Mammalian Cell Hybrids Research Articles

Genome-wide analysis of hepatic gene silencing in mammalian cell hybrids

Silencing of tissue-specific gene expression in mammalian somatic cell hybrids is a well-documented epigenetic phenomenon which is both profound (involving a large number of genes) and enigmatic. Our aim was to utilize whole-genome microarray analyses to determine the true extent of gene silencing on a genomic level. By comparing gene expression profiles of hepatoma × fibroblast cell hybrids with those of parental cells, we have identified over 300 liver-enriched genes that are repressed at least 5-fold in the cell hybrids, the majority of which are repressed at least 10-fold. Also, we identify nearly 200 fibroblast-enriched genes that are repressed at least 5-fold. Silenced hepatic genes include several that encode transcription factors and proteins involved in signal transduction pathways. These data suggest that extensive reprogramming occurs in cell hybrids, leading to a nearly global (although not complete) loss of tissue-specific gene expression.

Genetic analysis of resistance to total bromodeoxyuridine substitution in mammalian cell hybrids

Somatic cell hybrids derived from the fusion of Chinese hamster ovary cells (CHO) and mutant Syrian hamster melanoma cells (2E) were tested for their ability to grow with all of the thymidine (dThd) in their DNA replaced with 5-bromo-2'-deoxyuridine (BrdU), a phenotypic capability of the 2E cells but not of the CHO cells. Under these conditions, the 2E cells survived and grew, all of the hybrid clones survived and grew to varying degrees, and the CHO cells did not survive at all. When 2E cells were tested, they were also found to be resistant to the toxic effects of BrdU substitution and white light irradiation, relative to CHO cells. Thus, when the DNAs of 2E and CHO cells were equally (50%) substituted with BrdU, and the two cell lines irradiated with identical doses of white light, the survival of CHO cells was reduced to less than 1% of that of unirradiated cells, while 40% of the 2E cells survived. The 2E x CHO hybrid clones were found to survive at values from 10% to 40% under these identical conditions. Thus, the phenotypic characteristics of 2E cells involving total substitution and resistance to the toxic effects of BrdU substitution and white light irradiation appear to be expressed in a codominant fashion in somatic cell hybrids.

Genomic instability in mammalian cell hybrids. IV. Is mutation frequency elevated in hybrid cells?

In this study, we set out to determine whether the mutation frequency in cell hybrids is increased over the frequencies in the two parental lines, and whether this increase is related to the evolutionary divergence of the cell parents. Two test loci were chosen: forward mutation at the HPRT locus and mutation to resistance to the drug emetine. We conclude that while some cell combinations do seem to produce hybrids with higher mutation frequencies, this is not consistently so, and, indeed, mutation rates in hybrids may be higher, lower or very similar to rates in the parental lines. Further, evolutionary divergence between the parental lines does not appear to correlate to mutation frequency in the hybrids.

Measurement of changes in cytoplasmic free CA2+ in fused cell hybrids.

The investigation of many problems in cell biology requires a method for studying chemical ‘events’ in living cells. For example, the measurement of cytosolic free Ca2+, using Ca2+ indicators in giant cells, has provided vital evidence for the role of this ion in the activation of cells by electrical and chemical stimuli1. However, the incorporation of Ca2+ indicators into small cells (diameter <20 µm), without causing irreversible damage, has proved extremely difficult1. Recently, an elegant new technique has been reported2 for entrapping within small cells a fluorescent Ca2+ indicator after hydrolysis within the cell of a membrane-permeant ester of the compound. We have developed a method, using Sendai virus, for fusing cells with erythrocyte ghosts containing photoprotein Ca2+ indicators and other membrane-impermeant chemiluminescent-labelled indicators3,4. Because of the sensitivity of chemiluminescent analysis, these indicators can be used at concentrations which do not significantly disturb the chemistry of the cells. The ultimate aim was to establish a cell system in which free Ca2+, oxygen radicals and cyclic nucleotides could be directly monitored. Here we report the incorporation of the photoprotein, obelin5, an indicator of free Ca2+, into small mammalian cell hybrids.

Histone gene expression and chromatin structure in mammalian cell hybrids.

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.

Genetic control of tumorigenicity in interspecific mammalian cell hybrids

The nature of genetic control of cellular malignancy was investigated by examining the tumorigenicity of a series of interspecific mouse-human cell hybrids in the athymic nude mouse. Two highly malignant but genetically distinct mouse cell lines, A9 and PG19, were hybridized with three normal human diploid fibroblast strains, and 19 independently arising hybrid clones were isolated. Each of these clones was capable of forming progressive lethal tumors in the nude mouse, and thus resembled the malignant parental mouse cells rather than the nonmalignant parental human cells. We failed to obtain any evidence for complete suppression of tumorigenicity in these cell hybrids. The absence of suppression was observed regardless of the extent and composition of the human chromosome complements retained in the hybrid clones; the results of detailed cytological and isoenzyme analyses would make it highly improbable that the observed lack of suppression was due to cellular selection in vivo for a more tumorigenic subpopulation in the injected hybrid cells. These data demonstrate that at least for the parental cell combinations used in this study, no human chromosome, when present singly in the mouse-human cell hybrids, can suppress the tumorigenic phenotype of the mouse cells. Our results are consistent with the view that the suppression of cellular malignancy previously demonstrated in intraspecific (mouse × mouse) somatic cell hybrids does not occur in interspecific (mouse-human) cell hybrids, or alternatively, genetic determinants located on two or more human chromosomes are required simultaneously to suppress the malignancy of the mouse cells in cell hybrids derived from malignant mouse cell and nonmalignant human cells.