DNA amplification and tumorigenicity of the human melanoma cell line MeWo

Homogeneously staining regions (HSRs) were found in hypodiploid cells (40%) of a subline of the human melanoma cell line, MeWo, (MeWo-C) but were absent from the hypotetraploid cells (60%). Another subline (MeWo-B) was also shown to contain two populations of cells, 70% hypodiploid and 30% hypotetraploid. None of the MeWo-B cells contained HSRs, but all four cell types from both sublines shared marker chromosomes indicating their common origin. The hypodiploid MeWo-B cells were karyotypically similar to the hypodiploid MeWo-C cells except for the presence of the HSRs in the latter. Both MeWo-C and MeWo-B sublines were injected into BALB/c nude mice. The MeWo-C cells were markedly more tumorigenic than MeWo-B cells as judged by tumor incidence, latency, average tumor size, and tumor take values. Cytogenetic and flow cytofluorometric analyses of the tumors induced by MeWo-C cells revealed a shift in the tumor cell population from 40% to greater than 90% HSR-containing hypodiploid cells during tumor growth. Hybridization of tumor DNA to a probe (D15Z1), the sequence of which is amplified in the HSRs, also indicated an increase in the proportion of HSR-bearing cells during tumor growth. No such selective advantage was found with the hypodiploid, HSR-lacking MeWo-B cells. The results suggest that HSRs found in the human melanoma line MeWo may confer enhanced tumorigenicity to the cells containing them.     

DNA amplification and metastasis of the human melanoma cell line MeWo

A positive correlation was found between the presence of amplified DNA in the form of homogeneously staining regions (HSRs), and the formation of spontaneous metastases by the human melanoma cell line MeWo. HSR+ and HSR- MeWo sublines and clones were injected s.c. into BALB/c nude mice. All MeWo lines produced primary tumors that were allowed to grow to a similar size before the animals were sacrificed and examined for the presence of metastatic nodules in the lungs and abdominal cavity. HSR+ lines gave extensive metastases (greater than 100 nodules) in the lungs and/or liver and abdomen in 18 of 19 animals. The HSR- MeWo lines were effectively nonmetastatic, producing metastases in 3 of 20 animals, two of which had only a single metastatic lung nodule each. Evidence for the presence of HSRs in the primary tumors and metastatic nodules was obtained by DNA dot-blot hybridization to a sequence (D15Z1) amplified in the HSRs, flow cytofluorometry for cellular DNA content, and quinacrine staining of metaphase spreads. The HSR+ clones also colonized the lung to a much greater extent than HSR- clones following i.v. injection. In addition, the HSR+ clone had a selective advantage in lung colonization, since i.v. injection of a 50:50 mixture of HSR+ and HSR- clones resulted in extensive metastases populated exclusively by HSR+ cells. The results suggest that DNA sequences amplified in the HSRs of human melanoma MeWo cells may confer enhanced metastatic properties to these cells.     

Multiple human chromosomes carrying tumor-suppressor functions for the mouse melanoma cell line B16-F10, identified by microcell-mediated chromosome transfer

Many tumor-suppressor genes are involved in the development and progression of cellular malignancy. To understand the functional role of tumor-suppressor genes in melanoma and to identify the human chromosome that carries these genes, we transferred individually each normal human chromosome, except for the Y chromosome, into the mouse melanoma cell line B16-F10, by microcell fusion. We examined the tumorigenicity of hybrid cells in nude mice and their in vitro growth properties. The introduction of human chromosomes 1 and 2 elicited a remarkable change in cell morphologic features, and cellular senescence was induced at seven to 10 population doublings. The growth rates of tumors derived from microcell hybrid clones containing introduced human chromosome 5, 7, 9, 10, 11, 13, 14, 15, 16, 19, 20, 21, 22, or X were significantly slower than that of the parental B16-F10 cells, whereas the introduction of other human chromosomes had no effect on the tumorigenicity of these cells. The majority of microcell hybrid clones that exhibited suppressed tumorigenicity also showed a moderate reduction in doubling time compared with B16-F10 cells. Microcell hybrid clones with an introduced human chromosome 5 showed complete suppression of in vitro-transformed phenotypes, including cell growth, saturation density, and colony-forming efficiency in soft agar. Thus, these results indicated the presence of many cell senescence-related genes and putative tumor-suppressor genes for the mouse melanoma cell line B16-F10 and showed in vitro that many tumor-suppressor genes control the phenotypes of transformed cells in the multistep process of neoplastic development.     

Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.     

Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-mediated chromosome transfer

Putative tumor suppressor genes can be mapped to specific chromosomes by the introduction of individual chromosomes derived from normal cells via microcell fusion. We have examined whether a highly malignant human uterine endometrial carcinoma cell line, HHUA, can be suppressed by only one normal chromosome or by multiple chromosomes. A library of mouse A9 clones containing different human chromosomes tagged with the pSV2-neo plasmid DNA were constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11, or 19 into the HHUA tumor cell line was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of a chromosome 19 had no effect on the tumorigenicity of the cells, whereas microcell-hybrid clones with an introduced chromosome 1, 6 or 9 were completely suppressed for tumorigenicity. A decrease in tumor-take incidence in some but not all clones was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 senesced and/or showed alterations in cellular morphology and transformed growth properties in vitro. No growth or morphology alterations were observed following transfer of the other chromosomes. These results may indicate that more than one chromosome carries a tumor suppressor gene(s) for this human uterine endometrial carcinoma cell line and support the hypothesis that multiple tumor suppressor genes control the tumorigenic phenotype in the multistep process of neoplastic development.     

Presence of two cytogenetic forms of amplified DNA: evidence for a role in tumor growth in an intraspecific mouse hybrid cell line

An intraspecific mouse hybrid epithelial cell line, F5/B, is described in which the homogeneously staining region (HSR)-containing marker chromosome from one parent is absent in about half of the cells. It is replaced in these cells by double minutes (DM), an alternative form of amplified DNA, which is liable to loss because of its instability at mitosis. DM probably arise from the breakdown of the HSR during clonal growth of F5/B. Subclones were derived possessing one or another cytogenetic feature, and their cloning efficiency in vitro and tumorigenicity in syngeneic animals were compared. There were no differences in in vitro tumorigenicity, but in vivo DM-containing subclones were significantly less tumorigenic than HSR-containing subclones or the F5/B parent hybrid. In tumors that developed after long latent periods, cells had increased numbers of DM compared with the inoculated population, demonstrating a selective advantage in vivo for cells with a high DM content. These results indicate a role for the amplified DNA in tumor growth.     

Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosomes 3 and 11 introduced via microcell-mediated chromosome transfer

To map tumor suppressor genes for lung adenocarcinomas, we introduced normal human chromosomes 3, 7, and 11 into the A549 tumor cell line by microcell-mediated chromosome transfer to test which chromosomes had the ability to suppress tumorigenicity. These human chromosomes, which contain the neomycin gene as a selectable marker, were transferred into A549 lung adenocarcinoma cells at frequencies of 0.3–1.8 × 10^?6. Two microcell hybrid clones with an introduced chromosome 3, two with an introduced chromosome 7, and six with an introduced chromosome 11 were isolated and examined for their growth properties and tumorigenicity in nude mice. Whereas parental A549 cells formed tumors with an average latency of 68 d, both microcell hybrids with an introduced chromosome 3 failed to form tumors for over 360 d. Similar tumorigenicity results were obtained when the clones were implanted into denuded tracheas, a more orthotopic transplantation site. The two clones with an introduced chromosome 7 were still tumorigenic; they formed tumors within 100–123 d after injection and grew progressively, although the tumors grew slightly slower than the parental cells did. Among the six clones with an introduced chromosome 11, one clone was still highly tumorigenic but did not contain an extra copy of an intact introduced chromosome 11. Three clones with a single intact copy of introduced chromosome 11 formed tumors with latency periods significantly longer than those of the parental cells. Two clones had two copies of the introduced chromosome 11, and both failed to form tumors within 1 yr of injection. These results indicate that chromosomes 3 and 11 can suppress the tumorigenicity of A549 lung adenocarcinoma cells.     

Suppression of tumorigenicity in hybrids of tumorigenic Chinese hamster cells and diploid mouse fibroblasts: dependence on the presence of at least three different mouse chromosomes and independence of hamster genome dosage

Somatic cell hybrids were generated between Chinese hamster cell lines (Cl-4 and TK 17-O) with a near-diploid number of partially abnormal chromosomes and embryonic mouse fibroblasts (BALB/c). Hybrids harboring a near-diploid, near-triploid, and near-tetraploid set of hamster chromosomes plus 22 to 30 mouse chromosomes were analyzed for the expression of the transformed or tumorigenic phenotype, respectively, indicated by their capacity to form colonies in soft agar and by tumor formation after s.c. injection into nude mice. The hybrids showed (partial) suppression of tumorigenicity and of anchorage independence. The minimum number of hybrid cells required to initiate tumor growth in nude mice was 100- to 50,000-fold higher, and the latency period was 3- to 6-fold longer in comparison with the highly tumorigenic parental hamster cells. Suppression of tumorigenicity was also found in intraspecific Chinese hamster hybrids involving tumorigenic cells (E 36-O and TK 17-O) and embryonic hamster fibroblasts. To identify those mouse chromosomes associated with suppression of tumorigenicity, we investigated the expression of mouse isozyme genes and the presence of mouse chromosomes in interspecific suppressed hybrids and their tumorigenic hybrids described previously. No single mouse chromosome, even if present in two copies, and no combination of two different mouse chromosomes was sufficient to suppress tumorigenicity in these hybrids. This conclusion is based on either the presence of these chromosomes in hybrids isolated from tumors or their absence in suppressed hybrids.     

Detailed marker chromosome analysis in cell line U-BLC1, established from transitional-cell carcinoma of the bladder

A permanent cell line, U-BLC1, was established from a primary transitional-cell carcinoma, TCC, of the urinary bladder. Karyotype analysis showed the line to be highly aberrant, with a near-triploid chromosome number of 68 to 73. Comparative genomic hybridization revealed some distinct differences between the primary tumor and the established cell line. Karyotype analysis showed 3 marker chromosomes with homogeneously staining regions, HSRs, in the cell line. The HSRs were isolated by microdissection and the microdissection probes were hybridized to normal metaphase chromosomes. The HSRs contain sequences known to be frequently involved in amplification in transitional-cell carcinoma of the bladder, 6p22, 7p11-p12, 9p23-pter, and one region not yet reported to be amplified in primary TCC of the bladder, 1p31-p32. A candidate-gene approach showed that in the region 7p11-p12 the EGFR locus is amplified and highly expressed.     

Transforming genes from familial adenomatous polyposis patient cells detected by a tumorigenicity assay

We tried to detect oncogenes associated with familial adenomatous polyposis by a tumorigenicity assay in nude mice. One polyp and two peripheral blood lymphocyte DNAs out of 12 samples from patients induced Alu-positive tumors. Lymphocyte DNAs from one of 5 healthy people also showed tumorigenic activity. The transforming genes of polyps from a patient and lymphocytes from a normal person were found to be the human N-ras gene. Since these N-ras genes were amplified in nude mouse tumors and did not show any alterations in the nucleotide sequences around codons 12 and 61, it is likely that the tumors were induced by the amplified normal N-ras genes. The transforming sequences from two patients' lymphocytes did not hybridize with 12 known oncogene probes, suggesting that these two genes are novel oncogenes or genes for which we have not yet examined the homology. One oncogene derived from a patient's lymphocytes was partially cloned and shown to be located on human chromosome 7. This gene did not hybridize with the met and erbB1 genes, which are potential oncogenes located on chromosome 7. These data indicate that this gene is a new oncogene.     

Tumorigenicity of human HT1080 fibrosarcoma X normal fibroblast hybrids: chromosome dosage dependency

The tumorigenic capacity of hybrids formed by fusion of the highly tumorigenic HT1080 human fibrosarcoma cell line with nontumorigenic normal fibroblasts was examined. The HT1080 also contains an activated N-ras oncogene. Near-tetraploid hybrids which contained an approximately complete chromosomal complement from both parental cells were nontumorigenic when 1 X 10(7) cells were injected s.c. into athymic (nude) mice, whereas the parental HT1080 cells produced tumors in 100% of the animals with no latency period following injection of 2 X 10(6) cells. Tumorigenic variants were obtained from these hybrids which had lost only a few chromosomes compared to cells from the nontumorigenic mass cultures. In addition, several near-hexaploid hybrids were obtained which contained approximately a double chromosomal complement from the HT1080 parental line and a single chromosomal complement from the normal fibroblasts. All of these near-hexaploid hybrids produce tumors in 100% of nude mice with no latency period. Our results indicate that tumorigenicity of these particular human malignant cells of mesenchymal origin can be suppressed when fused with normal diploid fibroblasts. In addition, the results suggest that tumorigenicity in this system is chromosomal dosage dependent, since a diploid chromosomal complement from normal fibroblasts is capable of suppressing the tumorigenicity of a near-diploid but not a near-tetraploid chromosomal complement from the tumorigenic HT1080 parent. Finally, the loss of chromosome 1 (the chromosome to which the N-ras oncogene has been assigned) as well as chromosome 4 was correlated with the reappearance of tumorigenicity in the rare variant populations from otherwise nontumorigenic near-tetraploid hybrid cultures. Our results also suggest the possibility that tumorigenicity in these hybrids may be a gene dosage effect involving the number of activated N-ras genes in the hybrids compared to the gene(s) controlling the suppression of the activated N-ras genes.     

High c-myc amplification level contributes to the tumorigenic phenotype of the human breast carcinoma cell line SW 613-S

The c-myc gene is amplified in the SW 613-S cell line which was established from a human breast carcinoma. This line is heterogeneous: it contains cells with a high level of amplification and carrying the extra copies of the c-myc gene in double minute chromosomes (DMs) and cells with few c-myc genes integrated into chromosomes. Clones with different levels of amplification and different cytological localization of the c-myc copies were isolated from the SW 613-S cell population. Those with a high level of amplification and expression of the c-myc gene were highly tumorigenic in nude mice whereas those with a low level were not. Introduction of c-myc gene copies by transfection into the cells of several non-tumorigenic clones restored the tumorigenic phenotype. Our results indicate that a high level of amplification of the c-myc gene is a requirement for the tumorigenicity of SW 613-S cells in animals.     

Suggestive evidence for functionally distinct, tumor-suppressor genes on chromosomes 1 and 11 for a human fibrosarcoma cell line, HT1080

One approach for identifying chromosomes which carry putative tumor-suppressor genes is the introduction of specific chromosomes into the tumor cells of interest. We examined the ability of human chromosomes derived from normal fibroblasts to suppress or modulate tumorigenicity in nude mice and the in vitro properties of HT1080, a human fibrosarcoma cell line. We first isolated mouse A9 cells containing a single human chromosome (1, 2, 7, 11, or 12) integrated with pSV2neo plasmid DNA. Following fusion of microcells from these A9 cells with the HT1080 cells, clones that were resistant to G418 were isolated and karyotypically analysed. Three of 4 microcell-hybrids with an introduced chromosome 1 were non-tumorigenic (#1-7, -8 and -13), whereas the parental HT1080 cells were highly tumorigenic. The other microcell-hybrid clone (#1-1) formed tumors, the cells of which had lost one copy of chromosome 1. Two clones from the #1-1 cells were isolated; one contained an extra copy of chromosome 1, and the other one did not. The former was non-tumorigenic and the latter was tumorigenic. The introduction of chromosome 11 also suppressed the tumorigenicity of HT1080 cells, while the introduction of other chromosomes, i.e., 2, 7, or 12, had minimal or no effect on the tumorigenicity of these cells. Cells from tumors formed by microcell-hybrids with the introduction of chromosome 2, 7, or 12 still contained the introduced chromosome. Interestingly, only the microcell-hybrids with an introduced chromosome 1 had an alteration in cellular morphology and modulation of in vitro transformed properties, i.e., cell-growth and saturation density in a medium containing 10% calf serum and cell-growth in soft-agar. Thus, the results indicate the presence of putative tumor-suppressor genes for HT1080 cells on chromosomes 1 and 11, and further suggest that the genes on these chromosomes control different neoplastic phenotypes.     

Trisomy of chromosome 9q: specific chromosome change associated with tumorigenicity during the process of X-ray-induced neoplastic transformation in golden hamster embryo cells

We have reported that trisomy of chromosome 7 is commonly observed in anchorage-independent clones isolated from X-irradiated golden hamster embryo cells. All 10 clones derived from different irradiated populations showed tumorigenicity when 1 x 10(7) cells were injected s.c. into nude mice (BALB/c, nu/nu). From karyotypic analysis, we found that 8 of 10 cells showed trisomy of chromosome 9. One cell line had a translocation between chromosomes 9q and 19q and trisomy of chromosome 7. The other cell line contained trisomy of chromosome 9 and a translocation between chromosomes 7q and 8q. Using Southern blot analysis, we observed no amplification of v-myc, v-Ha-ras, v-Ki-ras or N-ras-related oncogenes. Furthermore, we could not detect either an increase in expression of v-myc- and v-Ha-ras-related genes or the activation of any oncogene, by the NIH 3T3 transfection assay. Our results suggest that trisomy of chromosome 7 is insufficient for the expression of tumorigenicity and that increased dosage of chromosome 9q may play an important role in the malignant progression of X-ray-induced neoplastic transformation.     

Homogeneously staining regions in 223 breast carcinomas: cytogenetic and clinicopathological correlations.

A correlation analysis was performed on 223 breast carcinomas to assess the relationships between gene amplification, karyotypic and clinicopathological features. Homogeneously staining region (HSR) is the most frequent form of amplification found in breast cancer. HSR-containing tumours accounted for 60% of the cases. Although up to 40% of tumours with slightly altered karyotype contained HSRs, an excess of HSRs was found within the tumours whose karyotype showed the highest rates of rearranged chromosomes. HSRs were also found to be particularly frequent in small tumours of high histological grade and with a low expression of progesterone receptors. An excess of HSRs seems to be observed in younger patients, however, significant correlation could be demonstrated only for patients below 55 years and below 60 years, compared with older ones. With a 120-month follow-up for 152 patients, a significant association between the presence of HSRs and a shortened overall survival was observed. Altogether, the presence of HSRs appears to be a good indicator of poor prognosis. Further studies are needed to determine whether amplification of specific genes or cell ability to amplify is the most important parameter for tumour progression.     

Tumorigenicity associated with chromosome 11p15 alterations in sv40-transformed human kidney-cells

Chromosome 11p15 has been suggested to be a potential site for a second Wilms' tumour gene (a childhood nephroblastoma). Human foetal kidney cells and normal kidney cells from Wilms' tumour patients were transformed with SV40 derivative vectors. As some of the cell lines progressed to tumorigenicity, we observed that chromosome 11p13, site of the WT1 suppressor gene, did not show any allelic loss. However, RFLP analysis showed that chromosome 11p15 was affected by allelic losses on different genes in some cell lines but not necessarily prior to the appearance of tumorigenicity. We also observed that the most aggressive cell Line (SVCU/NK), derived from the normal kidney cells of a Wilms' tumour patient, showed increased expression of c-Ha-ras proto-oncogene at later passage and in the tumour tissue extracted from nude mice. Finally we report a lack of tumour suppression activity of one cell line SVT1B6/NK, when fused with the tumorigenic G401 cell line (the latter has been used in tumour suppression experiments as a Wilms' tumour cell line before being identified as a Rhabdoid tumour cell line). These experiments are consistent with the existence of a suppressor gene at chromosome 11p15.     

Cytogenetic and molecular characterization of tumors in nude mice derived from a multidrug-resistant human leukemia cell line

We have evaluated whether selection of a human tumor leukemic line for resistance to vinblastine (Velban; VLB) alters its tumorigenicity. To address this question, CEM and CEM/VLB100 cells [which express the multiple drug-resistant (MDR) phenotype via amplification of the P-glycoprotein gene] were characterized by several techniques including chromosome banding, in situ hybridization, Southern blotting, RNA dot blotting, in vitro drug sensitivity, and tumorigenicity in nude mice. Analysis of the chromosome banding patterns of both drug-sensitive CEM cells and the MDR CEM/VLB100 cells revealed that the two lines differed primarily by the presence of a large metacentric marker chromosome associated with the acquisition of VLB resistance. In situ hybridization of a P-glycoprotein complementary DNA to metaphase chromosomes showed that the amplified P-glycoprotein genes in the CEM/VLB100 cell line were localized to this large marker. Tumorigenicity of both the CEM and CEM/VLB100 cell lines was measured after injection of 10(7) cells/nude mouse. The results showed that 4 of 4 drug-sensitive and 4 of 5 drug-resistant cell lines formed tumors in 5-10 wk. By comparison with the parental line, three of the four tumors arising from the CEM/VLB100 line retained their drug-resistance properties as measured by vinblastine resistance in vitro and elevated P-glycoprotein mRNA expression associated with P-glycoprotein gene amplification. In addition, tumors retaining the MDR phenotype also retained the large metacentric marker chromosome. One tumor arising from CEM/VLB100 reverted to the drug-sensitive phenotype, with a resultant decrease in P-glycoprotein mRNA expression and loss of P-glycoprotein gene amplification. This revertant was also missing the large metacentric marker present in all cells from the CEM/VLB100 parent. Our experiments show that the acquisition of the MDR phenotype resulting from overexpression of P-glycoprotein in the plasma membrane does not effect the tumorigenicity of human CEM cells.     

Coamplification of 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in methotrexate-resistant human leukemia cell lines

Methotrexate (MTX)-resistant K562 human myelocytic leukemia sublines with 20- and 200-fold amplified dihydrofolate reductase (DHFR) genes localized to homogeneously staining regions (HSRs) on the long arms of chromosomes 5, 6, and 19 were used to examine whether other genes mapping close to the DHFR genes were coamplified. The gene for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, located on chromosome 5q13.3-14, was coamplified 4-14-fold, corresponding to the levels of resistance exhibited by these cells. Similar observations were made with a MTX-resistant subline of the promyelocytic leukemia cell line, HL-60R, with 200 gene copies of DHFR. These observations indicate a tight linkage of DHFR and HMG-CoA genes on chromosome 5q.     

Characterization of the human mandibular osteoblastic osteosarcoma cell line HOSM-2 after long-term culture

We have been subculturing a human mandible-derived osteosarcoma cell line (HOSM-2) for approximately 15 years, and have compared the characters of early generations, which did not exhibit tumorigenicity, to those in the later generations. The shape and doubling time of the cells did not change during long-term culture. The number of chromosomes, however, changed from 59-81 in the 6th generation (modal number: 70) to 54-59 (modal number: 56 and 57), and the chromosomal structure also changed. In addition, the cell line in the later generations showed tumorigenicity in nude mice, and Codon 306 of the p53 gene was mutated to a stop codon due to a point mutation. HOSM-2 cells expressed osteoblast markers, thus confirming them to be osteoblastic osteosarcoma cells. These results showed that changes in certain genes in the HOSM-2 cells led to tumorigenicity in nude mice following long-term culture. In addition, as a mandible-derived cell line with characteristics different from those of limb-derived osteosarcoma cell lines, HOSM-2 cells may be a valuable model for mandibular osteosarcoma and osteoblasts.     

Novel tumor suppressor locus in human chromosome region 3p14.2.

BACKGROUND: Alterations of chromosome region 3p14 are observed in numerous human malignancies. Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation. METHODS: YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice. RESULTS: We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro. Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs. Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3. CONCLUSION: These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2. Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations.