Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM: To investigate SBA2 expression in CRC cell lines and surgical specimens of CRC and autologous healthy mucosa. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples. Normalization of the results was achieved by simultaneous amplification of beta-actin as an internal control. RESULTS: In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and beta-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts P【0.01). SBA2 expression was significantly (0.01】P 【 0.05) correlated with the grade of differentiation in CRC, with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in non-metastasis samples 0.01】P【0.05). CONCLUSION: SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.

Mcl-1 mediates cytokine deprivation induced apoptosis of human myeloma cell line XG-7

OBJECTIVE: To investigate apoptosis in XG-7, a human myeloma cell line, induced by IL-6 deprivation and the function of three anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-kappa(L), Mcl-1) in the apoptotic process. METHODS: Apoptosis in XG-7 myeloma cells induced by IL-6 withdrawal was determined by flow cytometry with propidium iodide (PI) nuclear staining. Expressions of three Bcl-2 proteins in XG-7 cells were monitored by immunoblotting assay. RESULTS: In the absence of IL-6 for a certain time, a significant percentage of apoptiotic XG-7 cells can be observed, as well as down-regulated expression of one of the three anti-apoptotic proteins (Mcl-1) in XG-7 cells. IL-6 re-stimulation in XG-7 cells following cytokine removal up-regulated the expression of Mcl-1 and inhibited cell apoptosis. CONCLUSION: Mcl-1,instead of Bcl-2 and Bcl-kappa(L), plays an important role in IL-6 deprivation induced apoptosis in XG-7 human myeloma cells.

Induction of cytotoxic T lymphocytes from the peripheral blood of a hepatocellular carcinoma patient using melanoma antigen-1 (MAGE-1) peptide

OBJECTIVE: To investigate the possibility of using melanoma antigen-1 (MAGE-1) peptide as a tumor vaccine to treat hepatocellular carcinoma (HCC). METHODS: The expressions of MAGE-1 in 8 HCC cell lines and in liver cancer tissue from a patient were detected using RT-PCR. The type of human leucocyte antigen I(HLA I) of both 8 HCC cell lines and peripheral blood mononuclear cells of the patient was detected using a microcytotoxicity method to screen out target cell lines for the cytotoxicity assay. Peripheral blood mononuclear cells from the HCC patient pulsed with an MAGE-1 peptide (NYKCRFPEI) were used as antigen presenting cells. Autogenous peripheral blood mononuclear cells were stimulated with antigen presenting cells every 7 days for 4 times to elicit cytotoxic T lymphocytes. The phenotype of effector cells was analyzed using flow cytometry. The cytotoxicity of effector cells was detected with a lactate dehydrogenase releasing assay. RESULTS: The expressions of both MAGE-1 and HLA-A24 were detected in BEL7405 cell line which were used as the positive target cell line in the cytotoxicity assay. The expression of MAGE-1 alone was detected in HLE, BEL7402, BEL7404, QGY7703 and SMMC7721 cell lines, and the expression of neither MAGE-1 nor HLA-A24 was shown in QGY 7701 and HpG2 cell lines. The last 7 cell lines could be used as negative target cell lines in the cytotoxicity assay. Peripheral blood mononuclear cells expanded 32 folds during 28-day culture. The ratio of CD3(+) T cells increased by 16% (from 54% to 70%), and the ratio of CD8(+) T cells increased by 20% (from 36% to 56%) during 28-day culture. When the ratio of effector cells to target cells was 10:1, effector cells exhibited 62.5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide (NYKCRFPEI) of MAGE-1 antigen, 40.25% cytotoxicity against BEL7405 cells, compared with 17.88% cytolysis observed against autogenous lymphoblasts, 19.55% against HLE cells, and 1.6% against QGY7701 cells. When the ratio of effector cells to target cells was 3.3:1, the cytotoxicity of effector cells against the peptide pulsed autogenous lymphoblasts was 53.6%, which was much higher against autogenous lymphoblasts, HLE cells and QGY7701 cells at 15.6%, 13% and 1%, respectively. CONCLUSION: The results demonstrate that cytotoxic T lymphocytes with the ability to specifically lyse target cells expressing both MAGE-1 and HLA-A24 could be successfully induced by the MAGE-1 peptide NYKCRFPEI in vitro. This indicates that a good result might be anticipated if this peptide is used as a tumor vaccine to treat HLA-A24 HCC patients.

Renal cell carcinoma related novel gene, GYLZ-RCC18: cloning and functional studies

OBJECTIVE: To clone the full length of renal cell carcinoma (RCC) related novel gene GYLZ-RCC18 and study its function. METHODS: SMART RACE technology was used to clone the full length of GYLZ-RCC18. RT-PCR was used to detect its expression in renal cell carcinoma tissue at different stages and grades. We transfected the antisense oligonucleotide of GYLZ-RCC18 to renal cell carcinoma cell line, GRC-1, and analyzed proliferation activity, growth rate, apoptosis, and mortality changes. RESULTS: The full length of GYLZ-RCC18 (GenBank accession number: BE825133) cDNA was about 3.5 kb. GYLZ-RCC18 had a higher expression in higher grades and stages of renal cell carcinoma than in lower ones. The expression of GYLZ-RCC18 in renal cell carcinoma was much higher than in normal kidney. After the transfection of GYLZ-RCC18 antisense oligonucleotide, the mortality of GRC-1 increased significantly, while proliferative activity and growth rate were substantially inhibited at the same time. The antisense oligonucleotide induced apoptosis of GRC-1 through the entire observation time. CONCLUSION: GYLZ-RCC18 is an important novel gene related to renal cell carcinoma. Overexpression of this gene results in higher growth and proliferative activity and has an antiapoptosis effect on renal cell carcinoma cells. Transfection of the antisense oligonucleotide may inhibit the generation and development of renal cell carcinoma.

Increased sensitivity of colorectal cancer cell lines with microsatellite instability to 5-fluorouracil in vitro

OBJECTIVE: To study the relationship between sensitivity to 5-FU and the status of a panel of microsatellite loci in three human colon cancer cell lines. METHODS: Cell viability in several concentrations of 5-FU was assessed by the MTT test. Expression of hMSH2 and hMLH1 in LoVo, SW480 and SW1116 cells were analyzed by immunocytochemical staining.Ten mononucleotide and dinucleotide microsatellite loci were analyzed by the PCR-SSLP-silver staining method. RESULTS: By MTT assay, it showed that LoVo cells were more sensitive than SW480 and SW1116 cells (0.8 micromol/L,2.2 micromol/L and 1.9 micromol/L, respectively, P

Effect of all-trans retinoic acid and arsenic trioxide on tissue factor expression in acute promyelocytic leukemia cells

OBJECTIVE: To study the effect of all-trans retinoic acid (ATRA) and arsenic troxide (As2O3) on tissue factor (TF) expression and procoagulant activity (PCA) of acute promyelocytic leukemia (APL) cells in vivo and in vitro. METHODS: PCA from freshly isolated APL blasts from APL patients treated with ATRA or As2O3 was detected using a one-stage clotting assay. TF antigen was detected by ELISA and TF mRNA by RT-PCR. The maturation sensitive (NB4) or resistant subclones (NB4-R1) of the promyelocytic NB4 cell line, as well as U937 cells infected with pMSCV-PML-RARa treated with or without ATRA or As2O3, were also examined. RESULTS: Both ATRA and As2O3 can down-regulate the TF antigen, its mRNA transcription and membrane PCA of APL cells in vivo and in vitro, in a time-dependent manner. The TF antigen level in PML-RARa + U937 cells was significantly higher than that in U937 cells infected with retrovirus vector. Both ATRA and As2O3 can also down-regulate the TF antigen in U937 cells transfected with or without PML-RARa. CONCLUSION: Tissue factor expression and PCA in APL cells may be down-regulated by ATRA and As2O3. By down-regulating TF expression, As2O3 might also be used to improve the DIC-related hemorrhage in APL. Our data indicate that elevated TF antigen in PML-RARa + U937 may be related to the fusion protein PML-RARa. The down-regulating effect of ATRA and As2O3 on TF expression in U937 cells might not involve this fusion protein.

Jianpijiedu Fang improves survival of hepatocarcinoma mice by affecting phosphatase and tensin homolog, phosphoinositide 3-kinase, and focal adhesion kinase

OBJECTIVE: To investigate the effect of Jianpijiedu Fang (JPJDF) on phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and focal adhesion kinase (FAK), and on the survival of hepatocellular carcinoma (HCC) nude mice. METHODS: Forty male nude mice were randomly divided into 4 groups. Human HCC tissue was implanted in the livers of three groups. After 24 h, the three groups were treated respectively with JPJDF (37.5 g/kg), saline (20 mL/kg) and Tegafur (FT-207, 160 mg/kg) once a day for 10 weeks. The control group without implanting the tissue was concurrently treated with saline (20 mL/kg). The survival data and body weight of all mice were recorded, and expression levels of PTEN, PI3K and FAK in normal tissue and cancer tissue of the livers were eval-uated with immunohistochemical method. RESULTS: The cumulative survival rate of the mice in the JPJDF group was higher than those of the other groups. The rate of weight loss was the lowest in JPJDF group. The survivability and weight loss rate in FT-207 group were the poorest in all groups. The expression intensity of PTEN was higher in normal tissues than in cancer tissues, and lower in the normal tissues of HCC models than in that of mice without HCC. The PTEN expression intensity in normal tissue and cancer tissue from mice treated with FT-207 were lower than that from the mice treated with JPJDF or saline.The expression intensity of PI3K was higher in cancer tissue than in normal tissue. The PI3K expression intensity was the lowest in normal tissue and cancer tissue from mice treated with JPJDF, and the intensity from mice treated with FT-207 was the highest. In mice treated with JPJDF, the expression intensity of FAK was higher in the normal tissue and lower in the cancer tissue than those of the other treatment groups. CONCLUSION: The mechanism accounting for the prolonged survival of HCC-bearing mice treated with JPJDF might be related to the reduction in weight loss and the benign regulation of PTEN, PI3K, and FAK.