Rejection of adenovirus infection is independent of coxsackie and adenovirus receptor expression in cisplatin-resistant human lung cancer cells.

The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity.

Co-expression of CXCR4 and CD133 proteins is associated with poor prognosis in stage II-III colon cancer patients.

Although CXCR4 and CD133 have been implicated in the metastatic process of malignant tumors, the clinicopathological significance of their expression in human colon cancer is not fully understood. The present study aimed to examine the expression of the CXCR4 and CD133 proteins in cases of stage II or III colon cancer and the related lymph nodes and to investigate the clinical and prognostic significance of these proteins in colon cancer. Immunohistochemical analysis was performed to examine CXCR4 and CD133 protein expression in paraffin-embedded stage II or III primary colon cancer tissues and matched lymph nodes. The correlation between the expression of the two proteins and clinicopathological parameters and the patient 5-year survival was analyzed. CXCR4 expression was detected in 74 of the 125 tumors (59.2%) and CD133 expression was detected in 45 (36.0%). The co-expression of CXCR4 and CD133 (both CXCR4 and CD133 were positive) was detected in 29 of the 125 tumors (23.2%). Compared with the other combinations, the co-expression of the CXCR4 and CD133 proteins was significantly associated with American Joint Committee on Cancer (AJCC) stage (P=0.029) and lymph node status (P=0.020). Log-rank analysis revealed that AJCC stage (P=0.014), lymph node status (P=0.011), CXCR4 expression (P=0.023), CD133 expression (P=0.034) and the co-expression of the CXCR4 and CD133 proteins (P=0.003) were significant prognostic indicators for the overall survival of patients. The results of the present study show that the co-expression of the CXCR4 and CD133 proteins is a risk factor for poor overall survival in stage II or III colon cancer patients, indicating that the co-expression of the CXCR4 and CD133 proteins contributes to the progression of colon cancer.

[Investigation on occupational exposure to 5-fluorouracil in pharmacy intravenous admixture service of a hospital].

OBJECTIVE: To investigate the level of occupational exposure to 5-fluorouracil (5-Fu) in the pharmacy intravenous admixture service (PIVAS) of a hospital, and identify the sources of 5-Fu contamination. METHODS: The 5-Fu concentrations in air, on the surface of different areas in PIVAS and personal protective equipments were detected using UV-vis spectrophotometry. RESULTS: The 5-Fu in air could not be detected. The 5-Fu concentrations on five different surfaces of biological safety cabinets were (22.00 +/- 6.35), (13.99 +/- 2.46), (14.13 +/- 0.72), (7.25 +/- 1.19) and (9.87 +/- 1.23) ng/cm2, respectively, which were significantly higher than those [(3.14 +/- 0.04), (5.43 +/- 0.65), (2.26 +/- 0.17), (2.26 +/- 0.17) and (3.63 +/- 0.46) ng/cm2] of corresponding controls (P < 0.05 or P < 0.01). The 5-Fu concentrations of the floor under cabinets [(18.19 +/- 5.22) ng/cm2], the floor in front of cabinets [(10.25 +/- 2.57)ng/cm2], the office floor [(11.64 +/- 2.53) ng/cm2], the terrace floor [(99.89 +/- 14.06 ) ng/cm2], the floor beside trash can in dressing room [(24.54 +/- 0.23) ng/cm2] were significantly higher than those of control [(3.36 +/- 0.11 ) ng/cm2] (P < 0.05 or P < 0.01). The 5-Fu concentrations of the tables in preparation room [(7.22 +/- l.04) ng/cm2] and the tables in office [(11.81 +/- 1.18) ng/cm2] were significantly higher than those of control [(5.56 +/- 0.14) ng/cm2] (P < 0.05 or P < 0.01). The 5-Fu concentrations of the indoor handle in preparation room were significantly higher than those of controls (P < 0.05 or P < 0.01). 5-Fu concentrations on the surfaces of outdoor handle and floor beside door in preparation room were not significantly increased compared with controls (P > 0.05). The 5-Fu concentrations on the surfaces of infusion bags, transfer box, transfer trays were significantly higher than those of controls (P < 0.05). The differences of 5-Fu concentrations between outer and inner masks and controls were not significant (P > 0.05). The 5-Fu concentrations of gloves of preparing and checking staffs were significantly higher than those of controls (P < 0.05 or P < 0.01). CONCLUSION: The preparing and checking process of 5-Fu and the treatment of medical wastes are major sources of 5-Fu contamination.

CT guided 125iodine seed implantation for portal vein tumor thrombus in primary hepatocellular carcinoma.

BACKGROUND: This study evaluated the clinical application of CT guided 125iodine implantation in patients with portal vein tumor thrombus in primary hepatocellular carcinoma. METHODS: The ten patients (9 males and 1 female, aged from 36 to 72 years) with portal vein tumor thrombus accompanying hepatocellular carcinoma had been treated with comprehensive therapy including surgery, transcatheter arterial chemoembolization, radiotherapy ablation, microwave ablation or percutaneous ethanol injection. The average diameter of each tumor thrombus was 21.5 mm x 30.5 mm. Seeds of 30 MBq 125I were implanted 5 mm apart within the portal vein tumor thrombus. The follow-up after 4 months included enhanced spiral CT. RESULTS: CT screening of the tumours indicated that 4 out of 10 patients showed complete response to the therapy, 5 partial response and 1 stable disease. Adverse effects included aggravated abdominal dropsy and temporarily increased transaminase, which were controlled by medical management. Severe complications such as haemorrhage, biliary fistula, hepatic abscess, pancreatic fistula and hepatic function failure were not observed. Implanted seeds migrated to lung and left hepatic lobe in 1 case. CONCLUSION: CT guided implantation of 125iodine seeds, can effectively treat portal vein tumor thrombus accompanying hepatocellular carcinoma with minimal damage and few complications.

Proteasome inhibitor MG-132 modifies coxsackie and adenovirus receptor expression in colon cancer cell line lovo.

The efficacy of adenovirus vector-based cancer gene therapy is controversial. Its uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Low transduction is believed to be one of the main barriers as the expression of CAR on tumor cells is frequently reduced. Increasing CAR expression on tumor cells thus offers a promising opportunity for more effective adenovirus based treatment. Expression of CAR in 62 cases of colon tumor specimens were examined with immunohistochemistry. To modify the CAR expression, the effects of proteasome inhibitor MG132 on CAR expression of colon cancer cell lines were determined by flow cytometry, RT-PCR, and western blot. To evaluate adenovirus transfer, we further used rAd.EGFP, rAd.p53, and oncolytic adenovirus to infect target cells. The CAR expression was significantly decreased in colon carcinomas, both in primary tumors and lymphonode metastasis. Though the deregulation of CAR occurred in early disease and showed no relationship with TNM stage, when primary tumors are more than 5 cm in diameter, this deregulation becomes more frequent. More importantly, proteasome inhibitor MG-132 could enhance CAR expression in colon carcinoma cell line lovo, accompanied with enhanced adenovirus transfer, target gene expression, and oncolysis. These data provide a rational basis for evaluation of CAR expression in tumors and pretreatment with CAR conditioner prior to adenovirus vector-based gene therapy.

Adenovirus-mediated recombinant human bone morphogenetic protein-7 expression promotes differentiation of human dental pulp cells.

Recombinant human bone morphogenetic protein-7 (BMP-7) has been shown to stimulate new reparative dentin formation in animal models. However, little is known about whether BMP-7 could promote the odontoblast-like differentiation and the formation of mineralized nodules in human dental pulp cells. Here, we reported that the infection with adenovirus-BMP-7 (Ad-BMP-7), a BMP-7-expressing adenoviral vector, induced the expression of BMP-7 in primarily cultured human dental pulp cells in the long term with little effect on their proliferation and viability. Importantly, BMP-7 expression significantly increased alkaline phosphatase activity and induced the dentin sialophosphoprotein expression in a dose- and time-dependent manner, suggesting that BMP-7 promoted the odontoblast differentiation. Furthermore, BMP-7 expression stimulated the formation of many mineralized dentin-like calcified nodules. Our data suggest that Ad-BMP-7-mediated BMP-7 expression can promote the differentiation of human pulp cells into odontoblast-like cells and mineralization in vitro, which may provide insight for the design of new gene therapy for the pulp capping in the clinic.

[Effects of adenovirus-mediated human bone morphogenetic protein-7 on proliferation and differentiation of human dental pulp cells].

OBJECTIVE: To investigate the effect of adenovirus expressing human bone morphogenetic protein-7 (hBMP-7) on proliferation and differentiation of human dental pulp cells. METHODS: The replication-deficient adenoviral vector encoding hBMP-7 gene was constructed by using homologous recombinant modality. The efficiency of transfection was evaluated by fluorescent microscopy and flow cytometry. The expression of hBMP-7 protein in adenovirus-infected dental pulp cells was determined by Western blot. The proliferation of cells was tested by MTT method, the activity of alkaline phosphatase was assayed, von Kossa staining was used to detect mineralized nodule formation, and the expression of DSPPmRNA in cells was detected using semi-quantitative RT-PCR. RESULTS: Green fluorescent protein was visible under fluorescent microscopy. Higher transfection efficiency (91.1 +/- 1.0)% could be obtained at MOI of 75. Western blot from dental pulp cells infected with Ad-hBMP-7 for 48h detected protein expression of a hBMP-7 gene. The activity of alkaline phosphatase in cells was significantly higher than those of the control groups (P < 0.05). The cells infected with Ad-hBMP-7 had the ability of mineralization. DSPP mRNA expression of cells was in a time- and dose- dependent manner. CONCLUSIONS: Ad-hBMP-7 can induce human pulp cells into odontoblasts, but has no obvious effect on their proliferation.

[Clinical characteristics and prognosis of primary nasal mucosal melanoma--a report of 44 cases].

BACKGROUND & OBJECTIVE: Primary mucosal malignant melanoma of the nasal cavity, paranasal sinuses, and nasopharynx is rare and current research data of this disease are mainly from western populations. This study was to analyze the clinical characteristics of this disease and prognositic factors. METHODS: From Jan. 1971 to Jul. 2005, 66 patients with primary nasal mucosal melanoma were treated in Cancer Center of Sun Yat-sen University, China. Demographics and baseline characteristics, treatments, recurrence, metastasis, and survival were documented in hospital records. All records of these patients were analyzed retrospectively. Kaplan-Meier method was used to calculate survival rate; Cox model was used to analyze prognostic factors. RESULTS: Among 44 evaluable cases, 37 were originated from the nasal cavity, 5 from the paranasal sinuses, and 2 from the nasopharynx. Cervical lymphadenopathy at initial presentation occurred in 12 patients. Of the 31 patients received operation-dominated treatment, 8 received adjuvant radiotherapy, 13 received adjuvant chemotherapy, and 6 received adjuvant non-specific immunotherapy. The median time of follow-up was 29 months. Local recurrence, cervical lymphadenopathy, and distant metastasis occurred in 24, 10, and 11 patients, respectively, during follow-up. The median survival time was 24 months and the 5-year survival time was 25%. Clinical stage affected prognosis, whereas age, gender, site, primary tumor mass, and adjuvant therapy were not correlated to survival status. CONCLUSION: Nasal mucosal melanoma has high incidence of local recurrence and distant metastasis, especially cervical lymphadenopathy. Clinical stage affects the prognosis.

[Sequence analysis of the CTL epitopes in transmembrane region of latent membrane protein 2 of Epstein-Barr virus derived from nasopharyngeal carcinoma cells].

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) cells expresses Epstein-Barr nuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1), and LMP2. LMP2 is an ideal target for immunotherapy because LMP2 mRNA is detected in 100% nasopharyngeal carcinoma cells and LMP2 protein shows stronger immunogenity than the rest 2 viral proteins. This study was to analyze the sequence of CTL epitopes in the transmembrane region of LMP2 to optimize LMP2-targeted immunotherapy. METHODS: Genomic DNA was extracted from 20 biopsies of NPC and 3 biopsies of normal nasopharynx from Cantonese. The transmembrane region of LMP2 gene was amplified with hemi-nest polymerase chain reaction (PCR), and then sequenced directly. RESULTS: As compared with prototype B95.8 cells, the transmembrane region of LMP2 gene, derived from Cantonese NPC and normal nasopharynx tissues, had 14 base pair substitutions, resulting in 6 amino acid substitutions. Among these substitutions, 3 changed amino acids were located in 4 HLA-restricted CTL epitopes (SSC, TYG, CLG, and VMS). Among these polymorphisms, the VMS variation was first identified. The sequence changes of the LMP2 derived from NPC was the same as those of the LMP2 from normal nasopharynx, indicating that those variations were due to geographic-associated polymorphisms rather than NPC-associated mutations. CONCLUSION: Polymorphisms of LMP2 exist in EBV derived from Cantonese, resulting in 4 CTL epitope variations, which implicates that the effect of LMP2 polymorphisms should be considered when LMP2-targeted vaccine is designed for immunotherapy.

The changes of CD4+CD25+/CD4+ proportion in spleen of tumor-bearing BALB/c mice.

CD4+CD25+ regulatory T lymphocytes (TR) constitute 5-10% of peripheral CD4+ T cells in naive mice and humans, and play an important role in controlling immune responses. Accumulating evidences show that TR cells are involved in some physiological processes and pathologic conditions such as autoimmune diseases, transplantation tolerance and cancer, and might be a promising therapeutic target for these diseases.To evaluate the change of CD4+CD25+ TR cells in mouse tumor models, CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing BABL/c mice were analyzed by flow cytometry using double staining with CD4 and CD25 antibodies.The proportion of CD4+CD25+/CD4+ in spleen lymphocytes was found to be higher than that in peripheral blood lymphocytes in normal mice. No difference was observed in the proportion in peripheral blood lymphocytes between tumor bearing mice and normal mice, while there was a significant increase in the proportion in spleen lymphocytes in tumor bearing mice as compared with normal mice. Moreover, the proportion increased in accordance with the increase in the tumor sizes. The increase in the proportion was due to the decrease in CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggests the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the TR in tumor mouse models, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes.