Ongoing DNA synthesis continues in young human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited

Yanishevsky, Rosalind M.; Stein, Gretchen H.

doi:10.1016/0014-4827(80)90290-6

Cited by 71 publications

(23 citation statements)

References 9 publications

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“…Indeed, hybrids derived from these fusions (with the exception of erythrocyte hybrids) have a normal chromosome constitution (7,27). On the other hand, the situation is different in heterokaryons formed between some proliferating cells and postreplicative "senescent" fibroblasts (31)(32)(33); in such heterokaryons, initiation of DNA synthesis [but not ongoing synthesis (32)(33)(34)] is inhibited by the postreplicative partner. We suggest that, similarly, initiation of DNA synthesis (or entry in mitosis) is inhibited in hepatocyte-hepatoma cell heterokaryons; the nondividing (Go) state of the hepatocytes (or other nondividing cells) would be maintained in such heterokaryons.…”

Section: Discussionmentioning

confidence: 99%

Control of serum protein production in hepatocyte hybridomas: immortalization and expression of normal hepatocyte genes.

Szpirer¹,

Szpirer²,

Wanson³

1980

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT"Hepatocyte hybridomas" have been isolated after fusion of adult hepatocytes and a-fetoprotein (AFP-producing mouse hepatoma cells. The yield of viable hybrid clones was low but could be increased by culturing the cells in the presence of insulin. On the basis of their chromosome constitution, the hybrids were classified into two groups characterized by either a single or a double set of mouse (hepatoma) chromosomes. The hybrids segregated rat chromosomes and thus constitute an excellent material for gene mapping studies in the rat.Most of the hepatocyte hybridomas retained the production of one or more rat serum proteins, indicating that the corresponding structural genes, contributed by the normal hepatocyte parent, have been immortalized and maintained in the active state after fusion. However, these hybrids do not produce rat AFP, although mouse AFP synthesis is maintained. This result strongly suggests that silent rat (hepatocyte) AFP genes coexist in hepatocyte hybridoma nuclei with active mouse (hepatoma) AFP genes. Finally, on the basis of certain proerties of these hepatocyte-hepatoma hybrids, we suggest that the nondividing state of the hepatocytes is actively controlled by a regulatory mechanism which prevents DNA synthesis or entry into mitosis or both. Hybrids formed between normal diploid cells and tumor cells of the same tissue origin (hybridomas) have proved useful for immortalizing certain functions expressed by normal differentiated cells, as shown by the numerous studies on the now classical "lymphocyte hybridomas" (1, 2). We have explored the possibility that differentiated liver functions could be immortalized by hybridization of normal hepatocytes with mouse hepatoma cells. In this communication, we report the successful isolation of interspecies "hepatocyte hybridomas" and show that these hybrids provide a tool for immortalizing normal liver functions.Hybridomas could also be valuable for analyzing the expression of differentiated functions subjected to developmental controls or to alterations associated with a malignant phenotype. This possibility has not yet been subjected to much investigation. Production of a-fetoprotein (AFP) is modulated during hepatic differentiation: AFP is produced actively by the fetal hepatocytes but only in trace amounts by the adult liver; and, in addition, it is synthesized by most hepatomas (3, 4). By fusing liver cells which differ in the expression of the AFP gene, it should be possible to explore the mechanisms controlling the expression of this gene. In previous studies, we have shown that hybrids formed between AFP-producing mouse hepatoma cells and non-AFP-producing rat hepatoma cells retain the capacity to secrete mouse AFP (5), unlike hybrids generated between AFP-producing mouse hepatoma cells and fibroblasts (6, 7). By fusing AFP-producing mouse hepatoma cells with adult ratThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U....

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Section: Discussionmentioning

confidence: 99%

Control of serum protein production in hepatocyte hybridomas: immortalization and expression of normal hepatocyte genes.

Szpirer¹,

Szpirer²,

Wanson³

1980

Proc. Natl. Acad. Sci. U.S.A.

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“…These two explanations are not mutually exclusive; for example, it is possible that as such cells reach late passage levels, they more readily enter a state of deep quiescence. If in vitro senescence turns out to be an independent variable in our results, it would represent a second parallel to the heterokaryon assay (6,20,23,26,40,45).…”

Section: Discussionmentioning

confidence: 99%

“…Genes have been cloned for several proteins possessing growth inhibitory activity, including transforming growth factor , which is bifunctional with respect to replication (8,29,44), and members of the interferon family (42); it seems likely, however, that there remains to be identified a much larger number of genes coding for growth inhibitory proteins. Important evidence for negative growth regulation has been obtained from studies of heterokaryons formed by fusion of normal replicating cells with either deeply quiescent cells or senescent cells (6,20,23,26,39,40,45). In such experiments, entry into the S phase of the cell cycle can be strongly inhibited by the nonreplicating partner.…”

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confidence: 99%

Specific growth inhibitory sequences in genomic DNA from quiescent human embryo fibroblasts.

Padmanabhan¹,

Howard²,

Howard³

1987

Mol. Cell. Biol.

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We used HeLa cells as recipients in a gene transfer assay to characterize DNA sequences that negatively regulate mammalian cell growth. In this assay, genomic DNA from quiescent human embryo fibroblasts was more inhibitory for HeLa replication than was DNA from either Escherichia coli or HeLa cells. Surprisingly, growth inhibitory activity depended on the growth state of the cells from which genomic DNA was prepared; it was strongest in DNA prepared from serum-deprived, quiescent embryo fibroblasts. This latter observation implies a role for DNA modification(s) in regulating the activity of the inhibitory sequences detected in our assay. The level of the observed growth inhibitory activity was sometimes high, suggesting that the relevant sequences may be abundantly represented in the mammalian genome. We speculate that these findings may provide new insights into the molecular mechanisms involved in cellular quiescence and in vitro senescence.Control of mammalian cell growth appears to be mediated by a balance between positive and negative stimuli. Members of the proto-oncogene family have been intensively studied as effectors of positive growth regulation (3). The molecular mechanisms mediating negative regulation of cell proliferation are, in comparison, less clearly defined. Genes have been cloned for several proteins possessing growth inhibitory activity, including transforming growth factor , which is bifunctional with respect to replication (8,29,44), and members of the interferon family (42); it seems likely, however, that there remains to be identified a much larger number of genes coding for growth inhibitory proteins. Important evidence for negative growth regulation has been obtained from studies of heterokaryons formed by fusion of normal replicating cells with either deeply quiescent cells or senescent cells (6,20,23,26,39,40,45). In such experiments, entry into the S phase of the cell cycle can be strongly inhibited by the nonreplicating partner. Additional evidence for negative growth control has been provided by experiments with somatic cell hybrids for which limitation of proliferative potential (5, 24, 25) and suppression of tumorigenicity (2,7,12,15,31,32,(36)(37)(38) have been demonstrated by a number of laboratories. Although the heterokaryon and somatic cell hybrid approaches have provided very important insights, they do not afford a straightforward means of obtaining molecular clones of the relevant genes.We

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“…Our procedures for cell culture, cell fusion, autoradiography, and identification of heterodikaryons have been described (2,3,5).…”

Section: Methodsmentioning

confidence: 99%

Carcinogen-transformed human cells are inhibited from entry into S phase by fusion to senescent cells but cells transformed by DNA tumor viruses overcome the inhibition.

Stein¹,

Yanishevsky²,

Gordon³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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With parameters of transformation other than immortality (3). Another logical possibility is that the ability to induce DNA synthesis in senescent HDC might be associated with viral transformation. Cells transformed by viruses may have gained a factor that overrides the inhibitory effect of senescent HDC, whereas carcinogen-transformed cells may have lost a normal factor necessary for the expression ofsenescence while retaining their sensitivity to the putative inhibitor when it is supplied to them in heterodikaryons. We have tested these hypotheses by fusing senescent HDC to a series of transformed human cells that have various transformed properties and were transformed in different ways. MATERIALS AND METHODSOur procedures for cell culture, cell fusion, autoradiography, and identification of heterodikaryons have been described (2,3,5).In these experiments, the senescent HDC were either IMR-90 or WI-38 fetal lung fibroblasts at 48-53 population doublings. Forty-eight hours before fusion, senescent HDC were labeled with both [3H]thymidine (0.5 ,Ci/ml; 1 Ci = 3.7 X 10'°b ecquerels) and 2-,um-diameter latex beads (Dow The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Ongoing DNA synthesis continues in young human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited

Cited by 71 publications

References 9 publications

Control of serum protein production in hepatocyte hybridomas: immortalization and expression of normal hepatocyte genes.

Control of serum protein production in hepatocyte hybridomas: immortalization and expression of normal hepatocyte genes.

Specific growth inhibitory sequences in genomic DNA from quiescent human embryo fibroblasts.

Carcinogen-transformed human cells are inhibited from entry into S phase by fusion to senescent cells but cells transformed by DNA tumor viruses overcome the inhibition.

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