Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of l-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an ≈2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.

Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09–0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-{N-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to ≈40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.

Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver

Cholesterol feeding reduces the mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway and the low density lipoprotein receptor in livers of hamsters. Here we show that cholesterol feeding also reduces the levels of the nuclear NH2-terminal domains of sterol regulatory element binding proteins (SREBPs), which activate transcription of sterol-regulated genes. We show that livers of hamsters, like those of mice and humans, predominantly produce SREBP-2 and the 1c isoform of SREBP-1. Both are produced as membrane-bound precursors that must be proteolyzed to release the transcriptionally active NH2-terminal domains. Diets containing 0.1% to 1.0% cholesterol decreased the amount of nuclear SREBP-1c without affecting the amount of the membrane precursor or its mRNA, suggesting that cholesterol inhibits the proteolytic processing of SREBP-1 in liver as it does in cultured cells. Cholesterol also appeared to reduce the proteolytic processing of SREBP-2. In addition, at high levels of dietary cholesterol the mRNA encoding SREBP-2 declined and the amount of the precursor also fell, suggesting that cholesterol accumulation also may inhibit transcription of the SREBP-2 gene. The high-cholesterol diets reduced the amount of low density lipoprotein receptor mRNA by 30% and produced a more profound 70–90% reduction in mRNAs encoding 3-hydroxy-3-methylglutaryl CoA synthase and reductase. Treatment with lovastatin and Colestipol, which increases hepatic demands for cholesterol, increased the amount of SREBP-2 mRNA as well as the precursor and nuclear forms of the protein. This treatment caused a reciprocal decline in SREBP-1c mRNA and protein. Considered together, these data suggest that SREBPs play important roles in controlling transcription of sterol-regulated genes in liver, as they do in cultured cells.

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool—as in speciation—aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

Arrest of neuronal migration by excitatory amino acids in hamster developing brain

The influence of the excitotoxic cascade on the developing brain was investigated using ibotenate, a glutamatergic agonist of both N-methyl-d-aspartate (NMDA) ionotropic receptors and metabotropic receptors. Injected in the neopallium of the golden hamster at the time of production of neurons normally destined for layers IV, III, and II, ibotenate induces arrests of migrating neurons at different distances from the germinative zone within the radial migratory corridors. The resulting cytoarchitectonic patterns include periventricular nodular heterotopias, subcortical band heterotopias, and intracortical arrests of migrating neurons. The radial glial cells and the extracellular matrix are free of detectable damage that could suggest a defect in their guiding role. The migration disorders are prevented by coinjection of dl-2-amino-7-phosphoheptanoic acid, an NMDA ionotropic antagonist, but are not prevented by coinjection of l(+)-2-amino-3-phosphonopropionic acid, a metabotropic antagonist. This implies that an excess of ionic influx through the NMDA channels of neurons alters the metabolic pathways supporting neuronal migration. Ibotenate, a unique molecular trigger of the excitotoxic cascade, produces a wide spectrum of abnormal neuronal migration patterns recognized in mammals, including the neocortical deviations encountered in the human brain.

Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure

Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase Vmax of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg2+, these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N-phosphonacetyl-l-aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.

Structure of the recombinant full-length hamster prion protein PrP(29–231): The N terminus is highly flexible

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrPC to the infectious scrapie form PrPSc; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29–231 [PrP(29–231)] is investigated by multidimensional heteronuclear NMR. Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29–231) contains three helices and possibly one short β-strand. Most striking is the random-coil nature of chemical shifts for residues 30–124 in the full-length PrP. Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121–231), the secondary structure boundaries of PrP(29–231) are different from those in mouse PrP(121–231) but similar to those found in the structure of Syrian hamster PrP(90–231). Comparison of resonance assignments of PrP(29–231) and PrP(90–231) indicates that there may be transient interactions between the additional residues and the structured core. Backbone dynamics studies done by using the heteronuclear [1H]-15N nuclear Overhauser effect indicate that almost half of PrP(29–231), residues 29–124, is highly flexible. This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of β-structure.

Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, δ-sarcoglycan, in hamster: An animal model of disrupted dystrophin-associated glycoprotein complex

Cardiomyopathy (CM) is a primary degenerative disease of myocardium and is traditionally categorized into hypertrophic and dilated CMs (HCM and DCM) according to its gross appearance. Cardiomyopathic hamster (CM hamster), a representative model of human hereditary CM, has HCM and DCM inbred sublines, both of which descend from the same ancestor. Herein we show that both HCM and DCM hamsters share a common defect in a gene for δ-sarcoglycan (δ-SG), the functional role of which is yet to be characterized. A breakpoint causing genomic deletion was found to be located at 6.1 kb 5′ upstream of the second exon of δ-SG gene, and its 5′ upstream region of more than 27.4 kb, including the authentic first exon of δ-SG gene, was deleted. This deletion included the major transcription initiation site, resulting in a deficiency of δ-SG transcripts with the consequent loss of δ-SG protein in all the CM hamsters, despite the fact that the protein coding region of δ-SG starting from the second exon was conserved in all the CM hamsters. We elucidated the molecular interaction of dystrophin-associated glycoproteins including δ-SG, by using an in vitro pull-down study and ligand overlay assay, which indicates the functional role of δ-SG in stabilizing sarcolemma. The present study not only identifies CM hamster as a valuable animal model for studying the function of δ-SG in vivo but also provides a genetic target for diagnosis and treatment of human CM.

Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells

We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.

High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.

Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes

CD22 is a B cell-restricted glycoprotein involved in signal transduction and modulation of cellular activation. It is also an I-type lectin (now designated Siglec-2), whose extracellular domain can specifically recognize α2–6-linked sialic acid (Sia) residues. This activity is postulated to mediate intercellular adhesion and/or to act as a coreceptor in antigen-induced B cell activation. However, studies with recombinant CD22 indicate that the lectin function can be inactivated by expression of α2–6-linked Sia residues on the same cell surface. To explore whether this masking phenomenon affects native CD22 on B cells, we first developed a probe to detect the lectin activity of recombinant CD22 expressed on Chinese hamster ovary cells (which have no endogenous α2–6-linked Sia residues). This probe is inactive against CD22-positive B lymphoma cells and Epstein–Barr virus-transformed lymphoblasts which express high levels of α2–6-linked Sia residues. Enzymatic desialylation unmasks the CD22 lectin activity, indicating that endogenous Sia residues block the CD22 lectin-binding site. Truncation of the side chains of cell surface Sia residues by mild periodate oxidation (known to abrogate Sia recognition by CD22) also had this unmasking effect, indicating that the effects of desialylation are not due to a loss of negative charge. Normal resting B cells from human peripheral blood gave similar findings. However, the lectin is partially unmasked during in vitro activation of these cells. Thus, the lectin activity of CD22 is restricted by endogenous sialylation in resting B cells and may be transiently unmasked during in vivo activation, perhaps to modulate intercellular or intracellular interactions at this critical stage in the humoral response.