Isolation and immunological characterization of an iron-regulated,transformation-sensitive cell surface protein of normal rat kidney cells

We have analyzed the surface proteins of cultured normal rat kidney (NRK) cells and virus-transfromed NRK cells subjected to iron deprivation. Such a treatment specifically induces two transformation-sensitive plasma membrane-associated glycoproteins with a subunit molecular wegiht of 160,000 (160 K) and 130,000 (130 K) daltons in NRK cells. In these cells the 160 K glycoprotein is readily available to lactoperoxidase-mediated iodination, and the 130 K is apparently inaccessible to iodination. Major differences were revealed when iodinated membrane proteins of normal and virus-transformed cells subjected to iron deprivation were compared. In Kirsten sarcoma virus-transformed NRK cells the 160 K glycoprotein was weakly labeled. In two clones of simian virus 40-transformed NRK cells the 160 K glycoprotein was weakly labeled or not at all. The 130 K glycoprotein was inaccessible to iodination in all the virus-transformed cell lines. The 160 K and 130 K glycoproteins were isolated form plasma membranes of NRK cells using preparative SDS gel electrophoresis. Antibodies generated against these glycoproteins stained the external surfaces of NRK cells and induced antigen redistribution. Evidence presented suggests that 160 K and 130 K are plasma membrane-associated procollagen molecules. A possible interaction of these proteins with transferrin is also described. The data suggest that these proteins may have an important role in the sequence of events leading to transformation.     

Enhancement of hormonal stimulation in intact cells. Potentiation of GTP-dependent activation of adenylate cyclase.

The ability of prostaglandin E1 (PGE1) and cholera toxin to increase cyclic AMP levels is potentiated 6-fold when normal rat kidney (NRK) cells are treated with picolinic acid or histidinol, or grown in isoleucine-deficient medium. The response to (-)-isoproterenol is increased 2-fold in NRK cells treated with picolinic acid but not in cells subjected to isoleucine deprivation. The increase in agonist responsiveness is time-dependent, reaches its maximum at 40 h, and is quickly reversed following removal of picolinic acid or addition of medium with normal amounts of isoleucine. The cholera toxin response is also increased about 7-fold in simian virus 40-transformed NRK cells and Moloney sarcoma virus-transformed NRK cells treated with picolinic acid. GTP-stimulated, but not fluoride-stimulated, adenylate cyclase activities are increased in membranes from NRK cells treated with picolinic acid or starved for isoleucine, indicating that the increased response is due, at least in part, to a specific potentiation of GTP-dependent functions of the adenylate cyclase system. The results demonstrate that GTP-dependent events in hormonal stimulation of adenylate cyclase can be altered in intact cells to modulate hormonal enhancement of cyclic AMP production.     

Sodium-n-butyrate induces secretion and substrate accumulation of p52 in Kirsten sarcoma virus-transformed rat kidney fibroblasts

1. A group of five transformation-responsive secreted proteins, ranging in molecular mass from 31 to 70 kDa, were identified in cultured normal rat kidney (NRK) fibroblasts. 2. One such protein (p52) was found to be a major secreted and substrate-attached component of NRK cells. 3. Kirsten sarcoma virus-transformed NRK cells failed to accumulate p52 in either the secreted or substrate-associated protein compartments; this protein was inducible, however, in transformed cells by culture in 2 mM sodium-n-butyrate. 4. Kinetics of p52 induction in transformed NRK cells, relative to the time course of increased cell spreading, and its enrichment in the substrate-associated protein fraction suggest that p52 might function in cell-substrate attachment.     

Differing sensitivity of tumor cells to apoptosis induced by iron deprivation in vitro

We studied the sensitivity of tumor cells to the induction of apoptosis by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Mouse 38C13 cells and human Raji cells die within 48 and 96 h of incubation in iron-deficient medium, respectively. On the contrary, mouse EL4 cells and human HeLa cells are completely resistant to the induction of death under the same experimental arrangement. Deoxyribonucleic acid fragmentation analysis by agarose gel electrophoresis as well as flow cytometric analysis after propidium iodide staining detected in 38C13 and Raji cells, but not in EL4 and HeLa cells, changes characteristic to apoptosis. The 38C13 cells, sensitive to iron deprivation, also displayed a similar degree of sensitivity to apoptosis induction by thiol deprivation (achieved by 2-mercaptoethanol withdrawal from the culture medium) as well as by rotenone (50 nM), hydroxyurea (50 microM), methotrexate (20 nM), and doxorubicin (100 nM). Raji cells shared with 38C13 cells a sensitivity to rotenone, methotrexate, doxorubicin, and, to a certain degree, to hydroxyurea. However, Raji cells were completely resistant to thiol deprivation. EI4 and HeLa cells, resistant to iron deprivation, also displayed a greater degree of resistance to most of the other apoptotic stimuli than did their sensitive counterparts. We conclude that some tumor cells in vitro are sensitive to apoptosis induction by iron deprivation, while other tumor cells are resistant. All the tumors found to be sensitive to iron deprivation in this study (four cell lines) are of hematopoietic origin. The mechanism of resistance to apoptosis induction by iron deprivation differs from the mechanism of resistance to thiol deprivation.     

Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli

Using biological iron chelators to control specifically iron availability to Escherichia coli K-12 in conjunction with radioactive pulse-labels, we examined the biosynthesis of six iron-regulated membrane proteins. Iron deprivation induced the synthesis of five proteins, which had molecular weights of 83,000 (83K), 81K (Fep), 78K (TonA), 74K (Cir), and 25K. The kinetics of induction were the same in entA and entA⁺ strains, but were affected by the initial iron availability in the media. Iron-poor cells induced rapidly (half-time, 10 min), whereas iron-rich cells began induction after a lag and showed a slower induction half-time (30 min). Within this general pattern of induction after iron deprivation, several different kinetic patterns were apparent. The 83K, 81K, and 74K proteins were coordinately controlled under all of the conditions examined. The 78K and 25K proteins were regulated differently. The synthesis of a previously unrecognized 90K inner membrane protein was inhibited by iron deprivation and stimulated by iron repletion. Both ferrichrome and ferric enterobactin completely repressed 81K and 74K synthesis when the siderophores were supplied at concentrations of 5 μM in vivo (half-time, 2.5 min). At concentrations less than 5 μM, however, both siderophores repressed synthesis only temporarily; the duration of repression was proportional to the amount of ferric siderophore added. The half-lives of the 81K and 74K mRNAs, as measured by rifampin treatment, were 1.2 and 1.6 min, respectively. The results of this study suggest that enteric bacteria are capable of instantaneously detecting and reacting to fluctuations in the extracellular iron concentration and that they store iron during periods of iron repletion for utilization during periods of iron stress. Neither iron storage nor iron regulation of envelope protein synthesis is dependent on the ability of the bacteria to form heme.     

Properties of a sarcoma-growth-factor-like peptide from cells transformed by a temperature-sensitive sarcoma virus

Serum-free conditioned media was collected from three sarcoma virus-transformed cell lines and an untransformed cell line. All three virally transformed lines produced and released growth factors into their serum-free media. The major activity in all cases, whether the cells were transformed by Moloney sarcoma virus (MSV) or Kirsten sarcoma virus (KiSV), or whether they were mouse or rat, was a sarcoma-growth-factor (SGF)-like activity with an apparent molecular weight of 10,000. The SGF-like pools from a Moloney sarcoma virus-transformed mouse 3T3 cell and a Kirsten sarcoma virus-transformed NRK cell were further purified by carboxymethyl cellulose chromatography. The elution profiles of these peptides were very similar. The serum-free conditioned media from the untransformed cells showed no detectable growth stimulating activity. The temperature sensitivity of an SGF-like growth factor from the supernate of a NRK cell transformed by a wild-type Kirsten sarcoma virus (KiSV) was compared with that of the SGF-like activity from the supernates of a NRK cell transformed by a ts-mutant of KiSV that is temperature sensitive with respect to transformation (ts-371 Cl 5). Neither the cells transformed by the wild-type sarcoma virus nor those transformed by the temperature sensitive virus released a SGF-like activity that was temperature sensitive under the conditions of the assays.     

Iron deprivation induces apoptosis via mitochondrial changes related to Bax translocation

In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we compared cells sensitive (38C13) and resistant (EL4) to apoptosis induced by iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. We detected the activation of caspase-3 as well as the activation of caspase-9 in sensitive cells but not in resistant cells under iron deprivation. Iron deprivation led to the release of cytochrome c from mitochondria into the cytosol only in sensitive cells but it did not affect the cytosolic localization of Apaf-1 in both sensitive and resistant cells. The mitochondrial membrane potential (_m) was dissipated within 24 h in sensitive cells due to iron deprivation. The antiapoptotic Bcl-2 protein was found to be associated with mitochondria in both sensitive and resistant cells and the association did not change under iron deprivation. On the other hand, under iron deprivation we detected translocation of the proapoptotic Bax protein from the cytosol to mitochondria in sensitive cells but not in resistant cells. Taken together, we suggest that iron deprivation induces apoptosis via mitochondrial changes concerning proapoptotic Bax translocation to mitochondria, collapse of the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and caspase-3.     

A multicopper ferroxidase involved in iron binding to transferrins in Dunaliella salina plasma membranes

The halotolerant alga Dunaliella salina is unique among plants in that it utilizes a transferrin (TTf) to mediate iron acquisition (Fisher, M., Zamir, A., and Pick, U. (1998) J. Biol. Chem. 273, 17553-17558). Two new proteins that are induced by iron deprivation were identified in plasma membranes of D. salina as follows: a multicopper ferroxidase termed D-Fox and an internally duplicated glycoprotein (p130B). D-Fox and p130B are accessible to glycolytic, proteolytic, and biotin surface tagging treatments, suggesting that they are surface-exposed glycoproteins. Induction of D-Fox was also manifested by ferroxidase activity in plasma membrane preparations. These results are puzzling because ferroxidases in yeast and in Chlamydomonas reinhardtii function in redox-mediated iron uptake, a mechanism that is not known to operate in D. salina. Two lines of evidence suggest that D-Fox and p130B interact with D. salina triplicated transferrin (TTf). First, chemical cross-linking combined with mass spectroscopy analysis showed that D-Fox and p130B associate with TTf and with another plasma membrane transferrin. Second, detergent-solubilized D-Fox and p130B comigrated on blue native gels with plasma membrane transferrins. 59Fe autoradiography indicated that this complex binds Fe3+ ions. Also, the induction of D-Fox and p130B is kinetically correlated with enhanced iron binding and uptake activities. These results suggest that D-Fox and p130B associate with plasma membrane transferrins forming a complex that enhances iron binding and iron uptake. We propose that the function of D-Fox in D. salina has been modified during evolution from redox-mediated to transferrin-mediated iron uptake, following a gene transfer event of transferrins from an ancestral animal cell.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.     

Electron spin resonance of growing normal and virus-transformed cells

A g = 2.003 ESR signal, attributed to a free radical localized in HeLa cell nuclei and mitochondria but absent in membranes and cytoplasm, has been studied as a function of the culture growth cycle in normal (NRK and 3T3) and virus transformed (NRK/RSV and 3T3/SV40) cells. For both these cell pairs, the signal is higher during the "lag" stage and lower during the "growth" stage. The average specific intensity of the signal in normal cells is about twice that in virus-transformed cells. However, the maximal point of resonance during the lag state is higher in transformed cells than in normal ones. The lag stage in NRK and NRK/RSV cells is much longer than in 3T3 and 3T3/SV40 cells, while the maximal value of the g = 2.003 ESR signal occurs, early in the lag stage of 3T3 and 3T3/SV40 cells and late in the lag stage of NRK and NRK/RSV cells.     

Sensitivity of cells to apoptosis induced by iron deprivation can be reversibly changed by iron availability

We tested the effect of iron deprivation on cell death induction in human Raji cells pre-adapted to differing availability of extracellular iron. Iron deprivation was achieved by incubation in a defined iron-free medium. Original Raji cells have previously been adapted to long-term culture in a defined medium with 5 microg/ml of iron-saturated human transferrin as a source of iron. Raji/lowFe cells were derived from original Raji cells by subsequent adaptation to culture in the medium with 50 microm ferric citrate as a source of iron. Raji/lowFe-re cells were derived from Raji/lowFe cells by re-adaptation to the transferrin-containing (5 microg/ml) medium. Iron deprivation induced cell death in both Raji cells and Raji/lowFe-re cells; that is, cells pre-adapted to a near optimum source of extracellular iron (5 microg/ml of transferrin). However, Raji/lowFe cells preadapted to a limited source of extracellular iron (50 microm ferric citrate) became resistant to the induction of cell death by iron deprivation. We demonstrated that cell death induction by iron deprivation in Raji cells correlates with the activation of executioner caspase-3 and the cleavage of caspase-3 substrate, poly-ADP ribose polymerase. Two other executioner caspases, caspase-7 and caspase-6, were not activated. Taken together, we suggest that in human Raji cells, iron deprivation induces apoptotic cell death related to caspase-3 activation. However, the sensitivity of the cells to death induction by iron deprivation can be reversibly changed by extracellular iron availability. The cells pre-adapted to a limited source of extracellular iron became resistant.     

Isolation of Immunogenic and Suppressogenic Glycoproteins from Adenovirus Type 12 Hamster Tumor Cells

Two types of glycoproteins were isolated from the membrane fraction of adenovirus type 12 (Ad12) hamster tumor cells by recovering detergent-solubilized glycoproteins using concanavalin A-affinity chromatography and gel filtration. One of the glycoproteins consisted of a polypeptide of 130,000 daltons (130K) with a pI value of 4.7–5.1, and the other consisted of a polypeptide of 18,500 daltons (18.5K) with a pi value of 6.3–6.6. The glycoproteins were immunologically different. The 18.5K glycoprotein induced in vivo resistance to tumor growth and anti-tumor cytotoxic T cells, while the 130K glycoprotein induced in vivo suppressor T cells which inhibited the activity of anti-tumor cytotoxic T cells.     

Modification of the calcium-independent mechanism of cell adhesion in transformed BHK cells

The calcium-independent mechanism of cell adhesion was studied in normal and polyoma virus-transformed BHK cells. The degree of Ca2+-independent adhesion was greatly reduced in pyBHK cells, whereas CA2+-dependent adhesion occurred to the same degree as in BHK cells. This decrease was shown not to be caused by simple masking of the adhesion sites or by their altered sensitivity to trypsin. Adhesion-blocking antibodies were used to identify molecules responsible for Ca2+-independent adhesion. The antibodies precipitated surface molecules specific for adhesion-competent cells. These have tentatively been named CIDSBHK and CIDSpyBHK. Both were glycoproteins with respective apparent molecular weights of 120K and 125K. CIDSpyBHK incorporated 3H-glucosamine more than CIDSBHK did. Possible modification of the Ca2+-independent adhesion mechanism in pyBHK cells is discussed.     

Iron deprivation induces apoptosis independently of p53 in human and murine tumour cells

Iron deprivation induces apoptosis in some sensitive cultured tumour cells, while other cells are resistant. In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we studied the expression of p53 and the expression of selected p53-regulated genes. To discriminate between changes coupled only with iron deprivation and changes involved in apoptosis induction by iron deprivation, we compared the expression of the genes in sensitive (human Raji, mouse 38C13) versus resistant (human HeLa, mouse EL4) cells under iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. The level of p53 mRNA decreased significantly under iron deprivation in sensitive cells, but it did not change in resistant cells. On the contrary, the level of the p53 protein under iron deprivation was slightly increased in sensitive cells while it was not changed in resistant cells. The activity of p53 was assessed by the expression of selected p53-regulated targets, i.e. p21(WAF1/CIP1) gene, mdm2, bcl-2 and bax. We did not detect any relevant change in mRNA levels as well as in protein levels of these genes under iron deprivation with the exception of p21(WAF1/CIP1). We detected a significant increase in the level of p21 mRNA in both (sensitive and resistant) mouse cell lines tested, however, we did not find any change in both (sensitive and resistant) human cell lines. Moreover, the p21(WAF1/CIP1) protein was accumulated in mouse-sensitive 38C13 cells under iron deprivation while all other cell lines tested, including human-sensitive cell line Raji, did not show any accumulation of p21(WAF1/CIP1) protein. It seems that the p21(WAF1/CIP1) mRNA, as well as protein accumulation, is not specifically coupled with apoptosis induction by iron deprivation and that it is rather cell-line specific. Taken together, we suggest that iron deprivation induces apoptosis at least in some cell types independently of the p53 pathway.     

The effect of cyclic AMP on the growth and morphology of a normal human fibroblast parent strain and its transformed progeny line.

A normal human diploid fibroblast cell strain, Lederle 130 (Led 130), and its virus-transformed progeny line, transformed Led 130, were subjected to 0.75 and 1.5 mM concentrations of adenosine-5'-monophosphate (AMP), cyclic AMP (cAMP) and dibutyryl cyclic AMP (Bt2cAMP). While cAMP was markedly inhibitory to neoplastic cells at 1.5 mM, Bt2-cAMP was even more effective at this concentration, producing 85% inhibition by 4 days and 91% inhibition by 6 days. Bt2-cAMP was the only nucleotide to reverse morphological transformation effects in the neoplastic fibroblasts. Normal fibroblasts were inhibited in growth rate to a comparable extent by all nucleotides, and were not altered morphologically.     

Iron induces Bcl-2 expression in human dermal microvascular endothelial cells

Iron is suspected to be involved in the induction and/or progression of various human tumors. The present study was designed to investigate the effects of iron on endothelial cells, keeping in mind that the homeostasis of microvessels plays a critical role in neo-angiogenesis. Applying a model of human dermal microvascular endothelial cell terminal differentiation and death induced by serum deprivation, we found that iron salts (iron chloride and ferric nitrilotriacetate) provided a survival advantage to endothelial cells. Using immunohistochemistry and Western Blot analysis, we found that the extended cellular life span induced by iron was paralleled by an increase of Bcl-2 protein expression. Taken together, these observations suggest that iron may give a survival advantage to endothelial cells and represent a novel mechanism through which iron may contribute to tumorigenesis.     

VDAC2 and aldolase A identified as membrane proteins of K562 cells with increased expression under iron deprivation

We have shown previously that iron deprivation significantly stimulates the uptake of non-transferrin ferric iron from ferric citrate by erythroleukemia K562 cells and that this stimulation depends on protein synthesis. However, we have not detected increased expression of any known iron transport protein (Kovar J. et al. (2006) Blood Cells Mol Dis 37:95–99). Therefore, in order to identify membrane proteins of K562 cells with increased expression under iron deprivation, we employed the isolation of membrane proteins by two-phase partitioning system, protein separation by high-resolution 2D electrophoresis, computer differential analysis, and tandem mass spectrometry. Employing these techniques we identified two proteins with statistically significant upregulation, i.e., aldolase A (ALDA) and voltage-dependent anion channel 2 (VDAC2). The upregulation of aldolase A and VDAC2 in K562 cells under iron deprivation was also confirmed by western blot analysis. This is the first time when the control of aldolase A and VDAC2 levels by iron status of the cell is demonstrated.     

Transforming growth factor-beta overrides the adhesion requirement for surface expression of alpha(5)beta(1) integrin in normal rat kidney fibroblasts. A necessary effect for induction of anchorage-independent growth.

We have previously shown that the expression of alpha(5)beta(1) integrin on the cell surface is dependent upon cell adhesion to the extracellular matrix, and we report here that transforming growth factor-beta (TGF-beta) overcomes this requirement in normal rat kidney (NRK) fibroblasts. Thus, suspended NRK cells treated with TGF-beta show levels of surface alpha(5)beta(1) integrin that are equivalent to those seen in adherent cells. Moreover, several experiments showed that this effect is necessary for the induction of anchorage-independent growth by TGF-beta. First, a kinetic analysis showed that surface expression of alpha(5)beta(1) integrin was restored in TGF-beta-treated NRK cells prior to the induction of anchorage-independent growth. Second, NRK cell mutants that have lost their TGF-beta requirement for surface expression of alpha(5)beta(1) integrin were anchorage-independent in the absence of TGF-beta. Third, an antisense oligonucleotide to the beta(1) integrin subunit or, fourth, stable expression of an alpha(5)-antisense cDNA blocked the ability of TGF-beta to stimulate anchorage-independent growth. Thus, TGF-beta overrides the adhesion requirement for surface expression of alpha(5)beta(1) integrin in NRK cells, and this effect is necessary for the induction of anchorage-independent growth.     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.