Reverse Transformation and Genome Exposure in the C6 Glial Tumor Cell Line

Abstract

Reexpression of growth control and differentiation in response to physiological inducers can be demonstrated in some malignant cell lines, showing that they are not irreversibly transformed. This switch in phenotype is likely to reflect a changing pattern of gene expression, but it has not been known whether such cellular transitions involve major or only minor modulation of chromatin structure. We have studied growth control and accessibility of chromatin to DNase I in C6 glioma cells subjected to different growth regimens using an in situ nick translation assay to label the most exposed regions of nuclear chromatin. In fibroblasts and primary glia, exposed chromatin was localized mainly at the nuclear lamina. This readily labeled DNA structure was largely lacking in the malignant C6 glioma. When C6 cells were treated with dibutyryl cyclic AMP, exposed chromatin was reestablished around the nuclear periphery. This restoration of a normal genome exposure pattern required cytoskeletal integrity. Thus large-scale nuclear reorganization events proceed in parallel with phenotypic normalization. The changes in cell morphology, growth control, cytoskeletal organization, and chromatin exposure and localization are similar to the reverse transformation reaction in CHO-K1 cells, which is also regulated by the cyclic nucleotide system. Hydrocortisone and dexamethasone also restored genome exposure in C6 but less markedly than cAMP derivatives. Diverse transformed cells can thus respond to growth control stimuli with similar nuclear restructuring events, which presumably underlie changes in gene expression. Reverse transformation and redifferentiation appear to be alternative terms describing essentially the same biological phenomenon.

Transposition of DNase hypersensitive chromatin to the nuclear periphery coincides temporally with nerve growth factor-induced up-regulation of gene expression in PC12 cells.

“…1 B'-E'), indicates that a spatial migration of DHC does occur. Moreover, in contrast to reverse transformed cells that show persistence of DHC in the nuclear interior, including a region surrounding the nucleolus (21,22,39), no such persistence of DHC in the nuclear interior was detectable in the cell type employed in the present study. In fact, the interior of nuclei, including those regions surrounding nucleoli and prominent in naive cells (Fig.…”

“…While an association exists between a change in the phenotype of a cell and its organization of DHC (16)(17)(18)(19)(20)(21)(22), the functional relevance and the mechanisms involved remains unclear. It is, however, probable that the topological organization of DHC is mediated by its attachment to the nuclear matrix (18).…”

“…Also, specific chromosomes and specific domains thereof, including centromeric DNA, transcribed sequences as well as aggregates of RNA processing factors, are arranged in distinct, spatial relationships (1, 10-13). Although transcription occurs throughout nuclei in some cell types (14,15), other evidence suggests that compartmentalization also applies to sites of transcription, represented by distinct, pan-nuclear foci of DNase I hypersensitive chromatin (DHC), a distribution that can be modulated by changes in cell function (16)(17)(18)(19)(20)(21)(22). Although DHC structurally represents all nucleosome-free DNA, regardless of transcriptional state, it does include transcriptionally active sequences (23,24).…”

“…Although transcription occurs throughout nuclei in some cell types (14,15), other evidence suggests that compartmentalization also applies to sites of transcription, represented by distinct, pan-nuclear foci of DNase I hypersensitive chromatin (DHC), a distribution that can be modulated by changes in cell function (16)(17)(18)(19)(20)(21)(22). Although DHC structurally represents all nucleosome-free DNA, regardless of transcriptional state, it does include transcriptionally active sequences (23,24).DHC (16,(18)(19)(20)(21)(22) and such nonexpressed sequences as centromeric DNA (25-28) and snRNP foci (8) change position under conditions that change gene expression. This indicates that changes in nuclear compartmentalization and gene expression may be functionally linked.…”

Preferential sites of early DNA cleavage in apoptosis and the pathway of nuclear damage

“…These sites most susceptible to early cleavage events are shown to correspond to "exposed" chromatin residing as perinucleolar and peripheral lamina allotments, which were previously shown to also be the domains most accessible to exogenously supplied DNase I (Krystosek and Puck 1990;Haag et al 1994). It is thus likely that the endonucleolytic attack in programmed cell death is not random but rather immediately destroys ac-Rat PC12 cells were obtained from Dr. Noboru Sueoka, University of Colorado, Boulder, and showed growth and neurite induction response similar to those reported at the time of establishment of the cell line (Greene and Tischler 1976).…”