[CANCER RESEARCH 54, 3173-3178, June 15, 1994] In Vivo Enhancement of Genomic Instability in Minisatellite Sequences of Mouse C3H/10T½ Cells Transformed in Vitro by X-Rays' Benoit Paquette and John B. Little2 Harvard School of Publk Health, Laboratory ofRadiobiology, Boston@Massachusetts 02115 ABSTRACT The level of genomlc instability was determined during tumor devel opment in vivo.Genomic rearrangements, a marker ofgenomlc instability, was measured mouse C3H/10T½ cells transfonned in vitro by X-rays with a DNA fingerprinting assay. Three transformed clones IsOlated from type ifi foci were divided into two groups Cells from the first group were [email protected]. into syngeneic and noaimmunosuppressed C3H mice. After 3 to 5 months, the tumors were excised, and the neoplastic cells were Isolated and subcloned. Cells from the second group were incubated in vitro for 25 passages (about 6 months) to approximate the number of cell divisions occurring in the tumor, and then they were subcloned. DNA was extracted from subclones grown in vitro and in vivo and analyzed with the DNA fingerprinting assay. A high frequency of genomic rearrangements (50-100%) was found in subclones derived from tumors that arose in vivo, whereas the frequency was very low (<10%) among subclones passaged in vitro, genetic suggesting that instability may be enhanced by factors present in the C3H mouse. In one clone (F-17) genomic Instability ap peat-edto be activated and down regulated. The high frequency of insta bility found in tumor cell subclones did not appear to result from an in vivo selection of a more tumorigenic subpopulatlon of cells present In the original clone prior to injection in the animal. This enhancement of genomic instability occurring in vivo could be required to complete the process of transformation to tumorigenicity and allow the neoplastic cells to adapt to a new environment. INTRODUCTION It is now widely accepted that the development of cancer pro ceeds by sequential steps from a normal cell to an invasive, metastatic tumor (1—4).Many genes are involved in this process including oncogenes, tumor suppressor genes, and genes involved in angiogenesis and the development of metastases. Since new oncogenes or tumor suppressor genes are still being discovered, it is likely that we are now observing only a fraction of the relevant mutations present in tumors (5). Nevertheless, the estimated num ber of mutations necessary for tumor development can vary from as many as 7 in the case of stomach cancer, up to 12 in the case of prostate cancer (6). Estimations based on spontaneous mutation rates in normal human cells cannot account for the large number of mutations found in an individual tumor cell (5). To explain the appearance of all these mutations in tumor cells, genomic instability has been proposed as a important cellular process in tumor heterogeneity and progression (2). The concept that genomic instability can be an important process in human cancer also arises from two other observations: (a) the incidence of most adult cancers exhibits an exponential rate of increase with age, suggesting a series of events each rendered more likely by the occurrence of a previous event (7); (b) cancer predisposition can be a heritable event as evidenced by the genetic chromosome instability syndromes, such as Bloom's syndrome (8), as well as ataxia-telangiectasia patients whose cells in vitro have an elevated frequency of translocations and a 10-fold increase in the loss of heterozygosity at the glycoprotein locus (9). Ataxia-telangiectasia patients rarely live beyond 30 years of age, and nearly 40% develop cancer (10). Genomic instability includes all mechanisms that accelerate the accumulation of damage or modifications to DNA, such as gene amplification, translocation, recombination, or point mutation. Many tumor cells exhibit rates of gene amplification, a type of genomic instability, several orders of magnitude greater than their normal counterparts (11, 12). Cells of high metastatic potential have been reported to exhibit a 7-fold increase in mutation rate compared to cells of low metastatic potential (13). The appearance of coincident muta tions in TK6 lymphoblast clones exposed to X-rays (14) and the acceleration of the spontaneous mutation rate at the hprt locus in mutant Chinese hamster cells also supports the concept of genomic instability (15). Although these data clearly indicate the existence of genomic instability, in vivo factors affecting its activation are not known, and a causal relationship between genomic instability and tumor progres sion remains to be established (16). The present study was thus designed to examine whether the genomic instability could be stim ulated by in vivo factors. The mouse fibroblast C3H/10T½ cell line was used as model because in vitro transformation of these cells is well documented (17—20),and their genomic instability can be mon itored by measuring the genomic rearrangements occurring in a spe cific minisatellite family with a DNA fingerprinting assay (21). The frequency of genomic rearrangements in the minisatellite family among clones growing as a tumor in syngeneic nonimmunosup pressed C3H mice was compared with that in clones that underwent approximately the same number of cell divisions in in vitro culture. The relationship between the level of genomic instability and the malignant potential of these transformed clones was also studied. MATERIALS AND METhODS Cells and Culture Conditions. Mouse C3H embryo-derived fibroblast cells (10T½,clone 8) characterized by Reznikoff et aL (17) were grown in Eagle's basal medium supplemented serum (Sigma Chemical with 10% heat-inactivated Co.), penicillin (50 units/mI), fetal bovine and streptomycin (50 p@g/ml). Thesecells wereaneuploidwith a stablemodeof 81 chromosomesand were used between passage 8—12. Extraction of GenomicDNA DNAwas extractedaccordingto a salting out procedure described by Miller et aL (22). Briefly, about 5 X i0@cells were trypsinized, washed in phosphate-buffered saline, and centrifuged a second time. The cell pellet was resuspended in 3 ml of a buffer containing 10 mM Tris-HCI, 400 mM sodium chloride, and 2 mp.i EDTA. To the cell suspension, 0.1 ml of SDS3 (20%) and 0.5 ml proteinase K (10 mg/rn]; DNase free) were added and incubated overnight at 37°Cor at 50°Cfor 3 h. The DNA was precipitated by the addition of 1.2 ml of 5 Msodium chloride. The tube was agitated by hand for 1 mm and centrifuged at 2500 rpm for 15 mm; the supematant was transferred to another tube. The DNA was precipitated with Received 2/3/94; accepted 4120/94. charges. This article must therefore be hereby marked advertisement in accordance with 2.5 volumes of 95% ethanol, the tube was gently inverted for 30 s, and the DNA was spooled out and air dried briefly. The DNA was dissolved in it 18U.S.C.Section1734solelyto indicatethisfact. buffer (10 mM Tris-HCI-1 mM EDTA, pH 8.0), and RNase A (0.05 ml of a Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page 1 This research was supported by Grants CA-47542 and ES-00002 from the NIH and a fellowshipto B. P. from“Lea Fondade Ia Rechercheen Santedu Québec.― 2 To whom requests for reprints should be addressed, at Harvard School of 3 The Public abbreviations used are: SDS, sodium dodecyl sulfate; SSC, standard citrate. Health, Laboratory of Radiobiology, 665 Huntington Avenue, Boston, MA 02115. 3173 Downloaded from cancerres.aacrjournals.org on April 23, 2015. © 1994 American Association for Cancer Research. saline IN VIVOGENOMICINSTABILITY 10 mg/mI solution) was added and incubated for 1 h at 37°C.The DNA was precipitated it a second time with ethanol as described above and redissolved in buffer. DNA Fingerprinting Analysis. Ten @gof each sample of DNA were digested by Hinfi according to the recommendations of the manufacturer (New •1 England Biolabs, Inc., Beverly, MA). Digested DNA was separated in 0.8% agarose gels (25 cm long). Electrophoresis was performed at 3.3 V/cm for 32 h; the gel was then soaked in 0.25 M HCI for 20 mm to induce chemical 9.4kb cleavage of the DNA and then in 0.4 MNaOH for 40 mm. Transfer of DNA -+ a1 onto Duralose-UV nitrocellulose membranes (Stratagene, La Jolla, CA) was performed according to the procedure described previously (23). The mem branes were baked for 2 h at 80°C;prehybridized for 3 h at 65°Cin 6 X SSC, 5 x Denhardt's, and 1% SDS; and hybridized for 16 h at 65°Cin 6 X SSC, 5 X Denhardt's solution, 1% SDS, 10% dextran sulfate, and 5 X 10@ cpm/ml r of 32P-labeledprobe M. The membranes were washed three times for 15 mm at room temperature in 1 X SSC-1% SDS and one to three times at 65°Cin r r 0.1% SSC-0.5% SDS. The washed filters were exposed at —70°C to a Fuji RX film with intensifying screens for 2 to 7 days. The plasmid carrying the multilocus multiallele probe M used for this study I was provided by Dr. Brian J. Ledwith (Merck Sharp and Dohme Research Laboratories, West Point, PA). Synthetically generated, probe M is based on a minisatellite sequence found in the mouse major histocompatibility complex (24) and is a tandem repeat of four nucleotides (52 X 5'-AGGC). @0 This probe was isolated from the plasmid as described previously (21). Fig. 1. Banding pattem of DNA extracted from the livers of normal (host) mice that was digested with Hinfi and analyzed on a DNA fingerprinting assay. Livers shown in Tumorigenicity. Tumorigenicitytestingofcells isolatedfromType IIIfoci and nontransformed C3H/10T½cells was carried out by inoculating 2 X 106 Lanes 3 and 4 were derived from host mice injected with the transformed clone shown or 2 X l0@cells s.c. in the dorsal region of syngeneic nonimmunosuppressed (X-ray-9 and F-17). C3H mice (C3H/HeNcr1BR; Charles River). Four to nine animals were in jected with each transformed cell clone. The number of progressively growing, nonregressing tumors that developed by 5 months after injection was scored. RESULTS DNA Fingerprinting Pattern from Normal C3H Mouse Cells. The tumors used for this study were obtained after s.c. injection of transformed 10T½cells in the back of C3H mice. The tumors were not invasive, since they grow as a separate mass of tissue isolated from the back muscles of the mice. Although some normal cells required for the development of the tumors were present, such as blood cells, the cell population in this type of tumor was clearly much more homogeneous than for an invasive human tumor or an animal tumor induced in vivo following exposure to a carcinogen. Neverthe less, the cells isolated from the tumors were cultured in vitro for 6 weeks prior to extraction of DNA for the purpose of eliminating most of the nontumor cells. In order to confirm that cells isolated from the tumor and subcloned were the indeed derived from the 10T½trans formed cells previously injected, their DNA fingerprinting pattern was compared to the banding pattern of DNA isolated from the liver of the host mouse. The DNA fingerprinting assay we used allows the detec tion of minisatellite families characterized by a tandem repeat of 4 to 10 nucleotides dispersed throughout the genome of the cell. Since each locus contains a different number of tandem repeats, digestion of the genomic DNA with a restriction enzyme gives specific fragments for each locus that can be easily visualized on an agarose gel. As these loci are highly polymorphic, the banding pattern for these fragments will vary from animal to animal. DNA derived from the livers of mice carrying tumors derived from the clones (X-ray-9 and F-17) as well as three additional mice was analyzed. The livers were excised and trypsinized, and the DNA from the single cell suspension was extracted as described in “Materialsand Methods.―After digestion ofthe DNA with Hinfi, the banding pattern of minisatellite sequences was obtained with the probe M. As is shown in Fig. 1, none of these band patterns matched the ones obtained with transformed 10T½cells (Figs. 2, 3, and 4). The DNA fingerprinting pattern of liver from the mouse carrying the tumor derived from clone X-ray-i I was not available. Thus, we cannot exclude the possibility that a banding pattern might belong to the normal host cells. Five new banding patterns were detected, which obviously cannot be caused by a contamination of tumor by normal mouse cells. Based on these data, we conclude that the modifications detected in the subclones of cells isolated from the tumors have occurred during development of the tumor and do not reflect contam ination with host cells. In Vivo Enhancement of Genomic Instability. This experiment was undertaken to determine whether genomic instability in 10T½ cells transformed in vitro by X-rays can be enhancement factors present in the host animal after injection in vivo. The three trans formed clones analyzed were isolated from type III foci. The trans formation of 10T½cells was performed in vitro with 6 Gy of X-rays according to the established protocol (18). In a previous study (21), genomic rearrangements in a family of minisatellite sequences hy bridized with probe M was used as a marker of genomic instability (21); 12 transformed clones were analyzed, five of which were tu morigenic in syngeneic and nonimmunosuppressed C3H mice. Tu morigenic clones with different levels of genomic instability were chosen for the present study. These clones are X-ray-9 and X-ray-il, which showed no detectable genomic instability, and clone F-17, which was the most unstable with four genomic rearrangements detected in cells isolated from the type III foci. As these rearrange ments occurred in most of the cells as determined by Southern blots (21), they presumably arose early in the process of transformation. Cells from each of these three transformed clones were divided into two groups. The first group was injected s.c. in syngeneic and non immunosuppressed C3H mice. After 3 to 5 months, the tumors were excised, and the tumor cells were isolated as described in “Materials and Methods.―The tumor cells were then cultured for 6 weeks to eliminate normal cells and subcloned into 96-well microtiter dishes. As controls, DNA was extracted from the cells of the second group immediately after their isolation from the type III foci. Additional cells were cultured in vitro for 25 passages (i.e., 6 months or about 125 population doublings) to approximate the number of cell divisions occurring in the tumor and then subcloned. The DNA of these sub clones isolated after 25 passages in vitro, as well as DNA from the 3174 Downloaded from cancerres.aacrjournals.org on April 23, 2015. © 1994 American Association for Cancer Research. @ @: @ @ @ @ w@ -%0 ,<;@a@ @O ‘0 @, %O -@ @p -@ — @o ‘P ‘P .M@ IN VIVO GENOMIC INSTABILITY IN VIVO iN VITRO ——— — 9.4 kb .@ Fig. 2. DNA fingerprinting of subclone X-ray-9 derived from transformed cells propagated in vitro and in vivo.In vitro (Lanes on left) transformed cells and subclones derived from tumor arising in vivo (Lanes on right). Lane 1, fingerprinting pattern of original clone X-ray-9. subclones derived from the tumors that developed in vivo, was ex tracted and analyzed with the DNA fingerprinting assay. This proce dure allowed: (a) detection of genomic instability after a long period of culture in vitro; and (b) determination of whether genomic insta biity is stimulated by factors present in the animal. Figs. 2, 3, and 4 illustrate the DNA fingerprinting analysis for the subclones derived from transformed clones X-ray-9, X-ray-li, and F-17, respectively. Following cultivation in vitro, no genomic rear rangements were detected for the subclones from X-ray-9 (Fig. 2); the same banding pattern was present in cells isolated immediately or after 25 passages in vitro. One of nine subclones from X-ray-i 1 (Fig. tion of genomic instability is not limited to a single pattern of genomic rearrangements. In order to eliminate the possibility that these data might be the result of in vivo selection of more transformed or more aggressive subpopulations of cells present before injection into the animal, the tumorigenic potential of randomly selected subclones derived from tumors that developed in vivo was compared to that of their original transformed clone. Since a subpopulation representing less than 1% of the cells in original transformed clones can be present in the cell population and not be detected with the DNA fingerprinting assay, mice were injected with 2 X iO@cells of in vivo subclones instead of 3) andoneof eightfromF-17 (Fig. 4) showeda genomicrearrange 2 X 106 cells for the initial injection (Table 2). The tumorigenicity of ment. In contrast to this low frequency of genomic rearrangements each in vivo subclone was tested in four mice. After 5 months, no following cultivation in vitro, new genomic rearrangements were tumor appeared for four of the five in vivo subclones tested as well as observed in 50—100%of the in vivo subclones studied (Table 1), and for the original clones injected at this same low cell number. Only one up to nine different banding patterns were detected (Fig. 2, 3, and 4). mouse of four injected with 2 X iO@cells of the subclones X-ray-il A-7 developed a tumor. These data indicate that the in vivo subclones In vivo subclones from X-ray-li showed the highest degree of genomic instability since five different banding patterns were ob tested do not have a higher tumorigenic potential than the original clones and suggest that the new pattern of genomic rearrangements served. These data indicate that: (a) new genomics rearrangements are not artifacts caused by an in vivo selection of a more aggressive have occurred during tumor development in vivo; and (b) the activa INVIVO IN ViTRO - .5 ... - @ 9.4 kb _— — — — — — — ——— — —— — @ I S — — S — I — S ba'@ I — S I I — — .I@@ii I — I — I — S — :. , _4 S @,, S @ — , — S S ba • I I — — — S S S S taJ S S @A S — — S S @S' )s. S — S )s. @J Fig. 3. DNA fingerprinting of in vitro and in vivo subclones derived from original clone X-ray-i 1 (Lane 1). 3175 Downloaded from cancerres.aacrjournals.org on April 23, 2015. © 1994 American Association for Cancer Research. IN VIVO GENOMIC INSTABILITY IN ViTRO @ 9.4kb — @ @ IN VIVO — bJ — t@J — UI 0% — — “J ) @ — S I t,I 0% @S, S S Fig. 4. DNA fmgerprinting of in vitro and in vivo subclones derived from original clone F-17 (Lane 1). and tumorigenic subpopulation of cells present in the original clone before the injection in the animal. Activity of Genomic Instability and Tumorigenic PotentiaL Data are summarized in Table 1 regarding the frequencies of genomic rearrangements in vitro and in vivo as well as the tumongenic poten tial reported previously for each of the transformed clones studied. As can be seen, clone X-ray-9 was the most stable after both in vitro and in vivo culture and was also the most tumorigenic since all four mice injected developed a tumor. On the other hand, clone F-i7 showed the opposite behavior. Four genomic rearrangements were previously detected for this transformed clone immediately after isolation (21), and all in vivo subclones showed new genomic rearrangements. Nev ertheless, only one mouse of eight injected with cells from original clone F-i7 has developed a tumor (Table 1). Clone X-ray-il showed an intermediate behavior. Modulation of the Level of Genomic Instability. Our data also indicate that the level of genomic instability can be activated and down regulated. In a previous study, four genomic rearrangements were detected early in the process of transformation for clone F-il. In the present study, we report that a further in vitro culturing of clone F-i7 for 6 months generated fewer genomic rearrangements. Indeed, seven of the eight subclones derived from the original clone F-il showed no detectable genomic instability. This finding is in contrast to the level of genomic instability detected among subclones derived from the tumor, thus after the growth of(culturing) clone F-il in vivo. All subclones derived from this tumor showed new genomic rear rangements. These data clearly indicate the possibility that the level of genomic instability may be modulated, at least for clone F-il. DISCUSSION We have demonstrated that the level of genomic instability in transformed C3H/iOT½ mouse cells can be modulated. These cells were transformed in vitro by X-rays and genomic rearrangements, a marker of genomic instability occurring after 25 subsequent passages Frequencyof genomicrearrangements among subclones― in vitro or during the tumor development in the C3H mouse, were Original detected by use of a DNA fingerprinting assay. This assay was used clones Inclone1'0/105/104/41/9iO/iO2/41/89/91/8 vitroIn vivoof original to measure genomic rearrangements in a specific family of minisat X-ray-9 ellite sequences dispersed throughout the genome with a multilocus X-ray-il F-i7 and multiallele probe identified as M. With this protocol, it was a Number of in vitro or in vivo subclones carrying at least one genomic rearrangement! possible to measure the incidence of genomic rearrangements inde number of subclones studied. pendent of the direct action of the carcinogen on DNA. b Number of animals with tumors/number of animals injected with 2 X i06 trans formedcells. It is interesting that very few new genomic rearrangements were observed after 25 passages in vitro (or 6 months in culture). No event was observed in X-ray-9 subclones, and only one subclone each from Table 2 Malignant potential of 10T½transformed clones and their related X-ray-il and F-il showed a genomic rearrangement. In contrast, we in vivo subclones have previously reported that a higher frequency of genomic rear rangements occurs in the first six cell divisions following the second Original 106 i0@ event of the transformation process induced in vitro by X-rays, i.e., at clonesInvivo subclonesTumorigenicitya2x cells injected2X injectedX-ray-9x-ray-9-A-3 cells the beginning of the formation of the type III foci (21). These data suggest, at least for clone F-il, that genomic instability may be 0/4 initially activated by X-rays and down regulated during the process of 0/4X-ray-ilx-ray-i x-ray-9-A-74/40/4 transformation in vitro. Additional experiments are required to sup 1-A-3 0/4 port this observation. x-ray-i i-A-7 1/4 x-ray-ii-A-iO2/40/4 0/4a The most important aspect of the present study is the observation Numbers ofanimals with a tumor/numberof animal injected. that genomic instability in cells transformed previously in vitro can be 3176 Table 1 Frequencies ofgenomic rearrangements among subclones derived from transformed cells after growth in vitro and in vivo and tumorigenicity initial transformed clone of the Downloaded from cancerres.aacrjournals.org on April 23, 2015. © 1994 American Association for Cancer Research. IN VIVO GENOMIC enhanced in vivo in the C3H mouse. After about the same duration of time and number of cell divisions as for the in vitro culture, new genomic rearrangements were observed in all the three clones tested, even in those found to be previously stable in vitro. Since the cells were previously exposed to X-rays in vitro, these new genomic rearrangements were induced in vivo after exposure to the carcinogen. Enhancement of the frequency of gene amplification and point mutations by toxic agents, two types of genomic instability, has already been reported for malignant cells in vitro. When incubated with N-(phosphonoacetyl)-L-asparate, highly tumorigenic cell lines amplified the genes that code for a multifunctional enzyme complex that contains carbamyl phosphate synthase, aspartate transcarbamy lase, and dihydro-orotase (CAD) activities, at a frequency of iO@, whereas in normal counterparts the event is undetectable by the method used in these studies (ii, 12). However, it was still not demonstrated that such enhancement of the activity of genomic insta biity can also occur in the animal and that this cellular process could play a role in tumor development (25). Why can genomic instability be enhanced in vivo in 10T½cells transformed in vitro by X-rays? One possible interpretation is that transformation was incomplete in vitro and additional genetic modi fications were required to obtain a fully tumorigenic cell. In this regard, the growth of weakly immunogenic tumors may actually be stimulated by an immune reaction (26). Such a process might be involved in the in vivo enhancement of genomic instability reported in this study, giving rise to a growth advantage that facilitates the development of a tumor. On the other hand, genomic instability can be an important part of a mechanism allowing the transformed cell to adapt to a new envi ronment. The cells that produced the tumor may have the ability to continue on with the genomic rearrangements, resulting in some malignant cells ultimately carrying the most favorable properties for their progression to metastatic cells and to evade host nonimmune and immune responses (21—34).In this regard, the capacity to modulate genomic instability could also be very important for the survival of the tumorigenic cell. An uncontrolled expression of genomic instability could be responsible for the accumulation of too many DNA modi fications that would eventually prove to be lethal such that the cell would die before forming a tumor, or conversely, that tumor regres sion might occur. This latter hypothesis can, at least in part, be supported by our results showing that clone F-il demonstrated the highest level of genomic rearrangement in vitro and in vivo but a lower tumorigenic potential. However, more research is still required to confirm this hypothesis. Another interesting aspect of genomic instability that we observed with our DNA fingerprinting assay is its specificity of action. In a previous report, four multilocus and multiallele probes detecting dif ferent minisatellite families dispersed through the genome were used to detect the genomic rearrangements induced in cells transformed by X-rays (21). Only the minisatellite family detected with the probe identified as M showed genomic rearrangements. This specificity cannot be attributed to direct action of the carcinogen, since X-rays interact essentially randomly within the genome. What cellular pro cess can account for the observations that genomic instability occurs in specific regions of the genome, and can be modulated during transformation and tumor development? Multiple genes show genomic stability functions, such as those controlling accurate dupli cation and distribution of DNA in progeny cells and the fidelity of repair (i6, 35). Recently, a mutation in genes controlling the cell cycle (36—37), such as the suppressor gene p.53, was correlated INSTABILITY age, they can only in part account for the modulation and accumula tion of mutations in a specific region of the genome. In conclusion, genomic instability appeared to be a early event in our cell system, occurring during the proliferation of cells surviving X-ray exposure; subsequently, the activity of this mechanism may be modulated to allow the cell to complete its transformation in vivo or to grow in this new environment. ACKNOWLEDGMENTS We thank Dr. Brian T. Ledwith for providing probe M. REFERENCES I. Foulds, L. The experimental study of tumor progression: a review. Cancer Res., 14: 317—339,1954. 2. Nowell, P. C. The clonal evolution of tumor cell populations. Science (Washington DC), 194: 23-28, 1976. 3. Pitot, H. C., Ooldsworthy, T., and Moran, S. 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