The transfer of human artificial chromosomes via cryopreserved microcells

Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at −80 °C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.     

Dysregulation of gene expression in the artificial human trisomy cells of chromosome 8 associated with transformed cell phenotypes

A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells) by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression.     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro.

Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome mumber between 49 and 52. Analysis of Wright's-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Artificial induction of chromosome aneuploidy in CHO cells alters their function as host cells

Chinese hamster ovary (CHO) cells are major host cells for biopharmaceuticals. During culture, the chromosome number of CHO cells alters spontaneously. Here, we investigated the effects of artificial changes in the chromosome number on productivity. When cell fusion between antibody-producing CHO-K1-derived cells was induced, we observed a wide range of aneuploidy that was not detected in controls. In particular, antibody productivities were high in clone-derived cell populations that retained a diverse chromosome number distribution. We also induced aneuploid cells using 3-aminobenzamide that causes chromosome non-disjunction. After induction of aneuploidy by 3-aminobenzamide, cells with an increased chromosome number were isolated, but cells with a decreased chromosome number could not be isolated. When antibody expression vectors were introduced into these isolated clones, productivity tended to increase in cells with an increased chromosome number. Further analysis was carried out by focusing on clone 5E8 with an average chromosome number of 37. When 5E8 cells were used as host, the productivity of multiple antibodies, including difficult-to-express antibodies, was improved compared with CHO-K1 cells. The copies of exogenous genes integrated into the genome were significantly increased in 5E8 cells. These findings expand the possibilities for host cell selection and contribute to the efficient construction of cell lines for recombinant protein production.     

Examining the link between chromosomal instability and aneuploidy in human cells

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule-chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise stable, near-diploid cells. However, chromosome missegregation compromises the proliferation of diploid cells, indicating that phenotypic changes that permit the propagation of nondiploid cells must combine with elevated chromosome missegregation rates to generate aneuploid cells with CIN.     

Differential Chromosome Conformations as Hallmarks of Cellular Identity Revealed by Mathematical Polymer Modeling

Inherently dynamic, chromosomes adopt many different conformations in response to DNA metabolism. Models of chromosome organization in the yeast nucleus obtained from genome-wide chromosome conformation data or biophysical simulations provide important insights into the average behavior but fail to reveal features from dynamic or transient events that are only visible in a fraction of cells at any given moment. We developed a method to determine chromosome conformation from relative positions of three fluorescently tagged DNA in living cells imaged in 3D. Cell type specific chromosome folding properties could be assigned based on positional combinations between three loci on yeast chromosome 3. We determined that the shorter left arm of chromosome 3 is extended in MATα cells, but can be crumpled in MAT a cells. Furthermore, we implemented a new mathematical model that provides for the first time an estimate of the relative physical constraint of three linked loci related to cellular identity. Variations in this estimate allowed us to predict functional consequences from chromatin structural alterations in asf1 and recombination enhancer deletion mutant cells. The computational method is applicable to identify and characterize dynamic chromosome conformations in any cell type.     

Somatic chromosomes of higher diptera

Summary The chromosome complements of eighty brain cells ofHylemya antiqua have been studied. The eighty cells were found in thirty-three larvae. Total complement length (TCL) is not randomly distributed among the larvae. Because there is an inverse correlation between chromosome length and width, it appears that in the cells studied the different chromosome lengths are partly expressions of different stages of metaphase contraction. It is suggested that synchronous division of cells still occurs in late larvae.The length of each chromosome arm is highly correlated with that of every other arm. It is possible that the correlations are complete but that inadequate technique causes the departures from completeness which are observed. The chromosome lengths are corrected slightly for distortions, but the corrections make very little difference in the correlation coefficients. There is a high value for the correlation between the correlation of two arm lengths and the sum of the two arm lengths. This is to be expected if the perfect correlation between all arm lengths is being obscured by errors of drawing and measurement.The autosomal arms have very similar coefficients of variation. The arm ratios (length of long arm divided by short arm) are not correlated with TCL or with each other, and arm ratio is randomly distributed among the larvae. The sex chromosomes have a smaller coefficient of variation than the autosomes, so that they are relatively large in small cells and relatively small in large cells.Twenty-two cells inHylemya fugax were measured. The autosomes also showed a high correlation between arm lengths. An entirely heterochromatic autosomal arm showed the same phenomenon of a low coefficient of variation which was shown by the heteropycnotic sex chromosomes inH. antiqua. The low variability of heterochromatic regions accompanied by an apparently non-random distribution of the TCL may produce an erroneous picture of the species complement when dealing with small numbers.It is suggested that for simplicity in using cytological observations of this sort for taxonomic purposes, the technique of measuring the percent TCL of a chromosome plus its arm ratio be replaced by the percent TCL of each arm plus the average length difference between the arms of each chromosome pair in units of percent TCL.     

'Sloughing-off' of heterochromatin in Werner's syndrome cells during high-temperature phosphate incubation

Previous investigations of cells undergoing rapid division revealed the presence of heterochromatic 'dots' in chromosomes as well as numerous chromocentres in interphase nuclei. Such structures were seen in human embryonic cells, as well as cells from organisms capable of regeneration, and cells from various malignancies. Cells with a reduced capacity for reproduction were found to be virtually devoid of nuclear chromocentres and chromosome dots after incubation in phosphate buffer at high temperature. The lack of heterochromatin in such cells (Werner's syndrome) thereby explained their reduced capacity for cell division and the resultant rapid rate of aging in individuals afflicted. Re-examination of such slides containing these cells revealed that chromocentres and chromosome dots were present initially, but the incubation process resulted in a 'sloughing-off' of such structures. The incubation process left these heterochromatic structures intact in malignant and control cells, inferring a link between cell proliferation and stable intact heterochromatin. These findings implicate heterochromatin as the object of the purported chromosomal instability factor characteristic of Werner's syndrome. The loss of heterochromatin did not result in chromosome breakage, suggesting that heterochromatin may not be an integral part of chromosome structure, but rather a surface feature or covering.     

Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70.

Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus.     

X-linked control of hemoglobin production in somatic hybrids of mouse erythroleukemic cells and mouse lymphoma or bone marrow cells

Somatic cell hybrids were generated by fusion of mouse erythroleukemic cells (clone 745) to mouse lymphoma cells or mouse bone marrow cells. The erythroleukemic cells have been shown previously to have a low basal level of erythroid differentiation which is markedly amplified when the cells are grown in medium containing dimethylsulfoxide (DMSO). Hybrid cells were examined for hemoglobin production by benzidine staining. Many hybrid clones were found in which hemoglobin production in response to DMSO was either abolished or greatly reduced. From these hybrids, subclones were isolated in which hemoglobin production was restored. Karyological and enzyme analysis showed that the restoration of hemoglobin production was associated with the loss of an X chromosome contributed by the nonerythroleukemic parent. Other subclones which retained an X chromosome continued to be inhibited for hemoglobin production. Analysis of other hybrid lines capable of a limited degree of erythroid differentiation indicated a quantitative inverse correlation between the proportion of cells bearing an X chromosome and the proportion of cells able to form hemoglobin. Finally, four hybrid lines having many cells without an X chromosome were grown in medium containing DMSO. This procedure led to the selection of hybrid sublines having a nondifferentiating phenotype and concomitantly having a greatly increased proportion of cells bearing an X chromosome. Thus three lines of evidence suggest that a locus (loci) on the X chromosome is capable of inhibiting the DMSO-inducible hemoglobin production of the erythroleukemic cells.     

Un ménage à quatre: the molecular biology of chromosome segregation in meiosis

Sexually reproducing organisms rely on the precise reduction of chromosome number during a specialized cell division called meiosis. Whereas mitosis produces diploid daughter cells from diploid cells, meiosis generates haploid gametes from diploid precursors. The molecular mechanisms controlling chromosome transmission during both divisions have started to be delineated. This review focuses on the four fundamental differences between mitotic and meiotic chromosome segregation that allow the ordered reduction of chromosome number in meiosis: (1) reciprocal recombination and formation of chiasmata between homologous chromosomes, (2) suppression of sister kinetochore biorientation, (3) protection of centromeric cohesion, and (4) inhibition of DNA replication between the two meiotic divisions.     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro

Summary Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome number between 49 and 52. Analysis of Wright’s-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10. This work was supported by grants-in-aid made available through the San Diego State University Foundation.     

Chromosome analysis of small and large L5178Y mouse lymphoma cell colonies: comparison of trifluorothymidine-resistant and unselected cell colonies from mutagen-treated and control cultures

Mutagenesis assays at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells frequently yield mutant colonies with a bimodal size distribution. The objectives of this study were to determine whether a relationship exists between mutant colony size and chromosomal aberrations and whether the colony-size distributions obtained from this assay can indicate the clastogenic activity of a test chemical. Cells from 8 different types of L5178Y mouse lymphoma cell colonies were examined for chromosomal abnormalities within 10 cell generations after colony isolation. The colonies included small (sigma) and large (lambda) unselected cell (UC) and trifluorothymidine-resistant (TFTr) colonies derived from TK +/- cell cultures treated with the solvent dimethyl sulfoxide (DMSO) or hycanthone methanesulfonate (HYC). Chromosome abnormalities were present in cells from 12% (7/60) of the UC colonies, but there was no apparent relationship between colony diameter and the presence of chromosomal abnormalities. Abnormalities affecting chromosome 11, which is believed to be the site of the TK gene, were not observed in cells from UC colonies. Abnormalities affecting chromosome 11 were observed only in cells from sigma-TFTr colonies irrespective of whether they were spontaneous (5/15 colonies) or induced by HYC (4/15 colonies). Overall, 30% (9/30) of sigma-TFTr colonies had cells with an abnormal chromosome 11 and 10% (3/30) had abnormalities affecting other chromosomes. Abnormalities affecting chromosome 11 were not observed in cells from lambda-TFTr colonies (0/30 colonies). The observation of only 30% of sigma-TFTr colonies with chromosome damage affecting chromosome 11 indicates that other mechanisms, in addition to chromosome damage at the level of resolution used in this study (i.e., 200-300 chromosome bands). contribute to small TFTr colony size.     

The Three-Dimensional Organization of Polytene Nuclei in Male Drosophila Melanogaster with Compound Xy or Ring X Chromosomes

The three-dimensional organization of polytene chromosomes within nuclei containing rearranged X chromosomes was examined in male Drosophila melanogaster. Salivary glands of third instar larvae containing either an inverted X chromosome (YSX.YL, In(1)EN/O) or a ring X chromosome (R(1) 2/BSYy+) were fixed, embedded, and serially sectioned. The nuclei in contiguous groups of cells were modeled and analyzed. We find that for both genotypes the three-dimensional behavior at each euchromatic locus is independent of the orientation of the chromosome on which it resides, independent of the behavior of loci not closely linked to it, and not similar in neighboring cells. The preference for right-handed chromosome coiling noted in previous studies is shown to be independent of homologous pairing. However, a relation between the extent of chromosome curvature and the handedness of chromosome coiling is present only in homologously paired chromosomes. The attached-XY chromosome has two previously undescribed behaviors: a nearly invariant association of the euchromatic side of the proximal heterochromatin/euchromatin junction with the nucleolus and a frequent failure of this site to attach to the chromocenter. The relative chromosome arm positions are often similar in several neighboring cells. The size of these patches of cells, assuming that they represent clones, indicates that such arrangements are at best quasi-stable: they may be maintained over at least one, but less than four, cell divisions. The observed nuclear organization in salivary glands is inconsistent with the idea that position in the polytene nucleus plays a major role in the normal genetic regulation of euchromatic loci.     

Y Chromosome aneuploidy,micronuclei, kinetochores and aging in men

This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant mcrease in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.     

High resistance of cultured Mongolian gerbil cells to X-ray-induced killing and chromosome aberrations.

The Mongolian gerbil (Meriones unguiculatus) is known to be one of the most radioresistant animals. We have examined the X-ray sensitivity of normal diploid fibroblasts from Mongolian gerbil embryos compared with those of cultured embryo cells obtained from various laboratory animals and a normal human. There was a wide difference in X-ray sensitivity for cell killing among different mammalian species. The D0 values for Mongolian gerbil cells ranged from 2.08 to 2.28 Gy, values which are twice as high as those for human cells. The mean D0 value for human cells was 1.06 Gy. Mouse, rat, Chinese hamster, and Syrian/golden hamster cells showed similar D0 values ranging from 1.30 to 1.56 Gy. When cells were irradiated with X rays, ten times more chromosome aberrations were detected in human cells than in Mongolian gerbil cells. The frequencies of chromosome aberrations in other rodent cells were between the values for cells from humans and those from gerbils. These data indicate that the Mongolian gerbil cells are resistant to X-ray-induced cell killing and chromosome aberrations, and that the radiation sensitivity of mammalian cells in primary culture may be reflected by their radioresistance in vivo.     

Sister chromatid exchange points in the heterochromatin and euchromatin regions of Chinese hedgehog chromosomes

Summary The Chinese hedgehog has a diploid chromosome number of 48 in which there are eleven pairs of telo- or subtelocentric autosomes, twelve pairs of meta- or submetacentric autosomes, a metacentric X chromosome and a telocentric Y chromosome. The heterochromatin is almost completely distributed in five large distal segments of chromosomes nos. 9 to 12 and no. 18. There is no positive C-band in the centromeres of the chromosomes except for the X chromosome which has a small, weakly stained C-band in the centromere. In Chinese hedgehog cells 52.1% of SCEs are found at the junction between the euchromatin and the heterochromatin, 39.5% in the heterochromatin and 8.4% in the auchromatin. The SCE number per unit C-band is double the SCE number per unit euchromatin. The SCE rate in the heterochromatin or euchromatin regions is not proportional to their chromosome length and can be quite different between different pairs of the chromosomes. Our results indicate that there is a non-uniform distribution of the SCEs in the Chinese hedgehog cells.     

Chromosome balance and the control of malignancy.

The Giemsa banding pattern of the chromosomes has been analyzed in a line of transformed golden hamster cells, revertant and re-revertant cells and their tumors. The transformed and re-revertant cells were malignant in vivo and had gained an additional chromosome 5(7). Revertants with a suppression of malignancy lost this additional chromosome 5(7) and gained an additional chromosome 7(2). The tumors produced by segregants from the revertant cells were malignant, although to a lower degree than transformed and re-revertant cells. These tumors had lost the additional chromosome 7(2) found in revertants and gained one or two 5(12) chromosomes. The results support the hypothesis that the balance between genes for expression and suppression controls malignancy. The data indicate that chromosome 7(2) carries genes for suppression and that chromosomes 5(7) and 5(12) carry genes for expression of malignancy. The genes on chromosome 5(7) seem to result in a greater degree of expression than the genes on chromosome 5(12). The chromosome balance that controlled malignancy in these cells, also controlled the expression and suppression of transformed properties in vitro.     

Role of polyamines during chromosome condensation of mammalian cells

The objective of the present study was to investigate the role of polyamines in the process of chromosome condensation. The phenomenon of premature chromosome condensation (PCC) involving fusion between mitotic and interphase cells was used as the assay system. The factors present in the mitotic cells would bring about the breakdown of the nuclear membrane and condensation of the interphase chromatin into chromosomes, similar to that which occurs during the initiation of mitosis. Alpha-difluoromethyl ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase was used to deplete both mitotic and interphase cells of polyamines. The results indicate that the polyamine depleted mitotic cells have a diminished ability to induce PCC. This inhibition could easily be reversed by exogenous addition of polyamines at the time of fusion. Furthermore, exogenously added polyamines hastened the entry of exponentially growing cells into mitosis. These observations suggest an essential role for polyamines during the process of chromosome condensation of mammalian cells.