Attempted hybridizations with senescent human fibroblasts

If the aging of diploid human fibroblasts reflects stable genetic or epigenetic changes which are few in number and different in different cells, then complementation could occur in hybrids between individual senescent cells. However neither pairs of aged fibroblasts nor even pairs of young and senescent fibroblasts produce viable hybrids under conditions known to promote cell fusion.     

Peroxisome assembly mutations in humans: structural heterogeneity in Zellweger syndrome.

Empty membrane ghosts of peroxisomes were found in fibroblasts from a patient with Zellweger's syndrome, a genetic disease of humans (Santos et al: Science 239:1536-1538, 1988). Import of soluble matrix proteins into the organelle was defective. We have now studied fibroblasts from seven patients representing five complementation groups of the syndrome (defined by complementation for peroxisome enzyme function). We find that empty peroxisome ghosts are present in all seven cell samples. Three patients, representing three complementation groups, give the same membrane pattern by immunofluorescence: few large ghosts. Three other patients, representing two complementation groups, give a second pattern: many large ghosts. The seventh patient's pattern is distinct. Thus, all seven of these patients exhibit Peroxisome IMport (PIM) mutations. Since membrane assembly occurs in these cells, the results indicate that biogenesis of organelle content and membrane proteins proceed by different mechanisms. Growth and division of the empty peroxisomal membrane must occur, but are modified by the mutations (ghost size and abundance vary). Cell fusion and immunofluorescence analyses of peroxisome size and catalase packaging formally demonstrate genetic complementation groups for peroxisome assembly in Zellweger syndrome.     

Mapping of the genes of some components of the electron transport chain (complex I) on the X chromosome of mammals

This paper describes genetic mapping studies with several respiration-deficient mutants of Chinese hamster fibroblasts which have a defect in complex I of the electron transport chain (NADH-coenzyme Q reductase). The mutations associated with two different complementation groups map on the X chromosome. In two cases (G14 and G20) karyotypic and isozyme analyses in hybrids have shown that a gene(s) on the mouse X chromosome complements the mutation(s) in the hamster cell mutant(s). A cosegregation analysis in hybrid cells has shown the corresponding genes to be linked to the HPRT genes (hamster-mouse hybrids of G14, and hamster-hamster hybrids for G14 and G20). By the same method the defective gene in a third mutant (G4) was also shown to be X-linked. A mutation representing a third complementation group (G11) was shown to be on an autosomal gene. These results provide an explanation for our observation that cells with recessive mutations in complementation groups I and II can be selected at relatively high frequencies.     

Juvenile sandhoff disease: Complementation tests with Sandhoff and Tay-Sachs disease using polyethylene glycol-induced cell fusion

Summary Juvenile Sandhoff, Sandhoff, and Tay-Sachs fibroblasts were mixed in paired combinations and treated with polyethylene glycol (PEG) to promote cell fusion. The hexosaminidase (hex) isozymes of PEG-treated mixed-cell cultures were determined and compared with those of untreated control cultures. Fusions involving juvenile Sandhoff and Sandhoff fibroblasts did not show an increase in either total hexosaminidase or heat-stable hex B. Fusions of juvenile Sandhoff (or Sandhoff) and Tay-Sachs fibroblasts showed an increase of heat-labile hex A. Thus, juvenile Sandhoff cells show complementation with Tay-Sachs cells but not Sandhoff cells. Consequently, the genetic defect in juvenile Sandhoff disease probably represents an allelic mutation of the gene that is defective in Sandhoff disease.     

Survival and DNA Repair of Somatic Cell Hybrids after Ultraviolet Irradiation

THE technique of somatic cell hybridization has opened up studies on genetic regulation¹ and human genetic analysis^2–5. Hybrid cells are isolated in conditions that select against parental cells while allowing hybrids to survive by genomic complementation. In xeroderma pigmentosum (XP), a human disease with an autosomal recessive defect in an early stage of DNA repair⁶, the skin is extremely sensitive to sunlight in vivo⁷ and skin fibroblasts show sharply reduced survival following ultraviolet irradiation in vitro^8,9. This communication concerns the use of ultraviolet irradiation in combination with a chemical method to produce hybrids between fibroblasts from XP and a hamster line, followed by analysis of these cells for their capacity to survive and repair DNA after exposure to ultraviolet. Methods for initiation and propagation of skin fibroblasts from two subjects, male and female siblings with XP, have been described⁸. Details on the origin of the TG2 line of golden hamster fibroblasts, which has a non-reverting mutation in the gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the general hybridization procedure¹⁰ and methods for cell survival and DNA repair by unscheduled synthesis⁸ were also described previously. Hybrids were produced by fusion with Sendai virus and selected by ultraviolet irradiation followed by culture on HAT medium (Fig. 1).     

Hybrids from fusion of normal human T lymphocytes with immortal human cells exhibit limited life span

A number of normal human cell types have been shown to exhibit cellular senescence in vitro. We and others had found that fusion of normal human fibroblasts with immortal human cells yielded hybrids having limited lifespan. This indicated that the phenotype of cellular senescence is dominant and that immortality results from recessive changes in genes involved in growth control. They also supported the hypothesis that senescence results from genetic mechanisms rather than random damage. Since T lymphocytes are a highly differentiated cell type, in contrast to fibroblasts, it was of interest to determine whether similar mechanisms caused senescence in the T cells. We therefore fused normal human T lymphocytes with an immortal human cell line to determine whether they could restore the senescent, nondividing phenotype in hybrids, as do normal human fibroblasts. Eleven of fifteen hybrid clones studied exhibited limited proliferative potential after achieving a range of population doubling similar to that observed in the cell fusion studies involving normal fibroblasts. These results provide evidence that cellular senescence in T lymphocytes occurs via genetic mechanisms.     

Complementation analysis in Fanconi anemia: assignment of the reference FA-H patient to group A

Fanconi anemia (FA) is an autosomal recessive disorder with diverse clinical symptoms and extensive genetic heterogeneity. Of eight FA genes that have been implicated on the basis of complementation studies, four have been identified and two have been mapped to different loci; the status of the genes supposed to be defective in groups B and H is uncertain. Here we present evidence indicating that the patient who has been the sole representative of the eighth complementation group (FA-H) in fact belongs to group FA-A. Previous exclusion from group A was apparently based on phenotypic reversion to wild-type rather than on genuine complementation in fusion hybrids. To avoid the pitfall of reversion, future assignment of patients with FA to new complementation groups should conform with more-stringent criteria. A new group should be based on at least two patients with FA whose cell lines are excluded from all known groups and that fail to complement each other in fusion hybrids, or, if only one such cell line were available, on a new complementing gene that carries pathogenic mutations in this cell line. On the basis of these criteria, the current number of complementation groups in FA is seven.     

Heterokaryons in the analysis of genes and gene regulation.

Cytological and chemical analysis of heterokaryons, the immediate product of cell fusion, offer new possibilities for studying the factors responsible for genetic regulation in eukaryotic cells. In comparison with proliferating cell hybrids the heterokaryon state offers the important advantage that a heterokaryon contains two complete genomes since chromosome loss does not occur, but since segregation and recombination are absent, heterokaryons cannot be used for gene mapping in the same way as proliferating cell hybrids. However, if two cell types carrying different genetic defects are fused the analysis can be used for studies of gene complementation. The biological information obtained with heterokaryons has emphasized the role of the cytoplasm in the control of nuclear activity. When a G1 nucleus is brought into contact with the cytoplasm of an S phase cell the G1 nucleus is stimulated to synthesize DNA. If the nucleus is brought into a mitotic cell, the chromatin of the G1 nucleus is forced to condense into prematurely condensed chromosomes. Inactive nuclei such as the dormant chick erythrocyte nucleus will be stimulated to initiate RNA and DNA synthesis when brought into contact with an active cytoplasm by cell fusion. Specific nuclear proteins have been shown to be responsible for this process of reactivation. Other inactive nuclei such as the nuclei of macrophages and spermatozoa have likewise been shown to be reactivated by fusion with active cells. The degree of activation in all of these cases appears to be determined by the state of the active cell. Inactive nuclei are activated to the same level as the active nucleus but seldom beyond this level. If differentiated cells are fused with undifferentiated cells, usually the differentiated character is lost rapidly after fusion. This observation is in agreement with several studies on proliferating cell hybrids indicating some type of negative control of differentiated properties. In heterokaryons obtained by fusion of cells of a similar type of histotypic differentiation usually coexpression of the differentiated markers is observed.     

Complementation studies with enucleated fibroblasts from different variants of beta-galactosidase deficiency.

Somatic cell hybridization of β-galactosidase fibroblasts derived from patients with the infantile type 1 and the adult type 4 G_M1-gangliosidosis results in restoration of the β-galactosidase activity. The kinetic properties of the enzyme activity in heterokaryons were found to be similar as in controls. Genetic complementation did not occur after inhibition of protein synthesis by cycloheximide indicating the necessity of de novo protein synthesis. When fibroblasts of the adult type 4 patient were enucleated and fused with cells from an infantile type 1 variant no restoration of β-galactosidase in the hybrids was observed. Fusion of enucleated type 1 cells with nucleated type 4 cells, however, did result in genetic complementation. The results obtained in this study and observations by others fit with the hypothesis of intergenic complementation. In heterokaryons the adult type 4 genome codes for normal β-galactosidase monomers and the infantile type 1 cells or cytoplasts provide a protein factor necessary for the hydrolytic activity and aggregation of the molecule.     

Evidence for at least eight Fanconi anemia genes.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.     

Bloom syndrome: a single complementation group defines patients of diverse ethnic origin.

Patients of diverse ethnic background were recruited in order to examine whether genetic heterogeneity could be demonstrated in Bloom syndrome (BS). Although most cells from BS patients exhibit high sister-chromatid exchange (SCE), lymphoid cells from some patients exhibit dimorphism for high and low SCE. We addressed the issue of dominance or recessivity of the low-SCE BS phenotype. A high-SCE lymphoblast line, HB1, was mutagenized, and a clone, HB10T, carrying the markers ouabain resistance and thioguanine resistance, was isolated to serve as a fusion parent. Two independent low-SCE BS lines were fused with HB10T, and hybrids were selected in HAT medium supplemented with ouabain. The hybrids, which were tetraploid, exhibited the expected phenotypes when exposed to ouabain and thioguanine. In every case, these hybrids had low SCE levels, establishing dominance of the low-SCE phenotype. The same methodology was also used to assess genetic heterogeneity in BS. A complementation analysis was carried out using high-SCE lymphoblast cell lines derived from BS patients. HB10T was fused with five other high-SCE BS lines. No correction of the high SCE characteristic of BS cells was seen in hybrid lines derived from patients of Ashkenazi Jewish, French-Canadian, Mennonite, or Japanese extraction. Thus, a single gene is responsible for the high-SCE phenotype in BS patients of diverse ethnic origin.     

A gene involved in control of human cellular senescence on human chromosome 1q.

Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, we introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras(+)-transformed derivative of TE85 (143B TK-), all of which were assigned to complementation group C. This chromosome 1 caused no change in proliferative potential of cell lines representing the other complementation groups. A derivative of human chromosome 1 that had lost most of the q arm by spontaneous deletion was unable to induce senescence in any of the immortal cell lines. This finding indicates that the q arm of human chromosome 1 carries a gene or set of genes which is altered in the cell lines assigned to complementation group C and is involved in the control of cellular senescence.     

Complementation analysis of xeroderma pigmentosum variants

DNA repair after UV exposure was studied in multinucleate cells, obtained after fusion of excision-defective and variant xeroderma pigmentosum fibroblasts. Optimal fusion conditions were determined, facilitating the measurement of DNA replication in heterokaryons. In unirradiated multikaryons, entry into the S phase was depressed, when compared with unfused cells. The extent of the depression of S phase entry was dependent on the fusion conditions. In heterokaryons obtained after fusion of XP variant (6 different strains) with excision-defective XP (three cell strains from complementation groups A, C and D) both unscheduled DNA synthesis and postreplication repair after UV irradiation were restored to normal levels. In contrast, complementation was not observed after pairwise fusion of the XP variant cell strains. These results suggest that the XP variants comprise a single complementation group, different from complementation groups A, C and D.     

Genetic complementation between UV-sensitive CHO mutants and xeroderma pigmentosum fibroblasts

The purpose of this study was to determine the feasibility of doing complementation analysis between DNA-repair mutants of CHO cells and human fibroblasts based on the recovery of hybrid cells resistant to DNA damage. Two UV-sensitive CHO mutant lines, UV20 and UV41, which belong to different genetic complementation groups, were fused with fibroblasts of xeroderma pigmentosum in various complementation groups. Selection for complementing hybrids was performed using a combination of ouabain to kill the XP cells and mitomycin C to kill the CHO mutants. Because the frequency of viable hybrid clones was generally < 10⁻⁶ and the frequency of revertants of each CHO mutant was 2×10⁻⁷, putative hybrids required verification. The hybrid character of clones was established by testing for the presence of human DNA in a dot-blot procedure.

Hybrid clones were obtained from 9 of the 10 different crosses involving 5 complementation groups of XP cells. The 4 attempted crosses with 2 other XP groups yielded no hybrid colonies. Thus, a definitive complementation analysis was not possible. Hybrids were evaluated for their UV resistance using a rapid assay that measures differential cytotoxicity (DC). All 9 hybrids were more resistant than the parental mutant CHO and XP cells, indicating that in each case complementation of the CHO repair defect by a human gene had occurred. 3 hybrids were analyzed for their UV-radiation survival curves and shown to be much more resistant that the CHO mutants but less resistant than normal CHO cells. With 2 of these hybrids, sensitive subclones, which had presumably lost the complementing gene, were found to have similar sensitivity to the parental CHO mutants. We conclude that the extremely low frequency of viable hybrids in this system limits the usefulness of the approach. The possibility remains that each of the nonhybridizing XP strains could be altered in the same locus as one of the CHO mutants.     

Complementation of multiple sulfatase deficiency in somatic cell hybrids

Multiple sulfatase deficiency (MSD) is an inherited disorder characterized by deficient activity of seven different sulfatases. Genetic complementation for steroid sulfatase (STS), arylsulfatase A, and N-acetylgalactosamine 6-SO4 sulfatase was demonstrated in somatic cell hybrids between MSD fibroblasts and mouse cells ( LA9 ) or Chinese hamster cells ( CHW ). In an electrophoretic system that separates human and rodent STS isozymes, enzyme from hybrids migrated as human enzyme. We concluded that the rodent cell complemented the MSD deficiency and allowed normal expression of the STS structural gene. Some MSD- LA9 hybrids showed significant levels of human arylsulfatase A activity, as shown by the immunoprecipitation of active enzyme by human-specific antiserum. Complementation was also suggested for N-acetylgalactosamine 6- sulfatate sulfatase (GalNAc-6S sulfatase) in several MSD- LA9 hybrids by the demonstration of a significant increase in activity (10-fold) over that of the GalNAc-6S sulfatase-deficient parental mouse and MSD cells. Thus, it was possible to demonstrate complementation for more than one sulfatase in a single MSD-rodent hybrid. Normal levels of sulfatase activity in hybrids indicate that the sulfatase structural genes are intact in MSD cells.     

Lack of complementation between xeroderma pigmentosum complementation groups D and H

Summary The construction of permanent hybrid cell lines between xeroderma pigmentosum (XP) cells from different complementation groups allows analysis not only of the degree of repair correction but also of the restoration of biological activity to the UV-irradiated cells. With use of an immortal human cell line (HD2) that expresses excision repair defects typical of XP group D, a series of permanent hybrid cells has been produced with XP cells from groups A to H. Excision repair, as measured by incision analysis and unscheduled DNA synthesis, is restored to normal or near normal levels in crosses involving HD2 and cells from XP groups A, B, C, E, F, G, and I. All these hybrids show complementation for the recovery of normal UV restistance. As expected, hybrids expressing poor incision and hypersensitivity to UV were produced in crosses between HD2 and XPD fibroblasts, but they were also produced without exception when XPH was the partner. In the permanent HD2 x XPD or XPH hybrids, analysis of incision capacity reveals abnormally low activity and therefore that there has been no complementation. The true hybrid nature of HD2 x XPH cells has been confirmed by HL-A and -B tissue typing; moreover, detailed kinetic analysis of incision in these cells shows that the XPH phenotype, rather than the XPD, is expressed, i.e. breaks accumulate at low UV fluence of 1 J/m². To help confirm these findings, another immortal XPD cell line was used in fusions involving HD2, XPH, or XPI. Cells resistant to ultraviolet were produced only with XPI fibroblasts. These data are discussed in terms of whether XPD and H mutations are likely to be allelic with respect to incision.     

Interaction of human and chick DNA repair functions in UV-irradiated xeroderma pigmentosum-chick erythrocyte heterokaryons

Fusion of chick erythrocytes with human primary fibroblasts results in the formation of heterokaryons in which the inactive chick nuclei become reactivated. The expression of chick DNA repair functions was investigated by the analysis of the DNA repair capacity after exposure to ultraviolet (UV) irradiation of such heterokaryons obtained after fusion of chick erythrocytes with normal human or xeroderma pigmentosum (XP) cells of complementation groups A, B, C and D. Unscheduled DNA synthesis (UDS) in normal human nuclei in these heterokaryons is suppressed during the first 2–4 days after fusion. The extent and duration of this suppression is positively correlated with the number of chick nuclei in the heterokaryons. Suppression is absent in heterokaryons obtained after fusion of chicken embryonic fibroblasts with XP cells (complementation group A and C).Restoration of DNA repair synthesis is found after fusion in XP nuclei of all complementation groups studied. It occurs rapidly in XP group A nuclei, starting one day after fusion and reaching near normal human levels after 5–8 days. In nuclei of the B, C and D group increased levels of UDS are found 5 days after fusion. At 8 days after fusion the UDS level is about 50% of that found in normal human nuclei. The pattern of UDS observed in the chick nuclei parallels that of the human counterpart in the fusion. A fast complementation pattern is also observed in chick fibroblast-XP group A heterokaryons resulting within 24 h in a UDS level comparable with that in chick fibroblast-normal human heterokaryons. In heterokaryons obtained after fusion of chick fibroblasts with XP group C cells UDS remains at the level of chick cells. These data suggest that reactivation of chick erythrocyte nuclei results in expression of repair functions which are able to complement the defects in the XP complementation groups A, B, C and D.     

Isolation and characterization of mouse FM3A cell mutants which are devoid of Newcastle disease virus receptors.

A method was developed to select host cell mutants which did not permit the replication of Newcastle disease virus (NDV), and 14 isolates of NDV-nonpermissive mutants of mouse FM3A cells were obtained. All these isolates were judged to be deficient in NDV receptors, since their ability to adsorb 3H-labeled NDV virions was markedly decreased. They were tested for genetic complementation in pairs by cell fusion and shown to fall into a single recessive complementation group, which was designated as Had-1. Vesicular stomatitis virus was able to replicate in this mutant to produce infectious progeny, but the glycoprotein of the released virion was abnormal in size, suggesting a defective processing of the asparagine-linked carbohydrate chains in the mutant cell. The Had-1 mutant was resistant to wheat germ agglutinin, but sensitive to a Griffonia simplicifolia lectin, GS-II, which recognizes terminal N-acetylglucosamine residues. The altered sensitivity to these plant lectins compared with that of the parental FM3A cells indicates that sialylated sugar chains on the cell surface are almost absent from the Had-1 cells, thereby rendering the cells NDV receptor deficient.     

Alteration of a nuclease in Fanconi anemia.

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.     

Tay-Sachs and Sandhoff''s disease: Intergenic complementation after somatic cell hybridization

Cultivated skin fibroblasts from patients with Tay-Sachs and Sandhoff's disease were fused and the isoenzymes of N-acetyl-β- -hexosaminidase were investigated after 2 and 7 days of subsequent cultivation. Enzyme analyses after heat inactivation showed a clear increase in the thermolabile component of hexosaminidase when compared with assays on fusion of each of the parental cell strains. Electrophoretic studies revealed that in cell homogenates prepared at various time intervals after cell fusion of Tay-Sachs with Sandhoff's fibroblasts, all three hexosaminidase isoenzymes were present, including hexosaminidase A which lacks in both parental cell strains. These results show that genetic complementation has occurred, which indicates that two different gene mutations are involved in these variants of GM2-gangliosidosis. The relevance of the data obtained for the elucidation of the molecular properties of the (iso)enzymes involved is discussed.