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.     

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.     

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.     

Suppression of tumorigenicity in somatic cell hybrids. II. Human chromosomes implicated as suppressors of tumorigenicity in hybrids with Chinese hamster ovary cells

Nontumorigenic diploid human cells were fused with tumorigenic Chinese hamster ovary cells (CHO), and the hybrids were tested for tumorigenicity to determine if specific human chromosomes are associated with suppression of tumorigenicity in cell hybrids. Chromosome complements of cells of 62 nontumorigenic and 45 tumorigenic hybrids (divided into those of low, medium, and high tumorigenicity) as well as 44 tumors derived from the tumorigenic hybrids were determined by both analysis of banded chromosomes and assays of gene markers. Although no single human chromosome was consistently associated with the suppressed phenotype, chromosome 2 was never found in tumor cells, and chromosomes 9, 10, 11, and 17 were found at very low incidences in tumor cells, which suggested that they carry tumorigenicity suppressor information. Since not all suppressed hybrids contained these chromosomes, it is likely that they suppressed tumorigenicity only in combination with each other or other chromosomes. Nine chromosomes in 12 pairwise combinations of nonhomologous chromosomes were not found in tumor cells and were found at an incidence of 5% or less in hybrids of both medium and high tumorigenicity. Other experiments implicated 11 of these combinations involving only 8 chromosomes (chromosomes 4, 7, 8, 9, 10, 11, 13, and 17) as those primarily involved in suppression. Whether chromosome 2 requires another chromosome to effect suppression could not be determined. Further evaluations of the implicated suppressors, including selection of tumorigenic segregants from a panel of suppressed hybrids, again implicated the same chromosomes and their combinations in suppression. Oncogenes have been mapped to many of these chromosomes, and they are frequently involved in tumor-type-specific numerical or structural abnormalities in human neoplasias. The combined evidence suggests that specific human chromosomes of a normal cell carry genes that can regulate several cell phenotypes necessary for the expression of tumorigenicity.     

Suppression of the tumorigenic phenotype of a rat liver epithelial tumor cell line by the p11.2–p12 region of human chromosome 11

Comparative chromosomal mapping studies and investigations of tumor-associated chromosomal abnormalities suggest that the development of hepatic tumors in humans and rats may share a common molecular mechanism that involves inactivation of the same tumor suppressor genes or common genetic loci. We investigated the potential of human chromosomes 2 and 11 to suppress the tumorigenic phenotype of rat liver epithelial tumor cell lines. These tumor cell lines (GN6TF and GP7TB) display elevated saturation densities in culture, efficiently form colonies in soft agar, and produce subcutaneous tumors in 100% of syngeneic rat hosts with short latency periods. Introduction of human chromosome 11 by microcell fusion markedly altered the tumorigenicity and the transformed phenotype of GN6TF cells. In contrast, the tumorigenic potential and phenotype of GP7TB cells was unaffected by the introduction of human chromosome 11, indicating that not all rat liver tumor cell lines can be suppressed by loci carried on this chromosome. Introduction of human chromosome 2 had little or no effect on the tumorigenicity or cellular phenotype of either tumor cell line, suggesting the involvement of chromosome 11–specific loci in the suppression of the GN6TF tumor cell line. The GN6TF-11^neo microcell hybrid cell lines displayed significantly reduced saturation densities in monolayer cultures, and their ability to grow in soft agar was completely inhibited. Although GN6TF-11^neo cells ultimately formed tumors in 80–100% of syngeneic rat hosts, the latency period for tumor formation was much longer. Molecular characterization of GN6TF-11^neo microcell hybrid cell lines indicated that some of the clonal lines had spontaneously lost significant portions of the introduced human chromosome, partially delineating the chromosomal location of the putative tumor suppressor locus to the region between the centromere and 11p12. Molecular examination of microcell hybrid–derived tumor cell lines further defined the minimal portion of human chromosome 11 capable of tumor suppression in this model system to the region 11p11.2-p12. © 1995 Wiley-Liss, Inc.     

Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa

We examined the ability of human chromosome 11 derived from normal fibroblast cells to suppress the tumorigenicity of SiHa cells, a human cervical tumor cell line. Using DNA transfection, the human chromosome was tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic G418), transferred to mouse A9 cells by cell hybridization and microcell transfer techniques, and then transferred to SiHa cells by microcell transfer. These procedures resulted in the appearance of 15 independent, G418-resistant clones, 5 of which had one or two extra copies of an intact human chromosome 11. In situ chromosomal hybridization of these clones with the pSV2neo plasmid revealed the presence of a neo-tagged human chromosome 11 in all of the five SiHa-microcell hybrids. Two SiHa-microcell hybrids that contained a single copy of neo-tagged human chromosome 12 were also isolated by the same methods. The tumorigenicities of SiHa clones with one or two extra copies of chromosome 11 (SiHa-11) were suppressed; four of the five SiHa-11 clones formed no tumors in nude mice, whereas both parental SiHa cells and SiHa cells with an extra chromosome 12 formed tumors within 30 d. One SiHa-11 cell clone formed a single tumor 90 d after injection. This rare tumor had lost one copy of chromosome 11 and rapidly formed tumors when reinjected. These results indicate that the introduction of a single copy of normal human chromosome 11, but not chromosome 12, suppresses the tumorigenicity of SiHa cells, indicating the presence on human chromosome 11 of a putative tumor-suppressor gene (or genes) for human cervical tumors.     

Suppression of tumorigenesis by the breast cancer cell line MCF-7 following transfer of a normal human chromosome 11.

Breast cancer development is associated with several genetic abnormalities. Loss of heterozygosity in the short arm of chromosome 11 has been observed in 30% of tumors. We found homozygosity at five chromosome 11 polymorphic loci in genomic DNA of the MCF-7 breast carcinoma cell line, suggesting a possible loss of one chromosome 11. We have studied the transformed and tumorigenic phenotypes of MCF-7 cells following introduction of a normal human chromosome 11 via microcell fusion. MCF-7/H11 cell hybrids, containing chromosome 11, showed in vitro characteristics similar to the parental cell line. However, tumorigenicity in athymic mice was completely suppressed. Since tumor formation by MCF-7 cells is estrogen dependent, we have analysed the expression of the estrogen receptor and of the estrogen-activated gene pS2. No difference was detected between the parental MCF-7 cells and the derived chromosome 11 cell hybrids, indicating that the mechanism of MCF-7 tumor suppression by chromosome 11-associated functions does not directly involve the estrogen/estrogen receptor molecular pathway.     

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.     

Introduction of normal chromosome 3p modulates the tumorigenicity of a human renal cell carcinoma cell line YCR

It has been suggested that loss and/or mutational inactivation of a gene or genes on the short arm of chromosome 3 (3p) may play a crucial role in the development of human renal cell carcinoma (RCC). If it is correct, the normal allele may carry suppressor activity for a tumor-associated phenotype(s). In order to test the hypothesis, we introduced a single chromosome containing 3p into a human renal cell carcinoma cell line YCR via microcell fusion, and examined tumorigenicity in nude mice and in vitro growth-properties. The following chromosomes derived from normal human fibroblasts were transferred to YCR or 6-thioguanine-resistant YCR cells: t(X;3) consisting of Xpter greater than Xq26::3p12 greater than 3pter, X, pSV2neo-tagged chromosome 11, and 3/t consisting of pSV2neo-tagged 3p and unknown segments. The introduction of t(X;3) or 3/t resulted in suppression of tumorigenicity or modulation of tumor-growth rate, whereas transfer of other chromosomes, i.e., X and 11, had no effect on tumorigenicity or tumor-growth rate of the cells. In vitro growth properties, i.e., cell-growth in medium containing 1% or 10% serum, growth in soft-agar and saturation density, were not correlated with the tumor-growth. In addition, the tumor-growth rate of 6-thioguanine-resistant segregants which have lost the t(X;3) became similar to that of the parental YCR cells. Thus, the introduction of 3p modulated at least the tumor-growth, indicating the presence on the 3p of a putative tumor-suppressor gene(s) for human RCC.     

Tumorigenic suppression of a human cutaneous squamous cell carcinoma cell line in the nude mouse skin graft assay.

The development of human squamous cell carcinomas has been associated with a number of genetic alterations involving chromosome 11, including cytogenetic and allelic deletions as well as amplification of genes in the 11q13 region. To determine the relevance of chromosome 11 in the formation of tumors of stratified squamous epithelial origin, we have introduced, via microcell fusion, a normal human chromosome 11 into the cutaneous squamous cell carcinoma cell line A3886TGc2. The ability of chromosome 11 to modulate the tumorigenicity of A3886TGc2 was evaluated first by inoculating cells s.c. in nude mice. All hybrids remained tumorigenic but exhibited longer tumor latencies than the parent, a result previously observed by other laboratories. We then tested our epidermally derived hybrids in the more physiologically relevant environment of the nude mouse skin graft system. The tumorigenic phenotype of three of four chromosome 11 hybrids placed into nude mouse skin grafts was completely suppressed. Polymerase chain reaction amplification of DNA from normal skin present at the suppressed graft sites failed to detect the introduced human cells. This information indicates that the normal skin is of mouse origin and suggests that the chromosome 11 microcell hybrids did not differentiate in vivo, but most likely failed to survive. We propose that external environmental factors present at the site of inoculation modulate the tumorigenic potential of these cells.     

Functional evidence for a novel human breast carcinoma metastasis suppressor, BRMS1, encoded at chromosome 11q13

We previously showed that introduction of a normal, neomycin-tagged human chromosome 11 reduces the metastatic capacity of MDA-MB-435 (435) human breast carcinoma cells by 70-90% without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 11. To identify the gene(s) responsible, differential display comparing chromosome 11-containing (neo11/ 435) and parental, metastatic cells was done. We describe the isolation and functional characterization of a full-length cDNA for one of the novel genes, designated breast-cancer metastasis suppressor 1 (BRMS1), which maps to human chromosome 11q13.1-q13.2. Stably transfected MDA-MB-435 and MDA-MB-231 breast carcinoma cells still form progressively growing, locally invasive tumors when injected into mammary fat pads but are significantly less metastatic to lungs and regional lymph nodes. These data provide compelling functional evidence that breast-cancer metastasis suppressor 1 is a novel mediator of metastasis suppression in human breast carcinoma.     

A human melanoma metastasis-suppressor locus maps to 6q16.3-q23

Loss, deletion or rearrangement along large portions of the long arm (q-arm) of chromosome 6 occurs in >80% of late-stage human melanomas, suggesting that genes controlling malignant characteristics are encoded there. Metastasis, but not tumorigenicity, was completely suppressed in the human melanoma cell line C8161 into which an additional intact chromosome 6 had been introduced by microcell-mediated chromosome transfer. Our objective was to refine the location of a putative metastasis suppressor gene. To do this, we transferred an intact (neo6) and a deletion variant [neo6qdel; neo6(del)(q16.3-q23)] of neomycin-tagged human chromosome 6 into metastatic C8161 subclone 9 (C8161.9) by MMCT. Single cell hybrid clones were selected in G-418 and isolated. Following verification that the hybrids retained the expected regions of chromosome 6 using a panel of polymorphic sequence-tagged sites, the hybrids were tested for tumorigenicity and metastasis in athymic mice. As reported previously, intact, normal chromosome 6 suppressed metastasis whether tumor cells were injected i.v. or into an orthotopic (i.e., intradermal) site. In contrast, metastasis was not suppressed in the neo6qdel hybrids. Tumorigenicity was unaffected in hybrids prepared with either chromosome 6 donor. These data strongly suggest that a human melanoma metastasis suppressor locus maps between 6q16.3-q23 ( approximately 40 cM).     

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.     

Functional evidence for the presence of tumor suppressor gene on chromosome 10p15 in human prostate cancers

Loss of heterozygosity on chromosome 10p was observed frequently in human prostate cancers. Studies have demonstrated that the introduction of the short arm of human chromosome 10 into a human prostate cancer cell line, PPC-1, by microcell-mediated chromosome transfer (MMCT), suppressed the malignant phenotype, suggesting the presence of a prostate tumor suppressor gene(s) within a region of 17 cM at distal 10p. To narrow down the candidate region harboring the tumor suppressor gene, a series of 10p fragments were transferred into PPC-1 cells by MMCT using a panel of hamster-human hybrid cells containing various portions of 10p. Four of the six hybrid cells obtained showed decreased tumorigenicity when injected subcutaneously into athymic nude mice. Tumors developed only at six of 40 injection sites for these four hybrid cells. In contrast, the other two hybrid cells, as well as parental PPC-1 cells, were judged to be fully tumorigenic because tumors appeared at a total 26 of 32 sites for the two hybrid cells and 15 of 16 sites for PPC-1. Allelotyping of 10p combined with fluorescence in situ hybridization in these hybrid cells suggested that a prostate tumor suppressor gene was located within a fragment of approximately 1.2 Mb flanked by D10S1172 and D10S226 on 10p15.1.     

Increased actin cable organization after single chromosome introduction: Association with suppression of in vitro cell growth rather than tumorigenic suppression

We previously showed that introduction of a single human chromosome 1, 6, or 9 derived from normal fibroblasts into HHUA endometrial carcinoma cells resulted in suppression of tumorigenicity. The tumorigenic suppression was accompanied by remarkable morphological changes in the microcell hybrids containing an extra copy of chromosome 1. The study presented here was undertaken to search for target cytoskeletal components affected by chromosome 1 transfer into endometrial carcinoma cells. We found that the microcell hybrids containing an extra copy of chromosome 1 were characterized by intracellular actin bundle formation and an excessive accumulation of actin and vinculin. The latter was a result of increased stabilization of the proteins. Additionally, chromosome 3 introduction into RCC23 human renal carcinoma cells resulted in prolongation of cell division and in senescence of a significant proportion of the microcell hybrids. In these microcell hybrids, the intracellular actin network was also reorganized, but the amounts of actin and vinculin protein were not increased. These findings suggest that the increased actin organization, which appeared not to cause tumorigenic suppression in the microcell hybrids, is associated with complementation of tumor suppressor genes and senescence by multiple mechanisms. © 1994 Wiley-Liss, Inc.     

Growth and transformation suppressor genes for BHK Syrian hamster cells on human chromosomes 1 and 11.

To map putative tumor suppressor genes for the near-diploid baby hamster kidney fibrosarcoma cell line BHK, we transferred five different normal human chromosomes (1, 3, 7, 11, and 12) into these tumor cells by microcell-mediated chromosome transfer. Transfer of human chromosome 1 into BHK cells resulted in suppression of cell growth both on plastic and in soft agar, indicating that chromosome 1 has a generalized effect on cell growth and thereby suppresses anchorage-independent growth. Selection against cells with an intact chromosome 1 was observed. In contrast, the introduction of chromosome 11 into BHK cells resulted in suppression of anchorage independence but not growth on plastic. Most chromosome-11 growth-suppressed BHK hybrids retained intact copies of human chromosome 11. Tumorigenic derivatives of chromosome 11 hybrids had lost this chromosome. Transfer of human chromosome 3, 7, or 12 into BHK cells did not correlate with growth suppression of BHK cells on plastic or in soft agar. Thus, we conclude that genes that suppress BHK-cell growth in general or in agar reside on human chromosomes 1 and 11, respectively.     

Identification of candidate liver tumor suppressor genes from human 11p11.2 by transcription mapping of microcell hybrid cell lines

Our previous studies utilized a microcell hybrid (MCH) cell line-based functional model of tumor suppression to localize a liver tumor suppressor to human chromosome 11, map the suppressor locus to a <1-Mb region within human 11p11.2, and identify a number of expressed sequence tags (ESTs) and genes that represent candidate liver tumor suppressor genes. The Human Genome Project has recently positioned a number of additional genes, ESTs, and predicted genes within the human 11p11.2 liver tumor suppressor region. In this study, we analyzed 26 ESTs and genes (known and predicted) that have been localized to human 11p11.2. Four of these ESTs/genes (FLJ23598, FLJ10450, KIAA1580, SYT13) mapped to the minimal tumor suppressor region of human 11p11.2, the smallest region conferring suppression of tumorigenicity in the MCH cell lines. Each of these ESTs/genes were expressed among an index panel of suppressed MCH cell lines (derived from GN6TF rat liver tumor cells), suggesting that these ESTs/genes represent excellent candidates for the human 11p11.2 liver tumor suppressor gene. To verify the candidate status of these sequences, 8 additional MCH cell lines (derived from GN3TG and GP10TA rat liver tumor cells) were analyzed. Three ESTs/genes (FLJ23598, FLJ10450, KIAA1580) proved to be less than ideal candidates, based upon their loss from suppressed MCH cell lines (DNA deletion), and/or their retention and expression in a non-suppressed MCH cell line. In contrast, SYT13 is present in the DNA from all suppressed MCH cell lines (n=10), and is deleted in a non-suppressed MCH cell line. Furthermore, SYT13 mRNA is expressed in 100% of suppressed cell lines, and is not expressed in the non-suppressed MCH cell line or in MCH-derived tumor cell lines (n=6). These results suggest that SYT13 is an excellent candidate for the human 11p11.2 liver tumor suppressor gene based upon its: i) location within the human 11p11.2 liver tumor suppressor region; ii) loss from the DNA of a non-suppressed MCH cell line that lacks the human 11p11.2 liver tumor suppressor region; iii) expression among suppressed MCH cell lines; and iv) lack of expression by MCH-derived tumor cell lines.     

Role of chromosome loss in ras/myc-induced Syrian hamster tumors

It has been shown previously that normal Syrian hamster embryo cells are neoplastically transformed by transfection with two cooperating oncogenes, v-myc plus v-Ha-ras. Karyotypic analyses of the cells from the tumors revealed a nonrandom chromosome change, monosomy of chromosome 15. In order to clarify the role of chromosome loss in these tumor cells with defined oncogene alterations, molecular and cytogenetic studies were performed on hybrids between normal Syrian hamster embryo cells and ras/myc tumor cells. Following fusion of the tumor cells with the normal cells which are not immortal, the majority of the cell hybrids senesced after less than or equal to 20 population doublings indicating that immortality was recessive. Some of the hybrids escaped senescence and grew indefinitely. These immortal hybrid cells retained the expected numbers of chromosome 15 indicating that escape from senescence did not involve loss of this chromosome. The tumorigenicity and anchorage-independent growth of the nonsenescent hybrids were still suppressed significantly. In these suppressed hybrid cells, RNAs complementary to the v-Ha-ras and v-myc oncogenes were expressed. Furthermore, radioimmune precipitation with a monoclonal antibody to p21ras of [35S]methionine-labeled cell extracts followed by polyacrylamide gel electrophoresis/sodium dodecyl sulfate electrophoresis showed that the suppressed hybrid cells contained high levels of the mutated ras protein. These results indicate that tumorigenicity is suppressed in the hybrids even though the oncogenes are expressed. When the hybrid cells were passaged, anchorage-independent variants appeared in the cultures. At this time, morphological changes occurred in the cultures and the cells were tumorigenic. Karyotypic analyses of the transformed segregants versus the parental hybrid cells revealed a nonrandom loss of one copy of chromosome 15 in the transformed segregants. No other nonrandom chromosome change was observed. These results suggest that the loss of chromosome 15 results in the loss of a cellular tumor suppressor gene which effects a phenotypic change necessary for expression of neoplastic transformation. In addition, the cellular factors responsible for the senescence of the hybrids may provide another mechanism involved in suppressing tumorigenicity.