Construction of a Representative Cdna Library from Prostatic Intraepithelial Neoplasia

ICANCERRESEARCH56. 5380-5383. December 1. 19961 Advances in Brief

Construction of a Representative cDNA Library from Prostatic Intraepithelial Neoplasia

David B. Krizman,' Rodrigo F. Chuaqw, Paul S. Meltzer, Jeffrey M. Trent, Paul H. Duray, W. Marston Linehan, Lance A. Liotta, and Michael R. Emmert-Buck

Laboratory of Cancer Genetics ID. B. K.. P. S. M., J. M. TI, National Centerfor Human Genome Research, Laboratory of Pathology (R. F. C., P. H. D., L A. L, M. R. E-B.J, and Surgical Branch fW. M. LI. National Cancer Institute, Bethesda, Maryland 20892

Abstract cDNA libraries corresponding to cells derived exclusively from a microscopic prostatic lesion (PIN). Our results indicate that construc We report the construction of a plasmid-based eDNA library made tion of cDNA libraries arising from cells of prostate-specific epithelial from microdissected cells derived from prostatic mtraepithelial neoplasia. origin can be achieved and should be of value in elucidating genes Total RNA was extracted and converted to blunt-ended, double-stranded eDNA by oligo(dT)-mediated reverse transcription followed by linker important in this disease. addition. A linker-specific primer with UDG.compatible ends was used to Materials and Methods amplify the eDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the Clinical Information and Microdissection. Tissue sample was obtained clones derived from either known genes, anonymous expressed sequence from a radical prostatectomy performed at the National Cancer Institute tags, or novel transcripts with very little redundancy of screened clones. (Bethesda, MD). Samples were snap frozen immediately after surgery and These results demonstrate the feasibility of constructing complex repre stored at â€”70Â°Cuntiluse. National Cancer Institute patient 1542 is a 47-year sentative eDNA libraries from specific microdissected cell populations old black male who presented with localized (clinical stage T2@)prostate that represent microscopic precursor stages of cancer progression. This cancer. The patient was clinically free of disease until 1 year postoperatively method should facilitate identification of transcripts specifically expressed when he developed a rapidly rising PSA and clinical evidence of recurrent in cells of a distinct histological origin and tumorlgemc stage. disease. Histopathological examination of the prostatectomy specimen showed Introduction a poorly differentiated adenocarcinoma (Gleason sum 8). Several small (<1 mm) discrete foci of PIN were present. cDNA libraries have proven essential to positional gene cloning Unstained l2-@tmfrozen tissue sections were dissected under microscopic efforts, differential gene expression studies, and EST2 sequencing! visualization as described previously (8, 9). A pure population of PIN cells was mapping projects. Traditionally, cDNA libraries are constructed from dissected. An adjacent H&E-stained section was used as a guide to ensure 1 to S ,.tg of purified mRNA obtained from either large heterogeneous accuracy of dissection. The PIN focus used for microdissection was selected based on defined histopathological criteria (10, 11). Approximately 2000â€” tissue samples or tissue culture cell lines (1â€”3).The technology of 5000 PIN cells were microdissected. cDNA library construction is relatively straightforward, given such RNA Extraction and Determination of RNA Integnty. A modified ver large amounts of starting material. However, because large tissue sion of the Stratageme RNA Microisolation (La Jolla, CA) procedure was used samples invariably contain substantial cellular heterogeneity, they are to isolate total cellular RNA. After resuspension of the RNA pellet, a DNase not a suitable source of starting material for generation of a represen step and reextractiomwas performed using the MessageClean protocol (Gene tational library from a distinct cell type. Also, because of limited Hunter, Nashville, TN) according to the manufacturer's recommendations.The tissue availability, precursor lesions of cancer (such as epithelial final RNA sample was resuspended in diethylpyrocarbomate-treated water dysplasia and in situ carcinoma) cannot be specifically studied from containing 0.5 units/id RNase inhibitor. bulk tissue specimens. Finally, it is not known whether gene expres The integrity of the RNA prep was determined with RT-PCR analysis of sion profiles of in vitro cell lines necessarily reflect the expression @3-actinmRNA.RT was perfonned utilizing the RNA map protocol according to the manufacturer's recommendations (GemeHunter),except 2.5 @zMrandom profiles of their tissues of origin, making this source of RNA also hexamers were used as primer. The final mixture was incubated as follows; problematic. Thus, it would be of considerable interest to generate 65Â°Cfor5 mm, 25Â°Cfor10mm, 1 j.tlMoloney murine leukemia virus reverse cDNA libraries directly from specific cell populations of various transcriptase added, 25Â°Cfor10 mm, 37Â°Cfor40 mm, and 94Â°Cfor5 mm. tissues to serve as reagents for studying in vivo gene expression. Each RT reaction generated 20 @.dofcDNA.Amplification ofactim mRNA was Although technologies designed to generate complex and represent performed using 1 @lofthe cDNA sample with actin-specific primers (Clon ative cDNA libraries from small amounts of total cellular RNA of a tech, Palo Alto, CA). The reaction was run twice to ensure reproducibility of given cell type have previously been developed (4â€”7),they would results and a negative reverse transcription reaction was run in parallel. benefit from pathology techniques enriching for the desired type of eDNA Synthesis and Library Construction. Double-strandedcDNAwas diseased cell. synthesized from 0.04 @gof total cellular RNA using the Superscript Choice We now report the feasibility of combining microdissection of System based on the RNase H-mediated second-strand replacement method (2), according to the manufacturer's recommendations, with some modifica specific cell populations with cDNA library construction to generate tions (Life Technologies, Inc., Gaithersburg, MD). RT was carried out in a l0-pJ volume reaction. A concentration of 50 ng/pi oligo(dT) was used for Received 8/9/96; accepted 10/I7/96. priming first-strand cDNA, and this reaction was carried out at 45Â°Cfor15ruin The costs of publication of this article were defrayed in part by the payment of page instead of at the recommended 37Â°Cfor 30 mm. The second-strand replace charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ment and EcoRI linker addition reactions were followed according to the I To whom requests for reprints should be addressed, at NCHGRINIH Building 49, manufacturer's suggestions. After linker addition, the double-stranded cDNA Room 4A15. 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 402-2015; Fax: was electrophoresed in 1.2% low-melt agarose and products from 0.3 to 2 kb (301) 402-3241. were excised with a scalpel blade. The agarose was melted at 65Â°Cfor15 rain, 2 The abbreviations used are: EST, expressed sequence tag; PIN, prostatic intraepithe hal meoplasia;RT, reverse transcription; PSA, prostate-specific antigen; dbEST, database digested overnight at 37Â°Cwith(3-agaroseenzyme according to the manufac of expressed sequence tags. turer's recommendations (New England BioLabs, Beverly, MA), phenol/ 5380

chloroform extracted, and ethanol precipitated. The cDNA pellet was resus Table 1 CumulativeclonesNo.sequence analysis of 154PiN-specific cDNA pended in 20 @lTEand stored at â€”20Â°C. clonesIdentification(n of A total of 5 pAof the cDNA was amplifiedby five cycles of PCR under totalRedundanciesNoinsert1610rRNA138.5Anonymous154)% of standard reaction conditions using 2.5 units of PerkimElmer-Cetus Taq po lymerase and 1.0 @.LMconcentrationof the linker-specific primer LINK (CUACUACUACUAAATrCGCGGCCGCGTCGAC) that also functions to eachKnown ESTs54352 ESTs 2X direct UDG cloning. The cycling conditions consisted of an initial 3-mm genes5334.5PSA 3X, ribosomal L29 2X, XUnique1812 cytochrome c oxidase 2 denaturing step at 94Â°Cfollowedby five cycles of94Â°Cfor15s, 55Â°Cfor15s, and 72Â°Cfor2 mm, with a final extension at 72Â°Cfor5 ruin.The PCR product Individual clone sequences were compared to sequence data bases using the BLAST was size selected by passing through a CHROMA SPIN-200 column to remove program. Identification groups are shown with total number of clones originating from the unincorporated primers, primer/primer artifacts, nucleotides, Taq, and buffers various groups calculated as well as the percentage of the total. The amount of redundancy according to the manufacturer's recommendations (Clontech). The flow is also shown for each group. Novel sequences showed no significant similarities to existing data base sequences; however, most demonstrated weak similarities to ESTs from through was ethanol precipitated, resuspended in 20 @.dofTE, and 6 @zl dbEST. nondirectionally cloned into the UDG cloning vector pAMPIO according to the manufacturer's recommendations (Life Technologies, Inc.). Recombinant clones were randomly picked for sequencing. Sequencing and Data Analysis. Clones were propagated and sequence Individual library subclones were picked at random for one pass ready DNA preparations made using the AGTC 96-well boiling lysis method sequencing using the dye-primer method. The sequences of 154 according to the manufacturer's recommendations (Advanced Genetic Tech subclones were determined and analyzed using BLASTN (13), with nologies Corp., Gaithersburg, MD). Dye primer cycle sequencing was carried the combined characterization of all 154 inserts shown in Table I. A out using the ABI Prism Ml3 forward primer reaction according to the total of 13 of 154 (8.5%) of the clones originated from rRNA, whereas manufacturer's recommendations (Applied Biosystems, Inc., Foster City, CA). Sequencing reactions were analyzed on an ABI 377 automatic sequencing 16 of 154 (10%) of the clones contained no insert. apparatus. Individual sequences were compared to various sequence data Analysis of the remaining 125 clones indicated they derived from bases, including dbEST (12), by the BLAST program accessed through the either known genes, anonymous ESTs, or novel transcripts. BLASTN National Center for Biotechnology Information at the following http server, analysis indicated that 53 of 154 (34.5%) clones contained inserts http:llwww. ncbi.nlm.nih.gov/ (13). from 46 different known genes. A number of housekeeping genes For determination of the size of cDNA inserts, a 96-well plate containing were identified including glutathione S-transferase, glyceraldehyde-3- clones picked at random was used as substrate for PCR analysis. A standard phosphate dehydrogenase, ubiquitin, and several ribosomal protein 20-pi PCR reaction was performed in each individual well in a Perkin genes. A number of identified genes indicates that the microdissected Elmer-Cetus 9600 thermal cycler using the LINK primer at a concentration of cells were of epithelial origin (epithelial cell growth regulator gene), 1 jLMwith 0.5 units of Taq/reaction. Cycling conditions were the same as described above for library amplification, except that 30 cycles were per derived from the prostate (prostatic acid phosphatase and PSA genes), formed. A total of 5 ,.ii from each reaction was analyzed on a 1.2% agarose gel and that they most likely were growing abnormally (acute lympho and product size was determined by comparison to a lOO-bpladder marker cytic leukemia-specific antigen and translationally controlled tumor (Life Technologies Inc.). protein genes). These results demonstrate construction of a cDNA library that is representative of the cells of origin. In addition, many Results of the library clones corresponding to known genes cross splice Total RNA was obtained from microdissected PIN cells and used junctions within cDNA sequences, demonstrating that the nucleic acid for cDNA library construction as described. The PIN focus used for used for oligo(dT)-initiated RT was mRNA and not contaminating microdissection is shown in Fig. 1 and the quality of recovered RNA DNA (data not shown). The known genes identified from this library was assessed using RT-PCR for (3-actin as described (data not shown). are listed in Table 2. A library consisting of approximately 220,000 clones was established. Further sequence analysis indicated that 54 of 154 (35%) clones derived from transcripts represented by anonymous human ESTs present in dbEST. The accession number of each EST identified is listed as follows: L3l980, H21l71, H15276, H50589, Hl8257, H80672, H03201, H59925, H77539, Hl60l8, H65440, H27988, N20576, N92608, N584l8, N27657, N93l8l, N56945, N53527, N92574, N67313, N80272, N80334, N42879, N48603, N2337l, N41712,W30809, W48653, W46778, W23697, W51810, W47 141, W46294, Wl8503, W04828, W15423, W42756, M62l6l, F1934l, Fl743l, D56690, D61l07, D80270, R64452, R59444, R96103, T6l257,T24l19,T16l47,T58539,andT97528.An additional18 of 154 (12%) clones contained novel sequences not significantly similar to any sequence found in the data bases, although 15 of 18 clones demonstrated weak similarities with many sequences in dbEST. Fur @ Â°---. â€¢Â°-@i:, .,.,.. @ . â€˜ â€¢, . thermore, the novel clones show no similarity to human specific .â€”.â€˜. repeats or any rRNA species, suggesting that they originated from authentic novel cellular transcripts. No clones were found to contain bacterial sequences nor were any chimeric clones identified. The amount of redundancy in clones originating from known genes,

@ Fi@ lli@H:i@l i@Ictl:I@ @t I\ â€˜JnJi iiit@tJi@cuci i @DNA ESTs, and novel sequences was also determined. Only three known @ Iibrar\ @r@:1u: @IhIc@ini@ (@ 1111flI11i\U1IIIIHI1IiftiHi@l\rmal genes were identified more than once. PSA was found three times stroma is seen at the upper and lower aspects of the figure. The glands within the PIN whereas the ribosomal protein L29 and cytochrome c oxidase tram focus show epithelial stratification, focal disruption of polarity, and nuclear overlapping. Scattered nuclei with prominent nucleoli are present. A basal cell layer is also present scripts were identified twice each. Likewise, there was very little consistent with an intraepithelial neoplasm. H&E stain, X200. redundancy of EST identification since only two ESTs were identified 5381

Table 2 Known genes iden:(fiedfrom this library is a change in the expression levels of particular genes. The determi PSA nation of altered tumor-specific patterns of gene expression would be L29 ribosomal protein useful in defining particular stages of tumorigenesis and potentially Prostatic acid phosphotase Ribosomal protein L37a could lead to improved diagnosis, identification of therapeutic targets, Acidic ribosomal protein P1 and better prognostic capability. glyceraldehyde-3-phosphate dehydrogenase MHC-G protein j3 subunit We initiated efforts to develop reagents to study gene expression AlP synthase @3 changes in tumor progression by integrating tissue microdissection Cell surface heparin-binding protein and cDNA library construction. We have previously demonstrated MHC class I HLA-ABC 8.2-kb differentiation factor successful RNA recovery from specific microdissected cell popula Ubiquitin tions and that this RNA can be amplified by RT-PCR for measure COX Vla-L cytochrome c oxidase Ribosomal protein L32 ment of known gene levels or in the search for novel disease associ Protein kinase C-@32 transcript ated genes.3@4Cell type-specific cDNA libraries have previously been Ribosomal protein L4l DNA-binding protein TAX REB 107 developed from as little starting material as a single ganglion or 10 HCG3 cells from a myeloma cell line (4, 5) and results indicate average Heavy neurofilamemt subunit (NH-H) Laminin-binding protein clonal insert sizes of 600 bp with an upper size limit of 1.5 kb. The a-fetoprotein enhancer binding protein amount of rRNA contamination and cloned primer artifacts was HLA-Cw6 minimal with concomitant maintenance of library complexity. In the Cytochrome C oxidase Stimulatory GTP-binding protein (GSA) present study, we constructed a cDNA library demonstrating an av BBCI erage insert size of 580 bp with a size range of 300-1200 bp. The Epithelium I and 2-epithelial cell growth regulator Glutaredoxin amount of contaminating rRNA clones in the present library was 13 of Sec6l -complex-j3subunit 154 clones or 8.5% of the total, which is slightly higher than previous Ribosomal protein 514 Translationally controlled tumor protein reports. Ataxia telangiectasia In general, cDNA library characterization has relied on hybridiza 23-kD highly basic protein tion of nylon filters containing randomly plated clones with specific Elongation factor EFIA 3-Hydroxy-3-methylglutaryl coenzyme probes to indicate library representation and redundancy. We chose to Ribosomal protein LI I characterize this library by sequencing a number of randomly picked PMI. phosphomannose isomerase CtBP independent clones. This proved to be a quick and efficient approach, LLrep3-ribosomal protein S4 since no data ambiguities due to subjective hybridization results were Glutathione S-transferase GTP-bimdingprotein-a subunit encountered and the origin of a large percentage of inserts was easily ALL antigen-CALLA determined. We analyzed 154 cloned inserts and unambiguously Cyclic nucleotide phosphodiesterase-HSPDE 3B Ribosomal protein L35 determined the origin of 88% of those inserts. We were also able to ID-myo-inositol triphosphate 3 kinase B isozyme demonstrate the remaining 12% most likely to be derived from novel Tumor necrosis factor type I receptor-associated protein transcripts not yet identified. In addition, no clones containing bacte NADH-ubiquinome oxidoreductase chain 4 rial sequence were found. The absolute number of chimeric clones could not be determined because each clone was sequenced only once twice each. These results demonstrate the complexity and relatively from a single direction; however, no chimeric clones were found with low redundancy of this cDNA library. the sequence that we were able to analyze. A single 96-well plate of randomly picked colonies was subjected A number of genes previously shown to be differentially expressed to PCR amplification to determine the average size of the inserts. in prostate cancer progression were not found in the present study Agarose gel electrophoresis indicated an average insert size of 580 bp including the putative cancer metastasis gene Kall (15), a novel (range, 250-1200 bp; data not shown). putative oncogene PTI-1 (16), the fusion gene tpc/hpr (17), and the human epitheial tropomyosin gene Tmel (18). The fact that any or all Discussion of these genes was not found is due to either a limited number of The multistep nature of tumorigenesis is reflected in the progressive cDNA clones actually sequenced or their down-regulation in PIN. stages identified by pathological study of tumors. Carcinomas pro This demonstrates the lack of functional data relevant to prostate gress through stages of dysplasia and in situ carcinoma prior to cancer biology provided by limited sequencing efforts and under developing into overt cancers. PIN is thought to be the precursor scores the need for further technologies aimed at obtaining such lesion to prostate cancer due to the spatial association and similar information. Future efforts include construction of similar cDNA histological features shared between PIN and invasive tumor cells. libraries representative of all stages of prostate cancer progression, PIN foci are discrete microscopic lesions which are generally less than large-scale sequencing of these cDNA libraries, and subsequent use of 1 mm in diameter. The specific genetic alterations which initiate PIN these libraries in subtractive hybridizations and cDNA microarray and those which cause progression to invasive cancer are largely technology to identify genes whose expression patterns are signifi unknown. Frequent loss of heterozygosity at chromosome 8p2l in cantly altered in prostate cancer initiation and progression. PIN indicates that inactivation of a tumor suppresser gene(s) in this region may be a fundamental event in the genesis of PIN and prostate cancer (14). However, much remains to be determined with respect to the genetic alterations in PIN. A more thorough understanding of 3 R. F. Chuaqui, C. R. Englert, S. Strup. C. D. Vocke, Z. Zhuang, P. H. Duray, D. G. Bostwick, W. M. Linehan, L. A. Liotta, and M. R. Emmert-Buck. Identification of a novel these genetic events will impact on our knowledge of prostate cancer gene up-regulated in clinically aggressive human prostate cancer, submitted for publica development as well as identify specific genetic changes in PIN that tion. 4 M. R. Emmert-Buck, R. F. Bonmer, P. D. Smith, R. F. Chuaqui, S. R. Goldstein, Z. are associated with progression to overt prostate cancer. Zhuang, R. A. Weiss, and L. A. Liotta. Laser capture microdissection (LCM), submitted Inherent in the progression from one pathological stage to the next for publication. 5382

Acknowledgments 9. Zhuang, Z., Bertheau, P., Emmert-Buck, M. R., Liotta, L. A., Gnarra, J., Linehan, W. M., and Lubensky, I. A. A mew microdissection technique for archival DNA We thank Dr. Marcelo Bento Soares for critical review and helpful com analysis of specific cell populations in lesions less than one millimeter in size. Am. J. Pathol., 146: 620â€”625, 1995. ments. We also thank Christianne Robbins, Bob Walker, John Leuders, and the 10. Bostwick, D. G., and Brawer, M. K. Prostatic intra-epithelial neoplasia and early National Center for Human Genome Research nucleic acid core facility for invasion in prostate cancer. Cancer (Phila.), 59: 788â€”794, 1987. sequencing. 11. McNeal, J. E., and Bostwick, D. G. Intraductal dysplasia: a premalignant lesion of the prostate. Hum. Pathol., 17: 64â€”71,1986. References 12. Boguski, M. S., Lowe, T. M., and Tolstoshev, C. M. dbEST- database for â€œexpressed sequence tags.â€•Nat. Gemet., 4: 332â€”333,1993. 1. Aviv, H., and Leder, P. Purification of biologically active globin messenger RNA by 13. Altschul, S. F., Gish, W., Miller W., Myers E. W., and Lipman, D. J. Basic local chromatography on oligothymidylic acid- cellulose. Proc. Natl. Acad. Sci. USA, 69: alignment search tool. J. Mol. Biol., 215: 403-410, 1990. 1408â€”1412,1972. 14. Emmert-Buck, M. R., Vocke, C. D., Pozzatti, R. 0., Duray, P. H., Jennings, S. B., 2. Okayama, H., and Berg, P. High-efficiency cloning of full-length cDNA. Mol. Cell. Florence, C. D., Zhuang, Z., Bostwick, D. G., Liotta, L. A., and Linehan, W. M. Biol.,2:161â€”170,1982. Allelic loss on chromosome 8pl2â€”2l in microdissected prostatic intraepithelial meo 3. Gubler, U., and Hoffman, B. J. A simple and very efficient method for generating plasia. Cancer Res., 55: 2959â€”2962, 1995. cDNA libraries. Gene, 25: 263â€”269,1983. 15. Dong, J-T., Lamb, P. W., Rinker-Schaeffer, C. W., Vukamovic J., Ichikawa, T., 4. Belyavsky, A., Vimogradova, T., and Rajewsky K. PCR-based cDNA library con Isaacs, J. T., and Barrett, J. C. Kall, a metastasis suppressor gene for prostate structiom: general cDNA libraries at the level of a few cells. Nucleic Acids Ret., I 7: 2919â€”2932,1989. cancer on human chromosome llpll.2. Science (Washington DC), 268: 884â€” 886, 1996. 5. Komeev, S., Blackshaw, S., and Davies, J. A. cDNA libraries from a few neural cells. Prog. Neurobiol., 42: 339â€”346, 1994. 16. Shen, R., Su, Z. Z., Olsson, C. A., and Fisher, P. B. Identification of the human 6. Lu, X., and Werner, D. Construction and quality of cDNA libraries prepared from prostatic carcinoma oncogene PTI-l by rapid expression cloning and differential cytoplasmic RNA not enriched in poly(A)@RNA. Gene, 71: 157â€”164,1988. RNA display. Proc. Natl. Acad. Sci. USA, 92: 6778â€”6782,1995. 7. Robinson, P. A., Marley J. J., and McGarva, J. Construction of cDNA libraries from 17. Veronese, M. L., Bullrich, F., Negrini, M., and Croce C. M. The t(6;l6)(p2l;q22) impure preparations of messenger RNA using RNase-H free Moloney-murine leuke chromosome translocation in the LNCaP prostate carcinoma cell lime results in a mis virus reverse transcriptase. Methods Mol. Cell. Biol., 3: 118â€”127,1992. tpc/hpr fusion gene. Cancer Res., 56: 728â€”732,1996. 8. Emmert-Buck, M. R., Roth, M. J., Zhuang, Z., Campo, E., Rozhin, J., Sloane, B. F., 18. Wang, F-L., Wang, Y., Wong, W-K., Liu, Y., Addivinola, J., Liamg, P., Chen, Lions, L. A., and Stetler-Stevenson, W. G. Increased gelatimase A (MMP-2) and L. B., Kantoff, P. W., and Pardee, A. B. Two differentially expressed genes in cathepsim B activity in invasive regions of human colon cancer samples. Am. J. normal human prostate tissue and in carcinoma. Cancer Res., 56: 3634â€”3637, Pathol., 145: 1285â€”1290,1994. 1996.

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David B. Krizman, Rodrigo F. Chuaqui, Paul S. Meltzer, et al.

Cancer Res 1996;56:5380-5383.

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