions of the Oct-1 protein were prominently localized in the nuclear periphery and colocalized with lamin B. During the cellular aging process, however, this subspecies of Oct-1 disappeared from the nuclear periphery. The cells lacking the nuclear peripheral Oct-1 protein exhibited strong collagenase expression and carried typical senescent morphologies. Concomitantly, the binding activity and the amount of nuclear Oct-1 protein were reduced in the aging process and resumed after immortalization. However, the whole cellular amounts of Oct-1 protein were not significantly changed during either process. Thus, the cellular aging-associated genes including the collagenase gene seemed to be derepressed by the dissociation of Oct-1 protein from the nuclear peripheral structure. Oct-1 may form a transcriptional repressive apparatus by anchoring nuclear matrix attachment regions onto the nuclear lamina in the nuclear periphery.