Identification of Several Human Homologs of Hamster DNA Damage-inducible Transcripts
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26720
ISSN1083-351X
AutoresM. Saeed Sheikh, France Carrier, Mathilda A. Papathanasiou, M. Christine Hollander, Qimin Zhan, Kelly J. Yu, Albert J. Fornace,
Tópico(s)RNA Interference and Gene Delivery
ResumoLow ratio hybridization subtraction technique was previously used in this laboratory to enrich and isolate a number of low abundance UV-inducible hamster transcripts (Fornace, A. J., Jr., Alamo, I. J., and Hollander, M. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8800–8804) that led to the identification and cloning of five important hamster and humanGADD genes (Fornace, A. J., Jr., Nebert, D. W., Hollander, M. C., Luethy, J. D., Papathanasiou, M., Fargnoli, J., and Holbrook, N. J. (1989) Mol. Cell. Biol.9, 4196–4203). In this study we have characterized the remaining DNA damage-inducible (DDI) transcripts. Of the 24 DDI clones, 3 clones (A13, A20, and A113) representing different regions of the same hamster cDNA exhibited near perfect homology to human p21 WAF1/CIP1 cDNA. The DDI clones A26, A88, and A99 displayed very high sequence homologies with the human proliferating nuclear antigen, rat translation initiation factor-5 (eIF-5), and human thrombomodulin, respectively, whereas clones A29 and A121 matched with express sequence tagged sequences of unknown identity. The DDI clones A18, 106, and A107 were different isolates of the same hamster cDNA (hereafter referred to as A18) and displayed high sequence homology with the members in the heterogeneous ribonucleoprotein (hnRNP) family. Using the hamster A18 partial-length cDNA as a probe, we screened human fibroblast cDNA library and isolated the corresponding full-length human cDNA. The deduced amino acid sequence revealed that the putative protein contains all the canonical features of a novel glycine-rich hnRNP. The A18 mRNA levels were specifically increased in response to DNA damage induced by UV irradiation or UV mimetic agents. Thus the putative A18 hnRNP is the first hnRNP whose mRNA is specifically regulated in response to UV-induced DNA damage; accordingly, it may play some role in repair of UV-type DNA damage. Low ratio hybridization subtraction technique was previously used in this laboratory to enrich and isolate a number of low abundance UV-inducible hamster transcripts (Fornace, A. J., Jr., Alamo, I. J., and Hollander, M. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8800–8804) that led to the identification and cloning of five important hamster and humanGADD genes (Fornace, A. J., Jr., Nebert, D. W., Hollander, M. C., Luethy, J. D., Papathanasiou, M., Fargnoli, J., and Holbrook, N. J. (1989) Mol. Cell. Biol.9, 4196–4203). In this study we have characterized the remaining DNA damage-inducible (DDI) transcripts. Of the 24 DDI clones, 3 clones (A13, A20, and A113) representing different regions of the same hamster cDNA exhibited near perfect homology to human p21 WAF1/CIP1 cDNA. The DDI clones A26, A88, and A99 displayed very high sequence homologies with the human proliferating nuclear antigen, rat translation initiation factor-5 (eIF-5), and human thrombomodulin, respectively, whereas clones A29 and A121 matched with express sequence tagged sequences of unknown identity. The DDI clones A18, 106, and A107 were different isolates of the same hamster cDNA (hereafter referred to as A18) and displayed high sequence homology with the members in the heterogeneous ribonucleoprotein (hnRNP) family. Using the hamster A18 partial-length cDNA as a probe, we screened human fibroblast cDNA library and isolated the corresponding full-length human cDNA. The deduced amino acid sequence revealed that the putative protein contains all the canonical features of a novel glycine-rich hnRNP. The A18 mRNA levels were specifically increased in response to DNA damage induced by UV irradiation or UV mimetic agents. Thus the putative A18 hnRNP is the first hnRNP whose mRNA is specifically regulated in response to UV-induced DNA damage; accordingly, it may play some role in repair of UV-type DNA damage. Both prokaryotic and eukaryotic cells are constantly exposed to endogenous as well as exogenous DNA damaging agents. DNA damage (genotoxic stress) is implicated not only in enhancing the rate of mutagenesis but has also been shown to invoke replication and transcription blocks (reviewed in Refs. 1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar and 2Hollander M.C. Fornace Jr., A.J. DNA Repair Mechanisms: Impact on Human Diseases and Cancer. Landes Co., Georgetown, TX1994: 221-237Google Scholar). UV irradiation and UV mimetic agents inflict DNA damage that is predominantly repaired via NER 1The abbreviations used are: NER, nucleotide excision repair; MMS, methylmethane sulfonate; AAAF,N-acetoxy-2-acetylaminofluorene; DDI, DNA damage-inducible; CHO, Chinese hamster ovary; EST, expressed sequence tagged; PCNA, proliferating cell nuclear antigen; eIF-5, eukaryotic translation initiation factor 5; RNP, ribonucleoprotein; hnRNP, heterogeneous RNP; RBD, RNA binding domain; ORF, open reading frame; kb, kilobase pair(s). pathway (reviewed in Ref. 2Hollander M.C. Fornace Jr., A.J. DNA Repair Mechanisms: Impact on Human Diseases and Cancer. Landes Co., Georgetown, TX1994: 221-237Google Scholar). DNA damage induced by base damaging agents such as MMS and H202, on the other hand, is repaired primarily by base excision repair pathway (reviewed in Ref. 1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar and 2Hollander M.C. Fornace Jr., A.J. DNA Repair Mechanisms: Impact on Human Diseases and Cancer. Landes Co., Georgetown, TX1994: 221-237Google Scholar). The cellular response to genotoxic stress is mediated via important genes that control a multitude of complex regulatory pathways. Although a number of mammalian genotoxic stress-inducible genes have been identified in recent years, the actual number of these genes is probably far greater (reviewed in Ref. 1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar). These genes have been implicated to play roles in a number of important cellular processes such as control of cell cycle progression, replication, transcription, signal transduction, DNA repair, and mutagenesis (reviewed in Ref.1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar). Low ratio hybridization subtraction technique is a highly sensitive method that allows for the enrichment of cDNAs of low abundance transcripts that are increased only a few-fold over the uninduced levels (3Fargnoli J. Holbrook N.J. Fornace Jr., A.J. Anal. Biochem. 1990; 187: 364-373Crossref PubMed Scopus (39) Google Scholar). This technique was used previously in this laboratory to enrich and isolate low abundance transcripts that were rapidly induced (within 4 h) by UV irradiation in CHO cells (4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). The cDNAs representing those UV-regulated, enriched transcripts were used to construct a DDI library (4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). Based on their response to genotoxic stress, these cDNA clones were later divided into two classes. Class I contained clones that were induced solely by UV irradiation and UV mimetic agents, whereas class II contained clones that were regulated by UV radiation and a host of other genotoxic agents producing base damage in DNA, such as MMS (4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). Upon further characterization of the isolated clones, five important GADD(growth arrest and DNAdamage-inducible) genes including GADD45, GADD153, GADD34, GADD33, and GADD7 were identified (5Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (649) Google Scholar,6Papathanasiou M.A. Kerr N.C. Robbins J.H. McBride O.W. Alamo I.J. Barrett S.F. Hickson I.D. Fornace Jr., A.J. Mol. Cell. Biol. 1991; 11: 1009-1016Crossref PubMed Scopus (250) Google Scholar). GADD45 is directly regulated by p53 at transcriptional level (7Kastan M.B. Zhan Q. El-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2930) Google Scholar) and appears to play a role in controlling cell proliferation (8Zhan Q. Lord K.A. Alamo I.J. Hollander M.C. Carrier F. Ron D. Kohn K.W. Hoffman B. Liebermann D.A. Fornace Jr., A.J. Mol. Cell. Biol. 1994; 14: 2361-2371Crossref PubMed Scopus (468) Google Scholar). Recently, the human GADD45 protein was also found to physically interact with proliferating cell nuclear antigen (PCNA) and p21 WAF1/CIP1 (9Smith M.L. Chen I. Zhan Q. Bae I. Chen C. Gilmer T. Kastan M.B. O'Connor P.M. Fornace Jr., A.J. Science. 1994; 266: 1376-1380Crossref PubMed Scopus (896) Google Scholar, 10Kearsy J.M. Coates P.J. Prescott A.R. Warbrick E. Hall P.A. Oncogene. 1995; 11: 1675-1683PubMed Google Scholar). The GADD153 gene codes for a protein that belongs to the C/EBP family of transcription factors (11Luethy J.D. Fargnoli J. Park J.S. Fornace Jr., A.J. Holbrook N.J. J. Biol. Chem. 1990; 265: 16521-16526Abstract Full Text PDF PubMed Google Scholar, reviewed in Ref. 1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar), whereas the GADD34 gene product appears to play a role in cell differentiation and perhaps in regulation of apoptosis (8Zhan Q. Lord K.A. Alamo I.J. Hollander M.C. Carrier F. Ron D. Kohn K.W. Hoffman B. Liebermann D.A. Fornace Jr., A.J. Mol. Cell. Biol. 1994; 14: 2361-2371Crossref PubMed Scopus (468) Google Scholar). The GADD33 is a hamster equivalent of a human cornifin gene and appears to regulate keratinocyte differentiation (1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar). Although the protein product of the GADD7 gene has not been detected and its transcript is probably not translated (12Hollander M.C. Alamo I. Fornace Jr., A.J. Nucleic Acids Res. 1996; 12: 387-395Google Scholar), enforced overexpression of exogenous GADD7 gene was sufficient to reduce the colony-forming efficiency of Chinese hamster ovary and RKO human colon carcinoma cells (12Hollander M.C. Alamo I. Fornace Jr., A.J. Nucleic Acids Res. 1996; 12: 387-395Google Scholar). The GADD genes are induced in response to various types of genotoxic and nongenotoxic stresses that elicit growth arrest (reviewed in Refs. 1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar and 2Hollander M.C. Fornace Jr., A.J. DNA Repair Mechanisms: Impact on Human Diseases and Cancer. Landes Co., Georgetown, TX1994: 221-237Google Scholar). Unlike theGADD genes, a number of other transcripts were variably induced only by genotoxic agents and were not modulated in response to nongenotoxic growth-arresting conditions (4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). The identity of these transcripts remains unknown. We undertook this study to further characterize the remaining DNA damage-inducible clones and to identify their human homologs. UV irradiation and/or MMS regulation of human homologs of hamster DDI transcripts were investigated in the following human cell lines: MCF-7, breast carcinoma cells; RKO, colon carcinoma cells; A549, lung carcinoma cells; H1299, lung carcinoma cells; HeLa, cervical carcinoma cells; Sk-N-SH, neuroblastoma cells; OVCAR, ovarian carcinoma cells; ML-1, myeloid leukemia cells; WMN, Burkitt's lymphoma cells; GM536, lymphoblastoma cells. Cells were irradiated with UV or γ-irradiation or treated with chemical agents as described previously (4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar, 5Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (649) Google Scholar, 6Papathanasiou M.A. Kerr N.C. Robbins J.H. McBride O.W. Alamo I.J. Barrett S.F. Hickson I.D. Fornace Jr., A.J. Mol. Cell. Biol. 1991; 11: 1009-1016Crossref PubMed Scopus (250) Google Scholar). The low ratio hybridization subtraction procedure has been described in detail elsewhere (3Fargnoli J. Holbrook N.J. Fornace Jr., A.J. Anal. Biochem. 1990; 187: 364-373Crossref PubMed Scopus (39) Google Scholar, 4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). Briefly, poly(A)+ RNA was extracted from cells 4 h after UV irradiation and subjected to cDNA synthesis. The cDNAs from irradiated cells were hybridized at a high Rot with poly(A)+ RNA from unirradiated cells. The single-stranded cDNA thus obtained was then hybridized to original poly(A)+ RNA obtained from irradiated cells, and the cDNA:RNA duplex was separated by hydroxylapatite column. The RNA was removed from the cDNA by alkali treatment, and the remaining single-stranded cDNAs were utilized as templates to synthesize second strand cDNAs using a mixture of random primers. The resulting double-stranded cDNAs were cloned into a plasmid pXF3 via GC-tailing (3Fargnoli J. Holbrook N.J. Fornace Jr., A.J. Anal. Biochem. 1990; 187: 364-373Crossref PubMed Scopus (39) Google Scholar, 4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar). For cloning of the human homolog of hamster A18, the Okayama-Berg GM637 human fibroblast cDNA library (13Chin D.J. Gil G. Russell D.W. Loscum L. Lusky K.L. Basu S.K. Okayama H. Berg P. Goldstein J.L. Brown M.S. Nature. 1984; 308: 613-617Crossref PubMed Scopus (155) Google Scholar) (kindly provided by H. Okayama) was screened using a partial-length hamster A18 cDNA. The DDI clones and the cDNAs representing human homologs of hamster A18 were sequenced by dideoxy chain termination method as described previously (4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar, 5Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (649) Google Scholar). Poly(A)+ RNA preparation, Northern blotting, and quantitative dot blot hybridization were performed essentially as described previously (14Hollander M.C. Fornace Jr., A.J. BioTechniques. 1990; 9: 174-179PubMed Google Scholar). For probe labeling, the appropriate cDNA inserts were excised from the plasmids and labeled using random primer methods (14Hollander M.C. Fornace Jr., A.J. BioTechniques. 1990; 9: 174-179PubMed Google Scholar, 15Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). The relative poly(A) content of each RNA sample was determined using a labeled polythymidylate probe, and the values were used to correct for variations in sample loading as described (14Hollander M.C. Fornace Jr., A.J. BioTechniques. 1990; 9: 174-179PubMed Google Scholar). For quantitation, the radioactive signals were either counted directly using a Betascope (Betagen), or the signals on the autoradiograms were measured by a densitometer. DNA extraction and Southern blot hybridization analysis were performed using standard protocols (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The following cDNA probes were used in this study: (a) cDNA inserts of DDI clones, (b) human equivalent of hamster A18 cloned in this study, (c) human EST sequences homologous to hamster DDI A29, A88, and A121 (obtained from IMAGE Consortium, LLNL), (d) human β-actin cDNA probe, as previously described (5Fornace Jr., A.J. Nebert D.W. Hollander M.C. Luethy J.D. Papathanasiou M. Fargnoli J. Holbrook N.J. Mol. Cell. Biol. 1989; 9: 4196-4203Crossref PubMed Scopus (649) Google Scholar). The A18 cDNA containing the ORF and 3′-untranslated region was polymerase chain reaction-amplified. The T7 promoter sequence was incorporated into the 5′ primer so that it was collinear with the A18 cDNA sequence starting with ATG. The polymerase chain reaction primer sequences are as follows: 5′ primer, 5′-GGATCCTAATACGACTCACTATAGGAACAGACCACCATGGCATCAGATGAAGGCA-3′; 3′ primer, 5′-CACAGCACAGATGGACGCA-3′. Aliquots ranging from 0.2 μg to 1 μg of the polymerase chain reaction-amplified A18 cDNA were subjected to in vitrotranscription/translation using TNT-coupled reticulocyte lysate system (Promega) and [35S]methionine (Amersham Life Science, Inc.) per manufacturer's instructions. Briefly, the reactions were performed in 50 μl for 90 min at 30 °C. Luciferase cDNA was used as a positive control, and reactions without DNA template were performed to determine the background incorporation of [35S]methionine. Five μl of each sample were analyzed on a 4–20% SDS-polyacrylamide gel. Gel was fixed, soaked for 30 min in an enhancer solution (Enlightening; Dupont), dried, and exposed to x-ray film. As described previously (3Fargnoli J. Holbrook N.J. Fornace Jr., A.J. Anal. Biochem. 1990; 187: 364-373Crossref PubMed Scopus (39) Google Scholar, 4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar), cDNA clones isolated after our hybridization subtraction procedure were only partial length, with an average insert size of 0.2–0.3 kb. In addition, many of these hamster cDNA sequences were from nontranslated portions of the UV-inducible transcripts and cross-hybridized poorly with human RNA. These partial-length cDNA clones were sequenced in their entirety and compared with all published sequences in the nucleotide and peptide sequence data bases using the BLAST network service at the National Center for Biotechnology Information. TableI illustrates the results of the homology searches. As reported previously, these clones can be divided into two classes; class I comprises the clones that are induced by UV irradiation, whereas the class II clones are UV irradiation- and MMS-inducible. Among the DDI clones shown in Table I, the clone A26 exhibited 96, 96, and 94% nucleotide sequence homology, respectively, with mouse, rat, and human cDNAs encoding PCNA (17Toschi L. Bravo R. J. Cell Biol. 1988; 107: 1623-1628Crossref PubMed Scopus (226) Google Scholar). The nucleotide sequence of A88 was 97% homologous to the rat cDNA encoding eukaryotic translation initiation factor-5 (eIF-5) and 91% homologous to human EST sequence bearing homology to rat eIF-5 cDNA (18Das K. Chevesich J. Maitra U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3058-3062Crossref PubMed Scopus (28) Google Scholar). The DDI clone A99 was 86 and 72% homologous to mouse and human thrombomodulin clones, respectively (19Shirai T. Shiojiri S. Ito H. Yamamoto S. Kusumoto H. Deyashiki Y. Maruyama I. Suzuki K. J. Biochem. 1988; 103: 281-285Crossref PubMed Scopus (46) Google Scholar). The DDI clones A113 as well as A13 and A20 each displayed 80, 85, and 80% homologies to mouse, rat, and human p21 WAF1/CIP1 cDNA sequences, respectively (20El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7951) Google Scholar, 21Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5245) Google Scholar). A18, A106, and A107 exhibited homology to nucleotide sequences encoding hnRNPs of diverse origin (22Cobianchi F. SenGupta D.N. Zmudzka B.Z. Wilson S.H. J. Biol. Chem. 1986; 261: 3536-3543Abstract Full Text PDF PubMed Google Scholar, 23Soulard M. Valle V.D. Siomi M.C. Pinol-Roma S. Codogno P. Bauvy C. Bellini M. Lacroix J.-C. Monod G. Dreyfuss G. Larsen C.-J. Nucleic Acids Res. 1993; 21: 4210-4217Crossref PubMed Scopus (132) Google Scholar, 24Swanson M.S. Nakagawa T.Y. LeVan K. Dreyfuss G. Mol. Cell. Biol. 1987; 7: 1731-1739Crossref PubMed Scopus (167) Google Scholar) as well as a number of unpublished EST sequences. The DDI clones A29 and A121 also displayed homology to EST sequences of unknown identity. An automated BLASTX search for the possible translation products of A29 and A121 from all reading frames did not reveal significant identities to any of the known amino acid sequences in the data bases. None of the remaining clones exhibited homology to any of the nucleotide or amino acid sequences deposited in these data bases. Fig.1 shows the nucleotide sequence alignment of various hamster DDI clones to their respective human homologs.Table IThe hamster DDI transcripts and their human homologsHamster DDI cloneHuman homologmRNA sizeInductionHamsterHumanHamsterHumanUVMMSUVMMSA18, A106, A107Novel hnRNP1.41.4+−+−A26PCNA1.31.4+−+A88eIF-53.7, 2.8, 22.8+−+−A29Unique (EST)4.84.4++++A99Thrombomodulin3.83.8++A113, A20, A13p21 WAF1/CIP122.2++++A121Unique (EST)1.41.5++++A50None0.8+−A70None4.3, 1.4+−A109None2.8, 1.4+−A143None3.7+−A144None1.9+−A162None0.6+−A8None3.1++A9None1.9++A15None2.1++A31None4.2, 3.5++A77None2.9, 2.3, 1.4++A94None10++A148None1.9, 1.1++Of the 24 DDI clones, 11 showing homology to known sequences in the data bases are listed above those that did not display homology to any known sequence. The transcript sizes of the hamster clones were determined by Northern blot hybridization performed on poly(A)+RNA, and where applicable the sizes of the corresponding human transcripts are also given for comparison. Open table in a new tab Of the 24 DDI clones, 11 showing homology to known sequences in the data bases are listed above those that did not display homology to any known sequence. The transcript sizes of the hamster clones were determined by Northern blot hybridization performed on poly(A)+RNA, and where applicable the sizes of the corresponding human transcripts are also given for comparison. PCNA, p21WAF1/CIP1, and thrombomodulin are among the known stress-inducible genes (25Zhang H. Xiong Y. Beach D. Mol. Biol. Cell. 1993; 4: 897-906Crossref PubMed Scopus (340) Google Scholar, 26Bae I. Fan S. Bhatia K. Kohn K.W. Fornace Jr., A.J. O'Connor P.M. Cancer Res. 1995; 55: 2387-2393PubMed Google Scholar, 27Conway E.M. Liu L. Nowakowski B. Steiner-Mosonyi M. Jackman R.W. J. Biol. Chem. 1994; 269: 22804-22810Abstract Full Text PDF PubMed Google Scholar). No information is available on the regulation of eIF-5 (A88 homolog), since only rat (18Das K. Chevesich J. Maitra U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3058-3062Crossref PubMed Scopus (28) Google Scholar) and yeast (28Chakravarti D. Maitra U. J. Biol. Chem. 1993; 268: 10524-10533Abstract Full Text PDF PubMed Google Scholar) eIF-5 cDNAs are cloned and sequenced, whereas the corresponding human cDNA remains to be cloned and sequenced. We next sought to investigate whether human homologs of hamster A88, A29, and A121 genes were also stress-inducible. In CHO cells, A88 is regulated by UV treatment, whereas the A29 and A121 are induced by UV and MMS. We exposed various human cell lines to UV irradiation, MMS, and γ-irradiation; poly(A)+ RNA was isolated and subjected to quantitative RNA blot hybridization using corresponding human cDNA probes. Our results (summarized in Table I) demonstrated that MMS induced the mRNA levels of human A29 and A121 in all the cell lines tested, whereas it had no effect on the expression of A88 (eIF-5 homolog). γ-Irradiation by contrast had no appreciable effect on the levels of these transcripts in any of the human cell lines tested (data not shown). The UV effects were, however, cell-type specific. Of the cell lines tested (mentioned under "Experimental Procedures"), UV treatment modestly enhanced the mRNA levels of human A88 (eIF-5) in H1299 human lung carcinoma and in GM536 human lymphoblastoma cells, whereas the A29 and A121 regulation was noted in ML-1 myeloid leukemia cell line and GM536 human lymphoblastoma cells. Both A29 and A121 were also regulated by UV irradiation and by MMS in CHO cells, whereas A88 was regulated only by UV irradiation and not by MMS in CHO cells. These results, therefore, demonstrate that the regulation of these genes in response to genotoxic stress is conserved in rodent and human (TableI). Northern blot analyses were performed to determine the sizes of these transcripts, and the overall results are summarized in TableI. A18, A106, and A107 were the three other DDI clones that belonged to class I and displayed nucleotide sequence homology to known sequences in the data bases. A comparison (using BLASTX search) of the predicted amino acids of the partial sequences of A18, A106, and A107 in all reading frames with known sequences in the data bases revealed high similarity with the hnRNPs of the RNP family (22Cobianchi F. SenGupta D.N. Zmudzka B.Z. Wilson S.H. J. Biol. Chem. 1986; 261: 3536-3543Abstract Full Text PDF PubMed Google Scholar, 23Soulard M. Valle V.D. Siomi M.C. Pinol-Roma S. Codogno P. Bauvy C. Bellini M. Lacroix J.-C. Monod G. Dreyfuss G. Larsen C.-J. Nucleic Acids Res. 1993; 21: 4210-4217Crossref PubMed Scopus (132) Google Scholar, 24Swanson M.S. Nakagawa T.Y. LeVan K. Dreyfuss G. Mol. Cell. Biol. 1987; 7: 1731-1739Crossref PubMed Scopus (167) Google Scholar). Further analysis revealed that these were three different isolates of the same cDNA and hereafter referred to as A18. The hnRNPs belong to a subgroup in a large family of the RNPs and exist in the nucleus in association with other proteins (22Cobianchi F. SenGupta D.N. Zmudzka B.Z. Wilson S.H. J. Biol. Chem. 1986; 261: 3536-3543Abstract Full Text PDF PubMed Google Scholar, 23Soulard M. Valle V.D. Siomi M.C. Pinol-Roma S. Codogno P. Bauvy C. Bellini M. Lacroix J.-C. Monod G. Dreyfuss G. Larsen C.-J. Nucleic Acids Res. 1993; 21: 4210-4217Crossref PubMed Scopus (132) Google Scholar, 24Swanson M.S. Nakagawa T.Y. LeVan K. Dreyfuss G. Mol. Cell. Biol. 1987; 7: 1731-1739Crossref PubMed Scopus (167) Google Scholar). These proteins form hnRNP complexes and are responsible for hnRNA processing (22Cobianchi F. SenGupta D.N. Zmudzka B.Z. Wilson S.H. J. Biol. Chem. 1986; 261: 3536-3543Abstract Full Text PDF PubMed Google Scholar, 23Soulard M. Valle V.D. Siomi M.C. Pinol-Roma S. Codogno P. Bauvy C. Bellini M. Lacroix J.-C. Monod G. Dreyfuss G. Larsen C.-J. Nucleic Acids Res. 1993; 21: 4210-4217Crossref PubMed Scopus (132) Google Scholar, 24Swanson M.S. Nakagawa T.Y. LeVan K. Dreyfuss G. Mol. Cell. Biol. 1987; 7: 1731-1739Crossref PubMed Scopus (167) Google Scholar). Isolation of three clones representing different regions of the same cDNA that did not cross-hybridize with any of the other isolated DDI clones coupled to the fact that none of the other clones exhibited homology to RNP family members suggested that the A18 mRNA might encode a novel hnRNP that is exclusively regulated by UV irradiation. The nucleotide and deduced amino acid sequences of A18 displayed a high degree of homology with the corresponding sequences of human and rat A1 hnRNP (22Cobianchi F. SenGupta D.N. Zmudzka B.Z. Wilson S.H. J. Biol. Chem. 1986; 261: 3536-3543Abstract Full Text PDF PubMed Google Scholar). However, in hamster cells the rat A1 hnRNP cDNA hybridized to a more abundant transcript of approximately 2 kb, which differs in size from that of A18 mRNA (1.4 kb), suggesting that the hamster DDI A18 clone does not represent hamster A1 hnRNP cDNA (22Cobianchi F. SenGupta D.N. Zmudzka B.Z. Wilson S.H. J. Biol. Chem. 1986; 261: 3536-3543Abstract Full Text PDF PubMed Google Scholar) but rather codes for a different hnRNP. Using hamster A18 and rat A1 hnRNP cDNAs as probes, we next investigated the effect of UV irradiation on the mRNA levels of A18 and A1 hnRNP in CHO cells. The results in Table II show that UV, near UV, and the UV mimetic agent AAAF up-regulated only A18 message and had no effect on A1 mRNA levels. Furthermore, only UV and UV mimetic agents enhanced the A18 transcript levels, whereas a number of other stress-inducing agents did not alter the A18 mRNA levels in these cells (Table II). It is interesting that the UV irradiation and AAAF-induced DNA damage is predominantly repaired via NER pathway (Ref.4Fornace Jr., A.J. Alamo I.J. Hollander M.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8800-8804Crossref PubMed Scopus (565) Google Scholar; see Refs. 1Fornace Jr., A.J. Annu. Rev. Genet. 1992; 26: 507-526Crossref PubMed Scopus (294) Google Scholar and 2Hollander M.C. Fornace Jr., A.J. DNA Repair Mechanisms: Impact on Human Diseases and Cancer. Landes Co., Georgetown, TX1994: 221-237Google Scholar for review). The specific induction of A18 mRNA exclusively by DNA damage that is repaired by NER is quite intriguing. To investigate the significance of the A18 gene in response to DNA damage that is repaired by NER and to investigate whether the A18 gene is also regulated in human cells, we sought to isolate and sequence the full-length human equivalent of hamster A18.Table IISpecific induction of DDI A18 mRNA by UV and UV mimetic AAAF in Chinese hamster ovary cellsAgentDoseDDI A18cDNA probeβ-actinA1 hnRNPUV14 J/m23.11.00.9Near UV300 J/m22.91.10.9AAAF20 μm3.01.01.1MMS100 μg/ml1.11.11.3200 μg/ml1.0ND2-aND, not determined.1.1N-methyl-N′-nitro-N-nitrosoguanidine10 μm1.10.81.030 μm1.00.90.9x-ray5 gray0.9ND0.940 gray1.1ND1.1Nitrogen mustard4 μm1.0ND0.68 μm1.00.91.040 μm1.41.01.0H2O20.4 mm1.30.91.1Bleomycin50 μg/ml1.2ND0.9Adriamycin0.4 μg/ml1.41.10.8Heat shock45.5 °C0.51.20.712-O-tetradecanoylphorbol-13-acetate20 ng/ml1.21.10.7Cells were ex
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