The novel gene LRP15 is regulated by DNA methylation and confers increased efficiency of DNA repair of ultraviolet-induced DNA damage.

LRP15 is a novel gene cloned from lymphocytic cells, and its function is still unknown. Bioinformatic data showed that LRP15 might be regulated by DNA methylation and had an important role in DNA repair. In this study, we investigate whether the expression of LRP15 is regulated by DNA methylation, and whether overexpression of LRP15 increases efficiency of DNA repair of UV-induced DNA damage in HeLa cells. The results showed (1) the promoter of LRP15 was hypermethylated in HeLa cells, resulting a silence of its expression. Gene expression was restored by a demethylating agent, 5-aza-2'-deoxycytidine, but not by a histone deacetylase inhibitor, trichostatin A; (2) overexpression of LRP15 inhibited HeLa cell proliferation, and the numbers of cells in the G2/M phase of the cell cycle in cells transfected with LRP15 increased about 10% compared with controls; (3) cyclin B1 level was much lower in cells overexpressing LRP15 than in control cells; and (4) after exposure to UV radiation, the LRP15-positive cells showed shorter comet tails compared with the LRP15-negative cells. From these results we conclude that the expression of LRP15 is controlled by methylation in its promoter in HeLa cells, and LRP15 confers resistance to UV damage and accelerates the DNA repair rate.

Methylation pattern of LRP15 gene in leukemia.

OBJECTIVE: To investigate the methylation status of LRP15 gene in acute leukemia (AL) patients and its role in the tumorigenesis. METHODS: The methylation of LRP15 promoter and first exon of bone marrow mononuclear cells in 73 patients with AL, 10 with chronic leukemia (CL), 9 with hematological benign diseases, and 20 healthy transplantation donors was analyzed by using methylation specific polymerase chain reaction. The methylation of LRP15 gene promoter and first exon in COS7, K562, and HL60 cell lines was also assayed. RESULTS: No LRP15 gene promoter methylation was detected in COS7 cell line. LRP15 gene promoter was methylated in K562 and HL60 cell lines. No deletion of LRP15 gene was detected in all samples. In nearly all French-American-British leukemia subtypes, we found that frequency of LRP15 methylation in adult patients with AL was 71.23% (52/73). There was no detectable methylation in any of the 20 healthy donors and 8 chronic myeloid leukemia patients. The difference in frequency of LRP15 methylation between AL patients and healthy donors or CL patients (10.00%, 1/10) was significant (P < 0.01). Hypermethylation of LRP15 gene was found in 57.14% (16/28) of newly diagnosed AL patients, 83.33% of relapsed AL patients respectively, which was significantly different (P < 0.05). We also demonstrated LRP15 methylation in 55.56% (5/9) adults with benign hematological diseases. CONCLUSIONS: LRP15 methylation changes are common abnormalities in leukemia. LRP15 is postulated to be a tumor suppressor gene.

[Analysis of LRP16 gene promoter activity].

The study was aimed to analyze the characteristics of LRP16 gene promoter and its activity in order to explore the possible regulation mechanism of LRP16 gene expression. A 2.6 kb genomic DNA sequence of LRP16 5'-end was obtained from NCBI by BLAST software. The 7 target sequences between 0.2 - 2.6 kb from a healthy blood donor DNA sample were amplified by PCR, then identified by DNA sequencing and semi-nest PCR. The verified sequences were analyzed on-line. The results showed that the 7 target sequences were about 400 bp different from each other. All 7 sequences were the same to these GenBank described. At last, all 7 promoter sequences were ligated with luciferase vector, and then the luciferase activity was analyzed in HeLa cells. A known gene promoter sequence can be freely obtained from NCBI database. It is concluded that LRP16 promoter is a standard type II promoter and its activity is strongest in the region from -200 to -600 bp.

[The methylation pattern of LRP15 gene in acute leukemia].

OBJECTIVE: To investigate the methylation status of LRP15 gene in acute leukemia (AL) and its role in tumorigenesis. METHODS: 73 cases of AL and 9 healthy subjects as well as COS7, K562 and HL60 cell lines were studied with methylation specific PCR (MSP). RESULTS: LRP15 was not detected in all the samples. No LRP15 methylation was detected in COS7, but LRP15 was methylated in K562 and HL60. In nearly all of the French-American-British leukemia subtypes, we found that the frequency of LRP15 methylation was 71.2% (52/73) of AL and none in the 9 healthy subjects. The difference in mean methylation for LRP15 between these two kinds of samples is statistically significant (P < 0.05). Hypermethylation of the LRP15 gene was found in 57.1% (16/28) of the newly diagnosed AL and 83.3% of the relapsed AL respectively; this is also statistically significant (P < 0.05). CONCLUSION: LRP15 methylation change is a common abnormality in leukemia and LRP15 is postulated to be a tumor suppressor gene.

[Analysis of the methylation in the promoter of LRP15 gene and its expression].

To study the methylation in the promoter of LRP15 gene and its relationship with gene expression and to explore the possible mechanism of regulating LRP15 gene methylation, the methylation in the promoter of LRP15 gene in K562 cell line was detected by MS-PCR. Then K562 was exposed to 5-aza-2'-deoxycytidine (CdR) and trichostatin (TSA), to determine whether the silencing of LRP15 gene by de novo methylation could be reversed. As a result, it was confirmed by MS-PCR that the promoter of LRP15 was hypermathylated in K562 cell line, and lost its transcription activity. After CdR, with or without TSA, the silencing of LRP15 gene by de novo methylation can be reversed. Observation demonstrated that the expression of LRP15 was controlled by methylation in its promoter in K562. It is suggested that methyltransferase inhibitor and deacetylase inhibitor may be effective agents in leukemia therapy.

[Effect of inhibitors for demethylation and histone deacetylase on proliferation of cell line K562 and expression of tumor related genes].

In order to observe the effect of inhibitors for demethylation and histone deacetylase on the growth of leukemia cell line K562 and the expressin of tumor related genes, the K562 cells were treated with 5-aza-2' deoxycytidine (DAC) and trichostatin A (TSA) in co-culture; the growth curves were observed; the cell cycle was detected by flow cytometry (FCM); the gene expression pattern before and after drug treatment was measured with Atlas7742-1 microarray. The results showed that the combination treatment of DAC and TSA inhibited the proliferation of K562 cells, the growth of most cells were stopped in G(1)/S phases after drug treatment, the gene expression after treatment was more than before, and a few gene expression were down-regulated. In conclusion, combination treatment of DAC and TSA had an inhibitive effect on the leukemia cell line K562, combination of DAC and TSA with microarray could be used for screening candidate genes inhibiting leukemia cells.

[Inhibitory effect of antisense human telomerase reverse transcriptase(hTERT) on telomerase activity of human pulmonary giant cell carcinoma cell line(PLA-801D)].

BACKGROUND & OBJECTIVE: Telomerase has been thought to play an important role in the carcinogenesis in recent years. Human telomerase reverse transcriptase (hTERT) is a limiting component for telomerase activity. This study was designed to explore the effect of transfection of the full-length cDNA of antisense hTERT on the malignant phenotype of human pulmonary giant cell carcinoma cell line (PLA-801D) and its potential role in the gene therapy for cancers. METHODS: An antisense hTERT cDNA eukaryotic expression vector pcDNA3.1(-)-hTERT including the full length of hTERT cDNA sequence was constructed using recombinant DNA technique and transfected into human pulmonary giant cell carcinoma cells (PLA-801D) with liposome. The effect of antisense hTERT on the cellular proliferation capacity of PLA-801D cells was analyzed by the growth curve. The expression of hTERT mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). The telomerase activity was determined by telomeric-repeat amplification protocol enzyme-linked immunoassay (TRAP-ELISA). RESULTS: Antisense pcDNA3.1 (-)-hTERT eukaryotic expression have been constructed and was successfully transfected into the PLA-801D cells. The growth speed of PLA-801D transfected with antisense hTERT was significantly inhibited compared with the control cells, and the hTERT mRNA expression was inhibited, the relatively expression was only 15.7% of control cells, and telomerase activity was down-regulated about 82.4%. CONCLUSION: Full-length antisense hTERT cDNA can suppress hTERT mRNA expression and telomerase activity, and restrict the growth speed of tumor cells.

[Cloning of the full length cDNA for a novel leukemia relapse-associated candidate gene LRP15].

To clone the full length cDNA of a novel leukemia relapse-associated candidate gene (LRP15), the human ESTs (Expression Sequence Tags) fragments obtained from electronic hybridization were assembled by a 1.8 kb DNA probe, which was only methylated in relapsed leukemia. Then the primers were designed for rapid amplification of cDNA end (RACE). Bioinformatic data of High Throughout Genomic Sequences (HTGS) and Serial Analysis of Gene Expression (SAGE) were used for chromosome localization and tissue expression analysis. The results showed that the full-length cDNA of the novel gene had an open reading frame of 780 bp encoding a 259 amino acid protein of unkown functions. LRP15 gene expressed in many different tissues was localized in chromosome 3p24. It is concluded that RACE is an effective method to clone novel genes and LRP15 may be a leukemia relapse-associated candidate gene playing an important role in carcinogenesis.

[Analysis of LRP15 Gene Expression Pattern and Its Expression in Leukemia Cells].

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.