Introduction of a normal human chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression.

The development of Wilms' tumor, a pediatric nephroblastoma, has been associated with a deletion in the p13 region of chromosome 11. The structure and function or functions of this deleted genetic material are unknown. The role of this deletion in the process of malignant transformation was investigated by introducing a normal human chromosome 11 into a Wilms' tumor cell line by means of the microcell transfer technique. These variant cells, derived by microcell hybridization, expressed similar transformed traits in culture as the parental cell line. Furthermore, expression of several proto-oncogenes by the parental cells was unaffected by the introduction of this chromosome. However, the ability of these cells to form tumors in nude mice was completely suppressed. Transfer of other chromosomes, namely X and 13, had no effect on the tumorigenicity of the Wilms' tumor cells. These studies provide support for the existence of genetic information on chromosome 11 which can control the malignant expression of Wilms' tumor cells.

Suppression of tumorigenicity in Wilms tumor by the p15.5-p14 region of chromosome 11.

Wilms tumor has been associated with genomic alterations at both the 11p13 and 11p15 regions. To differentiate between the involvement of these two loci, a chromosome 11 was constructed that had one or the other region deleted, and this chromosome was introduced into the tumorigenic Wilms tumor cell line G401. When assayed for tumor-forming activity in nude mice, the 11p13-deleted, but not the 11p15.5-p14.1-deleted chromosome, retained its ability to suppress tumor formation. These results provide in vivo functional evidence for the existence of a second genetic locus (WT2) involved in suppressing the tumorigenic phenotype of Wilms tumor.

Human cell hybrids: analysis of transformation and tumorigenicity.

Intraspecific human-human cell hybrids provide a stable model system with which to investigate the genetic control of transformed and tumorigenic phenotypes. Using this system it has been shown that these phenotypes are under separate genetic control. Furthermore, the tumorigenic phenotype can be complemented by fusion of different tumorigenic cells, resulting in nontumorigenic hybrids. This system also provides information on the control of differentiated function. Molecular cytogenetic techniques should reveal the nature of the chromosomal control of neoplastic transformation.

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.

Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta.

Transforming growth factor beta (TGF beta) is a potent inhibitor of epithelial cell proliferation. A nontumorigenic epidermal growth factor (EGF)-dependent epithelial cell line, BALB/MK, is reversibly growth arrested by TGF beta. TGF beta will also abrogate EGF-stimulated mitogenesis of quiescent BALB/MK cells. Increased levels of calcium (greater than 1.0 mM) will induce differentiation in BALB/MK cells; in contrast, TGF beta-mediated growth inhibition does not result in induction of terminal differentiation. In the present study, the effects of TGF beta and calcium on growth factor-inducible gene expression were examined. TGF beta markedly decreased c-myc and KC gene expression in rapidly growing BALB/MK cells and reduced the EGF induction of c-myc and KC in a quiescent population of cells. TGF beta exerted its control over c-myc expression at a posttranscriptional level, and this inhibitory effect was dependent on protein synthesis. TGF beta had no effect on c-fos gene expression, whereas 1.5 mM calcium attenuated EGF-induced c-fos expression in quiescent cells. Expression of beta-actin, however, was slightly increased in both rapidly growing and EGF-restimulated quiescent BALB/MK cells treated with TGF beta. Thus, in this system, TGF beta selectively reduced expression of certain genes associated with cell proliferation (c-myc and KC), and at least part of the TGF beta effect was at a posttranscriptional level.

Complementation of the tumorigenic phenotype in human cell hybrids.

Several combinations of human tumor cell lines were fused together. Hybrid populations formed from fusions between carcinoma x carcinoma and carcinoma x lymphoblastoid remained highly tumorigenic. However, suppression of tumorigenic potential was seen in carcinoma x sarcoma and carcinoma x melanoma hybrid populations. These results suggest that complementation of the tumorigenic phenotype can occur and that at least 2 different complementation groups may be involved.

KiSS-1, a novel human malignant melanoma metastasis-suppressor gene.

BACKGROUND: Microcell-mediated transfer of chromosome 6 into human C8161 and MelJuSo melanoma cell suppresses their ability to metastasize by at least 95% without affecting their tumorigenicity. This observation demonstrates that the ability to metastasize is a phenotype distinct from tumor formation and suggests that tumorigenic cells acquire metastatic capability only after accumulating additional genetic defects. These results also imply that mutations of genes on chromosome 6 are among those late genetic changes responsible for metastatic potential. They further suggest that a melanoma metastasis-suppressor gene(s) is encoded on chromosome 6 or is regulated by genes on chromosome 6. PURPOSE: Our objective was to identify the gene(s) responsible for the suppression of metastasis in chromosome 6/melanoma cell hybrids. METHODS: A modified subtractive hybridization technique was used to compare the expression of messenger RNAs (mRNAs), via an analysis of complementary DNAs (cDNAs), in metastatic cells (C8161 or MelJuSo) and nonmetastatic hybrid clones (neo6/C8161 or neo6/MelJuSo). RESULTS: A novel cDNA, designated KiSS-1, was isolated from malignant melanoma cells that had been suppressed for metastatic potential by the introduction of human chromosome 6. Northern blot analyses comparing mRNAs from a panel of human melanoma cells revealed that KiSS-1 mRNA expression occurred only in nonmetastatic melanoma cells. Expression of this mRNA in normal heart, brain, liver, lung, and skeletal muscle was undetectable by northern blot analysis. Weak expression was found in the kidney and pancreas, but the highest expression was observed in the placenta. The KiSS-1 cDNA encodes a predominantly hydrophilic, 164 amino acid protein with a polyproline-rich domain indicative of an SH3 ligand (binds to the homology 3 domain of the oncoprotein Src) and a putative protein kinase C-alpha phosphorylation site. Transfection of a full-length KiSS-1 cDNA into C8161 melanoma cells suppressed metastasis in an expression-dependent manner. CONCLUSIONS: These data strongly suggest that KiSS-1 expression may suppress the metastatic potential of malignant melanoma cells. IMPLICATIONS: KiSS-1 may be a useful marker for distinguishing metastatic melanomas from nonmetastatic melanomas.

Localization and characterization of a chromosome 11 tumor suppressor gene using organotypic raft cultures.

The development and progression of human cancer often involves the inactivation of tumor suppressor gene function. Alterations in human chromosome 11 during the development of human cutaneous squamous cell carcinoma suggest the presence of a tumor suppressor gene on this chromosome. Moreover, previous studies in our laboratory demonstrated the presence of a functional tumor suppressor gene on chromosome 11 for the human cutaneous squamous cell carcinoma cell line A388.6TG.c2. In this investigation, we have used organotypic culturing of epithelial cells as a novel in vitro assay for tumor suppression. A388.6TG.c2 and control cells form an abnormal stratified epithelium of 8-12 layers when cultivated on organotypic rafts. In contrast, the chromosome 11 microcell hybrids, HMC 100p4B and HMC 100p5A, form an epithelium of only two to three cell layers. This in vitro growth suppression of the chromosome 11 microcell hybrids in the organotypic rafts correlates well with our previous in vivo skin graft experiments. Comparison of the proliferation and apoptotic indices of cell lines grown on the organotypic rafts suggests that the tumor suppressor gene on chromosome 11 has restricted the ability of the microcell hybrids to stratify but has not significantly altered their ability to undergo cell division or programmed cell death. Furthermore, flow cytometric analysis of cells grown on organotypic raft cultures suggests that the chromosome 11 microcell hybrids are actively progressing through the cell cycle rather than arrested in a particular stage. We have used this novel application of organotypic raft cultures to further localize the chromosome 11 tumor suppressor gene. Introduction of a single der(11)t(X;11) chromosome lacking most of the long arm of chromosome 11 into A388.6TG.c2 does not affect growth on organotypic raft cultures. These data suggest the tumor suppressor gene maps to the long arm of chromosome 11 in the region of 11q13-qter.

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.

Tumorigenicity and oncogene expression in pediatric cancers.

Cytogenetic and epidemiological studies of pediatric cancers have implicated a loss of genetic information in the development of these tumors. In contrast, other studies have shown that activation of endogenous oncogenes is a common event in these cancer cells. The technique of somatic cell hybridization provides a model for investigating the interaction between loss of genetic elements and oncogene activation in pediatric cancers. A variety of human-human cell hybrids were formed between a tumorigenic adult carcinoma and representative tumorigenic pediatric cell lines. All hybrid cells were completely suppressed for tumor-forming ability when assayed in nu/nu (nude) mice. When the expression of the N-myc, c-myc, and sis oncogenes and tumorigenicity were examined in the same hybrid cells, no correlation was found, suggesting that the expression of these oncogenes in these hybrid cells did not appear to be controlled by putative "tumor suppressor" genes. Thus, tumorigenicity behaves as a recessive genetic trait in pediatric cancers. Furthermore, different genetic elements may be lost during tumor development of adult cancers as opposed to pediatric cancers.

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.

p53-dependent p21 induction following gamma-irradiation without concomitant p53 induction in a human peripheral neuroepithelioma cell line.

We previously generated cell hybrids between a derivative of the E6-containing HeLa cell line and a p53 null peripheral neuroepithelioma (PNET) cell line. Although p53 protein from the hybrids was genotypically wild type, it did not demonstrate wild-type behavior. Therefore, in the present study, we introduced wild-type p53 into the PNET parent to investigate whether p53 retained wild-type function within this cell line. Although the p53 null PNET parent lacked detectable p21 protein, introduction of wild-type p53 resulted in a detectable expression of p21 protein in all clones tested, suggestive of wild-type p53 function. In addition, p53 expression was necessary for induction of p21 in response to irradiation, and, furthermore, we show this induction to occur at the transcriptional level. Although introduction of wild-type p53 seems to be responsible for p21 induction, the overall protein levels of p53 were not induced. The involvement of p53 in up-regulating p21 is further substantiated by the observation that p21 up-regulation was dependent on the introduction of the wild-type protein. Our results suggest that wild-type p53 is capable of up-regulating p21 in response to DNA damage in the absence of p53 induction.

Genomic organization of the human p57KIP2 gene and its analysis in the G401 Wilms' tumor assay.

The p57KIP2 gene encodes an inhibitor of cyclin-dependent kinase activity, which negatively regulates cell cycle progression. The human p57 gene is located in 11p15.5, a region of DNA frequently altered in neoplasia. We have isolated a human genomic clone and mapped the p57 gene to a 2.2-kb region between D11S648 and D11S679. Sequence analysis revealed that the coding DNA of the human p57 gene is divided by 0.5-kb intron. A second intron was detected in the 3' untranslated region, indicating that the human p57 gene contains at least three exons. Our previous work with somatic cell hybrids mapped a tumor suppressor gene for the G401 Wilms' tumor cell line to a approximately 500-kb region of 11p15.5 that includes p57. Northern blot analysis detected a 0.8-kb p57 transcript in several of the G401 hybrid lines. However, p57 expression did not correlate with tumor suppression. These results suggest that p57 is not responsible for the tumor suppression observed in our somatic cell hybrid assay.

Growth modulation of mouse keratinocytes by transforming growth factors.

The effects of exogenously added transforming growth factor (TGF alpha and TGF beta on the growth of BALB/MK cells were examined. TGF alpha supplanted the epidermal growth factor (EGF) requirement in these cells. In contrast, TGF beta reversibly inhibited the growth of BALB/MK cells by abrogating the stimulatory actions of EGF or TGF alpha. The inhibitory effects of TGF beta appeared to be mediated by events distal to EGF ligand-receptor interactions. Growth inhibition of BALB/MK cells by TGF beta did not result in the induction of differentiation. This finding is different from the growth inhibition of these cells induced by elevated calcium levels (1.5 mM) which was tightly coupled to terminal differentiation. The BALB/MK cells were found to express TGF alpha mRNA, as well as TGF beta mRNA and protein. In addition, TGF alpha, as well as EGF, enhanced TGF alpha gene expression. These studies suggest a role for endogenous TGFs in regulating BALB/MK proliferation. TGF alpha provides a positive growth signal, while TGF beta is a potent inhibitor of growth even in the presence of such positive modulators as TGF alpha and EGF.

Suppression of MDA-MB-435 breast carcinoma cell metastasis following the introduction of human chromosome 11.

To determine the relevance of genetic information on chromosome 11 in the development of metastatic breast tumors, we introduced a normal human chromosome 11 into the highly metastatic MDA-MB-435 breast carcinoma cell line via the microcell-mediated chromosome transfer technique. Although the MDA-MB-435 recipient cell line and four randomly selected microcell hybrid clones remained tumorigenic in nude mice, the hybrids were >95% suppressed for metastasis to lung and regional lymph nodes (p<0.01). We also tested whether chromosome 6 harbors a metastasis-suppressor gene for breast cancer as observed previously for human melanoma. Grouped together, the four neo6 microcell hybrids had no statistically significant reduction in the incidence or number of lung or lymph node metastases compared to the weakly metastatic, subcloned parent cell line, MDA-MB-453.7. Expression of nm23-H1 (NME1), a known metastasis-suppressor gene in this breast cancer cell line, did not correlate with metastasis suppression in the microcell hybrids. These results further demonstrate that control of metastasis is molecularly distinct from tumorigenic potential. They also indicate that chromosome 11 encodes a metastasis-suppressor gene for human breast cancer.

Cyclin-dependent kinase inhibitor p57KIP2 in soft tissue sarcomas and Wilms'tumors.

Mammalian cyclin-dependent kinase inhibitors fall into two families, the INK4 and the CIP/KIP. The CIP/KIP family comprises three structurally related members, including p21CiP1/WAF1, p27KIP1, and p57KIP2. These proteins are all capable of inhibiting the progression of the cell cycle by binding and inhibiting G(1) cyclin/cyclin-dependent kinase complexes. In humans, p57KIP2 is expressed specifically in skeletal muscle, heart, brain, kidney, and lung. Human KIP2 resides in 11p15.5, a chromosomal region that is a common site for loss of heterozygosity in certain sarcomas, Wilms' tumors, and tumors associated with the Beckwith-Wiedemann syndrome. Because of the function, selective expression, and chromosomal location of p57KIP2, we undertook the present study to search for potential mutations of KIP2 in a cohort of 126 tumors composed of 75 soft tissue sarcomas and 51 Wilms' tumors. The KIP2 gene was characterized by Southern blot, comparative multiplex PCR, PCR -single-strand conformational polymorphism, and DNA sequencing assays in these neoplasms. Deletions of the KIP2 gene or point mutations at the region encoding the cyclin-dependent kinase inhibitory domain were not found in the tumors analyzed. The absence of KIP2 mutations might indicate that these tumors arise due to defects at a closely linked but separate locus. Alternatively, similarly to the mouse homologue, inactivation of KIP2 could occur via genomic imprinting.

Inactivation of human SRBC, located within the 11p15.5-p15.4 tumor suppressor region, in breast and lung cancers.

A cDNA clone encoding human SRBC [serum deprivation response factor (sdr)-related gene product that binds to c-kinase] was isolated in a yeast two-hybrid screening, with amino acids 1-304 of BRCA1 as the probe. The human SRBC gene (hSRBC) was mapped to chromosome region 11p15.5-p15.4, close to marker D11S1323, at which frequent loss of heterozygosity (LOH) has been observed in sporadic breast, lung, ovarian, and other types of adult cancers as well as childhood tumors. hSRBC-coding region mutations including frame shift and truncation mutations were detected in a few ovarian and lung cancer cell lines. More significantly, the expression of hSRBC protein was down-regulated in a large fraction [30 (70%) of 43] of breast, lung, and ovarian cancer cell lines, whereas strong expression of hSRBC protein was detected in normal mammary and lung epithelial cells. The down-regulation of hSRBC expression in cancer cells was associated with hypermethylation of CpG dinucleotides in its promoter region, and 3 (60%) of 5 primary breast tumors and 11 (79%) of 14 primary lung tumors were also found to be hypermethylated. Treatment of breast cancer MCF7 cells with 5'azacytidine and Trichostatin A resulted in expression of hSRBC, confirming DNA methylation as the mode of inactivation. Our results suggest that epigenetic or mutational inactivation of hSRBC may contribute to the pathogenesis of several types of human cancers, marking hSRBC as a candidate tumor suppressor gene.

A functional analysis of tumor suppressor activity for peripheral neuroepitheliomas by monochromosome transfer.

The microcell hybridization technique provides a powerful method for the identification and characterization of tumor suppressor genes. By introducing chromosomes from a normal human cell into a tumor cell, several studies have presented functional evidence for the presence of tumor suppressor activity. In order to map the location(s) of functional tumor suppressor gene(s) for peripheral neuroepithelioma (PNET) cells, we have used the microcell hybridization technique to transfer three individual human chromosomes into three different PNET cell lines, A673, SK-N-MC and TC32. We could not isolate microcell hybrids from one of the cell lines as the transferred chromosome tended to fragment upon transfer. Introduction of chromosome 13 into the remaining two cell lines caused a marked inhibition of in vitro and in vivo growth. Chromosome 11 appeared to harbor a functional tumor suppressor gene while transfer of chromosome 17 caused a suppression of growth in culture, presumably due to the presence of the p53 tumor suppressor gene. Thus, each cell line showed a different response to the introduction of normal genetic information suggesting diverse genetic abnormalities among these tumors of similar histological or origin.

Alteration of hSNF5/INI1/BAF47 detected in rhabdoid cell lines and primary rhabdomyosarcomas but not Wilms' tumors.

The organization of genomic DNA into chromatin aids in the regulation of gene expression by limiting the access of transcriptional binding domains. The SWI/SNF family of chromatin-remodeling complexes, which are conserved from yeast to humans, open the chromatin to facilitate the transcriptional machinery to access their targets. The gene encoding the BAF47/hSNF5 subunit of the complex has been found mutated in both rhabdoid cell lines and in primary rhabdoid tumors. Since the pediatric tumors rhabdomyosarcoma (RMS) and Wilms' tumor (WT) share a similar genetic link with rhabdoid tumors, it was hypothesized that they may also show alterations of the BAF47 gene. Using primary tumors, the BAF47 protein was detected in all WT but less than 75% of the RMS tested. In cell lines, the BAF47 protein was missing in all rhabdoid cell lines and one RMS cell line. Analysis of sample DNA displayed either a mutation or deletion of the BAF47 gene in all samples negative for the protein. Several other subunits of the human SWI/SNF complex, including BRG1 which is the subunit directly interacting with the Rb tumor suppressor gene, were detected in all tumor samples. Alteration of BAF47 may be a genetic marker associated with the poor prognosis seen in all rhabdoid tumors but only some RMS.

Interactions of growth factors and retroviral oncogenes with mitogenic signal transduction pathways of Balb/MK keratinocytes.

Balb/MK epidermal keratinocytes require epidermal growth factor (EGF) for growth in serum-containing medium and terminally differentiate in response to high Ca++ concentration. Several oncogenic retroviruses have been shown to relieve the EGF requirement and to block calcium-induced terminal differentiation. We developed a chemically defined medium to investigate the minimum growth factor requirements for Balb/MK cells, as well as how such requirements might be altered by retroviral oncogenes. In this medium insulin, apparently acting as IGF-1, and EGF supported cell growth in a manner comparable to serum and EGF. Acidic as well as basic fibroblast growth factor (FGF) substituted for EGF but not insulin in supporting Balb/MK proliferation. Infection with retroviruses containing v-ras oncogenes (v-H-ras, v-Ki-ras), oncogenes derived from growth factor receptors (v-erbB, v-fms) or the v-mos oncogene permitted growth in defined medium containing insulin but lacking EGF. The v-fgr oncogene, a member of the src subfamily, was unique in conferring independence from both insulin and EGF. Our findings establish the applicability of this system for biologic assay of epithelial cell growth factors as well as identification of specific growth factor requirements that can be altered or complemented by the actions of specific oncogenes.