Disease relevance of CAD

• Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant [1].
• Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix [1].
• Genomic fragments of the human CAD gene have been obtained by screening a human genomic library in bacteriophage lambda using a Syrian hamster cDNA clone as a probe [2].
• In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation [3].
• QoL improves during the OTIs, there is reduced toxicity, and a positive affect on bone density compared with CAD therapy [4].

High impact information on CAD

• The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase [5].
• In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD) [6].
• At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells [7].
• CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3) [7].
• We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating [7].

Biological context of CAD

• The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells [7].
• The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular [8].
• Thus, the nuclear import of CAD appears to promote optimal cell growth [9].
• The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase [9].
• The activation and subsequent down-regulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian pyrimidine biosynthesis [10].

Anatomical context of CAD

• Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared [7].
• CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively [7].
• From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes [7].
• Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix [9].
• Withdrawal of interleukin-3 or incubation with staurosporine (STS) or doxorubicin (Dox) resulted in proteolytic cleavage of CAD in a myeloid precursor cell line (32D) or in a cell line over-expressing CAD [11].

Associations of CAD with chemical compounds

• CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells [8].
• Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation [1].
• In mammals, this enzyme along with carbamyl phosphate synthetase II and dihydroorotase (DHOase) is encoded by a single gene called CAD [12].
• The potential advantages of IAD over CAD therapy are an improved QoL, a prolonged period of androgen dependence, a reduced incidence of the side-effects normally associated with AD therapy, and a decrease in the cost of care [4].

Other interactions of CAD

• In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated [9].
• Nuclear localization and mitogen-activated protein kinase phosphorylation of the multifunctional protein CAD [9].
• Once activated, pyrimidine biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase [10].
• On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus [13].

Analytical, diagnostic and therapeutic context of CAD

• Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting [1].
• These human genomic clones have been used to assign the CAD gene to human chromosome 2 using in situ hybridization to human metaphase chromosomes and Southern blot hybridization analysis of DNA isolated from a panel of Chinese hamster/human hybrid cells [2].
• A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR) [14].
• Further investigations of IAD are ongoing in randomized, controlled, phase III trials in the USA, Canada and Europe. Most of these studies are not yet mature and final results are awaited; however, interim analyses from some studies suggest IAD and CAD therapy are equally effective in terms of progression-free survival [4].

References