The purpose of this study was to construct a recombinant conditionally replicating adenovirus (CRAd) expressing programmed cell death 5 (pdcd5). Pdcd5 gene was inserted in the E3 region of SG600-a CRAd in which the key genes for virus replication E1a and E1b were controlled under the human telomerase reverse transcriptase promoter (hTERTp) and the hypoxia response element (HRE) respectively, and with a deletion of 24 nucleotides within CR2 region of E1a. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and polymerase chain reaction (PCR). The infection efficiencies of a recombined virus carrying enhanced green fluorescent protein (EGFP) in leukemic cell lines were observed by using fluorescence microscope. The relative pdcd5 expression levels of K562 after being infected with SG611-pdcd5 were detected by real-time quantitative PCR. The results showed that the construction of SG611-pdcd5 was completed and confirmed. Pdcd5, hTERTp, HRE, skeleton and fiber11 of recombinant adenovirus SG611-pdcd5 were successfully amplified. The infection efficiencies of SG611-EGFP were all above 70% in both leukemic K562 and MEG-01 cell lines. SG611-pdcd5 expressed pdcd5 with high efficiency in leukemic cells as compared with Ad-pdcd5 or SG611 (p < 0.001). The expression level of pdcd5 increased gradually along with the increase of MOI. It is concluded that the triple-regulated adenovirus of SG611-pdcd5 containing the pro-apopro-tic gene pdcd5 has been successfully established with high pdcd5 expression level in leukemic cells, indicating that the recombinant adenovirus, SG611-pdcd5, promises further development of targeted tumor gene therapy.
Adenoviridae ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Genetic Therapy ; methods ; Neoplasm Proteins ; genetics ; Oncolytic Viruses ; genetics ; Promoter Regions, Genetic ; Telomerase ; genetics

OBJECTIVETo investigate the effect of siRNA targeting Livin and Survivin gene simultaneously on the proliferation and apoptosis of human colon cancer cells.

METHODSSiRNA recombinant expression vectors targeting Livin and Survivin gene simultaneously were constructed and transfected into human colon cancer cell line Lovo. The effects of siRNA recombinant expression vector on Lovo cells were detected by RT-PCR, Western blot, MTT reduction assay and flow cytometry.

RESULTSIt was confirmed by restriction endonuclease and sequence analysis that siRNA recombinant expression vector targeting Livin and Survivin gene simultaneously was constructed successfully. The suppressive rates of siRNA targeting Livin and Survivin gene simultaneously on Livin mRNA and protein expression were 27.9% and 22.3% respectively, and those on Survivin mRNA and protein expression were 32.2% and 40.9% respectively. The survival rate of cancer cells was decreased whereas the apoptotic rate was increased, but the coordinate repression was weaker than Livin and Survivin RNA interference alone.

CONCLUSIONSsiRNA targeting Livin and Survivin gene simultaneously can decrease the expression of Livin and Survivin gene, suppress cell proliferation and induce cell apoptosis in human colon cancer. The coordinate repression was weaker than Livin and Survivin RNA interference alone.

Adaptor Proteins, Signal Transducing ; genetics ; Apoptosis ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; pathology ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; RNA, Small Interfering

To explore the possible function of LRP15 gene in carcinogenesis and its significance in the classification and prognosis of leukemia, the expression pattern of LRP15 in normal tissues, tumor tissues and cell lines was detected with SAGE and gene expression database provided by NCBI and NCI respectively. RT-PCR was used to detect the expression of LRP15 in leukemia patients. The results showed that LRP15 was expressed in different tissues and tumor cell lines, the positive rate of LRP15 in immature blood cells was higher than that of mature blood cells and the positive rate of M(1), M(2) and M(3) was higher than that of other AML subtypes (P < 0.01), the expression of LRP15 in refractory leukemia was higher than that of de novo leukemia. The results suggest that LRP15 may play an important role in carcinogenesis, AML classification and acute leukemia prognosis.
Cell Line, Tumor ; Gene Expression Profiling ; Humans ; Leukemia ; etiology ; genetics ; Neoplasm Proteins ; Proteins ; genetics

Objective: To investigate the clinicopathological features, immunophenotype and prognosis of nuclear protein in testis (NUT) midline carcinoma. Methods: Twenty-four resection cases of NUT midline carcinoma diagnosed at the Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China from January 2018 to September 2022, were collected, and retrospectively analyzed for their clinicopathological characteristics. Relevant literature was reviewed. Results: All 24 cases of NUT midline carcinoma occurred in the chest or head and neck, including 14 men and 10 women, with a median age of 40 years. Histological examination showed that the tumors were poorly differentiated, with solid nested or sheet-like arrangement, small to medium-sized cells, sparse cytoplasm and coarse granular chromatin, including 5 cases with abrupt squamous epithelial differentiation. Immunohistochemistry showed that all 24 cases were positive for NUT protein, while 16 cases were p63 positive, 19 cases were p40 positive, 15 out of 18 cases were CK5/6 positive. Follow-up data were obtained for 21 patients (follow-up time range, 1-21 months), of which 11 survived, 10 died, and 3 were lost to follow-up. Conclusions: NUT midline carcinoma is a rare and highly aggressive malignancy with unique histological, immunophenotypic and molecular features. It has a poor prognosis.
Male ; Humans ; Female ; Adult ; Neoplasm Proteins/genetics* ; Nuclear Proteins/genetics* ; Retrospective Studies ; Carcinoma/surgery* ; Testicular Neoplasms

OBJECTIVETo study the expression of prostate stem cell antigen (PSCA) at protein and mRNA levels in invasive micropapillary carcinoma of the breast (IMPC) and to analyze the relationship between PSCA expression and clinicopathologic features.

METHODSThe expression of PSCA protein was analyzed by immunohistochemistry (LSAB) in 66 cases of IMPC and 67 cases of invasive ductal carcinoma, not otherwise specified (IDC-NOS). The association between PSCA expression and clinicopathologic features was also analyzed in IMPC. Furthermore, RT-PCR was used to detect PSCA mRNA in 10 cases of primary IMPC and 10 cases of primary IDC-NOS with paired normal breast tissues, each from the same subject.

RESULTSImmunohistochemical analysis revealed the overexpression of PSCA in 47 of 66 (71.2%) cases of IMPC and 35 of 67 (52.2%) IDC-NOS. Statistical analysis showed a significant difference of PSCA expression between IMPC and IDC-NOS (P = 0.024). In IMPC, the expression of PSCA was correlated with lymph nodes metastasis (P = 0.039). RT-PCR showed the mRNA level of PSCA was significantly higher in primary IMPC and IDC-NOS tissue than that in paired normal breast tissue (7/10 and 5/10, respectively), and it was also significantly higher in primary IMPC tissue than that in IDC-NOS tissue.

CONCLUSIONPSCA might play an important role in lymph node metastasis in IMPC.

Antigens, Neoplasm ; genetics ; metabolism ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Papillary ; genetics ; metabolism ; pathology ; Female ; GPI-Linked Proteins ; genetics ; metabolism ; Humans ; Lymphatic Metastasis ; Neoplasm Invasiveness ; Neoplasm Proteins ; genetics ; metabolism ; Neoplasm Staging ; RNA, Messenger ; metabolism

HCAP1 is a novel hepatic cancer related gene located on human chromosome 17p13.3. The loss of heterozygosity occurred at 17p13.3 in various human cancers. In order to investigate the effects of exogenous HCAP1 gene products on cell proliferation of T lymphoma Jurkat cell line, HCAP1 gene! was transfected into Jurkat cells mediated by liposome, and the cells stably expressing exogenous HCAP1 were screened with G418. The effects of HCAP1 products on cell proliferation were assessed by viable cell count, cell growth curve and colony formation assay in soft agar. The results showed that the HCAP1 transgenic Jurkat cells displayed slow growth rate, extended doubling time and reduced colony formation capability, as compared with the cells transfected with pBK/CMV empty vector (P < 0.01). It is concluded that exogenous HCAP1 gene products could inhibit the proliferation of Jurkat cells.
Carcinoma, Hepatocellular ; genetics ; Cell Division ; Humans ; Jurkat Cells ; Liver Neoplasms ; genetics ; Neoplasm Proteins ; genetics ; Peptides ; Transfection

Acetyltransferases ; genetics ; Breast Neoplasms ; genetics ; Female ; GPI-Linked Proteins ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; Membrane Glycoproteins ; genetics ; Neoplasm Metastasis ; genetics ; physiopathology ; S100 Proteins ; genetics ; Transcription Factors ; genetics

This study was aimed to observe the effects of siRNA on Livin expression and function in K562 cells. Livin siRNA were designed and synthesized, then were transfected into K562 cells by using AMAXA nucle transfactor. Expressions of Livin mRNA and protein in transfected K562 cells was detected by RT-PCR and Western blot respectively. Non-transfected cells were used as control. The enhanced green fluorescent protein plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Cell apoptosis was measured by flow cytometry with Annexin V-FITC/PI double staining. The results showed that the transfection efficiency of electroporation method was about 50. The synthesized siRNA inhibited livin expression at both mRNA and protein levels. The rate of K562 cell apoptosis increased from (9.63 ± 0.89) in control group to (12.07 ± 1.39) and (27.41 ± 2.30) at 24 h and 48 h after transfection, respectively (P < 0.05). It is concluded that the siRNA can inhibit anti-apoptosis of livin gene via down-regulating livin gene expression, which may provide the new method for anti-leukemia study.
Adaptor Proteins, Signal Transducing ; genetics ; Apoptosis ; genetics ; Gene Expression Regulation, Leukemic ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; K562 Cells ; Neoplasm Proteins ; genetics ; RNA, Small Interfering ; genetics

OBJECTIVETo detect tumor cells in the peripheral blood of patients with hepatocellular carcinoma (HCC) by using the mRNA of the MAGE-1 and MAGE-3 genes as specific tumor markers.

METHODSPeripheral blood was obtained from 25 HCC patients and 20 healthy volunteers. The mRNA of the MAGE-1 and MAGE-3 genes in the peripheral blood mononuclear cells (PBMCs) was detected by nested RT-PCR. The MAGE-1 and MAGE-3 transcripts in the tumor tissues of these HCC patients were also detected by RT-PCR.

RESULTSOf the 25 HCC patients, MAGE-1 and MAGE-3 mRNA were positive in 44% (11/25) and 36% (9/25) of PBMCs respectively, and in 68% (17/25) and 56% (14/25) of HCC tissues respectively. In the PBMCs of the 25 HCC patients, 16 (64%) samples were detected to express at least one type of MAGE mRNA. MAGE mRNA were not detected in the PBMCs from the patients whose tumors did not express the MAGE genes, nor in the PBMCs from the 20 healthy donors. The positive rate of MAGE mRNA in the PBMCs was closely correlated with the TNM stages and the diameter of tumors, but there was no correlation between the positive rate of MAGE mRNA in PBMCs and tumor differentiation degree or serum alpha-FP level. Of 9 HCC patients whose serum alpha-FP was normal or slightly elevated (< 50 ng/ml), 6 were MAGE-1 and/or MAGE-3 mRNA positive in their PBMCs.

CONCLUSIONMAGE-1 and MAGE-3 mRNA could be specifically detected with high percentage in the PBMCs of HCC patients by our method. They can be used as specific tumor markers for the detection of the circulating HCC cells, and the detection results may be helpful to evaluate the prognosis of HCC patients.

Antigens, Neoplasm ; Carcinoma, Hepatocellular ; genetics ; Humans ; Leukocytes, Mononuclear ; Liver Neoplasms ; genetics ; Melanoma-Specific Antigens ; Neoplasm Proteins ; RNA, Messenger ; genetics

OBJECTIVETo construct a recombinant adenovirus vector bearing dual-survivin short hairpin RNA (shRNA).

METHODSDual-survivin shRNA was designed and synthesized respectively, both inserted into adenovirus DNA. The recombinant adenovirus vector was confirmed via both sequencing and restriction digestion analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenoviruses.

RESULTSThe recombinant adenovirus vector was constructed and the target sequence was obtained.

CONCLUSIONThe construction of the recombinant adenovirus vector provides a basis for the research of potential gene therapy for prostate cancer.

Adenoviridae ; genetics ; Cell Line ; DNA Restriction Enzymes ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Polymerase Chain Reaction ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection