TOB1 Research Articles

TOB1 inhibits the gastric cancer progression by focal adhesion pathway and ERK pathway based on transcriptional and metabolic sequencing

Gastric cancer is one of the most malignant digestive tract tumors worldwide and its progression is associated with gene expression and metabolic alteration. We revealed that the gastric cancer patients with lower expression level of TOB1 exhibited poorer overall survivals according to the data in Kaplan–Meier Plotter. The unphosphorylated TOB1 protein which is effective expressed lower in gastric cancer cells. The gastric cancer cells with TOB1 gene depletion performed higher abilities of proliferation, migration and invasion and lower ability of apoptosis in vitro. The TOB1 gene depletion also promoted the tumorigenesis of gastric cancer cells in vivo. The gastric cancer cells with TOB1 gene overexpression had the converse behaviors. The transcriptional and metabolic sequencing was performed. The analyzation results showed that genes correlate-expressed with TOB1 gene were enriched in the pathways related to ERK pathway, including focal adhesion pathway, which was verified using real-time quantitative PCR. After inhibiting ERK pathway, the proliferation, colony formation and migration abilities were reduced in gastric cancer cells with low phosphorylated TOB1 protein expression level. Moreover, Pearson correlation analysis was adopted to further analyze the correlation of enriched metabolic products and differentially expressed genes. The expression of Choline, UDP-N-acetylglucosamine, Adenosine and GMP were related to the function of TOB1. This study demonstrates the genes and metabolites related to focal adhesion pathway and ERK pathway are the potential diagnosis and therapeutic targets to gastric cancer with TOB1 depletion.

TOB1 modulates neutrophil phenotypes to influence gastric cancer progression and immunotherapy efficacy.

The ErbB-2.1(TOB1) signaling transducer protein is a tumor-suppressive protein that actively suppresses the malignant phenotype of gastric cancer cells. Yet, TOB1 negatively regulates the activation and growth of different immune cells. Understanding the expression and role of TOB1 in the gastric cancer immune environment is crucial to maximize its potential in targeted immunotherapy. This study employed multiplex immunofluorescence analysis to precisely delineate and quantify the expression of TOB1 in immune cells within gastric cancer tissue microarrays. Univariate and multivariate Cox analyses were performed to assess the influence of clinical-pathological parameters, immune cells, TOB1, and double-positive cells on the prognosis of gastric cancer patients. Subsequent experiments included co-culture assays of si-TOB1-transfected neutrophils with AGS or HGC-27 cells, along with EdU, invasion, migration assays, and bioinformatics analyses, aimed at elucidating the mechanisms through which TOB1 in neutrophils impacts the prognosis of gastric cancer patients. We remarkably revealed that TOB1 exhibits varying expression levels in both the nucleus (nTOB1) and cytoplasm (cTOB1) of diverse immune cell populations, including CD8+ T cells, CD66b+ neutrophils, FOXP3+ Tregs, CD20+ B cells, CD4+ T cells, and CD68+ macrophages within gastric cancer and paracancerous tissues. Significantly, TOB1 was notably concentrated in CD66b+ neutrophils. Survival analysis showed that a higher density of cTOB1/nTOB1+CD66b+ neutrophils was linked to a better prognosis. Subsequent experiments revealed that, following stimulation with the supernatant of tumor tissue culture, the levels of TOB1 protein and mRNA in neutrophils decreased, accompanied by enhanced apoptosis. HL-60 cells were successfully induced to neutrophil-like cells by DMSO. Neutrophils-like cells with attenuated TOB1 gene expression by si-TOB1 demonstrated heightened apoptosis, consequently fostering a malignant phenotype in AGS and HCG-27 cells upon co-cultivation. The subsequent analysis of the datasets from TCGA and TIMER2 revealed that patients with high levels of TOB1 combined neutrophils showed better immunotherapy response. This study significantly advances our comprehension of TOB1's role within the immune microenvironment of gastric cancer, offering promising therapeutic targets for immunotherapy in this context.

Expression of DNAJB9 and some other genes is more sensitive to SWCNTs in normal human astrocytes than glioblastoma cells.

Objective. Single-walled carbon nanotubes (SWCNTs) are considered to be one of the nanomaterials attractive for biomedical applications, particularly in the health sciences as imaging probes and drug carriers, especially in the field of cancer therapy. The increasing exploitation of nanotubes necessitates a comprehensive evaluation of the potential impact of these nanomaterials, which purposefully accumulate in the cell nucleus, on the human health and the function of the genome in the normal and tumor tissues. The aim of this study was to investigate the sensitivity of the expression of DNAJB9 and some other genes associated with the endoplasmic reticulum (ER) stress and cell proliferation to low doses of SWCNTs in normal human astrocytes (NHA/TS) and glioblastoma cells (U87MG) with and without an inhibition of ERN1 signaling pathway of the ER stress. Methods. Normal human astrocytes, line NHA/TS and U87 glioblastoma cells stable transfected by empty vector or dnERN1 (dominant-negative construct of ERN1) were exposed to low doses of SWCNTs (2 and 8 ng/ml) for 24 h. RNA was extracted from the cells and used for cDNA synthesis. The expression levels of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 mRNAs were measured by a quantitative polymerase chain reaction and normalized to ACTB mRNA. Results. It was found that the low doses of SWCNTs up-regulated the expression of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 genes in normal human astrocytes in dose-dependent (2 and 8 ng/ml) and gene-specific manner. These nanotubes also increased the expression of most studied genes in control (transfected by empty vector) U87 glioblastoma cells, but with much lesser extent than in NHA/TS. However, the expression of CLU gene in control U87 glioblastoma cells treated with SWCNTs was down-regulated in a dose-dependent manner. Furthermore, the expression of TOB1 and P4HA2 genes did not significantly change in these glioblastoma cells treated by lower dose of SWCNTs only. At the same time, inhibition of ERN1 signaling pathway of ER stress in U87 glioblastoma cells led mainly to a stronger resistance of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, and P4HA2 gene expression to both doses of SWCNTs. Conclusion. The data obtained demonstrate that the low doses of SWCNTs disturbed the genome functions by changing the levels of key regulatory gene expressions in gene-specific and dose-dependent manner, but their impact was much stronger in the normal human astrocytes in comparison with the tumor cells. It is possible that ER stress, which is constantly present in tumor cells and responsible for multiple resistances, also created a partial resistance to the SWCNTs action. Low doses of SWCNTs induced more pronounced changes in the expression of diverse genes in the normal human astrocytes compared to glioblastoma cells indicating for a possible both genotoxic and neurotoxic effects with a greater extent in the normal cells.

The Role of rs4626 and rs7221352 Polymorphisms on the TOB1 Gene in Turkish Relapsing-Remitting Multiple Sclerosis Patients

The Role of rs4626 and rs7221352 Polymorphisms on the TOB1 Gene in Turkish Relapsing-Remitting Multiple Sclerosis Patients

GRAPHENE OXIDE AFFECT THE EXPRESSION OF PROLIFERATION RELATED GENES AND microRNA IN NORMAL HUMAN ASTROCYTES

Aim. In this study we investigate the impact of low doses of graphene oxide on the expression of key regulatory genes which control cell proliferation as well as microRNAs in normal human astrocytes. Methods. The expression level of genes related to cell proliferation was studied by real-time qPCR in normal human astrocytes line NHA/TS (Cambrex Bio Science, Walkersville, MD, USA) using SYBRGreen Mix and specific for each mRNA forward and reverse primers. These astrocytes were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Graphene oxide (2 mg/ml, dispersion in water) was received from Sigma-Aldrich Chemie GmbH, Germany. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). For polyadenylation and reverse transcription of miRNAs we used Mir-X miRNA First-Strand Synthesis Kit (Takara, Japan). The expression level of microRNAs was studied by real-time qPCR using SYBRGreen Mix and specific for each miRNA forward primers and universal reverse primer. For normalization of microRNA expressions the level of U6 RNA expression was used. Results. It was shown that the expression level of TOB1, HSPA5, EDEM1, MYBL1, and MYBL2 significantly increased in normal human astrocytes line NHA/TS, which were treated with graphene oxide (1 and 4 ng/ml of medium) for 24 hrs. Up-regulation of these genes expression was dose-dependent: bigger dose of graphene oxide (4 ng/ml of medium) introduced more significant changes in the expression of all these genes. Furthermore, bioinformatics analysis of 3′-untranslated regions of mRNA allowed identifying binding sites of microRNA: miR-19a for MYBL1, miR-143 for MYBL2 and miR-182 for TOB1. It was also shown that the expression of all these microRNA significantly down-regulated by graphene oxide, supporting the idea of both post-transcriptional and transcriptional regulation of MYBL1, MYBL2 and TOB1 gene expressions. Conclusions. Graphene oxide significantly disturbs genome stability by up-regulation of the expression of key regulatory genes and down-regulation of microRNA.

Key Regulatory Differentially Expressed Genes in the Blood of Atrial Septal Defect Children Treated With Occlusion Devices.

Atrial septal defects (ASDs) are the most common types of cardiac septal defects in congenital heart defects. In addition to traditional therapy, interventional closure has become the main treatment method. However, the molecular events and mechanisms underlying the repair progress by occlusion device remain unknown. In this study, we aimed to characterize differentially expressed genes (DEGs) in the blood of patients treated with occlusion devices (metal or poly-L-lactic acid devices) using RNA-sequencing, and further validated them by qRT-PCR analysis to finally determine the expression of key mediating genes after closure of ASD treatment. The result showed that total 1,045 genes and 1,523 genes were expressed differently with significance in metal and poly-L-lactic acid devices treatment, respectively. The 115 overlap genes from the different sub-analyses are illustrated. The similarities and differences in gene expression reflect that the body response process involved after interventional therapy for ASDs has both different parts that do not overlap and the same part that crosses. The same portion of body response regulatory genes are key regulatory genes expressed in the blood of patients with ASDs treated with closure devices. The gene ontology enrichment analysis showed that biological processes affected in metal device therapy are immune response with CXCR4 genes and poly-L-lactic acid device treatment, and the key pathways are nuclear-transcribed mRNA catabolic process and proteins targeting endoplasmic reticulum process with ribosomal proteins (such as RPS26). We confirmed that CXCR4, TOB1, and DDIT4 gene expression are significantly downregulated toward the pre-therapy level after the post-treatment in both therapy groups by qRT-PCR. Our study suggests that the potential role of CXCR4, DDIT4, and TOB1 may be key regulatory genes in the process of endothelialization in the repair progress of ASDs, providing molecular insights into this progress for future studies.

Expression of TOB/BTG family members in patients with inflammatory bowel disease.

In recent years, the role of anti-proliferative TOB proteins in the regulation of immune response by inhibiting T cell activation has been demonstrated. Nevertheless, no previous studies have explored their expression in patients with IBD. The aim of the study was to characterize the gene and protein expression of the TOB/BTG family in intestinal tissue of patients with IBD. This is an observational and cross-sectional study that included 63 IBD patients. Gene expression of TOB/BTG family was measured by RT-PCR. Protein expression of TOB/CD16 and BTG/Ki-67 was evaluated by immunohistochemistry. TOB/BTG family mRNAs were detected and quantitated by RT-qPCR in rectal and ileum biopsies from UC patients and CD patients, respectively, and non-inflammatory control tissues. Results showed that TOB1 and BTG1 gene expression was decreased in the colonic mucosa from patients with UC compared with the control group. The TOB2 and BTG2 genes were over-expressed in the colonic mucosa of patients with UC in remission compared with the active UC and control group. The high TOB2 gene expression was associated with histological remission (P=.01). TOB1/CD16, TOB2/CD16, BTG1/Ki-67, BTG2/Ki-67 and BTG4/Ki-67 single and double positive cells were mostly NK, macrophages, epithelial cells, connective tissue cells and perivascular inflammatory infiltrates in tissues from patients with UC and CD. This is the first depiction of the TOB/BTG family gene and protein expression in rectal and ileum tissues by a CD16+ subpopulation in IBD.

MiR-32-5p Regulates Radiosensitization, Migration And Invasion Of Colorectal Cancer Cells By Targeting TOB1 Gene.

BackgroundMicroRNAs (miRNAs) play key roles in the development and progression of various cancers. However, the precise functions and regulation mechanisms of miRNAs in human tumors remain elusive.MethodsQuantitative real time-PCR (qRT-PCR) was performed to detect the level of miR-32-5p in colorectal cancer tissues. The relationships between miR-32-5p level with clinicopathological characteristics and prognosis were analyzed. The miR-32-5p inhibitor was employed to knock down the expression of miR-32-5p. The overexpression plasmid and si-RNA targeting TOB1 were generated. Clone formation assays under radiant exposure were used to evaluate the radiosensitization. Transwell migration and invasion were employed to test the ability of cell migration and invasion. Luciferase reporter assays were used to confirm the regulation of miR-32-5p on the expression of TOB1. Rescue experiments were conducted to investigate the effects of TOB1 on the functions of miR-32-5p.ResultsIn this study, we found that miR-32-5p was significantly upregulated in colorectal cancer tissues compared with adjacent normal tissues. The level of miR-32-5p was positively correlated with tumor differentiation and metastasis. Log-rank tests showed that high level of miR-32-5p was significantly correlated with poor overall survival and disease-free survival. Anti-miR-32-5p remarkably enhanced the radiosensitivity and inhibited migration and invasion of colorectal cancer cells. In addition, overexpression of TOB1 obviously increased the radiosensitivity and inhibited migration and invasion of colorectal cancer cells. Moreover, bioinformatics analysis and luciferase reporter assays demonstrated that miR-32-5p suppressed the expression of TOB1 through directly binding to the 3ʹ-UTR of TOB1 mRNA. Rescue experiments indicated that miR-32-5p regulated the radiosensitivity, migration and invasion of colorectal cancer cells through inhibiting TOB1 expression.ConclusionThis study suggested that miR-32-5p may serve as a prognostic and therapeutic target for colorectal cancer, and downregulation of miR-32-5p enhanced the radiosensitivity and inhibited migration and invasion through promoting TOB1 expression.

Association between the SNPs of the TOB1 gene and gastric cancer risk in the Chinese Han population of northeast China.

The TOB1 (ErbB-2,1) gene is an anti-proliferative factor that has the potential to regulate cell growth and encodes a member of the transducer of erbB-2/B-cell translocation gene protein. The association between the polymorphisms of the TOB1 gene and gastric cancer (GC) risk is still unclear. In this study, 506 GC cases and 548 healthy controls (HCs) were collected to evaluate the association between the eleven SNPs (rs35220381, rs12950561, rs7221352, rs61482741, rs9303568, rs34700818, rs12949115, rs9903822, rs12601477, rs11656976 and rs4626) of the TOB1 gene and GC risk in the population of northeast China. The results showed that there were significant associations of haplotype GCCTTGC, haplotype ATCTTGG, and haplotype GCCACGC with GC risk (P < 0.05, P < 0.001, and P <0.001, respectively). The association between rs12601477 GA+AA genotypes and GC risk was significant among individuals older than 58 (adjusted OR=1.53, 95% CI=1.05-2.22, P< 0.05). The association between rs4626 AG+GG genotypes and GC risk was significant among individuals older than 58 (adjusted OR=1.54, 95% CI = 1.03-2.28, P<0.05). The rs34700818 CT+TT genotypes were associated with a significantly increased risk of T3-T4 (CT+TT vs CC, adjusted OR=1.71, 95% CI= 1.01-2.88, P<0.05) and TNM stage II (CT+TT vs CC, adjusted OR=2.40, 95% CI =1.27-4.52, P<0.01). The rs61482741 CG+GG genotypes were also associated with a significantly increased risk of T3-T4 (CG+GG vs CC, adjusted OR=1.71, 95% CI = 1.01-2.88, P<0.05) and TNM stage II (CG+GG vs CC, adjusted OR=2.40, 95% CI=1.27-4.52, P<0.01). The results suggest that four SNPs (rs12601477, rs4626, rs34700818 and rs61482741) of the TOB1 gene play an important role in the occurrence and development of GC in the Chinese Han population of northeast China.

Tob1 is expressed in developing and adult gonads and is associated with the P-body marker, Dcp2.

Tob1 is a member of the BTG/TOB family of proteins with established antiproliferative function. In Danio rerio and Xenopus laevis, the Tob1 gene is expressed from the one-cell stage through to early gastrula stages, followed in later development by discrete expression in many tissues including the notochord and somites. In both mouse and human, Tob1 is expressed in many adult tissues including the testis and ovary; however, the specific cell types are unknown. We examine Tob1 gene expression in mouse in developing germ cells and in sorted male germ cells (gonocytes, spermatogonia, pachytene spermatocytes and round spermatids) by reverse transcription and droplet digital polymerase chain reaction (RT-ddPCR) and in adult ovary and testis by immunofluorescence with anti-Tob1 protein staining. By RT-ddPCR, Tob1 expression was low in developing male germ cells but was highly expressed in round spermatids. In developing female germ cells undergoing entry into meiosis, it increased 10-fold. Tob1 was also highly expressed in round spermatids and in oocytes in all stages of folliculogenesis. Notably, a marker for P-bodies, Dcp-2, was also highly expressed in round spermatids and all oocyte stages examined. The cytoplasmic presence of Tob1 protein in round spermatids and oocytes and the association of Tob1 protein with Dcp2 in both cell types suggest that Tob1 protein plays a role in post-transcriptional mechanisms.

Abstract 2042: TOB1 promotes cell survival in estrogen-independent estrogen receptor-positive breast cancer.

Abstract Antiestrogens are the most widely administered endocrine agents for the treatment of estrogen receptor (ER)-expressing breast cancers, such as tamoxifen, fulvestrant and aromatase inhibitor. However, most initially responsive breast tumors acquire resistance and become estrogen-independent. To find potential therapeutic target(s) against drug resistance, we performed siRNA library screening studies to identify cell survival determinants in estrogen independent cell lines from 631 ER network related genes. LCC1 and LCC9 cells, derivatives of MCF-7, are estrogen-independent. LCC1 cells are sensitive to fulvestrant and tamoxifen. LCC9 cells are resistant to fulvestrant and cross-resistant to tamoxifen. A number of genes selectively support the survival of estrogen-independent tumor cells, including TOB1 (Transducer of ErbB2). TOB1 gene knockdown significantly inhibited (&amp;gt;50%) estrogen-independent cell growth in LCC1 and LCC9 cells, but had much less effect on the survival of MCF-7 cells (10%). These cell lines have similar levels of Tob1 protein. TOB1 knockdown did not cause apoptosis or autophagy. DNA replication was reduced and G1 to S phase transition was blocked by TOB1 knockdown in the estrogen-independent cell lines. Next, we studied cell-signaling pathways that regulate cell cycle transition. Cyclin D1 was upregulated and activated by TOB1 knockdown in estrogen-independent cells, but not in MCF-7 cells. Therefore, cyclin D1 might be involved in G1/S transition blockage induced by Tob1 inhibition. TOB1 knockdown also activated cell cycle checkpoint kinase 2 (Chk2) and induced DNA damage. Our data demonstrated that Tob1 promotes cell survival after ER+ breast cancer cells acquire estrogen-independence, which might be mediated by cyclin D1. Citation Format: Yong-Wei Zhang, Rochelle Nasto, Robert Clarke, Louis M. Weiner. TOB1 promotes cell survival in estrogen-independent estrogen receptor-positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2042. doi:10.1158/1538-7445.AM2013-2042

The Relationship Between the Regulation of TOB1 Gene with Cell Proliferation, Apoptosis and Cell Cycle in BCR-ABL Positive Cells

AbstractAbstract 5125The TOB1 gene is a transcription factor responsible for the transduction of the gene ERBB2. It is a member of a family of cell suppressor proliferation proteins called TOB/BTG1 family; also, this gene operates on the inhibition of neoplastic transformation. The TOB1 gene presents a decreased expression in several types of cancer such as lung, breast, thyroid and stomach cancer. However, the function of this gene in chronic myeloid leukemia (CML) remains unknown. Aiming to evaluate the inhibition of gene TOB1 into BCR-ABL positive cells and trying to elucidate the molecular mechanisms associated with the inhibition of this gene in the CML we proceed to a more detailed study of this gene. The inhibition of this gene in K562 cells was performed using specific lentivirus. The effect of silencing TOB1 in the proliferation of K562 cells was assessed by the MTT assay after 48 hours of culture; in shTOB1 the proliferation was increased in comparison with shControl cells. To evaluate the synergistic effect between the inhibition of kinase tyrosine activity of BCR-ABL and the inhibition of TOB1 we performed a treatment with different concentrations of imatinib (0. 1, 0. 5 and 1μM), but we observed the decrease in cell proliferation of shTOB1 cells to similar levels of shControl cells only at the 1μM concentration. Therefore, the TOB1 silencing increased the proliferation of K562 cells without an additional effect of a treatment with Imatinib. To analyze the clonogenicity, we performed a formation of colonies assay, in methylcellulose, to determine whether silencing TOB1 could cause a change in the clonal growth of positive BCR-ABL cells. There was no significant change in the number of colonies that grew in cell culture shTOB1 compared to shControl cells. These results suggest that silencing TOB1 in K562 cells may not change the clonogenicity. In the assessment of cell cycle, the flow cytometry analysis revealed a significant accumulation of K562 cells in S phase, with consequent reduction of cells in the G2 phase of the cell cycle in cells shTOB1 compared to cells shControl. The TOB1 gene silencing in K562 cells kept the cells in the S phase and prevented the entry of cells in the G2 phase showing that the inhibition of gene TOB1 induced an increase in proliferation of K562 BCR-ABL cells. The level of apoptosis was assessed by flow cytometry after labeling the cells with anexin-V/PI. The Imatinib treatment presented dose-response in the induction of apoptosis as expected. However, a cumulative effect with TOB1 silencing was not observed. Furthermore, the apoptosis was also assessed by assays of caspases 3, 8 and 9, which showed an increase of the caspase activity of shControl cells in relation of the shTOB1 cells, showing that inhibition of this gene also changes the level of apoptosis. These results corroborate the literature data that report the relationship of this tumour suppressor gene in signalling pathways related to angiogenesis, carcinogenesis, apoptosis and metastasis. When we relate the results obtained with the LMC, we can consider the possibility of TOB1 regulation changes be related to modification of important signalling pathways such as AKT, PI3K, STAT3 and STAT5, among others. Furthermore, the inhibition of TOB1 may be related with an increase on the number of BCR-ABL positive cells and subsequent disease progression. In conclusion, this study confirmed literature data showing that TOB1 gene works as a tumour suppressor protein in cells of many types of cancer. From this work we can infer that in CML the expression of this gene is transformed, resulting in changing of the capacity of induction of apoptosis, decrease tumour necrosis and increase cell proliferation. This work was supported by FAPESP and INCT. Disclosures:No relevant conflicts of interest to declare.

The role of the TOB1 gene in growth suppression of hepatocellular carcinoma

The TOB1 gene, mapped on 17q21, is a member of the BTG/Tob family. In breast cancer it has been identified as a candidate tumor suppressor gene. However, whether TOB1 is a bona fide tumor suppressor and downregulated in hepatocellular carcinoma (HCC) remains unclear. In addition, whether its expression is regulated through methylation requires investigation. In the present study, we therefore analyzed the expression of TOB1 in HCC and its methylation levels in human HCC and breast cancer. No significant difference in the expression levels of TOB1 was observed between tumor tissues and adjacent normal tissues in HCC. Quantitative methylation analysis by MassArray revealed no significant differences at single CpG sites or in the global promoter region, and all these CpG sites shared a similar methylation pattern in HCC and breast cancer. Moreover, 5-aza-2'-deoxycytidine treatment of three tumor cell lines did not cause elevation of TOB1 mRNA in HepG2 cell lines. Based on these data, we speculate that TOB1 may be a candidate non-tumor suppressor gene in HCC. Furthermore, the clinical outcome was not correlated with TOB1 expression or expression rate. In addition, TOB1 expression or expression rate was not correlated with the overall survival (OS) rates or cumulative recurrence rates. Taken together, we suggest that TOB1 does not act as a tumor suppressor in HCC.

Novel Sequence Variations in the CER1 Gene Are Strongly Associated with Low Bone Mineral Density and Risk of Osteoporotic Fracture in Postmenopausal Women

Osteoporosis is a common skeletal disease characterized by a combination of low bone mass and increased fragility. In this case-control study, we investigated the possible association of two novel candidate genes, CER1 and TOB1, with bone mineral density (BMD) and fragility risk in 300 postmenopausal women of Hellenic origin. The entire CER1 and TOB1 gene sequences were amplified and resequenced to assess whether there is a correlation between these genes and BMD. We identified 26 variants in both genes. Statistical analysis did not reveal any correlation between TOB1 and osteoporosis. However, CER1 genetic analysis indicated that five polymorphisms, c.194C>G, c.507+506G>T, c.508-182A>G, c.531A>G, and c.*121T>C, were correlated, with a mean T score ≤-2.2. In particular, the greater number of vertebral fractures was found in patients with osteoporosis carrying the G allele of c.531A>G SNP (p = 0.015). When multiple logistic regression analysis was performed, only the c.507+506G>T polymorphism was independently associated with hip fractures or the presence of any fracture (OR = 6.95, p = 0.016, and OR = 5.33, p < 0.001, respectively). These results suggest that CER1 gene variations play a significant role in determining BMD and vertebral or hip fractures, which might be helpful in clinical practice to identify patients with increased fracture risk.

Molecular Characterization of Tob1 in Muscle Development in Pigs

Cell proliferation is an important biological process during myogenesis. Tob1 encoded a member of the Tob/BTG family of anti-proliferative proteins. Our previous LongSAGE (Long Serial Analysis of Gene Expression) analysis suggested that Tob1 was differentially expressed during prenatal skeletal muscle development. In this study, we isolated and characterized the swine Tob1 gene. Subsequently, we examined Tob1 chromosome assignment, subcellular localization and dynamic expression profile in prenatal skeletal muscle (33, 65 and 90 days post-conception, dpc) from Landrace (lean-type) and Tongcheng pigs (obese-type). The Tob1 gene was mapped to pig chromosome 12 (SSC12). The Tob1 protein was distributed throughout the nucleus and cytoplasm of PK15 cells. During prenatal skeletal muscle development, Tob1 was up-regulated and highly expressed in skeletal muscle at 90 dpc in Tongcheng pigs but peaked at 65 dpc in Landrace pigs. This result suggested that there were different proliferation patterns during myogenesis between Tongcheng and Landrace pigs. During postnatal skeletal muscle development, the expression of Tob1 increased with aging, indicating that the proliferation potential of myoblasts decreased in postnatal muscle development. In tissues of adult wuzhishan miniature pigs, the Tob1 gene was highly expressed in skeletal muscle. The expression of Tob1 was significantly increased at day 6 during C2C12 differentiation time, suggesting a possible role in skeletal muscle development. Therefore, this study indicated that Tob1 perhaps played an important role in skeletal muscle development.

Adenovirus-mediated expression of Tob1 sensitizes breast cancer cells to ionizing radiation1

To investigate the effect of the Tob1 gene, a member of the Transducing Molecule of ErbB2/B-cell Translocation Ggene (TOB/BTG) family, by using the adenovirus-mediated expression of Tob1 on radiosensitivity in a human breast cancer cell line MDA-MB-231. Cell survival was determined by clonogenic assay. Apoptosis was evaluated by DNA fragmentation gel electrophoresis and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Protein expression was analyzed by Western blot assay and DNA repair was measured by a host cell reactivation assay. We demonstrated that pre-irradiation treatment with Ad5-Tob1 significantly increased radiosensitivity, accompanying the increased induction of apoptosis and the repression of DNA damage repair. Furthermore, Ad5-Tob1-mediated radiosensitivity correlates with the upregulation of the pro-apoptotic protein Bax and the downregulation of several DNA double strand break repair proteins, including DNA-dependent protein kinases, Ku70 and Ku80, and X-ray-sensitive complementation group 4. Tob1, as a new radiosensitizer, is a new target in the radiotherapy of breast cancer via increasing apoptosis and suppressing DNA repair.

Cloning and expression analysis of zebrafish tob1 gene.

Many members of the Btg/Tob family, which possess anti-proliferation activity, have been identified in mammals but their physiological functions are poorly understood. We have identified a zebrafish tob1 gene, which encodes a peptide of 322 amino acids. The expression pattern of zebrafish tob1 during early embryogenesis is examined by RT-PCR and whole-mount in situ hybridization. The maternal tob1 transcript is abundant in fertilized eggs and blastulas. The expression of tob1 ceases during midgastrulation and early segmentation, when it is expressed and restricted to the notochord and somites. On day 2, expression occurs in the pectoral fin bud but not in other tissues. It will be interesting to investigate whether Tob1 is required for controlling cell proliferation during vertebrate embryogenesis.

A Rapid Chairside Test for the Severity of Periodontal Disease Using Gingival Fluid

A study was done in an attempt to develop a simple test for the severity of periodontal disease. Gingival fluid collected on filter paper was tested for protein content, and the resulting color was compared to standard color filters. Tissue was excised and prepared for histologic examination. The inflammatory cell infiltrate on each slide was graded on a scale of zero to three. Zero was least and three was the highest number of inflammatory cells. The results indicate that the white cell infiltrate graded 0 or 1 on the histologic inflammatory index has a color index of B 1/8 TO B1 whereas the tissue graded 2 or 3 HII has a range of B2 to B6 on the color scale. This test can give the dental practitioner a general idea of the severity of the inflammation.