The retinoblastoma susceptibility gene (Rb) participates in controlling the G1/S-phase transition, pre-
Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10-to 12-fold and 4-to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/-cell lines exhibited a 2-to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3-to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantimetabolites VP-16, cisplatin, and doxorubicin. A gel mobility-shift assay showed that parental SaOS-2 cells had increased levels of free E2F compared to the Rb-reconstituted SaOS-2 cells. These results indicate that pRB defective cells may have decreased sensitivity to growth inhibition by target enzymes encoded by genes whose transcription is enhanced by E2F proteins and suggest mechanisms of interaction between cytotoxic agents and genes involved in cell cycle progression.polyglutamate formation or impaired uptake (17). The fifth cell line (HS-18), although able to transport MTX and make long-chain polyglutamates comparable to the MTX-sensitive HT-1080 fibrosarcoma cell line, had an increased level of the MTX target enzyme DHFR compared to the other cell lines, without evidence of DHFR gene amplification. The subsequent finding that this cell line lacked both copies of chromosome 13, which contains the gene for pRB (18), led us to investigate further the relationship between pRB and resistance to MTX as well as FdUrd, antimetabolite drugs that inhibit enzymes involved in DNA replication.This study addresses the relationship between absent or abnormal pRB and sensitivity to drugs that target DHFR and TS. We have compared the growth inhibition of a human sarcoma cell line containing pRB (HT-1080) with cell lines either lacking pRB (HS-18) or having a truncated pRB incapable of binding E2F (SaOS-2). We have also obtained viable SaOS-2 sublines stably transfected with Rb after G-418 selection (see below). Cells lacking functional pRB were more resistant to growth inhibition by MTX and FdUrd compared to pRB-expressing cell lines. In cell lines lacking pRB, levels of DHFR and TS enzyme activity as well as mRNA expression were higher. Furthermore, cell lines lacking pRB had increases in free E2F as measured by gel-retardation assays.A wide variety of human cancers demonstrate loss or mutation in the tumor suppressor gene p53 and the retinoblastoma tumor suppressor gene (Rb) (reviewed in refs. 1 and 2). Rb encodes a nuclear phosphoprotein...
Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide:L-glutamine amido-ligase (ADPforming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA.The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition ofthe DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domainspecific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetaseDHOase-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus. The kinetics, pH dependence, and inhibition of mammalian DHOase have been extensively studied (6-15). Inactivation by cysteine (11) and diethyl thiopyrocarbonate (12) lead to the suggestion that a zinc ion and a histidine side chain, Saul
Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance ofADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.Amplification and consequent overexpression of oncogenes and growth factor receptor genes occurs in many human and rodent cancer cells in vivo. Amplification of a large number of target genes has also been shown to underlie the drug resistance of many mammalian cell lines propagated in vitro (see references 4, 5, 9, 43, and 44 for recent reviews). It is reasonable to infer that oncogene amplification contributes to tumor cell growth or survival since recent studies reveal that reducing the expression of oncogenes by antisense strategies (51) or by elimination of amplified oncogenes (49) reduces tumorigenicity.In many cases, the initial molecular intermediates of gene amplification are genetically unstable (see reference 26 for an early example and reference 50 for a review) and consist of chromosome fragments lacking centromeres (see reference 52 for a recent study and reference 53 for a review). These fragments often appear as heterogeneously sized, paired chromatin bodies called double minute chromosomes (DMs) (3,10,17,39). DMs can be formed by chromosome breakage (40, 52), resulting in the generation and subsequent oligomerization of submicroscopic circular precursors referred to as episomes or amplisomes (7,13,37,38,48,52), or by the generation of microscopically visible fragments in a single step (34,40,52 amplification suggests either that such structures are preferred intermediates or that they offer more substantial survival advantages than chromosomal amplicons in vivo (50).The common occurrence of DMs and their submicroscopic precursor...
The first three steps in mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein designated CAD. Regions of the single 240.kDa polypeptide chain are folded into separate structural domains that have discrete functions. Previous studies suggested that CAD forms predominantly trimers. The trimers are found to be in slow equilibrium with hexamers and higher oligomers com (3), while a distinct 44-kDa species, the DHOase domain, catalyzes the DHOase reaction (4).The protein is an oligomer composed of multiple copies of the 240-kDa polypeptide. Trimers, hexamers, nonamers, and higher oligomeric forms have been reported (1). These authors found that the predominant form is the trimer, and that this species is in slow equilibrium with lower concentrations of hexamer and much smaller, but significant amounts, of the larger oligomers. All of these species are composed of multiples of three polypeptides, suggesting that the trimer is the fundamental oligomeric unit. There is evidence (1, 5) that the oligomeric structure is extremely sensitive to proteases and that nicking the polypeptide results in a marked reduction in the molecular weight of the protomer.We have examined the oligomeric structure of CAD prepared under conditions that eliminate nicking of the polypeptide chain (6, 7 MA) and removed from the surface by incubation in 0.5 mM EDTA in phosphate-buffered saline at 370C for 5 min. CAD was isolated by the method of Coleman et al. (1) and stored at -700C at a concentration of 3 mg/ml in 20 mM Tris/50 mM KCI/4 mM glutamine/4 mM aspartate/0.1 mM EDTA/1 mM dithiothreitol/5% glycerol/30% dimethyl sulfoxide, pH 7.4.Chemical Crosslinking. The crosslinking reaction was initiated by the addition of dimethyl suberimidate (Pierce) to CAD in storage buffer to give a final concentration of 0.3 mg of dimethyl suberimidate and 0.2 mg of CAD per ml (1, 9). The pH of the reaction mixture was 9.2. The reaction was carried out at room temperature, and aliquots were removed at timed intervals from 10 to 180 min and overnight. The crosslinking was quenched by the addition of 1 M glycine to a concentration of 0.1 M. The crosslinked species were analyzed by electrophoresis on NaDodSO4/composite 2.2% acrylamide-0.5% agarose tube gels (1, 10) with the Weber and Osborn (11) phosphate buffer system. Electrophoresis was carried out for 7 hr at a constant current of 8 mA per tube. The gels were stained with Coomassie blue and scanned with a Helena Laboratories (Beaumont, TX) Quick Scan densitometer.Sucrose Density Centrifugation of CAD. The buffer exchange method described by Penefsky (12) was used to remove the dimethyl sulfoxide and glycerol from CAD samples used for sucrose gradient centrifugation. The protein at 2.0-2.5 mg/ml (0.20 ml; 0.4-0.5 mg in 0.05 M Tris/0.05 M glycine, pH 8.7) was layered onto 5.0 ml of 5-20% linear sucrose gradients (Schwarz/Mann density gradient grade) in 0.05 M Tris glycine (pH 8.7) and then centrifuged for 0.5-4 hr at 189,000 x g in a Beckman L5-65 ultracentrifuge (at ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.