[CANCER RESEARCH 46, 6174-6179. December 1986] Implication of Chromosome 11 in the Suppression of Neoplastic Expression in Human Cell Hybrids1

Eri S. Srivatsan, William F. Benedict, and Eric J. Stanbridge2

Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92717 [E. S. S., E. J, SJ, and Division of Hematology-Oncology, Children 's Hospital of Los Angeles, Los Angeles, California 90027 [W. F. B.J

ABSTRACT cies hybrids. Intraspecies hybrids, most notably human-human cell hybrids, are much more stable (9) and have been used to Cytogenetic analyses of intraspecies human HeLa x fibroblast hybrid identify the loss of certain specific chromosomes that correlate cell populations have provided tentative evidence for the correlation of with reexpression of tumorigenicity in malignant x normal loss of a single copy of chromosomes 11 and 14 with reexpression of tumorigenicity. In this study paired combinations of nontumorigenic and human cell hybrids (8). tumorigenic segregant HeLa x fibroblast hybrid cells from two inde In our previous studies (8), as well as those of others (2), pendent fusion events were examined for the presence or absence of detailed cytogenetic analyses of paired combinations of sup normal chromosomes 11 and 14. In human hybrid cell lines the parental pressed and tumorigenic segregant HeLa x human fibroblast origin of chromosomes can be distinguished on the basis of restriction cell populations indicated that chromsome 11 and possibly fragment length polymorphisms. Genes for c-Ha-ros, insulin, and apoli- other chromosomes, in particular chromosome 14, were impli poprotein A-l on chromosome 11 and a polymorphic locus AVVIDI on cated in the control of tumorigenic expression. Unfortunately, chromosome 14 were used as Southern hybridization probes. Analysis of it is not possible by conventional cytogenetics to determine the DNA from the parental fibroblast and HeLa cell lines and their nontu parental origin of the specific chromosomes that were missing morigenic and tumorigenic hybrids showed the loss of a fibroblast chro in the tumorigenic segregants. mosome 11 in four of the tumorigenic segregants and a HeLa chromosome The availability of chromosome-specific RFLP1 probes (10) 11 in a fifth hybrid cell line. This latter segregant has, interestingly, also lost a copy of chromosome 14 of fibroblast origin. There was no obvious has now made it possible to identify specific chromosomes from correlation of loss of a copy of normal chromosome 14 and reexpression different individuals and, thereby, the presence or absence of of tumorigenicity in any of the other hybrid cell populations. such chromosomes in somatic cell hybrids. We have used this Our conclusion from these observations is that gene(s) that map to approach to analyze a series of intraspecific HeLa x fibroblast normal chromosome 11 might be involved in control of tumorigenic human cell hybrids using RFLP probes specific for chromo expression in these human hybrid cells. somes 11 and 14.

INTRODUCTION MATERIALS AND METHODS Somatic cell hybrids, generated by fusing together two or more different cells of the same or different species, have been Cell Lines and Plasmids. The parental cell lines and the tumorigenic and nontumorigenic hybrids derived from fibroblast and HeLa fusions extremely useful in the study of the genetic analysis of malig nancy (reviewed in Refs. 1-3). In these studies the general are presented in Table 1. All cell lines were tested for tumorigenic potential at the time of the RFLP analysis studies. CGL series of approach has been to fuse a cancer cell with a normal cell and hybrids and the ESH5 series of hybrids were all derived from a common determine whether or not the resulting hybrid is tumorigenic. ancestral clone of HeLa x fibroblast, but each of the tumorigenic hybrid Both intraspecies and interspecies cell hybrids have been used clones represents an independent segregational event. For those cell (4-7). The general conclusion from these somatic cell hybrid lines where a detailed cytogenetic analysis was performed (8), cell studies is that tumorigenicity is initially suppressed in malig populations which had been frozen down at the time of that analysis nant x normal cell hybrids, and tumorigenic segregants arise were reconstituted and used within a few population doublings for the after varying intervals of time. The appearance of tumorigenic studies reported here. Identical hybrid-specific chromosome markers segregants is usually correlated with loss of chromosomes from were found in metaphases from both ESH39E and ESH39L, confirm ing that this tumorigenic segregant was derived from the nontumori the hybrid cell population in question (5, 7, 8). genic hybrid parent (data not shown). Fibroblast cells were grown in The most reasonable explanation for suppression of tumori Dulbecco's modified Eagle's medium containing nonessential amino genicity in somatic cell hybrids is that gene(s) located on chro- acids and 10% fetal calf serum. HeLa cells and the hybrid cell lines mosome(s) of the normal parental cell are able to control were grown in minimal essential medium containing nonessential tumorigenic behavior of the malignant cell when introduced amino acids and 10% fetal calf serum. into that cell via cell fusion. Similarly, reexpression of tumori Four RFLP probes were used in this study (Table 2). Three of the genicity would presumably result from loss of those specific probes were complementary to DNA sequences found on chromosome normal chromosome(s) from the hybrid cell via chromosome 11: a 6.6-kilobase fragment containing the c-Ha-ras-1 gene, which is nondisjunction or some other segregating event. Identification the cellular homologue of v-Ha-roj (11); phins310, which contains the of these normal "tumor-suppressor" chromosomes, however, 0.88-kilobase human insulin gene ligated to pBR322 at the Pvull site has not been easy. In interspecies human-rodent cell hybrids, (12); and pSV2-gpt-A-l, which contains the 2.2-kilobase human apo- lipoprotein A-l gene ligated to PSV2-gpt at the Pstl site (13). The where parental chromosomes would be most easily identified, fourth probe, pAWlOl, is a plasmili containing a 16-kilobase DNA suppression of tumorigenicity is usually only transient (2), due, fragment ligated to pBR322 at the EcoRl site (14). A function for this in large part, to the chromosomal instability of these interspe- DNA fragment has not yet been ascertained. This probe is specific for chromosome 14. Received 3/5/86; revised 6/11/86, 8/25/86; accepted 9/3/86. A fourth chromosome 11-specific probe, pHBl, which contains the 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 4.4-kilobase human /?-globin gene ligated to pBR322 at the Pstl site accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by NIH Grant CA-19401, and Grant 1475 from the Council for 3The abbreviations used are: RFLP, restriction fragment length polymor Tobacco Research. phism; SET buffer, 150 mM Tris-HCl (pH 8.0):5 HIMEDTA:750 mM NaCl; SDS, 1To whom requests for reprints should be addressed. sodium dodecyl sulfate. 6174 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. CHROMOSOME 11 AND CONTROL OF TUMORIGENICITY

(15), was used in an initial screen, but informative RFLP data were not (Fig. \A). We interpret from these data that HeLa is ho- forthcoming (data not shown), and this probe was not studied further. moyzgous and the two fibroblast parents heterozygous for the Plasm ids were grown in Escherichia coli strain HB101 (recA), am c-Ha-ras alíeleunder these particular restriction endonuclease plified by chloramphenicol and purified by ethidium bromide-CsCl digestion conditions. Thus, the discriminating RFLP fragments density gradent centrifugation (16). Cellular DNAs were prepared here are the GM-77-specific, 4.0- and 2.5-kilobase bands and according to Jolly et al. (17). Nick translation of the probe DNA was IMR-90-specific, 2.5-kilobase band relative to HeLa. The HeLa performed according to Feinberg and Vogelstein ( 18). 3.0-kilobase fragment is unique in crosses with fibroblast GM- Blot Hybridization Analysis. Genomic DNA from each parental cell 77, but is shared with fibroblast IMR-90. The 2.3-kilobase line and the hybrid cell lines was digested with the restriction enzymes Tail, Pvull, Pstl, or EcoRl according to the RFLP being analyzed fragment is common to all three parental cell lines. Other minor (Table 2). Each DNA sample was digested in a buffer containing 33 common bands are present but uninformative. The insulin mM Tris-HCl (pH 7.8), 60 mM potassium acetate, 10 mM MgCl, and probe, another chromosome 11 RFLP probe used in this anal 1 mM dithiothreitol at 37°Covernight for all the enzymes except Taq\. ysis, hybridized to a single fragment 2.3 kilobases in size in The Taq\ digestion was conducted in the same manner but performed Southern blots of the fibroblast DNAs and to a 0.75-kilobase at 65°Covernight. Digested DNAs were then mixed with 5% glycerol fragment of HeLa DNA (Fig. \B). These DNAs were digested and bromophenol blue (0.5 jig/ml) and loaded onto 0.8% agarose gels. with Pvull. Electrophoresis was carried out in Tris acetate buffer (pH 7.85) [40 The chromosome 14-specific probe, pAWlOl, hybridized to mM Tris acetate (pH 7.85): 1 mM EDTA] at 0.25 mA/cm for 8 h. DNA fragments of 20 and 17 kilobases in size with fibroblast GM- fragments were then transferred onto nitrocellulose filters by the method of Southern (19). Filters were hybridized to heat-denatured 77 DNA, 18.5 and 17 kilobases in size with fibroblast IMR-90 32P-labeled probe DNA (1 x IO6cpm/ml; specific activity, 5 to 9 x 10" DNA, and to a single 17-kilobase fragment with HeLa DNA cpm//jg of DNA) at 67°Covernight. Hybridization was performed in 5 (Fig. 1C). Again, this is suggestive of homozygosity in HeLa x SET buffer containing 0.02% each of bovine serum albumin, Ficoll, and heterozygosity in the fibroblasts for this particular marker and polyvinyl pyrrolidone; 0.5% SDS; 10% dextan sulfate; and heat- under the conditions studied. denatured salmon sperm DNA (100 Mg/ml) as carrier. Filters were RFLP Analysis of the Hybrid Cell Lines. Having established washed in 2 x SET buffer (pH 8.0) containing 0.2% SDS for 2 h at 67°C,in 0.2 x SET buffer containing 0.1% SDS at 65°Cfor 2 h, and the presence of RFLPs in parental fibroblast and HeLa DNAs, we used the four probes to analyze the DNA from tumorigenic in 3 mM Tris base solution (pH 8.0) at room temperature. Filters were dried and exposed to Kodak XAR-5 film with an intensifying screen at and nontumorigenic hybrid cell lines. Densitometric tracings -70°C for periods of 2 to 10 days. Densitometric tracings of the were performed as needed to compare the strength of the autoradiographs were performed with an LKB densitometer. signals. Chromosome 11 Probes. Examination of the cellular DNA of the nontumorigenic hybrid cell lines CGL2 and ESH541E, RESULTS digested with Taql and hybridized with the c-Ha-ras gene probe, revealed the presence of the fibroblast-derived, 4.0- and 2.5- RFLP Analysis of Parental Cell Lines. Genomic DNAs from kilobase alíelesand the HeLa 3.0-kilobase alíele(Fig. 2). In the parental cell lines GM-77, IMR-90, and D98/AH-2 (HeLa) contrast, the RFLP profiles of the tumorigenic segregants were digested with the appropriate restriction enzymes. South CGL4 and ESH541L showed the loss of the fibroblast alíeles ern blot analysis, using RFLP probes specific for chromosomes represented by the 2.5-kilobase and 4.0-kilobase fragments, 11 and 14, revealed useful unique polymorphic fragments in respectively. These gels show unequivocally that there has been the fibroblast and HeLa DNAs with all of the probes (Fig. 1). loss of a fibroblast chromosome 11 c-Ha-ras alíelefrom each Parental DNAs digested with Taq\ and hybridized with the c- Ha-ras gene probe exhibited a useful degree of polymorphism. of the tumorigenic segregants and presumably reflect the loss of a single copy of fibroblast chromosome 11 from these cells. Fragment lengths of 4.0, 2.5, and 2.3 kilobases were detected in fibroblast GM-77 DNA; 3.0, 2.5, and 2.3 kilobases in fibro Another feature of interest in this result is that it would seem blast IMR-90 DNA; and 3.0 and 2.3 kilobases in HeLa DNA that one copy of a fibroblast chromosome 11 is lost from CGL4 (represented by the 2.5-kilobase alíele),and the other copy of a fibroblast chromosome 11 (represented by the 4.0-kilobase al 1Parental Table of celllinesHybrid íele)islost from the ESH541L cells. linesNontumorigenic Three other HeLa x GM-77 hybrid lines were examined: the lines Tumorigenic nontumorigenic CGL1 cell line and two independent tumori HeLa)GM-77(fìbroblastx hybridshybridsCGL1 genic segregants (CGL3 and ESH5-136SA). As seen in Fig. 3 xD98/AH-2IMR-90 CGL3 ESH5-I36SA the tumorigenic ESH5-136SA cell line again unequivocally CGL2 CGL4 showed the loss of a fibroblast alíele(the2.5-kilobase fragment), ESH54IEESH541LESH39E indicating that a single copy of normal chromosome 11 was x D98/AH 2Description ESH39L lost from this tumorigenic segregant. Conversely, there was no Subclone C5 Subclone 13SA loss of either the 2.5- or the 4.0-kilobase alíelefrom the CGL3 Subclone C 14 Subclone 16SA DNA. There is some suggestion that the band representing the

Table 2 Restriction fragment length polymorphisms The numbers of constant and polymorphic fragments were derived from previous studies (12-14) and the present analysis. of of poly Constant morphic Genec-Ha-rof enzymetaql fragments30 fragments3 locationllpI3 kilobases Insulin |)liins Mli Pvu\l 31 Ilpl5 Apolipoprotein A-l" pSV2-gpt-A-l Pstl 1 Ilql3 AW 101Probe6.6 pAWlOlRestriction EcoRlNo. 0No. 8Chromosomal 14q32 °S. K Karathanasis. personal communication (apolipoprotein A-1 polymorphism with the Pstl enzyme). 6175 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. CHROMOSOME 11 AND CONTROL OF TUMORIGENICITY

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Fig. I. KH.I' profiles of parental cell DNAs. In A the DNA samples were digested with Taq\ and hybridized to the chromosome 230- 11-specific c-Ha-ras probe. In B DNA samples were digested with /'i-nl I and hybridized to the phins310 probe (chromosome 11 specific). In 4.4- 9.4- C DNA samples were digested with EcoRl and hybridized to the chromosome 14-specific 6.7- pAWlOl probe. See text for experimental de tails. X DNA digested with IHml(\\ was used for molecular size markers which are indicated 2.3- 4.4- on the left of each panel (size in kilobases). Arrowheads on the right of each panel indicate 2.0- informative RFLP bands.

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Fig. 2. RFLP analysis of hybrid cell DNAs with the chromosome 11-specific c-Ha-ra.v probe. DNA samples were digested with Taq\. Both fibroblast parent RFLP alíelesare present in the nontumorigenic hybrids CGL2 and ESH541E. The 2.5-kilobase fibroblast alíeleis lost from tumorigenic segregant CGL4, and the 4.0-kilobase fibroblast alíeleislost from tumorigenic segregant ESHS41L.

HeLa 3.0-kilobase alíeleis lighter relative to the 4.0- and 2.5- kilobase bands in the CGL3 lane compared to the CGL1 lane. Fig. 3. RFLP analysis of hybrid cell DNAs with the chromosome 1l-specilic This would indicate loss of a HeLa chromosome 11 in this c-Ha-ras probe. Both fibroblast parent RFLP alíelesare present in the nontu tumorigenic cell line. morigenic hybrid CGLI. Both alíelesarealso present in the tumorigenic segregant Further supporting evidence for the loss of a HeLa c-Ha-ras CGL3. The fibroblast 2.5-kilobase alíelehas been lost from the tumorigenic segregant ESH136SA. alíelefrom CGL3 was obtained by hybridizing /Vj/II-digested DNA with the human insulin gene probe phins310. This probe proved to be useful, since it hybridized to fragments unique to cell DNAs contained both the 2.3- and 0.75-kilobase fragments, both fibroblast and HeLa DNAs (Fig. IB). densitometric analysis of the bands from the autoradiograph Fig. 4 illustrates the RFLP analysis of CGL1 and CGL3. illustrated in Fig. 4 revealed 2.3:0.75 ratios consistent with loss Although the nontumorigenic and tumorigenic segregant hybrid of a single copy of HeLa chromosome 11 in the tumorigenic 6176 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. CHROMOSOME 11 AND CONTROL OF TUMORIGENICITY o co O) _l LU O O 00 OC O o o O) O) SE CO CO

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Fig. 4. RFLP analysis of hybrid cell DNAs with the chromosome 11-specific Fig. 5. RFLP analysis of ESH39 hybrid cell DNAs with the chromosome II- phins310 probe. The nontumorigenic hybrid CGL1 contains both the fibroblast specific c-Ha-roi probe. The nontumorigenic ESH39E hybrid contains the fibro- 2.3-kilobase and the HeLa 0.75-kilobase alíeles.Both alíelesare also present in blast-specific 2.5-kilobase alíele.This alíeleisabsent in the tumorigenic segregan! the tumorigenic segregan! CGL3, but the intensity of the 0.75-kilobase alíeleis ESH39L. reduced, this being suggestive of a loss of one of the HeLa alíeles.See Table 4 for densitometric measurement of this gel. was further indicated with the use of the apolipoprotein A-l Table 3 Densitometric analysis of the insulin RFLP alíelesin parental and hybrid cell DNA gene as probe. This probe has been mapped to the long arm of Densitometric measurements were made from the autoradiograph illustrated chromosome 11 (13). Digestion of DNAs with Pstl and hybrid in Fig. 4. ization to the A-l probe showed the presence of 2.2- and 3.5- intensity2.3 kilobase alíelesinthe IMR90 fibroblast parent and a single 2.2- kilo- 0.75 kil- of intensities kilobase alíelein HeLa (Fig. 6). The nontumorigenic ESH39E CelllineGM-77 basesobases1.70 (2.3/0.75kilobases)NA" subclones C5 and Cl4 possess both 2.2- and 3.5-kilobase bands. 0 D98/AH-2 0 0.65 NA However, both subclones (13SA and 16SA) of the tumorigenic CGL1 5.52 2.65 2.08 segregan! ESH39L reveal the loss of the fibroblast 3.5-kilobase CGL3Band 3.48 1.09Ratio 3.20 alíele.Thus, in this IMR90 x HeLa hybrid cell system we have " NA, not applicable. shown the loss of fibroblast-specific alíeleson both the short arm and the long arm of chromosome 11 in the tumorigenic segregant (Table 3). Specifically, the ratio of 3.20 for CGL3 is segregants, a finding consistent with the loss of the entire consistent with the loss of one HeLa chromosome 11 compared chromosome from the tumorigenic hybrids. to the nontumorigenic CGL1. Densitometric readings of three Chromosome 14 Probe. Only one chromosome 14-specific separate Southern blots confirmed these findings (data not RFLP probe was available for this study, the anonymous frag shown). Examination of a series of HeLa x IMR-90 hybrids also ment pAWlOl, the prototypic RFLP probe (14). As outlined in Fig. 1C HeLa is homozygous with a single 17-kilobase band, revealed the loss of a normal fibroblast chromosome 11 from whereas fibroblast GM-77 is heterozygous, sharing a 17-kilo the tumorigenic segregant hybrid cells. DNA digested with Taq\ and hybridized to the c-Ha-ras probe showed that the only base band in common with HeLa plus a unique 20-kilobase discriminating fibroblast-specific RFLP fragment in the non band. Similarly fibroblast IMR-90 is heterozygous, sharing a tumorigenic ESH39E cell line is the 2.5-kilobase one (Fig. 5). 17-kilobase band with HeLa and possessing a unique 18.5- This fragment is not seen in the tumorigenic segregant ESH39L kilobase band. Analysis of the hybrid cell populations showed and is, therefore, suggestive of a loss of a copy of a fibroblast that CGL3 was the only hybrid to show the loss of a fibroblastic chromosome 11. alíele,aGM-77-specific 20-kilobase fragment (Fig. 7). In all of Loss of a fibroblast chromosome 11 from each of 2 subclones the other hybrids examined there was no obvious loss of either of the tumorigenic segregant ESH39L (clones 16SA and 13SA) HeLa or fibroblast chromosome 14 alíeles(Fig. 7). 6177 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. CHROMOSOME 11 AND CONTROL OF TUMORIGENICITY

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o 0» Fig. 6. RFLP analysis of ESH39 hybrid cell DNAs with the chromosome 11- III spccific apolipoprotein A-l probe. The nontumorigenic ESH39E subclones C5 00 K and C14 both show the presence of the fibroblast 3.5-kilobase alíele.This alíele 0) a» a» has been lost from both tumorigenic subclones I6SA and I3SA. O n n

DISCUSSION

Detailed cytogenetic analysis of HeLa x fibroblast hybrids -23.0 had previously implicated chromosomes 11 and 14 in control of tumorigenic expression (8). Independent analyses by Klinger (2) had also implicated chromosome 11 as well as other chro mosomes, not including chromosome 14. The tentative impli cation of certain specific chromosomes in control of tumori -9.4 genic expression of human cell hybrids was based upon the number of copies of a given chromosome per metaphase spread -6.7 using trypsin-Giemsa banding techniques. The loss of specific chromosomes was noted but, using this technique, the parental origin of the chromosomes could not be ascertained. In this present paper we have utilized the technique of RFLP analysis, as originally conceived by Botstein and coworkers (10). Using three probes for RFLPs on chromosome 11 and one on chromosome 14 we have been able to strengthen the evidence for implication of chromosome 11 in control of tu morigenic expression. Four of the five tumorigenic segregan! HeLa x fibroblast hybrid cell lines studied have lost at least one human fibroblast Fig. 7. RFLP analysis of hybrid cell DNAs with the chromosome 14-specific pAWlOl probe. A, analysis of HeLa x GM77 hybrids. Tumorigenic segregan! copy of chromosome 11. The data obtained with cell lines CGL3 has lost the fibroblast 20-kilobase alíele.None of the other hybrids shows CGL4 and ESH541L (Fig. 2) would also suggest that the loss the obvious loss of any alíele.B. analysis of HeLa x IMR-90 hybrids. None of of either copy of normal fibroblast chromosome 11, either alone the hybrids shows the loss of either HeLa or fibroblast alíeles. or in concert with some other chromosome(s), is sufficient to allow reexpression of tumorigenicity. This would indicate that, Determination of such events will require a more extensive at least with respect to chromosome 11, suppression of tumor RFLP analysis of regions spanning the entire chromosome 11, igenicity may be controlled by chromosome dosage effects. a strategy that may be possible using probes that detect highly It is unclear at this time why the cell line CGL3 did not variable tandem repeat "minisatellite" regions (20). conform to the predicted loss of a normal fibroblast chromo Analysis of the data derived from the RFLP analysis with the some 11. There is the remote possibility that homologous pAWlOl chromosome 14-specific probe showed that unequiv recombination between HeLa and fibroblast chromosome 11 ocal loss of a fibroblast alíelefrom the tumorigenic segregants regions has occurred that results in the transfer of fibroblast occurred only in the CGL3 cell line. In the remainder of the "tumor-suppressor" sequences to the HeLa chromosome 11. tumorigenic segregants no loss of any alíelewas obvious. This 6178 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. CHROMOSOME 11 AND CONTROL OF TUMORIGENICITY result suggests that generally loss of a normal fibroblast chro transformation and tumorigenicity. Science (Wash. DC), 2/5: 252-259, 1982. mosome 14 is not necessary in order to reestablish the tumori- 4. Harris, H., Miller, O. J., Klein, G., Worst, P., and Tachibana, T. Suppression genie potential of HeLa x fibroblast hybrids. of malignancy by cell fusion. Nature (Lond.), 223: 363-368, 1969. As outlined in the "Introduction" we focused on RFLP 5. Klinger, H. P. Suppression of tumorigenicity in somatic cell hybrids. Cyto genet. Cell Genet., 27: 254-256, 1980. probes that recognize chromosomes 11 and 14, because earlier 6. Stanbridge, E. J. Suppression of malignancy in human cells. Nature (Lond.), cytogenetic evaluations (8) had implicated these chromosomes 260: 17-20, 1976. 7. Stoler, A., and Bouck, N. Identification of a single chromosome in the normal in control of tumorigenic expression in HeLa x fibroblast human genome essential for suppression of hamster cell transformation. hybrids. Although our present study continues to support the Proc. Nati. Acad. Sci. USA, 82: 570-574, 1985. notion that chromosome 11 is involved and chromosome 14 is 8. Stanbridge. E. J., Flandermeyer, R. R., Daniels, D. W., and Nelson-Rees, W. A. Specific chromosome loss associated with the expression of tumori not, it obviously provides no data regarding the possible control genicity in human cell hybrids. Somat. Cell Genet., 7: 699-712, 1981. that other normal chromosomes may exert. It does, however, 9. Stanbridge, E. J., and Wilkinson. J. Analysis of malignancy in human cells: malignant and transformed phenotypes are under separate genetic control. indicate that further study is warranted of gene(s) present on Proc. Nati. Acad. Sci. USA, 75: 1466-1469, 1978. chromosome 11 that regulate tumorigenic expression in these 10. Botstein, D., White, R., Skolnick, M.. and Davis, R. W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. cell hybrids. Ann. J. Hum. Genet., 32: 314-331, 1980. The results of these investigations are compatible with the 11. Der, C. J., Krontiris, T. G., and Cooper, G. M. Transforming genes of human notion that the genome of normal cells contains DNA se bladder and lung carcinoma cell lines are homologous to the ras genes of quences capable of controlling tumorigenic expression of cancer Harvey and Kirstin sarcoma viruses. Proc. Nati. Acad. Sci. USA, 79: 3637- cells when introduced by cell fusion. These "tumor-suppressor" 3640, 1982. 12. Bell, G. I., Selby, M. J., and Rutter, W. J. The highly polymorphic region sequences (21) may be analogous to putative regulatory se near the human insulin gene is composed of simple tandemly repeating quences seen in retinoblastoma (22) and Wilms' tumors (23- sequences. Nature (Lond.), 295: 31-35, 1982. 13. Karathanasis, S. K., Zannis, V. I., and Breslow, J. L. Isolation and charac 26). In these situations RFLP analysis has confirmed that terization of the human apolipoprotein A-l gene. Proc. Nati. Acad. Sci. neoplastic expression is correlated with the deletion of DNA USA, «0:6147-6151, 1983. 14. Wyman, A. R., and White, R. A highly polymorphic locus in human DNA. sequences on chromosome 13 in the case of retinoblastoma and Proc. Nati. Acad. Sci. USA, 77: 6754-6758, 1980. chromosome 11 in the case of Wilms—aniridia. 15. Huttner, K. M., Scangos. G. A., and Ruddle. F. H. DNA-mediated gene transfer of a circular plasmid into murine cells. Proc. Nati. Acad. Sci. USA, Given that a gene(s) that maps to chromosome 11 is capable 7(5:5820-5824, 1979. of suppressing tumorigenicity it should be possible to clone and 16. Clewell, D. B., and Helinski, D. R. Supercoiled circular DNA-protein com characterize the relevant DNA sequence(s). However, the prob plex in /•:.coli:purification and induced conversion to an open circular DNA lem of isolating tumor-suppressor or "antioncogenes" (21, 27) form. Proc. Nati. Acad. Sci. USA. 62: 1159-1166, 1969. 17. Jolly, D. G.. Esty, A., Bernard. H. U., and Friedmann, T. Isolation of a is not as straightforward as that of isolating transforming genomic clone partially encoding human hypoxanthine phosphoribosyl trans- oncogenes (28-30). Identification of oncogenes involves assay ferase. Proc. Nati. Acad. Sci. USA, 79: 5038-5041, 1982. for transformed foci on a background of contact-inhibited cells. 18. Feinberg, A. P., and Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., In searching for DNA sequences that suppress tumorigenicity, 137: 266-267, 1983. one is looking for the rare transfected cell that is nontumori- 19. Southern. E. M. Detection of sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol.. 98: 503-517, 1975. genic but still transformed on a background of transformed, 20. Jeffreys, A. J., Wilson. V., and Thein, S. L. Hyper-variable "minisatellite" tumorigenic cells. In order to overcome this problem we have regions in human DNA. Nature (Lond.), 314: 67-73. 1985. 21. Stanbridge, E. J. A case for human tumor-suppressor genes. Bioessays, 3: developed the technique of transferring single specific chro 252-256, 1985. mosomes to recipient cells via microcell transfer (31 ). It is now 22. Murphree, A. L., and Benedict. W. F. Retinoblastoma: clues to human possible to transfer a single copy of a fibroblast chromosome oncogenesis. Science (Wash. DC). 223: 1028-1033, 1984. 23. Pearson, E. R., Vogelstein, B., and Feinberg, A. P. Somatic deletion and 11 to a HeLa cell. These studies are in progress, and it will be duplication of genes on chromosome 11 in W'ilm's tumor. Nature (Lond.), interesting to see what effect the presence of a single normal 309:176-178, 1984. 24. Koufos, A., Hansen, M. F., Lampkin, B. C., Workman, M. L., Copeland, N. chromosome 11 will have on the neoplastic state of HeLa cells. G., Jenkins, N. A., and Cavenee, W. K. Loss of alíelesat loci on human chromosome 11 during genesis of Wilm's tumor. Nature (Lond.), 309: 170- 172, 1984. ACKNOWLEDGMENTS 25. Orkin, S. H.. Goldman, D. S., and Sallen, S. E. Development of homozygosity for chromosome lip markers in Wilm's tumor. Natue (Lond.), 309: 172- We thank Dr. Ted Friedmann, Dr. Channing Der, Dr. Graham Bell, 174, 1984. 26. Reeve, A. E., Housiaux. P. J., Gardner, R. J. M., Chewings, W. E., Grindley, Dr. Sotirios Karathanasis, and Dr. Ray White for the gift of chromo R. M., and Millow, L. J. Loss of Harvey raíalíeleinsporadic Wilm's tumor. some probes and Dr. Subal Chattopadhyay for technical assistance. Nature (Lond.), 309: 174-176, 1984. 27. Knudson, A. G., Jr. Hereditary cancer, oncogenes, and antioncogenes. Cancer Res..«: 1437-1443, 1985. ADDENDUM 28. Chang, E. H., Furth, M. E., Scolnick, E. M., and Lowy, D. R. Tumorigenic transformation of mammalian cells induced by a normal human gene ho After this paper was submitted for publication, Kaelbling and Klinger mologous to the oncogene of Harvey murine sarcoma virus. Nature (Lond.), (32) reported essentially the same findings with unrelated HeLa x 297:479-483, 1982. 29. Krontiris, T. G., and Cooper, G. M. Transforming activity of human tumor fibroblast hybrid cells. DNA. Proc. Nati. Acad. Sci. USA. 78: 1181-1184, 1981. 30. Murray, M. J., Shilo, B. 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6179 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research. Implication of Chromosome 11 in the Suppression of Neoplastic Expression in Human Cell Hybrids

Eri S. Srivatsan, William F. Benedict and Eric J. Stanbridge

Cancer Res 1986;46:6174-6179.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1986 American Association for Cancer Research.