MOLECULAR CARCINOGENESIS 4:297-307 (1991)
Isolation and Partial Characterization of a Transformation-Associated Sequence From Human Nasopharyngeal Carcinoma Ya Cao, Yi Sun, Sylvie Poirier, Dolores Winterstein, Glenn Hegamyer, John Seed, Sacha Malin, and Nancy H. Colburn' Laboratoryof Viral Carcinogenesis (YC, Se GH, JS, SM, NHC) and Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp (YS, DW), National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland A transforming activity associated with Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 DNA has been identified by transfer into nontransformed promotion-sensitive mouse JB6(P+) C141 cells. To clone this transformation-associated sequence, w e carried out three cycles of transfection, followed by cloning of anchorageindependent transformants in soft agar. A tertiary CNE/JB6 clonal transfectant cell line 625 whose DNA showed transforming activity, as indicated in b o t h soft-agar assay and nude-mice implantation, was used to make a genomic library in t h e vector A dash. Using the human repeated sequence Blur 8 to screen t h e library, w e obtained 10 human Alu-positive clones. A cloned Alu-positive insert of 16 kbp, CNE 323, was characterized in detail. CNE 323 transferred moderate transforming activity when introduced into JB6 P+ cells and showed no homology to Ha-, Ki-, or N-ras genes; human promotion sensitivity genes; src, myb,jun, myc, fos, r a t or int-2 oncogenes; or epidermal growth factor receptor. The isolated CNE 323 DNA sequence appeared to preserve t h e genomic structure o f t h e original sequence found in CNEz cells and in nude mouse tumors induced b y CNE2cells or by CNE/JB6 transfectant cells, indicating t h a t t h e cloned NPC sequence was activated during NPC carcinogenesis and not during transfection or construction of t h e library, and t h a t t h e cloned sequence or a larger sequence of which it was part played a role in t u m o r formation. Finally, w e identified a 1.3-kb mRNA t h a t hybridizes to a subclone of t h e 16-kb NPC sequence in CNE2 cell poly (A)+ RNA. Key words: Human nasopharyngeal carcinoma, oncogene, JB6 cell transfection assay INTRODUCTION Nasopharyngeal carcinoma (NPC) is a common disease in Southern China and some other areas of the world [I-21. In addition to the well-established association of NPC with Epstein-Barr virus (EBV) (3-51, certain dietary and environmental factors have been associated with NPC [6-91. It has been proposed (3,6-91 that initiation of NPC requires EBV expression, but induction of preneoplastic events and maintenance of tumor cell phenotype require critical cellular genes. The DNA of Chinese NPC cell line CNEz has shown two biological activities on transfection into mouse JB6 cells. The first is sensitivity to TPA-induced promotion of neoplastic transformation promotion-sensitive (P') detected in JB6 promotion-insensitive(P-) recipients) [lo].The DNA sequences in CNE2 cells that show homology to mouse pro-1 and that transfer susceptibility to tumor promoterinduced neoplastic transformation to mouse JB6 P - cells have been cloned and characterized [I1,I21. The second biological activity is transforming activity (Tx) detected in 186 P + recipients (13,141. Since CNEz cellular DNA harbors both P+ and Tx sequences, the NPC model offers the possibility of studying, in a human system, cooperation between promotion sensitivity genes and transforming genes. The present report describes the molecular cloning of a human CNE2 transformation-associated sePUBLISHED 1991 WILEY-LISS, INC.
quence. The data indicate that this NPC sequence is different from a number of known oncogenes and mouse and human pro-I sequences. MATERIALS AND METHODS Reagents Restriction enzymes were obtained from Boehringer Mannheim (Indianapolis, IN), Bethesda Research Laboratory (Gaithersburg, MD), and New England Biolabs (Commack, NY). T4 DNA ligase, cloning vector A dash, Gigapack Gold packaging extract, and the p2392 Escherichia cob cells (selection cells) were obtained from Stratagene Inc. (La Jolla, CA). LipofectinTM reagent was supplied by BRL. The oligolabeling kit was purchased from Pharmacia LKB Nuclear (Gaithersburg,MD). BA85 nitrocellulose paper was obtained from Schleicher & Schuell, Inc. (Keene, NH). [32PldCTPwas supplied by Amersham Corp. (Arlington Heights, IL). 'Corresponding author: Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201. Abbreviations: EBV, Epstein-Barr virus; EDTA. ethyleneciaminetetraacetic acid; EGF, epidermal growth factor; FBS, fetal bovine serum; LMP. latent membrane protein; LTR, long terminal repeat; NPC, nasopharyngeal carcinoma; P+, promotion sensitive; P-, promotion insensitive; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; Tx, transforming activity.
298
CAO ETAL.
Oncogene Clones pc-fos (human)-1, pHEl (human c-raf-I), and phSR1 (human c-myc) were purchased from the American Type Culture Collection (Rockville, MD); pbxepidermal growth factor (EGF) receptor was a gift from Dr. J. Schlessinger (New York University Medical Center) I151; pKC 3.2 (int-2) was a gift from Dr. Clive Dickson (Imperial Cancer Research Fund Laboratories, London, England) [I61; RSV-c-jun was a gift from Dr. Michael Karin (University of California, San Diego) [17]; pHA15d (human Alu Blur 2 and 8) was a gift from Dr. Pete Rogan (BRI, NCI-FCRDC) [18]; pSVZne0 was a gift from Southern and Berg [19l,and BNLF-1 (LMP) was a gift from Dr. Bill Sagden (Univ. of Wisconsin) [30,31]. Cell Lines Two Chinese NPC cell lines were established from two different patients and designated CNEl and CNE2 [ZO]. Both lines were maintained by weekly passages in basal medium Eagle’s (BME) containing 7.5% each of fetal bovine serum (FBS) and newborn calf serum. The average population doubling time for CNE, and CNE2cells was 19 and 21 h, respectively. Each of these cell lines gives rise to squamous cell carcinomas when injected into nude mice. Screening of CNE, and CNE2cells for the presence of EBV nuclear antigens gave negative results (E. Kieff and N. Colburn, data not shown). A CNEz cell line cloned from soft agar, CNE2 A1 5 (passage 6), was the DNA source for cloning the transforming gene. The JB6 clone 41 (186 C141) cell line, derived originally from a primary mouse epidermal culture, was grown on BME containing 5% FBS as described previously [21,22]. JB6 C141 5A was obtained by nonselective cloning from the JB6 C141 cell line that shows P + activity after exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA). JB6 C141 5A was used as the recipient cell for transfection. Assay for Transforming Activity of DNA The JB6 C141 system was used to detect transforming sequences as described by Colburn et at. [13]. Briefly, CNE2 A1 5 genomic DNA (1 5 pg) was sheared and transfected as a calcium phosphate precipitate into 2 x l o 5JB6 C141 5A cells, which were then subcultured 1 d after transfection. Three days later, 5 x 1O4 cells or 2.4 x 1O4 cells were suspended in 0.33% agar containing 5% FBS in 100-mm dishes or 60-mm dishes, respectively. Colonies of greater than eight cells were scored after 12 d. Values for negative controls (no DNA or recipient-cell DNA) were subtracted to give the anchorage-independent colony number. The largest colonies were cloned from agar and grown as transfectant clonal cell lines. The DNA from these clones was then extracted and purified for the next round of transfection assay. CNE 323 or its cloned 16-kb insert was cotransfected with pSVZfleOinto JB6 C141 recipient cells with lipofectin as recommended by the supplier, using recipient cell DNA as carrier. Neo-resistant cells were selected by growth in 5% Eagle’s minimum essential medium (EMEM) containing G418 (500 pg/mL) for 2 w k with the
medium changed every 3 d. 3.6 x 1O4 resistant cells were seeded in 0.33% agar to test their ability to grow anchorage independently as described above. Assay for Neoplastically Transformed Phenotype To test the transfectantclonal cell lines for transformed phe notype,2 x 104cellsfrom passages3and4weresuspended in 0.33% agar containing 10% FBS in a 60-mm dish. After 14 d, colonies of greater than eight cells were counted. Construction and Screening of t h e CNE2/JB6 Genomic Library A genomic library was constructed from tertiary transfectant clonal cell line 625 DNA. The DNA was partially digested with Mbol, and fragments of 9-22 kb were isolated by centrifugation through sucrose gradients. A dash vector arms (1 0 pg) were digested with Xhol, purified, partially filled in with dTTPand dCTe and ligated to the9-22-kb fraction of 625 transfectant DNA partially filled in with dGTP and dATP using T4 DNA ligase (a Sall restriction site was therefore generated, which facilitated the separation of inserts from the vector). The ligation mixture was packagedusing Gigapack Gold packaging extract (Stratagene Inc.) and selected by transformation of p2392 E. colicells. The total unamplified library size was 8 x l o 5 recombinant phages. After amplification by the plate lysates procedure, the library titer was 1.4 x 10” pfu/mL. The probe used for screening the library was a Blur 8 0.3-kb human repeatsequence insert [I81; the plaque hybridization and washes were done at high stringency (68°C for hybridization, 52°C inO.1 x standardsalinecitrate(SSC)/O.lO/O sodiumdodecyl sulfate (SDS) for washes) on duplicate filters. Restriction and Hybridization Mapping of CNE 323 A human Ah-positive clone, designated CNE 323, was digested with Sall to completion. The 16-kb insert was gel purified and then mapped by cutting with various restriction enzymes or pairs of enzymes. Restriction sites were assigned based upon the agarose-gel banding pattern of the enzyme digests. To more accurately define the restriction sites, the insert was end labeled with 32P using polynucleotide kinase, cut with restriction enzymes, and run on an agarose gel that was dried, and exposed directly to x-ray film. This provided us with the accurate location of the end sites for each enzyme used. A human Alu probe (Blur 8) was used to detect the Ah-positive fragment by Southern hybridization. High-Molecular-Weight Genomic DNA Purification and Southern Blot Analysis High-molecular-weight genomic DNA from cultured cells and nude mice tumors (2-3 pooled) was isolated by proteinase K digestion, phenol extraction, and ethanol precipitation as previously described (231. The resulting DNA was further purified through a cesium chloride gradient. Briefly, solid CsCl (1 g/mL) and ethidium bromide (2 mg) were added to TE buffer (10 rnM Tris-HCI, pH 8.0, 1 m M ethylenediaminetetraacetic acid (EDTA) containing DNA. The solution was centrifuged at 45000 rpm for 36 h at 15°C
299
A TRANSFORMATION-ASSOCIATEDSEQUENCE IN NPC
in a 50 Ti rotor. The DNA was collected as a viscous band by puncturing the side of the tube with a needle. Ethidium bromide was removed by several extractions with an equal volume of CsCI-saturated isopropanol, followed by exhaustive dialysis against TE buffer. The DNA was then precipitated with sodium acetate and ethanol and dissolved in TE buffer for Southern analysis. For Southern blot analysis, the oncogene plasmid DNA (quantity adjusted to yield 2 p g of insert) or genomic DNA (20 pg) was digested with various restriction enzymes to completion, electrophoresed on an 0.8% agarose gel, and transferred to nitrocellulose filters. Oncogene blots were then hybridized to either a 16-kb Sall insert from the CNE 323 A dash clone or a 3.2-kb EcoRl human Alu fragment (Blur 2 and 8) [ 181that was labeled with [32P]dCTPby random priming. Two human Ah-negative internal fragments (a 2.8-kb EcoRl fragment and a 3.0-kb EcoRI/Xhol fragment) from CNEz clone 323 were hybridized to genomic blots after labeling by random priming. All hybridizations (usingprobesat2-5 x 108cpm/pg,0.5-2 x 106cpm/mL) were carried out at 68°C in 5 x SSC, 5 x Denhardt's sohtion, and 50 mg/mL salmon sperm DNA for 16-20 h. Posthybridization washes were performed at 52°C in 0.1 x SSC/O.l YO SDS for high stringency and at 52°C in 5 x SSC/O.l YO SDS for low stringency. Filters were exposed to Kodak XAR-5 film using intensifying screens at - 70°C. RESULTS Detection of CNEz DNA Transforming Activity in JB6 P' Mouse Epidermal Cells but n o t in NIH 3T3 Cells The DNA of a variety of tumors contains oncogenes that can transform NIH 3T3 cells in culture into neoplastic cells. Many of these genes identified by the NIH 3T3 focus assay are rasfamily genes [e.g., see refs. 24-27], We addressed at the outset whether the CNE-cell DNA contains rastransforming activity as indicated by the NIH 3T3 focus assay. After testing at least 100 p g of DNA transfected at 20 pg/dish, no foci were observed for DNAs from CNE1, CNE2, CNEz A1 5 (a CNE2 clonal cell line), or two primary CNE2/JB6 transfectants [141. Control DNAs from a colon carcinoma DNA-NIH transfectant cell line (SW480-NIH) harboring Ki-ras (251and the DNA from a human tumor DNANIH transfectant cell line (MT181 -NIH) 1261 harboring activated N-ras produced 5-1 0 foci per 100 p g DNA. These results suggested that CNE DNA transforming activity cannot be detected in the NIH 3T3 focus assay. Whether detection could be achieved using NIH 3T3 recipients and an anchorage-independence or tumorigenicity endpoint is unknown. Using JB6 C141 P' cells as recipients, we successfully detected CNE DNA transforming activity. As shown in Table 1, CNE, and CNE2DNA can transfer anchorage-independent growth to preneoplastic JB6 C141 P+ cells. This degree of transformation is comparable to that seen after transfection of DNA from TPA-transformed mouse 186 cell lines [ 131. In contrast with NPC DNA, 186 C141-recipient DNA and human placental DNA (data not shown) did not show transforming activity in this assay.
Table 1. Transfer o f Anchorage-Independent Transforming Activity by Human CNE Cell DNAs* DNA transferred into C141P+ Origin of Cells DNA source CNEl CNEz CNEZ A1 5 C141
Differentiated EBV-NPC cell line Differentiated EBV-NPC cell line Clonal cell line of CNEz Mouse epidermal JB6 preneoplasticcell line
Anchorageindependent colonies/ 5 x 1o4 cells 23
?
3
21 f 2 20
?
6
Isolation and Partial Characterization of a Transformation-Associated Sequence From Human Nasopharyngeal Carcinoma Ya Cao, Yi Sun, Sylvie Poirier, Dolores Winterstein, Glenn Hegamyer, John Seed, Sacha Malin, and Nancy H. Colburn' Laboratoryof Viral Carcinogenesis (YC, Se GH, JS, SM, NHC) and Biological Carcinogenesis and Development Program, Program Resources, Inc./DynCorp (YS, DW), National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland A transforming activity associated with Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 DNA has been identified by transfer into nontransformed promotion-sensitive mouse JB6(P+) C141 cells. To clone this transformation-associated sequence, w e carried out three cycles of transfection, followed by cloning of anchorageindependent transformants in soft agar. A tertiary CNE/JB6 clonal transfectant cell line 625 whose DNA showed transforming activity, as indicated in b o t h soft-agar assay and nude-mice implantation, was used to make a genomic library in t h e vector A dash. Using the human repeated sequence Blur 8 to screen t h e library, w e obtained 10 human Alu-positive clones. A cloned Alu-positive insert of 16 kbp, CNE 323, was characterized in detail. CNE 323 transferred moderate transforming activity when introduced into JB6 P+ cells and showed no homology to Ha-, Ki-, or N-ras genes; human promotion sensitivity genes; src, myb,jun, myc, fos, r a t or int-2 oncogenes; or epidermal growth factor receptor. The isolated CNE 323 DNA sequence appeared to preserve t h e genomic structure o f t h e original sequence found in CNEz cells and in nude mouse tumors induced b y CNE2cells or by CNE/JB6 transfectant cells, indicating t h a t t h e cloned NPC sequence was activated during NPC carcinogenesis and not during transfection or construction of t h e library, and t h a t t h e cloned sequence or a larger sequence of which it was part played a role in t u m o r formation. Finally, w e identified a 1.3-kb mRNA t h a t hybridizes to a subclone of t h e 16-kb NPC sequence in CNE2 cell poly (A)+ RNA. Key words: Human nasopharyngeal carcinoma, oncogene, JB6 cell transfection assay INTRODUCTION Nasopharyngeal carcinoma (NPC) is a common disease in Southern China and some other areas of the world [I-21. In addition to the well-established association of NPC with Epstein-Barr virus (EBV) (3-51, certain dietary and environmental factors have been associated with NPC [6-91. It has been proposed (3,6-91 that initiation of NPC requires EBV expression, but induction of preneoplastic events and maintenance of tumor cell phenotype require critical cellular genes. The DNA of Chinese NPC cell line CNEz has shown two biological activities on transfection into mouse JB6 cells. The first is sensitivity to TPA-induced promotion of neoplastic transformation promotion-sensitive (P') detected in JB6 promotion-insensitive(P-) recipients) [lo].The DNA sequences in CNE2 cells that show homology to mouse pro-1 and that transfer susceptibility to tumor promoterinduced neoplastic transformation to mouse JB6 P - cells have been cloned and characterized [I1,I21. The second biological activity is transforming activity (Tx) detected in 186 P + recipients (13,141. Since CNEz cellular DNA harbors both P+ and Tx sequences, the NPC model offers the possibility of studying, in a human system, cooperation between promotion sensitivity genes and transforming genes. The present report describes the molecular cloning of a human CNE2 transformation-associated sePUBLISHED 1991 WILEY-LISS, INC.
quence. The data indicate that this NPC sequence is different from a number of known oncogenes and mouse and human pro-I sequences. MATERIALS AND METHODS Reagents Restriction enzymes were obtained from Boehringer Mannheim (Indianapolis, IN), Bethesda Research Laboratory (Gaithersburg, MD), and New England Biolabs (Commack, NY). T4 DNA ligase, cloning vector A dash, Gigapack Gold packaging extract, and the p2392 Escherichia cob cells (selection cells) were obtained from Stratagene Inc. (La Jolla, CA). LipofectinTM reagent was supplied by BRL. The oligolabeling kit was purchased from Pharmacia LKB Nuclear (Gaithersburg,MD). BA85 nitrocellulose paper was obtained from Schleicher & Schuell, Inc. (Keene, NH). [32PldCTPwas supplied by Amersham Corp. (Arlington Heights, IL). 'Corresponding author: Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201. Abbreviations: EBV, Epstein-Barr virus; EDTA. ethyleneciaminetetraacetic acid; EGF, epidermal growth factor; FBS, fetal bovine serum; LMP. latent membrane protein; LTR, long terminal repeat; NPC, nasopharyngeal carcinoma; P+, promotion sensitive; P-, promotion insensitive; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; Tx, transforming activity.
298
CAO ETAL.
Oncogene Clones pc-fos (human)-1, pHEl (human c-raf-I), and phSR1 (human c-myc) were purchased from the American Type Culture Collection (Rockville, MD); pbxepidermal growth factor (EGF) receptor was a gift from Dr. J. Schlessinger (New York University Medical Center) I151; pKC 3.2 (int-2) was a gift from Dr. Clive Dickson (Imperial Cancer Research Fund Laboratories, London, England) [I61; RSV-c-jun was a gift from Dr. Michael Karin (University of California, San Diego) [17]; pHA15d (human Alu Blur 2 and 8) was a gift from Dr. Pete Rogan (BRI, NCI-FCRDC) [18]; pSVZne0 was a gift from Southern and Berg [19l,and BNLF-1 (LMP) was a gift from Dr. Bill Sagden (Univ. of Wisconsin) [30,31]. Cell Lines Two Chinese NPC cell lines were established from two different patients and designated CNEl and CNE2 [ZO]. Both lines were maintained by weekly passages in basal medium Eagle’s (BME) containing 7.5% each of fetal bovine serum (FBS) and newborn calf serum. The average population doubling time for CNE, and CNE2cells was 19 and 21 h, respectively. Each of these cell lines gives rise to squamous cell carcinomas when injected into nude mice. Screening of CNE, and CNE2cells for the presence of EBV nuclear antigens gave negative results (E. Kieff and N. Colburn, data not shown). A CNEz cell line cloned from soft agar, CNE2 A1 5 (passage 6), was the DNA source for cloning the transforming gene. The JB6 clone 41 (186 C141) cell line, derived originally from a primary mouse epidermal culture, was grown on BME containing 5% FBS as described previously [21,22]. JB6 C141 5A was obtained by nonselective cloning from the JB6 C141 cell line that shows P + activity after exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA). JB6 C141 5A was used as the recipient cell for transfection. Assay for Transforming Activity of DNA The JB6 C141 system was used to detect transforming sequences as described by Colburn et at. [13]. Briefly, CNE2 A1 5 genomic DNA (1 5 pg) was sheared and transfected as a calcium phosphate precipitate into 2 x l o 5JB6 C141 5A cells, which were then subcultured 1 d after transfection. Three days later, 5 x 1O4 cells or 2.4 x 1O4 cells were suspended in 0.33% agar containing 5% FBS in 100-mm dishes or 60-mm dishes, respectively. Colonies of greater than eight cells were scored after 12 d. Values for negative controls (no DNA or recipient-cell DNA) were subtracted to give the anchorage-independent colony number. The largest colonies were cloned from agar and grown as transfectant clonal cell lines. The DNA from these clones was then extracted and purified for the next round of transfection assay. CNE 323 or its cloned 16-kb insert was cotransfected with pSVZfleOinto JB6 C141 recipient cells with lipofectin as recommended by the supplier, using recipient cell DNA as carrier. Neo-resistant cells were selected by growth in 5% Eagle’s minimum essential medium (EMEM) containing G418 (500 pg/mL) for 2 w k with the
medium changed every 3 d. 3.6 x 1O4 resistant cells were seeded in 0.33% agar to test their ability to grow anchorage independently as described above. Assay for Neoplastically Transformed Phenotype To test the transfectantclonal cell lines for transformed phe notype,2 x 104cellsfrom passages3and4weresuspended in 0.33% agar containing 10% FBS in a 60-mm dish. After 14 d, colonies of greater than eight cells were counted. Construction and Screening of t h e CNE2/JB6 Genomic Library A genomic library was constructed from tertiary transfectant clonal cell line 625 DNA. The DNA was partially digested with Mbol, and fragments of 9-22 kb were isolated by centrifugation through sucrose gradients. A dash vector arms (1 0 pg) were digested with Xhol, purified, partially filled in with dTTPand dCTe and ligated to the9-22-kb fraction of 625 transfectant DNA partially filled in with dGTP and dATP using T4 DNA ligase (a Sall restriction site was therefore generated, which facilitated the separation of inserts from the vector). The ligation mixture was packagedusing Gigapack Gold packaging extract (Stratagene Inc.) and selected by transformation of p2392 E. colicells. The total unamplified library size was 8 x l o 5 recombinant phages. After amplification by the plate lysates procedure, the library titer was 1.4 x 10” pfu/mL. The probe used for screening the library was a Blur 8 0.3-kb human repeatsequence insert [I81; the plaque hybridization and washes were done at high stringency (68°C for hybridization, 52°C inO.1 x standardsalinecitrate(SSC)/O.lO/O sodiumdodecyl sulfate (SDS) for washes) on duplicate filters. Restriction and Hybridization Mapping of CNE 323 A human Ah-positive clone, designated CNE 323, was digested with Sall to completion. The 16-kb insert was gel purified and then mapped by cutting with various restriction enzymes or pairs of enzymes. Restriction sites were assigned based upon the agarose-gel banding pattern of the enzyme digests. To more accurately define the restriction sites, the insert was end labeled with 32P using polynucleotide kinase, cut with restriction enzymes, and run on an agarose gel that was dried, and exposed directly to x-ray film. This provided us with the accurate location of the end sites for each enzyme used. A human Alu probe (Blur 8) was used to detect the Ah-positive fragment by Southern hybridization. High-Molecular-Weight Genomic DNA Purification and Southern Blot Analysis High-molecular-weight genomic DNA from cultured cells and nude mice tumors (2-3 pooled) was isolated by proteinase K digestion, phenol extraction, and ethanol precipitation as previously described (231. The resulting DNA was further purified through a cesium chloride gradient. Briefly, solid CsCl (1 g/mL) and ethidium bromide (2 mg) were added to TE buffer (10 rnM Tris-HCI, pH 8.0, 1 m M ethylenediaminetetraacetic acid (EDTA) containing DNA. The solution was centrifuged at 45000 rpm for 36 h at 15°C
299
A TRANSFORMATION-ASSOCIATEDSEQUENCE IN NPC
in a 50 Ti rotor. The DNA was collected as a viscous band by puncturing the side of the tube with a needle. Ethidium bromide was removed by several extractions with an equal volume of CsCI-saturated isopropanol, followed by exhaustive dialysis against TE buffer. The DNA was then precipitated with sodium acetate and ethanol and dissolved in TE buffer for Southern analysis. For Southern blot analysis, the oncogene plasmid DNA (quantity adjusted to yield 2 p g of insert) or genomic DNA (20 pg) was digested with various restriction enzymes to completion, electrophoresed on an 0.8% agarose gel, and transferred to nitrocellulose filters. Oncogene blots were then hybridized to either a 16-kb Sall insert from the CNE 323 A dash clone or a 3.2-kb EcoRl human Alu fragment (Blur 2 and 8) [ 181that was labeled with [32P]dCTPby random priming. Two human Ah-negative internal fragments (a 2.8-kb EcoRl fragment and a 3.0-kb EcoRI/Xhol fragment) from CNEz clone 323 were hybridized to genomic blots after labeling by random priming. All hybridizations (usingprobesat2-5 x 108cpm/pg,0.5-2 x 106cpm/mL) were carried out at 68°C in 5 x SSC, 5 x Denhardt's sohtion, and 50 mg/mL salmon sperm DNA for 16-20 h. Posthybridization washes were performed at 52°C in 0.1 x SSC/O.l YO SDS for high stringency and at 52°C in 5 x SSC/O.l YO SDS for low stringency. Filters were exposed to Kodak XAR-5 film using intensifying screens at - 70°C. RESULTS Detection of CNEz DNA Transforming Activity in JB6 P' Mouse Epidermal Cells but n o t in NIH 3T3 Cells The DNA of a variety of tumors contains oncogenes that can transform NIH 3T3 cells in culture into neoplastic cells. Many of these genes identified by the NIH 3T3 focus assay are rasfamily genes [e.g., see refs. 24-27], We addressed at the outset whether the CNE-cell DNA contains rastransforming activity as indicated by the NIH 3T3 focus assay. After testing at least 100 p g of DNA transfected at 20 pg/dish, no foci were observed for DNAs from CNE1, CNE2, CNEz A1 5 (a CNE2 clonal cell line), or two primary CNE2/JB6 transfectants [141. Control DNAs from a colon carcinoma DNA-NIH transfectant cell line (SW480-NIH) harboring Ki-ras (251and the DNA from a human tumor DNANIH transfectant cell line (MT181 -NIH) 1261 harboring activated N-ras produced 5-1 0 foci per 100 p g DNA. These results suggested that CNE DNA transforming activity cannot be detected in the NIH 3T3 focus assay. Whether detection could be achieved using NIH 3T3 recipients and an anchorage-independence or tumorigenicity endpoint is unknown. Using JB6 C141 P' cells as recipients, we successfully detected CNE DNA transforming activity. As shown in Table 1, CNE, and CNE2DNA can transfer anchorage-independent growth to preneoplastic JB6 C141 P+ cells. This degree of transformation is comparable to that seen after transfection of DNA from TPA-transformed mouse 186 cell lines [ 131. In contrast with NPC DNA, 186 C141-recipient DNA and human placental DNA (data not shown) did not show transforming activity in this assay.
Table 1. Transfer o f Anchorage-Independent Transforming Activity by Human CNE Cell DNAs* DNA transferred into C141P+ Origin of Cells DNA source CNEl CNEz CNEZ A1 5 C141
Differentiated EBV-NPC cell line Differentiated EBV-NPC cell line Clonal cell line of CNEz Mouse epidermal JB6 preneoplasticcell line
Anchorageindependent colonies/ 5 x 1o4 cells 23
?
3
21 f 2 20
?
6
