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Genetic heterogeneity of chromosome 11 associated with tumorigenicity in HeLa D98-OR cells
ELSEVIER
Genetic Heterogeneity of Chromosome 11 Associated with Tumorigenicity in HeLa D98-OR Cells Izumi Horikawa, Aikou Okamoto, Jun Yokota, and Mitsuo Oshimura
ABSTRACT: D98-OR is a tumorigenic subline of HeLa cells. We isolated nine subclones from D98-OR and examined their tumorigenicity in nude mice. Three, two, and four subclones were highly, weakly, and nontumorigenic, respectively. While they all contained two copies of intact chromosome 11, restriction fragment length polymorphism (RFLP) analysis revealed that the allelic composition of this chromosome differed among them. The highly tumorigenic subclones were heterozygous for the 1 l p and 1 l q loci, whereas those that were weakly or nontumorigenic were homozygous. Thus, the loss of one chromosome 11 with the duplication of another associated with the reduced tumorigenicity. Taken together with previous reports, our results indicate that a putative tumor suppressor gene on chromosome 11 controls tumorigenic expression in a gene dosage-dependent manner, and most importantly, suggested that the functional inactivation of the gene requires only a "one-hit" mutation.
INTRODUCTION HeLa (human cervical cancer cells) have been used as a malignant counterpart for the generation of whole cell hybrids with normal cells [1-4], and as recipients for microcell-mediated chromosome transfer [5]. These studies have suggested that normal chromosome 11 carries a tumor suppressor gene for cervical carcinogenesis. It is generally thought that the functional loss of tumor suppressor genes requires "two hits," most commonly a mutation of one allele and the following exclusion of another wild-type allele [6-9]. The suppression of tumorigenicity by a single normal chromosome 11 via microcell fusion [5, 10] might be brought about by complementing the "two hit" defect. However, in nontumorigenic HeLa x normal fibroblast hybrids, the loss of a single copy of normal fibroblast-derived chromosome 11 was sufficient to generate tumorigenic segregants [1-4]. This implied that the tumorigenic phenoty2e could be expressed even in the presence of the wild-type suppressor gene, contrary to the "two-hit" theory. A low incidence of LOH (loss of heterozygosity) on chromosome 11 in cervical cancers [11] also did not indicate inactivation by a "two-hit" mechanism.
From the Department oJ Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago (I. H., M. 0.); and the Biology Division, National Cancer Center Research Institute, Tokyo (A. 0., J. Y.), Japan. Address reprint request,J to: Mitsuo Oshimura, Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishi-machi 86, Yonago 683, Japan. Received Jan uary 26, 1995; accepted June 6, 1995.
Cancer Genet Cytogenet 85:97-100 (1995) © Elsevier Science Inc., 1995 655 Avenue of the Americas, New York, NY 10010
In this study, we describe the heterogeneity of the tumorigenic phenotype and of the chromosome 11-allelic composition in the HeLa subline, D98-OR [12]. Our findings support the notion of a gene dosage-dependent effect of a tumor suppressor gene, whose normal function can be inactivated by a "one-hit" mutation. MATERIALS AND METHODS Cells and Cultures D98-OR, an ouabain-resistant mutant of hypoxanthine phosphoribosyl transferase-deficient HeLa cells [12], was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum. To isolate subclones, D98-OR cells (1 × 102) were plated in a 90-ram dish. Nine subclones (termed Scl-9) were randomly selected 3 weeks later, and were expanded. Before experiments, all subclones were confirmed to be resistant to 6-thioguanine (10 ~g/mL) and ouabain (5 x 10 -7 M). The cells were checked for mycoplasma contamination and found to be negative. Assays of Tumorigenicity in Nude Mice Cells (1 x 107 per site) were suspended in 0.2 mL of serum-free DMEM and subcutaneously inoculated into 4-week-old nude mice (ICR nu/nu), which were periodically examined for up to 8 weeks. Chromosome Analyses Karyotypes were analyzed by means of quinacrine banding. Ten or more well-banded metaphases from parental D98-OR cells and subclones were analyzed.
0165-4608/95/$9.50 SSDI 0165-4608(95)00147-H
98 Restriction Fragment Length Polymorphism (RFLP) Analyses High-molecular-weight DNA (10 p.g) was digested with the restriction enzyme (MspI or BamH1), fractionated by 0.8% agarose gel electrophoresis, and transferred to a nylon membrane (Hybond-N, Amersham). The membrane was hybridized to a 32p-labeled probe prepared by random priming [13], then washed and autoradiographed. The polymorphic probes p32-1 (DllS16 locus, 11p13) [14] and SS6 (INT2 locus, 11q13) [15] can detect three allelic types of MspI fragments (11.0 kb, 7.6/6.0/4.0 kb, and 6.0/4.0/3.0 kb) and two types of BamH1 fragments (8.4 kb and 5.6/2.8 kb), respectively.
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RESULTS Nine subclones (Scl-9) were isolated from D98-OR cells. Their characteristic marker chromosomes, in addition to their resistance to both 6-thioguanine and ouabain, confirmed that they were derived from parental D98-OR cells. All subclones showed a parental-like morphology, and had similar in vitro growth properties to those of parental D98-OR cells (population doubling time, 19-22 hours; colony-forming efficiency in soft agar, 40-60%). Furthermore, they all, like parental D98-OR, carried human papillomavirus type 18 (HPV18) sequence of the same integration profile, and expressed a similar amount of its mRNA in vitro (data not shown). The tumorigenicity of these subclones is represented in Figure 1. Three subclones (Sc2, -3, and -6) formed more rapidly growing tumors than those from parental D98-OR. Two (Sc4 and -8) formed much more slowly growing tumors. The tumors from Sc7 regressed after 4 weeks, and disappeared. The remainder (Scl, -5, and -9) formed no palpable tumors for up to 8 weeks. The intermediate tumorigenicity of the parental cells most likely represented a mixture of these highly, weakly, and nontumorigenic cells. Chromosome analyses did not distinguish between subclones with different tumorigenic phenotypes (karyotypes not shown). All subclones had a similar number of chromosomes (59-62 in modes), as well as parental D98-OR (61 in mode). No specific differences in normal or marker chromosomes correlated with tumorigenic phenotypes were identified, although there were nonspecific variations among subclones, and even within each subclone. All metaphases examined contained two copies of intact chromosome 11. For a more precise examination of chromosome 11, which supposedly carries a putative tumor suppressor gene (1-5, 10], we performed RFLP analyses (Fig. 2). Probe p32-1 (DllS16, 11p13) hybridized to heterozygous allelic fragments of 7.6/6.0/4.0 kb and 6.0/4.0/3.0 kb in highly tumorigenic subclones (Sc2, -3, and -6). In contrast, this probe hybridized to a homozygous allele (7.6/6.0/4.0-kb fragments) in weakly or nontumorigenic subclones (Scl, -4, -5, -7, -8, and -9). Similarly, when probe SS6 (INT2, 11q13) was used, highly tumorigenic subclones were heterozygous (8.4-kb fragment and 5.6/2.8-kb fragments), and weakly or nontumorigenic subclones were homozygous
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Weeks after inoculation Figure 1 Tumor-growth curves of parental D98-OR cells and their subclones (Sc). Cells (1 × 107 per site) were inoculated subcutaneously into nude mice, which were examined weekly. Tumor volume was calculated using the following formula: 1/2 × (long diameter) × (short diameter) 2. Each curve shows average tumor volumes from four sites (subclones) or 11 sites (D98-OR). (5.6/2.8-kb fragments alone). For both loci, parental D98-OR showed the mixed feature of heterozygous and homozygous cells. DISCUSSION Studies of HeLa x normal fibroblast hybrids [1-4] and HeLa- or SiHa-normal chromosome 11 microcell hybrids [5, 10] have led to a cervical cancer-suppressor gene being mapped on chromosome 11. Here, we presented an excellent correlation between tumorigenic phenotypes and the allelic composition of chromosome 11. Cells containing two copies of chromosome 11 from different origins had a highly tumorigenic phenotype, whereas those containing two chromsomes 11 of the same origin were weakly or nontumorigenic. This also suggested that chromosome 11 carries a gene that controls tumorigenic expression. However, the conventional notion of tumor suppressor genes, namely the "two-hit" theory [6-9], cannot compatibly explain our results. In this theory, the LOH, which is the most common second hit for excluding a wild-type allele
Chromosome 11 in HeLa Cells
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Figure 2 RFLP analyses of chromosome 11 in parental D98-OR cells and their subclones (Sc). A) MspI-digested DNAs were hybridized with probe p32-1 (DllS16 locus, 11p13}. B) BamHIdigested DNAs were hybridized with probe SS6 (INT2 locus, 11q13). Fragment sizes are irdicated on the left. The comparison between ethidium bromide-stained gels and autoradiographs confirmed that all cell lines contained two copies for both loci. of t u m o r suppressor genes, s h o u l d promote the malignant p h e n o t y p e [7, 8]. This was not true of c h r o m o s o m e 11 in D98-OR cells. The m a r k e d r e d u c t i o n of tumorigenicity in the h o m o z y g o u s s u b c l o n e s i n d i c a t e d that the LOH in D98-OR cells e x c l u d e d a m u t a t e d gene and d u p l i c a t e d a w i l d type (HeLa-Homo in Fig. 3). F r o m another perspective, the c h r o m o s o m e 11-heterozygous cells carrying both m u t a t e d and w i l d - t y p e genes fully expressed the tumorigenic p h e n o t y p e (HeLa in Fig. 3), suggesting that a single m u t a t i o n ("one hit" sequestered the n o r m a l gene function despite the presence of a w i l d - t y p e allele. One possible m e c h a n i s m is that a m u t a t e d gene product abrogates the wild-tylpe function in a d o m i n a n t negative manner, as reported for p53 gene [16, 17]. If so, the s u p p r e s s i o n by transfer of a n o r m a l c h r o m o s o m e 11 w o u l d indicate that a single m u t a t i o n d i d not overcome two copies of w i l d type (HeLa-#11 in Fig. 3). However, since the tumorigenic segregant from w h o l e cell hybrids (HeLa x N-Tu in Fig. 3) also contained a single m u t a t e d and two w i l d - t y p e genes, this notion cannot be generalized:
Figure 3 Interpretation of the results from this study, whole cell fusion, and chromosome transfer experiments. Chromosome 11 carrying a mutated or wild-type HeLa-suppressor gene is shown as M or WT. The expression (Tu) or suppression (Sup) of tumorigenicity in each cell is indicated in parenthesis. HeLa × N, whole cell hybrid between HeLa and normal fibroblast; HeLa × N-Tu, tumorigenic segregant from HeLa × N; HeLa-#11, HeLa containing an extra copy of normal chromosome 11 via microcell transfer; HeLa-Homo, chromosome 11-homozygous cells in this study. WT/C ratio indicates the copy numbers of wild-type suppressor gene per cell, where a whole cell hybrid is regarded as two cells. The gene d o s a g e - d e p e n d e n t effect of a HeLa-suppressor gene has been i m p l i e d s o m e w h a t by the observation that tumorigenic segregants in cell hybrid experiments (HeLa × N-Tu in Fig. 3) retained one copy of normal fibroblastderived chromosome 11 [1-3, 18]. A s s u m i n g "one-hit" inactivation, the gene d o s a g e - d e p e n d e n t effect can explain both our and other results (Fig. 3). The copy numbers of the w i l d - t y p e gene per cell (WT/C ratio, where we regard a w h o l e cell h y b r i d as two cells) correlated with the expression or suppression of tumorigenicity. WT/C = 1 is not sufficient for suppression (HeLa and HeLa × N-Tu). In contrast, the tumorigenicity is s u p p r e s s e d w h e n WT/C = 1.5 (HeLa x N) or 2 (HeLa-Homo a n d HeLa-#11). We cannot c o m p l e t e l y rule out the involvement of activated oncogenes or putative tumor suppressor genes on other chromosomes. It is also u n k n o w n w h y the cells preferentially lost a m u t a t e d allele and d u p l i c a t e d a w i l d - t y p e allele during in vitro culture. However, we propose the hypothesis of the "one-hit" inactivation of a HeLa-suppressor gene on c h r o m o s o m e 11. This premise fits previous results using w h o l e cell and microcell hybrids. The "one-hit" hypothesis also agrees with the failure to detect an LOH on c h r o m o s o m e 11 in cervical cancers [11]. A n u m b e r of t u m o r suppressor genes, w h i c h can be functionally inactivated by "one hit," m a y r e m a i n to be identified. We thank Dr. Bernard E. Weissman, The University of North Carolina, for providing D98-OR cells. We also thank Drs. H. Kugoh and M. Katoh for helpful discussions, and M. Suzuki for technical assistance. This study was supported in part by a Grant-in-Aid for Cancer
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Research from the Ministry of Education, Science and Culture, Japan, and a Grant-in-Aid for a Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan.
REFERENCES 1. Kaelbling M, Klinger HP (1986): Suppression of tumorigenicity in somatic cell hybrids. III. Cosegregation of human chromosome 11 of a normal cell and suppression of tumorigenicity in intraspecies hybrids of normal diploid × malignant cells. Cytogenet Cell Genet 41:65-70. 2. Klinger HP, Kaelbling M (1986): Suppression of tumorigenicity in somatic cell hybrids. IV. Chromosomes of normal human cells associated with suppression of tumorigenicity in hybrids with D98AH2 carcinoma cells. Cytogenet Cell Genet 42:225-235. 3. Srivatsan ES, Benedict WF, Stanbridge EJ (1986): Implication of chromosome 11 in the suppression of neoplastic expression in human cell hybrids. Cancer Res 46:6174-6179. 4. Misra BC, Srivatsan ES (1989): Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Am J Hum Genet 45:565-577. 5. Saxon PJ, Srivatsan ES, Stanbridge EJ (1986): Introduction of human chromosome 11 via microcell transfer controls tumorigenic expression of HeLa cells. EMBO J 5:3461-3466. 6. Knudson AG (1971): Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820-823. 7. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL (1983): Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305:779-784. 8. Cavenee WK, Hansen MF, Nordenskjold M, Kock E, Maumenee I, Squire JA, Phillips RA, Gallie BL (1985): Genetic origin of mutations predisposing to retinoblastoma. Science 228: 501-503.
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9. Knudson AG (1985): Hereditary cancer, oncogenes, and antioncogenes. Cancer Res 45:1437-1443. 10. Koi M, Morita H, Yamada H, Satoh H, Barrett JC, Oshimura M (1989): Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa. Mol Carcinogen 2:12-21. 11. Yokota J, Tsukada Y, Nakajima T, Gotoh M, Shimosato Y, Mori N, Tsunokawa Y, Sugimura T, Terada M (1989): Loss of heterozygosity on the short arm of chromosome 3 in carcinoma of the uterine cervix. Cancer Res 49:3598-3601. 12. Weissman B, Stanbridge EJ (1980): Characterization of ouabain resistant, hypoxanthine phosphoribosyl transferase deficient human ceils and their usefulness as a general method for the production of human cell hybrids. Cytogenet Cell Genet 28:227-239. 13. Feinberg AP, Vogelstein B (1983): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266-267. 14. Feder J, Yen L, Wijsman E, Wang L, Wilkins L, Schroder J, Spurr N, Cann H, Blumenberg M, Cavalli-Sforza LL (1985): A systematic approach for detecting high-frequency restriction fragment length polymorphisms using large genomic probes. Am J Hum Genet 37:635-649. 15. Casey G, Smith R, McGillivray D, Peters G, Dixon C (1986): Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene. Mol Cell Biol 6:502-510. 16. Levine AJ, Momand J, Finlay CA (1991): The p53 tumour suppressor gene. Nature 351:453-456. 17. Unger T, Mietz JA, Scheffner M, Yee CL, Howley PM (1993): Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression. Mol Cell Biol 13:51865194. 18. Harris, H (1988): The analysis of malignancy by cell fusion: The position in 1988. Cancer Res 48:3302-3306.
Genetic Heterogeneity of Chromosome 11 Associated with Tumorigenicity in HeLa D98-OR Cells Izumi Horikawa, Aikou Okamoto, Jun Yokota, and Mitsuo Oshimura
ABSTRACT: D98-OR is a tumorigenic subline of HeLa cells. We isolated nine subclones from D98-OR and examined their tumorigenicity in nude mice. Three, two, and four subclones were highly, weakly, and nontumorigenic, respectively. While they all contained two copies of intact chromosome 11, restriction fragment length polymorphism (RFLP) analysis revealed that the allelic composition of this chromosome differed among them. The highly tumorigenic subclones were heterozygous for the 1 l p and 1 l q loci, whereas those that were weakly or nontumorigenic were homozygous. Thus, the loss of one chromosome 11 with the duplication of another associated with the reduced tumorigenicity. Taken together with previous reports, our results indicate that a putative tumor suppressor gene on chromosome 11 controls tumorigenic expression in a gene dosage-dependent manner, and most importantly, suggested that the functional inactivation of the gene requires only a "one-hit" mutation.
INTRODUCTION HeLa (human cervical cancer cells) have been used as a malignant counterpart for the generation of whole cell hybrids with normal cells [1-4], and as recipients for microcell-mediated chromosome transfer [5]. These studies have suggested that normal chromosome 11 carries a tumor suppressor gene for cervical carcinogenesis. It is generally thought that the functional loss of tumor suppressor genes requires "two hits," most commonly a mutation of one allele and the following exclusion of another wild-type allele [6-9]. The suppression of tumorigenicity by a single normal chromosome 11 via microcell fusion [5, 10] might be brought about by complementing the "two hit" defect. However, in nontumorigenic HeLa x normal fibroblast hybrids, the loss of a single copy of normal fibroblast-derived chromosome 11 was sufficient to generate tumorigenic segregants [1-4]. This implied that the tumorigenic phenoty2e could be expressed even in the presence of the wild-type suppressor gene, contrary to the "two-hit" theory. A low incidence of LOH (loss of heterozygosity) on chromosome 11 in cervical cancers [11] also did not indicate inactivation by a "two-hit" mechanism.
From the Department oJ Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago (I. H., M. 0.); and the Biology Division, National Cancer Center Research Institute, Tokyo (A. 0., J. Y.), Japan. Address reprint request,J to: Mitsuo Oshimura, Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishi-machi 86, Yonago 683, Japan. Received Jan uary 26, 1995; accepted June 6, 1995.
Cancer Genet Cytogenet 85:97-100 (1995) © Elsevier Science Inc., 1995 655 Avenue of the Americas, New York, NY 10010
In this study, we describe the heterogeneity of the tumorigenic phenotype and of the chromosome 11-allelic composition in the HeLa subline, D98-OR [12]. Our findings support the notion of a gene dosage-dependent effect of a tumor suppressor gene, whose normal function can be inactivated by a "one-hit" mutation. MATERIALS AND METHODS Cells and Cultures D98-OR, an ouabain-resistant mutant of hypoxanthine phosphoribosyl transferase-deficient HeLa cells [12], was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum. To isolate subclones, D98-OR cells (1 × 102) were plated in a 90-ram dish. Nine subclones (termed Scl-9) were randomly selected 3 weeks later, and were expanded. Before experiments, all subclones were confirmed to be resistant to 6-thioguanine (10 ~g/mL) and ouabain (5 x 10 -7 M). The cells were checked for mycoplasma contamination and found to be negative. Assays of Tumorigenicity in Nude Mice Cells (1 x 107 per site) were suspended in 0.2 mL of serum-free DMEM and subcutaneously inoculated into 4-week-old nude mice (ICR nu/nu), which were periodically examined for up to 8 weeks. Chromosome Analyses Karyotypes were analyzed by means of quinacrine banding. Ten or more well-banded metaphases from parental D98-OR cells and subclones were analyzed.
0165-4608/95/$9.50 SSDI 0165-4608(95)00147-H
98 Restriction Fragment Length Polymorphism (RFLP) Analyses High-molecular-weight DNA (10 p.g) was digested with the restriction enzyme (MspI or BamH1), fractionated by 0.8% agarose gel electrophoresis, and transferred to a nylon membrane (Hybond-N, Amersham). The membrane was hybridized to a 32p-labeled probe prepared by random priming [13], then washed and autoradiographed. The polymorphic probes p32-1 (DllS16 locus, 11p13) [14] and SS6 (INT2 locus, 11q13) [15] can detect three allelic types of MspI fragments (11.0 kb, 7.6/6.0/4.0 kb, and 6.0/4.0/3.0 kb) and two types of BamH1 fragments (8.4 kb and 5.6/2.8 kb), respectively.
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RESULTS Nine subclones (Scl-9) were isolated from D98-OR cells. Their characteristic marker chromosomes, in addition to their resistance to both 6-thioguanine and ouabain, confirmed that they were derived from parental D98-OR cells. All subclones showed a parental-like morphology, and had similar in vitro growth properties to those of parental D98-OR cells (population doubling time, 19-22 hours; colony-forming efficiency in soft agar, 40-60%). Furthermore, they all, like parental D98-OR, carried human papillomavirus type 18 (HPV18) sequence of the same integration profile, and expressed a similar amount of its mRNA in vitro (data not shown). The tumorigenicity of these subclones is represented in Figure 1. Three subclones (Sc2, -3, and -6) formed more rapidly growing tumors than those from parental D98-OR. Two (Sc4 and -8) formed much more slowly growing tumors. The tumors from Sc7 regressed after 4 weeks, and disappeared. The remainder (Scl, -5, and -9) formed no palpable tumors for up to 8 weeks. The intermediate tumorigenicity of the parental cells most likely represented a mixture of these highly, weakly, and nontumorigenic cells. Chromosome analyses did not distinguish between subclones with different tumorigenic phenotypes (karyotypes not shown). All subclones had a similar number of chromosomes (59-62 in modes), as well as parental D98-OR (61 in mode). No specific differences in normal or marker chromosomes correlated with tumorigenic phenotypes were identified, although there were nonspecific variations among subclones, and even within each subclone. All metaphases examined contained two copies of intact chromosome 11. For a more precise examination of chromosome 11, which supposedly carries a putative tumor suppressor gene (1-5, 10], we performed RFLP analyses (Fig. 2). Probe p32-1 (DllS16, 11p13) hybridized to heterozygous allelic fragments of 7.6/6.0/4.0 kb and 6.0/4.0/3.0 kb in highly tumorigenic subclones (Sc2, -3, and -6). In contrast, this probe hybridized to a homozygous allele (7.6/6.0/4.0-kb fragments) in weakly or nontumorigenic subclones (Scl, -4, -5, -7, -8, and -9). Similarly, when probe SS6 (INT2, 11q13) was used, highly tumorigenic subclones were heterozygous (8.4-kb fragment and 5.6/2.8-kb fragments), and weakly or nontumorigenic subclones were homozygous
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Weeks after inoculation Figure 1 Tumor-growth curves of parental D98-OR cells and their subclones (Sc). Cells (1 × 107 per site) were inoculated subcutaneously into nude mice, which were examined weekly. Tumor volume was calculated using the following formula: 1/2 × (long diameter) × (short diameter) 2. Each curve shows average tumor volumes from four sites (subclones) or 11 sites (D98-OR). (5.6/2.8-kb fragments alone). For both loci, parental D98-OR showed the mixed feature of heterozygous and homozygous cells. DISCUSSION Studies of HeLa x normal fibroblast hybrids [1-4] and HeLa- or SiHa-normal chromosome 11 microcell hybrids [5, 10] have led to a cervical cancer-suppressor gene being mapped on chromosome 11. Here, we presented an excellent correlation between tumorigenic phenotypes and the allelic composition of chromosome 11. Cells containing two copies of chromosome 11 from different origins had a highly tumorigenic phenotype, whereas those containing two chromsomes 11 of the same origin were weakly or nontumorigenic. This also suggested that chromosome 11 carries a gene that controls tumorigenic expression. However, the conventional notion of tumor suppressor genes, namely the "two-hit" theory [6-9], cannot compatibly explain our results. In this theory, the LOH, which is the most common second hit for excluding a wild-type allele
Chromosome 11 in HeLa Cells
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SS6 (iNT2, 11q13)
Figure 2 RFLP analyses of chromosome 11 in parental D98-OR cells and their subclones (Sc). A) MspI-digested DNAs were hybridized with probe p32-1 (DllS16 locus, 11p13}. B) BamHIdigested DNAs were hybridized with probe SS6 (INT2 locus, 11q13). Fragment sizes are irdicated on the left. The comparison between ethidium bromide-stained gels and autoradiographs confirmed that all cell lines contained two copies for both loci. of t u m o r suppressor genes, s h o u l d promote the malignant p h e n o t y p e [7, 8]. This was not true of c h r o m o s o m e 11 in D98-OR cells. The m a r k e d r e d u c t i o n of tumorigenicity in the h o m o z y g o u s s u b c l o n e s i n d i c a t e d that the LOH in D98-OR cells e x c l u d e d a m u t a t e d gene and d u p l i c a t e d a w i l d type (HeLa-Homo in Fig. 3). F r o m another perspective, the c h r o m o s o m e 11-heterozygous cells carrying both m u t a t e d and w i l d - t y p e genes fully expressed the tumorigenic p h e n o t y p e (HeLa in Fig. 3), suggesting that a single m u t a t i o n ("one hit" sequestered the n o r m a l gene function despite the presence of a w i l d - t y p e allele. One possible m e c h a n i s m is that a m u t a t e d gene product abrogates the wild-tylpe function in a d o m i n a n t negative manner, as reported for p53 gene [16, 17]. If so, the s u p p r e s s i o n by transfer of a n o r m a l c h r o m o s o m e 11 w o u l d indicate that a single m u t a t i o n d i d not overcome two copies of w i l d type (HeLa-#11 in Fig. 3). However, since the tumorigenic segregant from w h o l e cell hybrids (HeLa x N-Tu in Fig. 3) also contained a single m u t a t e d and two w i l d - t y p e genes, this notion cannot be generalized:
Figure 3 Interpretation of the results from this study, whole cell fusion, and chromosome transfer experiments. Chromosome 11 carrying a mutated or wild-type HeLa-suppressor gene is shown as M or WT. The expression (Tu) or suppression (Sup) of tumorigenicity in each cell is indicated in parenthesis. HeLa × N, whole cell hybrid between HeLa and normal fibroblast; HeLa × N-Tu, tumorigenic segregant from HeLa × N; HeLa-#11, HeLa containing an extra copy of normal chromosome 11 via microcell transfer; HeLa-Homo, chromosome 11-homozygous cells in this study. WT/C ratio indicates the copy numbers of wild-type suppressor gene per cell, where a whole cell hybrid is regarded as two cells. The gene d o s a g e - d e p e n d e n t effect of a HeLa-suppressor gene has been i m p l i e d s o m e w h a t by the observation that tumorigenic segregants in cell hybrid experiments (HeLa × N-Tu in Fig. 3) retained one copy of normal fibroblastderived chromosome 11 [1-3, 18]. A s s u m i n g "one-hit" inactivation, the gene d o s a g e - d e p e n d e n t effect can explain both our and other results (Fig. 3). The copy numbers of the w i l d - t y p e gene per cell (WT/C ratio, where we regard a w h o l e cell h y b r i d as two cells) correlated with the expression or suppression of tumorigenicity. WT/C = 1 is not sufficient for suppression (HeLa and HeLa × N-Tu). In contrast, the tumorigenicity is s u p p r e s s e d w h e n WT/C = 1.5 (HeLa x N) or 2 (HeLa-Homo a n d HeLa-#11). We cannot c o m p l e t e l y rule out the involvement of activated oncogenes or putative tumor suppressor genes on other chromosomes. It is also u n k n o w n w h y the cells preferentially lost a m u t a t e d allele and d u p l i c a t e d a w i l d - t y p e allele during in vitro culture. However, we propose the hypothesis of the "one-hit" inactivation of a HeLa-suppressor gene on c h r o m o s o m e 11. This premise fits previous results using w h o l e cell and microcell hybrids. The "one-hit" hypothesis also agrees with the failure to detect an LOH on c h r o m o s o m e 11 in cervical cancers [11]. A n u m b e r of t u m o r suppressor genes, w h i c h can be functionally inactivated by "one hit," m a y r e m a i n to be identified. We thank Dr. Bernard E. Weissman, The University of North Carolina, for providing D98-OR cells. We also thank Drs. H. Kugoh and M. Katoh for helpful discussions, and M. Suzuki for technical assistance. This study was supported in part by a Grant-in-Aid for Cancer
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Research from the Ministry of Education, Science and Culture, Japan, and a Grant-in-Aid for a Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan.
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