1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐Resistant, Flat‐Cell PC12 Variants Having a Partial Loss of Transformed Phenotype

Abstract

We have cloned and characterized two variants of PC12 cells. MPT1 cells were selected by their resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and variant 2068 was isolated nonselectively as a large, flat-cell variant commonly occurring in PC12 cultures. Variant 2068 cells also exhibit resistance to MPTP. Karyotype analysis revealed that these variants are true derivatives of wild-type PC12 cells; however, each variant is tetraploid, whereas the wild-type parent is diploid. The two variants contain an altered level and composition of lactate dehydrogenase isoenzymes, which could account for a previously described difference in lactate metabolism. Both variants exhibit a partial loss of transformed phenotype in culture in that they are nonrefractile, grow in monolayers, and fail to multiply in soft agar. We suggest that this alteration in transformed phenotype may result in altered mitochondria and lactate dehydrogenase and thus account for their resistance to MPTP. Compared with wild-type PC12 cells, MPT1 cells have a decreased level of fos mRNA and an increased level of myc mRNA; the latter results from an increased level of transcription of exon 1 of the myc gene. Studies with hybrid cells obtained by fusing MPT1 cells with wild-type-like cells show that most, but not all, of the parameters of the MPT1 phenotype predominate.

Differential Expression of Markers and Activities in a Group of PC12 Nerve Cell Clones

“…Interestingly, one of the two clones that failed to modify its phenotype after NGF was the granule-free clone 27. The latter, however, did not resemble some of the previously described NGFunresponsive clones, characterized by a flat, undifferentiated appearance (Andersen et al, 1990), but exhibited in contrast a few neurites independently of NGF treatment.…”

“…Peculiar clones can in fact offer advantages for the study of individual molecules and/or functions, an approach already widely employed in the PC12 field (see, among others, Greene et al, 1986;Cahill et al, 1989;Katoh-Semba et a/. , 1989;Ariga et al, 1989;Andersen et al, 1990;Altin et al, 1991;Baetge and Hammang, 1991;Loeb et al, 1991;Oohira et al, 1991;Zacchetti er al., 1991;Clementi et al, 1992). In addition, the parallel characterization of numerous clones can reveal information about complex problems, ranging from the coordinated expression of different molecules to the appreciation of their physiological role.…”

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

“…Neurite-promoting activity 6B3 is a PC12 flat-cell variant (Andersen et al, 1990) that had been transfected with the pMTEJ plasmid (Trimble etaaL, 1986). This plasmid contains the cDNA for Ha-ras under transcriptional control of the metallothionein promoter.…”

“…Our laboratory has isolated a flat cell PC1 2 variant (Andersen et al, 1990) that continues to multiply and only extends short processes when it expresses active Harvey (Ha) ras after transfection of the gene (Rozenberg, Bitler, and Howard, manuscript in preparation). In a search for an autocrine factor that might cause the extension of processes from the transfected variant, we have identified a 5000-Da polypeptide that induces neurite outgrowth from wild-type PC12 cells.…”

PC12‐E2 cells: A stable variant with altered responses to growth factor stimulation

“…As a result, the cells acquire a neuronal morphology and biochemical characteristics including increases in cell size (due to neurite outgrowth) and changes in cytoplasmic organization, electrical excitability, and cholinergic activities (Greene and Tischler, 1982). Since the initial isolation, PC12 cells, as well as numerous sublines, have been used to examine various cellular processes such as ion fluxes and catecholamine transport in addition to neuronal differentiation (Andersen et al, 1990;Bothwell et al, 1980;Bitler et al, 1986;Fujita et al, 1989;Ramachandran et al, 1993). Furthermore, both spontaneous variants and chemically mutagenized cells, that display defects in receptor and second messenger systems, have been quite valuable in dissecting the involvement of signal transduction pathways in the differentiative actions of neurotrophic factors (Green et al, 1986;Sano and Kitajima, 1992).…”