Introduction of Chromosome 7 Suppresses Telomerase with Shortening of Telomeres in a Human Mesothelial Cell Line

Introduction of human chromosome 7 by microcell-mediated chromosome transfer induced senescence in a telomerase-positive human mesothelial cell line, MeT5A. In microcell hybrids which underwent senescence, telomerase activity was decreased before entering senescence and telomeric sequences were shortened as cell division proceeded. Concomitantly, expression of the gene encoding telomerase catalytic subunit was abolished, whereas the genes encoding the RNA component of telomerase and its associated protein TEP1 were not affected. In revertants which arose from such microcell hybrids, telomerase activity was restored and the telomeric sequences were elongated. In microcell hybrids which showed no growth arrest, telomerase activity was unaltered. These results suggest that a putative mortality gene on chromosome 7 negatively regulates the telomere maintenance mechanism in MeT5A.     

Integration of Ecogpt and SV40 early region sequences into human chromosome 17: a dominant selection system in whole cell and microcell human-mouse hybrids

The dominant selectable gene, Ecogpt, has been introduced, by the calcium phosphate precipitation technique, into normal human fibroblasts, along with the SV40 early region genes. In one transfectant clone, integration of these sequences into human chromosome 17 was demonstrated by the construction of human-mouse somatic cell hybrids, selected for by growth in medium containing mycophenolic acid and xanthine. A whole cell hybrid, made between the human transfectant and a mouse L cell, was used as donor of the Ecogpt-carrying human chromosome 17 to 'tribrids' growing in suspension, made by whole cell fusion between a mouse thymoma cell line, and to microcell hybrids made with a mouse teratocarcinoma cell line. Two tribrids contained karyotypically normal human chromosomes 17 and a small number of other human chromosomes, while a third tribrid had a portion of the long arm of chromosome 17 translocated to mouse as its only human genetic material. Two independent microcell hybrids contained a normal chromosome 17 and no other human chromosome on a mouse teratocarcinoma background. These experiments demonstrate the ability to construct human-mouse somatic cell hybrids using a dominant selection system. By applying this approach it should be possible to select for a wide range of different human chromosomes in whole cell and microcell hybrids. In particular, transfer of single human chromosomes to mouse teratocarcinoma cells will allow examination of developmentally regulated human gene sequences after differentiation of such hybrids.     

Extinction of Albumin Gene Expression in a Panel of Human Chromosome 2 Microcell Hybrids

Expression of the serum albumin gene is extinguished in rat hepatoma microcell hybrids that retain mouse chromosome 1. These data define atrans-dominant extinguisher locus,Tse-2,on mouse chromosome 1. To localize the human TSE2 locus, we prepared and characterized rat/human microcell hybrids that contained either human chromosome 1 or chromosome 2, the genetic homologues of mouse chromosome 1. Rat hepatoma microcell hybrids retaining a derivative human chromosome 1 [der 1 t(1;17)(p34.3;q11.2)] expressed their serum albumin genes at levels similar to those of parental hepatoma cells. In contrast, microcell transfer of human chromosome 2 into rat hepatoma recipients produced karyotypically heterogeneous collections of hybrid clones, some of which displayed dramatic albumin extinction phenotypes. For example, albumin mRNA levels in several extinguished microcell hybrids were reduced at least 500-fold, similar to albumin mRNA levels in hepatoma × fibroblast whole-cell hybrids. Expression of several other liver genes, including α1-antitrypsin, aldolase B, alcohol dehydrogenase, and phosphoenolpyruvate carboxykinase, was also affected in some of the microcell hybrids, but expression of these genes was not concordant with expression of albumin. Hybrid segregants were prepared from the albumin-extinguished hybrids, and reexpression of albumin mRNA and protein was observed in sublines that had lost or fragmented human chromosome 2. Finally, expression of mRNAs encoding the liver-enrichedtransactivators HNF-1, HNF-4, HNF-3α, and HNF-3β was not affected in any of the chromosome 2-containing hybrids. These data define and map a genetic locus on human chromosome 2 that extinguishes albumin gene expression intrans,and they suggest that TSE2-mediated extinction is independent of HNF-1, -4, -3α, and -3β expression.     

Chromosome 7 suppresses indefinite division of nontumorigenic immortalized human fibroblast cell lines KMST-6 and SUSM-1.

Using nontumorigenic immortalized human cell lines KMST-6 (KMST) and SUSM-1 (SUSM), we attempted to identify the chromosome that carries a putative senescence-related gene(s). These cell lines are the only ones that have been established independently from normal human diploid fibroblasts following in vitro mutagenesis. We first examined restriction fragment length polymorphisms on each chromosome of these immortalized cell lines and their parental cell lines and found specific chromosomal alterations common to these cell lines (a loss of heterozygosity in KMST and a deletion in SUSM) on the long arm of chromosome 7. In addition to these, we also found that introduction of chromosome 7 into these cell lines by means of microcell fusion resulted in the cessation of cell division, giving rise to cells resembling cells in senescence. Introduction of other chromosomes, such as chromosomes 1 and 11, on which losses of heterozygosity were also detected in one of the cell lines (KMST), to either KMST or SUSM cells or of chromosome 7 to several tumor-derived cell lines had no effect on their division potential. These results strongly suggest that a gene(s) affecting limited-division potential or senescence of normal human fibroblasts is located on chromosome 7, probably at the long arm of the chromosome, representing the first case in which a specific chromosome reverses the immortal phenotype of otherwise normal human cell lines.     

Lack of tumor suppression but induced loss of copies of indigenous chromosome 10 in vitro following microcell-mediated transfer of a deleted human der(9)t(X;9) chromosome to Syrian hamster BHK-191-5C cells

We have previously shown that microcell-mediated transfer of a der(9)t(X;9) chromosome, containing an almost complete human chromosome (HSA) 9 derived from the human fibroblast strain GM0705, into the Syrian hamster (Mesocricetus auratus) cell line BHK-191-5C suppressed the anchorage independence and tumorigenicity of the hybrids. Transfer of a normal HSA X did not have any effect on these phenotypes. Although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells, all hybrids retaining the der(9) chromosome were near-tetraploid, in contrast to hybrids retaining a normal X chromosome. In the present study, we have generated microcell hybrids by transferring another der(9)t(X;9) chromosome derived from the human fibroblast strain GM01429. This derivative chromosome contained a deletion on the short arm of HSA 9 and was also missing the distal part of the long arm of HSA 9 due to the involvement in a reciprocal (constitutive) translocation of this chromosome with HSA X. Cytogenetic analysis showed that all hybrid clones were near-tetraploid, confirming our previous finding. We also observed that the introduction of the deleted der(9) chromosome forced the hybrids to lose Syrian hamster chromosome 10. A soft agar test and nude mice assay indicated that none of the hybrids was suppressed for either anchorage independent growth or tumor formation. These data suggest that there is an antagonistic relationship between growth-promoting genes and antiproliferative genes. The observed dosage effects of both growth-promoting and growth-suppressing genes indicate that cellular growth may be a quantitative trait.     

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.     

Effect of Telomeres on the Interphase Location of Adjacent Regions of the Human X Chromosome

Chromosomes occupy specific nonrandom domains in the interphase nucleus of eukaryotic cells. We have used a Chinese hamster-human somatic cell hybrid line containing a single human X chromosome to study the interphase distribution of the Xp telomere using fluorescent in situ hybridization and optical sectioning. A derivative cell line in which the X chromosome has been broken at Xq22-24 and healed by the addition of cloned human telomeric sequences was also studied to determine if introduction of these sequences at a previously interstitial site changed its location in interphase. The endogenous Xp telomere occupies a specific, nonrandom, internal domain. Introduction of a telomere at a previously interstitial site did not alter the interphase nuclear location of that site. The results suggest that nonrandom interphase location of telomeres may not be determined solely by the DNA sequence of the telomere.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, γ-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

The human MYOD1 transgene is suppressed by 5-bromodeoxyuridine in mouse myoblasts

5-Bromodeoxyuridine (BrdU) immediately and clearly suppresses expression of the mouse Myod1 and human MYOD1 genes in myoblastic cells. Despite various studies, its molecular mechanism remains unknown. We failed to identify a BrdU-responsive element of the genes in experiments in which reporter constructs containing known regulatory sequences were transferred to mouse C2C12 myoblasts. Therefore, we transferred human chromosome 11 containing the MYOD1 gene to the cells by microcell-mediated chromosome transfer. In the resulting microcell hybrids, BrdU suppressed expression of the transgene, as determined by quantitative real-time RT-PCR analysis. We then transfected human PAC clones containing the MYOD1 gene to the cells. In the resulting transfectants, BrdU suppressed the transgene similarly. Deletion analysis suggested that a BrdU-responsive element or chromatin structure exists between 24 and 47 kb upstream of the gene. These results are the first demonstrating BrdU-responsiveness of a transgene for the known BrdU-responsive genes and facilitating determination of its precise responsible structure.     

Rescue of the HNF4 → HNF1α Pathway in Hepatoma Variant Cells Containing Human Chromosome 12

Expression of liver-enrichedtrans-acting hepatocyte nuclear factors 1α (HNF1α) and 4 (HNF4) is correlated with the hepatic phenotype in cultured rat hepatoma cells. We have used a hepatoma variant cell line, H11, that specifically lacks the HNF4 → HNF1α pathway as a model to understand mechanisms controlling hepatic gene expression. We have introduced randomly marked human chromosomes into H11 cells and have isolated a number of microcell hybrids that have rescued hepatic gene expression, including HNF4, HNF1α, and α1-antitrypsin. Chromosomal analysis of cell hybrids showed that the rescued hepatic phenotype correlated closely with the presence of human chromosome 12p sequences. Although the gene encoding HNF1α is located on chromosome 12q24, its retention was not required to rescue the hepatic phenotype. Thus, we suggest that a locus on human chromosome 12p plays an important role in maintenance of hepatic gene expression through activation of the HNF4 → HNF1α pathway.     

Amplification of repeated DNA sequences in wheat X rye hybrids regenerated from tissue culture

Summary Previous C-banding analysis of wheat (Triticum aestivum)X rye (Secale cereale) hybrids regenerated from tissue culture revealed enlarged C-bands in some rye chromosomes, but the molecular nature of the change was not determined. In situ hybridization using two DNA probes containing repeated sequences from rye telomeric heterochromatin was conducted on these wheatX rye hybrids and their progeny to investigate the occurrence of amplification in repeated sequences. Clones pSC 74 and pSC 119, which contain sequences from the 480-bp and 120-bp repeated DNA families of rye, respectively, were used as probes. Amplification of 480-bp repeated sequences in the short arm telomere of chromosome 7R was detected in three wheatxrye hybrids and their progeny. The amplified 480-bp sequences were detected by an enlarged hybridization site for pSC 74 at the 7RS telomere, and by the appearance at this same telomeric site of an unlabeled, blue chromosome segment in an otherwise completely brown chromosome hybridizing entirely to the biotin-labeled pSC 119 probe. This variant form of chromosome 7R was not observed in several Chaupon plants, or in the other hybrids derived from the same embryos, indicating the origin of the change in tissue culture. The amplified sequences were inherited up to at least three generations. Deletions and translocations were also observed.Contribution No. 87-9-J, Kansas Agricultural Experiment Station, Kansas State University     

Functional complementation of the radiation-sensitive mutant M10 cell line by human chromosome 5

The mouse lymphoma (L5178Y) cell mutant M10 is defective in rejoining DNA double-strand breaks and is hypersensitive to ionizing radiation. The introduction of human chromosome 5 into M10 cells by microcell mediated chromosome transfer complemented the ionizing-radiation hypersensitivity defect of this cell line. The presence of chromosome 5 in the microcell hybrids was shown using PCR with chromosome-specific primers and fluorescence in situ hybridization. From this data we conclude that the gene that corrects the radiation hypersensitivity of M10 cells is located on chromosome 5 and tentatively assigned to the 5q14 to 5pter region. We designate this gene XRCC4L.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2.

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, gamma-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

Long-range chromatin reorganization of the human serpin gene cluster at 14q32.1 accompanies gene activation and extinction in microcell hybrids

The genes encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of six serine protease inhibitor (serpin) genes located on human chromosome 14q32.1. Both genes are actively transcribed in the liver and in human hepatoma cells, but they are not expressed in most other cell types. In this study we mapped DNase I-hypersensitive sites (DHSs) in an approximately 130-kb region of 14q32.1 that includes both genes. The distributions of DHSs in expressing (HepG2) vs nonexpressing (HeLa S3) cells were very different: HepG2 cells displayed 29 DHSs in this interval, but only 7 of those sites were present in HeLa cells. To determine the chromatin organization of activated or extinguished serpin alleles, we transferred human chromosome 14 into rat hepatoma cells or fibroblasts, respectively. Human alpha1AT and CBG gene expression was activated in rat hepatoma microcell hybrids containing human chromosome 14, but extinguished in rat fibroblast hybrids with the same genotype. DHS mapping in these microcell hybrids demonstrated that the chromatin structure of the entire 130-kb region was reorganized in microcell hybrids, and the distributions of DHSs in activated and extinguished alleles recapitulated those of expressing and nonexpressing cells, respectively. Thus, microcell hybrids provide a system in which reproducible changes in gene activity and long-range chromatin organization can be induced experimentally. This provides a basis for studying the effects of targeted modifications of the alpha1AT and CBG loci on the regulation of gene activity and chromatin structure.     

Evidence for suppression of cellular growth in vitro and selection against the indigenous mouse X chromosome in A9 cell hybrids after microcell-mediated transfer of an X from other mammalian species

Introduction of a human or Syrian hamster X chromosome (derived from BHK-191-5C cell hybrids) into tumorigenic mouse A9 cells via microcell fusion induced changes in cellular morphology and a retardation of cellular growth. The suppression of growth of the hybrids could be abolished, however, by daily changes of medium containing 20% serum. G-banding analysis showed the absence of a single, cytogenetically identifiable, indigenous X chromosome (marker Z) in two of four hybrid clones after an X chromosome was transferred from either hamster or human cells. All hybrids were tumorigenic when tested in nude mice. Together, these data suggest that the loss of the mouse X chromosome took place probably because of growth inhibitory effects imposed on hybrid cells due to the increase in X chromosome dosage. In addition, our results show a lack of association between the phenotype of cellular growth suppression in vitro and the phenotype of suppression of tumorigenicity in vivo.     

Introduction of new genetic markers on human chromosomes

The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter----3p12::Xq26----Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions (HAT medium) for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. In order to introduce a second selectable genetic marker to the t(X;3) chromosome, A9(GM0439)-1 cells were transfected with pcDneo plasmid DNA. Colonies resistant to both G418 and HAT medium (G418r/HATr) were selected. To obtain A9 cells that contained a t(X;3) chromosome with an integrated neo gene, the microcell transfer step was repeated and doubly resistant cells were selected. G418r/HATr colonies arose at a frequently of 0.09 to 0.23 x 10(-6) per recipient cell. Of seven primary microcell hybrid clones, four yielded G418r/HATr clones at a detectable frequency (0.09 to 3.4 x 10(-6)) after a second round of microcell transfer. Doubly resistant cells were not observed after microcell chromosome transfers from three clones, presumably because the markers were on different chromosomes. The secondary G418r/HATr microcell hybrids contained at least one copy of the human t(X;3) chromosome and in situ hybridization with one of these clones confirmed the presence of a neo-tagged t(X;3) human chromosome. These results demonstrate that microcell chromosome transfer can be used to select chromosomes containing multiple markers.     

DNA-mediated transfer of the mouse gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells: No integration of the transferred gene at its homologous site in the host genome

Summary An established Chinese hamster cell line was fused with microcells isolated from phenotypically stable transferent mouse cells which contained a mouse transgenome coding for an abnormal form of mouse hypoxanthine phosphoribosyltransferase (HPRT, EC. No. 2.4.2.8) (Willecke et al. 1979). Two hybrids were isolated which expressed the abnormal form of mouse HPRT but no mouse -galactosidase (GALA, EC. No. 3.2.1.22). In one of these microcell hybrids the abnormal HPRT activity segregated under counter-selective conditions with mouse chromosome 3. No mouse chromosome or additional mouse gene marker was found in the second microcell hybrid, possibly because of breakage and/or rearrangement of the integrated transgenome during the isolation of this hybrid. We conclude from these results that the transferred mouse HPRT gene in a phenotypically stable clone is not integrated at its homologous site on the host X chromosome. Rather, the transgenome is probably integrated into mouse chromosome 3, possibly due to homologies in repeated DNA sequences which may occur in the transgenome and which are interspersed at many sites in the host genome.