Analysis of the Functional Role of Chromosome 10 Loss in Human Glioblastomas1

Home , Chromosome 10, Chromosome 11, Chromosome 4

(CANCER RESEARCH 53. 5043-5050. October 15. 1993] Analysis of the Functional Role of Chromosome 10 Loss in Human Glioblastomas1

Mark A. Pershouse, Elton Stubblefield, Azra Hadi, Ann M. Killary, W. K. Alfred Yung, and Peter A. Steck2

Departments of Neuro-Oncohfy /M. A. P., A. H., W. K. A. Y., P. A. S.], Genetics Â¡E.S.Â¡,and Laboratory Medicine Â¡A.M. K.Â¡,and The Brain Tumor Center Â¡M.A. P., A. H.. W. K. A. Y., P. A. S./, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT tumor suppressor gene in neoplasia. However, the frequent loss of an entire copy of chromosome 10 or large segments of the chromosome Molecular and cytogenetic analyses of primary brain tumors have has made localization of regions harboring a suspected tumor sup shown that losses on chromosome 10 occur very frequently in human glioblastoma multiforme suggesting the presence of a glioma-associated pressor gene difficult to identify. Various studies have identified sev eral potential regions of consistent loss: lOp (14), l()p to 10q23 (12) tumor suppressor gene on this chromosome. To examine this hypothesis, a copy of chromosome 10 derived from a human fibroblast cell line was and 10q22 to qter (13, 14). However, these suspected regions are introduced into the human glioma cell line I 251 by microcell-mediated based on relatively few informative cases. The lack of common re chromosomal transfer. A human chromosome 2 was also independently gions of structural chromosomal abnormalities or chromosomal alter introduced into I 251 cells. The presence of novel chromosomes or chro ations has slowed progress in the search for the tumor suppressor mosomal fragments was confirmed by molecular and karyotypic analyses. gene(s) on chromosome 10 in GBMs. The hybrid clones containing a transferred chromosome 10 exhibited a One approach to demonstrating the existence and possible chromo suppression of their transformed and tumorigenic phenotype in vivo and somal localization of tumor suppressor genes involves introduction of in vitro, whereas cells containing a transferred chromosome 2 failed to a specific chromosome or chromosomal fragment into the appropriate alter their phenotype. The hybrid cells containing a transferred chromo tumor cells by microcell-mediated chromosomal transfer (15-24). The some 10 displayed a significant decrease in their saturation density and an altered cellular morphology at high cell density but only a slight decrease resulting hybrid cells are then examined for the expression of an in their exponential growth rate. A dramatic decrease was observed in the altered tumorigenic phenotype compared with the parental tumor ability of cells with an introduced chromosome 10 to grow in soft agarose. cells. This approach has been used to functionally demonstrate that the The introduction of chromosome 10 completely suppressed tumor forma majority of chromosomes previously implicated through molecular tion when the hybrid cells were injected into nude mice. These findings and cytogenetic studies do contain genes that suppress the tumorige- indicate that chromosome 10 harbors a tumor suppressor gene that is nicity of various types of human tumor cells (15-19). Fragmentation directly involved in glioma oncogenesis. of the chromosome, followed by insertion of the fragments individu ally, has then served to localize the suspected tumor-suppressive re INTRODUCTION gion (16). However, not all candidate chromosomes or chromosomal GBM1 is the most frequent and malignant primary brain tumor with regions suppress growth when introduced into tumor cells (22). This a median posttreatment survival of less than 1 year. Evidence for the observation suggests the need to investigate other possible roles for activation of oncogenes and loss of function of tumor suppressor deleted or mutated genes, such as promoting differentiation or medi genes in this disease has been suggested from various studies. Fre ating the invasive properties of various cancers. quent nonrandom cytogenetic alterations observed in GBMs include In the current study, we examined the possible biological signifi an increased copy number of chromosome 7, alterations in chromo cance of the observed deletions on chromosome 10 by introducing chromosome 10 into a glioma cell line, U251, by microcell-mediated somes 9 and 22, and partial deletions in or loss of an entire copy of chromosome 10 (1, 2). Molecular analyses of GBMs have revealed the chromosomal transfer. The presence of novel chromosome 10 material amplification and rearrangement of the epidermal growth factor re was demonstrated by cytogenetic and molecular methods. Further ceptor gene, which maps to chromosome 7, as well as alterations to its more, the hybrid cells containing chromosome 10 suppressed their protein product (3, 4). Additionally, RFLP analyses have identified ability to grow in soft agar and in nude mice compared with U251 loss of heterozygosity for specific alÃeleson chromosome 9 near the cells and hybrids containing a transferred chromosome 2. The results interferon a and ÃŸgene locus (5-7), deletions and mutations in 17p suggest that chromosome 10 contains a tumor suppressor gene that inhibits both the in vitro and in vivo tumorigenic phenotype of GBM- near or at the p53 gene locus (8, 9), and extensive losses involving chromosome 10 (10-14). derived cells. The deletions on chromosome 10 appear to be the most frequent chromosomal alteration observed in GBMs with 60 to 95% of infor MATERIALS AND METHODS mative cases exhibiting loss of heterozygosity ( 1, 10, 13,14). In recent studies, approximately two-thirds of the GBMs examined had lost an Cells and Cell Culture. The U251-MG cells (referred to as U251) were entire copy of chromosome 10 and another 25% had lost large regions obtained from American Type Culture Collection (Rockvillc. MD) and were of the chromosome (13, 14). Consistent cytogenetic abnormalities and originally derived from a male patient with a GBM (25). Two different mouse allelic losses implicate the possible localization and involvement of a A9 somatic cell hybrids containing individual human chromosome 2 [HA(2)A cells] or chromosome IO (HA-38 cells) were obtained from Dr. Killary lo be used as the microcell donors. Both human chromosomes were from a diploid Received 5/27/93; accepted 8/11/93. human fibroblast cell line and had been tagged with the neomycin resistance 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 accordance with gene by a process similar to those described previously (26, 27). About one- 18 U.S.C. Section 1734 solely to indicate this fact. fifth of the HA-38 cells were also observed to contain a copy of human 1Supported in part by National Cancer Institute Grants ROI CA56041, R55 CA5604I, chromosome 22. HA(1())A cells were subsequently derived from HA-38 cells and ROI CA51I48; a grant from The Gilland Foundation: and Cancer Center Core Grant NCI CA-16672. by microcell mediated chromosomal transfer into A9 cells and were shown to 2 To whom requests for reprints should he addressed, at Department of Neuro-Oncol- contain only a single copy of human chromosome 10. All cell lines were routinely grown in Dulhecco's modified minimal essen ogy. The University of Texas M. D. Anderson Cancer Center. Box 316. 1515 Holcomhe BlOd., Houston, TX 77030. tial medium:Ham's F-12 medium (1:1) and supplemented with 5% fetal bovine ' The abbreviations used are: GBM, glioblastoma multiforme: RFLP. restriction frag ment length polymorphism; DME. Dulbecco's modified minimal essential medium; SSC, serum (Hyclone Laboratories, Logan. UT) with no antibiotics. The HA(2)A, standard saline citrate; SDS, sodium dodccyl sulfate; PCR. polymcrasc chain reaction; HA(10)A, and HA-38 cells were maintained in the presence of 400 /J.g/ml of FISH, fluorescent in .v/'/whybridization. G418 (GIBCO, Grand Island, NY). Cells were harvested with 0.25% trypsin 5043

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1993 American Association for Cancer Research. CHROMOSOME 10 LOSS IN OLIOMAS and 2 min EDTA in Ca2+- and Mg2+-free phosphate-buffered saline and cell Tumorigenicity Assays. To determine tumorigenic potential, cells at pas numbers were determined using a hemacytometer. The hybrid clones contain sages 8-12 were trypsinized and assayed for viability by trypan blue exclusion, ing chromosome 10 were subcultured at a ratio of approximately 1:4. while the and 2 X IO6or 10 X IO6cells in 0.1 ml of serum-free DME:F-12 medium were parental U251 and hybrid cells with chromosome 2 were subcultured at 1:10. inoculated s.c. into 5-6-week old athymic BALB/c-;iw//iM (nude) mice. Ani Anchorage-independent colony forming assays in semisolid medium were mals were examined for tumor formation weekly for over 1 year. Tumors and performed as described previously (28). The cells were routinely monitored for injection sites, in the case of no macroscopically observable tumor, were the absence of Mycoplasma contamination. examined and sterilely dissected from euthanized animals. Portions of tumor Microcell-mediated Chromosomal Transfer. Microcells bearing chromo were cultured in DME:F-12 medium containing 5% fetal bovine serum. Tu somes from the donor cell lines HA-38 and HA(2)A were generated by minor mors formed from hybrid cells containing an introduced chromosome 2 tagged modifications of the procedure described previously (29, 30). Briefly, mitotic with the neomycin resistance gene were cultured in the presence of G418 (200 cells were obtained from log-phase donor cells exposed to Colcemid (0.1 /Â¿g/ml),and cellular viability was determined. fig/ml) for 24 h and then the mitotic cells were physically dislodged and collected by centrifugation at 200 x g for 4 min. The cell pellet was resus- RESULTS pended in Percolliculture medium (1:1) containing cytochalasin B to a final concentration of 20 (xg/ml. The gradient mixture (40 ml total) was centrifuged at 43,000 X g for 70 min at 37Â°C.The portion of the gradient between the clear To assess the possible biological significance of the observed ge netic alterations in chromosome 10 in gliomas, a human chromosome medium top and the Percoli lower layer was removed, diluted 1:4 with serum- 10 was introduced into U251 cells by microcell-mediated chromo free DME:F-12 medium, and filtered through a 3-fim Nuclepore filter (Pleas- somal transfer. Chromosome 2 was independently transferred into anton. CA) with stirring. The purified microcells were collected by centrifu gation at 200 X g for 10 min and resuspended in 4 ml of phytohemagglutinin U251 cells as a control chromosome transfer. Seven U251 hybrid P (250 p.g/ml; Difco, Detroit, MI) in Hanks' balanced salt solution. The clones were isolated by selection with G418 (200 fig/ml) following microccll suspension was then added to the recipient cell monolayer (U251 the transfer of human chromosome 10. Five clones were derived from cells), which was devoid of medium. After a 20-min incubation, the microcell- the microcell mediated transfers of chromosome 2. The individual containing solution was removed and 2 ml of polyethylene glycol (PEG 1500: clones formed colonies 5-7 weeks after selection with G418. Boehringer Mannheim, Indianapolis, IN) was added to the flask for 1 min at Several independent approaches were used to examine the insertion room temperature. The culture was then gently washed 3 times with serum-free of the transferred chromosome in addition to the ability of the hybrid DME:F-12. and growth medium was added. The microcell hybrids were al to proliferate in G418 at concentrations up to 500 /ng/ml. Cytogenetic lowed to remain in nonselective medium for 1-3 days followed by addition of analysis of the parental U251 cells showed a mean number of chro G418 (100-200 /ig/ml) to the recipient cell flasks. Resistant colonies appeared mosomes of 63/cell, of which 5-7 were marker chromosomes (Table 5-7 weeks later, were expanded in the presence of G418. and were then further 1). The hybrids all exhibited a mean number of chromosomes similar characterized. Cytogenetic Analysis and in Situ Hybridization. Cells were subcultured to that of the parental cells with the exception of U251.N10.4 which into flasks 1 day before harvest to assure log phase growth. Bromodeoxyuri- had a mean of 96 chromosomes. Two karyotypically normal copies of dine (0.3 fig/ml) was added 5 h before chromosomal preparation by hypotonie chromosome 10 were observed Â¡nU251 cells, although the original treatment and fixation in methanohacidic acid (3:1) (31). Chromosomal karyotypic analysis of U251 cells revealed only one copy of chromo spreads were prepared and banded using the trypsin-Giemsa technique as some 10 (25). However, our U251 cells by in vitro passage 18 were described previously (32). At least ten chromosomal spreads were analyzed for observed to have two copies of chromosome 10. Additionally, no each hybrid clone. RFLP markers for chromosome 10 were shown to be polymorphic in situ hybridization using biotinylated DNA probes for chromosome spe (see below), suggesting that duplication of chromosome 10 had oc cific human Alu sequences, chromosome-specific centromeric probe D10Z1 curred in these cells. In Cytogenetic analyses on the hybrid cells, (Oncor, Gaithersburg. MD), or total mouse Cot 1 DNA(GIBCO, Gaithersburg, karyotypes of four of the clones (N1Ãœ.2,N10.3, N10.5, and N10.6) MD) was performed as described previously (33) with propidium iodide coun- tcrstaining. The chromosome-specific human Alu sequences were generated showed a consistent increase in the copy number of chromosome 10 (~80% of metaphases examined; range, 95 to 60%). The other hy- using Alu primers (34) from the DNA of HA(2)A and HA(1())A cells. The probes were biotinylated by random hexamer-primed synthesis in the presence of biotin-16-dUTP(35). Table 1 Cylogenelic and FISH analysis of recipient ami hybrid cells RFLP Analysis. Southern blot analysis was performed using chromosome- specific polymorphic markers in order to document specific portions of the transferred chromosomes that were retained by the hybrid cells. High molecu no.of 10fragments/percellc(not lar weight DNA of the cell lines and clones was prepared, digested with ofchromosomes(range)63intactchromosome10Â°23-42-42-32-32-32-32-4%ofmetaphaseswithadditionalchromosome10*HA'68954072922737Markerchromosome appropriate restriction enzymes, fractionated by 0.8% agarose gel electropho- inU251)-'1(0)1-3(1)2(0)y2(2)3-Â» resis, and transferred to nylon membranes (36, 37). Probes used included CelllineU251U251.N10.2U251.N10.3U251.N10.4U251.N10.5U251.N10.6U251.N10.7U251.N10.8Meanno. D10S24, D10S28, D10S30, D10S15, D10S5, D10S22, PLAU, D10S14, (53-69)58 D10S4, GLUD, D10S20, CYP2C, D10S27, D10S25, and D10S6 on chromo (52-66)57 some 10, and D2S48 and D2S49 on chromosome 2 (38). Probes were radio- (52-66)96(76-106)61 actively labeled by random hexanucleotide priming (35) in the presence of [12P]dCTP. Blots were hybridized at 42Â°Cfor 16-24 h with 32P-labeled probes (55-67)60 in 50% formamide, 10 x Denhardt's, 5 x SSC, 1% SDS, and 0.2 mg/ml (54-65)61 (2)2-3 (53-65)62 (2)2^t(2) sheared salmon sperm DNA. The membranes were sequentially washed in 6 X (52-69)Copy SSC 0.1% SDS (23Â°C,30 min); 1 x SSC 0.1% SDS (37Â°C,30 min); and 0.1 "The number of chromosome 10 fragments/metaphase spread was determined hy x SSC, 1% SDS (65Â°C, 60 min), prior to autoradiography. Alternatively, FISH analysis, which was in agreement with G handing using chromosome 10 specific microsatellite repeat DNA polymorphisms were examined by PCR (39). PCR Alu-PCR probes. h Frequency of metaphases examined per hybrid clone that exhibited an increase in primers (mapped pairs) were obtained from the American Type Culture Col copy number of intact chromosome 10 as determined by FISH analysis. lection or the Genetic Institute (Huntsville, AL): D2S72, CRYG1, and GCG for ' Chromosome 10-associated material was determined by FISH analysis using chro chromosome 2; D10S89, D10S110, D10S88, D10S168, and D10S169 for mosome 10-specific Alu-PCR biolinylated probes. Each derivative chromosome contain chromosome 10; and D22S264, D22S258, and D22S259 for chromosome 22. ing a fragment of chromosome 10 was determined. rf Numbers in parentheses, novel chromosome 10 derivatives not found in U251 cells. The PCR products were labeled with [ÃŽ2P)ATPasdescribed (40) followed by ''NA, not applicable. resolution of the polymorphic markers by nondenaturing polyacrylamide gel 'Does not include multiple translocations observed in approximately 10% of meta electrophoresis on DNA sequencing apparatus and autoradiography. phases. 5044

Fig. 1. Results of fluorescent in situ hybridization with hiotinylated human chromosome 10 specific inter-Alu-PCR probes on metaphasc spreads of (A ) U25I, (B) U25I.N10.3, (C) U251.N10.5, and (O) U251.NIÃœ.6cells. Solid arrows, intact chromosome 10; open arrows, fragments of chromosome 10 observed in most cells; A. marker chromosome 10 fragment. All the chromosome 10-containing hybrid cells exhibited an additional copy of chromosome 10. novel fragments of chromosome 10, or both.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1993 American Association for Cancer Research. CHROMOSOME II) LOSS IN OLIOMAS brids did not exhibit a consistent gain (<60% of metaphases), al O D10S24 though all clones had some metaphases that displayed an increase in p15 â€¢¿D10S28 copy number of chromosome 10. p14 â€¢¿D10S89 To further clarify the distribution of chromosome 10-associated p13 â€¢¿D10Z1 DNA retained in the various hybrids, FISH was performed on meta- pi2 O DIOSSO phase spreads of the hybrid cells using biotinylated chromosome 10 specific inter-Alu-PCR products (Fig. 1). The Alu-PCR products were D10S15 generated using human specific Alu primers (34) and HA(10)A DNA. D10S5 No PCR products were observed using mouse A9 DNA as a template. D10S22 Results of the FISH analysis confirmed the cytogenetic analysis re qZ1 PLAU sults showing that four hybrids (N10.2, N10.3, N10.5 and N 10.6) that D10S14 D10S4 showed a gain of a copy of chromosome 10 consistent with the q22 cytogenetic analysis (Table 1; Fig. l, B and C ). The other three D10S91 hybrids showed a gain of an apparently intact chromosome 10 in q23 D10S110 27-40% of the metaphases examined (Table 1). D10S168 Novel chromosome 10-containing fragments were observed in the q24 D10S169 vast majority of hybrid and parental cells examined. A marker chro q25 CYP2C mosome with a segment of chromosome 10 inserted into the middle of q26 D10S27 a marker chromosome was observed in the parental cells and the D10S25 majority of the hybrid cells illustrated in Fig. 1. Various other novel DIOS6 fragments were seen in the different hybrid cells containing a trans ferred chromosome 10 (Fig. IÃŸ).Three of the fragments appeared to O - Non-informative be found only in individual clones, while two other fragments were O - Gain in some clones represented in several clones. The majority of novel derivatives ap peared to contain only chromosome 10-associated material as assessed â€¢¿-Gain in all clones by chromosome painting. However, chromosomal translocations were Fig. 3. Allclic gains among the hybrid cells showing regions of consistent gain of observed in several metaphase spreads, particularly in clone N 10.4. heterozygosity of loci on chromosome 10. Two regions of consistent gain were determined [â€¢)one on the p arm and one on the q arm. The clones that consistently failed to display an increase in the copy number of chromosome 10 exhibited a high frequency of chromosome 10 fragments. The hybrid N 10.6 displayed both a high frequency of a hybrids. Three other lOq loci (D10S5, D10S25, and D10S110) were gain of an intact copy of chromosome 10 and novel fragments (Table gained or showed only partial gains in certain clones (Fig. 3). The 1). This analysis showed that all the clones displayed additional chro partial gains would reflect the genetic instability and chromosomal mosome 10 material, either an entire chromosome or novel fragments fragmentation observed in the various clones (Table 1, Fig. 1). Loss of not found in the parental cells. all three loci was observed only in clone N 10.8. In contrast, the clones To further document the transfer of regions of chromosome 10, that cytogenetically had an additional copy of chromosome 10 showed RFLP and microsatellite repeat polymorphism analyses were per gains at all the loci. The remainder of the probes were uninformative. formed using a variety of human chromosome-specific polymorphic The parental U251 cells exhibited no chromosome 10 polymorphic DNA markers (Fig. 2). That several informative loci were observed alÃeles,suggesting that the two copies of chromosome 10 were iden illustrates the introduction of certain regions of chromosome 10 into tical. Three polymorphic markers (D22S258, D22S257, and hybrid clones (Fig. 3). Two 10p loci, D10S28 and DÃŒOS89,andtwo D22S264) that showed different polymorphic alÃelesbetween HA-38 lOq loci, CYP2C and D WS 169, were gained in all the 10 containing and U251 cells exhibited no gain in any of the hybrid cells. These results indicate that the human chromosome 22 observed in some of 123456789 10 the HA-38 cells was not transferred or retained in any of the clones. as- Â«TÂ»- pÂ»- - - The hybrids containing chromosome 2 were similarly examined. Chromosome painting with chromosome 2-specific inter-Alu-PCR ^ D10S28 probes showed that the hybrid cells contained an additional copy of 2.4- â€”¿ - - chromosome 2. RFLP analysis of informative probes (D2S48, D2S49, â€¢¿ D2S72, and GCG) showed a gain of polymorphism at markers on both 4.5- - - Â«--Â«Â» arms of the chromosome (data not shown). The combination of these as- *Â»â€¢¿Â»- D10S5 analyses suggests that both hybrids contained a transferred copy of chromosome 2. To screen for the presence of mouse chromosomes or chromosomal fragments, metaphase spreads were hybridized to biotinylated mouse 1.0- â€¢¿â€¢D10S15 Cot 1 DNA, followed by treatment with fluorescein isothiocyanate- avidin. Control spreads from A9 hybrids containing a human chro mosome revealed differential staining of the mouse and human chro Fig. 2. Analysis of gain of RFLP alÃelesin hybrid cells compared with recipient parental U251 and HA-3X donor cells by Southern hint hybridization. Samples of parental mosomes. For all the hybrid cells examined, no hybridization to and hybrid cells DNA (20 Â¿ig)were digested with 7ii

m S&SfÃ¬*/''

Fig. 4. Morphologies of (A) U251 cells, and hybrid cells containing an inserted chromosome 10, (fl) U251.N10.2. (C) U251.N10.6. and (O) U251.N10.8. The cells were grown to near their saturation densities. compared with the parental cells. Most of the hybrid clones also recipient cells (27, 41). The N 10.3 cells exhibited the most consistent displayed an increase in cell size (Fig. 4). At confluence, the U251 gain of chromosome 10-associated material and they also showed the cells formed tightly packed monolayers such that the individual cells slowest growth rate. were difficult to distinguish (Fig. 4). The hybrid cells, however, ex We examined the ability of the cells to form colonies under an hibited a variety of morphologies and processes and individual cells chorage- independent conditions. The U251 cells exhibited a colony were clearly visible. The hybrid cells containing chromosome 2 had a forming ability of approximately 10-35%, depending on seeding den morphology similar to that of the parental U251 cells. sity (Table 3). The hybrids containing chromosome 2 showed a com The differences in morphology were also reflected in the saturation parable level. In contrast, chromosome 10 hybrid cells had a signifi density of the hybrid cells. All the hybrid cells containing chromo cantly reduced colony-forming capability [between 6- and 500-fold some 10 had a significantly lower saturation density than did the lower (Table 3)]. Furthermore, the colonies that did form were of parental cells or the hybrid cells containing chromosome 2 (Table 2). significantly smaller diameter compared to the control cells (Fig. 5). The exponential growth rates of the hybrid cells, with either intro The U251.N10.5 cells exhibited an intermediate colony forming abil duced chromosome, was decreased compared with that of the parental ity. However, some of the colonies that did form had a diameter cells. This observation is in agreement with other studies showing that similar to that of the parental cells. Preliminary analyses of the U251. the addition of chromosome(s) may decrease the growth rate of the N10.5 cells suggest that this clone is now composed of several sub- populations, some of which contain a fragmented chromosome IO.4 Table 2 In vitro characteristics of hybrid cells TÃ¼morigenicity. Parental U251 cells and U251 hybrid cells con taining a neomycin-tagged chromosome 2 or 10 were assayed for their density time tumorigenic potential by s.c. injection into the flank of athymic nude (X104/cm2)264.41.6237.8118.75.62229Doubling(h)"364X6557414249463841 CelllineU251U251.N10.2U251.N10.3U251.NK1.4U251.N10.5U251.N10.6U251.N10.7U251.N10.8U251.N2.1U251.N2.2ChromosomeaddedNAIII11)111111III11)HI22Saturation mice (Table 4). The parental U251 cells and hybrid containing chro mosome 2 formed tumor in 100% of the animals given injections. The hybrid cells with chromosome 2 exhibited a slight increase in the latency period to form clearly identifiable tumors (0.5 cm3) which may reflect a slight decrease in exponential growth rate of these cells (Table 2). When tumor cells were cultured in vitro from the mice inoculated with the hybrid cells containing chromosome 2, the cells were able to proliferate in the presence of G418 (2(X) Â¿ig/ml),sug-

" Doubling time was determined from cells in exponential phase of growth. 4 M. A. Pershouse and P. A. Steck, unpublished observation.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1993 American Association for Cancer Research. CHROMOSOME 10 LOSS IN OLIOMAS gesting the retention of the marked chromosome in these cells. We Table 4 Tumorigenicity in nude mice ofU251 parental and hybrid cells containing observed no tumor formation in any of the mice inoculated with chromosome 2 or 10 Cell hybrid cells containing chromosome 10 (Table 4). The U251.N10.5 (days)"73NTJNTNTNTNT63NTNTNTNTNT7770Tumorigenicity*5/50/50/50/40/40/5'4/40/40/411/40/40/44/44/4 cells, which exhibited some colony-forming ability in soft agarose, lineExperiment 1'U251U251.N10.2U251.N10.3U251.N10.6U251.N10.7U251.N10.8Experiment also failed to form tumors. Furthermore, residual hybrid tumor cells were not detected at the injection sites, suggesting the regression of the hybrid cells rather than a failure to grow (42). Tissue was obtained at the injection sites and processed for PCR analysis using human Alu primers; human-specific sequences were inconsistently visualized. One assay was conducted over a period of 1 year with no tumor 2U251U251.NH1.2U251.N10.3U251.N10.5U251.N10.6U251.N10.8U25I.N2.1U251.N2.2Latency formation, indicating that the altered tumorigenicity was not a result

Table 3 Anchorage-independent colony formal ion No. of colonies0 (initial seeding density) I)-11358 CelllineU251U251.N10.2U251.N10.3U251.N10.4U251.N10.5U251.N10.6U251.N10.7U25I.N10.8U251.N2.1U251.N2.2X103366" Average number of days for tumors to grow to approximately 0.5 cm1 within each Â±17*7Â±63Â±2'I9554743313228321625292 Â±21127 group. h Tumor formation of hybrid (passage 8â€”12)orparental cells injected in female nu/nu Â±63 mice 6-9 wk old. Â±14Â±2207 '' For experiment 1, 2 X IO"1cells in 0.2 ml were injected s.c. into the flank of nude mice, one injection per mouse. For experiment 2, 10 X 10h cells in 0.15 ml were injected Â±10724 into the mice. Â±1035 d NT, no tumor detected. The assay time for experiment 1 was approximately 4(X) days; Â±2259 experiment 2 was for at least 1(X)days. Â±171233 '' One mouse was observed to have a tumor in a regional lymph node upon pathological examination (see text). Â±1271197 17xl Â±233 "The effects of seeding density on the growth of human U251 and hybrid cells of the decreased growth rates observed in some of the chromosome 10 containing inserted chromosome 2 or 10 in soft agarose. The cells were grown for 6-7 wk containing hybrids. Additionally, varying the number of cells injected and rcfed weekly. Similar results were obtained in several independent assays. h Mean Â±SDfor each clone. did not alter the tumorigenicity of the clones, although a difference in the latency period was observed. In one U251.N10.8-injected mouse, a tumor bearing regional lymph node (~1 mm3) was observed only upon autopsy. The histolÃ³gica! makeup of the tumor cells was similar to that of the parental tumor. Attempts to grow the tumor cells were not successful; therefore, the genetic status of these tumor cells could not be determined.

DISCUSSION Several previous studies have documented the capacity of microcell-mediated chromosomal transfer to generate strong biological evi dence for tumor suppressive activity associated with certain chromo somes or chromosomal fragments in a variety of human cancers (16-24). In the present study, we have demonstrated that the intro duction of a chromosome 10 derived from human fibroblast cells into a human glioma cell line (U251) suppressed the tumorigenicity of the hybrid cells. The hybrid cells, which contained either an entire chro mosome 10 or fragments of chromosome 10, all exhibited an inhibi tion of the malignant phenotype shown by the parental cells, as de termined by their inability to form tumors in nude mice. These hybrid cells also appeared to regress in nude mice with no evidence of tumor formation over a period of 1 year. This suppression could not be attributed to the transfer of nonspecific chromosomal material because introduction of human chromosome 2 failed to alter the tumorigenic phenotype of these hybrid cells. The decrease in tumorigenicity and the transformed phenotype of the hybrid cells appeared to correlate in this experimental system. Cytogenetic analysis of the parental U251 cells and the hybrid cells revealed heterogeneity in the karyotypes, as has been shown previ ously for these cells (25). From 5 to 10 marker chromosomes were observed in the various cells, although most of the cells exhibited a mean number of chromosomes of about 61, except N10.4 cells, which Fig. 5. Colony-forming ability of (A) U251 cells and hybrid cells containing an were near tetraploid. Four of the hybrid cell clones displayed an inserted chromosome 10, (B) U251.NK). 2. (C) U251.NI0.6. and (D) U251.N10.8. Note the differences in the quantity and size of colonies. Hybrid cells with a transferred increase of a complete copy of chromosome 10 in the majority of their chromosome 2 formed colonies similar to those formed by the parental U251 cells. cells. However, all hybrids remained G418 resistant, exhibited a sup- 5048

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1993 American Association for Cancer Research. CHROMOSOME lÃ¼LOSS IN OLIOMAS pressed tumorigenic phenotype, and showed a dramatic decrease in reminiscent of the morphology of astrocytic cells suggests a possible their anchorage-independent colony forming ability. The FISH analy induction of differentiation following the transfer of chromosome 10 sis revealed the presence of additional copies of chromosome 10 or (20, 43). However, the parental U251 and hybrid cells all expressed fragments of chromosome 10 in all the hybrid cells. The hybrid cells glial fibrillary acidic protein, a marker of astrocytic differentiation; that failed to gain a complete copy of chromosome 10 gained novel therefore, the differentiation of these cells was not altered as measured fragments of the chromosome not observed in the parental cells. Some by this criterion. hybrid cells gained both an entire copy and fragments of chromosome This study shows that the introduction of chromosome 10 into 10. However, all cells contained at least two intact copies of chromo human glioma cells suppresses both the transformed and tumorigenic some 10. The molecular analysis suggests that the transferred chro phenotype. The hybrid cells were viable and continued to proliferate mosome was the one that underwent fragmentation or rearrangement, with a slight decrease in their growth rates. The paucity of informative inasmuch as various hybrid cells showed incomplete gain of poly alÃeleshas made localization of the suppressor region difficult, how morphic alÃeles.The instability and fragmentation of transferred chro ever, we are further investigating clones of U251.N10.5 cells that mosomes have been observed previously in several systems (20, 43- appear to have fragmented the introduced chromosome and have 45). Inherent karyotypic heterogeneity of glioblastoma cells has partially regained their transformed phenotype. The N 10.5 cells also previously been reported (1, 2, 13, 14) with an associated increase in illustrate that the other chromosome 10 containing hybrids retained genetic instability of glioblastoma compared with the lower grade the suppressive region(s), even though some of the hybrids failed to brain tumors (10). retain an intact copy of chromosome 10; the nonsuppressed N 10.5 RFLP analyses exhibited two regions of consistent gain, one each clones were easily identified by their colony-forming ability. Neither on the short and long arms of chromosome 10 (using probes D10S28/ the other chromosome 10- containing hybrids nor the majority of D10S89 and D10S169/CYP2C, respectively). The hybrid cells that N10.5 cells formed colonies of significant size compared with the gained an entire copy of chromosome 10 exhibited gains at all infor nonsuppressed parental and chromosome 2-containing hybrids. Fur mative loci. Preliminary results of FISH analyses suggests that the thermore, preliminary studies suggest that the colony-forming U251. neomycin resistance gene is integrated in the short arm of chromo N10.5 subclones form tumors in nude mice, emphasizing the corre some 10, which may reflect the retention of the loci on the p arm in lation between tumorigenicity and the ability to grow in soft agar in all the hybrids. However, the possible location of a tumor suppressor this system. gene could not be ascertained from this analysis because many of the examined loci were uninformative. In contrast, three loci (D10S5, REFERENCES D10S25, and D10S110) exhibited gains or partial gains in only some 1. Bigner, S. H., Mark, J.. and Bigner, D. D. 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Mark A. Pershouse, Elton Stubblefield, Azra Hadi, et al.

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