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Purpose: Tumor fibroblasts (TF) have been suggested to play an essential role in the complex process of tumor-stroma interactions and tumorigenesis. The aim of the present study was to investigate the specific role of TF in the esophageal cancer microenvironment.Experimental Design: An Affymetrix expression microarray was used to compare gene expression profiles between six pairs of TFs and normal fibroblasts from esophageal squamous cell carcinoma (ESCC). Differentially expressed genes were identified, and a subset was evaluated by quantitative real-time PCR and immunohistochemistry.Results: About 43% (126 of 292) of known deregulated genes in TFs were associated with cell proliferation, extracellular matrix remodeling, and immune response. Up-regulation of fibroblast growth factor receptor 2 (FGFR2), which showed the most significant change, was detected in all six tested TFs compared with their paired normal fibroblasts. A further study found that FGFR2-positive fibroblasts were only observed inside the tumor tissues and not in tumor-surrounding stromal tissues, suggesting that FGFR2 could be used as a TF-specific marker in ESCC. Moreover, the conditioned medium from TFs was found to be able to promote ESCC tumor cell growth, migration, and invasion in vitro.Conclusions: Our study provides new candidate genes for the esophageal cancer microenvironment. Based on our results, we hypothesize that FGFR2(+)-TFs might provide cancer cells with a suitable microenvironment via secretion of proteins that could promote cancer development and progression through stimulation of cancer cell proliferation, induction of angiogenesis, inhibition of cell adhesion, enhancement of cell mobility, and promotion of the epithelial-mesenchymal transition.

Supplementary Data from Fibroblast Growth Factor Receptor 2–Positive Fibroblasts Provide a Suitable Microenvironment for Tumor Development and Progression in Esophageal Carcinoma

Purpose To investigate whether the PAX6, Lumican, and MYOC genes are related to high myopia in Han Chinese since the association between these genes and high myopia is unclear in this patient population. Methods Peripheral venous blood samples were collected for DNA extraction from 220 subjects with high myopia (refractive error ≤-10.00 D) vs. normal controls among the Han Chinese of Northeastern China. Mass spectrometry was applied to detect 10 SNP loci of the PAX6, Lumican, and MYOC genes. The candidate region was analyzed using case-control correlation analysis. The χ(2) test was used to analyze the allele and genotype frequencies in the myopic group vs. the control group. Haploview software was used for haplotype analysis. Results The χ(2) test was used to compare the allele and genotype frequencies of SNPs in patients and control subjects and the results showed that ten SNPs of the PAX6, Lumican, and MYOC genes were not significantly associated with high myopia. Conclusions Our results confirm that the PAX6, Lumican, and MYOC genes were not associated with high myopia in the Han Chinese in Northeastern China.

We investigated the mechanism by which endothelial cells (ECs) resist various forms of physical stress using an experimental system consisting of rat arterial EC sheets. Formation of actin stress fibers (SFs) and expression of endothelial heat-shock stress proteins (HSPs) in response to mechanical stretch stress were assessed by immunofluorescence microscopy. Stretch stimulation increased expression of HSPs 25 and 70, but not that of HSP 90. Treatment with SB203580, a p38 MAP kinase inhibitor that acts upstream of the HSP 25 activation cascade, or with geldanamycin, an inhibitor of HSP 90, had no effect on the SF formation response to mechanical stretch stress. In contrast, treatment with quercetin, an HSP 70 inhibitor, inhibited both upregulation of endothelial HSP 70 and formation of SFs in response to tensile stress. In addition, treatment of stretched ECs with cytochalasin D, which disrupts SF formation, did not adversely affect stretch-induced upregulation of endothelial HSP 70. Our data suggest that endothelial HSP 70 plays an important role in inducing SF formation in response to tensile stress.

In this study, we investigated heat shock protein (HSP) expression and stress fiber (SF) formation in endothelial cells (ECs) within the arterial vascular tree of adult rats under normal physiological conditions. Using quantitative immunofluorescence microscopy, we found no significant differences in expression of HSPs 25, 60, 70, and 90 among ECs in the straight portions of rat arteries. In these regions, ECs appeared spindle-shaped and contained short bundles of central SFs. In contrast, ECs in the curved portions or the branch sites of the arteries, exhibited striking differences in HSP expression. ECs with higher HSP expression were localized at the lesser curvature in the curved portions or the distal site of the branch ostia. Moreover, the ECs became polygonal and contained irregular central SFs at the lesser curvature. At the branch sites, downstream ECs became spear-shaped and contained long, thick bundles of central SFs. Curved portions or branch sites are the regions of disturbed flow at which early atherosclerotic lesions are often found. Our results demonstrate these positional differences in HSP expression associated with changes in SF formation within the arterial vascular tree under non-pathological conditions. Our study provides basic information for understanding stress responses via HSP expression and SF formation in vascular ECs and the pathogenesis of atherosclerotic disease.

Arsenic is a multi-system toxicant. However, the mechanism of arsenic toxicity is not fully clarified and few effective protein biomarkers could be used for arsenic poisoning. This study was to investigate the differentially expressed proteins in the serum of rats subchronically exposed to arsenic. Sixty male rats were randomly divided into four groups, and the dose of sodium arsenite in drinking water for each group was 0, 2, 10, and 50 mg L−1, respectively. The exposure lasted for 12 weeks. An Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic approach was used to identify the differentially expressed proteins in serum between control and 50 mg L−1 groups. A total of 201 serum proteins were identified by iTRAQ, of which 12 were significantly changed by arsenic exposure with two up-regulated and ten down-regulated proteins. One down-regulated protein 14-3-3 ζ, an abundant protein expressed in the brain, was verified by ELISA using serum samples and by immunohistochemical, real time PCR, and western blot methods using brain tissues in four groups. Our work provided valuable insight into the serum protein changes in rats exposed to arsenic, and indicated that 14-3-3 ζ may serve as a useful biomarker for nervous damage caused by arsenic poisoning.

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 micromol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.

To detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).Stat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed.Suppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012).In a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.

6-Hydroxydopamine (6-OHDA) is a widely used dopaminergic neurotoxin that leads to cell apoptosis in vivo and in vitro, and is a widely accepted experimental model of neurodegeneration in Parkinson's disease. However, the molecular mechanisms responsible for 6-OHDA-induced cell apoptosis are unclear. We found that the treatment of PC12 cells with 6-OHDA resulted in a significant decrease in cell viability and elevated apoptosis as detected by MTT assay, Hoechst 33258 staining, and flow cytometry. In addition, 6-OHDA induced a time-dependent phosphorylation of ERK1/2 at Thr-202/Tyr-204 and of Raf-1 at Ser-338, but a decreased level of Raf-1 phosphorylation at Ser-259. Phosphorylation of ERK1/2 at Thr-202/Tyr-204 and Raf-1 at Ser-338 were inhibited by the Raf-1 inhibitor GW5074, while the ERK1/2 pathway inhibitor U0126 decreased phosphorylation of ERK1/2. Furthermore, 6-OHDA-induced PC12 cells apoptosis was suppressed by GW5074 and U0126. Our results suggest that GW5074 and U0126 act as neuroprotants against 6-OHDA toxicity in PC12 cells by modulating Raf-1/ERK1/2 signaling systems.

Our aim was to study calcium overload-induced apoptosis and its relation to reactive oxygen species (ROS) in rat C6 glioma cells after sonodynamic treatment (SDT).Hematoporphyrin monomethyl ether (HMME) was used as the sonosensitizer. The concentration of intracellular Ca(2+) ([Ca(2+)](i)) was measured by fluorometry. Apoptosis and necrosis rates were evaluated by a flow cytometry. Moreover, sarcoplasmic reticulum Ca(2+) -ATPase (SERCA(2)), cytochrome c (cyto-c) and cleaved caspase-3 were investigated by immunoblotting.Our study indicated that [Ca(2 +)](i) and ROS increased in cells of SDT group, the apoptosis rate, quantity of cyto-c and cleaved caspase-3 markedly increased after SDT. Furthermore, N-Acetyl-L-cysteine (NAC) or 1,2-bisethane-N,N,N',N'-tetraacetic acid tetrakis ester (BAPTA-AM) could decrease the apoptosis rate, the release of cyto-c and cleaved caspase-3 in SDT group, SERCA(2) degradation was found in SDT group and could also be prevented by the addition of NAC.Our results show that HMME-SDT can induce C6 cell death through both necrosis and apoptosis. ROS in C6 cells play a decisive role in HMME-SDT-induced cell death. The endoplasmic reticulum (ER) may be a major target of HMME-SDT, ROS can induce SERCA(2) degradation, causing the elevation of [Ca(2+)](i).

Purpose Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo. Methods A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 μg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days. Results Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed. Conclusions Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.

To investigate the inhibitory effects of amniotic membrane, polylactic acid membrane and chitosan membrane on scar formation following trabeculectomy.A total of 24 New Zealand white rabbits (48 eyes) were randomly divided into 4 groups: amniotic membrane group, polylactic acid membrane group, chitosan membrane group, and control group, with 6 rabbits (12 eyes) in each group. The left eyes underwent routine trabeculectomy, and the right eyes were considered as controls. Amniotic membrane, polylactic acid membrane and chitosan membrane were respectively installed under sclera flap in three groups, but any treatment was not applied in control group. Intraocular pressure, conjunctival filtering bleb, and anterior chamber inflammation responses were monitored at day 1, 3, 7, 14, 28 and 56 post-operatively. Eyeball tissue underwent histopathological examination at day 56 post-operatively.Fibrocytes and inflammatory cells were reduced in amniotic membrane, polylactic acid membrane and chitosan membrane groups compared to that in control group. At day 1 post-operatively, intraocular pressure was decreased in three membrane groups compared to that in control group. At day 14 post-operatively, the intraocular pressure was decreased significantly, while it of three membrane groups was significantly lower than that of preoperative (P0.01). There were no significant differences among three membrane groups (P0.05). Filtering bleb of four groups was clearly observed at day 7 post-operatively, but there was no significant difference in pair-wise comparison. At day 28 and 56 post-operatively, filtering bleb in control group was significantly narrowed compared to that in three membrane groups (P0.05), but there was no significant difference in pair-wise comparison of three membrane groups.All amniotic membrane, polylactic acid membrane and chitosan membrane can effectively inhibit scar formation following trabeculectomy, the effect of amniotic membrane is the best.

To investigate the inhibitory effect of transforming growth factor (TGF)-β₁ on oral squamous cell carcinoma (OSCC) Tb cell line.Cell counting method was used to examine the inhibitory effect of TGF-β₁ on Tb cell and flow cytometry (FCM) assay performed to measure the changes of cell cycle. Superarray was used to screen the changing expression of genes in TGF-β₁/Smads signaling pathway.RT-PCR method was used to detect the results of Superarray.TGF-β₁ showed significant inhibiting effect on OSCC Tb cell line. TGF-β₁ blocked the cell cycle at G₁ phase. The expression level of activin receptor-like kinase-1 (ACVRL-1), anti-mullerian hirmine (AMH), cyclim-dependent kinase inhibitor-2B (CDKN-2B) and transforming growth factor-beta-indnced factor (TGIF) was higer in the cells treated with TGF-β₁ than in control, while TDGF-1 expression was down-regulated. ACVRL-1 and CDKN-2B gene expression was consistent with the results of Superarray.TGF-β₁ can inhibit the growth of OSCC Tb cell line. The mechanism may be related to the regulation of cell cycle and the expression of ACVRL-1 and CDKN-2B in TGF-β₁-Smads signaling pathway.

Tumour suppressor ING4 is one of ING family genes, which are involved in cell cycle arrest, gene transcription regulation, DNA repair and apoptosis. ING4 inhibition has been reported in various tumours, including gliomas, breast tumours, and stomach adenocarcinoma. The aim was to evaluate ING4 expression in lung cancers.By immunohistochemistry of 246 lung tumour tissues, reduced ING4 nuclear and cytoplasmic expression were both revealed in lung cancer and associated with tumour grade. Interestingly, compared with normal tissues, we found more tumours with ING4 expression in the cytoplasm higher than in the nucleus. Nuclear ING4 inhibition correlated with the tumour stage and lymph node metastasis. Consistent with these findings, semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting demonstrated decreased ING4 mRNA and expression in 100% (50/50) tumour tissues. Furthermore, ING4 expression was lower in grade III than in grades I-II tumours. Reduced ING4 mRNA correlated with lymph node metastasis.Our results indicate that overall inhibition of ING4 expression and ING4 expression higher in cytoplasm than in nucleus of tumour cells may be involved in the initiation and progression of lung cancers, and thus, analysis for ING4 expression may be useful as a clinical diagnostic and prognostic tool for lung cancer.

Colorectal cancer has a high cure rate if it can be detected early. Identifying and understanding the genes involved may enable early diagnosis and reduce mortality. The aim of our study was to investigate the correlation between the expression of ING4 and the pathological features in patients with colorectal cancer. We assayed ING4 protein expression levels in tumor samples from 97 patients diagnosed with colorectal cancer between January 2001 and January 2002. The patients received no other treatments except surgery. ING4 protein expression was downregulated in adenoma relative to normal mucosa and further reduced in colorectal cancer tissues. Furthermore, the suppression of ING4 expression was also related to the more advanced Dukes’ stages. We observed that ING4 expression levels in patients with lymphatic metastasis were lower than those without metastasis. Together, our results indicate that ING4 play a role in colorectal carcinoma progression.

The aim of this study was to investigate the expression of transforming growth factor-beta1 (TGF-beta1) and its signaling pathway molecules in oral squamous cell carcinoma (OSCC) and analyze the association between these factors and genesis and metastasis of OSCC.The express of TGF-beta1, TbetaRI, TbetaRII and Smad4, a pivotal downstream molecule of its signaling, in 10 normal oral mucosa tissues and 108 OSCC was detected by SP immunohistochemistry, and thier correlation with genesis and metastasis of OSCC were assessed.The expressions of TbetaRII and Smad4 were lower in the tumors (34.3%, 38.9%) than those in the normal oral epithelium (80.0%, 100.0%, P0.05). The positive expression rates of TGF-beta1 and TbetaRI in the normal oral epithelium and OSCC were not significantly different (P0.05). There was an inverse correlation between TGF-beta1, Smad4, TbetaRII, TbetaRI expression and clinical stages (P0.01). The expression of TGF-beta1 was related with histological differentiation and tumor localization (P0.05). There was a relationship beteween Smad4 expression and histological differentiation and lymph node metastasis (P0.05). The expression of TbetaRII in the samples with lymph node metastasis was less than that in the ones without lymph node metastasis (P0.01), although there was no association between expression of TbetaRII and lymph node metastasis status.There is an important relationship between the abnormal TGF-beta1/Smad4 signal pathway and genesis and development of OSCC, while the low expressed Smad4 and TbetaRII may promote the metastasis of OSCC.

The autosomal dominant form of retinitis pigmentosa (ADRP) can be caused by mutations in 14 genes and further loci remains to be identified. This study was intended to identify mutations in a Chinese pedigree with ADRP.A large Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to genes known to be responsible for ADRP were selected for linkage analysis. Exons along with adjacent splice junctions of PRPF31 were amplified by polymerase chain reaction (PCR) and screened by direct sequencing.The caused gene of ADRP was mapped to 19q13.4 between markers D19S572 and D19S877, with a maximum LOD score of 3.01 at marker D19S418 (recombination fraction = 0).The affected gene linked to the 19q13.4 in a Chinese family with ADRP, which is different from other mutations at the same loci in other Chinese families.

To study the apoptotic effects of arsenic trioxide on human coronary smooth muscle cells (HCSMCs).HCSMCs were cultured and randomly divided into 5 groups to be treated by arsenic trioxide of the concentrations 1.0, 2.0, 3.0, 4.0, and 5.0 micromol/L for 48 h. Cell growth curve was drawn by MTT method. DNA electrophoresis was used to observe the apoptosis. Western blotting was conducted to examine the protein expression of Bax, an apoptosis-promoting gene, and Bcl-2, an apoptosis-inhibiting gene. Other HCSMCs were cultured with 4.0 micromol/L arsenic trioxide for 48 h, then transmission electron microscopy was used to observe the ultra-structure and TUNEL was used to detect the percentage of apoptotic cells. HCSMCs not treated with arsenic trioxide were used as control group.Arsenic trioxide of different concentrations inhibited the proliferation of HCSMCs dose and time-dependently. When the concentration of arsenic trioxide was 5.0 micromol/L the number of living cells was (4.41 +/- 0.10) x 10(5)/ml, significantly lower than that of the control group [(30.11 +/- 0.93) x 10(5)/ml, P0.05]. Apoptosis bodies were observed under the transmission electron microscope. DNA electrophoresis showed hazy ladders, especially when the concentrations were 3.0 - 4.0 micromol/L. When the concentration was 5.0 micromol/L the number of necrotic cells increased remarkably and apoptosis became less significant. TUNEL showed that the apoptotic cells increased from 16.0% +/- 3.1% to 38.7% +/- 2.7% (P0.05). MTT test showed that arsenic trioxide decreased the absorbance of HCSMCs dose and time-dependently. Western blotting showed that the Bcl-2 expression was decreased and the expression of Bax increased after treatment of arsenic trioxide.Arsenic trioxide exerts an apoptotic effect on HCSMCs.

Aim of the present study was to investigate the effect of chronic trimetazidine treatment on atrial energy metabolism and endothelial function in a canine model of chronic atrial fibrillation (AF).Eighteen canines were randomly divided into sham-operated group (n = 6), atrial pacing group (n = 6), and trimetazidine group (n = 6). In atrial pacing group and trimetazidine group, dogs were atrial paced at 400 beats per minutes for 6 weeks. Trimetazidine at 5 mgxkg(-1)xd(-1) was given one day before rapid atrial pacing for 6 weeks. Creatine phosphate (CrP), adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in atrial tissue were analyzed by high-performance liquid chromatography. Total adenosine (TAN) was calculated. The expression of endothelial nitric oxide synthase (eNOS) in atrial tissue was determined by Western blot and immunohistochemical staining. In addition, plasma levels of von Willebrand factor (vWF) was quantified with enzyme-linked immunoadsorbent assay and NO(2)(-)/NO(3)(-) (NOx) was determined by nitrate reductase method.Atrial CrP (P0.01) and CrP/ATP were significantly decreased in paced atrium compared to atrium from sham-operated group (P0.05) while ATP, ADP, AMP and TAN remained unchanged (all P0.05). Plasma vWF was significantly increased and plasma NOx significantly decreased in paced animals compared to sham-operated animals. Atrial expression of eNOS was also significantly reduced in paced animals (P0.01). Trimetazidine treatment did not alter the contents of CrP, ATP, ADP, AMP and TAN, but significantly increased atrial eNOS expression (P0.05), decreased plasma vWF (P0.01) and increased plasma NOx concentration.Trimetazidine treatment affect chronic AF induced disturbance in energy metabolism but may improve endothelial function through a NOx depended manner.

To evaluate the anti-angiogenic activity of peptide 21 obtained by modification of tumstatin, and its inhibitory effect on the growth and metastasis of human ovarian cancer transplanted in nude mice.The peptide 21 was purified by affinity chromatography. Human ovarian cancer SKOV3 cells were inoculated in nude mice and the transplanted tumor was treated with the peptide 21 to observe the tumor growth and metastasis. The microvessel density (MVD) and immunohistochemical staining index of PCNA, VEGF and MMP-2 and TIMP-2 were performed to assess the inhibitory effect of the peptide 21.In the nude mice at 21 days after peptide 21 treatment, the inhibition rate of tumor growth was 53.17%, the tumor microvessel density was significantly reduced (P0.05), the expression of PCNA, VEGF and MMP-2 were significantly lower (P0.01), and TIMP-2 expression was significantly higher (P0.01) in comparison with that of control group.The peptide 21 generated in this study has a significant anti-angiogenetic activity, showing significant inhibitory effect on the growth of human ovarian cancer transplanted in nude mice. The mechanism of its inhibitory action on ovarian cancer growth may be mediated by reduction of neovascularization and reduction of expression of angiogenetic factors.

To investigate the genetic polymorphism of 9 short tandem repeats (STR) gene loci, namely CSFIPO, TPOX, TH01, D16S539, D7S820, D13S317, F13A01, FESFPS and vWA in Chinese Korean population in Mudajiang area.Amplified fragment length polymorphism (Amp-FLP) method was used to get the allele frequency distribution.The genotype distributions of the 9 STR loci are conformed to Hardy-Weinberg equilibrium by chi(2) test analysis. The total accord frequency, the accumulated total discrimination power and the the accumulative excluding probability of paternity were calculated.The result suggested that all 9 gene loci have high power of excluding probability of paternity and individual identification. They can be used in paternity testing and individual identification for forensic medicine. The gene frequencies of CSFIPO, TPOX and TP01 gene loci have significant differences between the Korean population in Mudanjiang area and those in Yanji area, but there is no difference in gene loci of D7S820, D17S317 and vWA.

To evaluate whether the clustering of risk factors, both environmental and genetic, increases the risk of essential hypertension (EH) and the accumulation of risk factors influences the blood pressure level in normotensives.On the basis of a prevalence survey, 501 subjects of Mongolian ethnicity (243 hypertensives and 258 normotensives) who were not related to each other were selected to conduct a case-control study. All subjects were interviewed with questionnaires and their blood specimens were collected. Renin gene insertion/deletion (I/D) polymorphism, a new genetic marker, was genotyped with PCR and polyacrylamide gel electrophoresis.Overweight, alcohol consumption, and renin gene I/D polymorphism were significant risk factors of EH (P0.05). The odds ratios (OR) for the number of risk factors were 2.39 (95%CI: 0.98-6.74) for one risk factor, 5.03 (95%CI: 2.06-14.18) for two, and 6.09 (95%CI: 1.85-22.38) for three respectively after adjusting for age and sex. In normotensives, age- and sex-adjusted mean blood pressures increased with more accumulation of risk factors. However, there were no significant differences among the different blood pressure levels according to the number of risk factors (P0.05).Overweight, alcohol consumption, and renin gene I/D polymorphism are risk factors of EH in the Mongolian ethnic population of China. The accumulation of the risk factors causes a sharp increase of the risk of EH.

The frequency of lactase persistence varies widely in human populations. Study showed that the T allele of a C/T transition 13910bp upstream from exon 1 of lactase gene (LCT) was completely associated with lactase persistence in a Finnish population. To evaluate if the frequency of -13910T allele was in concordance with the lactase persistence in northern Chinese populations, in this study, we used Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) to detect the lactase -13910T allelic frequency in 5 northern Chinese populations for the first time. Results showed that the T allele frequency was low in these populations and that it did not match the lactase persistence phenotype in these populations. Therefore the -13910T allelic frequency can not serve as a predictor of the lactase persistence in these populations and this suggests the existence of other possible mechanisms of lactose tolerance in Chinese populations.

To study the impact of arsenic trioxide (As2O3) on human colorectal carcinoma LS-174T cells and their activity of telomerase.LS-174T cells and xenograft model of nude mice were treated with As2O3. The inhibitory effect of As2O3 on survival of LS-174T cells was determined by MTT assay. Apoptosis was determined by electron microscopy and fluorescence microscopy. Cell cycle was assessed by flow cytometry. Telomerase activity in LS-174T cells was determined by PCR-ELISA kit.With the increasing concentration of As2O3, the ratio of living cells to dead cells decreased significantly, and the IC50 value was 5.23 micromol/L. Apoptosis curve appeared after 24 h and cells turned to apoptosis in a time-dependent manner. As2O3 inhibited the telomerase activity in cell extraction, obviously in a concentration-dependent and time-dependent manner. Inhibitiory effect of As2O3 on xenograft model of nude mice was observed by tumor volume and weight measurement, showing a significant difference between As2O3 and control groups (P0.05).Both the experiments in vitro and in vivo showed an inhibitory effect of As2O3 on colonrectal cancer S-174T cell growth, probably by induction of apoptosis and inhibition of telomerase activity.

To investigate the effect of trichostatin A(TSA) on SGC- 7901 cells.Cytotoxicity and cell viability of gastric cancer cell line SGC- 7901 were assayed by MTT method. Morphologic assessment of apoptosis was performed with fluorescence microscope. Cell cycle and apoptosis rate were analyzed by flow cytometry. Histone H3 acetylation was detected by Western blot.TSA showed apparently cytotoxicity in SGC- 7901 cells. The growth curve showed the growth ratio decreased with the increase of TSA concentration. Apoptosis rate were significantly different between TSA treated group(75 ng/ml for 72 h)and control group (P0.05). Morphologic changes of apoptosis including nuclear chromatin condensation and fluorescence strength were observed with fluorescence microscope.TSA treatment (75 ng/ml for 72 h) sensitively induced apoptosis in the cell,which was demonstrated by the migration of many cells to the sub- G1 phase,the reduction of G1- phase cells and the increment of apoptosis rate (29.54%) in flow cytometric analysis. The expression of acetylated histone H3 was increased in TSA group(75 ng/ml) for 48 h compared with control group by Western blot.TSA can induce SGC- 7901 cell apoptosis. The expression of acetylated histone H3 may contribute to the apoptosis.

Y-chromosome short tandem repeats (STRs) are potentially useful for forensic, anthropological and evolutionary studies. In this study we chose the loci DYS 19, DYS 388, DYS 389 I, DYS 389 II, DYS 390, DYS 391, DYS 392, DYS 393, DYS 425 and DYS 426. Blood samples were taken from 46 unrelated male individuals from Fujian Han and 43 unrelated males from Sichuan Han in China. DNA was extracted by conventional chelex extraction procedure. PCR was carried out in two multiplex reactions. Fragment analysis was conducted on an ABI PRISM 310 Genetic Analyzer. Allele frequency distributions and discrimination indices were calculated, and the two populations were tested for genetic differences by means of analysis of molecular variance (AMOVA). Here we obtained 75 Y-STR haplotypes and the haplotype diversity for the complete haplotype was 0.9884 in Fujian Han and 0.9967 in Sichuan Han. A larger genetic difference became apparent between the two populations that belong to the Sino-Tibetan speaking populations.

To map the gene responsible for autosomal dominant congenital sutural cataract of a Chinese family.Peripheral blood samples were collected from the members in a family with autosomal dominant congenital sutural cataract. The genomic DNA was extracted from the blood samples. A genescan was performed using approximately 400 microsatellite markers (ABI). Linkage analysis was processed by Linkage software package to define the location of mutation gene. High density primers labeled with fluorescent stain for the positive region were adopted for fine targeting, and haplotype analysis was processed using Cyrillic software.The maximum two-point LOD scores was obtained at D3S1279, Z(max) = 2.32 (theta(max) = 0.00). After fine targeting and haplotype analysis, the gene was located within a 38.6 cM region between D3S1267 and D3S1614.The gene responsible for congenital sutural cataract in a Chinese pedigree is located at chromosome 3q21.1 - q26.2 within a 38.6 cM region.

Mutations in the tumor suppressor gene transforming growth factor beta (TGFB) Type II receptor (TGFBR2) are frequently found in many cancers with microsatellite instability, but are less common in lung cancer. In the present study, we looked for mutations in TGFBR2 in nonsmall cell lung carcinoma (NSCLC) cells and tissues. A novel homozygous microdeletion (c.492_507del) was identified in two cell lines derived from the same giant cell carcinoma (GCC) and was confirmed in the corresponding tumor tissues. Furthermore, a heterozygous c.492_507del was found in the germ-line of one patient, as well as in the other GCC cases and some large cell carcinomas (LCC) but not in other subtypes of NSCLC. The 16 bp-microdeletion introduced a premature stop codon at positions 590-592 of the cDNA, resulting in a truncated TGFBR2 protein with a mutated transmembrane domain and loss of kinase domain. The GCC cells were characterized as being unresponsive to TGFB induction both in growth inhibition and stimulation of extracellular matrix protein. Moreover, after the reconstitution of wild-type TGFBR2 expression, the sensitivity to TGFB was restored. Therefore, mutated TGFBR2 seems to play an important role in the abrogation of TGFB signal transduction in GCC cells.

To investigate the polymorphism of DYS287 among 28 ethnic populations in 9 provinces of China.YAP element was detected by Touchdown PCR amplification and 2% agarose gel electrophoresis.YAP+ frequencies in these ethnic populations were as follows: Zang 36.7%, Tu 23.8%, Yi 18.4%, Pumi 11.3%, Tajik 7.4%, Bai 6.7%, Jino 5.1%, Shandong Han 4%, Mulao 2.7%, and Maonan 1.3%. The rest ethnic populations in our study, including Gansu Han, Yunnan Han, Zhuangzu, Daizu, Lizu, Nuzu, Lisu, Naxi, Lahu, Dulong, Hani, Shezu, Weiwuer, Sala, Kerkizi, Dongxiang, Vazu, and Korea didn't carry YAP + element.Zangzu, Tuzu, Yizu, Pumi, Jino, and Baizu, which belong to Sino-Tibetan language family, carry a high YAP + frequency. Sala, Tuzu, and Tajik, regarded as Central Asia by origin in history and linguistics, also have a high YAP + frequency. Mulao and Maonan, which origin from "Baiyue" ancient ethnic groups, also have a considerable YAP + frequency.

To study the effect of RAB5A gene on microfilaments in human lung adenocarcinoma cells, AGZY83-a cells were stained with fluorescein isothiocyanate (FITC)-phalloidin. Microfilament bundles in RAB5A gene over-expressed AGZY83-a cells were shown to be denser than in control cells using confocal laser scanning microscope (CLSM). According to the analysis of Tumor Metastasis Microarray on RAB5A gene, three genes related to the regulation of cytoskeleton were identified, including NM23H1, Rac1 (downregulated in RAB5A over expressing cells), and S100A4 (upregulated in RAB5A overexpressing cells). Previous studies demonstrated that S100A4 gene functioned to suppress the expression of NM23H1 gene. To test if this elevated expression of S100A4 results in the down-regulation of NM23H1, RNAi was applied to silence the expression of S100A4 in AGZY83-a cells. Our data indicated that expression of NM23H1 was increased following the inhibition of S100A4 expression. Altogether, the results indicated that RAB5A gene was involved in the suppression of expression of NM23H1 by promoting the expression of S100A4.

To investigate if microdeletion of chromosome 22q11 is an epidemiologically important cause of congenital heart disease (CHD), we studied 25 cases with CHD phenotypes. Venous blood samples were tested by fluorescence in situ hybridization (FISH) for microdeletion of 22q11. Among 23 cases with simple CHD, 19 were shown not to have microdeletion of 22q11 and the other 4 cases were shown to have 22q11 microdeletion. Microdeletion of 22q11 was found in 2 cases with Tetralogy of Fallot (TOF) accompanied by multiple malformations. The results suggested that microdeletion of 22q11 was associated with CHD.

To identify the relationship between p53-dependent apoptosis associated genes and tumor metastasis.mRNA differential display (mRNA DD) was adopted for gene cloning after the different metastatic potential lung cancer cell lines were infected by Adv-p53 (a reconstructed adenovirus encoding wild type p53 gene). RT-PCR, Northern blot and Western blot assays were used to confirm the result from mRNA DD.After induction by p53 gene, the ANNEXIN A2 gene had differential expression in the cell lines; its level was down regulated in all the cells infected by Adv-p53 gene, especially in the Anip973 cell lines with high metastatic potential. RT-PCR, Northern blot and Western blot assays confirmed the consequence.The experimental data suggest that the ANNEXIN A2 gene may relate to cellular apoptosis induced by p53 gene. The affirmative relationship between ANNEXIN A2 gene and p53 needs further investigation.

To study the role of rab5a gene in the mechanism of tumor metastasis, rab5a gene was stable transfected into AGZY83-a cell, a lung adenocarcinoma cell line with low metastatic potential. Superarray was applied to study the effect of rab5a on the differential expression of genes related to tumor metastasis. Five differentially expressed genes were obtained, including one up-regulated gene s100a4 and 4 down-regulated genes, nm23a, rac1, cst3 and col4a2. These results were confirmed at both the RNA and protein levels. We concluded that rab5a promoted tumor metastasis by affecting multiple genes in several pathways involved in tumor metastasis.

The immortal lymphoblastoid cell lines were established by EBV transformation of B cells and addition of cyclosporin A to inhibit the activity of T cells. In the present study,549 immortal cell lines of different ethnic groups of Hazak, Manchu, Korea, Hozhe, Mongolia, Sibe, Hui, Puyi, Han in Fujian and Han in Sichuan were established. Through our research,we found it is harmful for B lymphocytes to transform if excessful leucocytes are inoculated.And it is crisis that cyclosporine A shoud be added the last. Our work is an important part of the research of human genome diversity for the exploration of the origin and evolution of different ethnic groups,and it also provides enough research materials for further studies.Moreover,we have sent 50 cell lines of Hozhe, Mongolia, Sibe, Daur, Oroqen to CEPH. Thus it is possible for us to utilize the genetic resources of CEPH freely.

In order to explore the association of apolipoprotein E (ApoE) gene polymorphism with cerebral infarction in type 2 diabetic patients of Han nationality in Northeast China , the genotypes of ApoE gene were analyzed by polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) in the 208 cases, including 69 cases in control (CON) group and 67 in type 2 diabetes mellitus (T2DM) group as well as 72 in type 2 diabetes mellitus with cerebral infarction (T2DMCI) group. Plasma lipid content in T2DMCI was also detected for 70 cases. The distribution of genotypes in ApoE gene,epsilon(2)epsilon(3),epsilon(3)epsilon(3) as well as epsilon(3)epsilon(4) was no significant difference in three groups (epsilon(2)epsilon(3) : 13.2%,epsilon(3)epsilon(3) : 67.6%,epsilon(3)epsilon(4) : 16.2%in CON group;epsilon(2)epsilon(3) : 19.4%,epsilon(3)epsilon(3): : 70.1%epsilon(3)epsilon(4) : 9%in T2DM group;epsilon(2)epsilon(3) : 15.2%,epsilon(3)epsilon(3) : 75%,epsilon(3)epsilon(4) : 4.2%in T2DMCI group). The allele frequencies of epsilon(2),epsilon(3) and epsilon(4) were not significantly different in the three groups, either(epsilon(2) : 9.6%,epsilon(3) : 82.4%,epsilon(4) : 8.1%in CON group; epsilon(2) :10.5%,epsilon(3) :84.3%,epsilon(4) : 5.2%in T2DM group; epsilon(2) :11.8%,epsilon(3) :84.7%,epsilon(4) : 3.5%in T2DMCI group). The levels of total cholesterol (TC), tryglyceride (TG), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were not significantly different among the different genotypes in T2DMCI group. The study confirmed that the polymorphisms of ApoE gene are neither associated with the T2DMCI, nor with the levels of plasma lipid in T2DMCI.

Resistance to the growth inhibitory effects of transforming growth factor-beta (TGF-beta) is a characteristic of many transformed cells. The purpose of this study was to determine the response of ovarian cancer cells to TGF-beta1 and to investigate the roles of components of the TGF-beta/Smads signaling pathway in carcinogenesis of ovarian cancer. Three ovarian cancer cell lines, HO-8910, HO-8910PM and SKOV3, were treated with TGF-beta1 and assayed for growth response by MTT assay. Furthermore, expression and subcellular localization of the components of TGF-beta/Smads signaling pathway in these cell lines in the absence or presence of TGF-beta1 were determined by RT-PCR and immunofluorescence analysis. We found that proliferation of SKOV3 cell was not significantly inhibited by TGF-beta1 while it expressed all components of the TGF-beta/Smads signaling pathway. After exposure to TGF-beta1, Smad7 protein in SKOV3 increased transiently and translocated to cytoplasm from nucleus while P-Smad2 translocated into nucleus from cytoplasm. Taken together, the results suggested that the TGF-beta/Smads signaling pathway remained functional in human ovarian cancer cells, HO-8910, HO-8910PM and SKOV3, and the abnormalities of the downstream effectors of Smads proteins might contribute to the resistance of SKOV3 cell to TGF-beta1.

The tumor suppressor gene p16, located on chromosome 9p21, encodes the cell cycle regulatory protein, p16. Inactivation of the p16 gene could lead to uncontrolled cell growth. It was examined that methylation of the p16 gene 5'CpG island of the tumor suppressor gene may be an important mechanism for transcriptional inactivation. In order to determine the role of methylation status of the 5'CpG island and abnormal expression of incarcinogenesis of endometrial carcinoma (EC), Methylation-Specific PCR (MSP) was used to determine the methylation status of p16 gene 5'CpG islands of 62 cases of EC. Loss or decrease of p16 expression was analyzed by immunohistochemistry (IHC) and homozygous deletion of exon1 (E1) and exon2 (E2) was determined by complex PCR. Ten specimens of normal tissues and adjacent tissues of tumor displayed no methylation and showed normal expression of p16. In E1 and E2 of the 62 EC, we found that 24.2% (15/62) were methylated, 54.8% (33/62) lost or reduced p16 expression, 16.1% (10/62) and 30.6% (19/62) had deletions of E1 and E2 respectively. There were 9.68% (6/62) and 46.6% (29/62) deletions of both or either of E1 and E2 respectively. Inactivation of p16 gene is a frequent event and positively correlated with pathological grades and clinical stages in EC. p16 gene methylation was an important event in the development of EC. MSP is an accurate and relatively simple method for evaluating the methylation status of a related gene.

To assess the role of microsatellite instability(MSI) on chromosome 19 in the carcinogenesis of gastric cancer. 26 microsatellite markers on chromosome 19 were amplified by Fluorescence-Labelled Primers Multiplex PCR in 44 paired gastric cancer tissues for MSI. The amplified fragments ran in PAGE gel by ABI 377 sequencer and the gel images were analyzed by GeneScan and Genotyper. Meanwhile, MSI of four of all microsatellite sites were also analyzed using PCR-SSLP-silver stain method. MSI was observed in 22 of 26(84.62%) microsatellite sites. D19S402 showed the highest frequency(22.73%). The total frequency of MSI was 8.3%. In 44 gastric cancer samples, 34(77.27%) cases showed MSI at one or more than one site. MSI, as a possible mechanism of carcinogenesis, might play an important role in human primary gastric cancer. MSI detections will become an integral part of the routine classification of all gastrointestinal tumors in the very near future.

Peptide nucleic acid (PNA) has many characteristics useful in molecular biology. This paper described an effective way to raise the cell ingestion rate of PNA so as to kill gastric cancer cells.Heteroduplexes of PNAs and oligonucleotides, wrapped by Lipofectamine 2000, were used to infect SGC7901 cells. The inhibitive effect of heteroduplexes was evaluated by analyzing cell clone forming and cell growth rate. Telomerase activity of SGC7901 cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and silver staining assay.PNAs showed a dose-dependent inhibition of cell proliferation. The percentage of proliferation inhibition was 99.4% after 7 days; the rate of cloning inhibition was 98.2% after 8 days; whereas for oligonucleotide groups, at the same concentration, the percentages were 50.1% and 67.5% respectively. Antisense PNA-DNA-Lipofectamine 2000 group (AP-D-L group) exhibited significantly different percentages from the control groups (P0.05). The test result indicated that telomerase activity of the AP-D-L group was inhibited (P0.05). At the same time, the impact on cell morphology was observed.The results showed that PNAs are potent antisense reagents. The telomerase-associated therapies are very promising for the treatment of malignant tumours.

To analyze the genetic polymorphism of 15 single nucleotide polymorphism (SNP) loci on the nonrecombining portion of the Y chromosome in 6 populations in China.Allelic specific polymerase chain reaction and 2% agarose gel electrophoresis and 6% PAGE were used to analyze the genetic polymorphism of 343 unrelated males, representing 6 populations in China, including Fujian Hans, Sichuan Hans, Mongolian, Hezhen, Sibo and Hui from the South, Northeast and Northwest.Thirty haplogroups were observed, and 3 of them (H15, H16, H18) were seen in all of the six populations. Although the heterozygosity levels of the Hezhen, Mongolian, Sibo populations are similar and those of the other 3 populations (Fujian Hans, Sichuan Hans, Hui) are similar, the pairwise differences among haplogroups are significant. Analysis of molecular variance (AMOVA) and principal component (PC) analysis of the haplogroup distributions suggested highly different allele diversity between group I including Hezhen, Mongolian, Sibo and group II including Hui, Fujian Hans, Sichuan Hans.The above analyses show more significant variance components in Northeast/South populations and clearly reveal the geographic genetic relationship among the six populations in the Northeast/Northwest/South. These results confirm the complexity of the genetic structure of Chinese populations and make a significant contribution for constructing the contemporary human gene pool and tracing genetic dispersal trail from Chinese populations.

Smad proteins transduce signals from transforming growth factor beta superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. TGF-beta/Smads signal pathway not only has transforming potential but can also drive tumourigenesis, malignant progression, invasion and metastasis of human cancers. Using the immuno-histochemistry, we investigate the expression and location of TGF-beta R II, Smad2, Smad4 and Smad7 in 20 lung cancer specimens and 8 lung cancer cell lines. The results suggest that aberrant smads protein expression is significantly related to lung cancer tumoruigenesis and progression. Interestingly, TGF-beta R II and Smad7 strongly express in high metastasis cell lines. High expression of TGF-beta R II and smad7 in the cell lines with high-metastatic potential showed a conceivable TGF-beta signal pathway independent Smads in the lung cancer, and that might mediate invasion and metastasis of lung cancer.

Atomic force microscopy (AFM) has many advantages in the study of biological samples, such as the convenient specimen preparation and the high resolution. In the present study, AFM was used to observe the double minute chromosomes (DMs) in mouse methotrexate-resistant cell line 3T3R500. AFM images were obtained by tapping mode, contact mode and later force mode of AFM. DMs were composed of two compact spheres connected with fibers. The number of DMs in the 3T3R500 cells increased with increasing levels of methotrexate (MTX) resistance. The data of the height and the underside diameter of the DMs were also obtained. The details of specimen preparation and scan mode selection of AFM were discussed. Our results show that AFM is a powerful method in the study of DMs.

To study the growth inhibitory effects of p21WAF1 and p53 overexpression in human lung adenocarcinoma cell line.The p21WAF1 and p53 gene were transfected respectively into a human lung adenocarcinoma cell line, GLC-82. Flow cytometry (FLC), transmission electron microscopy (EM) and TUNEL technique were used to evaluate cell growth and identify apoptosis.The GLC-82 transfected by p21 plasmid showed increased cell number in G1 phase of cell cycle, decreased proliferation potential and decreased cloning efficiency. Apoptosis have not been detected neither on EM nor by TUNEL technique, whereas the GLC-82 infected by Ad-p53 showed significantly decreased proliferation potential and some of them even died, in addition apoptosis was confirmed by TUNEL technique.The results indicate that p21WAF1 and p53 can inhibit proliferation; p53 also can induce apoptosis of lung adenocarcinoma cell. Therefore, these two genes should have a wide application in gene therapy of tumors in future.

To investigate the sequence of amyloid fibrils (BRI) gene and its expression in two lung adenocarcinoma cell lines AGZY83-a and Anip973 with the same tumor origin but different metastatic potential.DNA sequencing, sequential G banding fluorescence in situ hybridization (FISH) and Northern blot were used to analyze the sequence and expression of BRI gene was in two lung adenocarcinoma cell lines with different metastatic potential.The expression of BRI gene was up-regulated in the highly metastatic cell line Anip973 and was down-regulated in the low metastatic cell line AGZY83-a from which the Anip973 was derived. FISH results disclosed that in the two cell lines, the same rearrangements existed in the chromosome region where BRI gene was located, but in Anip973, amplification took place in the chromosome region where BRI gene was located. DNA sequencing results showed different mutations in the 5' untranslated region of BRI gene in the two cell lines.The above results revealed that there was no relation between BRI gene differential expression and rearrangements of chromosome. The amplification of the chromosome region where BRI gene was located and the different mutations in the 5' untranslated region of BRI gene probably contributed to the differential expression.

Gene amplification is a common mechanism that contributes to the drug resistance. To explore the molecular genetic background related to the MTX resistance in the mouse MTX-resistant cells, differential PCR was used to determine the amplification and overexpression of DHFR gene. In addition, the correlations between c-myc, p53 status and dhfr amplification were studied. Amplification and overexpression of dhfr suggested its role in MTX-resistant cells. However, no amplification and overexpression of c-myc were detected. On the other hand, no alteration of p53 copy number was found. The increased mRNA level of p53 suggested the normal function of p53. These results implicated the status of c-myc and p53 had no correlation with dhfr amplification, therefore some other molecular genetic alterations may exist to permit the dhfr amplification in MTX-resistant cells.