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.     

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.     

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.     

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.     

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.     

Establishment of Cell Lines with High and Low Metastatic Potential from A549 Human Lung Adenocarcinoma

This article reports the establishment of variant cell lines with high and low metastatic potential by the dilution plating technique. Two clones with high metastatic potential, 2S Lu-4 and 11S Lu-1 and two clones with low metastatic potential, 8S and 16S were established from A549 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low- metastatic cell lines did not produce lung metastasis after injection into the tail vein of 5-week-old BALB/c nude mice. The primary tumors produced by the two high-metastatic cells grew fast and showed enhanced angiogenesis. The high-metastatic cells were small and flat-shaped, while the low- metastatic cells were large and flat-shaped. When the four variant cell lines and original A549 cells were embedded in collagen gels, the colonies of 2S Lu-4, 11S Lu-1 and A549 grew actively, whereas almost of all the colonies of 8S and 16S did not survive after 35 days in culture. These four cloned cell lines originated from heterogeneous populations of the parental A549 cells should be an excellent tool for studying the process of metastasis of lung cancer.     

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.     

Suppression of tumorigenicity of a Wilms' tumor cell line is associated with a decrease in synthesis of two proteins.

Wilms' tumor has been associated with deletions in two loci on chromosome 11, and the introduction of a translocated human chromosome [t(X;11)] into a Wilms' tumor cell line (G401.6TG.6) by microcell hybridization suppresses tumor formation in nude mice. The tumorigenic phenotype is restored in segregants of these microcell hybrids, in which the introduced chromosome is lost. We have used ultrahigh-resolution 'giant' two-dimensional gel electrophoresis of metabolically labeled cellular proteins and in vitro translation products of isolated mRNA to identify changes in cellular gene expression that occur in these cell lines. The changes in gene expression associated with these chromosomal manipulations per se are quite minimal. However, we have identified two proteins (p16 and p28) whose synthesis is consistently decreased in three non-tumorigenic (suppressed) microcell hybrid clones relative to parental and segregant tumorigenic lines. They are also decreased at the level of mRNA in at least two of the non-tumorigenic clones. The decrease of these proteins represents markers of the suppressed phenotype, and their down-regulation may conceivably mediate the suppression of tumorigenicity.     

Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line (A549)

The morphology, cell growth, antigenic expression and tumorigenicity of cell subpopulations from the A549 lung adenocarcinoma isolated by Percoll gradient separation have been analysed. Four subpopulations were obtained (subpopulations A, B, C and D). Immunocytochemical analysis of several antigens was performed with monoclonal antibodies (MAbs): MUC1 mucin (C595, HMFG1 and HMFG2), MUC5B (PANH2); gp230 (PANH4); carbohydrate antigens including sialyl Lewis x (KM93), Tn antigen (83D4), Lewis y (C14); 5, 6, 8, 17 and 19 cytokeratins and p53. The cell population D tended to form cell aggregates that piled up on the monolayer similar to overgrowth cultures of the A549 parental cell line, whereas A, B and C cell subpopulations formed well spread monolayers. Both parental A549 and subpopulation D secreted abundant mucus. The topographic distribution and secretion production were correlated with tumorigenic assays since only subpopulation D grew in nude mice exhibiting reduced latency period; these characteristics correlated with the fast growth of the subpopulation D in vitro. Immunocytochemical analysis demonstrated that subpopulation D showed greater expression of MUC1 mucin and carbohydrate antigens such as Tn antigen, sialyl Lewis x and Lewis y and less expression of cytokeratins, p53, MUC5B and gp230; conversely, subpopulations A, B and C showed the opposite antigenic profile. Our results illustrate heterogeneity in the A549 cell line; subpopulations A, B and C retained characteristics of more differentiated adenocarcinoma while subpopulation D displayed features of a less differentiated tumor line.     

Establishment of cell lines with high and low metastatic potential from A549 human lung adenocarcinoma.

This article reports the establishment of variant cell lines with high and low metastatic potential by the dilution plating technique. Two clones with high metastatic potential, 2S Lu-4 and 11S Lu-1 and two clones with low metastatic potential, 8S and 16S were established from A549 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low-metastatic cell lines did not produce lung metastasis after injection into the tail vein of 5-week-old BALB / c nude mice. The primary tumors produced by the two high-metastatic cells grew fast and showed enhanced angiogenesis. The high-metastatic cells were small and flat-shaped, while the low-metastatic cells were large and flat-shaped. When the four variant cell lines and original A549 cells were embedded in collagen gels, the colonies of 2S Lu-4, 11S Lu-1 and A549 grew actively, whereas almost of all the colonies of 8S and 16S did not survive after 35 days in culture. These four cloned cell lines originated from heterogeneous populations of the parental A549 cells should be an excellent tool for studying the process of metastasis of lung cancer.     

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.     

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.     

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.     

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 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.     

Normal human chromosome 1 carries suppressor activity for various phenotypes of a Kirsten murine sarcoma virus-transformed NIH/3T3 cell line.

In order to identify chromosomes that carry putative tumor-suppressor genes for the various phenotypes of Kirsten sarcoma virus-transformed NIH/3T3 (DT) cells, we performed microcell-mediated chromosome transfer into DT cells. We first isolated mouse A9 clones, containing a single human chromosome 1, 11 or 12 tagged with pSV2-neo plasmid DNA. Then, chromosome 1, 11 or 12 was transferred from the A9 clones into DT cells by microcell fusion. The growth rate, colony-forming ability in soft agar and tumorigenicity of the DT cells were controlled by chromosome 1, but not by chromosome 11 or 12, indicating that normal human chromosome 1 carries a putative tumor-suppressor gene(s) that affects various transformed phenotypes of DT cells.     

Construction of mouse A9 clones containing a single human chromosome tagged with neomycin-resistance gene via microcell fusion

Normal human fibroblasts (MRC-5 or NTI-4) were transfected with pSV2-neo plasmid DNA. Fifty G418-resistant fibroblast clones were isolated and independently fused to mouse A9 cells. The cell hybrids were selected and isolated in the medium containing G418 plus ouabain. Since micronuclei were more efficiently induced in these hybrids compared to parental human fibroblasts by colcemid treatment, the transfer of neo-tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell fusion. Two hundred A9 microcell hybrids were isolated and karyotyped. Among them, thirteen microcell clones, each containing a single human chromosome 1, 2, 5, 6, 7, 8, 10, 11, 12, 15, 18, 19 or 20 were established. Isozyme analyses conformed the presence of each human chromosome in these A9 microcell clones. The results of Southern blot and chromosomal in situ hybridization analyses indicate that the human chromosomes in these clones were tagged with pSV2-neo plasmid DNA.     

Relationship between tumorigenicity and the dosage of lymphoma- vs. normal-parent-derived chromosome 15 in somatic cell hybrids between lymphoma cells with rearranged pvt-1 gene and normal cells

Somatic cell hybrids were generated between YACUT, a doubly drug-resistant subline of YAC-1 (a Moloney-virus-induced T-cell lymphoma of strain A/Sn origin with 2 proviral insertions near the pvt-1 locus) and normal diploid fibroblasts of CBAT6T6 origin. Three independent fusions were performed. Three uncloned hybrid cultures and 9 independently-derived clones were tested for tumorigenicity by the inoculation of graded cell numbers into syngeneic hosts. One of 3 uncloned hybrid cultures and 3 of 9 clones were weakly tumorigenic (take incidence 0%), and 1 of 3 uncloned hybrid cultures and 6 clones were highly tumorigenic (take incidence greater than 80%). One weakly tumorigenic hybrid and 3 weakly tumorigenic clones carried 3 copies of the tumor-derived chromosome 15 and 2 copies of the normal fibroblast-derived t(14;15) chromosomes. In contrast, 2 highly malignant hybrid clones lost one copy of the normal-fibroblast-derived t(14;15), but contained increased numbers (3.44-4.44) of the tumor-derived chromosome 15. Four tumorigenic segregants selected from the weakly tumorigenic fibroblast hybrids by in vivo inoculation showed the same cytogenetic change as the highly tumorigenic hybrid clones, in that the ratio of the normal:tumor-derived chromosomes 15 changed from 1.18-1.55 to 4.11-5.71. Tumorigenicity was thus associated with a modified balance between the tumor vs. the normal-parent-derived 15-chromosomes. Instead of the usual 3:2 ratio, the tumor-derived 15-chromosomes increased disproportionately, whereas the relative number of the normal-parent-derived 15-chromosome decreased, as a rule. These results suggest that amplification of the lymphoma-derived chromosome 15 favors tumorigenicity, but that this effect is counteracted by some influence emanating from the normal-parent-derived homologous chromosome.     

Genetic factors and suppression of metastatic ability of prostatic cancer

Progression of prostatic cancer from nonmetastatic to high metastatic ability may involve the loss of a metastasis suppressor gene. To test this possibility, nonmetastatic and highly metastatic Dunning rat prostatic cancer cells were fused. Hybrid clones were isolated which conserved the chromosomes from their parental cells. When these hybrids were injected into animals, none developed distant metastases. When these nonmetastatic primary tumors were passaged in vivo, occasional animals developed distant metastases. Cytogenetic analysis of eight of these metastatic revertants demonstrated a consistent loss of a copy of a normal chromosome 2. Although previous studies have demonstrated that specific chromosomes can inhibit tumorigenicity in cell fusion experiments, this is the first study to show that prostatic cancer metastasis is associated with the loss of a specific chromosome. Furthermore, these studies suggest that a metastasis suppressor gene for rat prostatic cancer is located on chromosome 2.