Targeting 4-1BB for tumor immunotherapy from bench to bedside.

Immune dysfunction has been proposed as a factor that may contribute to disease progression. Emerging evidence suggests that immunotherapy aims to abolish cancer progression by modulating the balance of the tumor microenvironment. 4-1BB (also known as CD137 and TNFRS9), a member of tumor necrosis factor receptor superfamily, has been validated as an extremely attractive and promising target for immunotherapy due to the upregulated expression in the tumor environment and its involvement in tumor progression. More importantly, 4-1BB-based immunotherapy approaches have manifested powerful antitumor effects in clinical trials targeting 4-1BB alone or in combination with other immune checkpoints. In this review, we will summarize the structure and expression of 4-1BB and its ligand, discuss the role of 4-1BB in the microenvironment and tumor progression, and update the development of drugs targeting 4-1BB. The purpose of the review is to furnish a comprehensive overview of the potential of 4-1BB as an immunotherapeutic target and to discuss recent advances and prospects for 4-1BB in cancer therapy.

Proteomic Analysis Reveals the Vital Role of Synaptic Plasticity in the Pathogenesis of Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is a chronic neurological disorder that is often resistant to antiepileptic drugs. The pathogenesis of TLE is extremely complicated and remains elusive. Understanding the molecular mechanisms underlying TLE is crucial for its diagnosis and treatment. In the present study, a lithium-pilocarpine-induced TLE model was employed to reveal the pathological changes of hippocampus in rats. Hippocampal samples were taken for proteomic analysis at 2 weeks after the onset of spontaneous seizure (a chronic stage of epileptogenesis). Isobaric tag for relative and absolute quantization (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique was applied for proteomic analysis of hippocampus. A total of 4173 proteins were identified from the hippocampi of epileptic rats and its control, of which 27 differentially expressed proteins (DEPs) were obtained with a fold change > 1.5 and P < 0.05. Bioinformatics analysis indicated 27 DEPs were mainly enriched in "regulation of synaptic plasticity and structure" and "calmodulin-dependent protein kinase activity," which implicate synaptic remodeling may play a vital role in the pathogenesis of TLE. Consequently, the synaptic plasticity-related proteins and synaptic structure were investigated to verify it. It has been demonstrated that CaMKII-α, CaMKII-ß, and GFAP were significant upregulated coincidently with proteomic analysis in the hippocampus of TLE rats. Moreover, the increased dendritic spines and hippocampal sclerosis further proved that synaptic plasticity involves in the development of TLE. The present study may help to understand the molecular mechanisms underlying epileptogenesis and provide a basis for further studies on synaptic plasticity in TLE.

Cytisine Exerts an Anti-Epileptic Effect via α7nAChRs in a Rat Model of Temporal Lobe Epilepsy.

Background and Purpose: Temporal lobe epilepsy (TLE) is a common chronic neurological disease that is often invulnerable to anti-epileptic drugs. Increasing data have demonstrated that acetylcholine (ACh) and cholinergic neurotransmission are involved in the pathophysiology of epilepsy. Cytisine, a full agonist of α7 nicotinic acetylcholine receptors (α7nAChRs) and a partial agonist of α4ß2nAChRs, has been widely applied for smoking cessation and has shown neuroprotection in neurological diseases. However, whether cytisine plays a role in treating TLE has not yet been determined. Experimental Approach: In this study, cytisine was injected intraperitoneally into pilocarpine-induced epileptic rats for three weeks. Alpha-bungarotoxin (α-bgt), a specific α7nAChR antagonist, was used to evaluate the mechanism of action of cytisine. Rats were assayed for the occurrence of seizures and cognitive function by video surveillance and Morris water maze. Hippocampal injuries and synaptic structure were assessed by Nissl staining and Golgi staining. Furthermore, levels of glutamate, γ-aminobutyric acid (GABA), ACh, and α7nAChRs were measured. Results: Cytisine significantly reduced seizures and hippocampal damage while improving cognition and inhibiting synaptic remodeling in TLE rats. Additionally, cytisine decreased glutamate levels without altering GABA levels, and increased ACh levels and α7nAChR expression in the hippocampi of TLE rats. α-bgt antagonized the above-mentioned effects of cytisine treatment. Conclusion and Implications: Taken together, these findings indicate that cytisine exerted an anti-epileptic and neuroprotective effect in TLE rats via activation of α7nAChRs, which was associated with a decrease in glutamate levels, inhibition of synaptic remodeling, and improvement of cholinergic transmission in the hippocampus. Hence, our findings not only suggest that cytisine represents a promising anti-epileptic drug, but provides evidence of α7nAChRs as a novel therapeutic target for TLE.

Current trends and future prediction of novel coronavirus disease (COVID-19) epidemic in China: a dynamical modeling analysis.

The novel coronavirus disease 2019 (COVID-19) infection broke out in December 2019 in Wuhan, and rapidly overspread 31 provinces in mainland China on 31 January 2020. In the face of the increasing number of daily confirmed infected cases, it has become a common concern and worthy of pondering when the infection will appear the turning points, what is the final size and when the infection would be ultimately controlled. Based on the current control measures, we proposed a dynamical transmission model with contact trace and quarantine and predicted the peak time and final size for daily confirmed infected cases by employing Markov Chain Monte Carlo algorithm. We estimate the basic reproductive number of COVID-19 is 5.78 (95%CI: 5.71-5.89). Under the current intervention before 31 January, the number of daily confirmed infected cases is expected to peak on around 11 February 2020 with the size of 4066 (95%CI: 3898-4472). The infection of COVID-19 might be controlled approximately after 18 May 2020. Reducing contact and increasing trace about the risk population are likely to be the present effective measures.

LXR agonists promote the proliferation of neural progenitor cells through MEK-ERK pathway.

The liver X receptors (LXRs) are transcriptional regulators of lipid homeostasis and may be critical for neurodegeneration and neurogenesis in vivo. However, it remains largely unknown about the role of LXRs and its agonists in the in vitro proliferation of neural progenitor cells (NPCs). Here we revealed for the first time that LXRs were markedly expressed in mouse NPCs and were critical for the in vitro proliferation. LXR agonists GW3965 and LXR623 promoted the proliferation of wildtype NPCs, but not NPCs from LXR double-knockout mice. Mechanistically, phosphorylation of MEK1/2 and ERK1/2 in NPCs was enhanced upon LXR agonist treatment, while abrogation of MEK/ERK phosphorylation by the inhibitors PD98059 and U0126 impaired the proliferation of wildtype NPCs in the presence or absence of LXR agonists. Collectively, our findings suggest that LXR agonists GW3965 and LXR623 can stimulate the NPC proliferation in LXR- and MEK/ERK-dependent manner.

PPARα Agonist Fenofibrate Ameliorates Learning and Memory Deficits in Rats Following Global Cerebral Ischemia.

Increasing evidence demonstrates that local inflammation contributes to neuronal death following cerebral ischemia. Peroxisome proliferator-activated receptor α (PPARα) activation has been reported to exhibit many pharmacological effects including anti-inflammatory functions. The aim of this study was to investigate the neuroprotective effects of PPARα agonist fenofibrate on the behavioral dysfunction induced by global cerebral ischemia/reperfusion (GCI/R) injury in rats. The present study showed that fenofibrate treatment significantly reduced hippocampal neuronal death, and improved memory impairment and hippocampal neurogenesis after GCI/R. Fenofibrate administration also inhibited GCI/R-induced over-activation of microglia but not astrocytes and prevented up-regulations of pro-inflammatory mediators in hippocampus. Further study demonstrated that treatment with fenofibrate suppressed GCI/R-induced activations of P65 NF-κB and P38 MAPK. Our data suggest that the PPARα agonist fenofibrate can exert functional recovery of memory deficits and neuroprotective effect against GCI/R in rats via triggering of neurogenesis and anti-inflammatory effect mediated by inhibiting activation of P65 NF-κB and P38 MAPK in the hippocampus, which can contribute to improvement in neurological deficits.

Valproic acid alleviates memory deficits and attenuates amyloid-ß deposition in transgenic mouse model of Alzheimer's disease.

In the brains of patients with Alzheimer's disease (AD) and transgenic AD mouse models, astrocytes and microglia activated by amyloid-ß (Aß) contribute to the inflammatory process that develops around injury in the brain. Valproic acid (VPA) has been shown to have anti-inflammatory function. The present study intended to explore the therapeutic effect of VPA on the neuropathology and memory deficits in APPswe/PS1ΔE9 (APP/PS1) transgenic mice. Here, we report that VPA-treated APP/PS1 mice markedly improved memory deficits and decreased Aß deposition compared with the vehicle-treated APP/PS1 mice. Moreover, the extensive astrogliosis and microgliosis as well as the increased expression in interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the hippocampus and cortex of APP/PS1 transgenic mice were significantly reduced following administration of VPA, which attenuated neuronal degeneration. Concomitantly, VPA alleviated the levels of p65 NF-κB phosphorylation and enhanced the levels of acetyl-H3, Bcl-2, and phospho-glycogen synthase kinase (GSK)-3ß that occurred in the hippocampus of APP/PS1 transgenic mice. These results demonstrate that VPA could significantly ameliorate spatial memory impairment and Aß deposition at least in part via the inhibition of inflammation, suggesting that administration of VPA could provide a therapeutic approach for AD.

[Research the biogeochemical processes of nutrients in Minjiang Estuary].

The variations in the concentration and distribution of nutrients and influencing factors in the Minjiang Estuary with a tidal cycle were investigated based on the data obtained during field observations in May 2007. The results showed the suspended sediment, dissolved inorganic nitrogen and silicate were opposite to the change of tidal, while the water level and salinity were consistent with tidal. The buffer mechanism of phosphate was controlled by suspended sand and water. The concentrations of silicate, phosphate and inorganic nitrogen were ranged 0.63-9.00 mg/L, 0.013-0.075 mg/L, 0.33-4.24 mg/L respectively. The contents of dissolved inorganic nitrogen in water mass increased remarkably comparing 1980s because of agriculture, industry and living. The research indicated that the nitrate and silicate were conservative, but phosphate was non-conservative in the biogeochemical processes of nutrients in Minjiang Estuary. The diluted water carried abundant inorganic nitrogen, silicate nutrients to Minjiang Estuary and thus phosphate was similar between diluted water and sea water. Based on the results of nutrient ratios, it was suggested that phosphate was a limiting factor for phytoplankton growth in the Minjiang Estuary.

[Genome DNA hypomethylation in the process of crystalline nickel-induced cell malignant transformation].

OBJECTIVE: To observe the effect of crystalline NiS on genome DNA methylation profile in in vitro cultured cells. METHODS: 16HBE Cells were treated with crystalline NiS at 0.25, 0.50, 1.00 and 2.00 µg/cm(2) for 24 h and three times at total. DAC treatment was given at 3 µmol/L for 72 h.5-mC immunofluorescence and SssI methyltransferase assay methods were applied to investigate if the hypomethylation of genome DNA involved. RESULTS: The results of 5-mC immunofluorescence showed that the fluorescence intensity of NiS-treated cells were decreased in some degree, and transformed cells were decreased dramatically. By the SssI methylase assay, an average of (81.9 ± 7.3)% methylated CpG were found in negative control cells. By contrast, (77.9 ± 6.2)%, (75.3 ± 6.8)%, (59.5 ± 4.9)%, (67.4 ± 5.1)% methylated CpG were observed in cells treated with NiS for three times at dosage of 0.25, 0.50, 1.00 and 2.00 µg/cm(2) which were abbreviated as NiS0.25, NiS0.50, NiS1.00, NiS2.00 respectively. The ANOVA analysis results showed that there was a significant difference in the 5 groups above (F = 124.95, P < 0.01). The results of Dunnett-t test showed that the methylated CpG of both group NiS1.00 and NiS2.00 were significantly decreased compared with the negative control group (t values were 7.64, 4.89 respectively, P < 0.01). For methylated CpG, (46.2 ± 4.1)% and (43.6% ± 4.3)% were observed in NiS-transformed cells (NSTC1 and NSTC2) which were dramatically decreased compared with the negative control group (t values were 12.79, 13.56 respectively, P < 0.01). CONCLUSION: Genomic DNA methylation levels were decreased during NiS induced malignant transformation.

[Relationship between polymerase eta expression and DNA damage-tolerance in human hepatic cells by hydroquinone].

OBJECTIVE: To investigate the effects of hydroquinone (HQ) on expression of Polymerase eta (Pol eta) and DNA damage in human hepatic cells (L-02), and to explore the role and possible mechanism of Pol eta involved in the process of DNA damage-tolerance. METHODS: After L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was detected by MTT assay; DNA impairment was detected by single cell gel electrophoresis (SCGE); Real-time fluorescent quantitative PCR and Western blotting methods were used to measure the expression of Pol eta at the mRNA and protein level in L-02 hepatic cells exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L). RESULTS: MTT assay showed that HQ with concentrations from 0 to 80 micromol/L had little effect on the survival rate of L-02 (P>0.05); whereas the survival rate of the group of 160 micromol/Lwas significantly higher than that of the control (P<0.01) after being treated with HQ for 24 h; the higher dose of HQ presented, the more degrees of DNA damage were produced. It was found that HQ in a low concentration (1-80 micromol/L) could induce the expression of Pol eta which was in proportion to the increasements of HQ concentration; the expression levels of mRNA and protein were reached to the maximum when treated with 80 micromol/L; the expression of Pol eta decreased (the relative quantity values were 2.32 +/- 0.16 and 1.20 respectively) once the concentration of HQ exceeded 160 micromol/L as compared with the group of 80 micromol/L, but it was higher than that of the control. CONCLUSION: This study suggested that Pol eta might involve in the process of DNA damage-tolerance induced by HQ in the hepatic cells.

Breast cancer resistance protein expression and 5-fluorouracil resistance.

OBJECTIVE: To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance. METHODS: MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immunohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens. RESULTS: MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P<0.05, n=3) in the cell model, while HPLC assay indicated that the intracellular retention dose of 5-Fu was significantly correlated with the expression of BCRP (r=-0.897, P<0.05, n=3). A total of 140 breast cancer tissue specimens were collected. BCRP-positive expression was detected in forty-seven specimens by both RT-PCR and IHC. As shown by MTT assay subsequently, the resistance index (RI) of 47 BCRP-positive breast cancer tissue specimens to 5-Fu was 7-12 times as high as that of adjacent normal tissue samples. BCRP expression was related to 5-Fu resistance (R2=0.8124, P<0.01). CONCLUSION: Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.

[Expression and sequence analysis of human MutS homologue 2 during different stages of human bronchial epithelial cells induced by cadmium chloride].

OBJECTIVE: To explore the expression and sequence of human MutS homologue 2 (hMSH2) during different stages of human bronchial epithelial (16HBE) cells induced by cadmium chloride (CdCl2). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining (SP method) were used to measure the hMSH2 mRNA and protein expression in 16HBE cells and its different passage cells treated by CdCl2 (the 5th, 15th, 35th passage, and neoplasm cells from nude mice's tumor tissue). hMSH2 exon 6, hMSH2 exon 7, hMSH2 exon 8, hMSH2 exon 9, hMSH2 exon 12 of the 16HBE cells and neoplasm cells from nude mice's tumor tissue were amplified by polymerase chain reactions (PCR). The amplified DNA strips were purified. Then the exons were detected by DNA analysis. RESULTS: During the passages of 16HBE cells treated with CdCl2, the expression of hMSH2 gene were decreased gradually. The hMSH2 gene mRNA and protein expression levels of the CdCl2 transformed 35th 16HBE cells and tumorigenic cells of nude mice significant decreased compared with non-transformed 16HBE cells (P < 0.01). In the tumorigenic cells of nude mice induced by CdCl2, there were thymine (T) deletion in 1st, 2nd and 7th site of hMSH2 exon 8, there were adenine (A) deletion in 20th and 182th site of hMSH2 exon 9, there were adenine (A) insertion in 241st site of hMSH2 exon 12. All the mutations were frame shift mutation. CONCLUSION: The expression decreased and the mutation of hMSH2 gene may be the possible carcinogenic mechanism for CdCl2.

Possible role of DNA polymerase beta in protecting human bronchial epithelial cells against cytotoxicity of hydroquinone.

OBJECTIVE: To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. METHODS: DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 micromol/L to 120 micromol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone. RESULTS: MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. CONCLUSIONS: Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

Alterations of FHIT gene and P16 gene in nickel transformed human bronchial epithelial cells.

OBJECTIVE: To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. METHODS: 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. RESULTS: NiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P < 0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. CONCLUSIONS: The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

[Construction of "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference].

OBJECTIVE: To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs). METHODS: According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing. RESULTS: The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment. CONCLUSION: The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.

[Identification of protein peroxiredoxin 2 related to crystalline NiS-induced neoplastic transformation].

OBJECTIVE: To provide evidence for illustrating the molecular mechanism of nickel carcinogenesis, and to identify the differential expression of protein in crystalline NiS-induced neoplastic transformation of human bronchial epithelial cell by proteomics technology. METHODS: Two dimensional electrophoresis (2-DE) and the ImageMaster 3.10 software were used to analyze the differential expression of protein, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) combined with database search was applied to identify protein peroxiredoxin 2 (PDX2) related to malignant transformation. RESULTS: The good 2-DE pattern including resolution and reproducibility was obtained. Nearly 700 expressed proteins per 2-D gel were isolated with molecular weights (MW) ranging from 14,400 to 94,000 KD and pI 3 - 10. A protein PDX2 with MW 21,890 KD, pI 5.66, which was highly expressed in malignantly transformed cell, was identified using MALDI-TOF-MS. CONCLUSION: PDX2 was involved in malignant transformation of human bronchial epithelial cell induced by crystalline nickel sulfide.

[Crystalline nickel sulfide-induced genomic instability in transformed human broncho-epithelial cells].

OBJECTIVE: To detect the genomic instability in the 16 human broncho-epithelial (16HBE) cells induced by crystalline nickel sulfide so as to provide the scientific basis for further study of nickel-induced cancer molecular mechanism. METHODS: To analyse the genomic instability in transformed 16HBE cells induced by crystalline nickel sulfide by random amplified polymorphic DNA (RAPD). RESULTS: All the 7 random primers selected could amplify 1 - 6 clear PCR bands. There were no significant differences between transformed 16HBE cells and negative control cells in the 4th, 5th, and 7th primers, but in the rest 4 primers there were significant differences, with special PCR bands for the same primer, indicating that genomic instability in transformed 16 HBE cells was induced by crystalline nickel sulfide. CONCLUSION: Crystalline nickel sulfide could induce genomic instability in 16HBE cells.

[Antitransforming activity of chlorophyllin against trans-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide].

OBJECTIVE: To study the inhibitory effect of chlorophyllin (CHL) on trans-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) induced malignant transformation in human bronchial epithelial cell line (16HBE). METHODS: 10, 50 or 100 micro mol/L CHL were added into the media during the cells transformation induced by BPDE, and the malignant degree of transformed cells were identified by the ConA agglutination test and the assay for anchorage-independent growth and tumorigenicity. RESULTS: After the cells were cultured for 25 times, the time of cells agglutination in groups treated with both CHL and BPDE was increased significantly; the colony formation efficiency in soft agar in groups treated with both CHL and BPDE (7.4 per thousand, 11.4 per thousand and 14.4 per thousand ) showed significant decrease (P < 0.05) in dose-dependent manner, as compared with that in group treated with BPDE alone (19.6 per thousand ). Cells treated with both CHL and BPDE or BPDE alone developed tumor in nude mice, a squamous carcinoma confirmed by histopathological examination. The volume of tumor in groups treated with both CHL and BPDE (0.43 +/- 0.13) cm(2), (0.22 +/- 0.04) cm(2) and (0.10 +/- 0.06) cm(3) was significantly smaller (P < 0.05) and dose-dependent, as compared with that in the group treated with BPDE alone (1.71 +/- 0.37) cm(3). CONCLUSION: CHL showed significant antitransforming ability in human bronchial epithelial cell line induced by BPDE.

[Alteration of FHIT gene and p16 gene in malignant transformed cells induced by crystalline nickel sulfide].

OBJECTIVE: To detect the alteration of fragile histidine triad (FHIT) gene and p16 gene during malignant transformation of immortal human bronchial epithelial cell line (16HBE) induced by crystalline nickel sulfide, and study the molecular mechanism of nickel carcinogenesis. METHODS: Malignant transformed cells and tumorigenic cells were examined for the alteration of FHIT gene and p16 gene by RT-PCR, DNA sequencing and silver staining PCR-SSCP. RESULTS: Compared with those of control 16HBE, neither mutation of exon2 or exon2-3, abnormal expression in p16 gene nor mutation of FHIT exon5, 6, 7 and 8, exon1-4 or exon5-9 were observed in transformed cells and tumorigenic cells. But aberrant transcript or FHIT gene expression loss were observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in FHIT gene, the deletion of exon6, exon7 and exon8 and an insertion of 36 bp sequence replacing exon6-8, was confirmed by sequencing. CONCLUSION: FHIT gene, not p16 gene, could play a definite role in nickel carcinogenesis. Alterations of FHIT gene induced by crystalline NiS could be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation, and FHIT gene could be one of the important target genes activated by exotic carcinogens.

[Study on lung carcinogenesis associated genes in human lung squamous cell carcinoma and malignant transformation of human bronchial epithelial cells induced by carcinogen].

OBJECTIVE: To investigate lung carcinogenesis associated genes in human lung squamous cell carcinoma and malignant transformation of human bronchial epithelial cells induced by chemical carcinogens with cDNA microarray. METHODS: The gene expression patterns were detected in all specimens by cDNA microarray which representing 4 096 different human genes. The differences in gene expression among 6 cases of human lung squamous cell carcinoma tissues and 6 normal lung tissues were analyzed. The different gene expression patterns between the normal human bronchial epithelial cell lines (16HBE) and the malignant transformation of human bronchial epithelial cells induced by Benzo(a)pyrene metabolite BPDE (anti-Benzo(a)pyrene diol-epoxide,BPDE) and crystalline nickel sulfide were also studied by that method. The similar changed genes among those gene expression patterns were identified as lung carcinogenesis associated genes. RESULTS: Among the 4096 genes of cDNA microarrays, there were 171 genes expressed differently among lung cancer tissues and normal lungs, 143 genes expressed differently between BPDE transformed cells and normal 16HBE cell lines, 151 genes differed between nickel sulfide transformed cells and normal 16HBE cell lines. By comparing the gene expression profiles, there were 89 similar changed genes which might be associated with human lung carcinogenesis, 39 of which were up regulated: 6 oncogenes, 4 cell cycle control genes, 6 cell proliferation genes, 8 metastasis genes, 3 neuroendocrine genes, 1 drug-resister gene, 1 anti-apoptosis gene, 1 oxidative gene and other 9 genes. 50 genes were down-regulated: 7 tumor suppression genes, 11 DNA repair genes, 1 antioxidant genes, 3 GST family genes, 3 cell framework genes, 2 apoptosis induced genes, 5 signal conduction genes, 5 cytokines and their receptor genes, 7 metabolization genes, 1 cell matrix genes, and other 5 genes. CONCLUSION: cDNA microarray can be applied to study gene expression profiles effectively and to screen human lung carcinogenesis associated genes.