SV40 transfection of human kidney epithelial cells and stability of chromosome 11

Simian virus 40 (SV40) transformation has been used in a variety of mammalian cells and has been shown to extend their life span. We therefore decided to apply these results to normal kidney and tumoral cells derived from Wilms' patients. Wilms' tumour, a nephroblastoma which presents in early childhood, has been linked to deletions and rearrangements in chromosome 11. Analysis of gene structure in the 11p13 locus involved in the development of the tumour has been restricted by the very short life-span of the tumoral cells in vitro. We transfected normal kidney WT/NK, tumoral WT/T cells and human foetal kidney HFK cells with 2 SV40-derived plasmids SV3neo (pBR322-SV40-containing neomycin bacterial gene) and SVori- (pMK-origin mutated SV40). We isolated a high number of SV40-transfected cell lines. The efficiency of transfection appeared to be extremely low in WT/T cells compared with HFK and even WT/NK. The life span of the cell lines was increased in relation to their untransfected homologues. However, in all of the cell lines except 3, senescence occurred, after crisis step or not. We looked at different markers associated with SV40 transformation of mammalian cells and specifically with the presence of SV40 T antigen in the cells and its consequences: AIG, tumorigenicity, expression and insertion in genomic DNA of SV40 T antigen. Genetic studies involving karyotypic and restriction fragment length polymorphism (RFLP) analysis demonstrated that, despite a frequent pseudo-diploidy, the cell lines derived have conserved the 11p13 locus.     

Role of oncogenes and tumour suppressor genes in human lung carcinogenesis.

Six families of activated protooncogenes, ras, raf, fur, neu, jun and myc have so far been associated with human lung cancer. Human bronchial epithelial cells in vitro are being used to investigate the functional role of these specific oncogenes and growth regulatory genes in carcinogenesis and tumour progression. When transferred into normal human bronchial epithelial cells by the highly efficient protoplast fusion method, the v-Ha-ras oncogene initiates a cascade of events leading to decreased responsiveness of these cells to inducers of squamous differentiation, aneuploidy and, less frequently, 'immortality' and tumorigenicity with metastasis in athymic nude mice. Transfection of the SV40 T antigen gene results in nontumorigenic cell lines that have a nearly normal pathway of terminal squamous differentiation and can be transformed into malignant cells by transfected Ha-ras, N-ras or Ki-ras. The combination of transfected c-myc and c-raf-1 also transforms human bronchial epithelial cells into neoplastic cells that exhibit some phenotypic traits found in small-cell carcinomas. These and other results indicate that proto-oncogenes dysregulate the pathways of growth and differentiation of human bronchial epithelial cells and play an important role in human carcinogenesis. Analyses of allelic deletion and somatic cell hybrids are being used to identify the chromosomal localization of tumour suppressor genes. We have examined 54 non-small-cell bronchogenic carcinomas with 13 polymorphic markers. Loss of heterozygosity was more frequent than among 23 squamous-cell carcinomas than among 23 adenocarcinomas or eight large-cell carcinomas. Loss of heterozygosity for chromosome 17p was found in 89% of cases of squamous-cell carcinoma and 18% of adenocarcinomas. Analysis of chromosome 11 for allelic deletions revealed two commonly deleted regions (11p13 and 11p15.5). Somatic cell hybrids between normal human bronchial epithelial cells and Hut292DM, a lung carcinoma cell line, had a finite lifespan in vitro and were nontumorigenic in athymic nude mice. Tumour suppressive effects of individual or combinations of specific human chromosomes on Hut292DM are being examined by formation of microcell-cell hybrids. Chromosome 11 has tumour suppressor activity in these hybrids. Both of these studies suggest that tumour suppressor genes play a dominant role in lung carcinogenesis and provide in-vitro model systems for isolating these genes by subtraction library and insertional mutagenesis techniques.     

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.     

Alteration of tumorigenicity in undifferentiated thyroid carcinoma cells by introduction of normal chromosome 11

The tumorigenicity of various cell lines has been shown to be suppressed by the introduction of chromosome 11 and other chromosomes via micro-cell-mediated chromosome transfer. In this study, we investigated whether a human undifferentiated thyroid carcinoma cell line, TTA-1, was suppressed by the introduction of normal human chromosome 11 or 10. Chromosome 10 or 11 was transferred from A₉ cells containing a single human chromosome 10 or 11 tagged with pSV₂-neo plasmid DNA into TTA-1 cells, by microcell fusion. The tumorigenicity of the TTA-1 cells and their colony-forming efficiency in soft agar were suppressed by chromosome 11, but not by chromosome 10. These results suggest that normal human chromosome 11 carries a putative tumor suppressor gene that affects the tumor-associated phenotypes of TTA-1 cells. © Wiley-Liss, Inc.     

Analysis of the candidate 8p21 tumour suppressor,BNIP3L,in breast and ovarian cancer

Loss of heterozygosity (LOH) on the short arm of chromosome 8, at 8p12-p23, is one of the most frequent genetic events in both breast and ovarian cancer, suggesting the location of a shared tumour suppressor gene. Microcell-mediated chromosome transfer of chromosome 8 suppresses tumorigenicity and growth of colorectal and prostate cancer cell lines, further supporting the presence of a tumour suppressor gene on 8p. We have taken a candidate gene approach to try to identify this tumour suppressor gene at 8p12-p23. BNIP3L, which has sequence homology to pro-apoptotic proteins and the ability to suppress colony formation in soft agar, is located at 8p21, within a region of ovarian cancer LOH, breast cancer LOH and prostate cancer metastasis suppression. BNIP3L expression was assessed by both RT-PCR and Northern blot analysis in breast and ovarian cancer cell lines and found to be expressed at similar levels relative to expression in their respective normal epithelial cell lines. Genetic analysis of BNIP3L in 40 primary ovarian and 25 primary breast tumours identified one somatic, intronic mutation in one ovarian tumour, as well as several polymorphisms, including one resulting in an amino-acid substitution. These data suggest that BNIP3L is unlikely to be the target of 8p LOH in ovarian or breast cancer.     

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.     

Cell types expressing the Wilms' tumour gene (WT1) in Wilms' tumours: implications for tumour histogenesis.

Wilms' tumour (nephroblastoma), a childhood embryonal kidney tumour, is believed to arise from malignant transformation of abnormally persistent metanephric blastemal cells. At a histological level, tumours show a remarkable mimicry of the normal nephrogenic pathway. There is histological and epidemiological evidence for at least two pathogenetic groupings within Wilms' tumour which may reflect different timings of the tumorigenic insult in this pathway and/or involvement of different genes. Tumorigenesis is thought to result from loss of function of a so-called tumour-suppressor gene which has an essential role in control of normal genitourinary development. Such a candidate, Wilms' tumour gene (WT1) mapping to chromosome 11p13, has been isolated and is known to be mutated in some tumours. We have examined the cell types expressing this gene in 32 Wilms' tumours and in nephroblastomatosis by in situ mRNA hybridization. Our results show that WT1 is expressed only in neoplastic structures whose normal counterparts also express the gene and that abnormally persistent high levels of expression are common in both these lesions. Thus, WT1 expression is a good marker for tumour differentiation and reveals how the normal pattern of differentiation is disrupted in Wilms' tumours. We postulate that mutation of the WT1 gene at the 11p13 locus results in Wilms' tumours associated with intralobar nephrogenic rests, which frequently show stromal-predominant histology. We have used our results and ideas to reinterpret current theories on tumour histogenesis and propose a model which explains how patterns of epithelial differentiation are disrupted in Wilms' tumour and how malignant stroma can result from mutation in WT1.     

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.     

Identification of a tumor suppressive critical region mapping to 3p14.2 in esophageal squamous cell carcinoma and studies of a candidate tumor suppressor gene, ADAMTS9

A gene critical to esophageal cancer has been identified. Functional studies using microcell-mediated chromosome transfer of intact and truncated donor chromosomes 3 into an esophageal cancer cell line and nude mouse tumorigenicity assays were used to identify a 1.61 Mb tumor suppressive critical region (CR) mapping to chromosome 3p14.2. This CR is bounded by D3S1600 and D3S1285 microsatellite markers. One candidate tumor suppressor gene, ADAMTS9, maps to this CR. Further studies showed normal expression levels of this gene in tumor-suppressed microcell hybrids, levels that were much higher than observed in the recipient cells. Complete loss or downregulation of ADAMTS9 gene expression was found in 15 out of 16 esophageal carcinoma cell lines. Promoter hypermethylation was detected in the cell lines that do not express this gene. Re-expression of ADAMTS9 was observed after demethylation drug treatment, confirming that hypermethylation is involved in gene downregulation. Downregulation of ADAMTS9 was also found in 43.5 and 47.6% of primary esophageal tumor tissues from Hong Kong and from the high-risk region of Henan, respectively. Thus, this study identifies and provides functional evidence for a CR associated with tumor suppression on 3p14.2 and provides the first evidence that ADAMTS9, mapping to this region, may contribute to esophageal cancer development.     

11q23.1 and 11q25-qter YACs suppress tumour growth in vivo

Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region. In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region. An 11q YAC contig was accessed and three YACs, one containing the candidate gene ATM at 11q23.1, and two contiguous YACs (overlapping for approximately 400-600 kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells. Selected A9 transfectant clones (and control untransfected and 'irrelevant' alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice. All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and 'irrelevant' YAC transfected A9 cells. These results define two discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a approximately 1.2 Mb region on 11q23.1 (containing the ATM locus) and another to a approximately 400-600 kb subterminal region on 11q25-qter.     

Methylation of chromosome 11p genes in wilms-tumor and kidney tissue

Three genes on the short arm of chromosome 11 (WT1, IGF2 and HRAS) were hypomethylated in Wilms' tumour tissue compared to normal kidney tissue, and one (CALCA) was hypermethylated. IGF2 and HRAS showed evidence of allele-specific methylation, which may indicate genomic imprinting of the 11p15 region in some kidney cells.     

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.     

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.     

Loss of heterozygosity for chromosome region 11p15 in Wilms' tumours is not related to HRAS gene transforming mutations

Although a candidate Wilms' tumour gene--WT1--has been identified in chromosome region 11p13, there is strong evidence from loss of heterozygosity studies suggesting that a second relevant gene is present in region 11p15. The Harvey-Ras proto-oncogene also lies in this region. In other types of tumours mutations in RAS genes have been associated with the development and/or progression of a number of tumour types. We therefore analysed the sequence of the Ras oncogene for possible mutations in six Wilms' tumours showing loss of heterozygosity for chromosome region 11p15. No tumour analysed showed HRAS sequence mutations. We conclude that loss of heterozygosity at 11p15 does not implicate HRAS mutations in the molecular pathogenesis of Wilms' tumour.     

Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome 11.

Previous studies using somatic cell hybridization of highly metastatic and nonmetastatic rat prostatic cancer cells demonstrated that the resultant hybrids were nonmetastatic if all of the parental chromosomes were retained. Somatic hybrid segregants which underwent nonrandom chromosomal losses reexpressed high metastatic ability. These results demonstrated that there are gene(s) the expression of which can suppress metastatic ability of prostatic cancer cells. To identify the location of homologous gene(s) in the human, specific human chromosomes were introduced into highly metastatic rat prostatic cancer cells using the microcell-mediated chromosome transfer. Introduction of human chromosome 11 into highly metastatic rat prostate cancer cells results in suppression of metastatic ability without suppression of the in vivo growth rate or tumorigenicity of the hybrid cells. Spontaneous deletion of portions of human chromosome 11 in some of the clones delineated the minimal portion of human chromosome 11 capable of suppressing prostatic cancer metastases as the region between 11p11.2-13 but not including the Wilms' tumor-1 locus.