[CANCER RESEARCH 58. 4233-42.17, October I. W8] Advances in Brief A Novel Candidate Oncogene, MCT-1, Is Involved in Cell Cycle Progression Misha Prosniak, Jamil Dierov, Kenji Okami, Brian Tilton, Brad Jameson, Basse! E. Sanava, and Ronald B. Gartenhaus1 Center for NeuroVirvlngy ami NeuroOncology. Allegheny University of the Health Sciences. Philadelphia. Pennsylvania 19102 IM. P.. J. D.. B. T., B. J.. B. E. S., K. B. G.I. and [)('¡>Ã-irlttient of Ololiiryn\>olos\-, Johns Hopkins Universit\. Baltimore. Maryland 21205 [K. O.¡ Abstract Using the arbitrarily primed-PCR (AP-PCR) assay to detect genetic ab normalities that occur in a panel of lymphoid cell lines, we identified an amplified stretch of genomic DNA that contained a putative open reading frame. Northern blot analysis with this genomic clone revealed widespread low level expression in normal human tissue. The full cDNA sequence was obtained with no significant homology to any known genes in the genome database. We termed this novel gene with multiple copies in a T-cell malig nancy as MCT-1. MCT-1 was localized to the long arm of chromosome Xq22-24 by flourescence in situ hybridization analysis. Although there was no significant homology at the primary sequence level, there was a limited degree of amino acid homology with a domain of cyclin H that appears to specify protein-protein complexes. This relationship between MCT-1 and cyclin H implied a potential role for MCT-1 in cell cycle regulation. Overex- pression oÃ- MCT-1 increased the proliferative rate of cells by decreasing the length of the Gt phase without a reciprocal increase in the S and G2-M phases. Recent work has established the role of cell cycle regulatory molecules in the development of certain human malignancies. Therefore, we investi gated the transforming ability of MCT-1 overexpression using soft agar growth assays and demonstrated that only A/CTW-overexpressing cells were able to establish colonies. Taken together, MCT-1 is a novel candidate onco gene with homology to a protein-protein binding domain of cyclin H. Introduction Most cancers have both random and nonrandom chromosomal abnormalities ( 1). Single copy losses and gains have been well char acterized in many malignancies (2). Many of these nonrandom chro mosomal abnormalities have been invaluable in identifying those genes involved in tumor formation. Gene amplification is often asso ciated with oncogenesis and drug resistance ( 1). The contribution of oncogene amplification to lymphoid neoplasms has not been well established (3). A PCR-based method for detecting genetic lesions is the AP-PCR2 assay (4-6). Different genomic DNA templates will generate electrophoretic patterns, referred to as DNA fingerprints, with a number of differences proportional to the degree of dissimi larity between the two genomes. Additionally, it is possible to detect losses or gains in the number of copies of a target genomic sequence by the differences in the intensity of the corresponding band. Because the AP-PCR method is quantitative as well as qualitative, it can also identify amplified regions of DNA as well. In the present study, we screened a panel of T-cell leukemia/lymphoma lines using AP-PCR and identified an amplified DNA sequence that encodes a novel gene with limited amino acid homology to a region of cyclin H that appears Received 6/26/98; accepted 8/11/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Center for NeuroVirology and NeuroOncology. Allegheny University of the Health Sciences, Broad and Vine. Philadelphia. PA 19102. Phone: (215) 762-3664; Fax: (215) 762-8404; E-mail: gartenhaus@auhs.edu. 2 The abbreviations used are: AP-PCR. arbitrarily-primed PCR: PBL. peripheral blood lymphocyte; RACE, rapid amplification of cDNA ends; GSP. gene-specific primer; CMV. cytomegalovirus. to specify protein-protein interactions. We termed this novel gene with multiple copies in a T-cell malignancy as MCT-l. Recent work has demonstrated that cell cycle regulatory molecules can participate in oncogenesis through amplification and overexpression (7-12). Our data demonstrate a shortened G, phase, decreased cell doubling time, and anchorage-independent growth in MC7-/-overexpressing cells, further supporting this important association of cell cycle regulatory molecules and tumorgenesis. Materials and Methods AP-PCR. Genomic DNA was prepared from all T-cell lines and normal PBL samples. All reactions were carried out in a 25-fi\ volume containing 10 ITIMTris-HCl (pH 8.3). 20(1 HIMeach deoxynucleotide triphosphates. 50 mvi KC1, 5 mM MgCU, 25 pmol of 10-mer arbitrary primer. 0.1 mg of DNA template, and 1 unit of Taq DNA polymerase (Fisher Biotech). A panel of IO-20-mer primers were labeled with T4 polynucleotide kinase and [•y-12P]ATP. All reactions were performed with a GeneAMP PCR System 9600 (Perkin-Elmer). The profile was as follows. The first five cycles of the temperature profile: denaturation at 95°Cfor 30 s, primer annealing at 25°Cfor 1 min. and extension for 1 min at 72°C.The last 25 cycles were: denaturation at 95°Cfor 30 s. primer annealing at 30°Cfor 30 s. and extension for 1 min at 72°C.PCR products were separated by electrophoresis in denaturing 8M urea/polyacrylamide gels, followed by autoradiography. Cloning and Sequencing of Genomic MCT-1 Sequences Amplified by AP-PCR. The band that appeared to be amplified in the HUT 78 lane relative to the corresponding bands from other cell lines and normal lymphocytes was isolated for cloning and sequencing. This band was excised from gels and incubated in 10 ml IX Assay Buffer A (Fisher Biotech) at 90°Cfor 10 min. Five fil of eluted DNA was reamplified with the same AP-PCR primer as before with MgCK concentration of 5 mM for 30 cycles at 30°C.The PCR product was analyzed in polyacrylamide gel to confirm its size and purity. The amplified DNA was cloned into compatible thymidine pMOSß/i/c T-vector (Amersham). The presence of an appropriate insert was determined using direct colony PCR with T7- and UI9-mer pMOSS/i/f-specific primers. Se quencing was performed using established methodology. Sequences obtained from several clones were compared with known sequences in the GenBank database using the BLASTn and BLASTx program (13). Isolation and Sequencing at MCT-1 cDNA. The full-length MCT-1 cDNA sequence was obtained by the RACE method as described previously 114). Briefly. 5' and 3' RACE PCRs were carried out using total RNA obtained from normal human PBLs. In the 3' RACE procedure, first-strand cDNA synthesis was initiated using a poly(T) adapter primer. The cDNA was amplified by PCR with the GSP (S'-GATCCTGTCAAAATAGTCCGATGC-S'I. and the abridged universal am plification primer was supplied by the manufacturer (Lite Technologies. Inc.). which is homologous to the adapter sequence used to prime first cDNA synthesis. In the 5' RACE protocol, first-strand cDNA was synthesized using GSP1 (5'- TCCAGGAGAAGTTAAGCCTG-3'). After adding a homopolymeric tail to the 3'-end of the cDNA. amplification was performed using a nested. GSP2 (5'- GACAGGATCTTTCTTAGGCATG-3') that anneals to a site located within the cDNA molecule and a novel deoxyinosine containing anchor primer supplied by the manufacturer (Life Technologies. Inc.). PCR reaction conditions were carried out as specified by the manufacturer (Life Technologies. Inc.). GSPs were de signed based on sequence data obtained from the original AP-PCR amplified fragment containing a putative open reading frame. Plasmid Construction, Cell Transfection. and Cell Culture. The full length MCT-1 cDNA expression vector (pCMV-MCT-l) was made using 4233