Dec 18, 1986 - The findings suggest that genes of the normal cell parent can affect proto-oncogene expression. Whether the genes affecting fos, ets2, and myb ...
MOLECULAR AND CELLULAR BIOLOGY, Aug. 1987, p. 2941-2946 0270-7306/87/082941-06$02.00/0 Copyright (C 1987, American Society for Microbiology
Vol. 7, No. 8
Levels of fos, ets2, and myb Proto-Oncogene RNAs Correlate with Segregation of Chromosome 11 of Normal Cells and with Suppression of Tumorigenicity in Human Cell Hybrids BRYAN M. O'HARA,lt HAROLD P. KLINGER,2 TOM CURRAN,3 YI-DU ZHANG,' AND DONALD G. BLAIR`* Laboratory of Molecuilar Oncology, Frederick Cancer Research Facility, National Cancer Instituite, Frederick, Maryland 21701-10131; Department of Genetics, Albert Einstein College of Medicine, Bronx, Neit' York 104612; and Department of Molecular Genetics, Roche Research Center, Nuitley, New Jersey 071103 Received 18 December 1986/Accepted 4 May 1987 The tumorigenicity in nude mice of human carcinoma-derived D98AH2 (D98) cells is suppressed when cell hybrids are made by fusing these cells with normal human diploid cells. Selection for hybrids that have segregated chromosomes results in the recovery of tumorigenic segregants. These segregants have all lost at least one copy of chromosome 11 of the diploid cell parent. Earlier we found that the parental D98 cells had detectable levels of mRNA specific for 13 of 21 proto-oncogenes examined. To determine if transregulation of proto-oncogenes by genes of the normal cell occurs in such hybrids, the steady-state levels of mRNA specific to 22 proto-oncogenes in the parental cells were compared with those of nontumorigenic D98 x human diploid hybrids as well as with those of their tumorigenic segregants and with the cells of the resulting tumors. The only chromosome consistently segregated in the latter was chromosome 11 of the diploid cell. fos and ets2 RNA levels and the amount of fos protein were consistently elevated in the segregants compared with amounts in the original hybrids. An unexpected finding was the inverse relationship for myb RNA that was barely detected in the parental D98 cells but was at least 10-fold elevated in hybrids that did not have segregated chromosomes compared with those that did. These patterns were evident in RNAs prepared from both subconfluent and confluent cell cultures. The findings suggest that genes of the normal cell parent can affect proto-oncogene expression. Whether the genes affecting fos, ets2, and myb RNA levels are on chromosome 11 and whether these alterations are causally related to the tumorigenic phenotype of the hybrids remain to be determined.
in the development of Wilms' tumor and other embryonal carcinomas (16, 17). An analogous situation may also exist in retinoblastoma, in which homozygous loss of a segment of chromosome 13, which has recently been cloned (8, 18), also appears to be an etiologic factor (1, 3). In addition, the existence of suppressor loci is compatible with the loss of heterozygosity found for a number of loci in various types of more common tumors, including bladder carcinomas and melanomas (6, 7, 31). We speculate that in nontumor cells these suppressor genes are critical in maintaining normal cell growth and differentiation. The important role oncogenes appear to play in malignant transformation and that of proto-oncogenes in controlling normal cell functions, where they are clearly under precise regulatory control, prompted us to determine if D98 x HD hybrids could be used to obtain information about control of these genes, primarily to determine if this regulatory information is located on the chromosomes of the normal cell parent, since we had previously found that the D98 parent of the hybrids expresses RNAs specific for a large number of oncogenes (24). The steady-state levels reported here of RNAs specific for 22 proto-oncogenes in a series of nontumorigenic hybrids, as well as in tumorigenic segregants of these hybrids that had lost at least one chromosome 11 of the normal cell parent and in cells of the resulting tumors, show that the levels of fos, ets2, and myb RNAs correlate with the segregation of chromosome 11 of the normal cell and with the tumorigenic phenotype of the hybrids. In subsequent sections, the term correlation will be used simply to describe this state without implying a causative relationship with tumorigenicity, which remains to be established.
Fusion of malignant human tumor cells with normal cells results in the suppression of the tumorigenic phenotype of the tumor cell. Chromosome and restriction-fragment-length polymorphic chromosome marker analysis of such intraspecies hybrids revealed cosegregation of specific chromosomes of the diploid cell, primarily chromosome 11, and suppression of the tumorigenic phenotype of hybrids of human HeLa carcinoma-derived D98AH2 (D98) cells fused with normal human diploid (HD) embryonic fibroblast-like cells (12, 14, 28). Furthermore, no heterozygosity could be detected in D98 cells for a large number of chromosome-11specific restriction-fragment-length polymorphic chromosome markers (13), suggesting that one copy of chromosome 11 had been lost and that the remaining homolog had been duplicated. This finding is consistent with the hypothesis that in this hybrid system there is a tumorigenicity suppressor gene(s) on the normal chromosome 11 that is nonfunctional in the D98 cells. This malfunction may be due to the deletion or mutation of one copy of the gene during the evolution of D98 cells. Loss of heterozygosity could then result in the phenotypic expression of this change. Further evidence in support of suppressor information being located on chromosome 11 was provided by Saxon et al. (26), who showed that transfer of a chromosome 11 from a normal cell into a D98 cell resulted in suppression. The putative suppressor gene(s) is perhaps analogous to those on chromosome 11 whose homozygous loss seems to be a critical event Corresponding author. t Present address: Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461. *
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MOL. CELL. BIOL.
TABLE 1. Chromosome 11 content and tumorigenicity of parental cells and hybrids in nude mice Cell or hybrid designation"
Avg no. of and origin chromosome 116
No. of mice with tumors/total of mice' no.
Time (wk) for tumor development
HD1799 normal fibroblast
2 (2)
0/5
D98
2
2/3
6-8
NT1.1 TU1.1.1 Tl.1.11
4.0 (2) 2.0 (1) 1.6 (1)
0/3 2/3 4/4
5 3
NT4.3 TU4.3.1 T4.3.1
4.0 (2) ND 3.0 (ND)
0/5 2/3 3/3
6-8 1
NT2.1 NT2.1.1
4.0 (2) 3.8 (2)
0/3 0/4
NT3.3
4.0 (2)
0/5
TU4.2 T4.2
3.7 (2) 2.6 (1)
1/4 5/5
7 4-6
a See Materials and Methods for system of designations. Each set of hybrids (same first number) represents an independent fusion event. NT4.3 and TU4.2, despite having been isolated from the same flask, appear different because they were picked soon after colonies had formed and because their tumorigenicity and karyotypes were very different. Cells, hybrids, and tumors are described in detail in reference 14. b The number in parentheses indicates the number of chromosomes derived from the normal diploid parent determined with restriction-fragment-length polymorphic chromosome marker analysis; some numbers are taken from an earlier report (12). ND, Not done. cTumorigenicity was determined by injecting 5 x 106 cells subcutaneously on the flanks of nude mice irradiated (500 rad) 1 day previously. Mice that did not develop tumors were maintained for up to 24 weeks.
MATERIALS AND METHODS
Cells. The hybrids of a previously described intraspecies human carcinoma (D98) x HD fusion series (designated D98 x HD1799) were used, including the parental D98 cells, derivatives of the HeLa cervical-carcinoma cell line, and the HD1799 normal human embryonic fibroblasts (Table 1). The methods for colony selection of nontumorigenic and tumorigenic hybrids were also described in these reports (12, 14). In this report, we use a decimal system for designating hybrids. NT designates nontumorigenic, TU designates tumorigenic, and T designates the cell cultures of tumors. The first number of a hybrid name identifies a cell fusion flask, the second represents a colony picked from that flask, and the third represents a single cell clone isolate of the colony. Those hybrids used in this study whose full chromosome content was described in our earlier reports (12, 14) were designated in those reports as follows: NT1.1 was 1F col 1, TU1.1.1 was 1F col 1 cn 1 TG, T.1.11 was T146, NT2.1 was 2U col 1, NT2.1.1 was 2U col 1 col 1, T4.2 was T70, and NT4.3 was 4F col 3. After backselection in 6-thioguanine or a period of in vitro culture and recloning, both tumorigenic and nontumorigenic derivatives were obtained. Cells of tumors resulting from the injection of hybrids into nude mice were grown for short periods in culture before RNA preparation. Tumorigenicity of all hybrids, reexamined at the time of RNA preparation, indicated that all of the hybrids had retained their original tumorigenicity phenotypes (14). The cell line designated SV80 is an SV40-transformed human fibroblast line (29). The hybrids and parental cells were
maintained in McCoy 5A medium with 15% fetal bovine serum, and the SV80 line was maintained in minimal essential medium with 10% calf serum. Nucleic acid analysis. Total cellular polyadenylated RNA was prepared and analyzed as described elsewhere (24). RNA was quantitated initially by hybridization to 3H-labeled poly(U) (2) and later by normalization to the intensities of the bands obtained on Northern blots (RNA blots) after hybridization with myc and an actin gene probes. Each method of normalization gave similar band intensities with a given proto-oncogene probe. Expression levels as percentages of myc or an actin gene were calculated from densitometric scans of autoradiograms (with preflashed film) of blots developed while in the linear phase of exposure. DNA was prepared and analyzed as described elsewhere (20). The majority of the hybridization probes have been described elsewhere (24). The rat P-actin cDNA was a gift from Steven Farmer (Boston University School of Medicine, Boston, Mass.); the 6K and H33 probes, specific for the E26 retroviral homologous loci of etsl and ets2 on human chromosomes 11 and 21, respectively, were gifts from Dennis Watson (30). The human heat shock cDNA probe, pURHS, was a gift from Joseph Nevins (The Rockefeller University, New York, N.Y.); and the murine ,B-2-microglobulin probe p929 was from David Seidman (25). Probes specific for human lymphocyte antigens A and B were gifts from Harry T. Orr (15). Protein analysis. Cells were plated at 106 per 35-mm petri dish, grown for 2 days, preincubated in methionine-free medium for 20 min, and labeled with 200 p.Ci of [35S]methionine per plate for 1 h. Lysis and immunoprecipitation with fos peptide M antiserum were as described elsewhere (5). RESULTS Levels of 19 oncogene RNAs and other RNAs that do not vary substantially or are not present in parental cells or hybrids. Chromosome 11 content and tumorigenicity of the parental cells and the hybrids are given in Table 1. There were no large variations in the RNA levels of an actin gene and 12 proto-oncogenes, including N-ras and src (not shown), in any of these hybrids. Table 2 gives the densitometrically determined relative RNA levels of nine protooncogenes of the parental cells and hybrids determined from autoradiograms of Northern blots and normalized as a percentage of the myc or actin gene RNA levels. Most of these oncogenes are expressed at substantial levels in the D98 cells. While the levels of some of the RNAs vary from one hybrid to another, there is no evident correlation between these levels and the chromosome content or tumorigenicity of the hybrids. The values in Table 2 that suggest a correlation for the smaller erbB transcript appear spurious because the lowest values for the transcript in the tumorigenic hybrids were equivalent to the higher values in the nontumorigenic hybrids, only one of the erbB transcripts showed this variation, and the levels of both transcripts are low, with the range of values in the tumorigenic hybrids not approaching that found in D98 cells. The sizes of all transcripts appear to be normal when compared with the previously published sizes (24 and references therein). Probes for B-lym, fes, fms, mos, N-myc, rel, or sis did not detect homologous RNA sequences in any of the hybrids or in D98 cells. Hybridization with these probes was followed in every case by hybridization with myc or actin gene probes to determine if hybridizable RNA remained on the blots in
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ONCOGENE RNA LEVELS IN CELL HYBRIDS
VOL. 7, 1987
TABLE 2. RNAs not correlating with segregation of chromosome 11 or with tumorigenicitya Probes compared
Relative RNA level (%) in:
RNA size (kb)
HD
D98
NT1.1
TU1.1.1
7.0 6.0
ND ND
8 36
5 25
erbAlmyc
6.7 3.2
ND ND
150 42
erbBImyc
10.0 5.5
ND ND
H-rasllA
1.3
K-ras2/myc
TU, T (avg _ SD)
NT (avg _ SD)
TU4.2 T4.2
NT3.3
T1.1.1
NT4.3
NT2.1
NT2.1.1
ND ND
ND ND
39 130
39 108
6 12
37 67
38 83
40 88
25 68
29 35
36
