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.

Methylation of O(6)-methylguanine-DNA methyltransferase gene is associated significantly with K-ras mutation, lymph node invasion, tumor staging, and disease free survival in patients with gastric carcinoma.

BACKGROUND: O(6)-methylguanine-DNA methyltransferase (MGMT) can remove O(6)alkylG DNA adducts. If they are not removed, then the adducts mispair with T during DNA replication, resulting in G-to-A mutation. Interrelations between MGMT gene inactivation by promoter methylation, K-ras mutation, and clinicopathologic features in patients with gastric carcinoma were studied. METHODS: Surgically removed tumor tissues from 79 patients were analyzed with MGMT methylation by genomic DNA modification and methylation specific polymerase chain reaction analysis, K-ras mutation by mutant allele specific amplification, TNM classification according to the International Union Against Cancer system, and MGMT protein expression by immunohistochemistry. RESULTS: MGMT-promoter methylation was found in 18 of 79 tumors. Among those 18 tumors, K-ras mutations were found in 33% and 11% of tumors at codons 12 and 13, respectively, corresponding to 20 times and 7 times greater rates of mutation compared with unmethylated tumors. MGMT methylation was associated significantly with lymph node invasion (P < 0.01), tumor stage (P < 0.03) and 5-year disease free survival (P < 0.02). MGMT protein expression was detected in intestinal metaplasia and adenocarcinoma samples, whereas no expression was detected in normal foveolar cells. CONCLUSIONS: MGMT-promoter methylation in patients with gastric carcinoma was associated significantly with point mutations of K-ras at codons 12 and 13, lymph node invasion, tumor stage, and disease free survival. These associations indicate a significant role of MGMT methylation during gastric carcinogenesis.

Sequential changes in hepatocarcinogenesis induced by diethylnitrosamine plus thioacetamide in Fischer 344 rats: induction of gankyrin expression in liver fibrosis, pRB degradation in cirrhosis, and methylation of p16(INK4A) exon 1 in hepatocellular carcinoma.

To clarify the sequential changes in pRB and p16 during different stages of hepatocarcinogenesis such as fibrosis, cirrhosis, hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC), male Fischer 344 rats were singly injected with diethylnitrosamine (DEN), immediately followed with phenobarbital for 1 wk and then thioacetamide (TAA) for 39 wk in drinking water. Rats were killed at 9, 20, 30, and 40 wk after DEN initiation and changes of pRB level, p16 gene hypermethylation, and in vivo gankyrin expression were examined. Histologic examination showed stepwise appearances of fibrosis, cirrhosis, HCA, and HCC at weeks 9, 20, 30, and 40, respectively. Hypermethylation of p16 exon 1 was not found until HCA but appeared in 50% of the rats with HCC accompanied by complete loss of its mRNA expression. The amount of glutathione S-transferase--gankyrin bound to pRB and pRB degradation in the liver depended on the concentration of gankyrin and incubation time. Gankyrin expression preceded pRB degradation in liver cirrhosis. In conclusion, gankyrin expression induced in liver fibrosis accelerated the degradation of pRB during liver cirrhosis, and inactivation of p16 exon 1 by DNA hypermethylation occurred during the progression of tumor cells to poorly differentiated HCC. Inactivation of pRB and/or p16 resulted in complete loss of regulation in the cell-division cycle during early and late stages, respectively, of hepatocarcinogenesis. Mol. Carcinog. 30:138--150, 2001.

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.

Subcellular redistribution of protein kinase C isozymes is associated with rat liver cirrhotic changes induced by carbon tetrachloride or thioacetamide.

BACKGROUND AND AIMS: Protein kinase C (PKC) plays a key role in the alteration of signal transduction in the liver, which may contribute to the development of liver cirrhosis. The aim of the present study was to examine the subcellular redistribution of PKC isozymes in rat liver cirrhosis, which is induced by two different cirrhotic chemical agents, carbon tetrachloride (CCl4) and thioacetamide (TAA). METHODS AND RESULTS: Thioacetamide and CCl4 were administered to rats for 8 and 30 weeks, respectively before rats were killed and autopsies performed at 9, 20 and 30 weeks later. The TAA induced a fibrotic pattern in the liver that differed from that produced by CCl4, notably in 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 that severe cirrhotic changes were present 9 weeks after the commencement of CCl4 treatment and 30 weeks after TAA treatment. DISCUSSION: When the subcellular redistribution of PKC isozymes (PKCalpha, -beta1, -delta, and -epsilon) was examined, all the PKC isozymes in CCl4-treated rats were found to be translocated to the membrane fraction, which may mean PKC activation, and then downregulated by proteolytic degradation after 9 weeks of treatment, which coincided with peak cirrhotic changes. All rats treated with CCl4 recovered to the control level after 20 weeks of treatment. In the case of TAA-treated rats, PKC isozymes were translocated to the particulate fraction of the liver after 9 weeks of treatment and this persisted in most of the rats for the duration of the experiment. CONCLUSIONS: From these results, it would appear that PKC translocation preceded morphologic changes, and that an altered subcellular distribution of the PKC isozyme may be associated with the response to liver damage and carcinogenesis.

Selective left-lobe atrophy by nodularin treatment accompanied by reduced protein phosphatase 1/2A and increased peroxisome proliferation in rat liver.

The effect of nodularin on selective atrophy of left lobes in the liver was investigated in F344 rats. Nodularin was injected for 10 weeks from the third week of initiation with saline or N-nitrosodiethylamine (DEN), grouped as S/N and D/N, respectively. Nodularin significantly decreased weights of left (LL) and caudate (CL) lobes but increased right (RL) and middle (ML) lobes in S/N rats. Activity of protein phosphatases [types 1 (PPI) and 2A (PP2A)] was more severely reduced in S/N than D/N rats; moreover, in LL compared with RL of S/N rats, activity was significantly inhibited by nodularin treatment from week 4, which corresponded to 2 weeks after nodularin injection. However, nodularin significantly induced peroxisomal palmitoyl-CoA oxidase and cytochrome P-450 4A1 expression in S/N compared with D/N rats. An effect of nodularin on apoptosis was evident since expression of Bcl-Xs was clearly induced in LL of S/N rats as opposed to various inductions of Bcl-XL. However, Bcl-XL in RL was persistently induced, with undetectable Bcl-Xs expression. These results demonstrate biochemical evidence of selective atrophy of LL by inhibition of PP1 and PP2A activity, increase of peroxisomal enzymes and induction of Bcl-Xs expression, in contrast to proliferation of RL in rats treated with nodularin alone. However, nodularin endowed DEN-altered hepatocytes with regenerating power and concomitant restoration of phosphatase activity as well as persistent expression of Bcl-XL in D/N rats.

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.

Phosphorylation of methylated-DNA-protein-cysteine S-methyltransferase at serine-204 significantly increases its resistance to proteolytic digestion.

In a previous paper [Lim, Park, Jee, Lee and Paik (1999) J. Cancer Res. Clin. Oncol. 125, 493-499], we showed two major forms of active DNA-6-O-methylguanine:protein-L-cysteine S-methyltransferase (MGMT; EC 2.1.1.63) in the liver with N-nitrosodiethylamine (DEN)-induced carcinogenesis: these were 26 and 24 kDa species. Here we show that a 2 kDa C-terminal fragment was cleaved from the 26 kDa species in vitro by thrombin or microsomal fractions isolated from DEN-treated rat livers. When Ser(204) of the 26 kDa protein was replaced with Ala by site-directed mutagenesis, phosphorylation of the protein was completely abolished, indicating Ser(204) to be the site of phosphorylation. We also show that the phosphorylation was performed by Ca(2+)-independent protein kinase isoenzymes, and that the phosphorylated rat MGMT protein was resistant to digestion by protease(s) whose activity was increased during DEN-induced hepatocarcinogenesis and also by digestion with endopeptidase Glu-C (V8 protease).

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 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.

Regulation of selection of liver nodules initiated with N-nitrosodiethylamine and promoted with nodularin injections in fischer 344 male rats by reciprocal expression of transforming growth factor-beta1 and its receptors.

To investigate how glutathione-S-transferase placental form (GST-P)+ hyperplastic nodules (HNs) are selected and to determine the driving force for progression or regression of HNs, changes in transforming growth factor-beta1 (TGF-beta) and its receptors were examined during hepatocarcinogenesis initiated by N-nitrosodiethylamine (DEN) and promoted by nodularin. The induction of TGF-beta1 expression in the GST-P+ HNs was dependent on nodularin injections for 10 wk, which started the third week after DEN initiation. The kinetics of TGF-beta1 induction during carcinogenesis were quite different from that of simple regeneration after partial hepatectomy (PH): hepatocytes initiated with DEN alone induced TGF-beta1 expression for 24 d, and subsequent stimulation by PH on the fourteenth day after DEN initiation super-induced TGF-beta1 mRNA (50 times that of the control level), as opposed to a transient expression for less than 5 d by PH alone. GST-P+ HNs did not express TGF-beta receptors I (RI) and II (RII) during the early stage of carcinogenesis, whereas the surrounding hepatocytes strongly expressed both of these receptors. On cessation of nodularin injection, however, the expression of RI and RII in the HNs changed significantly: RII+ nodules appeared, and the number and area of RII+/- nodules were significantly increased at 10 wk after the cessation. These findings indicate that induction of TGF-beta expression in GST-P+ HNs might be a strong selection pressure that allows outgrowth of RII- nodules during liver carcinogenesis.

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.

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.

Effect of nodularin on the expression of glutathione S-transferase placental form and proliferating cell nuclear antigen in N-nitrosodiethylamine initiated hepatocarcinogenesis in the male Fischer 344 rat.

The tumor-promoting effect of nodularin during carcinogenesis was investigated. Male Fischer 344 rats were injected with nodularin for 10 weeks from week 3 after N-nitrosodiethylamine initiation without partial hepatectomy. Rats were further maintained for 10 weeks after the cessation of nodularin and were periodically killed. In contrast to the minimal foci in the DEN and nodularin alone groups, treatment with DEN and nodularin produced four kinds of nodules with eosinophilic, clear, mixed and basophilic cells. After the cessation of nodularin, the maximally increased number, but not the area, of glutathione S-transferase placental form-positive [GST-P(+)] nodules at week 12 decreased significantly and the appearance of two types of hyperplastic nodules was noted by GST-P immunostaining; homogeneously stained dense nodules (DN) and heterogeneously stained pale nodules (PN), which appeared only after the cessation of nodularin. DN were well circumscribed by enzyme-altered cells, as opposed to poorly in PN. Moreover, normal-appearing hepatocytes replaced the enzyme-altered cells in PN. In contrast to the higher PCNA index in GST-P(+) DN, the background level returned to that of the control at week 15. PCNA indices in DN were significantly higher than in PN, which were still higher than the control, indicating that nodularin affected the PCNA index differentially in the altered and unaltered hepatocytes. However, nodularin without DEN initiation significantly increased the PCNA index through initial cell death and subsequent hepatocyte proliferation. These results suggest that: (i) nodularin has a promoting effect by inducing hepatocyte proliferation in both enzyme-altered hyperplastic nodules and the surrounding parenchyma; (ii) proliferation is transient in background cells but not in enzyme-altered hepatocytes; (iii) GST-P(+) DN can be regarded as progressive and GST-P(+) PN as regressive, revealed by both immunohistochemistry and PCNA index.

Induction of growth inhibition of 293 cells by downregulation of the cyclin E and cyclin-dependent kinase 4 proteins due to overexpression of TIS21.

We earlier reported that TIS21 mRNA expression was markedly decreased in A549 and NCIH69 human lung cancer cells and in thymic carcinoma tissues obtained from transgenic mice containing simian virus 40 large T antigen (J Cancer Res Clin Oncol 121:279-284, 1995). To determine how TIS21 inhibits growth, we made 293 cells that constitutively expressed TIS21 protein. The constitutive TIS21 expresser lines C9 and C11 grew to a lower saturation density than did those in the vector-transfected clones (V7 and V10) and antisense-transfected clones (AS1 and AS4), and the size of the C9 and C11 cells increased significantly after transfection with TIS21 cDNA. The serum-stimulated cell cycle was analyzed by fluorescence-activated cell sorting after double thymidine treatment; V10 progressed normally through the cell division cycle, but C9 and C11 cells accumulated continuously in G1 phase until 36 h after treatment. On the other hand, the progression of cells that had already entered to S or G2/M phase was not inhibited. When cell-cycle regulatory proteins were measured, C9 and C11 cells showed significantly reduced synthesis of cyclin E and cyclin-dependent kinase (cdk) 4 as well as a decrease in cyclin E-associated cdk activity. These observations led us to conclude that TIS21 overexpression in G1 phase decreased the amounts of cyclin E and cdk4, thereby decreasing the activity of cdks at the G1-S transition.

Enzymatic methylation of recombinant TIS21 protein-arginine residues.

Recombinant TIS21 protein was overexpressed in Escherichia coli harboring the expression vector plasmid pQE-30 carrying the TIS21 cDNA coding sequence containing an extra 120 nucleotides upstream. Employing this protein consisting of 158 amino acid residues of the main chain plus 40 residues of the fusion peptide. It was found that one of the protein methylase I group [S-adenosylmethionine:nuclear protein/histone-arginine N-methyltransferase; BC 2.1.1.23; J. Biol. Chem., 269, 1075 (1994)] methylated this protein. The methylation products were identified as guanidino-N-methylated arginines. Some of the kinetics of the reaction are described.