[Comparison of allelic frequencies of 15 short tandem repeat loci between chronic myeloid leukemia patients and non-related healthy individuals].

OBJECTIVE: To compare allelic frequencies of 15 short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) between chronic myeloid leukemia (CML) patients and non-related healthy individuals from Changzhou region in order to predict genes related with the CML. METHODS: Blood samples were collected from 745 healthy subjects and 132 CML patients with complete remission. Genotypes were determined with gene scan technology and multiplex PCR with fluorescence-labeled primers. Allelic polymorphisms of 15 STR loci were compared between the two groups. Potential genes related with CML were predicted with statistical analysis of differences in allelic frequencies. RESULTS: Allelic frequencies of 3 loci, including CSF1PO, vWA and TPOX, showed a significant difference (P<0.05) between the two groups. CONCLUSION: CSF1PO, vWA and TPOX loci may be related with CML, albeit that the exact biologic mechanisms is unclear.

Identification and validation of Kallikrein-ralated peptidase 11 as a novel prognostic marker of gastric cancer based on immunohistochemistry.

BACKGROUND AND OBJECTIVES: It is important to identify and validate the differentially expressed genes in gastric cancer to screen diagnostic and/or prognostic tumor markers. METHODS: cDNA expression microarray, gene set enrichment analysis, and bioinformatics approaches were used to screen the differentially expressed genes between gastric cancer tissues and adjacent non-cancerous mucosa. A novel candidate prognostic marker, Kallikrein-related peptidase 11 (KLK11), was validated in 400 Chinese gastric cancer patients. KLK11 expression in gastric cancer tissues was detected using real-time PCR and Western blot. KLK11 protein expression was further analyzed by immunostaining on tissue microarray, followed with clinicopathological significance and survival analysis. RESULTS: KLK11 expression was significantly decreased in gastric cancer compared with that in normal gastric mucosa (P<0.001). Furthermore, KLK11 expression was much lower in poorly differentiated cancer samples than that in well-differentiated group (P<0.01). Survival analysis showed that negative KLK11 expression was associated with nearly fivefold increased risk of distant metastasis after curative gastrectomy (HR 4.65, P<0.01). Multivariate Cox regression analysis showed that KLK11 expression emerged as a significant independent prognostic factor for disease-free survival and overall survival (P<0.05). CONCLUSIONS: The results indicated that KLK11 expression was decreased in gastric cancer and might serve as a novel independent prognostic marker.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.

[The Mutated Gene Sequenced by NGS and the Correlation of Their Coexistent Mutual Exclusiveness in NPM1 Mutated Acute Myeloid Leukemia].

OBJECTIVE: To analyze the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) detected by next generation sequencing (NGS) and their coexistence and mutual exclusivity relationship in the AML subtype. METHODS: The NGS data based on 112 genes related to blood disease in 238 newly diagnosed patients with NPM1mut were collected. The χ2 test and non-parametric test were used to analyze the distribution correlation between the genes in the mutational spectrum. RESULTS: Among all the patients, at least one co-mutation was detected out. The median number per case of the mutated genes, including NPM1mut was 4.5 (range 2－14), among them, there were 5.0 (range 2－10) for NPM1mut/FLT3-ITD+ and 4.0 (range 2－14) for NPM1mut/FLT3-ITD- cases, but it was no significant difference between the two groups (P=0.378). A total of 240 NPM1 mutational events were detected out in entire 238 NPM1mut patients, of which 10 (4.2%) were missense mutations, and were all found in NPM1mut/FLT3-ITD- patients. Most (9/10, 90%) of these NPM1 missense mutations were accompanied by AML subtype-defining cytogenetic or molecular abnormalities, of which 7 patients were in low risk or 2 in high risk. The most common NPM1mut coexisting mutations were DNMT3A (104, 43.7%), followed were FLT3-ITD (95, 39.9%) and FAT1 (57, 23.9%), FLT3-ITD and DNMT3A showed significant coexistence (P=0.005). FLT3-ITD showed significantly reciprocal exclusivity with FLT3-nonITD (P<0.001), NRAS (P<0.001), PTPN11 (P=0.017) and IDH1 (P=0.005), and showed an exclusivity inclination with KRAS (P=0.073). In addition, FLT3-nonITD along with KRAS (P=0.035), NRAS along with KRAS (P=0.008) and PTPN11 (P=0.039) coexisted significantly. CONCLUSION: Prognoses of AML involving less common NPM1 missense mutations should be stated on a case by case basis. The mutational landscape and co-occurrence and mutual exclusivity correlations of NPM1mut AML provide a mechanism explaining biological diversity and clinical heterogeneity in this AML subset.

[Application of 1D wavelet analysis in detecting species of harmful algae blooms with absorption spectra of phytoplankton].

In the present study, the feasibility of using wavelet analysis to extract the eigen spectra from the absorption spectra of phytoplankton for species detection was investigated. Thirteen absorption spectra taken from single species cultures, representing four divisions (Dinophyta, Bacillariophyta, Haptophyta, and Chlorophyta), six genus (Gymnodinium, Prorocentrum, Skeletonema, Guinardia, Phaeocystis, and Prasinophyte) and seven species (Karenia mikimotoi, Karenia brevis, Prorocentrum minimum, Skeletonema costatuma, Guinardia delicatula, Phaeocystis globosa, and Pyramimonas parkeae), were used. First, the 1D wavelet analysis with five levels was applied to the thirteen absorption spectra, so each spectrum was decomposed with 5 levels. The 5th level component of low frequency corresponds to the background without information for species detection, and 1st and 2nd level component of high frequency is the random noise. The other levels (3rd to 5th) of high frequency are the useful information, and the sum of levels (3rd to 5th) of high frequency was retained as the eigen spectra for species detection. Second, the clustering analysis was used to the eigen spectra for examining the performance of the wavelet analysis in extracting species information. The clustering results were compared with the known species class information, and the results show that the 13 absorption spectra are correctly classified at the level of division, genus and species. This means that the wavelet analysis has good performance in extracting the eigen spectra for species detection. However, the above results were obtained with only limited species, and the more species data are required to identify the extensive validity of the conclusion.

Refined mapping of loss of heterozygosity on 1q31.1-32.1 in sporadic colorectal carcinoma.

AIM: To explore precise deleted regions and screen the candidate tumor suppressor genes related to sporadic colorectal carcinoma. METHODS: Six markers on 1q31.1-32.1 were chosen. These polymorphic microsatellite markers in 83 colorectal cancer patients tumor and normal DNA were analyzed via PCR. PCR products were electrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for Loss of heterozygosity (LOH) scanning and analysis. Comparison between LOH frequency and clinicopathological factors was performed by c2 test. RESULTS: 1q31.1-32.1 exhibited higher LOH frequency in colorectal carcinoma. The average LOH frequency of 1q31.1-32.1 was 23.0%, with the highest frequency of 36.7% (18/49) at D1S2622, and the lowest of 16.4% (11/67) at D1S412, respectively. A minimal region of frequent deletion was located within a 2 cM genomic segment at D1S413-D1S2622 (1q31.3-32.1). There was no significant association between LOH of each marker on 1q31.1-32.1 and the clinicopathological data (patient sex, age, tumor size, growth pattern or Dukes stage), which indicated that on 1q31.1-32.1, LOH was a common phenomenon in all kinds of sporadic colorectal carcinoma. CONCLUSION: Through our refined deletion mapping, the critical and precise deleted region was located within 2 cM chromosomal segment encompassing 2 loci (D1S413, D1S2622). No significant association was found between LOH and clinicopathologic features in 1q31.1-32.1.

[Elimination of leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes specifically targeting WT1-derived peptide: an experimental study in vitro].

OBJECTIVE: To investigate the effects of targeting elimination of the leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes (CTLs) specifically against Wilms tumor gene (WT1)-derived peptide. METHODS: A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A * 0201-binding anchor motifs was synthesized. The suspended cells were collected from the culture of the peripheral blood mononuclear cells and divided into 2 groups: Group D (pure T cells) and Group C (IL2 + T cells). The dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A* 0201-positive healthy donor were repeatedly loaded with WT1 peptide so as to elicit cytotoxic T cells (CTLs) specifically for WT1 peptide, and restricted by HLA-A * 0201 (Group A). The specific lysis effects of WT1 peptide specific CTLs upon leukemic bone marrow CD34+ progenitor cells positively expressing WT1 (3 being HLA-A * 0201(+) and 3 being HLA-A*0201(-)), peripheral blood CD34+ cells from healthy persons (2 being HLA-A * 0201(+) and 1 being HLA-A * 0201(-)), and leukemia cells of the lines of NB4 (HLA-A * 0201(+)/WT1(+)), U937 (HLA-A * 0201(+)/WT1(-)), and K562 (HLA-A * 0201(-)/WT1(+)) were measured by methyl thiazolyl tetrazolium (MTT) chromatometry assay. The colony-forming unit-granulocyte macrophage (CFU-GM) forming capability of the leukemic bone marrow CD34+ progenitor cells and peripheral blood CD34+ cells from healthy persons treated with WT1 peptide specific CTLs were also determined by methylcellulose medium colony-forming unit assay. The CTLs elicited by DCs without WT1 peptide loading(Group B)and T cells cultured with IL-2 (Group C) were taken as controls. RESULTS: The CTLs specific for WT1 peptide and restricted by HLA-A * 0201 (Group A) exerted a specific lysis upon the 3 cases with HLA-A * 0201(+)/WT1(+) leukemic bone marrow CD34+ progenitor cells and NB4 leukemia cells, the specific lysis levels were 55.3% +/- 2.8%, 67.1% +/- 3.2%, 49.4% +/- 3.8% and 55.0% +/- 3.7% respectively, all significantly higher than those upon the 3 cases with HLA-A * 0201(-)/WT1(+) leukemic bone marrow CD34+ progenitor cells, normal healthy peripheral blood CD34+ cells of the healthy persons, and leukemia cell of the lines K562 and U937 (the specific lysis levels were all < 20%). The specific lysis level of Group A CTLs was significantly higher than those of Group B and Group C CTLs (both P < 0.01). The relative colony formation rates of WT1-CTLs in Group A upon the 2 cases with HLA-A * 0201(+)/WT1(+) leukemic CD34+ progenitor cells were 17.8% +/- 4.0% and 20.8% +/- 3.41% respectively, both significantly lower than those of the none WT1-CTLs in Group B (88.9% +/- 3.4% and 91.8% +/- 5.7% respectively, both P < 0.01). There was no significant difference in the relative colony formation rate upon HLA-A * 0201(+)/WT1(-) leukemic bone marrow CD34+ progenitor cells and the HLA-A * 0201(-)/WT1(-) or HLA-A * 0201(+)/WT1(-) normal peripheral blood CD34+ progenitor cells between the CTLs of Groups A and B. CONCLUSIONS: The CTLs specific for WT1 and restricted by HLA-A * 0201 can exert specific lysis upon leukemic CD34+ progenitor cells highly expressing WT1 gene, and can inhibit the CFU-GM colony formation of such leukemic CD34+ progenitor cells. The product of expression of WT1 gene may be used as a target in the leukemic CD34+ cells.

[Prognostic Significance of the Percentage of Blasts with CD34+/CD38low/-/CD123+ Phenotype in Acute Myeloid Leukemias].

OBJECTIVE: To investigate the percentage of blasts with the CD34+/CD38low/-/CD123+ phenotype in de novo acute myeloid leukemia (AML) patients and analyse its correlation with prognosis. METHODS: The percentage of CD34+/CD38low/-/CD123+ cells in the blast population of 148 newly diagnosed patients with AML was determined by using flow cytometry and its correlation with complete response, disease-free survival and overall survival were evaluated. RESULTS: The median percentage of CD34+/CD38low/-/CD123+ cells in newly diagnosed patients was 2.8% (ranged from 0.01 to 67%). The high expression of CD34+/CD38low/-/CD123+ in AML patients positively correlated with the NPM1 wild-type (χ2=5.194,P<0.05), but did not relate with the positive FLT3-ITD mutations (χ2=0.418,P>0.05). Further multivariable analysis showed that the higher expression of the CD34+/CD38low/-/CD123+ was associated with lower complete remission (P<0.05), worse disease-free survival(P<0.01) and shorter overall survival(P<0.01) in AML patients. CONCLUSION: The percentage of CD34+/CD38low/-/CD123+ cells at diagnosis significantly correlates with the response to treatment and survival. This prognostic marker may be used to rapidly identify the risk of treatment failure in clinical practice.

[Refined mapping of loss of heterozygosity of 1p36.33-36.31 in sporadic colorectal carcinoma].

OBJECTIVE: To screen the candidate tumor suppressor gene (TSG) related to colorectal cancer (CRC). METHODS: Seven fluorescent labeled polymorphic markers (1p36.33-36.31) were analyzed in the samples of tissues of CRC and adjacent normal tissues obtained during operation performed on 83 CRC patients, 40 males and 43 females, aged 31 - 84. The PCR products were electrophoresed. The softwares Genescan 3.1 and Genotype 2.1 were used for loss of heterozygosity (LOH) scanning and analysis. Comparison between the LOH frequency and the clinicopathological factors was performed by Chi-square test. RESULTS: The average LOH frequency in the 1p36.33-36.31 was 31.47%, with the highest LOH frequency of 47.22% at the D1S243 locus and the lowest LOH frequency of 7.35% at the locus D1S1347. Two obvious high LOH regions were detected: D1S1347 locus (1p36.33, about 1 cM) and a region between D1S468 and D1S2660 (1p36.32-36.31 about 3 cM). The LOH rats of the loci in 1p36.33-36.31 region were not correlated with the age and sex of the patient, and the size, growth pattern, and Dukes stage of the tumor. CONCLUSION: Two obvious high frequency LOH regions, 1p36.32-36.31 and 1p36.33 have been detected in sporadic CRC where tumor-suppressor-gene (s) may exist.

Decitabine enhances bortezomib treatment in RPMI 8226 multiple myeloma cells.

The present study investigated the interactions between decitabine (DAC) and bortezomib (BTZ) in RPMI 8226 multiple myeloma (MM) cells. Cells were exposed to DAC alone and in combination with BTZ for 48 h. A Cell Counting Kit­8 assay was performed to assess the rate of proliferation inhibition in the cells. Cell apoptosis was investigated by Annexin V-fluorescein isothiocyanate and propidium iodide staining. Flow cytometry was used to detect the different cell cycle stages. Western blotting was performed to analyze the protein expression levels of poly(ADP­ribose) polymerase 1 (PARP­1), caspase­3, ­9 and DNA (cytosine­5­)­methyltransferase 1 (DNMT1). Reverse transcription­quantitative polymerase chain reaction was used to assess DNMT1 gene expression. The combination of DAC and BTZ increased the proliferation inhibition, apoptotic rate and G0­G1 arrest compared with use of a single therapeutic agent. In addition, the combination treatment enhanced PARP­1 cleavage, caspase­3 and caspase­9 activation and downregulated the protein and mRNA expression levels of DNMT1. Therefore, the current study determined that the combination of BTZ and the epigenetic agent DAC may be a novel therapeutic strategy to improve the efficacy of BTZ in patients with MM.

Detailed deletion mapping of loss of heterozygosity on 22q13 in sporadic colorectal cancer.

AIM: Both development and progression of malignancies occur as a multistep process, requiring the activation of oncogenes and the inactivation of several tumor suppressor genes. The loss of heterozygosity (LOH) of tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer (CRC). In this study, we analyzed the LOH of seven loci on chromosome 22q13 in an effort to identify candidate tumor suppressor genes involved in colorectal carcinogenesis. METHODS: Matched tumor and normal tissue DNA were analyzed by PCR using fluorescence-labeled polymorphic microsatellite markers in 83 CRC patients. PCR products were eletrophoresed and LOH was determined by calculating the peak height acquired through computer software. Comparisons between LOH frequency and clinicopathological features were performed by chi2 test. P<0.05 was considered as statistical significance. RESULTS: The average LOH frequency of chromosome 22q13 was 28.38%. The highest LOH frequency was 64.71% on D22S1160 locus, and the lowest was 21.43% on D22S1141 locus. We detected two obvious minimal deletion regions: one between markers D22S1171 and D22S274, the other flanked by markers D22S1160 and D22S1149, each about 2.7 and 1.8 cm, respectively. None had lost in all informative loci. LOH frequency on D22S1171 is 50% on distal colon, which was higher than that on proximal one (P = 0.020); on D22S114 locus, none LOH event occurred in patients with liver metastasis, whilst 46.94% occurred in patients without liver metastasis (P = 0.008); on D22S1160 locus, LOH frequency in lymph nodes metastasis patients was 83.33%, which was much higher than 43.75% without lymph nodes metastasis ones (P = 0.016). There was no statistical significance between clinicopathological features and other loci. CONCLUSION: This study provides evidence of two minimal deletion regions, which may harbor putative tumor suppressor genes related to progression and metastasis in sporadic colorectal carcinoma on chromosome 22q13.

[Immunotherapy for leukemia cells by using cytotoxic T lymphocyte specifically against WT1-derived peptide: an experimental study in vitro].

OBJECTIVE: To investigate the effect of targeting immunotherapy for leukemia cells by using cytotoxic T lymphocyte (CTL) specifically against WT (Wilm's tumor) 1-derived peptide. METHODS: A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A*0201-binding anchor motifs was synthesized. Dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A*0201-positive healthy donor were cultured and divided into 2 groups: experimental group to be loaded with Wilms' tumor 1 (WT1) peptide so as to elicit CTL's specifically for WT1 peptide, restricted by HLA-A*0201, and control group. Six days later rhTNF-alpha was added for 3 days more to promote the maturation of DCs. Before loading of WT1 peptide and 2 days after the addition of rhTNF-alpha direct immunofluorescence labeling method was used with PE or FITC labeled mono-antibodies to detect the expression of the surface antigens: CD83, CD1alpha, CD80, CD86, CD14, and HLA-DR. DCs suspended and attached to wall were collected and then divided into 2 groups: pure T cell group (group D) to be cultured in culture medium without IL-2, and IL-2 + T cell group (Group C) to be cultured in 1640 culture medium with IL-2. Eight days later the T cells of Group C were co-cultured with the DCs of the experimental group (WTI peptide + DC + IL-2 + T cells, Group A) or the DCs of the control group (DC + IL-2 + T cells, Group B). Five days later the killing activity was detected. The CTLs of Groups A, B, and C at logarithmic growth phase were mixed with leukemic cells of the lines: NB4/WT1D, NB4WT1A, NB4 (all HLA-A*0201 +, WT1 +), U937 (HLA-A*0201 +, WTl -), and K562 (HLA-A*0201 -, WTI +), and mononuclear cells of the bone marrow of leukemic patients at different effector cell-target cell of 20:1 and 10:1. MTT method was used to examine the killing rate of CTL to the target cells. RESULTS: (1) The killing rates of Group A cells to NB4/WT1 D, NB4WTA, and NB4, leukemic cells were 60.4% +/- 3.1%, 56.4% +/- 5.7%, and 55.0% +/- 3.7%, all significantly higher than those of the Group B cells (10.9% +/- 1.6%, 11.1% +/- 2.7%, and 11.9% +/- 2.5%), and those of Group C cells (9.1% +/- 1.0%, 9.2% +/- 1.7%, and 9.4% +/- 1.8%) (all P < 0.01). There were no significant differences in the killing rates to U937 and K562 leukemic cells among the 3 groups. (2) When the effect-target ratio was 20: 2, the killing activity of the CTLs of Group A to the HLA-A*0201 +, WT1 + NB4/WT1 D, NB4/WT1A and NB4 leukemic cells was significantly higher than those to the HLA-A*0201 +, WT1 - U937 cells and the HLA-A*0201 -, WT1 + K562 cells (both P < 0.001), however, not significantly different from that to the U937 and K562 cells. (3) There were no significant differences in the killing activity of Group A cells to NB4/WT1D, NB4/WT1A, and NB4 cells (P = 0. 065, P = 0.621). (4) When the effect-target ratio was 10:2, the killing rates of Group A cells to the NB4/ WT1D, NB4/WT1A, and NB4 cells were 45.9% +/- 3.9%, 43.9% +/- 3.7%, and 44.1% +/- 3.2% respectively, all significantly lower than those when the effect-target ratio was 20:1 (all P < 0.01). CONCLUSION: CTLs specific for WT1 and restricted by HLA-A*0201 exert specific lysis upon leukemia cell lines and primary leukemia cells, but not upon normal hematopoietic cells, which provides a rationale for developing a strategy of WT1 peptide-based adoptive T-cell therapy and vaccination for human leukemia and solid tumors.

[Refined mapping of loss of heterzygosity on 10q23-24 in sporadic colorectal carcinoma].

OBJECTIVE: Loss of heterozygosity (LOH) of tumor suppressor gene (TSG) is believed to play a key role in carcinogenesis of colorectal cancer (CRC). In this study, we performed refined mapping of LOH on 10q23-24 region and screened the candidate TSG related to sporadic CRC. METHODS: Seven fluorescent labeled polymorphic markers (encompassing D10S185 locus) were analyzed in 83 cases of colorectal carcinoma and normal tissues by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. GeneScan 3.1 and Genotyper 2.1 softwares were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological factors were performed by Chi-square test. RESULTS: The average LOH frequency was 36.11%. The highest frequency of LOH (D10S583 locus) and the lowest (D10S205 locus) were 54.84% and 21.3%, respectively. Two obviously high LOH regions were detected: one between D10S583 locus and D10S185 locus (about 0.9 cm, 10q23.33); another flanked by D10S1709 and D10S1265 locus (about 1.5 cm, 10q24.2-24.31). Furthermore, significant difference was observed between LOH frequency and Dukes stages only on D10S1265 locus. CONCLUSION: Two obviously high frequency LOH regions, 10q23.33 and 10q24.2-24.31, were detected in sporadic CRC. Besides PTEN gene, the above two regions may harbor candidate TSG involved in development and progression of sporadic CRC.

Overexpression of HOXA10 promotes gastric cancer cells proliferation and HOXA10(+)/CD44(+) is potential prognostic biomarker for gastric cancer.

Gastric cancer (GC) is a malignant cancer with poor prognosis. This study aims to investigate the roles of homeobox A10 (HOXA10) in GC and the correlations between HOXA10/CD44 expression and GC prognosis. Based on qRT-PCR and Western Blot analyses in 50 pairs of fresh GC samples and adjacent normal samples, it is identified that HOXA10 was significantly up-regulated in GC tissues at mRNA and protein levels. Cell proliferation, migration, and invasion were enhanced in GC cells with overexpressed HOXA10, while inhibited in cells with silenced HOXA10. Through IPA software, HOXA10 was predicted to interact with CD44 via MSN, which was preliminarily confirmed by using Western Blot. Through immunohistochemistry and tissue microarray (N=264), it is found that HOXA10 expression was significantly correlated with tumor size (P=0.011) and CD44 expression (P<0.001), while CD44 expression was significantly correlated with tumor size (P<0.001), depth of tumor invasion (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P=0.001), UICC stage (P<0.001), histological differentiation (P<0.001), and HOXA10 expression (P<0.001). Additionally, the over-all survival and disease-free survival of HOXA10(+)/CD44(+) patients were dramatically decreased in comparison with that of HOXA10(+)/CD44(-), HOXA10(-)/CD44(+), or HOXA10(-)/CD44(-) patients (P<0.001), suggesting that the combinatory expression of HOXA10 and CD44 was correlated with poor GC prognosis. In conclusion, HOXA10 and CD44 might play roles in GC tumorigenesis, metastasis, and invasion. HOXA10(+)/CD44(+) expression might serve as a prognostic biomarker for GC, which needs more studies to validate.

[MicroRNA-10a expression in FAB different subtype of acute myeloid leukemia and its relationship with drug resistance].

OBJECTIVE: This study was to investigate the expression of miR-10a in the different FAB subtype of acute myeloid leukemia (AML) and its relationship with drug resistance. METHODS: Forty de novo patients with AML, 16 patients with non-malignant hematologic disease and three AML cell lines HL-60, U937 and HL-60/ADR were enrolled in this study, the MiR-10a expression in bone marrow mononuclear cells of above-mentioned patients and 3 AML cell lines was detected by TaqMan RT-PCR. The correlation of miR-10a with clinicopathological factors of AML patients was analyzed. RESULTS: The miR-10a expression level in HL-60 cell line was higher than that in U937 cell line (P = 0.039). And its expression level in de novo AML patients was higher than that in patients with non-malignant hematologic disease (P < 0.01). FAB-AML-M3 patients exhibited higher expression of miR-10a than that in M1, M2 and M4 (P < 0.05); HL-60/ADR cell line showed higher miR-10a expression than that in HL-60 cell line (P < 0.01) . Except M3, the patients without CR (non-CR) after the first cycle of chemotherapy showed a higher level of miR-10a as compared with CR patients (P < 0.01). CONCLUSION: The high expression of miR-10a may be closely related to over-proliferation of promyelocyte and drug resistance of acute myeloid leukemia cells, except M3.

Loss of heterozygosity on chromosome 1 in sporadic colorectal carcinoma.

AIM: Loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer. When it occurs at a tumor suppressor gene locus with abnormal allele, neoplastic transformation happens. In this study, we analyzed the LOH at 21 loci on chromosome 1 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis. METHODS: Twenty-one polymorphic micro-satellite DNA markers were analyzed with PCR both in 83 cases of colorectal cancer and in normal tissues. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. chi2 test was used to compare LOH frequency with clinicopathological data. P<0.05 was considered as statistically significant. RESULTS: The average LOH frequency of chromosome 1, short arm and long arm was 19.83%, 18.00% and 21.66%, respectively. The 2 highest LOH loci with a frequency of 36.54% and 32.50% were identified on D1S468 (1p36.33-p36.31) and D1S413 (1q31.3), respectively. On D1S2726 locus, LOH frequency of rectal cancer was 28.57% (6/21), which was higher than that of colon cancer (0.00%, 0/33) (P=0.002), suggesting that the mechanism of carcinogenesis was different in both groups. CONCLUSION: Putative tumor suppressor genes on chromosome 1 may relate to sporadic colorectal carcinomas. Tumor-suppressor-genes might locate on 1p36.33-36.31 and/or 1q31.3.

Refinement of heterozygosity loss on chromosome 5p15 in sporadic colorectal cancer.

AIM: To refine the loss of heterozygosity on chromosome 5p15 and to identify the new tumor suppressor gene (s) in colorectal tumorigenesis. METHODS: Sixteen polymorphic microsatellite markers were analyzed on chromosome 5 and another 6 markers were applied on chromosome 5p15 in 83 cases of colorectal and normal DNA by PCR. PCR products were electrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. RESULTS: We observed 2 distinct regions of frequent allelic deletions on Chromosome 5, at D5S416 on 5p15 and D5S428-D5S410 on 5q. Another 6 polymorphric microsatellite markers were applied to 5p15 and the minimal region of frequent loss of heterozygosity was established on 5p15 spanning the D5S416 locus. CONCLUSION: Through our detailed deletion mapping studies, we have found a critical and precise location of 5p deletions, 5p15.2-5p15.3, which must contain one or more unknown tumor suppressor gene (s) of colorectal cancer.

Expression of telomerase activity and oxidative stress in human hepatocellular carcinoma with cirrhosis.

AIM: To study the expression and significance of telomerase activity and oxidative stress in hepatocellular carcinoma (HCC) with cirrhosis. METHODS: In this study, TRAP-ELISA assay was used to determine telomerase activity in 21 cases of HCC as well as in 23 cases of hepatic cirrhosis. Malondialdehyde (MDA), glutathione S-transferase (GST) and total anti-oxidative capacity (T-AOC) were also examined in the same samples with human MDA, GST and T-AOC kits. RESULTS: Eighteen of 21 cases of HCC were found to have increased telomerase activity, whereas only three of the 23 non-cancerous cirrhotic samples were found to have weak telomerase activity, and the difference was significant (P<0.001). No significant difference in telomerase activity was detected according to different tumor size, tumor stage, histological grade, HBsAg, contents of albumin, bilirubin, ALT, AFP, r-GT and platelet. There were significant differences between HCC and cirrhosis in the expression of MDA, GST and T-AOC respectively. Telomerase activity correlated positively with the content of MDA (P<0.05). CONCLUSION: Telomerase activation is the early event of carcinogenesis, which is not correlated with clinicopathological factors of HCC. The dysfunction of the anti-oxidative system is closely correlated with the progression from cirrhosis to hepatocellular carcinoma. Oxidative stress may contribute partly to telomerase activation.

Loss of heterozygosity on long arm of chromosome 22 in sporadic colorectal carcinoma.

AIM: The loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer. In this study, we analyzed the LOH at 5 loci on the long arm of chromosome 22 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis. METHODS: Five polymorphic microsatellite markers were analyzed in 83 cases of colorectal and normal DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer; Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological data were performed by chi(2) test. P<0.05 was considered as statistically significant. RESULTS: The average LOH frequency on chromosome 22q was 28.38 %. The region between markers D22S280 and D22S274 (22q12.2-q13.33) exhibited relatively high LOH frequency. The two highest LOH loci with frequencies of 35.09 % and 34.04 % was identified on D22S280 (22q12.2-12.3) and D22S274 (22q13.32-13.33). 8 cases showed LOH at all informative loci, suggesting that one chromosome 22q had been completely lost. On D22S274 locus, LOH frequency of rectal cancer was 50 % (9/18), which was higher than that of proximal colon cancer (12 %, 2/17) (P=0.018). The frequency of distal colon cancer was 42 % (5/12), also higher than that of proximal colon cancer. But there was no statistical significance. Putting both the tumors in distal colon and rectum together into consideration, the frequency, 47 % (14/30), was higher than that of proximal colon cancer (P=0.015),suggesting the mechanism of carcinogenisis was different in both groups. CONCLUSIONS: This study provided evidence for the involvement of putative tumor suppressor genes related to the sporadic colorectal carcinoma on chromosome 22q. The tumor-suppressor-gene(s) might locate on the 22q12.2-12.3 and/or 22q13.32-13.33.

[Effect of p16 gene on carcinogenesis of hepatitis B virus related hepatocellular carcinoma].

OBJECTIVE: To investigate the relation between p16 gene expression and the carcinogenesis and progress of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). METHODS: In 35 specimens of HCC tissue and the adjacent liver tissue, the integration of HBV X gene was detected by polymerase chain reaction (PCR) and Southern blot. The point mutation of exon-1alpha, 2 and 3 of p16 gene were detected by PCR-single strand conformation polymorphism (SSCP). The expression of p16 mRNA and p16 protein was detected by RT-PCR and Western blot. RESULTS: The integration of X gene correlated with the expression loss of p16 mRNA and p16 protein in HCC (P < 0.05). The expression loss rates of p16 protein in HCC and adjacent tissues were 62.9% (22/35) and 40.0% (14/35) with significant difference (P < 0.05). The expression loss of p16 protein in HCC correlated with the differentiation degrees of HCC and the infiltration of tumor cells (P < 0.05). CONCLUSION: The integration of X gene correlates with the expression loss of p16 protein. The alteration of p16 gene, playing an important role in all stages of hepatocarcinogenesis, correlates with the progress and invasion of hepatocellular carcinoma.