MTA1 Protein Research Articles

Overexpression of MTA1 promotes invasiveness and metastasis of ovarian cancer cells

To investigate the effect of metastasis-associated gene MTA1 on proliferation and invasion potential of ovarian cancer cell line A2780. The eukaryotic expressing vector pcDNA3. 1-MTA1 was introduced into A2780 cells by gene transfection in vitro. The MTA1 mRNA and protein level in cancer cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blot, respectively. The growth activities of cancer cells were detected by trypan blue stain method. The clone formation assay in soft agar was used to observe the proliferation of cancer cells. Wound healing assay and Transwell assay were used to evaluate migration and invasion abilities of cancer cells. And the protein level of bcl-xL in ovarian cancer cells was measured by immunohistochemistry and western blot. The TUNEL assay was performed to study the apoptosis of tumor cells. Seventy-two hours after transfection, the MTA1 expression increased significantly (P<0.01). The up-regulation of MTA1 did not affect the growth activities of cancer cells (P>0.05), but it promoted clone formation, migration and invasion abilities of cancer cells (P<0.01). The cellular expression of bcl-xL increased 65.22%, with a PI value of (71.64±5.96)%. With the up-regulation of MTA1 and bcl-xL level, the apoptotic rate of A2780 cell was decreased. MTA1 gene plays an important role in progression and metastasis of ovarian cancers, which provides an ideal strategy for gene therapy of ovarian cancers.

The MTA family proteins as novel histone H3 binding proteins

BackgroundThe nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined.ResultsIn this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells.ConclusionsTogether our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

MTA1 induced angiogenesis, migration and tumor growth is inhibited by Glycyrrhiza glabra

Angiogenesis is an important host process that interacts with cancer cells to promote growth, invasion and metastasis. Vascular endothelial growth factor (VEGF) and Metastasis Associated protein (MTA1) are known to play a major role in angiogenesis. The recombinant VEGF and MTA1 proteins were used to induce proliferation and cell migration in MDA-MB-231 cells. Here we investigated the antiangiogenic and antitumor activity of G glabra F6 (G1) on VEGF and MTA1 induced angiogenesis. The angio inhibitory activity of G.glabra F6 (G1) was confirmed by its inhibition of angiogenesis in in vivo assays, peritoneal and chorioallantoic membrane assay. Reduction in the levels of the cytokine VEGF and microvessel density count in the peritoneum of mice treated withG.glabra F6 (G1) indicated that the plant extract decreased VEGF production. It also inhibits the neovascularization in CAM induced by VEGF and MTA1. These findings not only suggest a potential role of VEGF or MTA1 in tumour angiogenesis but it is also an effective beginning to explore mechanism of metastasis and cancer therapy strategy targeting MTA1. Our results suggest that the extract from the roots of G.glabra may be a potential supplemental source for cancer therapy.

MTA1 of the MTA (metastasis-associated) gene family and its encoded proteins: molecular and regulatory functions and role in human cancer progression

MTA1 (a metastasis-associated gene) is a newly discovere d human gene (residing on chromsome 14q32.3) that belongs to a family of cancer progression-related genes ( MTA ). The mRNA product of MTA1 along with its protein product MTA1 have been reported to be over-expressed in a wide variety of animal and human tumors. For example, the expression of MTA1 and its encoded protein MTA1 correlates with the malignant properties of many human cancers, including cancers of the breast, colon, stomach, li ver, prostate and others. The MTA proteins have bee n shown to be ubiquitinated transcriptional co-repressors that fu nction in histone deacetylation and are part of the NuRD complex, a nucleosome remodeling and histone deacetylating complex whose stability appears to be regulated by ubi quitinated MTA1 binding to E3 ubiquitin ligase constitutive ph otomorphogenesis protein-1 (COP1). The MTA1 protein plays an essential role in c-MYC-mediated cell transformatio n, and its expression correlates with mammary gland tumor formation. In the latter, MTA1 helps convert mammary cells to more aggressive phenotypes by repression of the estrogen receptor (ER) via trans-activation through deacetylation of chromatin in the ER-responsive el ement of ERresponsive genes. Another member of the MTA family, MTA3, is induced by estrogen and represses the exp ression of the transcriptional repressor Snail, a master regul ator of epithelial to mesenchymal transformation, r esulting in the expression of the cell adhesion molecule E-cadherin and maintenance of a differentiated, normal epithel ial phenotype in mammary cells. An important activity mediated by both MTA1 and MTA2 is deacylation and inactivation of tumor suppressor p53protein, in part by controlling its s tability by inhibiting ubiquitination, leading to i nhibition of growth arrest and apoptosis. Another factor deacetylated a nd stabilized by MTA1 NuRD complex is hypoxia-inducible factor1α (HIF-1α), which is involved in angiogenesis. Therefore, th e MTA proteins represent a possible set of master c oregulatory molecules involved in the carcinogenesis and progression of various malignant tumors. As su ch, they could be important new tools for cancer diagnosis and tre atment.

Silencing MTA1 by RNAi reverses adhesion, migration and invasiveness of cervical cancer cells (SiHa) via altered expression of p53, and E-cadherin/β-catenin complex

It has been reported that metastasis-associated gene 1 (Mta1) is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.

Relation between prognosis and expression of metastasis-associated protein 1 in stage I non-small cell lung cancer

Metastasis-associated protein 1 (MTA1 protein) has been reported to be correlated with the biological behavior and prognosis of several malignant carcinomas. We hypothesized that stage I non-small cell lung cancer (NSCLC) patients with MTA1 protein overexpression would be more likely to have a poor prognosis. Therefore, we tested the expression of MTA1 protein in 60 stage I NSCLC and 30 paracarcinous normal lung tissues using the streptavidin-perosidase method. The Kaplan-Meier method was used to calculate the survival rate, and Cox regression analysis was performed to identify prognostic risk factors. MTA1 protein overexpression was detected in 22 stage I NSCLC tissues in this study. Tumor differentiation and tumor diameter were significantly associated with MTA1 protein overexpression, while not correlated with age, sex, pathological type or smoking status. The five-year survival rate of patients with MTA1 protein overexpression was significantly lower than that of those without expression (40.9% vs. 84.1%; P<0.001). The results of multivariate analysis confirmed that MTA1 protein overexpression was an independent prognostic factor (risk ratio=5.23, P=0.007). These findings demonstrated MTA1 might be a prognostic factor in NSCLC.

Regulation of NF-κB Circuitry by a Component of the Nucleosome Remodeling and Deacetylase Complex Controls Inflammatory Response Homeostasis

The MTA1 coregulator (metastatic tumor antigen 1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, has been intimately linked with human cancer, but its role in inflammatory responses remains unknown. Here, we discovered that MTA1 is a target of inflammation, and stimulation of macrophages with Escherichia coli lipopolysaccharide (LPS) stimulates MTA1 transcription via the NF-kappaB pathway. Unexpectedly, we found that MTA1 depletion in LPS-stimulated macrophages impairs NF-kappaB signaling and expression of inflammatory molecules. MTA1 itself acts as a transcriptional coactivator of inflammatory cytokines in LPS-stimulated macrophages, and in contrast, it acts as a corepressor in resting primary macrophages as its depletion induced cytokine expression. LPS stimulates S-nitrosylation of histone deacetylase 2 (HDAC2) and interferes with its binding to MTA1, which, in turn, resulted in the loss of corepressor behavior of MTA1.HDAC complex in activated macrophages. Consequently, the net levels of inflammatory cytokines in LPS-stimulated macrophages from MTA1(-/-) mice were high compared with wild-type mice. Accordingly, MTA1(-/-) mice were much more susceptible than control mice to septic shock induced by LPS, revealing that MTA1 protects mice from deregulated host inflammatory response. These findings reveal a previously unrecognized, critical homeostatic role of MTA1, both as a target and as a component of the NF-kappaB circuitry, in the regulation of inflammatory responses.

Truncated MTA‐1: A Pitfall in ELISA‐Based Immunoassay of HTLV‐1 Infection

HTLV-1 causes adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Recombinant envelope glycoprotein is used in production of diagnostic enzyme-linked immunosorbent assay (ELISA) kit. There are some reports that a significant percentage of Iranian HTLV-1 infected patients showed no seroreactivity with MTA-1 peptide, while HTLV-1 had been confirmed by PCR detection methods or ELISA kits containing a cocktail of HTLV-1 specific peptides. This report describes experiments designed to determine whether some discrepancies between ELISA and PCR results could be due to truncation of immunodominant epitopes using immunoassay method. We have cloned the MTA-1 epitope of env gene from HTLV-1 in NotI/NdeI sites of pET22b(+) expression vector. Sequencing analysis of recombinant plasmids revealed an insertion of a cytosine in position 271 causing a stop codon in the MTA-1 protein translation. SDS-PAGE analysis also failed to reveal the presence of the desired protein. Subjects with a mutant HTLV-1 env gene were shown to be seronegative using ELISA, but positive with PCR.

Correlation of appearance of metastasis-associated protein1 (Mta1) with spermatogenesis in developing mouse testis

Mta1, a representative of the MTA gene family, is believed to be involved in the metastasis of malignant tumors. However, a systematic study of its physiological function has not been performed. It has been found in normal mouse organs at relatively low levels, except for in testis, suggesting a potential function in the male reproductive system. In order to explore the role of Mta1 protein during spermatogenesis, its expression in adult mouse testis was compared with that in developing mouse testis and in testis from adult mice treated with methoxyacetic acid, which selectively depletes primary spermatocytes. Quantitative analysis revealed that Mta1 protein gradually increased in the testis from 14 days postnatally. Immunolocalization analysis demonstrated strong signals in the seminiferous tubules, and Mta1 was predominantly present in the nucleus of primary spermatocytes and spermatogonia from 14 days postnatally. The most intensive staining was located in the nucleus of pachytene spermatocytes in mature testes. The expression pattern of Mta1 during spermatogenesis was also shown to be stage-specific by immunohistochemistry analysis. Finally, dramatic loss of Mta1 expression from pachytene spermatocytes was observed in the spermatogenic-arrested adult mouse testis. These results collectively demonstrate that Mta1 appears during postnatal testis development and suggest that this expression may be crucial for spermatogenesis.

Utility of molecular genetic signatures in the delineation of gastric neoplasia

Current techniques to define gastric neoplasia are limited but molecular genetic signatures can categorize tumors and provide biological rationale for predicting clinical behavior. We identified three gene signatures: Chromogranin A (CgA), MAGE-D2 (adhesion), and MTA1 (metastasis) that define gastrointestinal (GI) carcinoids and hypothesize that their expression can delineate gastric neoplasia. This strategy provides a molecular basis to define neuroendocrine gastric carcinoids (GCs), neuronal stromal tumors (GISTs), or epithelial cell (gastric adenocarcinomas [GCAs])-derived tumors. Total RNA was isolated from 38 GCs: Type I/II (n = 7), Type III/IV (n = 6), GISTs (n = 12), GCAs (n = 13), and normal mucosa (n = 12). Quantitative reverse transcriptase polymerase chain reaction (Q RT-PCR) gene expression was quantified against glyseraldehyde-3-phosphate dehydrogenase (GAPDH) and CgA and MTA1 protein expression levels were analyzed by immunohistochemical analyses of a gastric neoplasia microarray. CgA was elevated in Type I/II (10-fold; P < .01) and Type III/IV (100-fold, P < .005), decreased in GISTs (100-fold, P < .03), and unchanged in GCAs. MAGE-D2 was 5-10-fold elevated (P < .05) in Type III/IV, GISTs, and GCAs but not in Type I/II tumors. MTA1 (> 5-fold, P < .01) was elevated in GCs (Type III/IV>I/II, P < .05), in GISTs (> 4-fold, P < .05), and GCAs. CgA protein levels were elevated in GCs (P < .005) but not in GISTs and GCAs. MTA1 levels were elevated in all tumors (P < .02) compared with normal, and especially with tumor invasion (P < .05). CgA discriminates GCs from other gastric neoplasms; overexpression of MAGE-D2 and MTA1 differentiate Type III/IV from Type I/II GCs. GISTs share similar expression patterns with Type III/IV GCs but have decreased CgA. MTA1 is a marker of tumor invasion.

Reduced MTA1 Expression by RNAi Inhibits in Vitro Invasion and Migration of Esophageal Squamous Cell Carcinoma Cell Line

To distinguish aggressive esophageal squamous cell carcinoma from indolent disease is the important clinical challenge. Studies have indicated that metastasis-associated gene 1(Mta1) played a role in the process of metastasis of carcinoma. The overexpression of Mta1 gene has been found in a variety of tumors. To identify the detailed roles of MTA1 protein in the carcinogenesis of esophageal squamous cell carcinoma, this study analyzed the pathological specimens on tissue microarray derived from 72 patients using immunohistochemistry. MTA1 expression increased in the nuclear with the development of esophageal squamous cell carcinoma from normal epithelial cell, dysplasia, to invasive cancer. In biological studies with human esophageal squamous cell carcinoma cell line, MTA1 plays its roles to promote cancer cell invasion, adhesion and movement. RNA interference (RNAi) against MTA1 decreased the malignant phenotypes. Gene microarray analysis revealed some metastasis-associated genes were altered by MTA1 RNAi. This study started an effective beginning to explore metastasis mechanisms and cancer gene therapy strategy targeting MTA1.

Overexpression of Metastatic Tumor Antigen in Osteosarcoma: Comparison between Conventional High-Grade and Central Low-Grade Osteosarcoma

The metastatic tumor antigen (MTA) gene is a recently identified metastasis-associated gene which has implications in the signal transduction or regulation of gene expression. However, the expression of MTA in osteosarcoma and its potential relationship with metastasis have not been examined, forming the basis of this study. We compared the expression levels of the MTA1 protein between 32 cases of high-grade osteosarcomas and 21 cases of low-grade osteosarcomas by immunohistochemistry. In addition, the mRNA expression levels of MTA1, 2, 3 in these osteosarcoma cell lines and control fibroblasts were evaluated by real-time quantitative PCR. MTA1 immunoreactivity was present in 81.25% of high-grade osteosarcoma specimens. Its expression was predominantly localized to the nucleus or cytoplasm of osteosarcoma cells. Thirteen (86.6%) of 15 patients who died of osteosarcomas displayed strong MTA1 expression. Both primary bone and pulmonary metastatic lesions exhibited MTA1 expression. All low-grade osteosarcomas were negative for MTA1 except for focal weak reactivity in two cases. The tested high-grade osteosarcoma cell lines showed marked amplification of MTA1 and MTA2 mRNA compared to control cells. These results suggest that MTA might be involved in the progression of high-grade osteosarcoma, particularly in hematogenous metastasis of osteosarcoma.

Expression of the metastasis-associated MTA1 protein and its relationship to deacetylation of the histone H4 in esophageal squamous cell carcinomas.

Metastasis-associated protein MTA1 and histone deacetylase form a protein complex with histone deacetylase activity that plays an important role in histone deacetylation, alteration of chromatin structure and transcriptional control. The precise role of the MTA1 protein in the malignant progression of human cancers remains unknown, however, especially its overexpression and relationship with histone acetylation/deacetylation in experimental and clinical tumors. The expression levels of MTA1 protein and the acetylation levels of histone H4 were examined in 70 cases of surgically resected esophageal squamous cell carcinomas, using immunohistochemistry. The intensities of immunostaining of MTA1 protein and acetylated histone H4 in carcinoma tissues (Ca) were compared to normal epithelium (N) contained in the same section. Thirty of 70 cases (42.9%) displayed overexpression of MTA1 protein (N < Ca). Cancers overexpressing MTA1 protein invaded deeper into the esophageal wall (p < 0.005) and showed significantly higher degrees of lymph node metastasis (p < 0.01), higher pathological stage, more lymphatic involvement and poorer prognosis (p < 0.05) than the remaining cases. The acetylation levels of histone H4 inversely correlated to the depth of cancer invasion and pathological stage (p < 0.05), and the patients with higher level of histone H4 acetylation had a better prognosis (p < 0.05). Furthermore, immunostaining patterns of MTA1 and acetylated histone H4 were inversely correlated (p < 0.001), demonstrating the relationship of deacetylation of histone H4 in MTA1-overexpressing carcinomas. In conclusion, the data suggest that the overexpression of MTA1 protein and acetylation level of histone H4 protein, both of which are closely related, might be useful predictors of malignant potential of esophageal squamous cell carcinomas. Thus, strategies involving inhibition of MTA1 function as well as inhibition of histone deacetylation could be novel approaches for the treatment of esophageal squamous cell carcinomas.

Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation.

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines, we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat mta1 gene, we cloned the human MTA1 gene and found it to be over-expressed in a variety of human cell lines (breast, ovarian, lung, gastric and colorectal cancer but not melanoma or sarcoma) and cancerous tissues (breast, esophageal, colorectal, gastric and pancreatic cancer). We found a close similarity between the human MTA1 and rat mta1 genes (88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively). Both genes encode novel proteins that contain a proline rich region (SH3-binding motif), a putative zinc finger motif, a leucine zipper motif and 5 copies of the SPXX motif found in gene regulatory proteins. Using Southern blot analysis the MTA1 gene was highly conserved, and using Northern blot analysis MTA1 transcripts were found in virtually all human cell lines (melanoma, breast, cervix and ovarian carcinoma cells and normal breast epithelial cells). However, the expression level of the MTA1 gene in normal breast epithelial cells was approximately 50% of that found in rapidly growing adenocarcinoma and atypical epithelial cell lines. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in inhibition of growth and invasion of human MDA-MB-231 breast cancer cells with relatively high MTA1 expression. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected with a mammalian expression vector containing a full-length MTA1 gene. Although some MTA1 protein was found in the cytoplasm, the vast majority of MTA1 protein was localized in the nucleus. Examination of recombinate MTA1 and related MTA2 proteins suggests that MTA1 protein is a histone deacetylase. It also appears to behave like a GATA-element transcription factor, since transfection of a GATA-element reporter into MTA1-expressing cells resulted in 10-20-fold increase in reporter expression over poorly MTA1-expressing cells. Since it was reported that nucleosome remodeling histone deacetylase complex (NuRD complex) involved in chromatin remodeling contains MTA1 protein and a MTA1-related protein (MTA2), we examined NuRD complexes for the presence of MTA1 protein and found an association of this protein with histone deacetylase. The results suggest that the MTA1 protein may serve multiple functions in cellular signaling, chromosome remodeling and transcription processes that are important in the progression, invasion and growth of metastatic epithelial cells.

Differential expression and subcellular distribution of the mouse metastasis-associated proteins Mta1 and Mta3

The human metastasis-associated gene ( MTA1) is overexpressed in cell lines and tissues representing metastatic tumors. Here we report cloning of the mouse Mta1 as well as a novel structurally related mouse gene, Mta3. The mouse Mta1 protein shares 94 and 59% homology to the human MTA1 and mouse Mta3 proteins, respectively. Northern blotting analysis using an Mta1 cDNA probe revealed a prevalent 3 kb hybridization signal in all mouse tissues except the skeletal muscle while a smaller ∼1.0 kb mRNA product was also detected in the heart. Mta3 transcripts (∼2 kb) were detected in most tissues with an additional ∼6.2 kb signal detected in the brain. In vitro transcription/translation of the full-length Mta1 and Mta3 cDNAs generated products of the expected molecular masses, i.e. 80 and 60 kDa, respectively. To assess subcellular localization, green fluorescence protein (GFP)-tagged expression constructs of Mta1 and Mta3 and various deletion constructs of GFP-Mta1 were transiently expressed in Balb/MK keratinocytes. GFP-Mta1 was found exclusively in the nucleus while GFP-Mta3 was present in both the nucleus and cytoplasm. Compared to Mta3, the carboxy terminal end of Mta1 contains an additional nuclear localization signal (NLS) and a proline-rich Src homology 3 (SH3) ligand. The results of transient expression experiments of various Mta1 fragments containing these domains in different combinations indicated that nuclear localization of Mta1 depended on the presence of at least one NLS and one SH3 binding site. These SH3 ligands appeared to be functional as they facilitated interaction with the adaptor protein, Grb2, and the Src-family tyrosine kinase, Fyn.

Tumor metastasis‐associated human MTA1 gene: Its deduced protein sequence, localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligonucleotides

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat MTA1 gene we cloned the human MTA1 gene and found it to be overexpressed in a variety of human cell lines. We found a close similarity between the human MTA1 and rat MTA1 genes, as shown by 88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively. Both genes encode novel proteins that contain a proline-rich region (SH3 binding motif), a putative zinc finger motif, a leucine zipper motif, and five copies of the SPXX motif often found in gene regulatory proteins. Using Southern blot analysis, the MTA1 gene was found to be highly conserved among all species examined; and using Northern blot analysis, MTA1 transcripts were found in virtually all cell lines of human origin that were analyzed, including melanoma and breast, cervix and ovarian carcinoma cells and normal breast epithelial cells. However, the expression level of the MTA1 gene in a normal breast epithelial cell was approximately 50% of that found in rapidly growing breast adenocarcinoma cell lines and an atypical mammary cell line. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in growth inhibition of human MDA-MB-231 breast cancer cells with relatively high expression of the MTA1 gene. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected using a mammalian expression vector containing the full-length MTA1 gene. The results suggest that the MTA1 protein may function in cellular signaling processes important in the progression and growth of cancer cells, possibly as a nuclear regulatory factor. J. Cell. Biochem. 79:202–212, 2000. © 2000 Wiley-Liss, Inc.

Tumor metastasis-associated humanMTA1 gene: Its deduced protein sequence, localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligonucleotides

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat MTA1 gene we cloned the human MTA1 gene and found it to be overexpressed in a variety of human cell lines. We found a close similarity between the human MTA1 and rat MTA1 genes, as shown by 88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively. Both genes encode novel proteins that contain a proline-rich region (SH3 binding motif), a putative zinc finger motif, a leucine zipper motif, and five copies of the SPXX motif often found in gene regulatory proteins. Using Southern blot analysis, the MTA1 gene was found to be highly conserved among all species examined; and using Northern blot analysis, MTA1 transcripts were found in virtually all cell lines of human origin that were analyzed, including melanoma and breast, cervix and ovarian carcinoma cells and normal breast epithelial cells. However, the expression level of the MTA1 gene in a normal breast epithelial cell was approximately 50% of that found in rapidly growing breast adenocarcinoma cell lines and an atypical mammary cell line. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in growth inhibition of human MDA-MB-231 breast cancer cells with relatively high expression of the MTA1 gene. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected using a mammalian expression vector containing the full-length MTA1 gene. The results suggest that the MTA1 protein may function in cellular signaling processes important in the progression and growth of cancer cells, possibly as a nuclear regulatory factor.

Sensitivity of Two Enzyme-linked Immunosorbent Assay Tests in Relation to Western Blot in Detecting Human T-Cell Lymphotropic Virus Types I and II Infection among HIV-1 Infected Patients from São Paulo, Brazil

We investigated the presence of human T-cell lymphotropic virus types I and II (HTLV-I and HTLV-II) infections, first searching for specific antibodies in 553 serum samples obtained from HIV-1-infected patients from São Paulo, Brazil. Sera were screened using two enzyme-linked immunosorbent assays (ELISAs): the ELISA-EM (ELISA HTLV-I/II, EMBRABIO, BR), which contains HTLV-I and HTLV-II lysates, and the ELISA-DB [ELISA HTLV-I/II, Diagnostic Biotechnology (DB), Singapore], which contains HTLV-I lysate, and HTLV-I and HTLV-II recombinant env proteins (MTA-1 and K55, respectively). Serum samples showing two positive and/or borderline results were confirmed by Western blot (WB 2.3, DB), which discriminates HTLV-I from HTLV-II. WB analyses disclosed 22 cases (4.0%) of HTLV-I and 34 (6.1%) of HTLV-II seroreactivity; 24 sera had indeterminate antibody profile (4.3%) and 2 specimens showed reactivity to both MTA-1 and K55 env proteins. Using stringent WB criteria and analyzing the population according to risk factors, the prevalence rates of HTLV-I and HTLV-II infections were 11.2% and 16.8% in IV drug users, 3.4% and 5.5% in heterosexual individuals, and 1.4% and 2.2% in homosexual/bisexual men, respectively. A comparison of ELISA and WB results disclosed that both ELISAs were highly sensitive in detecting HTLV-I antibodies, whereas the ELISA-DB showed 82% sensitivity and the ELISA-EM 100% sensitivity in detecting HTLV-II antibodies. PCR analyses conducted on 37 representative cells samples confirmed the presence of HTLV proviral DNA in the majority of concordant serological cases, except in one, which was HTLV-I infected and seroreacted with K55 protein of HTLV-II. Indeed, after PCR, one case of HTLV-I infection and HTLV-II coinfection, and 30% of WB-seroindeterminate or inconclusive cases infected with HTLV-II could be detected. Our data stress high prevalences of both HTLV-I and HTLV-II infections in HIV-1 coinfected i.v. drug users from São Paulo, and suggests that ELISA kits containing only K55 protein as the HTLV-II-specific antigen, may not have the appropriate sensitivity for the detection of HTLV-II infection in this geographic region, pointing out the need of improved screening tests to be used in Brazil.

A novel candidate metastasis-associated gene, mta1, differentially expressed in highly metastatic mammary adenocarcinoma cell lines. cDNA cloning, expression, and protein analyses.

To understand the genes involved in breast cancer invasion and metastasis, we analyzed a novel candidate metastasis-associated gene, mta1, which was isolated by differential cDNA library screening using the 13762NF rat mammary adenocarcinoma metastatic system. Northern blot analyses showed that the mRNA expression level of the mta1 gene was 4-fold higher in the highly metastatic cell line MTLn3 than in the nonmetastatic cell line MTC.4. The mta1 gene was expressed in various normal rat organs, especially in the testis, suggesting its essential normal function. The mRNA expression levels of the human homologue of this gene also correlated with the metastatic potential in two human breast cancer metastatic systems. The full-length mta1 cDNA sequence contained an open reading frame encoding a protein of 703 amino acid residues, and sequence analysis by data base homology search indicated that mta1 is a novel gene. The Mta1 protein contained several possible phosphorylation sites, and a proline-rich amino acid stretch at the carboxyl-terminal end completely matched the consensus sequence for the src homology 3 domain-binding motif. Using antibodies raised against glutathione S-transferase-Mta1 fusion protein or a synthetic oligopeptide, Western blots showed that the molecular mass of the Mta1 protein was approximately 80 kDa, and the levels of the Mta1 protein also correlated with the metastatic potential, results similar to those obtained from the Northern analyses. Thus, the novel gene mta1 may encode a molecule that is functional in normal cells as well as in breast cancer invasion and metastasis.