Expression of a constitutively active Ca2+/calmodulin-dependent kinase in Aspergillus nidulans spores prevents germination and entry into the cell cycle.

The unique gene for Ca2+/calmodulin-dependent protein kinase (CaMK) has been shown to be essential in Aspergillus nidulans. Disruption of the gene prevents entry of spores into the nuclear division cycle. Here we show that expression of a constitutively active form of CaMK also prevents spores from entering the first S phase in response to a germinating stimulus. Expression of the constitutively active kinase induces premature activation of NIMEcyclin B/NIMXcdc2 in G0/G1. As NIMXcdc2 is present in spores, the elevation of maturation promotion factor activity may be secondary to the early production of NIMEcyclin B or post-translation modification of maturation promotion factor. The expression of the constitutively active CaMK also results in the appearance of NIMA kinase activity within 1 h of the germinating signal. These results support the contention that the activities of maturation promotion factor and NIMA are coincidentally regulated in A. nidulans and suggest that the unscheduled appearance of one or both of these activities may be sufficient to prevent A. nidulans spores from entering into DNA synthesis.

Ca(2+)/calmodulin-dependent kinase is essential for both growth and nuclear division in Aspergillus nidulans.

The calmodulin gene has been shown to be essential for cell cycle progression in a number of eukaryotic organisms. In vertebrates and Aspergillus nidulans the calmodulin dependence also requires calcium. We demonstrate that the unique gene encoding a multifunctional calcium/calmodulin-dependent protein kinase (CaMK) is also essential in A. nidulans. This enzyme is required both for the nuclear division cycle and for hyphal growth, because spores containing the disrupted gene arrest with a single nucleus and fail to extend a germ tube. A strain conditional for the expression of CaMK was created. When grown under conditions that resulted in a 90% decrease in the enzyme, both nuclear division and growth were markedly slowed. The CaMK seems to be important for progression from G2 to mitosis.

Role of Ca++/calmodulin binding proteins in Aspergillus nidulans cell cycle regulation.

The goal of this review is to summarise the current knowledge concerning the targets of Ca++/calmodulin that are essential for cell cycle progression in lower eukaryotes. Emphasis is placed on Aspergillus nidulans since this is the only organism to date shown to posses essential Ca++ dependent calmodulin activated enzymes. Two such enzymes are the calmodulin activated protein phosphatase, calcineurin and the calmodulin dependent protein kinase. These proteins, each the product of a unique gene, are required for progression of quiescent spores into the proliferative cycle and also for execution of the nuclear division cycle in exponentially growing germlings.

GTP depletion induced by IMP dehydrogenase inhibitors blocks RNA-primed DNA synthesis.

Inhibitors of IMP dehydrogenase (EC 1.2.1.14), including mizoribine (Bredinin) and mycophenolic acid, have significant antitumor and immunosuppressive activities. Studies were aimed at determining the mechanism by which intracellular GTP depletion induced by these agents results in inhibition of DNA synthesis. Incubation of human CEM leukemia cells for 2 hr with IC50 concentrations of either mizoribine (4 microM) or mycophenolic acid (0.5 microM) reduced cellular GTP levels an average of 68% or 58%, respectively, compared with the levels in control cells. Under similar conditions, mizoribine and mycophenolic acid decreased the amount of [3H]adenosine incorporated into primer RNA by 75% and 70%, respectively, relative to the untreated controls, but had no significant effect on total RNA synthesis. Repletion of the guanine nucleotide pools by coincubation of CEM cells with guanosine plus 8-aminoguanosine prevented both the inhibition of primer RNA synthesis and the inhibition of tumor cell growth induced by these agents. Additional studies demonstrated that GTP depletion alone was capable of directly inducing inhibition of primer RNA synthesis. Primer RNA synthesis was inhibited an average of 84% in whole-cell lysates that lacked GTP but contained all remaining ribo- and deoxyribonucleoside triphosphates. On an M13 DNA template, RNA-primed DNA synthesis catalyzed by the purified complex of DNA primase (EC 2.7.7.6) and DNA polymerase alpha (EC 2.7.7.7) was decreased an average of 70% in the absence of GTP, compared with synthesis in the presence of 0.5 mM GTP. These results provide evidence that mizoribine and mycophenolic acid inhibit DNA replication by inducing GTP depletion, which suppresses the synthesis of RNA-primed DNA intermediates.

Effects of human T lymphocyte activation on inosine monophosphate dehydrogenase expression.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the first step in the formation of guanine ribonucleotides from inosine monophosphate and the activity of this enzyme appears to be essential for cell proliferation. Inhibitors of IMPDH have been demonstrated to be effective immunosuppressive agents and to inhibit T cell activation in vitro. IMPDH activity results from the expression of two different genes (types I and II) that encode protein subunits of identical size with 84% amino acid identity. To determine the relative contribution of the expression of these two genes to T cell activation, we have examined the effects of T cell stimulation on IMPDH activity, mRNA levels, and protein. The stimulation of isolated peripheral blood CD28+ T cells with PMA and ionomycin causes a 15-fold increase in IMPDH activity over a 72-h period. This is associated with a 10-fold increase in type II mRNA levels at 48 h. Type I mRNA is expressed at very low levels in resting T cells, but increases 10-fold by 24 h after stimulation. The type I cDNA probe also detects a second larger mRNA species of 4.0 kb that is not detectable in a variety of normal tissues or in a panel of leukemic cell lines. RNase protection assays using RNA probes corresponding to the entire coding region of the type I enzyme reveal a single protected fragment, demonstrating that the 4.0-kb message is the result of alternate splicing in the 5' or 3' untranslated regions or the use of an alternative polyadenylation site. Western blot analysis demonstrates a concomitant increase in total IMPDH protein on T cell activation, although posttranslational modifications do not allow the distinction between type I and type II on isoelectric focusing gels. We conclude that the induction of both type I and type II IMPDH contribute significantly to the T cell proliferative response. Both enzymes therefore should be considered important targets for immunosuppressive therapy.

Type I inosine monophosphate dehydrogenase: evidence for a single functional gene in mammalian species.

Human inosine monophosphate dehydrogenase activity is the result of the expression of two independent but closely related genes, termed IMPDH type I and type II. We have documented the presence of multiple, processed pseudogenes of type I IMPDH in human and Rhesus monkey genomic DNA, as well as a single functional gene encoding low levels of type I mRNA in human brain, heart, kidney and placenta. Single copy genes for each IMPDH isoenzyme were also found in rat, mouse, dog, cow, and chicken DNA and distinct mRNA species for type I and type II were identified by Northern blots in mouse and hamster RNA. Northern blot analysis of chicken RNA revealed a single mRNA species that hybridized to human IMPDH type I and II probes. These data document the high degree of evolutionary conservation of these two genes among mammals.

IMP dehydrogenase inhibitors as immunomodulators.

IMP dehydrogenase is a key enzyme in the de novo pathway of purine biosynthesis and is responsible for catalyzing the first step in the formation of guanine ribonucleotides from inosine monophosphate. Mizoribine, an immunosuppressive agent in wide-spread clinical use in Japan, has been demonstrated to inhibit this enzyme. We have investigated the effects of mizoribine on human T cell activation. Stimulation of purified human peripheral blood T lymphocytes with phorbol ester and ionomycin leads to a five-fold increase in guanine ribonucleotide levels over 72 hours. The addition of mizoribine to these cultures at concentrations that are achieved in vivo leads to a dose-dependent inhibition of proliferation and concomitant 50% decrease in guanine ribonucleotide levels, an effect that is reversible with the addition of guanosine, which repletes the GTP pool. Similar effects are seen with direct stimulation via the CD3/T cell receptor complex. Inhibition of proliferation occurs at the G1/S interface of the cell cycle and is additive to that produced by cyclosporine. In order to determine whether inhibition of IMP dehydrogenase is a common mechanism of immunosuppression for drugs such as azathioprine and 6-mercaptopurine that interfere with purine biosynthesis, we compared the effects of these agents on the metabolism of purified T lymphocytes. The results of these studies demonstrate that mizoribine and mycophenolic acid, a highly specific inhibitor of IMP dehydrogenase, inhibit proliferation directly by the depletion of guanine ribonucleotides; 6-mercaptopurine, on the other hand, has a mixed effect on adenine and guanine ribonucleotide pools, whereas azathioprine inhibits proliferation by a mechanism completely independent of its effects on the purine metabolic pathway. We conclude from these studies that inhibitors of IMP dehydrogenase have potential as specific immunosuppressive agents.

Regulation of human deoxycytidine kinase expression.

The human deoxycytidine kinase gene is a single copy gene and is comprised of seven exons that are spread over more than 34 kb of the genome. The 5'-flanking region is highly G/C rich and does not contain CAAT or TATA boxes. This region, when cloned into a recorder gene construct containing the chloramphenicol acetyltransferase gene, is capable of mediating CAT activity in human lymphoid cell lines and appears to have greater activity in human T, as compared to B, lymphoblast cell lines. The expression of the gene at the mRNA level does not appear to be cell-cycle regulated in that the levels of mRNA in human peripheral blood T lymphocytes remain constant as the cells progress from a resting to a proliferating state. Since this enzyme catalyzes the conversion of a number of chemotherapeutic agents to their corresponding monophosphate form and is thus essential for their activation, it will be important to define further the genetic elements which regulate the expression of this gene.

Comparison of the effects of mizoribine with those of azathioprine, 6-mercaptopurine, and mycophenolic acid on T lymphocyte proliferation and purine ribonucleotide metabolism.

The immunosuppressive drug mizoribine has been demonstrated to inhibit T lymphocyte proliferation by depleting these cells of guanine ribonucleotides as a consequence of inhibiting the enzyme inosine monophosphate (IMP) dehydrogenase. Because the immunosuppressive agents azathioprine and 6-mercaptopurine (6MP) are both converted to the IMP analog 6-thio-IMP, we postulated that these drugs might inhibit T cell activation and/or proliferation by a similar mechanism. Incubation of isolated peripheral blood T cells with either mizoribine or the selective IMP dehydrogenase inhibitor mycophenolic acid caused a dose-dependent inhibition of T cell proliferation, which was reversible with the addition of 50 microM guanosine to replete guanine ribonucleotide pools. In contrast, guanosine exacerbated the inhibition of proliferation induced by azathioprine and restored proliferation at IC50 concentrations of 6MP by only 10%. Complete restoration of proliferation in the presence of 6MP, but not azathioprine, was achieved with the addition of adenine. The inhibitory effects of azathioprine, as well as those of mizoribine, 6MP, and mycophenolic acid, were identical in cells stimulated with antibody to the T cell receptor and in cells stimulated with phorbol ester and ionomycin. We conclude from these studies that mizoribine selectively inhibits guanine ribonucleotide formation in purified T cells, whereas the effect of 6MP appears to be more dependent on adenine ribonucleotide depletion. Azathioprine, on the other hand, inhibits proliferation by a mechanism independent of purine ribonucleotide depletion. None of these agents inhibits T cell proliferation by interfering with signal transduction mediated by the T cell receptor. Inhibition of guanine ribonucleotide biosynthesis appears to be a novel and perhaps more selective mechanism of inhibiting T cell proliferative responses after T cell activation.