[ RESEARCH 56. 2904-2907. July I, 1996] Advances in Brief

H19 Overexpression in Atypical Multidrug-resistant Cells Associated with Expression of a 95-Kilodalton Membrane Glycoprotein1

L. Austin Doyle,2 Weidong Yang, Arun K. Rishi, Yongming Gao, and Douglas D. Ross2

University of Maryland Cancer Center, and Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland. 21201

Abstract differentially expressed in p95-overexpressing multidrug-resistant cells to elucidate the molecular mechanisms responsible for the drug The multidrug resistance phenotype of human breast carcinoma Ml 1 - resistance manifested by these cells. 7/AdrVp cells is characterized by overexpression of a 95-kilodalton membrane glycoprotein (p95), accompanied by a marked reduction in Materials and Methods intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein. We discovered that the Cell Lines. MCF-7 breast carcinoma cells, their drug-resistant subline mRNA of the H19 gene is overexpressed in MCF-7/AdrVp cells relative to MCF-7/AdrVp, and a drug-sensitive MCF-7/AdrVp revertant subline, obtained parental MCF-7 cells or drug-sensitive MCF-7/AdrVp revertant cells. H19 from Dr. Antonio Fojo (Medicine Branch, National Cancer Institute), were is an imprinted gene with an important role in fetal differentiation, as well maintained in culture as described previously (2, 5). The NCI-HI688 small cell as a postulated function as a tumor suppressor gene. Another p95-over- lung cancer cell line was obtained from Dr. Adi F. Gazdar (then at the National expressing multidrug-resistant cell line, human lung carcinoma NCI- Cancer Institute Navy Medical Oncology Branch) and was cultured as de H1688, also displays high levels of 1119 mRNA. In contrast, several scribed previously (6). The MCF-7/AdrVp subline was continuously main multidrug-resistant cell lines that overexpress P-glycoprotein or the mul tained in the presence of 100 ng/ml doxorubicin (Pharmacia Adria, Dublin. tidrug resistance protein do not have higher levels of H19 mRNA than OH) and 5 jig/ml verapamil (Sigma Chemical Co., St. Louis, MO). The HL-60 their drug-sensitive counterparts. This is the first report of 1119 gene human leukemia cell line, its Pgp-overexpressing, vincristine-resistant subline overexpression accompanying any form of drug resistance. The associa HL-60/Vinc, and its MRP-overexpressing, doxorubicin-resistant subline HL- tion of 1111'and p95 in drug resistance warrants further 60/Adr were obtained from Dr. Melvin Center (Kansas State University; Ref. study. 7). The small cell lung cancer cell line UMCC-1 and its etoposide-resistant subline UMCC-1/VP, which overexpresses MRP. were developed in our Introduction laboratories at the University of Maryland Cancer Center and have been described previously (8). The human breast carcinoma subline MCF-7/AdrVp displays a Construction and Screening of a cDNA Library. A cDNA library was novel MDR3 phenotype that is distinguished by a unique pattern of created using RNA isolated from NCI-HI688 cells and the multifunctional drug cross-resistance as well as greatly diminished accumulation and A-Zap Express vector (Stratagene, Inc., La Jolla, CA). The Àlibrary was used retention of daunorubicin in the absence of overexpression of the for expression cloning with a picoBlue immunoscreening kit (Stratagene) and that encode MRP or Pgp (1-3). MCF-7/AdrVp cells were a preadsorbed rabbit antiserum against gel-purified p95 protein as described derived from MCF-7 cells by selection with doxorubicin in the pres previously (5). Insert DNA from positive colonies was recovered by secondary ence of verapamil, an inhibitor of Pgp-mediated drug transport. The and tertiary screening to pick isolated plaques. The cDNA inserts from positive plaques was excised in vivo from the phage in the form of the neomycin- MCF-7/AdrVp subline was found to overexpress a 95-kDa membrane resistant pBK-CMV phagemid, following the manufacturer's protocols. Se glycoprotein termed p95 ( 1). If MCF-7/AdrVp cells are removed from quencing of the cDNAs was performed with an automated DNA sequencer the selective pressure of doxorubicin, they revert to drug sensitivity, (Perkin Elmer, Inc., Foster City, CA). All DNA sequences were confirmed by and their expression of p95 diminishes to that of parental cells. High sequencing in the reverse direction. levels of p95 expression have been observed in clinical samples of Southern and Northern Blot Analysis. Southern and Northern blotting drug-refractory human (1) and lung cancer (4), including with 32P-labeled cDNA probes was as described previously (4-6). a multidrug-resistant human small cell lung carcinoma cell line, RT-PCR Assay for H19. Primers were designed that span exons 3 and 4 and introns 2-4 of the W/9gene (sense, bp 1407-1422, 5'-CCGGCCTTCCT- NCI-HI688, which has no demonstrable overexpression of Pgp or GAACA; antisense, bp 1580-1600, 5'-TTCCGATGGTGTCTTTGATGT). MRP. We have observed that p95 expression is detectable in blast Therefore, this RT-PCR assay is useful in distinguishing the cDNA amplifi cells from 30% of de novo cases of acute myeloid leukemia, and that cation product from that formed from contaminating genomic DNA in the this expression correlates with lower accumulation and retention of assays. The conditions for the RT-PCR assay, with 35 PCR cycles, were the daunorubicin in these cells (2). We have also found that p95 is an same as those used for detection of MRP or MDR1 mRNA transcripts, as TV-linked sialoglycoprotein with a 35-kDa polypeptide core (5). The described previously (9). Detection of ß-actinby RT-PCR was also as de present report arises from studies designed to discover genes that are scribed previously (9). Gene Database Analysis. Analysis of DNA sequences was accomplished Received 2/14/96; accepted 5/15/96. using the GenBank and EMBL nucleotide sequence databases. These were The costs of publication of this article were defrayed in part by the payment of page accessed using the Wisconsin Software Package version 8 (Genetics Computer charges. This article must therefore be hereby marked advertisement in accordance with Group, Madison, WI), which are available through the Supercomputing Facil 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by National Cancer Institute Grants RO1 C A52178 and RO1 ity of the Frederick Cancer Research Center (Frederick, MD). CA40188. a U.S. Department of Veterans Affairs merit review grant (D. D. R.). a grant from Pharmacia and Upjohn, Inc., and a grant from the Charlotte Geyer Foundation. Results and Discussion 2 To whom requests for reprints should be addressed, at University of Maryland Cancer Center, 22 South Greene Street, Baltimore, MD 21201. Phone: (410) 328-3685; Fax: (410) To clone the genes responsible for the MDR associated with p95 328-6559. 3 The abbreviations used are: MDR. multidrug resistance; RT, reverse ; overexpression, a cDNA library was created from NCI-HI688 cells, Pgp, P-glycoprotein; MRP, multidrug resistance protein; kDa, kilodalton. packaged in a A-phage vector (Stratagene), and used for expression 2904

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. H/9 OVEREXPRESSION IN MDR CELLS cloning, using antisera to p95 (5). Fifteen phage plaques reacted with 59 1798421 p95 antiserum, and phages from these plaques were isolated by l l * TT1 TI/ replating and secondary screening. The cDNA inserts were excised H19 and used as probes for Northern blotting of RNA from MCF-7, mRNA MCF-7/AdrVp, MCF-7/AdrVp revenant, and NCI-HI688 cells. Only 1 GCH1663 ¿1 two of the cDNA probes selectively hybridized with RNA from the A2 drug-resistant cell lines MCF-7/AdrVp and NCI-H1688; one of these, cDNA a 2.2-kb cDNA species initially termed clone A2, was even more highly overexpressed in the NCI-HI688 cell line than in the MCF-7/ 5' CGGCACGAGATGT •¿*• AdrVp line (Fig. 1), consistent with the level of expression of p95 in Fig. 2. Comparison of nucleotide sequences derived from the A2 cDN A and the human these cell lines (2, 5). The clone A2 cDNA probe hybridized with a HI9 mRNA. The A2 cDNA is missing Ihe first 59 bp of exon 1 of the HI9 mRNA but 2.4-kb mRNA species by Northern analysis (Fig. 1). Southern blot has a 5' non-/// 9 13-mer sequence appended. The A2 cDNA is also missing the first 107 analysis of genomic DNA digested with EcoRI and BamHl, using bp of exon 4 of the HI 9 gene. clone A2 as a probe, did not reveal amplification of the gene repre sented by clone A2 in either of the drug-resistant cell lines compared mRNA. The 2.4-kb mRNA species hybridizing with the A2 cDNA with parental MCF-7 or revenant MCF-7/AdrVp cells (data not probe is consistent with the previously published size of H19 mRNA shown). (11). A search of the confirmed sequence of clone A2 cDNA using the The H19 gene was discovered in differential hybridization screens GenBank and EMBL databases revealed it to be almost identical to of fetal mouse livers in efforts to find genes that were coordinately the published sequence of mRNA of the human H19 gene (10). The regulated with the a-fetoprotein gene during fetal differentiation (12, A2 cDNA was 2179 bp in length, compared with 2332 bp for the 13). The protein encoded by the murine HI 9 gene was not apparent full-length human H19 cDNA (10). As depicted in Fig. 2, the A2 from its sequence because of frequent stop codons in all three reading cDNA is missing the first 59 bp of exon 1 oíH19. However, there is frames. The human H19 mRNA sequence contains 21 AUG codons, one non-///9 sequence at the 5' end of A2. This 13-mer sequence, but most of these initiators are followed shortly thereafter by termi 5'-CGGCACGAGATGT, does not correspond to the linker arms of nators, with no open reading frame spanning the exon junctions in the the A-Zap system and is of unknown significance. Three nucleotide mRNA (10). Nine of the AUG codons are shared with the mouse gene changes in A2 are noted in comparison to the published H19 sequence but there is no open reading frame that is conserved along its entire (Fig. 2). In addition, the A2 cDNA has also lost the first 107 of the 123 length between the two species. Analysis of the human and murine bp on H19 exon 4, (bp 1844-1950 in the published H19 sequence), HI 9 genes demonstrates an overall high degree of homology, but suggesting that clone A2 may represent a splice variant of H19 neither species has an open reading frame capable of producing the 35-kDa polypeptide core of p95. H19 mRNA has not been detected in large polysomes but, rather, is sequestered in monosomes and/or /• ribonucleoprotein particles (10). Therefore, it has been suggested that ^r ^* the gene may function directly as the RNA component of a ribonu f cleoprotein and that the RNA, rather than the protein, is the active è^ *?S// # gene product (10). Our analysis of all reading frames of the sequence s s of clone A2 cDNA revealed that the deletion in exon 4 did not lead to the creation of an extended open reading frame, making it unlikely that p95 represents a protein product of the H19 gene. Although the relationship between H19 and p95 is unclear, the longest open reading frame in exon 1 of the human H19 gene is longer than predicted by chance alone and could encode a 22-kDa protein (10). A between peptides encoded by the H19 and p95 genes could have been identified by our antisera; alternatively, the isolation of the A2 clone may have been fortuitous. 2.4 kb - -A2 To investigate the significance of the truncated H19 mRNA repre •¿ sented by clone A2, we designed PCR primers that spanned exons 3 and 4 as well as introns 2-4 of the H19 gene. These primers were chosen to enable the assay to distinguish the desired product from that formed from contaminating genomic DNA in RT-PCR applications and to differentiate between the full-length mRNA for H19 and the exon 4-truncated form represented by clone A2. With these primers, the full-length H19 transcript gives a 297-bp product, whereas the truncated H19 form represented by clone A2 would give a 190-bp product (297 - 107). We found that the drug-resistant NCI-HI688 1.9kb- -18S and MCF-7/AdrVp cells overexpress the full-length H19 transcript rather than the truncated A2 species when compared with the MCF-7 and the MCF-7/AdrVp revenant cell lines (Fig. 3). The faint 190-bp product corresponding to the A2 cDNA is detectable to an equal Fig. 1. Northern analysis with A2 cDNA as a probe. Total cellular RNA (20 /ig) from extent in all cell lines (Fig. 3). Furthermore, NCI-H1688 cells display each cell line was separated on a 1.2% agarose gel, transferred to a polyvinylidene relatively higher expression of the full-length H19 mRNA than do difluoride membrane, and hybridized with the radiolabeled A2 cDNA probe. The blot was later stripped and rehybridized with a control 18S rRNA probe. Lane 1, MCF-7 cells; Lane MCF-7/AdrVp cells, consistent with the Northern blot pattern oíHI9 2, MCF-7/AdrVp cells; Lane 3. MCF-7/AdrVp revenant cells: Lane 4. NCI-HI688 cells. expression in these cell lines (Fig. 1). 2905

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Fig. 3. RT-PCR assay for H19 mRNA tran scripts. A, with primers for H19 described in "Ma terials and Methods": B. with j3-actin primers (9). Total RNA was isolated from each cell line. One fig cellular RNA was used in each RT reaction mixture, except in the control (No RNA). in which no RNA was added. Each resulting cDNA was B amplified with primer pairs, as described in "Ma terials and Methods". The RT and PCR conditions were as reported previously for determination of MRP and MDR1 expression by RT-PCR (9). A2, - 242 bp our cDNA A2; the PCR assay for A2 was done using the primers for HIV. PÌ4,plasmidcontaining a full-length cDNA of MDR1. The PCR reaction for PI4 used AfDR/-specific primers, which yielded a 190-bp PCR product, as displayed above (9). Product size was additionally determined with - 297 bp markers consisting of pGEM-3 DNA digested with - 190 bp «infl. foal, and Sin I and XI74 DNA digested with Hue\\\. respectively (Promega. Madison WI). The expected amplification product for ß-actinis 242 bp in length.

We have used the A2 cDNA as a probe to examine whether the HÌ9 and MCF-7/AdrVp cells is potentially significant as a marker and gene is overexpressed in other multidrug-resistant cells that do not possibly a mediator of the MDR phenotype associated with p95 overexpress p95. We have performed Northern blot analysis with overexpression. The HI 9 gene plays an important role in embryonal RNA from HL-60/Adr (7) and UMCC-1/VP (8) cells, two cell lines growth and differentiation: it is genomically imprinted and regulates that are amplified for and overexpress the MRP gene, and HL-60/Vinc the expression of insulin-like growth factor 2(11, 14-18). Although cells (7), which display MDR1 gene amplification and overexpress the activation of H19 in MCF-7/AdrVp cells could represent a loss of Pgp. These lines, and their parental drug-sensitive lines, did not imprinting, preliminary studies of the methylation of H19 in MCF-7 demonstrate detectable Hi 9 expression following 72 h of autoradio- and MCF-7/AdrVp DNA restricted with Msp\ and Hpall isozymes do graphie exposure (Fig. 4). In contrast, the NCI-HI688 control line was not reveal decreased methylation in the MCF-7/AdrVp subline (data strongly positive. These data suggest a connection between H19 gene not shown). H19 has been postulated to function as a tumor suppressor overexpression and the form of MDR associated with p95 overex- gene involved in the oncogenesis of Wilms' tumor and other pression. because of a highly selective loss of the active maternal alíeleof H19 This report is the first to link overexpression of the H19 gene with in tumors (19). Transfection of embryonal tumor cells with H19 MDR. Our observed overexpression of H19 in both the NCI-HI688 expression constructs has been shown directly to abrogate clonoge- nicity in soft agar and tumorigenicity in nude mice, suggesting a tumor suppressor activity of the H19 gene product (20). It is possible that the tumor suppressor function of HI 9 may induce the reverse- transformed phenotype described for certain multidrug-resistant cells (21). The induction of HI9 expression has been demonstrated in rat vascular smooth muscle cells after mechanical injury, suggesting that a similar induction might occur with exposure to cytotoxic drugs (22). To examine whether H19 expression in MCF-7/AdrVp cells might have been related to the concomitant exposure of the cells to vera pamil, we performed HÌ9Northern analysis of RNA from MCF-7 cells cultured for 4 or 8 weeks in 5 /XMverapamil (data not shown). No induction of H19 expression by verapamil was detectable, suggesting that exposure to the cytotoxic drug doxorubicin is responsible for relative H19 overexpression in the MCF-7/AdrVp subline.

2.4 kb The hypotheses that HÌ9expression confers drug resistance, in duces p95 expression, and confers a reverse-transformed phenotype in neoplastic cells are testable with cellular transfection studies using H19 expression constructs; these are currently in progress in our laboratories. In any event, study of the gross overexpression of a tumor suppressor gene regulated by genetic imprinting in a unique model of cancer MDR is likely to add to our knowledge of cancer drug resistance.

Acknowledgments Fig. 4. HÌ9Northern analysis with parental and multidrug-resistant sublines. using the A2 cDNA as a probe. H19 mRNA expression was examined in the human small cell lung We gratefully acknowledge the gift of cells and valuable discussions pro cancer cell line UMCC-1 and its etoposide-resistant subline UMCC-1/VP, which over- expresses MRP. Also tested were the human acute leukemia cell line HL-60. its resistant vided by Drs. Melvin Center, Adi Gazdar. Antonio Fojo, and Susan Bates. We sublines HL-60/Vinc and HL-60/Adr. which overexpress MDR1 and MRP. respectively, thank Florence Wade and Helen Spiker for expert help preparing the manu and HL-60/Vinc and HL-60/Adr revenant sublines. script. We also thank Dr. Nicholas Ambulos, Jr.. and Carol Robey (University 2906

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. HI9 OVEREXPRESSION IN MDR CELLS of Maryland Biopolymer Laboratory Shared Resource) for their assistance protein (MRP) mRNA in blast cells from acute myeloid leukemia (AML1 patients. with DNA sequencing. We thank Dr. Sunil Gupta. Pharmacia, and Upjohn. Leukemia (Baltimore), 10: 48-55, 19%. 10. Brannan. C. I.. Dees. E. C.. Ingram, R. S., and Tilghman, S. M. The product of the Inc. for their interest and partial support of these studies. H¡9gene may function as an RNA. Mol. Cell. Biol.. 10: 28-36, 1990. 11. Kondo, M., Suzuki, H., Ueda. R.. Osada. H., Takagi. K., Toshitada, T., and References Takahashi. T. Frequent loss of imprinting of the H19 gene is often associated with its overexpression in human lung cancers. Oncogene, IO: 1193-1198, 1995. 1. Chen, Y-N.. Mickley, L. A., Schwartz, A. M., Acton. E. M.. Hwang. J., and Fojo, 12. Pachnis, V.. Belayew, A., and Tilghman. S. M. A unlinked to o-fetoprotein A. T. Characterization of Adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein. J. Biol. under the control of the murine raf/Rif genes. Proc. Nati. Acad. Sci. USA, 81: Chem.. 265.- 10073-10080, 1990. 5523-5527, 1984. 13. Pachnis, V.. Brannan. C. I., and Tilghman. S. M. The structure and expression of a 2. Doyle, L. A., Ross, D. D., Sridhara, R., Fojo, A. T., Kaufmann, S. H., Lee. E. J., novel gene activated by early mouse embryogenesis. EMBO J.. 7: 673-681, 1988. and Schiffer, C. A. Expression of a 95 kDa membrane protein is associated with low daunorubicin accumulation in leukaemic blast cells. Br. J. Cancer 71: 52-58, 14. Li, E.. Beard. C., and Jaenisch. R. Role for DNA methylation in . Nature (Lond.). 366: 362-365. 1993. 1995. 3. Lee, J. S.. Dickstein. B., and Bates. S. E. Reduced drug accumulation without 15. Bartolomei. M. S., Webber, A. L.. Brunkow, M. E., and Tilghman, S. M. Epigenetic P-glycoprotein (Pgp) expression in MCF-7 AdrVp cells. Proc. Am. Assoc. Cancer mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev., 7: 1663-1673, 1993. Res.. 35: 2128. 1994. 4. Doyle. L. A., Kaufmann. S. H.. Fojo, A. T.. Bailey. C. L.. and Gazdar, A. F. A novel 16. Zemel, S., Bartolomei. M. S., and Tilghman, S. M. Physical linkage of two mam malian imprinted genes. HI9 and insulin-like growth factor 2. Nat. Genet.. 2: 61-65. 95 kilodalton membrane polypeptide associated with lung cancer drug resistance. Lung Cancer. 9: 317-326. 1993. 1992. 5. Doyle, L. A.. Gao, Y., Yang. W., and Ross. D. D. Characterization of a 95 kilodallon 17. Brunkow, M. E., and Tilghman, S. M. Ectopie expression of the HI9 gene in mice membrane glycoprotein associated with multi-drug resistance. Int. J. Cancer. 62. causes prenatal lethality. Genes Dev.. 5: 1092-1101. 1991. 593-598. 1995. 18. Leibovitch. M. P.. Solhonne, B., Guillier. M., Verelle. P.. and Leibovitch. S. A. Direct 6. Gazdar, A. F.. Steinberg. S. M.. Russell. E. K.. Linnoila. R. !.. Oie, H. K.. Ghosh, relationship between the expression of tumor suppressor HI9 mRNA and c-moj B. C., Cotelingen. J. D.. Johnson. B. E.. Minna. J. D.. and Ihde. D. C. Correlation of proto-oncogene during myogenesis. Oncogene. IO: 251-260. 1995. in vitro drug-sensitivity testing results with response to chemotherapy and survival in 19. Zhang, Y.. Shields. T., Crenshaw, T., Hao, Y., Moulton. T., and Tycko, B. Imprinting extensive-stage small cell lung cancer: a prospective clinical trial. J. Nati. Cancer of human HI9: -specific CpG methylation. loss of the active alíelein Wilms Inst., 82: 117-124. 1990. tumor and potential for somatic alíeleswitching. Am. J. Hum. Genet.. 53: 113-124, 7. Marquardt, D., McCrone, S., and Center. M. Mechanisms of mulinimi: resistance in 1993. HL60 cells: detection of resistance-associated proteins with antibodies against syn 20. Hao. Y.. Crenshaw, T.. Moulton. T., Newcomb. E.. and Tycko. B. Tumour-suppressor thetic peptides that correspond to the deduced sequence of P-glycoprotein. Cancer activity of HI9 RNA. Nature (Lond.), 365: 764-767. 1993. Res.. 50: 1246-1430. 1990. 21. Biedler. J. L.. Riehm. H.. Peterson. R. H.. and Spengler. B. A. Membrane-mediated 8. Doyle, L. A.. Ross, D. D.. Ordonez. J. V.. Yang. W.. Tong. Y.. Belani. C. P.. and drug resistance and phenotypic reversion to normal growth behavior of Chinese Gulheil, J. C. An etoposide-resistant lung cancer subline overexpresses the multidrug hamster cells. J. Nati. Cancer Inst.. 55: 671-680, 1975. resistance-associated protein. Br. J. Cancer. 72: 535-542. 1995. 22. Kim. D. K.. Zhang. L.. Dzau, V. J.. and Pratt. R. E. H19. a developmentally regulated 9. Ross. D. D., Doyle, L. A., Schiffer, C. A., Lee, E. J., Grant, C. E., Cole, S. P. C.. gene, is reexpressed in rat vascular smooth muscle cells after injury. J. Clin. Invest.. Deeley, R. G., Yang. W.. and Tong. Y. Expression of multidnig resistance-associated 93: 355-360, 1994.

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L. Austin Doyle, Weidong Yang, Arun K. Rishi, et al.

Cancer Res 1996;56:2904-2907.

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