An automatic measurement method for the response of Caenorhabditis elegans to chemicals.

BACKGROUND: Caenorhabditis elegans is a widely used model animal. Chemotaxis assay is one of the experiments that study the effects of different chemicals on nematodes. It is mainly used to study the effects of different chemicals on the perception behavior of nematodes. By conducting this experiment, not only can the neurotoxicity of chemicals be reflected, but also the impact of chemicals on physiological functions regulated by the nervous system, such as nematode feeding behavior and basic motor ability. OBJECTIVE: The experiment of detecting the response of nematode to chemicals is also a common method of chemical toxicity testing based on nematode models. In the analysis of worm tendency behavior, manual operations are generally used. Manually processing a large number of worms under a microscope is very time-consuming and labor-intensive. The current quantitative methods for nematode chemotaxis experiments are not only time-consuming and labor-intensive, but also biased in experimental results due to differences in judgment standards among experimenters. The automatic and efficient quantification method for nematode chemotaxis experiments is a very important technical difficulty in the field of nematode experiments. METHODS: Here, we have designed an automatic quantification method for nematode chemotaxis experiments by incorporating image acquisition and processing techniques into the nematode experiment. RESULTS: The experimental results show that the Pearson correlation coefficient between manual and automatic counting results is 0.978. CONCLUSION: This proves the effectiveness of our method. Applying the automatic measurement method to replace manual counting by the experimenter can improve work efficiency, and reduce errors in human counting operations.

Genotoxicity Evaluation of Titanium Dioxide Nanoparticles In Vivo and In Vitro: A Meta-Analysis.

BACKGROUND: Recent studies have raised concerns about genotoxic effects associated with titanium dioxide nanoparticles (TiO2 NPs), which are commonly used. This meta-analysis aims to investigate the potential genotoxicity of TiO2 NPs and explore influencing factors. METHODS: This study systematically searched Chinese and English literature. The literature underwent quality evaluation, including reliability evaluation using the toxicological data reliability assessment method and relevance evaluation using routine evaluation forms. Meta-analysis and subgroup analyses were performed using R software, with the standardized mean difference (SMD) as the combined effect value. RESULTS: A total of 26 studies met the inclusion criteria and passed the quality assessment. Meta-analysis results indicated that the SMD for each genotoxic endpoint was greater than 0. This finding implies a significant association between TiO2 NP treatment and DNA damage and chromosome damage both in vivo and in vitro and gene mutation in vitro. Subgroup analysis revealed that short-term exposure to TiO2 NPs increased DNA damage. Rats and cancer cells exhibited heightened susceptibility to DNA damage triggered by TiO2 NPs (p < 0.05). CONCLUSIONS: TiO2 NPs could induce genotoxicity, including DNA damage, chromosomal damage, and in vitro gene mutations. The mechanism of DNA damage response plays a key role in the genotoxicity induced by TiO2 NPs.

Investigation on the potential adverse outcome pathway of the sensitive endpoint for nephrotoxicity induced by gardenia yellow based on an integrated strategy using bioinformatics analysis and in vitro testing validation.

To explore the potential the adverse outcome pathway of Gardenia Yellow (GY)-induced sensitive endpoint for nephrotoxicity, an integrated strategy was applied in the present study. Using bioinformatic analysis, based on the constructed Protein-protein interaction networks, Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis on the core target network were performed to illustrate the potential gene targets and signal pathways. Then, the most enriched pathway was validated with Cell counting kit-8 assays and Western blot analysis in embryonic kidney epithelial 293 cell models. According to the findings, GY may interact with 321 targets related to the endpoint. The five targets on the top ranking in the PPI network were STAT3, SRC, HRAS, AKT1, EP300. Among them, PI3K/Akt was the most enriched pathway. In vitro testing showed that GY exerted a proliferative effect on the cell variability in a dose-dependent manner. GY at concentration of 1000 µg/ml and stimulation for 30 min can significantly enhance the expression of phosphorylated Akt. Thus, after the quantitative weight of evidence evaluation, Akt phosphorylation induced PI3K/Akt activation was speculated as a molecular initiating event leading to a proliferative and inflammatory response in renal tubular epithelial cells.

An automatic recognition method of nematode survival rate based on bright field and dark field experimental images.

BACKGROUND: The survival rate of experimental animals is a very important index in chemical toxicity evaluation experiments. The calculation of nematode survival rate is used in many experiments. OBJECTIVE: Traditional survival rate quantification methods require manual counting. This is a time-consuming and laborious work when using 384-well plate for high-throughput chemical toxicity assessment experiments. At present, there is a great need for an automatic method to identify the survival rate of nematodes in the experiment of chemical toxicity evaluation. METHODS: We designed an automatic nematode survival rate recognition method by combining the bright field experimental image of nematodes and the dark field image of nematodes which is captured after adding Propidium Iodide dye, and used it to calculate the nematode survival rate in different chemical environments. Experiment results show that the survival rate obtained by our automatic counting method is very similar to the survival rate obtained by manual counting. RESULTS: Through several different chemical experiments, we can see that chemicals with different toxicity have different effects on the survival rate of nematodes. And the survival rate of nematodes under different chemical concentrations has an obvious gradient trend from high concentration to low concentration. In addition, our method can quantify the motility of nematodes. There are also significant differences in the motility of nematodes cultured in different chemical environments. Moreover, the nematode motility under different chemical concentrations showed an obvious gradient change trend from high concentration to low concentration. CONCLUSION: Our study provides an accurate and efficient nematode survival rate recognition method for chemical toxicology research.

PM2.5 induce lifespan reduction, insulin/IGF-1 signaling pathway disruption and lipid metabolism disorder in Caenorhabditis elegans.

Introduction: Exposure to fine particulate matter (PM), especially PM2.5, can induce various adverse health effects in populations, including diseases and premature death, but the mechanism of its toxicity is largely unknown. Methods: Water-soluble components of PM2.5 (WS-PM2.5) were collected in the north of China in winter, and combined in two groups with the final concentrations of 94 µg/mL (CL group, AQI ≤ 100) and 119 µg/mL (CH group, 100 < AQI ≤ 200), respectively. The acute and long-term toxic effects of WS-PM2.5 samples were evaluated in several aspects such as development, lifespan, healthspan (locomotion behavior, heat stress tolerance, lipofucin). DAF mutants and genes were applied to verify the action of IIS pathway in WS-PM2.5 induced-effects. RNA-Sequencing was performed to elucidate the molecular mechanisms, as well as ROS production and Oil red O staining were also served as means of mechanism exploration. Results: Body length and lifespan were shortened by exposure to WS-PM2.5. Healthspan of nematodes revealed adverse effects evaluated by head thrash, body bend, pharyngeal pump, as well as intestinal lipofuscin accumulation and survival time under heat stress. The abbreviated lifespan of daf-2(e1370) strain and reduced expression level of daf-16 and hsp-16.2 indicated that IIS pathway might be involved in the mechanism. Thirty-five abnormally expressed genes screened out by RNA-Sequencing techniques, were functionally enriched in lipid/lipid metabolism and transport, and may contribute substantially to the regulation of PM2.5 induced adverse effects in nematodes. Conclusion: WS-PM2.5 exposure induce varying degrees of toxic effects, such as body development, shorten lifespan and healthspan. The IIS pathway and lipid metabolism/transport were disturbed by WS-PM2.5 during WS-PM2.5 exposure, suggesting their regulatory role in lifespan determination.

A repeated dose 28-day oral toxicity study of sodium dehydroacetate (DHA-S) in Wistar rats.

Sodium dehydroacetate (DHA-S) is a food additive and preservative. The present study was conducted to investigate the potential toxicity of repeated oral doses of DHA-S. DHA-S was administered orally by gavage to Wistar rats at doses of 0, 50, 100, or 200 mg/kg BW/day for 28 days, after which growth indicators, clinical pathology, organ weights, and histopathology were determined. Body weight and food consumption were significantly reduced at doses of 100 or 200 mg/kg BW, and some hematological indexes and organ weight were significantly affected, particularly in female rats. At a dose of 200 mg/kg BW, the blood coagulation activities were significantly reduced in female rats. At a dose of 100 or 200 mg/kg BW, the main blood biochemical parameters of both sexes were obviously affected. Similar histological changes in the hepatic and renal tissues were observed in both the treated (200 mg/kg BW DHA-S) and control animals. Female rats were more susceptible to most of the toxic effects caused by DHA-S, which further indicating a gender difference in the toxic phenotype profile of rats. Based on these results, the no observed adverse effect level (NOAEL) of DHA-S was determined to be 50 mg/kg BW/day in rats.

Effects of commercial beverages on the neurobehavioral motility of Caenorhabditis elegans.

To study the effects of different types of commercially available drinks/beverages on neurobehavior using the model organism C. elegans, and critically review their potential health hazards. Eighteen kinds of beverages from the supermarket were randomly selected and grouped into seven categories namely functional beverage, tea beverage, plant protein beverage, fruit juice beverage, dairy beverage, carbonated beverage and coffee beverage. The pH value, specific gravity and osmotic pressure were also examined. The L4 stage N2 worms were exposed to different concentration of tested beverages (0, 62.5, 125, 250 and 500 µL/mL) for 24 h to measure the survival rate and locomotory behavior such as head thrashing, body bending as well as pharyngeal pumping. All the 18 beverages tested did not induce any visible lethal effects in the nematodes. However, exposure to different types of tested beverages exhibited different effects on the behavioral ability of C. elegans: (1) sports functional beverage and herbal tea drink accelerated the head thrashing and body bending of nematodes when compared to the control group (P < 0.05). (2) The vibration frequency of the pharyngeal pump of nematodes was significantly accelerated after treated with three plant protein beverages (almond milk, coconut milk and milk tea) and dairy products A and B (P < 0.05), and decelerated after treatment with other tested beverages. (3) Carbonated beverage significantly inhibits the head thrashing, body bending and pharyngeal pumping vibration (P < 0.05). Our results indicate that 18 kinds of popular beverages in the market have different influence on the neurobehavior in C. elegans, which may be related to their different components or properties. Further research would be required to conduct a systematic analysis of the effect of beverages by appropriate kinds, taking into consideration other endpoints such as reproduction, lifespan and molecular stress response, etc., and to elucidate the mechanism for its potential health hazards.

Effects of lanthanum nitrate on behavioral disorder, neuronal damage and gene expression in different developmental stages of Caenorhabditis elegans.

Rare earth elements (REEs) are widely used in the industry, agriculture, biomedicine, aerospace, etc, and have been shown to pose toxic effects on animals, as such, studies focusing on their biomedical properties are gaining wide attention. However, environmental and population health risks of REEs are still not very clear. Also, the REEs damage to the nervous system and related molecular mechanisms needs further research. In this study, the L1 and L4 stages of the model organism Caenorhabditis elegans were used to evaluate the effects and possible neurotoxic mechanism of lanthanum(III) nitrate hexahydrate (La(NO3)3·6H2O). For the L1 and L4 stage worms, the 48-h median lethal concentrations (LC50s) of La(NO3)3·6H2O were 93.163 and 648.0 mg/L respectively. Our results show that La(NO3)3·6H2O induces growth inhibition and defects in behavior such as body length, body width, body bending frequency, head thrashing frequency and pharyngeal pumping frequency at the L1 and L4 stages in C. elegans. The L1 stage is more sensitive to the toxicity of lanthanum than the L4 stage worms. Using transgenic strains (BZ555, EG1285 and NL5901), we found that La(NO3)3·6H2O caused the loss or break of soma and dendrite neurons in L1 and L4 stages; and α-synuclein aggregation in L1 stage, indicating that Lanthanum can cause toxic damage to dopaminergic and GABAergic neurons. Mechanistically, La(NO3)3·6H2O exposure inhibited or activated the neurotransmitter transporters and receptors (glutamate, serotonin and dopamine) in C. elegans, which regulate behavior and movement functions. Furthermore, significant increase in the production of reactive oxygen species (ROS) was found in the L4 stage C. elegans exposed to La(NO3)3·6H2O. Altogether, our data show that exposure to lanthanum can cause neuronal toxic damage and behavioral defects in C. elegans, and provide basic information for understanding the neurotoxic effect mechanism and environmental health risks of rare earth elements.

Comet Assay Evaluation of Lanthanum Nitrate DNA Damage in C57-ras Transgenic Mice.

Due to the wide application of rare-earth elements (REEs) in the last decades, lanthanum has increasingly entered the environment and has gradually accumulated in the human body through the food chain. Lanthanum is worth paying attention in terms of food safety. Although the genotoxicity of lanthanum has been studied in vitro, data on its DNA damage in vivo rodent are limited, moreover, which have also presented some controversy. This study aimed to conduct an in vivo rodent alkaline comet assay, and as a companion test to the lanthanum nitrate carcinogenicity test. We conducted an oral gavage experiment for 180 days (26 weeks) to test for the persistence of DNA damage of long-term low-dose accumulation of lanthanum nitrate (12.5, 25, and 50 mg/kg body weight), in F1 hybrid C57-ras transgenic mice (CB6F1) by using alkaline comet assay in the blood and liver. The comet assay revealed that all the tested concentrations of lanthanum nitrate did not induce DNA damage in any of the tissues investigated, whereas DNA damage was induced in the positive control group. These results could indicate that lanthanum nitrate can accumulate in tissues and organs of the mice after exposure, and does not possess DNA damage in C57-ras transgenic mice after repeated treatments at oral doses up to 50 mg/kg·BW for 26 weeks; also, it did not cause pathological changes in the liver of the mice.

Synthetic peptide, Ala-Arg-Glu-Gly-Glu-Met, abolishes pro-proliferative and anti-apoptotic effects of high glucose in vascular smooth muscle cells.

Apoptosis plays a critical role in normal vascular development and atherosclerosis. However, high glucose has been reported to generate a certain level of ROS that can inhibit vascular smooth muscle cell (VSMC) apoptosis, with the underlying mechanism remaining unclear. In this study, a synthetic peptide AREGEM (Ala-Arg-Glu-Gly-Glu-Met) exhibited antioxidative effects and was used to investigate its function in VSMCs during hyperglycaemia. MTT assay results demonstrated that AREGEM significantly attenuated high glucose-induced VSMCs proliferation. Flow cytometry displayed that high glucose levels inhibited cell apoptosis, whereas this effect was attenuated by pre-incubation with AREGEM. In addition, the 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe assay further demonstrated that AREGEM reduced intracellular ROS accumulation in VSMCs. Furthermore, this peptide was able to prevent the decrease of caspase-3 activity and the increase of the ratio of Bcl-2/Bax protein in VSMCs exposed to high glucose. These findings demonstrated that AREGEM is able to abolish the effects of high glucose in VSMCs; therefore, this peptide can be a potential candidate to develop a novel strategy for curing diabetic related diseases.

Pathogenesis of emerging severe fever with thrombocytopenia syndrome virus in C57/BL6 mouse model.

The discovery of an emerging viral disease, severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV), has prompted the need to understand pathogenesis of SFTSV. We are unique in establishing an infectious model of SFTS in C57/BL6 mice, resulting in hallmark symptoms of thrombocytopenia and leukocytopenia. Viral RNA and histopathological changes were identified in the spleen, liver, and kidney. However, viral replication was only found in the spleen, which suggested the spleen to be the principle target organ of SFTSV. Moreover, the number of macrophages and platelets were largely increased in the spleen, and SFTSV colocalized with platelets in cytoplasm of macrophages in the red pulp of the spleen. In vitro cellular assays further revealed that SFTSV adhered to mouse platelets and facilitated the phagocytosis of platelets by mouse primary macrophages, which in combination with in vivo findings, suggests that SFTSV-induced thrombocytopenia is caused by clearance of circulating virus-bound platelets by splenic macrophages. Thus, this study has elucidated the pathogenic mechanisms of thrombocytopenia in a mouse model resembling human SFTS disease.

Suppression of AMPK activation via S485 phosphorylation by IGF-I during hyperglycemia is mediated by AKT activation in vascular smooth muscle cells.

As a metabolic sensor, the serine/threonine protein kinase AMP-activated protein kinase (AMPK) promotes the adaptation of cells to signals arising from nutrients, hormones, and growth factors. The ability of IGF-I to stimulate protein synthesis is suppressed by AMPK, therefore, these studies were undertaken to determine whether IGF-I modulates AMPK activity. IGF-I dose-dependently suppressed phosphorylation of AMPK T172, and it stimulated AMPK S485 phosphorylation in vascular smooth muscle cells (VSMC). To determine whether stimulation of AMPK S485 phosphorylation was mediating this response, VSMC were transduced with a mutant AMPKα (AMPK S485A). Expression of this altered form inhibited the ability of IGF-I to suppress AMPK T172 activation, which resulted in inhibition of IGF-I-stimulated phosphorylation of P70S6 kinase. In contrast, expression of an AMPK S485D mutant resulted in constitutive suppression of AMPK activity and was associated with increased IGF-I-stimulated P70S6K phosphorylation and protein synthesis. The addition of a specific AKT inhibitor or expression of an AKT1 short hairpin RNA inhibited AMPK S485 phosphorylation, and it attenuated the IGF-I-induced decrease in AMPK T172 phosphorylation. Exposure to high glucose concentrations suppressed AMPK activity and stimulated S485 phosphorylation, and IGF-I stimulated a further increase in S485 phosphorylation and AMPK T172 suppression. We conclude that AMPK S485 phosphorylation negatively regulates AMPK activity by modulating the T172 phosphorylation response to high glucose and IGF-I. IGF-I stimulates S485 phosphorylation through AKT1. The results suggest that AMPK plays an inhibitory role in modulating IGF-I-stimulated protein synthesis and that IGF-I must down-regulate AMPK activity to induce an optimal anabolic response.

AMP-activated protein kinase inhibits IGF-I signaling and protein synthesis in vascular smooth muscle cells via stimulation of insulin receptor substrate 1 S794 and tuberous sclerosis 2 S1345 phosphorylation.

AMP-activated protein kinase (AMPK) inhibits IGF-I actions, but the mechanism by which AMPK functions is undefined. This study identified signaling events that were induced by AMPK that mediated inhibition of IGF-I-stimulated phosphoinosotide-3-kinase (PI3K) pathway activation. The AMPK activator metformin stimulated AMPK Thr172 phosphorylation and inhibited IGF-I-stimulated phosphorylation of Akt/tuberous sclerosis 2 (TSC2)/mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K). Expression of constitutively active forms of AMPK suppressed IGF-I-stimulated activation of Akt/TSC2/mTOR/p70S6K and protein synthesis, whereas AMPK knockdown resulted in enhanced responses to IGF-I. To determine the mechanism by which AMPK inhibited IGF-I signaling, the role of insulin receptor substrate-1 (IRS-1) was examined. Both metformin and constitutively activated AMPK enhanced phosphorylation of IRS-1 Ser794, which led to decreased IRS-1 tyrosine phosphorylation and recruitment of the p85 subunit of PI3K. Overexpression of IRS-1 S794A was associated with increased IGF-I-stimulated IRS-1 tyrosine phosphorylation, p85 association, and protein synthesis. To determine whether other signaling molecules mediated the effect of AMPK, TSC2 function was examined. Cells overexpressing TSC2/S1345A (the site of AMPK phosphorylation) were less responsive to metformin-induced inhibition of p70S6 kinase. These findings are relevant to whole animal physiology because administration of metformin to mice resulted in inhibition of IGF-I-stimulated phosphorylation of Akt/mTOR/p70S6K. In conclusion, AMPK functions to inhibit IGF-I-stimulated PI3K pathway activation through stimulation of IRS-1 serine 794 phosphorylation. Because IGF-I is an important stimulant of the anabolic response, this effect of AMPK could account for part of its inhibitory effect on protein synthesis, thus allowing more efficient energy use by other cellular processes.

[Biological implications of the inhibition of survivin by RNA interference in human androgen-independent prostate carcinoma with highly metastatic potential].

OBJECTIVE: To determine the expression level of survivin in androgen-independent prostate carcinoma, and to investigate the biological role of survivin in invasion and metastasis of androgen-independent prostate carcinoma. METHODS: Highly metastatic prostatic cancer cell line PC-3M-1E8 was stably transfected with pSilencer plasmid targeting survivin expression by RNA interference. The biological effects were observed, including anchorage-independent growth, in vitro invasion by soft agar colony formation and Boyden chamber assay, and also in vivo tumorigenesis in nude mice. Cell cycle and apoptosis indices were evaluated by flow cytometry and Western blot analysis of bioactive fragments of caspase 3. RESULTS: The expression of survivin in transfected PC-3M-1E8 cells was markedly depressed at both mRNA and protein levels (about 78% to 80%) as compared with control. The growth of tumor cells was retarded by anchorage-independent growth assay. The survivin transfectants formed smaller and fewer colonies (14.33 +/- 3.51) than the negative (52.33 +/- 6.81) and blank controls (54.00 +/- 6.00). Inhibition of survivin expression was correlated with enhanced apoptosis of tumor cells (percentages of apoptotic cells of the negative control, blank control and experimental groups were 5.88 +/- 0.99, 6.97 +/- 1.60, 16.40 +/- 1.95 respectively), along with an increased expression of activated caspase 3, and cell cycle inhibition at G(0)/G(1) phase (the relative number of cells at G(0)/G(1) phase were 43.65 +/- 3.44, 43.59 +/- 1.83 and 52.71 +/- 1.10, respectively). In addition, multinucleated giant cells were observed along with a marked inhibition of invasion as reflected by fewer penetrating cells by Boyden chamber assay (46.07 +/- 9.97, 47.87 +/- 9.58 and 38.67 +/- 6.59, respectively). CONCLUSIONS: Survivin expression is high in androgen-independent prostate cancer cells and likely may be related to the apoptosis, growth and invasion of the tumor cells. Targeting the survivin pathway by RNA interference appears to be a promising approach for clinical treatment of androgen-independent prostate cancer.

[Monoclonal antibody against G3BP: preparation, characterization and its application in analysis of human tumors].

OBJECTIVE: To better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors. METHODS: By using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining. RESULTS: A hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression. CONCLUSIONS: The G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.

YMDD variants of HBV DNA polymerase gene: rapid detection and clinicopathological analysis with long-term lamivudine therapy after liver transplantation.

AIM: To look for a rapid low-cost technique for the detection of HBV variants. METHODS: Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene. RESULTS: YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT. CONCLUSION: PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.

[Monoclonal antibodies against human tumor metastasis suppressor gene-1 TMSG-1: preparation, characterization and application].

OBJECTIVE: In order to clarify the exact molecular weight of tumor metastasis suppressor gene-1 (TMSG-1) protein and its cellular localization, a monoclonal antibody against TMSG-1 was prepared, characterized and applied to evaluate the metastatic potential of human tumors. METHODS: A dominant epitope-TMSG-1(15)-derived from TMSG-1 was synthesized based on Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen preparation was used to immunize BAL B/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, which were further characterized by western blotting and immunohistochemical staining. RESULTS: One hybridoma cell line secreting anti-TMSG-1 antibody, designated as C8, was eventually established after primary ELISA screening, followed by rapid limited dilution procedure. It was confirmed that C8 was of IgM isotype. Result of competitive inhibition assay showed that the antibody was TMSG-1 specific. Using this antibody, an expected protein band of about 45,000 (relative molecular mass) was detected in the non-metastatic variants PC(3)-2B4 and PG-LH7 cells by Western blotting, but not in the isogenetic metastatic variants of PC3-1E8 and PG-BE1 cells. Immunohistochemistry using C8 showed a positive staining of cell membrane and cytoplasm of 2B4 and LH7 cells, whereas 1E8 and BE1 cells were non-reactive. Immunostaining using C8 of paraffin sections of 52 breast carcinomas and 41 colon cancers demonstrated a strong positivity in non-metastatic tumors, but none to weakly reactive in metastatic tumors (P < 0.05). CONCLUSION: C8 monoclonal antibody against the synthetic peptide is TMSG-1 specific and is effective for Western blot and immunohistochemistry assays to detect TMSG-1 expression in cancer cells. TMSG-1 protein is about 45 000 (relative molecular mass) at cell membrane and cytoplasm of tumor cells. Expression of TMSG-1 protein correlates well, inversely with the tumor metastatic potential.

[Thymosin beta10 expression and actin filament organization in tumor cell lines with different metastatic potential].

OBJECTIVE: To investigate the expression of thymosin beta10 (Tbeta10) and related changes of actin filament organization in human tumor cell lines with different metastatic potential. METHODS: Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the expression of Tbeta10 mRNA detected by northern-blot and its peptide by immunohistochemical staining. The filamentous actin (F-actin) was stained with TRITC-phalloidin to detect changes in actin organization. RESULTS: In comparison with the non and/or weakly metastatic counterparts, Tbeta10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. TRITC-phalloidin staining revealed less actin bundles and a fuzzy network of shorter filaments in the highly metastatic tumor cells, while in the non and/or weakly metastatic cancer cell lines, there were thick and orderly arranged actin filaments. CONCLUSIONS: Tbeta10 levels correlate positively with the metastatic phenotype in human tumors currently examined. The increased metastatic potential of tumor cells is accompanied by the loss of F-actin and poorly organized actin skeleton. There is a consistent correlation between the elevated Tbeta10 expression and the disrupted actin skeleton.

Differential thymosin beta 10 expression levels and actin filament organization in tumor cell lines with different metastatic potential.

BACKGROUND: To investigate the differential expression levels of thymosin beta 10 (T beta 10) and the corresponding changes of actin filament organization in human tumor cell lines with different metastatic potential. METHODS: Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the amount of T beta 10 mRNAs by Northern blot and for their peptide expression levels by immunohistochemistry. The filamentous actin (F-actin) was observed by staining of TRITC-phalloidin to detect changes in actin organization. RESULTS: In comparison with non-/weakly metastatic counterparts, T beta 10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. Staining of TRITC-phalloidin revealed less actin bundles, a fuzzy network of shorter filaments and some F-actin aggregates in the highly metastatic tumor cells. Meanwhile, the actin filaments were robust and orderly arranged in the non-/weakly metastatic cancer cell lines. CONCLUSION: T beta 10 levels correlate positively with the metastatic capacity in human tumors currently examined. The increasing metastatic potential of tumor cells is accompanied by a loss of F-actin, poorly arranged actin skeleton organizations and presence of F-actin aggregates. There is a consistent correlation between the elevated T beta 10 expression and the disrupted actin skeleton.

[The effects of TMSG-1 gene transfection on metastatic phenotype of pg cancer cells].

OBJECTIVE: To observe the relationship between TMSG-1 gene and tumor metastatic phenotype. METHODS: TMSG-1 cDNA fragment which contained full length open reading frame of TMSG-1 gene was cloned into pcDNA3 plasmid to reconstruct sense and antisense eukaryotic expression plasmids of TMSG-1 gene containing neo selection marker. Both sense and antisense eukaryotic expression plasmids of TMSG-1 gene were transfected into the highly metastatic subclone PG-BE1 by LipofectAMINE method and the positive clones were selected by G418. RT-PCR was used to examine the expression level of the transfected gene and the changes of biological characteristics were checked by a series of in vitro and in vivo assays. RESULTS: The results showed that higher expression levels of TMSG-1 gene in BE1-S cells (cells transfected by sense TMSG-1 cDNA) than the control BE1 cells and BE1-V cells (cells transfected by pcDNA3 plasmid). The expression levels of TMSG-1 gene in BE1-AS cells (cells transfected by antisense TMSG-1 cDNA) were lower than those of the control BE1 cells and BE1-V cells. Compared with the control BE1 cells and BE1-V cells, BE1-AS cells grew more rapidly, and produced more foci in soft agar. Although the BE1-S cells did not reveal significant different growth capacity, the infiltrating ability and colony formation potential of BE1-S cells were decreased, compared with the control BE1 cells and BE1-V cells. Flow cytometry showed higher percentage of BE1-S cells in G0G1 phase than that of BE1 cells, and the presence of apoptotic peak in BE1-S cells. CONCLUSION: The results suggest TMSG-1 gene may represent a tumor metastasis suppressor gene.