Identification of genetic determinants responsible for the rapid immunosuppressive activity and the low leukemogenic potential of a variant of Friend leukemia virus, FIS-2.

An immunosuppressive variant of Friend murine leukemia virus (F-MuLV), FIS-2, induces suppression of the primary antibody response against sheep erythrocytes (SRBC) in adult NMRI mice more efficiently than the prototype F-MuLV clone 57 (cl.57). It is, however, less potent than F-MuLV cl.57 in inducing erythroleukemia upon inoculation into newborn NMRI mice. Nucleotide sequence analysis shows a high degree of homology between the two viruses. Single point mutations are scattered over both the gag and the env encoding regions. The most notable mutations are the deletion of one direct repeat and a few single point mutations occurring in the binding sites for cellular transcriptional factors in the FIS-2 long terminal repeat region (LTR). To define the genetic determinants responsible for the pathogenic properties of FIS-2, we constructed six chimeras between FIS-2 and F-MuLV cl.57. Adult mice were infected with the chimeras, and their primary antibody responses against SRBC were investigated. The results showed that the fragment encompassing the FIS-2 env encoding region SU is responsible for the increased immunosuppressive activity in adult mice. A leukemogenicity assay was also performed by infecting newborn mice with the chimeras. Consistent with the previous studies, it showed that the deletion of one direct repeat in the FIS-2 LTR is responsible for the long latent period of erythroleukemia induced by FIS-2 in newborn-inoculated mice. However, studies of cell type-specific transcriptional activities of FIS-2 and F-MuLV cl.57 LTRs using LTR-chloramphenicol acetyltransferase constructs showed that the deletion of one direct repeat does not reduce the transcriptional activity of the FIS-2 LTR. The activity is either comparable to or higher than the transcriptional activity of the F-MuLV cl.57 LTR in the different cell lines that we used, even in an erythroleukemia cell line. It seems that the high transcriptional strength of the FIS-2 LTR is not sufficient to give FIS-2 a high leukemogenic effect. This suggestion is inconsistent with the previous suggestion that the transcriptional strength of an LTR in a given cell type is correlated with the leukemogenic potential in the corresponding tissue. In other words, these data indicate that the direct repeats in the F-MuLV LTR may play other roles besides transcriptional enhancer in the leukemogenesis of F-MuLV.

Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes.

cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.

Elevated cAMP gives short-term inhibition and long-term stimulation of hepatocyte DNA replication: roles of the cAMP-dependent protein kinase subunits.

The study reports the role of the isozyme forms (cA-PKI and cA-PKII) and subunits (R and C) of cAMP-dependent protein kinase in mediating the acute depression of hepatocyte DNA replication by elevated cAMP. Combinations of cAMP analogs preferentially activating cA-PKI or II showed that either isozyme could inhibit DNA replication. The effects of glucagon and cAMP analogs were counteracted by the cAMP antagonist RpcAMPS, implicating the necessity for cA-PK dissociation in cAMP action. The effect of elevated cAMP was mimicked by microinjected C subunit, but not by the RI subunit of cA-PK. Hepatocytes under continuous cAMP challenge more than regained their replicative activity. This tardive stimulatory effect of cAMP was enhanced by insulin and blocked by dexamethasone, and was preceded by downregulation of cA-PK. In conclusion, a burst of cAMP acutely inhibits hepatocyte G1/S transition in late G1 regardless of hormonal state. In the presence of high glucocorticoid/low insulin the inhibition persists. At high insulin/low glucocorticoid the inhibitory phase is followed by a prolonged stimulation of DNA replication. Downregulation of endogenous cA-PK is a mechanism for escape from the inhibitory action of highly elevated cAMP.