Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.     

Gene amplification in Chinese hamster DNA repair deficient mutants

In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2–5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.     

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.     

Inefficient growth arrest in response to dNTP starvation stimulates gene amplification through bridge-breakage-fusion cycles.

Cells often acquire resistance to the antiproliferative agents methotrexate (MTX) or N-phosphonacetyl-L-aspartate (PALA) through amplification of genes encoding the target enzymes dihydrofolate reductase or carbamylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), respectively. We showed previously that Syrian hamster BHK cells resistant to selective concentrations of PALA (approximately 3 x ID50) arise at a rate of approximately 10(-4) per cell per generation and contain amplifications of the CAD gene as ladder-like structures on one of the two B9 chromosomes, where CAD is normally located. We now find that BHK cells resistant to high concentrations of PALA (approximately 15 x ID50) appear only after prior exposure to selective concentrations of PALA for approximately 72 h. Furthermore, in contrast to untreated cells, BHK cells pretreated with selective concentrations of MTX give colonies in high concentrations of PALA, and cells pretreated with selective concentrations of PALA give colonies in high concentrations of MTX or 5-fluorouracil. As judged by measuring numbers of cells and metaphase cell pairs, BHK cells do not arrest completely when starved for pyrimidine nucleotides by treatment with selective concentrations of PALA for up to 72 h. We propose that DNA damage, caused when cells fail to stop DNA synthesis promptly under conditions of dNTP starvation, stimulates amplification throughout the genome by mechanisms--such as bridge-breakage-fusion cycles--that are triggered by broken DNA. Amplified CAD genes were analyzed by fluorescence in situ hybridization both in cells where amplification was induced by PALA pretreatment and in cells in which the amplification occurred spontaneously, before selection with PALA. The ladder-like structures that result from bridge-breakage-fusion cycles were observed in both cases.     

Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells.

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.     

Overexpression of the 18 kDa and 22/24 kDa FGF-2 isoforms results in differential drug resistance and amplification potential

We investigated the role of low molecular weight (LMW) and high molecular weight (HMW) isoforms of basic fibroblast growth factor 2 (FGF-2) in the expression of transformation-related phenotypic alterations, drug sensitivity modulation, and gene amplification potential. For this purpose, we used NIH 3T3 and A31 cells transfected with different cDNA FGF-2 constructs allowing expression of the different proteins. Both cell lines showed marked phenotypic alterations when expressing the LMW FGF-2 or the four HMW FGF-2 isoforms: they acquired a transformed morphology, grew at higher saturation densities in 10% serum, and exhibited anchorage-independent growth and increased invasive potential. However, HMW FGF-2-expressing cells also grew in 1% serum and their invasive potential was lower than in cells expressing all FGF-2 forms or LMW FGF-2 alone. We have grown the different cell lines under a selective pressure of N-(phosphonacetyl)-l-aspartate (PALA), a drug which specifically inhibits the aspartate transcarbamylase activity of the multifunctional carbamyl-P-synthetase/aspartate transcarbamylase/dihydro-orotase genes (CAD) enzyme (and thus inhibits de novo pyrimidine biosynthesis) and selects for cells with amplified copies of the CAD gene. Our results demonstrate that aberrant expression of the LMW FGF-2 and/or HMW FGF-2 isoforms differently modulates drug resistance and gene amplification properties in the NIH 3T3 and A31 cell lines by differential amplification of the CAD gene. Coexpression of all isoforms appears to be necessary to obtain cumulative effects and nuclear-targeted HMW FGF-2 has a pivotal role in such a cooperation.     

Hamster cells with increased rates of DNA amplification, a new phenotype

Baby hamster kidney (BHK) cells selected simultaneously with N-phosphonacetyl-L-aspartate (PALA) and methotrexate (MTX) gave rise to doubly resistant colonies at frequencies 20 to 260 times greater than the product of the independent frequencies found with PALA or MTX alone. Double resistance was due to amplification of both target genes, CAD and DHFR. Four independent doubly resistant "MP" lines were selected and characterized. Cells resistant to coformycin, pyrazofurin, or ouabain were generated from all four MP lines at rates up to 25 times greater than the rates for BHK cells. These three drugs select cells that have amplified the genes for their target enzymes. Therefore, we conclude that the four MP lines have an amplificator phenotype. All four grew much more slowly than BHK cells, indicating that the amplificator phenotype may be linked to significant defects in metabolism or cell division.     

Gamma-ray and hydrogen peroxide induction of gene amplification in hamster cells deficient in DNA double strand break repair

To investigate the role of DNA double strand breaks (DSBs) and of their repair in gene amplification, we analyzed this process in the V3 Chinese hamster cell line and in the parental line AA8, after exposure to gamma-rays and to hydrogen peroxide (H2O2). V3 is defective in DSB repair because of a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene, a gene involved in the non-homologous end-joining pathway. As a measure of gene amplification we used the frequency of colonies resistant to N-(phosphonacetyl)-L-aspartate (PALA), since in rodent cells PALA resistance is mainly achieved through the amplification of the CAD (carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase) gene. After treatment with different doses of gamma-rays and of H2O2, we found a dose related increase in the frequency of gene amplification and of chromosome aberrations. When the same doses of damaging agents were used, these increments were higher in V3 than in AA8. These results indicate that DSBs that are not efficiently repaired can be responsible for the induction of gene amplification. H2O2 stimulates gene amplification as well as gamma-rays, however, at similar levels of amplification induction, chromosome damage was about 50% lower. This suggests that gene amplification can be induced by H2O2 through pathways alternative to a direct DNA damage. Stimulation of gene amplification by H2O2, which is one of the products of the aerobic metabolism, supports the hypothesis that cellular metabolic products themselves can be a source of genome instability.     

Down-regulation of Betaig-h3 gene is involved in the tumorigenesis in human bronchial epithelial cells induced by heavy-ion radiation

High-energy (HZE) heavy ions, when compared to low-LET radiation, are highly effective in inducing gene mutation, chromosomal aberrations and neoplastic transformation. However, the underlying molecular mechanisms are not clearly understood. We have recently shown that the down-regulation of Betaig-h3 expression is causally linked to the tumorigenic phenotype of papillomavirus-immortalized human bronchial epithelial (BEP2D) cells treated with high-LET alpha-particle radiation. Using the BEP2D cell culture system, a radiation-induced transformation model has been established by a single 60-cGy dose of (56)Fe heavy-ion radiation. To determine whether the Betaig-h3 gene is involved in (56)Fe ion-induced tumorigenesis, the expression levels of the Betaig-h3 gene in tumorigenic cell lines and the ability of in vivo tumor suppression through the reintroduction of the Betaig-h3 gene in tumorigenic cells were determined. We found that the expression level of this gene is markedly decreased in three tumorigenic cell lines ((56)FeT1-T3) compared with parental BEP2D cells. Ectopic expression of its cDNA in the (56)FeT2 tumorigenic cells significantly suppressed their tumorigenicity. Although biologically active TGFB1 is elevated in two of three tumorigenic cell lines, all these cell lines are resistant to the induction of Betaig-h3 expression by incubating the transformed cells with exogenous TGFB1 relative to control cells. Our data strongly suggest that down-regulation of Betaig-h3 expression results from the defect in the TGFB1 signaling pathway and plays a pivotal role in the tumorigenic process induced by (56)Fe heavy-ion radiation.     

Chromosome rearrangements associated with CAD gene amplification. Experiments with cell hybrids.

Resistance to phosphonacetyl-L-aspartate (PALA) is caused by CAD gene amplification. The marker chromosome of a PALA-resistant cell line containing a homogeneously staining region with amplified CAD gene was introduced into PALA-sensitive Chinese hamster cells by microcell-mediated chromosome transfer. Two monochromosomal hybrids containing the marker chromosome in addition to the normal chromosome complement of sensitive cells and 1 tetraploid hybrid containing the complete genomes of donor (resistant) and recipient (sensitive) cells were studied in detail. It was shown that (i) the presence of the marker chromosome was both a necessary and a sufficient condition for the expression of the PALA-resistant phenotype; (ii) the marker chromosome underwent rearrangements in the monochromosomal hybrids, with preferential loss of non-amplified chromosomal regions, while it was not rearranged in the tetraploid hybrid; (iii) unlike the original PALA-resistant cells obtained after long-term selection in the presence of PALA, the PALA-resistant hybrids did not show chromosomal aberrations of other than the marker chromosome. This result indicates that chromosomal aberrations may be due to the selective procedure and is not an inherent property of cells containing amplified genes.     

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.     

Decreased transforming growth factor-beta response and binding in insulin-independent teratoma-derived cell lines with increased tumorigenic properties.

The mouse C3H teratoma-derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin-independent cell line called 1246-3A was isolated. It was found that, in contrast to 1246 cells, 1246-3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 10(6) cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor alpha- and beta-like polypeptides which stimulate their proliferation. Highly tumorigenic insulin-independent cell lines able to give rise to tumor when injected at a density of 10(4) cells/mouse were isolated by using an in vitro-in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF-beta 1. The binding of TGF-beta 1 to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF-beta 1 to the cells. However, the decreased number of cell surface TGF-beta 1 binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF-beta protein by the tumorigenic cells, as all of TGF-beta produced by the cells was in a latent form. Affinity cross-linking experiments indicated that the 1246 cell line displayed several TGF-beta cross-linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross-linked bands, particularly of the 92 and 70 kDa species.     

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.     

Nuclear control of tumorigenicity in cells reconstructed by PEG-induced fusion of cell fragments

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.     

Amplification of the rudimentary gene in a PALA-resistant Drosophila cell line

In Drosophila melanogaster the rudimentary locus encodes for a multifunctional protein catalyzing the first three enzymatic activities of pyrimidine biosynthesis. Cell lines were selected which were resistant to PALA (N-(phosphonoacetyl)-L-aspartate), a specific inhibitor of aspartate transcarbamylase, the second enzyme of this pathway. In a cell line where the enzyme production is increased 5 times, Southern blot analyses show that the rudimentary gene and surrounding regions are amplified about 5 times. In this case gene amplification could therefore account for the observed enzyme overproduction.     

Aberrant Expression of Basic Fibroblast Growth Factor in NIH-3T3 Cells Alters Drug Resistance and Gene Amplification Potential

We have investigated the genetic stability of NIH-3T3 cells transfected with sequences coding for basic fibroblast growth factor (bFGF) by determining drug resistance and gene amplification potential. Colony-forming experiments and fluctuation analyses showed that the frequency and rate of resistance to N -(phosphonacetyl)-L-aspartate (PALA) was dramatically elevated in cells transfected with either the normal bFGF coding sequence that lacks a known signal for secretion or a chimeric bFGF sequence that targets the growth factor to the secretory pathway. Basic FGF-transfected cells that grew in the presence of PALA were found to possess an amplification of the CAD gene, which codes for a multifunctional protein involved in pyrimidine biosynthesis and is the site of action for PALA. The observation that these alterations occur in cells transfected with a bFGF sequence, without a conventional signal sequence for secretion, suggests an intracrine as opposed to autocrine mechanism of action. The results describe a new function for this growth factor and suggest a novel role for aberrant expression of bFGF in mechanisms of tumor progression.     

Evaluation of tumorigenic potential of high yielding cloned MDCK cells for live-attenuated influenza vaccine using in vitro growth characteristics,metastatic gene expression and in vivo nude mice model

Several mammalian cell lines, including Madin–Darby canine kidney (MDCK) cells have been approved by regulators for manufacturing of human vaccines. A new MDCK 9B9-1E4 cloned cell line has been created which is capable of producing live attenuated influenza vaccine (LAIV) with high yield. This cell line was shown to be non tumorigenic in eight week old adult athymic nude mouse model. This property is desirable for vaccine production and is unique to this cell line and is not known to be shared by other MDCK cell lines that are currently used for vaccine production. This significant difference in tumorigenic phenotype required further characterization of this cell line to ensure its safety for use in vaccine production. This is particularly important for LAIV production where it is not possible to incorporate a virus inactivation and/or removal step during manufacturing. Characterization of this cell line included extensive adventitious agent testing, tumorigenicity and oncogenicity assessment studies. Here, we describe the development of tumorigenic MDCK cell lines for use as positive controls and in vitro methods to aid in the evaluation of the tumorigenicity of MDCK 9B9-1E4 cloned cells. Tumorigenic MDCK cells were successfully generated following Hras and cMyc oncogene transfection of MDCK 9B9-1E4 cloned cells. In this study we demonstrate the lack of tumorigenic potential of the MDCK 9B9-1E4 cloned cell line in adult athymic nude mice model.     

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.