A comparison of the interaction of Chinese hamster fibroblasts with human and bovine transferrins, and conalbumin (chick-egg transferrin)

The iron requirement of a cell line of Chinese hamster fibroblasts is met more efficiently by human transferrin than by bovine transferrin or conalbumin. One possible explanation is that the binding of these transferrins to the Chinese hamster V79 cells may differ. Binding studies now show that the affinity of V79 cells for human transferrin is about 40 times greater than for bovine transferrin. Conalbumin has no detectable affinity for the human transferrin binding sites. Human apotransferrin has approximately one-sixth the affinity for the transferrin binding sites. The binding constant for the relation of human transferrin with the V79 cell is about 2.3·10⁶1· mole⁻¹, and the approximate number of binding sites per cell is 9 · 10⁵.     

Comparative aspects of transferrin-reticulocyte interactions: membrane receptors and iron uptake

1. A comparative study was made of transferrin and iron uptake by rabbit, rat and human reticulocytes and chick embryo erythrocytes from rabbit, rat, human, chicken and porcine transferrins, human lactoferrin and chicken conalbumin. 2. Three methods were used, viz. direct and competitive uptake studies of transferrin and iron by the four species of cells, and competitive studies of transferrin binding by solubilized membrane receptors (rabbit reticulocytes only). 3. Methods were devised to analyse the data so as to obtain indices of relatedness or relative affinities of each type of heterologous transferrin in rates of iron uptake found with transferrin and cells from various species are largely due to variation in the affinity of cellular receptors for different transferrins. 5. It is concluded that the procedure used in this investigation allow the assessment of phylogenetic relationships and evolutionary trends obtained by structural studies of proteins.     

The effect of heterologous transferrin on the uptake of iron and haem synthesis by bone marrow cells

The initial process of transfer of extracellular iron to the haem-synthesizing mitochondria of immature erythroid cells is the association of iron-transferrin with the cell membrane. When rat bone marrow cells were incubated in the presence of iron bound to rat transferrin, iron uptake was higher than in the presence of iron bound to heterologous transferrin. The relative activities of the various isolated transferrins towards rat transferrin were found to be approximately 0.3, 0.8, 0.1 and 0.04 for rabbit, human, bovine and fish (tench, Tinca tinca) transferrin, respectively, and 0.7, 0.7 and 0.15 for mouse, guinea pig and calf serum, respectively, as compared with rat serum. Although great difference exist in cellular uptake of iron bound to different species of transferrin, the subcellular distribution of ⁵⁹Fe was quite similar. In all cases about 60% of the radioactivity taken up by the cells could be recovered in the haemin fraction and only about 15% in each the membrane and the non-haem soluble cell fraction. Similar results were obtained with guinea pig bone marrow cells.From the results of the experiments presented it might be concluded that the species of transferrin plays an important role during the initial stages of iron uptake by bone marrow cells, whereas the intracellular iron transfer process is not influenced by the species of transferrin.     

A structural comparison of human serum transferrin and human lactoferrin

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.     

A comparison of the structure and properties of human, rat and rabbit serum transferrin

1. The chemical and physical properties of human, rat and rabbit serum transferrins were compared. 2. The proteins were found to differ in heat stability, iron release, their behaviour during electrophoresis in SDS polyacrylamide gels, and their sulphur amino acid content. 3. The results are discussed in relation to the possible role of disulphide bridges in maintaining the shape and flexibility of the three transferrins, and in their appearance during the evolution of the transferrin family.     

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 chinese hamster cells

Summary A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed “growth inhibiting factor” (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity CIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.     

The nucleotide sequence of rabbit liver transferrin cDNA

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.     

Effects of iron binding agents on Saccharomyces cerevisiae growth and cytochrome P450 content

The yeast Saccharomyces cerevisiae Y222 was studied in the presence of the following iron-binding agents: Desferal, dipyridyl, and human and bovine transferrins. We report that cell growth and lanosterol 14 alpha-demethylase cytochrome P450 are not affected by Desferal but that dipyridyl and serum transferrins decrease the cytochrome P450 content of the yeast. Paradoxically, while both human and bovine transferrins reduce cytochrome P450 content, only bovine transferrin appears to affect cell growth in this strain. No evidence for siderophore production by this strain was found under low iron conditions.     

Specificity of hepatic iron uptake from plasma transferrin in the rat.

1. The role of specific interaction between transferrin and its receptors in iron uptake by the liver in vivo was investigated using 59Fe-125I-labelled transferrins from several animal species, and adult and 15-day rats. Transferrin-free hepatic uptake of 59Fe was measured 2 or 0.5 hr after intravenous injection of the transferrins. 2. Rat, rabbit and human transferrins gave high and approximately equal levels of hepatic iron uptake while transferrins from a marsupial (Sentonix brachyurus), lizard, crocodile, toad and fish gave very low uptake values. Chicken ovotransferrin resulted in higher uptake than with any other species of transferrin. 3. Iron uptake by the femurs (as a sample of bone marrow erythroid tissue) and, in another group of 19-day pregnant animals by the placentas and fetuses, was also measured, for comparison with the liver results. The pattern of uptake from the different transferrins was found to be similar to that of iron uptake by the liver except that with femurs, placentas and fetuses ovotransferrin gave low values comparable to those of the other non-mammalian species. 4. It is concluded that iron uptake by the liver from plasma transferrin in vivo is largely or completely dependent on specific transferrin-receptor interaction. The high hepatic uptake of iron from ovotransferrin was probably mediated by the asialoglycoprotein receptors on hepatocytes.     

The effect of hexose on chloramphenicol sensitivity and resistance in Chinese hamster cells.

The toxicity and extent of growth inhibition produced by chloramphenicol (CAP) in CAP^s Chinese hamster cells (line V79-5) was found to be dependent on the type and concentration of hexose in the medium. In high levels of glucose (6.5 mM), cultures of CAP^s cells underwent 7–8 population doublings in the presence of 100 μg/ml CAP and viability then dropped rapidly. In contrast, lower concentrations of glucose (1.0 mM) permitted only limited growth (2–3 doublings) in the presence of 100 μg/ml CAP, but the cells remained viable and apparently quiescent for prolonged periods of time. The growth potential of V79-5 cells in CAP appeared specifically dependent on glucose, as mannose and galactose could not substitute for glucose. The toxicity of CAP to these cells seemed to be determined primarily by the number of cell doublings in the presence of the drug. A CAP^R derivative of V79-5, designated 5-3, was analyzed in order to determine whether the requirement for glucose for cell growth in the presence of CAP also occurred in cells that were isolated as resistant to the drug. In order to rigorously control the hexose in the medium, some experiments were performed with medium containing dialysed, instead of whole, fetal calf serum. It was seen that the growth of the CAP^R line in the presence (but not the absence) of 100 μg/ml CAP was dependent on glucose in the medium. Thus, resistance to CAP in these cells appears to be a conditional state, dependent on glucose for expression. Furthermore, the glucose auxotrophy of these cells in the presence of CAP suggests that CAP is still affecting some activities in cells isolated as CAP^R.     

Iron uptake by Chinese hamster fibroblasts from human transferrin

The manner of uptake or iron by Chinese hamster fibroblasts, type DON, from human transferrin was investigated by means of replacement studies, in which the cells that were incubated with ¹²⁵I-labelled human transferrin were chased with non-radioactive transferrin for only a few minutes. The results did not support the reversible endocytosis hypothesis for the uptake of iron from transferrin. The uptake of iron measured as ⁵⁹Fe during several cell divisions was found to be a function of time and cell number. It was found that the total uptake of iron in the harvests was directly proportional to the incubation, and that the uptake per 10⁶ cells levelled off in the course of time.     

Iron metabolism pathways in the rat hepatocyte

A small to moderate inhibitory effect of iron uptake by isolated rat hepatocytes in short-term studies was seen with oxidative phosphorylation and electron transport inhibitors, and no inhibition by agents affecting pinocytosis. Intracellular transferrin was able to donate iron to the small-molecular weight iron pool, and the latter was able to transfer, by a process not requiring energy or movement of serum transferrin, iron to ferritin. Serum transferrin was not able to lose iron to any cytosol components. Reducing agents were not able to abstract iron from rat serum transferrin to any great extent. It is concluded that iron is taken up by the rat hepatocyte from serum transferrin by a process not requiring energy or movement of serum transferrin into the cell interior; and that intracellular transferrin is involved in acquiring iron from serum transferrin at the cell surface, with iron then being transferred to the small-molecular weight iron pool and hence to ferritin. It is also proposed that intracellular transferrins may have the general function of interacting with serum transferrin at cell surfaces.     

Induction of micronuclei by 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in Chinese hamster V79MZh1A1 cells

The clastogenicity/aneugenicity of N-heterocyclic polycyclic aromatic pollutant 7H-dibenzo[c,g]carbazole (DBC) and its two synthetic derivatives N-methyl DBC (MeDBC) and 5,9-dimethyl DBC (diMeDBC) was evaluated in the genetically engineered Chinese hamster V79 cell line V79MZh1A1 with stable expression of human cytochrome P4501A1 and in the parental V79MZ cell line without any cytochrome P450 activity. While none of the three carbazoles changed significantly the level of micronuclei in the parental V79MZ cells, a variable, but statistically significant rise of micronucleus frequencies was assessed in V79MZh1A1 cells. DBC induced dose-dependent increase in the number of micronuclei at harvest times of 24 and 48h and MeDBC at sampling time of 48h in V79MZh1A1 cells in comparison to untreated cells, however, no significant time-dependent increase in micronucleus frequencies was found. The use of the antikinetochore immunostaining revealed that DBC and MeDBC induced approximately equal levels of both kinetochore positive (C+) and kinetochore negative (C-) micronuclei. DiMeDBC, a strict hepatocarcinogen, did not manifest any effect on micronucleus induction in V79MZh1A1 cells.These studies suggest that genetically engineered Chinese hamster V79 cell lines expressing individual CYP cDNAs are a useful in vitro model for evaluation the role of particular cytochromes P450 in biotransformation of DBC and its tissue and organ specific derivatives.     

转铁蛋白结构与功能的研究——骆驼血清转铁蛋自的分离纯化及其含单一铁结合部位的结构域的制备和鉴定

分离纯化获得的骆驼血清转铁蛋白由分子量为73,000和63,000两个组分组成。两者至少N-端五肽顺序相同(Met-Pro-Asp-Lys-Thr)。骆驼血清转铁蛋白在生理pH下不能与人胎盘转铁蛋白受体结合。用胰蛋白酶酶解骆驼转铁蛋白可以同时得到两个合单一铁结合部位的结构域,分别来自转铁蛋白分子的N-端称N-端结构域(分子量34,700和40,700)和C-端称C-端结构域(分子量35,100)。在上述结果的基础上指出并讨论了反刍动物转铁蛋白在结构和功能上存在更多的共同性,而与其它哺乳动物的转铁蛋白有着明显的区别。     

A genetic analysis of the adenine phosphoribosyl transferase locus in Chinese hamster V79-AP4 cells: relevance to mutagenesis studies

The chromosomal location of the autosomal locus aprt has been investigated in the permanent Chinese hamster cell line V79-AP4 by standard somatic cell genetics methodologies. Aprt is functionally dizygous in V79-AP4 and the 2 alleles map on 2 chromosome 3 homologs, in agreement with the chromosome assignment of the gene in Chinese hamster primary cells. Chromosome G-banding and a Southern blot analysis of V79-AP4 DNA, using as a probe the cloned Chinese hamster aprt gene, have not revealed any structural alteration at either of the 2 aprt alleles. One of the chromosomes 3 has, however, a terminal deletion in its long arm and is therefore morphologically marked. These findings could make V79-AP4 an interesting cell system for the study of mutational mechanisms at the aprt locus in Chinese hamster.     

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin.

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin has been studied by analyzing the distribution of iron between the chelator and the proteins as a function of both ligand concentration and transferrin saturation. The kinetics of iron removal by 3-hydroxypyridin-4-ones from both transferrins is slow; in ovotransferrin it appears to be monophasic, in contrast to that observed for serum transferrin. After 24 hours incubation at a 40:1 chelator:protein molar ratio, the percentage of iron removed from Fe(III)-ovotransferrin is 50%-60%, and is somewhat higher in the case of serum transferrin, in line with the respective affinity constants for the metal. The 3-hydroxypyridin-2-ones and the 3-hydroxypyran-4-ones, both of which have lower affinities for Fe(III), remove smaller proportions of the metal. The percentage of desaturation obtained with bidentate and hexadentate pyridinones appears to be similar for both transferrin classes at chelator:protein molar ratios from 40:1. The degree of transferrin saturation influences the extent of chelator mediated iron mobilization in the case of serum transferrin, but not of ovotransferrin. 59Fe competition studies demonstrate that bidentate pyridin-4-ones are capable of donating iron to serum apotransferrin; the relative concentrations of ligand and protein influence the distribution of iron because their effective binding constants (at pH 7.4) for Fe(III) are similar.     

The action of AF 2 on cultured hamster and human cells under aerobic and hypoxic conditions

AF 2 (2-(2-furyl)-3-(5-nitro-furyl)acrylamide) was toxic to Chinese hamster V 79 cells and normal human fibroblasts in aerobic media. However, the toxicity of the drug was increased many times by hypoxia. Similarly, the frequency of AF 2-induced azaguanine- and ouabain-resistant mutants of V 79 cells was much higher in hypoxia than under aerobic conditions. Both hamster V 79 cells and human fibroblasts metabolized AF 2 and other nitrofurans rapidly only under hypoxic conditions. Human fibroblasts were more sensitive to AF 2 both under aerobic conditions and in hypoxia than were V 79 cells under similar conditions. The Chinese hamster cells consistently gave survival curves with marked shoulders while human cells did not. Aerobic cultures of fibroblasts derived from xeroderma pigmentosum (XP) patients were markedly sensitive to AF 2 while fibroblasts from two ataxia telangeictasia patients had normal sensitivity. Under hypoxic conditions the sensitivity of both types of cells was increased but the XP line remained 5--10-fold more sensitive than normal or ataxia cells. These results suggest that the DNA lesions produced by AF 2 may be regarded as similar to those produced by ultraviolet light, at least in terms of their repairability in human cells.     

Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro.

Following suggestions that transferrin present in fetal-bovine serum, a common supplement used in tissue-culture media, may not bind well to human cells, we have isolated the protein and investigated its interaction with both human and bovine cells. Bovine transferrin bound to a human cell line, K562, at 4 degrees C with a kd of 590 nM, whereas human transferrin bound with a kd of 3.57 nM, a 165-fold difference. With a bovine cell line, NBL4, bovine transferrin bound with the higher affinity, kd 9.09 nM, whereas human transferrin bound with a kd of 41.7 nM, only a 5-fold difference. These values were reflected in an 8.6-fold difference in the rate of iron delivery by the two proteins to human cells, whereas delivery to bovine cells was the same. Nevertheless, the bovine transferrin was taken up by the human cells by a specific receptor-mediated process. Human cells cultured in bovine diferric transferrin at 40 micrograms/ml, the concentration expected in the presence of 10% fetal-bovine serum, failed to thrive, whereas cells cultured in the presence of human transferrin proliferated normally. These results suggest that growth of human cells in bovine serum could give rise to a cellular iron deficiency, which may in turn lead to the selection of clones of cells adapted for survival with less iron. This has important consequences for the use of such cells as models, since they may have aberrant iron-dependent pathways and perhaps other unknown alterations in cell function.     

Comparison of amphibian whole-cell and cell-free activity towards different transferrins.

1. A cell-free preparation from immature-erythrocytes of the bullfrog (Rana catesbeiana) was about equally active towards iron bound to homologous, rabbit or human transferrin. 2. In contrast with the cell-free system, whole immature erythrocytes exhibited optimum, intermediate or low uptake of iron from the above and from other transferrins.     

Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyltransferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts

The results presented in this communication demonstrate that hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA can be expressed in both Chinese hamster and human fibroblasts deficient in the endogenous gene product at levels permitting normal growth of the transformants. All the elements necessary for this expression are present in a pBR322-derived plasmid containing HPRT cDNA coding sequence and a retroviral long terminal repeat. These molecules function in both species investigated and, at least in the case of the Chinese hamster transformants, are efficient at the single copy level. Although the effects of the presence of intron sequences and a polyadenylation signal within the plasmids have yet to be evaluated, these studies demonstrate that neither is an absolute requirement for expression of HPRT cDNA sequences in cultured mammalian cells. We describe the construction of recombinant plasmids containing wild type human or Chinese hamster HPRT cDNA sequences in tandem with a retroviral LTR which confer the HPRT+ phenotype in HPRT-deficient V79 and Lesch-Nyhan fibroblasts. Both stable and unstable transformants, that expressed HPRT mRNA and protein, were isolated at high frequency.