[Isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris in Yunnan Safari Park].

OBJECTIVE: We studied the isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris by using culture-dependent approaches. METHODS: Fresh fecal samples of healthy Panthera tigris tigris were collected from Yunnan Safari Park. Pretreatment of the samples, isolation media and inhibitors were tested for actinobacteria isolation. 16S rRNA genes of actinobacteria were sequenced and subjected to phylogenetic analysis. RESULTS: The abundance of culturable actinobacteria was 1.10 x 10(8) cfu/g colony forming units (CFU) per gram of feces (wet weight). We obtained 110 purified cultural actinobacterium strains. The analysis based on 16S rRNA gene sequences showed that these strains were distributed in 10 different families and 12 genera of actinobacteria at least, and most of them were non-filamentous, such as Arthrobacter, Dietzia, Kocuria, Corynebacterium and Microbacterium. Streptomyces was the mainly classical filamentous actinobacteria, and up to 64% of total. CONCLUSION: Drying and heating up the fecal samples can greatly increase the rate of the actinobacteria. Many kinds of inhibitors and chemical defined media are suitable for isolation of fecal actinobacteria. The culturable actinobacteria are abundant in Panthera tigris tigris feces. Our study found an effective method to isolate animals' fecal actinobacteria and it's useful for studying and exploiting animals' fecal actinobacteria.

Enteractinococcus coprophilus gen. nov., sp. nov., of the family Micrococcaceae, isolated from Panthera tigris amoyensis faeces, and transfer of Yaniella fodinae Dhanjal et al. 2011 to the genus Enteractinococcus as Enteractinococcus fodinae comb. nov.

A novel actinobacterium, designated strain YIM 100590(T), was isolated from Panthera tigris amoyensis faeces collected from Yunnan Wild Animal Park in Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequence data showed that strain YIM 100590(T) is a member of the family Micrococcaceae. Cells were coccoid to oval (0.7-1.5 µm in diameter) occurring singly or in clusters. Growth was observed at 10-37 °C (optimum 28 °C) and at pH 7.0-11.0 (optimum pH 8.0). The major fatty acids were iso-C(15:0) (32.22%), anteiso-C(15:0) (31.64%) and iso-C(16:0) (17.38%). The peptidoglycan was of A4α type (L-Lys-Gly-L-Glu). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, dimannosyl diacylglycerol, an unknown glycolipid and two unknown phospholipids. The quinone system comprised menaquinones MK-7 (91.9%) and MK-8 (8.3%). The DNA G+C content of strain YIM 100590(T) was 56.2 mol%. Chemotaxonomic data indicated that the strain belongs to the family Micrococcaceae. On the basis of morphological and chemotaxonomic data and phylogenetic analysis, strain YIM 100590(T) is considered to represent a novel species of a new genus within the family Micrococcaceae, for which the name Enteractinococcus coprophilus gen. nov., sp. nov. is proposed. The type strain of Enteractinococcus coprophilus is YIM 100590(T) (=DSM 24083(T)=JCM 17352(T)). Yaniella fodinae DSM 22966(T) was transferred to the new genus as Enteractinococcus fodinae comb. nov. (type strain G5(T)=DSM 22966(T)=JCM 17931(T)=MTCC 9846(T)).

[Diversity and bioactivity of culturable actinobacteria from animal feces].

OBJECTIVES: In order to provide new source for discovering new lead compounds of drugs and other products, the diversity and some bioactivities of culturable actinobacteria in animal feces were studied. METHODS: Five animals' fecal samples were collected from Yunnan Wild Animal Park. The pure cultures of actinobacteria were isolated from these samples by using 5 different media. The 16S rRNA gene sequences of 119 selected strains were determined; the phylogenetic analysis was carried out; and antimicrobial and anti-tumor activities were determined by using agar diffusion method, tumor cell lines k562and HL60 respectively. RESULTS: In total 20 genera of actinobacteria from the 5 animals' feces were identified. Many strains inhibited Bacillus subtilis, Staphylococcus lentus, Mycobacterium tuberculosis, Candida albicans and Aspergillus niger. Some strains presented antitumor activities. Some known secondary metabolites and Sannastatin, a novel macrolactam polyketide glycoside with bioactivities, were isolated and identified. CONCLUSION: Fecal actinobacteria are a new potential source for discovering drug lead and other industry products.

Nonomuraea soli sp. nov., an actinomycete isolated from soil.

A straight-chain, spore-forming actinobacterium, strain YIM 120770(T), was isolated from soil. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparisons revealed that the isolate represents a distinct cluster within the clade comprising the genus Nonomuraea and is related most closely to Nonomuraea rhizophila YIM 67092(T) (96.5% similarity). Cells of strain YIM 120770(T) grew in the presence of 0-3% (w/v) NaCl, at 15-37 °C and at pH 7.0-8.0. The diagnostic amino acid was meso-diaminopimelic acid, cell hydrolysates contained madurose, glucose, mannose, ribose and galactose, the predominant cellular fatty acids were 10-methyl C(17:0) and iso-C(16:0), and the DNA G+C content was 66.4 mol%, data consistent with affiliation of strain YIM 120770(T) to the genus Nonomuraea. Strain YIM 120770(T) shared low levels of 16S rRNA gene sequence similarity (<97%) with the type strains of recognized species of the genus Nonomuraea and could be differentiated from its closest phylogenetic relative based on phenotypic characteristics. These results suggested that strain YIM 120770(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea soli sp. nov. is proposed. The type strain is YIM 120770(T) (=DSM 45533(T)=JCM 17347(T)).

Methylobacterium soli sp. nov. a methanol-utilizing bacterium isolated from the forest soil.

A Gram-negative, pink-pigmented, non-spore-forming rod shaped, methanol-utilizing bacterium, strain YIM 48816(T), was isolated from forest soil collected from Sichuan province, China. Strain YIM 48816(T) can grow at 4-37 °C, pH 5.0-7.0 and 0% NaCl (w/v). Based on 16S rRNA gene sequence similarity studies, it belonged to the genus Methylobacterium, and formed a phyletic line. The 16S rRNA gene sequence similarities were 96.2% to Methylobacterium mesophilicum DSM 1708(T) and 96.0% to Methylobacterium brachiatum DSM 19569(T), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 96.0%. The major menaquinones detected were Q-10 (97.14%) and Q-9 (2.86%). The major fatty acids were C18:1 ω7c (80.84%). The DNA G + C content was 66.2 mol%. It is apparent from the genotypic and phenotypic data that strain YIM 48816(T) belongs to a novel species of the genus Methylobacterium, for which the name Methylobacterium soli sp. nov. is proposed. The type strain is YIM 48816(T) (CCTCC AA 208027(T) = KCTC 22810(T)).

Planosporangium mesophilum sp. nov., isolated from rhizosphere soil of Bletilla striata.

A Gram-stain-positive, non-motile actinomycete, designated strain YIM 48875(T), was isolated from rhizosphere soil of Bletilla striata and its taxonomic position was established by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence data showed that strain YIM 48875(T) belonged to the genus Planosporangium, supported by a bootstrap value of 100 %. Cells of strain YIM 48875(T) showed two kinds of sporangia, which also supported its classification in the genus Planosporangium. Strain YIM 48875(T) grew optimally at 28 °C, at pH 6.0-8.0 and in the presence of 2 % (w/v) NaCl. The level of 16S rRNA gene sequence similarity between strain YIM 48875(T) and Planosporangium flavigriseum YIM 46034(T) was 98.6 %. Strain YIM 48875(T) exhibited a quinone system with menaquinones MK-9(H(4)), MK-9(H(6)) and MK-9(H(8)) as the predominant compounds, a polar lipid profile comprising diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol mannoside and the major fatty acids iso-C(15 : 0) and iso-C(16 : 0); these data were markedly different from those for P. flavigriseum YIM 46034(T). The level of DNA-DNA relatedness between strain YIM 48875(T) and P. flavigriseum YIM 46034(T) was 45.5 %. It is apparent from the genotypic and phenotypic data that strain YIM 48875(T) represents a novel species of the genus Planosporangium, for which the name Planosporangium mesophilum sp. nov. is proposed. The type strain is YIM 48875(T) ( = CCTCC AA 209049(T)  = KCTC 19779(T)).

[Diversity of cultured actinomycete in the Baltic Sea].

OBJECTIVE: Actinomycetes (actinobacteria) are getting more and more recognized as a natural source for new drug exploration. In order to find new lead compounds, the diversity and selected bioactivities of cultured actinomycetes in the Baltic Sea (Germany) were investigated. METHODS: One hundred sediment samples were collected from south of the Baltic Sea, of which 809 purified cultures of actinomycetes were obtained by using 7 media. The phylogenetic analysis of 280 selected strains based on 16S rRNA gene sequences was carried out. In addition, activities of 21 enzymes, which play a role in metabolic processes, and anti-microbial activities were determined. RESULTS: Fifteen genera and eight possible new species of actinobacteria were identified. Members of 3 genera were not isolated from marine habitats before. Of the 280 strains 21% and 20% inhibited the growth of Bacillus subtilis and Staphylococcus lentus, respectively. More than 75% of the strains exhibited 8 types of enzymatic activities, including esterase lipase (C8), catalase and naphthol-AS-BI-phosphohydrolase. CONCLUSION: Baltic Sea provides a rich diversity of actinobacteria regarding the phylogenetic analysis and the biological activities. Research and utilization of marine actinomycetes should be strengthened.

[Diversity and bioactivity of cultured actinomycetes in Sichuan and Yunnan area].

OBJECTIVE: In order to discover new leader compounds, the diversity and some bioactivities of cultured actinomycetes from Sichuan and Yunnan were studied. METHODS: In total 250 soil samples were collected from forest of Huangjin, Emei and Qingcheng Mountains, Jiuzhaigou in Sichuan, and Xishuangbanna in Yunnan. Actinomycetes in these samples were isolated and identified. Bioactivities of isolated strains were determined. RESULTS: In total 2676 strains of actinomycetes were isolated from these samples. The 16S rRNA gene sequences of 98 selected strains were determined, and the phylogenetic analysis was carried out. 13, 5, 9 and 20 genera were identified from Forest of Huangjin, Emei and Qingcheng Mountains (one sample area), Jiuzhaigou, and Xishuangbanna respectively. The diversity of Xishuangbanna was the richest. That of Emei and Qingcheng Mountains was monotone, and only five genera were isolated. Antimicrobial activities of 169 selected strains against 11 bacteria and fungi were tested using agar well diffusion method, and genes of type I and II polyketide synthases (PKS I, PKS II), non-ribosomal peptide synthase (NRPS) and polygene cytochrome P450 hydroxylase (CYP) were detected by PCR. High rate of antimicrobial activity and the synthesis genes of four antibiotic existed in these actinomyctes. CONCLUSION: In sample-collecting areas, the poorer human beings disturbance, the richer the diversity of actinomycetes. For isolation of actinomycetes, author advocate using of " extreme" conditions, although it may got small number of actinomycetes, but the proportion of unknown actinomycetes was greater. Gram-negative bacteria and fungi could be inhibited by adding inhibitors in media.

[Ultrasonic treatment of soil samples for actinomycete isolation].

OBJECTIVE: We used ultrasonic to treat soil samples to isolate rare actinomycetes strains. METHODS: We collected and suspended soil samples from mix tropical-rain-forest in Xishuangbanna. Farther, we treated the soil suspension with ultrasound for 10 to 120 s and used the plate dilution method to isolate actinomycetes. After got pure colonies, we sequenced their 16S rRNA gene sequences and calculated to put in phylogenetic analysis. The isolates were identified to the genus. We treated 10 kinds of common and identified Streptomyces with 1 - 5 min using ultrasonic, and then were cultured to measure their survival rate. RESULTS: The soil suspensions treated with different times by the ultrasonic, actinomycetes gradual increased in the number and types. Ultrasonic treating of known Streptomyces with 0-5 min, there was no significant effect on the survival number of Streptomyces. CONCLUSION: The ultrasonic treating with soil suspensions for 40 s can significantly increase the total number of actinomycetes and the types of rare actinomycetes. Thereby, it is an economic and simple method to apparently increase the types of rare actinomycetes in the isolation.

12-Lipoxygenase and the regulation of hypoxia-inducible factor in prostate cancer cells.

12-Lipoxygenase, an arachidonic acid metabolizing enzyme of the lipoxygenase pathway, has been implicated as a major factor in promoting prostate cancer progression and metastasis. The ability of 12-LOX to aggravate the disease was linked to its proangiogenic role. Recent studies clearly demonstrated that 12-LOX enhances the expression and secretion of the angiogenic factor, vascular endothelial growth factor (VEGF) thus providing a direct link between this enzyme and its angiogenic properties. In the present study we have investigated the relationship between 12-LOX and hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor involved in the regulation of VEGF expression under hypoxic conditions in solid tumors. Our findings have revealed that HIF-1 is one of the target transcription factors regulated by 12-LOX and 12(S)-HETE, in hypoxic tumor cells of the prostate. Regulation of HIF-1alpha by 12-LOX adds to the complexity of pathways mediated by this enzyme in promoting prostate cancer angiogenesis and metastasis. We have evidence that 12-LOX increases the protein level, mRNA, and functional activity of HIF-1alpha under hypoxic conditions, one of the mechanisms by which it upregulates VEGF secretion and activity.

Significance of SHP-1 and SHP-2 expression in human papillomavirus infected Condyloma acuminatum and cervical cancer.

Human papillomaviruses (HPVs) are a group of DNA viruses that infect the skin and mucous membranes. Type HPV6/11 is closely related to Condyloma acuminatum, while HPV16/18 is the principal cause of cervical cancer. In this study, we examined the expression of protein tyrosine phosphatases SHP-1 and SHP-2 in Condyloma acuminatum, cervical cancer and the relationship between SHP-1/SHP2 expression and HPV infection. Forty Condyloma acuminatum cases, 20 cervical cancer cases and 20 normal human foreskins were examined for HPV infection by in situ hybridization and the expression of SHP-1 and SHP-2 were examined by immunohistochemistry. Results demonstrated that positive expression rates of HPV6/11, HPV16/18, and HPV31/33 were 98%, 10%, and 7.5% in Condyloma acuminatum, 10%, 85%, and 25% in cervical cancer. Only one normal foreskin demonstrated positive staining for HPV16/18. Positive expression rates of SHP-1 and SHP-2 were 80% and 85% in Condyloma acuminatum, 85% and 90% in cervical cancer. The SHP-1 and SHP-2 expressions were mainly distributed in the prickle layer of Condyloma acuminatum and were diffusely distributed in cervical cancer cells. Only 35% and 30% of foreskins demonstrated weak staining in the basal layer cells. There were statistically significant correlations among the infection of HPV and the expression of SHP-1 and SHP-2 in both Condyloma acuminatum and cervical cancer (P < 0.05). SHP-1 expression has a positive correlation with SHP-2 expression. Our results demonstrate putative roles of SHP-1 and SHP-2 in the progression of both Condyloma acuminatum and cervical cancer after HPV infection.

A naturally occurring truncated beta3 integrin in tumor cells: native anti-integrin involved in tumor cell motility.

Alternatively spliced integrins may play an important role in integrin mediated tumor cell adhesion, spreading, and migration. Here we report in human tumor cells a naturally occurring alternatively spliced variant of the beta3 integrin [i.e., truncated (tr) beta3] that lacked a cytoplasmic and a transmembrane domain. The presence of trbeta3 was demonstrated at the mRNA level by RT-PCR, cloning, and sequencing; at the protein level by immunohistochemistry and Western Blotting. The alternately spliced beta3 integrin was detected in human prostate carcinomas, breast carcinomas, and melanoma cells. Expression in vivo was confirmed by immunohistochemistry with an antibody to trbeta3 that does not recognize wild type beta3. Tumor cells secreted this protein and deposited it on the extracellular matrix. Secreted trbeta3 inhibited adhesion of melanoma and prostate cancer cells to fibronectin and vitronectin, which was partially reversed by adsorption of trbeta3 from the media. Confocal microscopy and time lapse live cell microscopy demonstrated that trbeta3 distributed to the trailing edge of migrating cells, which may represent an alternative cell detachment mechanism in these cells. Results suggest that trbeta3 may act as an anti-integrin and play a crucial role in cell migration, which is an important process in tumor invasion and metastasis.

Metallothionein as a prognostic biomarker in breast cancer.

Breast cancer is the most common cancer in women, with a general upward trend in incidence. Basic and clinical breast cancer research has continued at a rapid pace, in the endeavor to understand the biology of the disease so as to improve management of patients. Besides traditional pathological indicators, expression of molecular markers in breast cancer has also been comprehensively investigated. This paper will focus on the prognostic utility of metallothioneins (MTs), a family of low molecular weight metal binding proteins encoded by at least 10 functional MT genes that are associated with cell proliferation in breast cancer. Evidence that MT is a potential prognostic biomarker for breast cancer is supported by many reports in the literature. Expression of the MT protein has been detected by immunohistochemistry in a significant portion of invasive ductal breast cancers. MT expression has also been well studied in association with traditional clinico-pathological parameters of breast cancers. Generally, higher MT expression in breast cancers is predictive of worse patient outcomes. The relationship of MT isoforms to histological grade, estrogen receptor (ER) status, and prognosis will also be discussed.

Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells.

12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.

Clinicopathological significance of metallothioneins in breast cancer.

Metallothioneins (MTs) are a family of metal binding proteins that play an important role in maintaining transition metal ion homoeostasis, redox balance in the cell and fundamental cellular processes such as proliferation and apoptosis. In humans, there are 4 groups of MT proteins which are encoded by 10 functional MT isoforms. In breast tissues, MT is primarily expressed in myoepithelial and malignant epithelial cells. Immunohistochemical studies have revealed that 26% to 100% of invasive ductal breast cancers express the MT protein. The MT-1F and MT-2A isoforms have been reported to be associated with higher histological grade in breast cancer, whereas higher MT-1E mRNA expression was found in estrogen receptor-negative tumors compared to their estrogen receptor-positive counterparts. A number of studies have shown that MT expression in breast cancer is associated with poorer prognosis. In addition, metallothionein expression may have a potential role in protecting the breast cancer cell from chemotherapeutic threats to survival.

Differential expression of metallothionein 1 and 2 isoforms in breast cancer lines with different invasive potential: identification of a novel nonsilent metallothionein-1H mutant variant.

Metallothionein (MT), a low-molecular weight protein with pleiotropic functions, is believed to play an important role in tumorigenesis. The aim of this study was to compare the expression of functional MT-1 and MT-2 mRNA isoforms in five breast cancer cell lines ranging from noninvasive MCF7 breast cancer cells to highly aggressive MDA-MB-231 breast cancer cells together with breast myoepithelial cells in vitro by conventional semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The MT-2A isoform was observed to be differentially upregulated in the invasive phenotype. The MT-1E isoform was found to be present in estrogen receptor-negative breast cancer cell lines (MDA-MB-231 and Hs578T) but not detectable in the estrogen receptor-positive cell lines (T47D, MCF7, and ZR75-1 cells). Only the myoepithelial cells exhibited the presence of the MT-1G transcript. Direct sequencing of the RT-PCR products revealed the occurrence of a variant MT-1H isoform with changes in amino acid residues in the protein sequence and notable differences in the predicted secondary protein structure. The observations in this study are relevant to the development of novel approaches to metastatic breast cancer disease, and may herald the search for novel MT mutants and the elucidation of their biological roles.

Expression of the FAT10 gene is highly upregulated in hepatocellular carcinoma and other gastrointestinal and gynecological cancers.

The ubiquitin-like modifier (UBL) family has recently generated much interest in the scientific community, as it is implicated to play important regulatory roles via novel protein-protein modification. FAT10 (diubiquitin) belongs to this family of proteins, comprising two ubiquitin-like moieties fused in tandem, and has been implicated to be involved in the maintenance of spindle integrity during mitosis. As FAT10 may play a role in the regulation of genomic stability, we examined if there is an association between FAT10 expression and hepatocellular carcinoma (HCC) or other cancers. Northern blot analyses revealed upregulation of FAT10 expression in the tumors of 90% of HCC patients. In situ hybridization as well as immunohistochemistry utilizing anti-FAT10 antibodies localized highest FAT10 expression in the nucleus of HCC hepatocytes rather than the surrounding immune and non-HCC cells. FAT10 expression was also found to be highly upregulated in other cancers of the gastrointestinal tract and female reproductive system. In conclusion, we demonstrated upregulation of FAT10 expression in various gastrointestinal and gynecological cancers. Its overexpression is unrelated to the general increase in protein synthesis or a general immune/inflammatory response to cancer. Rather, FAT10 may modulate tumorigenesis through its reported interaction with the MAD2 spindle-assembly checkpoint protein.

Progesterone induces cellular differentiation in MDA-MB-231 breast cancer cells transfected with progesterone receptor complementary DNA.

Progesterone is an important regulator of growth and differentiation in breast tissues. In this study, the effect of progesterone on cell differentiation was evaluated in the estrogen receptor-negative and progesterone receptor (PR)-negative MDA-MB-231 cell line which was transfected with PR-complementary DNA. Morphological changes were analyzed at the ultrastructural level by scanning and transmission electron microscopy. Progesterone-treated PR-transfected cells exhibited a more protracted and well spread morphology with an increase in organelles such as mitochondria and rough endoplasmic reticulum as compared to the rounded form of control vehicle (0.1% ethanol)-treated PR-transfected cells. Vehicle and progesterone-treated MDA-MB-231 cells transfected with the pSG5 plasmid (transfection control cells) had similar rounded morphology as control vehicle-treated PR-transfected cells. Immunofluorescence staining revealed that expression of E-cadherin, a differentiation marker, was more prominent in progesterone-treated cells. Expression of keratin and vimentin but not beta-catenin was up-regulated in progesterone treated cells when evaluated by immunoblotting. As signal transducers and activators of transcription (STAT) molecules have been implicated in mammary differentiation, we analyzed the expression of Stat 1, 3, 5a, and 5b proteins and found a significant up-regulation of the Stat 5b protein in progesterone-treated cells. We have provided in vitro evidence of the close association of PR with differentiation in breast cancer. It is likely that the Stat 5b protein may play a major role in progesterone-induced differentiation in breast cancer cells.

Metallothionein 2A expression is associated with cell proliferation in breast cancer.

Metallothioneins (MTs) belong to a family of cysteine-rich, metal-binding intracellular proteins, which have been linked with cell proliferation. In this study, expression levels of the 8 known MT-1 and MT-2 functional isoforms in human invasive ductal breast cancer specimens were determined by RT-PCR. The expression profiles of the MT protein and MT-2A mRNA were further evaluated in 79 cases of human invasive ductal breast carcinoma by immunohistochemistry and in situ hybridization, and correlated with cancer cell proliferation (determined by Ki-67 nuclear antigen immunolabeling). MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X and MT-2A but not MT-1B, were detected in breast cancer tissue samples. The MT-2A mRNA transcript was the highest among all the isoforms detected. A positive correlation was observed between MT-2A mRNA and MT protein expression with Ki-67 labeling (P = 0.0003 and P < 0.0001, respectively) but not with apoptosis (P = 0.1244 and P = 0.8189, respectively). Co-localization of the MT protein and Ki-67 nuclear antigen in breast cancer cells was demonstrated by double immunofluorescence staining. There was also significantly higher MT protein and MT-2A mRNA expression in histological grade 3 tumors than in histological grade 1 and 2 tumors. The finding that MT 2A appears to be the main isoform associated with cell proliferation in invasive ductal breast cancer tissues, may have therapeutic implications.