Implication of Chromosome 11 in the Suppression of Neoplastic Expression in Human Cell Hybrids1
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[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 alÃeleunder 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.