Lia Menapace

The effects of corticotrophin (ACTH1−24), cyclic AMP and TPA (12-0-tetradecanoyl phorbol-13-acetate) on DNA replication and proliferation of primary rabbit adrenocortical cells in a synthetic medium

ACTH1-24 stimulated the parenchymal cells in cultures of rat adrenal cortex in serum-free synthetic HiWoBa 2000 medium to replicate DNA, enter mitosis and divide. But ACTHs principal mediator, cyclic AMP, was not a complete mitogen: the adenylate cyclase-stimulating cholera toxin and dibutyryl cyclic AMP stimulated parenchymal cells to replicate DNA but not to enter mitosis. Thus, there must have been an additional mediator of the response to ACTH1-24 that enabled the parenchymal cells to enter mitosis. This additional mediator might have been protein kinase C because a protein kinase C activator and cyclic AMP elevator, TPA, stimulated the adrenocortical parenchymal cells to replicate DNA, enter mitosis and divide.

The exposure of carcinogen-initiated primary neonatal rat hepatocytes to tumor promoters modulates both the transcripts and the enzymatic activity of nuclear poly(ADP-ribose) polymerase

Four tumor promoters, i.e. PB, TPA, NAF, and DDT, added singly to a calcium-deprived synthetic medium, elicited early and late mitogenic effects and concurrent surges of nuclear poly(ADP-ribose) polymerase (pADPRP) activity in primary neonatal rat hepatocytes mutagenized with an intra-uterine dose of DMN. These actions were fully abated by the pADPRP inhibitor 3-MBA. Conversely, EGF only acted as a full mitogen when mediums calcium was at physiological levels, and its effects could not be blocked by 3-MBA. The same tumor promoters, but not EGF, also evoked a swift and lingering amplification of pADPRP transcripts in DMN-initiated hepatocytes kept in low-calcium medium. Hence, a coordinated modulation of both pADPRP transcripts and activity by xenobiotics is likely to be involved in the clonal expansion of early preneoplastic hepatocytes.

Primary tissue culture of normal adult rabbit adrenocortical cells fromzonae fasciculata andreticularis

Parenchymal cells isolated from decapsulated adrenal cortices of adult New Zealand white female rabbits by means of enzymatic (trypsin plus collagenase and hyaluronidase) predigestion at 37° C followed by mechanical dissociation within a calcium (Ca2+)-free buffer were set up in primary monolayer cultures onto very thin, porous polyethylene discs floating on the top of a growth medium contained inside the wells of a plastic cluster plate. In this setting, adrenal cortical cells, being at the interface between air and growth medium, benefit from both the respiratory and metabolic standpoint. Therefore, they fully respond to anabolic and growth-promoting stimuli which may increase cellular oxygen consumption significantly. Hence, this in vitro model is well suited for studying the fairly complex regulatory mechanisms of mammalian adult adrenal cortical cell proliferation and differentiation, as it allows one to take full advantage of an artificially controlled environment and of completely synthetic growth media.

Morphology, growth, and gene expression in five newly isolated murine hepatocellular tumor cell lines

Five murine hepatocellular tumor cell lines (HepM-1-5) were isolated and grown in a synthetic medium added with hormones, growth factors and/or serum. The morphology of these lines ranged from a nearly homogeneous epithelial-like shape (HepM-2) to a stromal appearance (HepM-1). The remaining lines displayed a mixed morphology. For their proliferation all of the cell lines retained a clear dependence on the extracellular calcium level and hormonal and/or serum growth factors and, rather homogeneously, they did not express the albumin, alpha-fetoprotein (with the exception of HepM-2 cells), tyrosine aminotransferase, and ornithine transcarbamylase genes, whereas they all exhibited discrete levels of the ornithine aminotransferase mRNA. Only HepM-3 and HepM-5 lines expressed the procollagen type I gene.

Tumor Promoters, But Not EGF, Increase Nuclear Poly(ADP-Ribose) Polymerase Gene Expression in Rat Hepatocytes Initiated in Utero with DMN and Cultured After Birth in Low-Calcium Synthetic Medium

Mixed, in vivo-in vitro,models of liver chemical carcinogenesis have been used only rarely [1,2]. Recently, we devised to utilize a new one of such mixed systems to clarify the role(s) played in the unfolding of liver malignancies by nuclear poly(ADP-ribosyl)ating reactions. Such metabolic events posttranslationally modify manifold nuclear protein species, including the enzyme itself, i.e. poly(ADP-ribose) polymerase (pADPRP), by which they are catalyzed [3]. Nuclear poly(ADP-ribosyl)ations are involved not only in DNA repair, but even in the modulation of gene expression related to normal and abnormal cell proliferation, as the occurrence of malignancy is prevented by established inhibitors of pADPRP [4,5]. An increased activity of nuclear pADPRP is indispensable for the multiplication of fully transformed hepatocytes [6,7]. Likewise, both partial hepatectomy and the administration of lead nitrate increase the activity and genetic expression of nuclear pADPRP in rat hepatocytes in vivo [8]. Previously, we showed that the activation of nuclear pADPRP played a pivotal role in the enactment of the mitogenic effects elicited by several tumor promoters administered to bona fide normal, primary neonatal rat hepatocytes [9,10]. In this communication we relate that the in utero initiation with a transplacental genotoxic carcinogen like dimethylnitrosamine (DMN) [11,12] and the in vitro postnatal promotion of the offspring’s initiated hepatocytes with divers xenobiotics [9,10] do increase the expression of the nuclear pADPRP gene.