Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.

Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.

We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.

Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.

Physical and functional mapping of a tumor suppressor locus for renal cell carcinoma within chromosome 3p12.

Using a functional genetic approach, we previously identified a novel genetic locus, NRC-1 (Nonpapillary Renal Cell Carcinoma 1), that mediated tumor suppression and rapid cell death of renal cell carcinoma (RCC) cells in vivo. For these experiments, a defined subchromosomal fragment of human chromosome 3p was transferred into a sporadic RCC cell line via microcell fusion, and microcell hybrid clones were tested for tumorigenicity in vivo. The results indicated functional evidence for a novel tumor suppressor locus within the 3p14-p12 interval known to contain the most common fragile site of the human genome (FRA3B), the FHIT gene, and the breakpoint region associated with the familial form of RCC. We now report the physical mapping of the NRC-1 critical region by detailed microsatellite analyses of novel microcell hybrid clones containing transferred fragments of chromosome 3p in the RCC cell background that were phenotypically suppressed or unsuppressed for tumorigenicity in vivo. The results limit the region containing the tumor suppressor locus within chromosome 3p12. The FHIT gene, FRA3B, and the familial RCC breakpoint region were excluded from the NRC-1 critical region. Furthermore, the NRC-1 locus falls within a well-characterized homozygous deletion region of 5-7 Mb associated with a small cell lung carcinoma cell line, U2020, suggesting that a more general tumor suppressor gene may reside in this region.

Analysis of the functional role of chromosome 10 loss in human glioblastomas.

Molecular and cytogenetic analyses of primary brain tumors have shown that losses on chromosome 10 occur very frequently in human glioblastoma multiforme suggesting the presence of a glioma-associated tumor suppressor gene on this chromosome. To examine this hypothesis, a copy of chromosome 10 derived from a human fibroblast cell line was introduced into the human glioma cell line U251 by microcell-mediated chromosomal transfer. A human chromosome 2 was also independently introduced into U251 cells. The presence of novel chromosomes or chromosomal fragments was confirmed by molecular and karyotypic analyses. The hybrid clones containing a transferred chromosome 10 exhibited a suppression of their transformed and tumorigenic phenotype in vivo and in vitro, whereas cells containing a transferred chromosome 2 failed to alter their phenotype. The hybrid cells containing a transferred chromosome 10 displayed a significant decrease in their saturation density and an altered cellular morphology at high cell density but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in the ability of cells with an introduced chromosome 10 to grow in soft agarose. The introduction of chromosome 10 completely suppressed tumor formation when the hybrid cells were injected into nude mice. These findings indicate that chromosome 10 harbors a tumor suppressor gene that is directly involved in glioma oncogenesis.

The genetic locus NRC-1 within chromosome 3p12 mediates tumor suppression in renal cell carcinoma independently of histological type, tumor microenvironment, and VHL mutation.

Human chromosome 3p cytogenetic abnormalities and loss of heterozygosity have been observed at high frequency in the nonpapillary form of sporadic renal cell carcinoma (RCC). The von Hippel-Lindau (VHL) gene has been identified as a tumor suppressor gene for RCC at 3p25, and functional studies as well as molecular genetic and cytogenetic analyses have suggested as many as two or three additional regions of 3p that could harbor tumor suppressor genes for sporadic RCC. We have previously functionally defined a novel genetic locus nonpapillary renal carcinoma-1 (NRC-1) within chromosome 3p12, distinct from the VHL gene, that mediates tumor suppression and rapid cell death of RCC cells in vivo. We now report the suppression of tumorigenicity of RCC cells in vivo after the transfer of a defined centric 3p fragment into different histological types of RCC. Results document the functional involvement of NRC-1 in not only different cell types of RCC (i.e., clear cell, mixed granular cell/clear cell, and sarcomatoid types) but also in papillary RCC, a less frequent histological type of RCC for which chromosome 3p LOH and genetic aberrations have only rarely been observed. We also report that the tumor suppression observed in functional genetic screens was independent of the microenvironment of the tumor, further supporting a role for NRC-1 as a more general mediator of in vivo growth control. Furthermore, this report demonstrates the first functional evidence for a VHL-independent pathway to tumorigenesis in the kidney via the genetic locus NRC-1.

A homozygous deletion on chromosome 3 in a small cell lung cancer cell line correlates with a region of tumor suppressor activity.

Small cell lung cancer (SCLC) tumors frequently display deletions on the short arm of chromosome 3 suggesting the existence of a 'tumor suppressor' gene within that region whose functional inactivation may be involved in tumorigenesis. Recently, a hybrid, HA(3)BB9F, was identified that contains a small fragment of human chromosome 3 of approximately 2 Mb on a mouse (A9) background (Killary et. al., 1992). This hybrid was utilized to define a functional tumor suppressor gene within 3p22-p21 which could suppress the tumorigenic properties of the mouse fibrosarcoma cell line. The existence of a tumor suppressor gene in the region 3p22-p21 is supported by the present report which describes the assessment of 89 SCLC and 32 non-SCLC lung cancer tumors and cell lines for the existence of a homozygous deletion(s) at 43 loci on the short arm of chromosome 3. One of the SCLC cell lines was found to harbor a homozygous deletion involving the loss of five markers on chromosome 3p. All five of the markers map to the region 3p21.3-p21.2 and four of the five markers are located within the chromosome 3 fragment exhibiting properties of tumor suppression in the HA(3)BB9F hybrid. The other tumors analysed all retained at least one copy of each of the markers assessed.

Functional evidence for an ovarian cancer tumor suppressor gene on chromosome 22 by microcell-mediated chromosome transfer.

The identity of many tumor suppressor genes important in epithelial ovarian cancer tumorigenesis remains unknown. In an effort to localize a novel tumor suppressor on chromosome 22, a psv2neo tagged human chromosome 22 was transferred into the malignant epithelial ovarian cancer cell line, SKOv-3, by microcell-mediated chromosome transfer. Complete suppression of the transformed phenotype was observed in 16 of 18 individual microcell hybrid clones as evidenced by the complete abrogation of cell growth under anchorage-independent conditions. In vitro doubling times were also dramatically reduced, as was the ability to form subcutaneous tumors in CD1 nu/nu mice. Only one polymorphic marker, D22S429, segregated with decreased transformation and tumorigenic potential, suggesting that an unrecognized tumor suppressor may localize to chromosome 22q11-q12. These data provide functional support for the presence of a novel tumor suppressor locus (or loci) on chromosome 22 that is important in ovarian cancer tumorigenesis.

An 80 Kb P1 clone from chromosome 3p21.3 suppresses tumor growth in vivo.

High frequencies of allelic loss on the short arm of chromosome 3 in small cell lung cancer (SCLC) and a number of other tumors suggest the existence of a tumor suppressor gene(s) within the deleted regions. Two small cell lung cancer lines, NCI H740 and GLC20, have been described which have homozygous deletions in the region 3p21.3. The deleted region overlaps with a 2 Mb fragment of human DNA present in the interspecies hybrid HA(3)BB9F, that suppresses tumor formation by mouse A9 fibrosarcoma cells. Human sequences from this cell hybrid were isolated using inter Alu PCR. From this starting point, a P1 contig was developed for the region of 450 Kb that is common to the homozygous deletions seen in the SCLC lines NCI H740 and GLC20 and is also present in HA(3)BB9F, the suppressed A9 hybrid. Individual P1 clones were assayed for their ability to suppress the tumorigenicity of the mouse fibrosarcoma cell line A9 as assayed by injection of transfected A9 cells into athymic nude mice. The introduction of one of the P1 clones into A9 cells resulted in suppression of tumor growth whereas two other P1 clones from the contig failed to suppress tumor formation in athymic nude mice. These data functionally delimit a tumor suppressor locus to a region of 80 kb within a P1 clone at 3p21.3.

Cytogenetic analysis of a primary salivary gland myoepithelioma.

Myoepithelioma, a rare benign salivary gland neoplasm, is a tumor composed entirely of myoepithelial cells. Unlike pleomorphic adenoma, these tumors lack any ductal epithelial differentiation, and manifest a minor stromal element. Previous cytogenetic and molecular genetic studies have mainly investigated pleomorphic adenomas and reported recurring specific chromosomal alterations at 8q12 and 12q13-q15 regions. The cell origin of these alterations, however, remains speculative. We report the cytogenetic analysis of a parotid myoepithelioma and discuss the putative origin for the cells with cytogenetic alterations. Our analysis shows 12q12 involved in a translocation with a previously unreported partner (1q), and nonrandom del(9)(q22.1q22.3) and del(13)(q12q22). Our results indicate that the myoepithelial cell is the source of those cells with chromosomal alterations, and that myoepithelioma shares 12q alterations reported in a subset of pleomorphic adenomas.

Concurrent cytogenetic, interphase fluorescence in situ hybridization and DNA flow cytometric analyses of a carcinoma ex-pleomorphic adenoma of parotid gland.

We report the cytogenetic, fluorescence in situ hybridization (FISH), and DNA ploidy analyses of a high grade carcinoma ex-pleomorphic adenoma of the submandibular gland. Our overall combined analyses showed a marked DNA aneuploidy and numerical abnormalities involving all chromosomes. Cytogenetic analysis revealed a near tetraploid modal chromosomal number with tetraploid loss of chromosomes Y, 1, 6, 9, 11, 14, 15, 17, and 19-21 and hypertetraploid gain of chromosomes 7, 8, and 22. The structural abnormalities included der(1;14)(q10;q10), del(6)(q15q34), +del(6)(q15q34), +der(8) t(1;8)(q12;q12.2),der(9;19)(q10;q10),add(14)(p11.2),i(20)(q10),der(21) t(8;21)(q11.2;q22.3),+der(21)t(8;21) (q11.2;q22.3). Interphase FISH of the primary and short-term cultured cells using directly labeled pericentromeric probes for chromosomes 6-12, 17, 18, and Y resulted in alterations corresponding to the cytogenetic findings. DNA ploidy analysis of both the primary and cultured tumor cells showed a hyperdiploid stemline with DNA indices of 2.6. The results indicate that: (1) marked numerical, structural chromosomal, and DNA content abnormalities are present in this tumor; and (2) alteration at 8q and 6q regions, together with previous results, suggest an association between these events and the development and/or progression of this tumor.

Limited nonrandom chromosomal aberrations in a recurrent adenoid cystic carcinoma of the parotid gland.

We present the cytogenetic, interphase fluorescence in-situ hybridization (FISH) and DNA content findings in a clinically aggressive adenoid cystic carcinoma (ADCC) of the parotid gland. The tumor manifested diploid chromosomal and DNA content by cytogenetic, interphase FISH and flow cytometry. G-banding analysis revealed inv(5)(p15.2q33) and t(6;15)(q25;q15) as the only structural alterations in all 30 metaphases examined. The limited structural abnormalities found in this recurrent lesion suggest that they may constitute a primary or early event in the development of this tumor. The involvement of 6q region in our tumor and in some of the previously reported ADCC supports the association between this region and the evolution of at least a subset of these tumors.

A mucoepidermoid carcinoma of minor salivary gland with t(11;19)(q21;p13.1) as the only karyotypic abnormality.

We present the cytogenetic analysis of a mucoepidermoid carcinoma of the minor salivary gland with t(11,19)(q21;p13.1) as the sole karyotypic abnormality. Our findings, along with those of previous reports, indicate that this translocation is an early and most likely a primary event in the development of a least a subset of these neoplasms.

Genotypic characterization of a primary mucoepidermoid carcinoma of the parotid gland by cytogenetic, fluorescence in situ hybridization, and DNA ploidy analysis.

We present a case of mucoepidermoid carcinoma with t(3;12)(q24;p13) and polysomy X by cytogenetic and fluorescence in situ hybridization (FISH) techniques. Flow cytometric DNA analysis of the primary tumor showed DNA aneuploidy and analysis of cultured tumor cells showed DNA diploidy indicating restricted growth of the diploid tumor cells in short-term tissue culture. Interphase cytogenetic analysis of chromosomes 1, 3, 6-12, 16-18, and X in the primary tumor showed polysomy 1, 9, 18, and X, monosomy 8 and 17, and disomy 3, 6, 7, 10-12, and 16. Except for chromosome X, other numerical chromosomal abnormalities were not detected by conventional cytogenetic analysis. Our combined approach allowed for better characterization of the genotypic features of this neoplasm.

Multiple unrelated translocations in a metastatic epimyoepithelial carcinoma of the parotid gland.

We report the cytogenetic, fluorescence in-situ hybridization (FISH) and DNA flow cytometric analysis of a rare metastatic epimyoepithelial carcinoma of the parotid gland to the lung with a clinical course of 29 years. DNA content and FISH analyses of tumor and short-term culture cells showed diploid DNA content and lack of numerical chromosomal abnormalities. Immunohistochemical analysis of the short-term culture cells showed predominantly keratin positive and sparse desmin staining supporting an epithelial rather than myoepithelial origin. Cytogenetic analysis showed 46,XY karyotype with clonal translocations of t(3;22)(q13.2;q13.1), t(1;7)(q21;q22), t(8;9)(p10;p10), and t(5;6) (q35;q21). Our findings indicate that these alterations developed in a diploid stemline during tumor progression and in the epithelial component of this tumor.

Dissociation of cytotoxicity and DNA cleavage activity induced by topoisomerase II-reactive intercalating agents in hamster-human somatic cell hybrids.

Previous studies using the mutant Chinese hamster ovary cell line VpmR-5 indicate that its resistance to epipodophyllotoxins and intercalating agents is likely to be mediated through a qualitative change in type II topoisomerase that confers resistance to drug-stimulated DNA cleavage activity. In a further investigation of the genetic basis of drug resistance in VpmR-5 cells, we fused a hypoxanthine-guanine phosphoribosyl transferase-deficient subline of VpmR-5 (Vtgm-6) with normal human lymphocytes and analyzed the resultant hybrid lines (HL) for altered drug sensitivity. In all, 3 of 16 hybrid clones exhibited partial reconstitution of sensitivity to etoposide, mitoxantrone, doxorubicin, and 5-iminodaunorubicin while retaining complete resistance to m-AMSA. However, enhanced sensitivity to drug-induced DNA cleavage activity was observed only for etoposide. Biochemical and molecular-marker analysis of the hybrids failed to identify human chromosome 17 (the provisional location of TOP2) or any other human chromosome that is consistently and uniquely associated with drug sensitivity. We therefore sought to verify the chromosomal assignment of TOP2 by Southern blot hybridization of TOP2 cDNA on a human hybrid mapping panel and confirmed its location on chromosome 17. However, no hybridizing sequence to the TOP2 cDNA was found in any of the 16 Vtgm-6 hybrid lines. Efforts to select more directly for human chromosome 17 VpmR-5 hybrids using microcell fusion of mouse A9 cells carrying human 17 linked to pSV2neo were unsuccessful. None of the five hybrid clones thus obtained had 17q markers, including the gene for TOP2. Although the mechanism underlying partial reversion to a drug-sensitive phenotype in the original Vtgm-6 hybrid lines has yet to be defined, the data obtained in these lines indicate that anthracycline- and anthracenedione-induced cytotoxic effects can be dissociated from DNA cleavage activity. This suggests that pathways distal to cleavable-complex formation or, alternatively, independent of interactions with topoisomerase II that involve other intracellular targets are important in mediating the cytotoxicity produced by these drugs.

Targeted disruption of Aurora A causes abnormal mitotic spindle assembly, chromosome misalignment and embryonic lethality.

Aurora A (also known as STK15/BTAK in humans), a putative oncoprotein naturally overexpressed in many human cancers, is a member of the conserved Aurora protein serine/threonine kinase family that is implicated in the regulation of G(2)-M phases of the cell cycle. In vitro studies utilizing antibody microinjection, siRNA silencing and small molecule inhibitors have indicated that Aurora A functions in early as well as late stages of mitosis. However, due to limitations in specificity of the techniques, exact functional roles of the kinase remain to be clearly elucidated. In order to identify the physiological functions in vivo, we have generated Aurora A null mouse embryos, which show severe defects at 3.5 d.p.c. (days post-coitus) morula/blastocyst stage and lethality before 8.5 d.p.c. Null embryos at 3.5 d.p.c. reveal growth retardation with cells in mitotic disarray manifesting disorganized spindle, misaligned and lagging chromosomes as well as micronucleated cells. These findings provide the first unequivocal genetic evidence for an essential physiological role of Aurora A in normal mitotic spindle assembly, chromosome alignment segregation and maintenance of viability in mammalian embryos.

A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells.

Extinction is an operational term that refers to the lack of expression of tissue-specific traits that is generally observed in hybrid cells formed by fusing dissimilar cell types. To define the genetic basis of this phenomenon, a series of rat hepatoma x mouse fibroblast hybrids has been isolated and characterized. We report here that the extinction of hepatic marker traits in these clones was strictly correlated with the retention of five particular fibroblast chromosomes (autosomes 8, 9, 10, 11, and 13). In order to dissect this correlation into its component parts, hepatoma microcell hybrids containing single, specific fibroblast chromosomes were constructed. Hepatoma clones retaining only fibroblast chromosome 11 were specifically extinguished for liver-specific tyrosine aminotransferase (TAT) expression, while expression of four other hepatic traits and of numerous constitutive markers was unaffected. Furthermore, removal of fibroblast chromosome 11 from the populations by back-selection resulted in reexpression of TAT activity to full parental levels. These data define and localize a genetic locus, tissue-specific extinguisher-1 (Tse-1), which regulates hepatic TAT expression in trans. We also provide evidence that human Tse-1 resides on the homologous chromosome (human chromosome 17), and that hybrids retaining active Tse-1 loci lack TAT-specific mRNA.

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity.

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.