Increase in nitrosourea resistance in mammalian cells by retrovirally mediated gene transfer of bacterial O6-alkylguanine-DNA alkyltransferase.

Maloney murine leukemia virus-based, replication-defective retroviral vectors containing the neomycin resistance gene (neo) were developed to transfer the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase, into mammalian cells. To optimize gene transfer and expression, the following promoters were linked to ada: the Maloney murine leukemia virus promoter within the long-terminal repeat, the Rous sarcoma virus promoter, the thymidine kinase promoter, or the human phosphoglycerate kinase promoter. Sequences were transfected into the helper virus-free retroviral packaging psi-2 cell line. Recombinant retroviruses were tested in CCL-1 cells, which, like most murine tissues, have low levels of alkyltransferase and are sensitive to 1,3-bis(2-chloroethyl)nitrosourea (BCNU), and in NIH-3T3 cells, which are BCNU resistant and have high levels of alkyltransferase. Lines infected with each of the four retroviruses were selected for neo expression and found to have intact proviral integration and ada gene expression. Alkyltransferase activity was greatest with retrovirus containing the Rous sarcoma virus-ada gene; infected NIH-3T3 cells had up to 2300 units of alkyltransferase/mg of protein compared with 151 units/mg of protein in control cells, and infected CCL-1 cells had up to 1231 units/mg of protein compared with 33 units/mg of protein in control cells. CCL-1 cells expressing ada were more resistant to BCNU cytotoxicity than were controls. However, NIH-3T3 cells expressing ada were only slightly more resistant to BCNU than controls, possibly because most of the ada protein was cytoplasmic rather than nuclear as suggested by immunohistochemical stain. These studies establish a series of retroviruses containing the bacterial ada gene, which efficiently infect mammalian cells. ada expression increases nitrosourea resistance in cells with low mammalian alkyltransferase activity.

High level, regulated expression of the chimeric P-enolpyruvate carboxykinase (GTP)-bacterial O6-alkylguanine-DNA alkyltransferase (ada) gene in transgenic mice.

Transgenic animals expressing genes capable of repairing DNA may be a valuable tool to study the effect of DNA-damaging agents on tissue-specific carcinogenesis. For this reason, we constructed a chimeric gene consisting of the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) gene linked to the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase and the polyadenylate region from the bovine growth hormone gene. The PEPCK promoter results in gene expression in liver and kidney and is induced by hormones, and its transcription is regulated by diet. The chimeric PEPCK ada gene was injected into the male pronucleus of fertilized eggs to produce transgenic mice. Six of 65 developing mice contained 5-10 copies of the intact trans gene per genome. Two founders transmitted the trans gene in a heterozygous manner, whereas 3 transmitted as germ line mosaics and 1 did not transmit to F1 offspring. All F1 offspring carrying the PEPCK ada trans gene expressed ada mRNA in liver and kidney and produced a functional alkyltransferase with a protein molecular weight of 39,000 originating from the bacterial gene. Total alkyltransferase activity was increased in the liver of F1 offspring from all founder mice, but offspring of only one founder had elevated renal alkyltransferase levels. A diet high in protein markedly increased ada mRNA and alkyltransferase activity within 1 week in both liver and kidney, whereas a high carbohydrate diet for 1 week markedly reduced expression of PEPCK ada and alkyltransferase levels. Nontransgenic animals were unaffected by these dietary manipulations. During induction with a high protein diet, hepatic alkyltransferase in transgenic mice was 16.6 +/- 1.5 units/micrograms DNA (mean +/- SE) compared to 5.3 +/- 0.6 units/micrograms DNA in control animals. This level of alkyltransferase is higher than that in any mammalian tissue noted previously except human liver. Transgenic animals expressing high levels of alkyltransferase should help define the role of DNA repair in protection from carcinogenesis induced by N-nitroso compounds.

Inhibition of breast cancer invasion by TIS21/BTG2/Pc3-Akt1-Sp1-Nox4 pathway targeting actin nucleators, mDia genes.

The mammalian homolog of Drosophila diaphanous (mDia), actin nucleator, has been known to participate in the process of invasion and metastasis of cancer cells via regulating a number of actin-related biological processes. We have previously reported that tumor suppressor TIS21(/BTG2/Pc3) (TIS21) inhibits invadopodia formation by downregulating reactive oxygen species (ROS) in MDA-MB-231 cells. We herein report that TIS21(/BTG2/Pc3) downregulates diaphanous-related formin (DRF) expression via reducing NADPH oxidase 4 (Nox4)-derived ROS generation by Akt1 activation and subsequently impairs invasion activity of the highly invasive breast cancer cells. Knockdown of Akt1 by RNA interference recovered the TIS21(/BTG2/Pc3)-inhibited F-actin remodeling and ROS generation by recovering Nox4 expression. Furthermore, Sp1-mediated Nox4 transcription was downregulated by TIS21(/BTG2/Pc3)-Akt1 signals, leading to the inhibition of cancer cell invasion via F-actin remodeling by mDia genes. To our best knowledge, this is the first study to show that TIS21(/BTG2/Pc3)-Akt1 inhibited Sp1-Nox4-ROS cascade, subsequently reducing invasion activity via inhibition of mDia family genes.

Biological methylation of myelin basic protein: enzymology and biological significance.

Myelin is a membrane characteristic of the nervous tissue and functions as an insulator to increase the velocity of the stimuli being transmitted between a nerve cell body and its target. Myelin isolated from human and bovine nervous tissue is composed of approximately 80% lipid and 20% protein, and 30% of the protein fraction constitutes myelin basic protein (MBP). MBP has an unusual amino acid at Res-107 as a mixture of NG-monomethylarginine and NG, N'G-dimethylarginine. The formation of these methylarginine derivatives is catalysed by one of the subtypes of protein methylase I, which specifically methylates Res-107 of this protein. Evidence is presented to demonstrate an involvement of this biological methylation in the integrity and maintenance of myelin.

Expression of rat BTG(3) gene, Rbtg3, is regulated by redox changes.

The Rbtg3 gene was isolated by PCR (polymerase chain reaction) cloning from the cDNA library of Rat1 fibroblasts that were stimulated with TPA (12-O-tetradecanoylphorbol-13-acetate) or various growth factors for 3h and was found to be a rat homologue of mouse BTG3 and human ANA genes. The Rbtg3 gene had unique DNA sequences in the 5'-UTR and 3'-UTR that contained four ATTTA and one TTATTTA(T/A)(T/A) nonamer motif, and also a polyA addition site. Nucleotide homology of Rbtg3 with BTG3 and ANA was 88.5 and 76.6%, respectively. Expression of Rbtg3 was investigated in SD rats as well as cell lines derived from mouse--SW3T3, NIH3T3 fibroblasts--and rat--Rat1, 3Y1 fibroblasts and PC12--cells. Rbtg3 was highly expressed in brain but barely in lung, kidney, thymus and spleen. The constitutive expression level was high in SW3T3, Rat1 and 3Y1 fibroblasts, but very low in NIH3T3 fibroblast and PC12 cells. However, in all cells tested, Rbtg3 was proved to be one of the primary response genes superinduced by TPA (50ng/ml)+cycloheximide (CHX, 10 microgram/ml). Expression of Rbtg3 was induced by H(2)O(2) (500mM) up to fourfold in PC12 cells and was blocked by pretreatment of NAC (N-acetyl-L-cysteine, 10mM). The induction was ninefold in 3Y1 fibroblasts by menadione (25mM) treatment for 1h, whereas it was reduced to a third of the control level in SW3T3 fibroblast by the same treatment. Rbtg3 was not expressed in NIH3T3 cells but minimally regulated by redox changes as compared with rapid and strong induction of TIS21/BTG2 mRNAs after TPA or H(2)O(2) stimulation. The above results indicate that Rbtg3 is one of many redox-regulated genes as well as a primary response gene.

Cytoplasmic retention of p-Erk1/2 and nuclear accumulation of actin proteins during cellular senescence in human diploid fibroblasts.

In order to investigate the role of signal transduction and the related changes of actin cytoskeleton organization in the process of cellular senescence, H-ras double mutants--V12S35, V12G37 and V12C40--proteins were expressed constitutively in human diploid fibroblast (HDF) cells by retrovirus infection at PD26. Constitutive expression of V12S35, V12G37 and V12C40 proteins induced premature senescence at PD38, PD47 and PD50, respectively, in contrast to the control cells at PD59. Premature senescence was evidenced by the slow cellular growth rate and SA-beta-galactosidase expression accompanied by morphological changes such as flat and large cell shape. Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells as well as V12S35 and V12G37 expressers were unable to export actin fibers from nucleus to cytoplasm, form stress fibers through the MAPK and Ral.GDS pathways. Perinuclear expression of Racl was prominent in the HDF cells and V12C40 expresser, while translocation of Racl from perinucleus to nucleus and strong expression of RhoA were observed in the V12S35 expresser. In summary, the induced premature senescence by H-ras double mutants were accompanied by nuclear accumulation of actin and Racl proteins, cytoplasmic retention of p-Erk1/2 and marked induction of RhoA expression mainly through dysregulation of the MEK pathway.

Expression of androgen receptor and its implication in hepatoma cells.

Hepatocellular carcinoma (HCC) in human and murine has been known to occur more frequently in male than female. Attempts have been made to find a possible mechanism of increased growth advantage in the HCC cells by testosterone (T). Treatment of HepG2 human HCC cells with T increased DNA synthesis by 30-50%, whereas T increased it by 10-30% in FaO (rat) cells. Androgen receptor (AR) levels in HepG2 and FaO cells were 94.1 fmol/mg protein and 55.0 fmol/mg protein, respectively. Expression of AR mRNA species was detected as about 10 kb (doublet) in HepG2. On the other hand, much less expression of AR mRNA was observed in FaO cells. AR signals around 28S rRNA (4.7 kb) and the small size RNAs detected in the total cellular RNAs were not found in the poly(A) + RNAs isolated from the cells. Contrary to the earlier observation by other investigators, expression of hepatic AR was not down-regulated by T until 72 h of treatment. These results demonstrate one of the modes of action by T for the preferential development of HCC in male.

Two synaptotagmin genes, Syt1 and Syt4, are differentially regulated in adult brain and during postnatal development following kainic acid-induced seizures.

The synaptotagmins together with other vesicle proteins are thought to be essential for the docking and/or fusion of synaptic vesicles with the plasma membrane that occurs following depolarization and calcium influx in presynatic terminals. Syt4, the fourth identified member of the synaptotagmin family, is inducible in PC12 cells by depolarization and secretagogues, and in limbic regions of the adult rat brain by kainic acid-induced seizures. In the present study, we examined the time course of the seizure-induced changes in the expression of Syt4 and Syt1, both in adult animals and during the postnatal period. Syt4 was transiently induced in several structures of the adult rat brain following seizure activity with peak inductions between 4 and 8 h and overal return to control values by 30 h. No induction was observed following seizure activity in 7-day-old animals. The brain regions most sensitive to increased induction were, in decreasing order of sensitivity, hippocampal pyramidal cells dentate granule cells and piriform cortex pyramidal cells. The brain areas showing the greatest Syt4 stimulation in adults were also the areas in which Syt4 was induced by seizures earlier in development. In contrast, Syt1 mRNA was depressed in adult brains following seizure activity, particularly in the dentate granule cells. Our results suggest that the differential regulation of different synaptotagmin genes following excessive neuronal activity might participate in rapid adaptation of subsequent transmitter release.

Differential expression of O6-methylguanine-DNA methyltransferase during diethylnitrosamine-induced carcinogenesis and liver regeneration in Sprague-Dawley male rats.

Differential expression of DNA-O6MeG: protein-L-cysteine S-methyltransferase (MGMT) activity and posttranslational modification of the protein during liver regeneration and carcinogenesis were compared in Sprague-Dawley male rats after partial hepatectomy and/or single i.p injection of diethylnitrosamine (DEN, 200 mg/kg). Regenerating hepatocytes after partial hepatectomy induced MGMT transiently within 3 days; however, the induction of MGMT was persistent for 2 weeks after DEN injection, and the combined treatment of DEN and partial hepatectomy maintained the elevated MGMT level for up to 4 weeks. The increased activity was transcriptionally regulated, when analyzed by Northern blot hybridization. The major active form of MGMT protein in the partially hepatectomized or DEN-treated rats was a 26-kDa or 24-kDa species respectively, which was confirmed by Western blot analysis and gel slice assay. The biological significance of the differential induction of MGMT during partial hepatectomy or DEN-induced carcinogenesis is not obvious; however, further studies on possible posttranslational modifications of MGMT protein might shed some light on the functional aspect of MGMT induction.

Differential expression of TIS21 and TIS1 genes in the various organs of Balb/c mice, thymic carcinoma tissues and human cancer cell lines.

As a part of a series of investigations on the functions of TIS21 and TIS1 genes, we measured in vivo 12-O-tetradecanoylphorbol-13-acetate (TPA) inducibility of primary response genes (TIS21, TIS8 and TIS1) in the Balb/c mice and the changes of TIS gene expression in thymic carcinoma tissues and A549 and NCIH69 human lung cancer cell lines. In vivo induction of the TIS genes (TIS21, -8 and -1) by intraperitoneal injection of TPA was dramatic only at the needle contact site, i.e. in the abdominal muscle, not in the thigh muscle. Expression of TIS21 and TIS1 in the Balb/c mice thymus, lung, stomach and spleen was very strong (Lim IK et al. 1994a), regardless of TPA injection. Thymic carcinoma tissues developed in SV40-T-antigen-containing transgenic mice did not express TIS21 and TIS1, and expressed TIS8 weakly. Interestingly, induction of TIS21 expression was obliterated in the human lung cancer cells; A549 cells completely lost the ability to express TIS21 after a combined treatment of TPA and cycloheximide. We also measured the induction of TIS genes by TPA and/or cycloheximide in Raw264.7 mouse macrophage cells and U937 human histiocytic lymphoma cells. However, the induction profile was quite different; repressed and deregulated expression in the U937 cells as compared to rapid and transient induction of TIS genes in the Raw264.7 cells. These data may suggest a repressed expression of TIS21 and TIS1 in the cancer tissues and cells derived from the organs that constitutively express TIS21 in mice and in human cancer cells.

Expression of the tumor necrosis factor alpha gene and early response genes by nodularin, a liver tumor promoter, in primary cultured rat hepatocytes.

Nodularin is a new liver carcinogen possessing a potent tumor-promoting activity in rat liver, mediated through inhibition of protein phosphatases 1 and 2A, and a weak initiating activity. Since we previously reported evidence that nodularin up-regulated expression of the tumor necrosis factor alpha gene (TNF alpha) and early-response genes in rat liver after its i.p. administration, and since TNF alpha had tumor-promoting activity in vitro, it is possible that TNF alpha itself is involved in liver tumor promotion. We investigated whether hepatocytes themselves induce expression of the TNF alpha gene and early-response genes in primary cultured rat hepatocytes treated with nodularin. Like nodularin, microcystin-LR, which is another liver tumor promoter belonging to the okadaic acid class, strongly induced TNF alpha gene expression in rat hepatocytes, as well as TNF alpha release from those cells into the medium. On the other hand, 12-O-tetradecanoylphorbol-13-acetate, which has been reported to induce no tumor promotion in rat liver, induced no apparent expression of the TNF alpha gene in primary cultured rat hepatocytes. As for the expression of early-response genes, 1 microM nodularin or microcystin-LR induced expression of the c-jun, jun B, jun D, c-fos, fos B and fra-1 genes in the hepatocytes, and the expression of these genes was prolonged up to 24 h, suggesting mRNA stabilization induced by inhibition of protein phosphatases 1 and 2A. This paper presents new evidence that the TNF alpha gene and early-response genes were expressed in hepatocytes treated with a liver tumor promoter.

Translocational inefficiency of intracellular proteins in senescence of human diploid fibroblasts.

In order to investigate signal transduction pathways and related changes of actin cytoskeleton organization in cellular senescence, H-ras double mutants--V12S35, V12G37, and V12C40--were constitutively expressed in human foreskin fibroblast (HDF). Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells, V12S35 and V12G37 expressers, revealed a failure to export actin fiber from nucleus to cytoplasm and also to form stress fibers. Perinuclear expression of Rac1 was prominent in the HDF cells and V12C40 expresser; however, in the V12S35 expresser, translocation of Rac1 from perinucleus to nucleus and strong expression of RhoA were obvious. In summary, the H-ras double mutant expressers induced premature senescence through the MEK pathway, accompanied by nuclear accumulation of actin and Rac1 proteins, cytoplasmic retention of p-Erk1/2, and marked induction of RhoA expression, suggesting the translocational inefficiency of the intracellular proteins in the senescent HDF cells.

Expression patterns of cell cycle-related proteins in a rat cirrhotic model induced by CCl4 or thioacetamide.

To analyze the aberrant expression of cell cycle-related proteins and their biological significance in relation to cirrhosis, we compared the cirrhotic patterns induced by two different types of cirrhotic agents, CCl4 and thioacetamide (TAA) in rats. CCl4 or TAA treatment was given to rats for 8 or 30 weeks, respectively, and the livers were removed at 9, 20, and 30 weeks after the experiment began. The TAA-induced fibrotic pattern was different from the CCl4-induced one, in terms of the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed severe cirrhotic changes at 9 weeks in CCl4-treated rats and at 30 weeks in TAA-treated rats. Immunoblotting for cyclin D1, E, A, B, and proliferating cell nuclear antigen (PCNA) and their counterpart protein kinases (CDK2, 4, and CDC2) showed significant overexpression in rats with severely cirrhotic livers. The p53 tumor suppressor protein increased dramatically in the CCl4-treated group, while it was not detected in the livers of TAA-treated rats. Upregulation of p21WAF1, a CDK inhibitory protein, was detected in TAA-treated rats, but not in CCl4-treated rats. Immunohistochemical data for cyclin D1, E, and PCNA were well correlated with immunoblotting data; these proteins were increased in hepatocytes surrounding the cirrhotic lesions, suggesting that hepatocyte regeneration is correlated with cell cycle-related protein expression in cirrhotic liver. In the TAA-treated rats, the expression of these proteins was increased both in hepatocytes and in ductule cells. Our data suggest that liver cirrhosis induced by CCl4 or TAA is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis.

Identification of highly methylated arginine residues in an endogenous 20-kDa polypeptide in cancer cells.

Enzymatic methylation of endogenous proteins in several cancer cell lines was investigated to understand a possible relationship between protein-arginine methylation and cellular proliferation. Cytosolic extracts prepared from several cancer cells (HeLa, HCT-48, A549, and HepG2) and incubated with S-adenosyl-L-[methyl-3H]methionine revealed an intensely [methyl-3H]-labeled 20-kDa polypeptide. On the other hand, cytosolic extracts prepared from normal colon cells did not show any methylation of the 20-kDa protein under identical conditions. To identify nature of the 20-kDa polypeptide, purified histones were methylated with HCT-48 cytosolic extracts and analyzed by SDS-PAGE. However, none of the histones comigrated with the methylated 20-kDa polypeptide, indicating that it is unlikely to be any of the histone subclasses. The [methyl-3H]group in the 20-kDa polypeptide was stable at pH 10-11 (37 degrees C for 30 min) and methylation was not stimulated by GTPgammaS (4 mM), thus the reaction is neither carboxyl methylesterification on isoaspartyl residues, nor on C-terminal farnesylated cysteine. The present study together with the previous identification of N(G)-methylated arginine residues in the HCT-48 cytosol fraction suggests that this novel endogenous 20-kDa arginine-methylation is a cellular proliferation-related posttranslational modification reaction.