Study On Biological Functions Of Human UHRF1 Gene

UHRF1 (ubiquitin-like protein containing PHD and RING domain 1) was first cloned and isolated in 1998, also named NP95 as a 95-kDa nuclear protein consisting of 782 amino acids (18 exons, spanning 60 kb) and is assigned to chromosome 19p13.3. The hUHRF1 open reading frame contains an unusual N-terminal domain that bears a striking resemblance to ubiqutin, a leucine zipper motif, a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, retinoblastoma protein (Rb)-binding motifs (331LMCDE335 and 725LCCQE729), a ring finger domain and the SRA-YDG domain.It has been found that mouse UHRF1 is strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. Although biological functions of hUHRF1 are unknown, previous studies have suggested that (i) hUHRF1 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events; (ii) hUHRF1 may be a growth-regulated gene, since its expression was regulated during the cell cycle, required for the G1/S transition, and specifically induced by E1A which can force postmitotic cells to proliferate, and it is a chromatin-associated ubiquitin ligase; (iii) hUHRF1 mRNA was also increased in the transformed BALB/3T3 cells, suggesting it may participate in the maintenance of the transformed phenotype; (iv) hUHRF1 might help recruit DNA-cytosine-5-methyltransferase 1 (DNMT1) to hemimethylated DNA to facilitate faithful maintenance of DNA methylation; and (v) mouse Np95-null (Np95-/-)embryonic stem cells are more sensitive toχrays, UV light, N-methyl-N′-nitro-N-nitrosoguanidine and hydroxyurea versus wild-type (Np95+/+) or Np95+/? embryonic stem cells. Furthermore, several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense human UHRF1 cDNA were more sensitive toχrays, UV light and hydroxyurea than the corresponding parental cells. Additionally, there was no significant redistribution of hUHRF1 foci shortly after DNA damage byγ-irradiation, but nodular hUHRF1 foci characteristically seen in G2 phase were also detected in G2-arrested cells followingγ-irradiation. These results indicate that hUHRF1 may play an important role in regulation of radiosensitivity. Taken together, the available studies indicate that hUHRF1 may be a putative oncogene, which will become a new target for cancer treatment.The current study was designed to determine the impact of overexpression of hUHRF1 by stable transfection of pcDNA3-hUHRF1 expression vector or adenoviral-mediated transduction of hUHRF1 (Ad5-UHRF1) on cell growth, radiosensitivity and migration as well as invasion in human breast cancer MDA-MB231 cells and human cervical cancer HeLa cells. The findings are summzied as following:(1) There is a higher expression of hUHRF1 in breast cancer tissues and breast cancer cell lines compare with normal breast tissues and normal cell lines;(2) Overexpression of hUHRF1 reduced cell doubling time or promoted cell growth in p53-mutant breast cancer cells lines, including MDA-MB-231 and BT-549, but not in wild-type p53 MCF-7 cells. This promotion of cell growth was associated with the regulation of cell cycle, reduction of apoptosis and reduction of apoptosis promoting protein Bax and cyclin D1 by hUHRF1 overexpression;(3) Overexpression of hUHRF1 reduced the radiosensitivity to ionzing radiation and UV, while knocking down hUHRF1 expression increased the radiosensitivity in MDA-MB-231 and HeLa cells. This radioresistance caused by hUHRF1 overexpression may be relative to (i) abolishing apoptosis induction by radiation; (ii) reducing Bax expression; (iii) reducing chromosome abbration caused radiation; (iv) affecting Ku70, Ku 80 and XRCC4 expression. Knocking-down XRCC4 expression reduced hUHRF1-mediated radioresistance.(4) Overexpression of hUHRF1 elevated cellular capability of invasion and migration in MDA-MB-231 cells. Although enhanced expression of hUHRF1 did not affect the expression of PTEN and Maspin, two anti-metastasis proteins. However, reduction of PTEN and Maspin will significantly affected the hUHRF1-mediated invasion . These results provide the first evidence that hUHRF1 is an important gene as a novel oncogene in cell growth, invasion, migration and radiosensitivity of cancer, at least human breasat cancer and cervical cancer cells.