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Biological Functions of Mammalian Nit1, the Counterpart of the Invertebrate NitFhit Rosetta Stone Protein, a Possible Tumor Suppressor

2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês

10.1074/jbc.m603590200

ISSN

1083-351X

Autores

Shuho Semba, Shuang‐Yin Han, Haiyan Qin, Kelly A. McCorkell, Dimitrios Iliopoulos, Yuri Pekarsky, Teresa Druck, Francesco Trapasso, Carlo M. Croce, Kay Huebner,

Tópico(s)

Cancer-related gene regulation

Resumo

The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1Marcotte E.M. Pellegrini M. Ng H.L. Rice D.W. Yeates T.O. Eisenberg D. Science. 1999; 285: 751-753Crossref PubMed Scopus (1356) Google Scholar) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies. The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1Marcotte E.M. Pellegrini M. Ng H.L. Rice D.W. Yeates T.O. Eisenberg D. Science. 1999; 285: 751-753Crossref PubMed Scopus (1356) Google Scholar) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies. The "Rosetta Stone" hypothesis was proposed for proteins consisting of fused domains in one organism that are expressed as separate polypeptides in other organisms (1Marcotte E.M. Pellegrini M. Ng H.L. Rice D.W. Yeates T.O. Eisenberg D. Science. 1999; 285: 751-753Crossref PubMed Scopus (1356) Google Scholar, 2Enright A.J. Iliopoulos I. Kyrpides N. Ouzounis C.A. Nature. 1999; 402: 86-90Crossref PubMed Scopus (880) Google Scholar). According to this theory, supported by bioinformatic and experimental evidence, the existence of a fusion protein in one genome powerfully predicts that the separate polypeptides function in the same cellular or biochemical pathway in other organisms (3Enright A.J. Ouzounis C.A. Genome Biol. 2001; 2 (Research 0034.1-0034.7)Crossref PubMed Google Scholar, 4Veitia R.A. Genome Biol. 2003; 3 (Interactions 1001.1-1001.3)Google Scholar). In a search for the tumor suppressor fragile histidine triad (FHIT) 2The abbreviations used are: FHIT, fragile histidine triad; ApnA, diadenosine polyphosphate; NMBA, N-nitrosomethylbenzylamine; MMC, mitomycin C; RT, reverse transcription. gene homolog in Drosophila melanogaster and Caenorhabditis elegans, NitFhit was cloned and identified as a fusion protein composed of a C-terminal Fhit domain and a domain related to plant and bacterial nitrilase (5Pekarsky Y. Campiglio M. Siprashvili Z. Druck T. Sedkov Y. Tillib S. Draganescu A. Wermuth P. Rothman J.H. Huebner K. Buchberg A.M. Mazo A. Brenner C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8744-8749Crossref PubMed Scopus (69) Google Scholar). The fusion of unrelated proteins is believed to indicate involvement in the same biological pathway if the distinct proteins have similar gene expression patterns. Indeed, murine Nit1 and Fhit have overlapping patterns of tissue-specific mRNA expression and were expected to be closely associated functionally (5Pekarsky Y. Campiglio M. Siprashvili Z. Druck T. Sedkov Y. Tillib S. Draganescu A. Wermuth P. Rothman J.H. Huebner K. Buchberg A.M. Mazo A. Brenner C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8744-8749Crossref PubMed Scopus (69) Google Scholar). FHIT is a tumor suppressor gene located at chromosome region 3p14.2, encompassing the most active common fragile site of the human genome, FRA3B (6Ohta M. Inoue H. Cotticelli M.G. Kastury K. Baffa R. Palazzo J. Siprashvili Z. Mori M. McCue P. Druck T. Croce C.M. Huebner K. Cell. 1996; 84: 587-597Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar, 7Huebner K. Croce C.M. Br. J. Cancer. 2003; 88: 1501-1506Crossref PubMed Scopus (108) Google Scholar); frequent deletion at the FHIT locus, aberrant transcripts, and promoter hypermethylation and resultant loss of Fhit expression have been associated with many types of human malignancies (6Ohta M. Inoue H. Cotticelli M.G. Kastury K. Baffa R. Palazzo J. Siprashvili Z. Mori M. McCue P. Druck T. Croce C.M. Huebner K. Cell. 1996; 84: 587-597Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar, 8Sozzi G. Veronese M.L. Negrini M. Baffa R. Cotticelli M.G. Inoue H. Tornielli S. Pilotti S. De Gregorio L. Pastorino U. Pierotti M.A. Ohta M. Huebner K. Croce C.M. Cell. 1996; 85: 17-26Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 9Croce C.M. Sozzi G. Huebner K. J. Clin. Oncol. 1999; 17: 1618-1624Crossref PubMed Google Scholar, 10Kuroki T. Trapasso F. Yendamuri S. Matsuyama A. Alder H. Mori M. Croce C.M. Cancer Res. 2003; 63: 3724-3728PubMed Google Scholar, 11Iliopoulos D. Guler G. Han S.Y. Johnston D. Druck T. McCorkell K.A. Palazzo J. McCue P.A. Baffa R. Huebner K. Oncogene. 2005; 24: 1625-1633Crossref PubMed Scopus (164) Google Scholar). Fhit is a member of the histidine triad nucleotide-binding protein superfamily, encoding a diadenosine polyphosphate (ApnA) hydrolase that cleaves substrates such as Ap3A to AMP plus the other nucleotide (12Barnes L.D. Garrison P.N. Siprashvili Z. Guranowski A. Robinson A.K. Ingran S.W. Croce C.M. Ohta M. Huebner K. Biochemistry. 1996; 35: 11529-11535Crossref PubMed Scopus (371) Google Scholar, 13Huebner K. Garrison P.N. Barnes L.D. Croce C.M. Annu. Rev. Genet. 1998; 32: 7-31Crossref PubMed Scopus (179) Google Scholar). Fhit hydrolytic activity is lost when histidine 96 is replaced with asparagine (12Barnes L.D. Garrison P.N. Siprashvili Z. Guranowski A. Robinson A.K. Ingran S.W. Croce C.M. Ohta M. Huebner K. Biochemistry. 1996; 35: 11529-11535Crossref PubMed Scopus (371) Google Scholar), but the enzymatically dead mutant Fhit protein also induced caspase-dependent apoptosis in cancer cells, confirming that Fhit Ap3A binding activity is necessary for the proapoptotic function of Fhit but that the hydrolytic activity is dispensable (14Trapasso F. Krakowiak A. Cesari R. Arkles J. Yendamuri S. Ishii H. Vecchione A. Kuroki T. Bieganowski T. Pace H.C. Huebner K. Croce C.M. Brenner C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1592-1597Crossref PubMed Scopus (68) Google Scholar, 15Semba S. Trapasso F. Fabbri M. McCorkell K.A. Volinia S. Druck T. Iliopoulos D. Pekarsky Y. Ishii H. Garrison P.N. Barnes L.D. Croce C.M. Huebner K. Oncogene. 2006; 25: 2860-2872Crossref PubMed Scopus (61) Google Scholar). Overexpression of FHIT by adenoviral gene delivery in human cancer cells suppressed cell growth and induced caspase-dependent apoptosis in vitro and in vivo (16Ji L. Fang B. Yen N. Minna J.D. Roth J.A. Cancer Res. 1999; 59: 3333-3339PubMed Google Scholar, 17Dumon K.R. Ishii H. Vecchione A. Trapasso F. Baldassarre G. Chakrani F. Druck T. Rosato E.F. Williams N.N. Baffa R. During M.J. Huebner K. Croce C.M. Cancer Res. 2001; 61: 4827-4836PubMed Google Scholar, 18Ishii H. Dumon K.R. Vecchione A. Trapasso F. Mimori K. Alder H. Mori M. Sozzi G. Baffa R. Huebner K. Croce C.M. Cancer Res. 2001; 61: 1578-1584PubMed Google Scholar, 19Roz L. Gramegna M. Ishii H. Croce C.M. Sozzi G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3615-3620Crossref PubMed Scopus (141) Google Scholar, 20Sevignani C. Calin G.A. Cesari R. Sarti M. Ishii H. Yendamuri S. Vecchione A. Trapasso F. Croce C.M. Cancer Res. 2003; 63: 1183-1187PubMed Google Scholar). In addition, Fhit-/- and Fhit+/- mice exhibited increased susceptibility to spontaneous tumors, and Fhit deficiency in mice enhanced sensitivity to the carcinogens N-nitrosomethylbenzylamine (NMBA) and dimethylnitrosamine (21Zanesi N. Fidanza V. Fong L.Y. Mancini R. Druck T. Valtieri M. Rudiger T. McCue P.A. Croce C.M. Huebner K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10250-10255Crossref PubMed Scopus (158) Google Scholar, 22Fujishita T. Doi Y. Sonoshita M. Hiai H. Oshima M. Huebner K. Croce C.M. Taketo M.M. Br. J. Cancer. 2004; 91: 1571-1574Crossref PubMed Scopus (20) Google Scholar, 23Zanesi N. Mancini R. Sevignani C. Vecchione A. Kaou M. Valtieri M. Calin G.A. Pekarsky Y. Gnarra J.R. Croce C.M. Huebner K. Cancer Res. 1995; 65: 6576-6582Crossref Scopus (28) Google Scholar). Fhit-deficient cells show altered sensitivity to DNA damage by mitomycin C (MMC), UVC, and ionizing radiation (24Ottey M. Han S.Y. Druck T. Barnoski B.L. McCorkell K.A. Croce C.M. Raventos-Suarez C. Fairchild C.R. Wang Y. Huebner K. Br. J. Cancer. 2004; 91: 1669-16677Crossref PubMed Scopus (53) Google Scholar, 25Hu B. Han S.Y. Wang X. Ottey M. Potoczek M.B. Dicker A. Huebner K. Wang Y. J. Cell Physiol. 2005; 202: 518-523Crossref PubMed Scopus (49) Google Scholar). These biological functions of Fhit are consistent with its tumor suppressor activity. In Arabidopsis, Nit family members (Nit1-3) are believed to convert indole-3-acetonitrile into indole-3-acetic acid, known as a plant growth hormone auxin, which plays an important role in development of the embryo, leaf formation, and root initiation (26Normanly J. Grisafi P. Fink G.R. Bartel B. Plant Cell. 1997; 9: 1781-1790Crossref PubMed Scopus (142) Google Scholar, 27Reinhardt D. Mandel T. Kuhlemeier C. Plant Cell. 2000; 12: 507-518Crossref PubMed Scopus (755) Google Scholar, 28Casimiro I. Marchant A. Bhalerao R.P. Beeclman T. Dhooge S. Swarup R. Graham N. Inze D. Sandberg G. Casero P.J. Bennett M. Plant Cell. 2001; 13: 843-852Crossref PubMed Scopus (793) Google Scholar). In addition, an apoptosis-inducing effect of Nit1 in plant wound and herbicide-induced cell death has been proposed (29Cutler S.R. Somerville C.R. BMC Plant Biol. 2005; 5: 4-18Crossref PubMed Scopus (36) Google Scholar). Structural analysis of C. elegans NitFhit protein, gene name nft-1, showed that NitFhit is a tetramer in which the Nit domains are localized at the center with Fhit dimers at the poles with Fhit nucleotide binding sites facing away from Nit and that recombinant D. melanogaster NitFhit was capable of cleaving Ap3A to AMP and ADP (30Pace H.C. Garrison P.N. Robinson A.K. Barnes L.D. Draganescu A. Rosler A. Blackburn G.M. Siprashvili Z. Croce C.M. Huebner K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5484-5489Crossref PubMed Scopus (133) Google Scholar, 31Pace H.C. Hodawadekar S.C. Draganescu A. Huang J. Bieganowski P. Pekarsky Y. Croce C.M. Brenner C. Curr. Biol. 2000; 10: 907-917Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The biological role of worm and fly Nit domains, as well as Nit1-interacting proteins and substrates, is unknown. In this study, we conducted diverse experiments in vitro and in vivo to elucidate the biological or physiological effects of Nit1 expression in mammalian cells and in mice. In particular, possible roles of Nit1 as a tumor suppressor were assessed on the basis of the previous data that supported Fhit-modulated cell cycle progression, apoptosis, and tumorigenesis in vitro and in vivo. Mouse Models and Genotyping Protocols—A conditional deletion construct for the murine Nit1 gene was designed to flank exon 4 through 7 with LoxP sites. Briefly, LoxP sites were put into intron 3 and intron 7 with a phosphoglycerate kinaseneomycin cassette, and a diphtheria toxin cassette was included in the construct for negative selection. This targeting vector was transfected into 129/SVJ embryonic stem cells, and after neomycin selection, embryonic stem clones with heterozygously recombined Nit1 alleles were identified by Southern blot. DNAs digested with BamHI and PvuII and blotted onto a nylon membrane were hybridized with 32P-labeled probes. Correctly targeted embryonic stem cell clones were injected into blastocysts. Germ line transmitters were obtained by crossing the chimeric mice with C57BL/B6 mice, which were then crossed with EIIa Cre transgenic mice (The Jackson Laboratory) to obtain mosaic mice. Segregation of the mosaic mice by crossing with C57BL/B6 mice resulted in Nit1fl/fl and Nit1fl/- mice. The whey acidic protein (Wap)-Cre transgenic mice (National Cancer Institute Mouse Models to Human Cancers Consortium, Rockville, MD) were mated with the Nit1fl/fl mice and ultimately produced Wap-Cre Nit1fl/fl mice. All animals used in the studies were treated humanely in accordance with federal guidelines and institutional policies. Screening of founder animals and their offspring was performed by genomic PCR with the following primer sets: Nit1 floxed/wild-type allele, 5′-GTTGGTCTAGCAATCTGTTATGA-3′ and 5′-GTGCTGGGATTAAAGGTGTGC-3′; Nit1 deletion allele, 5′-GTACCGGATACCGATTACTTCGA-3′ and 5′-GTGCTGGGATTAAAGGTGTGCA-3′; Wap-Cre transgene, 5′-TAGAGCTGTGCCAGCCTCTTC-3′ and 5′-CATCACTCGTTGCATCGACC-3′. Genotypes of Nit1fl/- and Nit1-/- mice obtained from the offspring of Nit1fl/fl mice were also confirmed by genomic PCR. Nit1fl/fl and Nit1-/- Mouse Kidney Cells and Cell Culture—Whole kidneys from a Nit1fl/fl mouse were dissected and placed in culture, and cells were grown and subcultured in minimal essential medium (Sigma) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. To generate Nit1-deficient (Nit1-/-) cells, Nit1fl/fl cells were infected with retrovirus-expressing Cre recombinase, constructed using pLNCX1-IRES-EGFP (Clontech). Viral infection efficiencies were determined by inspection of enhanced green fluorescent protein expression and fluorescence-activated cell sorter-based counting. A549 (Fhit-positive) and H1299 (Fhit-negative) human lung cancer cells (15Semba S. Trapasso F. Fabbri M. McCorkell K.A. Volinia S. Druck T. Iliopoulos D. Pekarsky Y. Ishii H. Garrison P.N. Barnes L.D. Croce C.M. Huebner K. Oncogene. 2006; 25: 2860-2872Crossref PubMed Scopus (61) Google Scholar) and human embryonic kidney 293 cells were maintained in minimal essential medium and Dulbecco's modified Eagle's medium (Sigma), respectively. Cell Viability Test, DNA Damage Induction, and Flow Cytometric Analyses—The ViCell cell counter (Beckman Coulter) was used for cell counting and viability tests (15Semba S. Trapasso F. Fabbri M. McCorkell K.A. Volinia S. Druck T. Iliopoulos D. Pekarsky Y. Ishii H. Garrison P.N. Barnes L.D. Croce C.M. Huebner K. Oncogene. 2006; 25: 2860-2872Crossref PubMed Scopus (61) Google Scholar). MMC treatment and UVC light exposure experiments were carried out as reported (24Ottey M. Han S.Y. Druck T. Barnoski B.L. McCorkell K.A. Croce C.M. Raventos-Suarez C. Fairchild C.R. Wang Y. Huebner K. Br. J. Cancer. 2004; 91: 1669-16677Crossref PubMed Scopus (53) Google Scholar). Cells were treated with medium supplemented with2or5 μm MMC (Fisher) or exposed to 30 or 60 J/m2 UVC (254 nm) irradiation. Viable cell numbers were estimated using a Cell Counting Kit-8 (Dojindo). To analyze cellular DNA content and cells positive for active caspase-3 with a fluorescence-activated cell sorter Calibur cytometer (BD Biosciences), cells were stained with propidium iodide and phycoerythrin-conjugated monoclonal anti-active (cleaved) caspase-3 (BD Biosciences), respectively (15Semba S. Trapasso F. Fabbri M. McCorkell K.A. Volinia S. Druck T. Iliopoulos D. Pekarsky Y. Ishii H. Garrison P.N. Barnes L.D. Croce C.M. Huebner K. Oncogene. 2006; 25: 2860-2872Crossref PubMed Scopus (61) Google Scholar). RT-PCR and Quantitative Real-time RT-PCR—Nit1 mRNA expression was confirmed by RT-PCR and quantitative real-time RT-PCR. Total RNAs were amplified with OneStep RT-PCR kit (Qiagen) and QuantiTect SYBR Green RT-PCR kit (Qiagen), using primers as follows: 5′-GTACTTTGTACTCAGCCCAG-3′ (forward) and 5′-CCATAGAGGTCAGGTCTGCG-3′ (reverse). Quantitative real-time RT-PCR analyses were performed using the iCycler multicolor real-time PCR detection system (Bio-Rad). The primer set for amplification of glyceraldehyde-3-phosphate dehydrogenase mRNA served as a housekeeping control gene (15Semba S. Trapasso F. Fabbri M. McCorkell K.A. Volinia S. Druck T. Iliopoulos D. Pekarsky Y. Ishii H. Garrison P.N. Barnes L.D. Croce C.M. Huebner K. Oncogene. 2006; 25: 2860-2872Crossref PubMed Scopus (61) Google Scholar), and the levels of Nit1 transcripts were determined by the ΔΔCt method (32Junttila T.T. Laato M. Vahlberg T. Soderstrom K.O. Visakorpi T. Isola J. Elenius K. Clin. Cancer Res. 2003; 9: 5346-5357PubMed Google Scholar). Western Blot Analysis—Preparation of total protein and the immunoblotting procedure used are described elsewhere (15Semba S. Trapasso F. Fabbri M. McCorkell K.A. Volinia S. Druck T. Iliopoulos D. Pekarsky Y. Ishii H. Garrison P.N. Barnes L.D. Croce C.M. Huebner K. Oncogene. 2006; 25: 2860-2872Crossref PubMed Scopus (61) Google Scholar). Total protein (40 μg) was separated on a polyacrylamide gel, transferred to a membrane, and probed with antisera against Fhit (33Fong L.Y. Fidanza V. Zanesi N. Lock L.F. Siracusa L.D. Mancini R. Siprashvili Z. Ottey M. Martin S.E. Druck T. McCue P.A. Croce C.M. Huebner K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4742-4747Crossref PubMed Scopus (187) Google Scholar), cyclin D1 (NeoMarkers), and other cell cycle markers, including cyclin B1, cyclin E, cdk2, cdc2, cdc25A, p21CIP1, p53, Chk1 (Santa Cruz), Cdk4, and p27KIP1 (BD Biosciences). Rabbit polyclonal Nit1 antiserum against human Nit1 C-terminal peptide (QHRRPDLYGSLGHPLS) was custom-made by Zymed Laboratories Inc. and shown to specifically detect both human and mouse Nit1 protein. Specificity was shown by immunoblot and immunohistochemistry using tissues from mice with homozygous deletion of the floxed NIT1 alleles. These tissues were shown to be negative for staining of the Nit1-sized band on immunoblots and negative for immunohistochemical staining relative to Nit1-positive controls, as seen below under "Results" and in supplemental Figs. S1 and S2. Tubulin (NeoMarkers) and green fluorescent protein (Molecular Probes) antibodies were used in detection of protein in loading controls. Recombinant Adenoviral Vector Construction—Adenoviruses carrying human recombinant NIT1 (Ad-NIT1) and FHIT (Ad-FHIT) were constructed as described previously (14Trapasso F. Krakowiak A. Cesari R. Arkles J. Yendamuri S. Ishii H. Vecchione A. Kuroki T. Bieganowski T. Pace H.C. Huebner K. Croce C.M. Brenner C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1592-1597Crossref PubMed Scopus (68) Google Scholar). The transfer vector pAdenoVator-CMV5-GFP (Qbiogene) carrying full-length human NIT1 or FHIT cDNAs and the AdVator ΔE1/E3 adenoviral genome construct were mixed and recombination performed in BJ5183 Escherichia coli. Resulting recombinant vectors were transfected into human embryonic kidney 293 cells to package viruses. Isolated single viral plaques were expanded and assessed for Nit1 or Fhit expression by Western blot. Ad-GFP virus was used as a nonspecific control (Qbiogene). Viral titers were determined by absorbance measurement, multiplicity of infection test, and tissue culture infectious dose 50 methods. Cells were incubated with adenoviral aliquots at a desired multiplicity of infection (0-100) for 4 h prior to addition of culture medium (>25 × volume of virus inoculum). Transfections—For transient expression of exogenous wild-type and mutant Nit1, Nit1-/- cells were transfected with pIRESneo vector (Clontech) encoding wild-type (pNit1) or C203A mutant Nit1 (pNit1-C203A). Transient transfection was conducted with Lipofectamine reagent (Invitrogen). At 48 h after transformation, cells were assessed for viability, active caspase-3, and expression of specific proteins. Mating Experiments for the Mammary Gland Development Study—Wap-Cre Nit1fl/fl and Nit1fl/fl matings were set up late in the afternoon and female mice checked for copulation plugs over the following few days to determine time of mating. The morning on which a copulation plug was observed was pregnancy day 0.5 and the female separated from the male. Mice were sacrificed for phenotypic analysis during pregnancy (P13, P16, P19) or lactation (L1, L4). For whole mount analysis of mammary glands, the fourth inguinal mammary glands were spread on glass, fixed in Carnoy's solution, defatted, and stained in carmine alum solution overnight. The tissue was then dehydrated and mounted on glass. The average numbers of branch points and total number of endbuds between nipple and endbuds were counted microscopically (34Deans A.J. Simpson K.J. Trivett M.K. Brown M.A. McArthur G.A. Oncogene. 2004; 23: 6136-6145Crossref PubMed Scopus (12) Google Scholar, 35Qi C. Kashireddy P. Zhu Y.T. Rao S.M. Zhu Y.-J. J. Biol. Chem. 2004; 279: 33696-33701Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). For histological examination, formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin or subjected to immunohistochemical analyses. Immunofluorescent and Immunohistochemical Analyses—Nit1fl/fl cells on coverslips and formalin-fixed, paraffin-embedded whole mount mouse embryo (E13) sections were used. Cells were incubated with the antiserum against Nit1 and goat Texas Red or fluorescein isothiocyanate-conjugated anti-rabbit IgG (Zymed Laboratories Inc.). Mounting medium containing 4′, 6-diamidino-2-phenylindole (Vector Laboratories) was used for nuclear staining. Immunohistochemical staining by a modified version of the immunoglobulin enzyme bridge technique was performed to detect Nit1 and cyclin D1 expression (15Semba S. Trapasso F. Fabbri M. McCorkell K.A. Volinia S. Druck T. Iliopoulos D. Pekarsky Y. Ishii H. Garrison P.N. Barnes L.D. Croce C.M. Huebner K. Oncogene. 2006; 25: 2860-2872Crossref PubMed Scopus (61) Google Scholar). Cyclin D1-positive cells in mammary ducts and lobules were counted in at least 10 high power fields (×400) for each section. For detection of apoptotic bodies, ApopTag Peroxidase kit (Chemicon) was used. Briefly, mouse mammary gland sections were pretreated with proteinase K and incubated with 2 N HCl. Terminal deoxynucleotidyl transferase enzyme was added to the pre-equilibrated cells. Antidigoxigenin peroxidase conjugate IgG and 3,3′-diaminobenzidine-tetrahydrochloride-dihydrate were added to the slides. Slides were counterstained with hematoxylin. The percent of ApopTag-positive cells was determined by counting cells in at least 10 high power fields. NMBA Carcinogenicity Study—Nit1fl/fl, Nit1fl/-, and Nit1-/- mice (15 each) were given intragastric doses of NMBA (Ash Stevens), 2 mg/kg body weight, a total of six times over a 3-week period. All mice were sacrificed 10 weeks after the final NMBA dose and examined for end-point tumor incidence (33Fong L.Y. Fidanza V. Zanesi N. Lock L.F. Siracusa L.D. Mancini R. Siprashvili Z. Ottey M. Martin S.E. Druck T. McCue P.A. Croce C.M. Huebner K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4742-4747Crossref PubMed Scopus (187) Google Scholar). At autopsy, whole stomachs were removed and opened longitudinally. The number of animals bearing tumors in the esophagus, the forestomach, and the squamocolumnar junction and number of papillomas and dysplasias (carcinomas in situ) were scored by histological examination. Fhit-/- mice, which were known to show enhanced susceptibility to NMBA-induced upper digestive tract tumors, were included in the study as controls. Statistical Analysis—p values calculated by the Student's two-sided t test were used to determine significance of differences in measurements of mammary gland developmental stages in the Nit1fl/fl and Wap-Cre Nit1fl/fl mice. Expression of Nit1 Protein in Normal Tissues—To study the function of Nit1 in murine homeostasis and embryogenesis, we initially examined Nit1 protein expression in adult mouse tissues and embryos. Nit1 expression was detected in most tissues and was particularly abundant in adult liver and kidney (Fig. 1, A and C) and in embryonic adrenal gland and skeletal muscle (Fig. 1D). In cultured mouse kidney cells, endogenous Nit1 was distributed mainly in the cytoplasm, weakly in nuclei, and, apparently, partially in mitochondria (Fig. 1B). Because the strongest case for Rosetta Stone proteins decoding real interactions can be made when the separate genes have similar gene expression patterns (1Marcotte E.M. Pellegrini M. Ng H.L. Rice D.W. Yeates T.O. Eisenberg D. Science. 1999; 285: 751-753Crossref PubMed Scopus (1356) Google Scholar), we compared expression patterns of Nit1 and Fhit in mouse tissues and found that the pattern of Nit1 expression in adult mouse tissues partially overlapped the Fhit pattern (Fig. 1A). Nit1 Deficiency Results in Up-regulated Growth, Increased Cyclin D1 Expression, and Resistance to DNA-damaging Agents—We generated conditional knock-out mice in which Nit1 coding exons 4-7 can be deleted through Cre-mediated recombination (Fig. 2A); Nit1fl/+ mice were mated and Nit1fl/fl mice obtained (Fig. 2, B and C). Mice with two floxed Nit1 alleles developed normally to adulthood; males and females were fertile, without phenotypic abnormalities, suggesting that insertion of the selectable marker did not affect transcriptional regulation of the Nit1 gene. To investigate the role of Nit1 in cultured cells, we established Nit1-/- cells by infecting Nit1fl/fl mouse kidney cells with a retrovirus expressing Cre recombinase. Homozygous genomic deletion of the targeted alleles, decreased levels of truncated Nit1 mRNA expression, and resultant loss of Nit1 protein expression were confirmed; Fhit expression was unaffected in Nit1 null cells (Fig. 2D). Nit1-/- cells exhibited round and compact shapes, loss of lobular structure, higher cell density with increased S and G2/M cell populations, and >3-fold higher clonogenicity in comparison with Nit1fl/fl cells (Fig. 3, A-C). To clarify mechanisms of acceleration of cell growth induced by Nit1 deficiency, Nit1fl/fl and Nit1-/- cell lysates were immunoblotted for assessment of expression of specific proteins. Deficiency of Nit1 expression caused higher cyclin D1 expression, whereas differences in the other cell cycle-associated proteins appeared minimal (Fig. 3D). Because Fhit modulates cell survival and resistance to MMC and UVC light (24Ottey M. Han S.Y. Druck T. Barnoski B.L. McCorkell K.A. Croce C.M. Raventos-Suarez C. Fairchild C.R. Wang Y. Huebner K. Br. J. Cancer. 2004; 91: 1669-16677Crossref PubMed Scopus (53) Google Scholar), we examined sensitivity to these DNA-damaging agents in cells with or without Nit1 expression. Enhanced survival of the Nit1-/- cells after treatment with MMC and UVC light was observed; these DNA-damaging agents seemed to influence cell viability in a dose-dependent manner (Fig. 3E). Mutation of the Nit1 Catalytic Site Did Not Alter Nit1-mediated Biological Effects—Previous reports have demonstrated induction of caspase-3-dependent apoptosis by Ad-FHIT-wild-type in human cancer cells (16Ji L. Fang B. Yen N. Minna J.D. Roth J.A. Cancer Res. 1999; 59: 3333-3339PubMed Google Scholar, 17Dumon K.R. Ishii H. Vecchione A. Trapasso F. Baldassarre G. Chakrani F. Druck T. Rosato E.F. Williams N.N. Baffa R. During M.J. Huebner K. Croce C.M. Cancer Res. 2001; 61: 4827-4836PubMed Google Scholar). However, Nit1 has not been shown to interact with Fhit nor has its biological activity been examined. Thus, we assessed the apoptosis-inducing effect of Ad-NIT1 and Ad-FHIT in cells with or without Nit1 expression. Recombinant protein expression and equivalent infectivity of the two viruses were confirmed by Western blot (data not shown). Irrespective of Nit1 expression, overexpression of NIT1 and FHIT by adenovirus-mediated gene delivery resulted in increased populations of active caspase-3 positive cells and cells with sub-G1 DNA content in a multiplicity of infection-dependent manner; Ad-FHIT-mediated induction of caspase-3-dependent apoptosis was more effective in Nit1fl/fl cells than in Nit1-/- cells (Fig. 4A). Conversely, we examined Ad-NIT1 effects in cells with or without Fhit expression. Ad-NIT1 could in

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