[In-vitro amplification of oval cells with preservation of stem cell phenotype].

OBJECTIVE: To explore cell culture techniques for amplification of oval cells with preservation simultaneously of the stem cell characteristics. METHODS: Oval cell line OC3 was cultured in RPMI 1640 supplemented with 15% fetal bovine serum and 20 µg/L EGF. Cells were harvested every 5 passages and were examined with biomarkers including OV-6, c-kit, gamma-glutamyl transpeptidase, placental form of glutathione-S-transferase (GST-P), pyruvate kinase M2, pyruvate kinase L and albumin using techniques including RT-PCR, immunocytochemistry, and enzymo-cytochemistry. RESULTS: OC3 cell lines could be amplified abundantly in-vitro associating with expression of infant liver cell markers at various level, including OV-6, c-kit, gamma-glutamyl transpeptidase, GST-P, pyruvate kinase M2, but no expression of mature hepatocyte markers detected including pyruvate kinase L and albumin. CONCLUSIONS: Amplification of OC3 cells with preservation of the stem cell phenotype and high proliferation index can be achieved up to the 79(th) passages by culturing in RPMI 1640 supplemented with 15% fetal bovine serum and 20 µg/L EGF.

Overexpression of EIF-5A2 is associated with metastasis of human colorectal carcinoma.

Our previous study has suggested an oncogenic role of eIF-5A2 in ovarian tumorigenesis. Abnormalities of eIF-5A2, however, in colorectal carcinoma are unclear. In this study, amplification and overexpression of eIF-5A2 in colorectal carcinoma were studied by fluorescence in situ hybridization and immunohistochemistry using colorectal carcinoma tissue microarrays, including 139 primary colorectal carcinomas and their adjacent normal mucosa, 22 paired premalignant adenomas, and 42 metastatic tumors. The immunohistochemistry results showed that overexpression of EIF-5A2 was detected in none of normal epithelial mucosa, 35.3% of colorectal adenomas, 53.2% of primary colorectal carcinomas, and 67.6% of metastases. Amplification of eIF-5A2 was detected in 15.8% (16/101) of informative colorectal carcinomas, and most of them showed overexpression of EIF-5A2. In primary colorectal carcinomas, the frequency of EIF-5A2 overexpression was significantly higher in colorectal carcinomas with lymphovascular invasion (61.2%) than that in colorectal carcinomas without lymphovascular invasion (36.6%, P < .05). In addition, significant positive associations were found between EIF-5A2 overexpression and the tumors' later pN and pM stages, as well as increased tumor cell proliferation (P < .05). These findings suggest that overexpression of EIF-5A2 in colorectal carcinomas may be important in the acquisition of a metastatic phenotype and plays an important role in colorectal carcinoma development and progression.

[Expression of membrane/soluble MHC class I chain-related molecule A and NKG2D receptor in osteosarcoma and its clinical significance].

OBJECTIVE: To study the expression of membrane MICA (mMICA), soluble MICA (sMICA) and NKG2D receptor in cases of osteosarcoma and to analyze its clinical significance. METHODS: Expression of mMICA in osteosarcoma tissue of 43 cases was detected with immunohistochemistry. Expression of NKG2D in peripheral blood lymphocytes of 16 cases was analyzed by flow cytometry. Serum level of soluble MICA (sMICA) was measured by ELISA. RESULTS: mMICA was widely expressed in osteosarcoma tissue (37/43). Expression of NKG2D in peripheral blood lymphocytes was significantly decreased. High levels of mMICA and NKG2D expression were associated with better differentiation and earlier tumor stage of osteosarcoma (P < 0.05). A significant increase in serum level of sMICA was demonstrated in patients with metastasis and advanced tumor. CONCLUSIONS: The mMICA expression in tumor tissue, NKG2D expression in peripheral lymphocytes and serum sMICA level correlate with the differentiation and stage of osteosarcoma. These parameters may thus represent potential diagnostic and prognostic markers in patients with osteosarcoma. Manipulation of the MICA-NKG2D pathway may become a target of immunotherapy for osteosarcoma.

[Nuclear and cytoplasmic expressions of survivin in glioma and their prognostic value].

OBJECTIVE: To investigate the nuclear and cytoplasmic expressions of survivin in human glioma, and their correlations to clinicopathological characteristics and prognosis. METHODS: A tissue microarray (TMA) based on 94 patients with glioma was constructed and then immunohistochemistry was used to examine the nuclear and cytoplasmic expressions of survivin in these glioma cohorts. Their correlations with clinicopathological characteristics and prognosis were analyzed. RESULTS: Among the 94 samples of glioma, nuclear survivin was detectable in 21 (22.3%) cases and cytoplasmic survivin in 82 cases (87.2%). The positive rate of nuclear expression of survivin was significantly lower in grade I - II gliomas than in the grade III - IV gliomas (7.3% vs 34.0%, P = 0.005). Statistically significant associations were observed between nuclear survivin and recurrence (P = 0.031). Cytoplasmic expression of survivin was not related to any clinicopathological characteristics. The 5-year overall survival rate and 3-year progression-free survival rate were significantly lower in the nuclear survivin-positive group than in nuclear survivin-negative group (0% vs 39.50%, P < 0.001; 0% vs 27.52%, P = 0.021). The 5-year overall survival rate and 3-year progression-free survival rate in the cytoplasmic positive-group and negative-group were 43.60% vs 13.85% (P = 0.134), and 23.88% vs 0% (P = 0.965) respectively. A multivariate analysis was conducted according to the Cox regression model. The significantly independent prognostic factors for overall survival were nuclear expression of survivin, grade and age. The relative risk of death in the patients whose tumors were survivin positive compared with those whose tumors were survivin negative was 3.847. CONCLUSION: Nuclear expression of survivin is correlated with glioma differentiation and recurrence. It is a negative prognostic factor for progression-free survival and overall survival in patients with glioma.

[The significance and characteristics of chromosomal abnormalities in patients with microsatellite and chromosome stable colorectal carcinoma].

OBJECTIVE: To investigate the clinico-pathological significance and characteristics of chromosomal abnormalities in microsatellite and chromosome stable (AMCS) colorectal carcinoma (CRC). METHODS: Flow cytometry, microsatellite instability analysis and immunohistochemistry methods were used to examine the DNA ploidy, microsatellite instability and expression of mismatch repair proteins (hMSH(2) and hMSH(1)) in 156 cases of CRCs, so as to select the MACS CRCs. Comparative genomic hybridization (CGH) method was subsequently performed to analyze the status of chromosomal abnormalities of MACS CRCs. The correlation between chromosomal changes of MACS CRC and patients clinico-pathological features was further evaluated. RESULTS: Of the 156 CRCs studied, fourty-one (26%) cases were observed both diploid/near diploid DNA content and stable microsatellite sequence and all these CRCs showed normal expression of hMSH(2) and hMSH(1) protein, and thus, defined as MACS CRC. Our CGH results showed that the overall mean numbers of chromosomal abnormality of 41 MACS CRCs was 9.6. The chromosomal arms with DNA amplification (frequency > or = 10%) were 20 q (68%), 13 q (56%), 7 q (49%), 13 p (39%), 20 p (37%), 8 q (30%), 1 q (22%), 11 q (15%), 16 q (12%) and 2 p, 4 q, 10 q (10%). The chromosomal arms with DNA deletion (frequency > or = 10%) were 18 q (63%), 8 p (51%), 17 p (37%), 1 p (30%), 3 p (26%), 4 p, 13 q, 14 (15%) and 21 q, Xp (10%). In addition, there was 79% and 82% MACS CRCs in Dukes'C/D stages observed amplification of 20 q and deletion of 18 q, respectively, the frequency was significantly higher that that in Dukes'A/B stages (46%, P = 0.038 and 21%, P = 0.0009). CONCLUSIONS: The overall number of chromosomal abnormalities in MACS CRCs is similar to that of chromosomal instable (CI) CRCs, but is higher than that of microsatellite instable (MSI) CRCs. The frequent deletion of 8 p in MACS CRCs might be a special molecular event that is different from CRCs in CI and MSI pathway. Both 20 q amplification and 18 q deletion are the most frequent chromosomal abnormalities in MACS CRC and are associated closely with tumor's malignant clinical phenotype. These chromosomal changes might play important roles the development and progression of MACS CRC.

[Induced differentiation of rat hepatic oval cells in-vitro by combined hepatocyte growth factor and epidermal growth factor treatment].

OBJECTIVE: To characterize the biologic featrues of hepatic oval cells and their protein expression profiles during induced differentiation in vitro. METHODS: Rat hepatic oval cells were treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro, followed by morphological and molecular marker assessment by electromicroscopy, immunocytochemistry, RT-PCR and protein expression chip technology. RESULTS: Ten weeks after induction, the levels of GST-P mRNA and M2-PK mRNA were significantly reduced, whereas those of ALB and CK18 were elevated. Significant variations of expression was seen in 8 protein species during the course of the induced differentiation. CONCLUSION: Combined EGF and HGF treatment in vitro induces cell differentiation of hepatic oval cells, a process in which 8 protein species may play some regulatory roles.

SRC-3/AIB1 protein and gene amplification levels in human esophageal squamous cell carcinomas.

It has been suggested that the steroid receptor coactivatior-3 (SRC-3) gene, also known as AIB1, ACTR, RAC3, p/CIP and TRAM-1, located at 20q12, plays an oncogenic role in several types of human cancers. In this study, we examined the encoded protein expression of SRC-3 and its copy number in 221 human esophageal squamous cell carcinomas (ESCCs). In this ESCC series, the overexpression and increased copy number of SRC-3 gene was detected in 46 and 13% of ESCCs, respectively. In addition, overexpression of SRC-3 was observed more frequently in primary ESCCs in late T stages (T3/T4) than that in earlier T1/T2 stages (P<0.05), but no significant association of expression of SRC-3 and status of lymph node metastases was observed (P>0.05). These results suggest that overexpression of SRC-3, caused by gene amplification/gain or other molecular mechanisms, might provide a selective advantage for the development and local invasion of certain subsets of ESCC. In addition, a significant correlation (P<0.05) of overexpression of SRC-3 with increased cell proliferation (through detection of Ki-67 expression) was observed in these ESCCs. These findings suggest a potential role of SRC-3 in the control of ESCC cell proliferation; such may be responsible, at least in part, for tumorigenesis and/or progression of ESCC.

[The relationship between expression of interleukin-8 and prognosis of breast cancer].

OBJECTIVE: To investigate the expression of interleukin-8 (IL-8) and its prognostic significance in breast cancer. METHODS: Expression of IL-8 in 113 breast cancers, 19 breast benign tumors and 20 breast normal tissues was examined by tissue microarray using immunohistochemistry, and the association of IL-8 expression with patient's clinico-pathological characteristics and prognosis was further analyzed. RESULTS: The positive rate of IL-8 expression in breast cancer was 27.4%, which was significantly higher than that in benign tumor and normal tissue of breast (P = 0.002). IL-8 expression related to histological type (P = 0.040) and lymph node status (P = 0.021). The expression of IL-8 was observed to correlate negatively with ER and PR status (P = 0.015 and P = 0.034), and correlate positively with C-erbB-2 status (P = 0.002). In addition, Kaplan-Meier curves of disease-free survival analysis showed a significant difference between IL-8 positive groups and negative group (P = 0.031). CONCLUSIONS: IL-8 might be a poor prognostic factor for human breast cancer, and also might be a novel molecular marker to predicate the occurrence and progression of breast cancer.

[Nuclear expression of Survivin in glioma and its correlation to prognosis].

BACKGROUND & OBJECTIVE: Survivin, an inhibitor of apoptosis protein, is correlated to aggressive behavior, resistance to chemotherapy and radiotherapy, and prognosis of human cancers. It is expressed in both nuclei and cytoplasm. Recent research revealed that the nuclear expression of survivin is an important prognostic factor of human cancers. This study was to investigate the nuclear expression of Survivin in human glioma, and its correlations to clinicopathologic characteristics and prognosis. METHODS: The nuclear expression of Survivin in 88 specimens of glioma (33 at grade I-II, and 55 at grade III-IV) was detected by tissue microarray and immunohistochemistry. Its correlations to clinicopathologic characteristics and prognosis were analyzed. RESULTS: The positive rate of nuclear Survivin was 27.3% in the whole group. It was significantly lower in grade I-II gliomas than in grade III-IV gliomas (12.1% vs. 36.4%, P=0.013). Nuclear expression of Survivin had no correlation to patient's age (P=0.053), sex (P=0.376), Karnofsky performance status (P=0.486), and clinical stage (P=0.359). The 5-year overall survival rate and 3-year disease-freely survival rate were significantly lower in nuclear Survivin-positive group than in nuclear Survivin-negative group (22.7% vs. 47.7%, P=0.005; 13.7% vs. 39.5%, P=0.015). The median survival was slightly lower in high nuclear Survivin expression group than in low nuclear Survivin expression group (14.9 months vs. 20.2 months, P=0.089). CONCLUSIONS: The nuclear expression of Survivin is related to the pathologic grade of glioma, and might be a negative prognostic factor of glioma.

Oncogenic role of clusterin overexpression in multistage colorectal tumorigenesis and progression.

AIM: To investigate the expression pattern of clusterin in colorectal adenoma-carcinoma-metastasis series, and to explore the potential role of clusterin in multistage colorectal tumorigenesis and progression. METHODS: A colorectal carcinoma (CRC)-tissue microarray (TMA), which contained 85 advanced CRCs including 43 cases of Dukes B, 21 of Dukes C and 21 of Dukes D tumors, were used for assessing the expression of clusterin (clone 41D) and tumor cell apoptotic index (AI) by immunohistochemistry and TUNEL assay, respectively. Moreover the potential correlation of clusterin expression with the patient's clinical-pathological features were also examined. RESULTS: The positive staining of clusterin in different colorectal tissues was primarily a cytoplasmic pattern. Cytoplasmic overexpression of clusterin was detected in none of the normal colorectal mucosa, 17% of the adenomas, 46% of the primary CRCs, and 57% of the CRC metastatic lesions. In addition, a significant positive correlation between overexpression of clusterin and advanced clinical (Dukes) stage was observed (P<0.01). Overexpression of cytoplasmic clusterin in CRCs was inversely correlated with tumor apoptotic index (P<0.01), indicating the anti-apoptotic function of cytoplasmic clusterin in CRCs. CONCLUSION: These data suggests that overexpression of cytoplasmic clusterin might be involved in the tumorigenesis and/or progression of CRCs. The anti-apoptotic function of cytoplasmic clusterin may be responsible, at least in part, for the development and biologically aggressive behavior of CRC.

Heterogeneous expression and association of beta-catenin, p16 and c-myc in multistage colorectal tumorigenesis and progression detected by tissue microarray.

Most colorectal carcinomas (CRCs) arise from adenomas through an archetypal pathogenic pathway, the adenoma-carcinoma-metastasis sequence. Aberrant expression of beta-catenin, p16, E-cadherin and c-myc appears to have played important roles in the development and/or progression of CRC, but their precise distribution pattern and associations in different pathologic loci along CRC's pathogenic pathway have not been thoroughly examined. In this study, a tissue microarray (TMA) containing 85 advanced CRCs in different Dukes stages was constructed. In each of 85 cases, tissue specimens from normal mucosa and primary carcinomas in different layers of the bowel wall were included in the TMA. Tissue specimens from matched adenoma, lymph node metastases and distant metastases were obtained from 22, 21 and 21 cases, respectively. Expression patterns of beta-catenin, p16, E-cadherin and c-myc were evaluated by immunohistochemistry. The results revealed that nuclear expression of beta-catenin, p16 and c-myc was quantitatively increased from normal mucosa to premalignant adenoma, primary carcinoma and lymph node metastatic carcinoma; the frequency of nuclear overexpression of beta-catenin and p16 in lymph node metastases was significantly higher than that in distant metastases (p < 0.05). These results suggest an association between nuclear overexpression of beta-catenin and/or p16 and CRC lymph node metastasis but not distant metastasis. The results also showed that correlative high nuclear expression of beta-catenin and c-myc was observed in primary carcinomas involving the serosa and lymph node metastases (p < 0.05) but not in other pathologic regions of CRCs, suggesting that the tumor microenvironment in different pathologic loci of colorectal tumorigenesis and progression may influence c-myc responsiveness to beta-catenin/Tcf activation.

[Expressions of CD44s, MMP-9, and Ki-67: possible association with invasion, metastasis, and recurrence of osteosarcoma].

BACKGROUND & OBJECTIVE: Recent research had showed that tumor cell adhesion molecular CD44 and matrix metalloproteinases (MMP) were expressed strongly in many tumors, and was associated closely with invasion and metastasis of the tumors. Ki-67 was one of the proliferative markers, which indicated the growth rate of tumor cells. However, the relationship among these markers and the invasion, metastasis, and recurrence of osteosarcoma was unclear yet. In this research the authors studied the expression of standard-type CD44(CD44s), MMP-9, and Ki-67 in osteosarcoma, and their relation to the invasion, metastasis, and recurrence of the tumor. METHODS: Immunohistochemistry staining(SP method) was used to detect CD44s, MMP-9, and Ki-67 in cases of osteosarcoma. Bone benign disease or normal connective tissue were used as the control. The results were treated with semi-quantitative method and analyzed by using non-parameter rank sum test. RESULTS: The positive rates of CD44s, MMP-9, and Ki-67 in osteosarcoma were 71.0%, 75.8%, and 35.5%, respectively, which were significantly higher than that in control tissue. The positive rate of Ki-67 in recurrent osteosarcoma was 81.8%, which was significantly higher than that in primary tumor. CD44s and Ki-67 positive rates were 88.9% and 66.7% respectively in osteosarcoma with lung metastasis, which were both significantly higher than that in osteosarcoma without lung metastasis. In poorly differentiated osteosarcoma positive rates of CD44s and MMP-9 were 76.3% and 79.7%, respectively, which were significantly higher than that in well differentiated tumor. Spearman correlation analysis proved that the expression of CD44s, MMP-9, and Ki-67 had significant relation to another. CONCLUSIONS: Increase of CD44s, MMP-9, and Ki-67 were involved in the growth and local invasion of osteosarcoma. The recurrence of osteosarcoma was associated with the proliferative rate of tumor cells. Whether there were early lung metastasis or not was affected by the amount of CD44s and the proliferative rate of the tumor cell. The poorer differentiation of osteosarcoma cells, the higher level of CD44s and MMP-9.