MTA1 Research Articles

Relationship between the expression of MTA-1 gene and the metastasis and invasion in human osteosarcoma

To compare the expression level of metastasis associated-1 (MTA1) gene in high and low metastatic human osteosarcoma cell lines and examine the relationship of MTA1 expression and the metastasis potentiality of osteosarcoma cells, the expression of MTA1 in MG-63 osteosarcoma cell lines with high and low metastasis potential was detected by semiquantitative TR-PCR. Boyden chamber invasion assay was used to evaluate the invasive capacity in vitro in two osteosarcoma cell lines. The low metastasis MG-63 cells were transfected with MTA1 full-length cDNA expression plasmid by lipofectamine and the changes of MTA1 expression and in vitro invasion potential were examined after the transfection. Our results showed that MG63 cell line with high metastasis potential expressed significantly higher MTA1 than that of MG63 cells with low metastasis as reavealed by RT-PCR. The invasion potential of low metastasis MG63 cell line was increased after MTA1 gene transfection. It is concluded that there may be a relationship between MTA 1 and invasive potentiality of human osteosarcoma cells, and the mechanism of MTA1 in osteosarcoma metastasis and its possible role in associated gene therapy deserve further study.

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.

Repression of 92-kDa Type IV Collagenase Expression by MTA1 Is Mediated through Direct Interactions with the Promoter via a Mechanism, Which Is Both Dependent on and Independent of Histone Deacetylation

Although the expression of the metastases-associated gene MTA1 correlates with tumor metastases, its role in regulating type IV collagenase expression is unknown. Enforced MTA1 expression in HT1080 cells reduced basal and 12-myristate 13-acetate-induced 92-kDa type IV collagenase (MMP-9) protein/mRNA levels. DNase I hypersensitivity and PstI accessibility assays revealed multiple regions of the MMP-9 promoter (-650/-450 and -120/+1), showing reduced hypersensitivity in the MTA1-expressing cells. Chromatin immunoprecipitation assays demonstrated MTA1 binding to the distal region, which spans several regulatory cis elements. Co-immunoprecipitation and chromatin immunoprecipitation assay experiments revealed histone deacetylase 2 (HDAC2)-MTA1 protein-protein interactions and the MTA1-dependent recruitment of HDAC2 to the distal MMP-9 promoter region, yielding diminished histone H3/H4 acetylation. However, HDAC2 binding and H3/H4 acetylation at the proximal MMP-9 region were unaffected by MTA1 expression. Furthermore, trichostatin treatment only partially relieved MTA1-repressed MMP-9 expression, indicating a HDAC-insensitive component possibly involv ing the nucleosome-remodeling Mi2 activity, which was recruited to the promoter by MTA1. In summary, (a) MMP-9 adds to a short list of MTA1-regulated genes, which so far only includes c-myc and pS2, and (b) MTA1 binds to the MMP-9 promoter, thereby repressing expression of this type IV collagenase via histone-dependent and independent mechanisms.

Expression of the MTA1 mRNA in advanced lung cancer

The MTA1 gene is a recently identified metastasis-associated gene which has been implicated in the signal transduction or regulation of gene expression. We examined the mRNA expression levels of the MTA1, the human homologue of the rat mta1 gene in non-small cell lung cancer (NSCLC). Expression of MTA1 messenger RNA was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in 74 non-small cell lung carcinoma samples using LightCycler. The data was analyzed in reference to clinicopathological data. There was no relationship between MTA1 gene expression and age and gender. MTA1/GAPDH mRNA level in stage II–IV NSCLC (3.465±3.675) was significantly higher than the level in stage I NSCLC (1.614±2.434, P=0.0153). MTA1/GAPDH mRNA levels in T4 NSCLC (4.377±4.169) was significantly higher than the level in T1 NSCLC (1.966±2.148, P=0.0351) and in T2 NSCLC (2.048±1.899, P=0.0269), respectively. MTA1/GAPDH mRNA level in NSCLC with lymph node metastasis (4.242±3.758) was significantly higher in NSCLC without lymph node metastasis ( P=0.0169). Our results show that the expression of the MTA1 gene is closely related to invasiveness and metastasis in NSCLC. The gene MTA1 could thus potentially provide information on the mechanism of cancer invasion and metastasis.

Expression of the MTA1 mRNA in thymoma patients

The MTA1 gene is a recently identified metastasis-associated gene which has been implicated in the signal transduction or regulation of gene expression. We examined the mRNA expression levels of the MTA1, the human homologue of the rat mta1 gene in thymoma. Expression of MTA1 mRNA was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in 30 thymoma samples using LightCycler. The data was analyzed in reference to clinicopathological data. There was no relationship between MTA1 gene expression and age and gender. MTA1/GAPDH mRNA level in stage IV thymoma (6.431±3.404) was significantly higher than the level in stage I thymoma (2.592±1.902, P=0.0081). There was a tendency towards higher MTA1/GAPDH mRNA level in stage IV thymoma when compared to stage II thymoma (3.746±3.292, P=0.072). Thus our results show that the expression of the MTA1 gene is closely related to invasiveness in thymoma. The gene MTA1 could potentially provide information on the mechanism of tumor invasion and metastasis.

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.

A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat.

Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas a cDNA coding for the 29 kDa protein (ZG29p) was identified by immunoscreening of a hormonally stimulated pancreas cDNA library. Western blot analysis suggests that ZG29p is a pancreas-specific protein and immunofluorescence shows that ZG29p is mainly associated with zymogen granules. Analysis of subcellular fraction applying immunoblotting revealed that ZG29p was localized mainly in the soluble fraction of zymogen granules and in a Golgi- and RER-enriched fraction, but was absent from the cytosol. In isolated zymogen granule content ZG29p was associated with protein complexes containing amylase as main constituent. The cDNA coding for ZG29p is homologous to the C-terminal region of the candidate metastasis-associated gene mta1. Northern blot analysis and RT-PCR showed that no MTA1 mRNA is present in pancreas from fasted rats and in the rat pancreas carcinoma cell line AR4-2J in its protodifferentiated state. Although no ZG29p specific mRNA was seen in the northern blot analysis, RT-PCR showed that ZG29p was expressed under both non-stimulated and stimulated conditions. The expression of MTA1 was up-regulated in the pancreas by endogenous cholecystokinin release and in AR4-2J after induction of cellular differentiation by dexamethasone. Western blotting and immunofluorescense studies indicated that MTA1p is localized in the nucleus in all tissues studied. Using genomic DNA in PCR analysis it was shown that two short introns are present flanking the sequences of the 5'end of ZG29p cDNA. One intron contains consensus elements required for pancreas specific transcription initiation, suggesting that MTA1 and ZG29 are differentially expressed by alternative transcription initiation in the pancreas. The localisation of MTA1p in the nucleus of most cell types could signify a general role in gene regulation, while the cell type specific and exclusive expression of ZG29p in pancreatic acinar cells could indicate a role in granule formation.

Overexpression of metastasis-associated MTA1 mRNA in invasive oesophageal carcinomas

The MTA1 gene is a recently identified novel candidate breast cancer metastasis-associated gene which has been implicated in the signal transduction or regulation of gene expression. We examined the mRNA expression levels of the MTA1, the human homologue of the rat mta1 gene in 47 surgically resected oesophageal squamous cell carcinomas by quantitative reverse transcription polymerase chain reaction. The relative overexpression of MTA1 mRNA (tumour/normal ratio ≥ 2) was observed in 16 out of 47 (34.0%) oesophageal carcinomas. Oesophageal tumours overexpressing MTA1 mRNA (T/N ratio ≥ 2) showed significantly higher frequencies of adventitial invasion (P < 0.05) and lymph node metastasis (P < 0.05), and tended to have a higher rate of lymphatic involvement than the remaining tumours. Thus, the data suggest that the MTA1 gene might play an important role in invasion and metastasis of oesophageal carcinomas. © 1999 Cancer Research Campaign

Overexpression of the MTA1 gene in gastrointestinal carcinomas: Correlation with invasion and metastasis

The mta1 gene is a recently identified novel candidate metastasis-associated gene. The deduced amino acid sequence contains an src homology-3 domain binding motif, a zinc finger motif and possible phosphorylation sites, suggesting that this gene is involved in signal transduction or regulation of gene expression. The purpose of our study was to examine the mRNA expression levels of the MTA1, the human homologue of the rat mta1 gene in colorectal and gastric carcinomas and thus to evaluate the relevance of the expression of this gene to human carcinoma progression. The expression of MTA1 mRNA in 36 colorectal and 34 gastric carcinoma samples was compared with that in corresponding normal mucosa tissues by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and the results were compared with clinico-pathologic data. A relative overexpression of MTA1 mRNA (tumor/normal ratio ≥2) was observed in 14 of 36 (38.9%) colorectal carcinomas and 13 of 34 (38.2%) gastric carcinomas. Clinico-pathologic correlations demonstrated that in colorectal carcinomas, tumors overexpressing MTA1 mRNA exhibited a significantly deeper wall invasion and a higher rate of metastasis to lymph nodes, and tended to be at an advanced Dukes' stage with frequent lymphatic involvement. In gastric carcinomas, the tumors overexpressing MTA1 mRNA showed significantly higher rates of serosal invasion and lymph node metastasis and tended to have a higher rate of vascular involvement. Our data suggest that overexpression of the MTA1 gene correlates with tumor invasion and the presence of metastases and that a high expression of MTA1 mRNA may be a potential indicator for assessing the malignant potential of colorectal and gastric carcinomas. Int. J. Cancer 74:459–463, 1997. © 1997 Wiley-Liss, Inc.

Overexpression of theMTA1 gene in gastrointestinal carcinomas: Correlation with invasion and metastasis

The mta1 gene is a recently identified novel candidate metastasis-associated gene. The deduced amino acid sequence contains an src homology-3 domain binding motif, a zinc finger motif and possible phosphorylation sites, suggesting that this gene is involved in signal transduction or regulation of gene expression. The purpose of our study was to examine the mRNA expression levels of the MTA1, the human homologue of the rat mta1 gene in colorectal and gastric carcinomas and thus to evaluate the relevance of the expression of this gene to human carcinoma progression. The expression of MTA1 mRNA in 36 colorectal and 34 gastric carcinoma samples was compared with that in corresponding normal mucosa tissues by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and the results were compared with clinico-pathologic data. A relative overexpression of MTA1 mRNA (tumor/normal ratio > or = 2) was observed in 14 of 36 (38.9%) colorectal carcinomas and 13 of 34 (38.2%) gastric carcinomas. Clinico-pathologic correlations demonstrated that in colorectal carcinomas, tumors overexpressing MTA1 mRNA exhibited a significantly deeper wall invasion and a higher rate of metastasis to lymph nodes, and tended to be at an advanced Dukes' stage with frequent lymphatic involvement. In gastric carcinomas, the tumors overexpressing MTA1 mRNA showed significantly higher rates of serosal invasion and lymph node metastasis and tended to have a higher rate of vascular involvement. Our data suggest that overexpression of the MTA1 gene correlates with tumor invasion and the presence of metastases and that a high expression of MTA1 mRNA may be a potential indicator for assessing the malignant potential of colorectal and gastric carcinomas.

Species-specific and mating type-specific DNA regions adjacent to mating type idiomorphs in the genus Neurospora.

Mating type idiomorphs control mating and subsequent sexual development in Neurospora crassa and were previously shown to be well conserved in other Neurospora species. The centromere-proximal flanks of the A and a idiomorphs, but not the distal flanks from representative heterothallic, pseudohomothallic, and homothallic Neurospora species contain apparent species-specific and/or mating type-specific sequences adjacent to the well-conserved idiomorphs. The variable flank is bordered by regions that are highly homologous in all species. The sequence of approximately 1 kb immediately flanking the conserved idiomorphs of each species was determined. Sequence identity between species ranged from 20% (essentially unrelated) to > 90%. By contrast, the mt-A1 gene shows 88-98% identity. Sequence and hybridization data also show that the centromere-proximal flanks are very different between the two mating types for N. intermedia, N. discreta, and N. tetrasperma, but not for N. sitophila and N. crassa. The data suggest a close evolutionary relationship between several of the species; this is suppported by phylogenetic analysis of their respective mt-A1 genes. The origin of the variable regions adjacent to the evolutionarily conserved mating type idiomorphs is unknown.

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.