BACKGROUNDThe formation and growth of tumors are related to the synthesis of the DNA. The enzyme ribonucleotide reductase (RR) is an enzyme that regulates the total rate of DNA synthesis and thus plays a pivotal role in cell growth. Catalytic subunit M2 (RRM2) is the main unit modulating the ribonucleotide reductase enzymatic activity. This study aimed to investigate the expression of RRM2 mRNA and protein in patients with ovarian cancer and its relevance to diagnosis and clinical outcome of the patients.

METHODSRRM2 mRNA levels and protein expression were detected in 98 ovarian specimens with immunohistochemistry and real-time quantitative polymerase chain reaction (PCR). Expression of the RRM2 protein and correlation of the RRM2 gene expression with clinical pathological features were analyzed. The Kaplan-Meier test was used for evaluating RRM2 expression and time to progression and survival. The Cox proportional model was used to analyze the risk factors in prognosis of patients.

RESULTSPositive RRM2 immunostaining was found in 43 of 62 (69.4%) patients with epithelial ovarian cancer, 10 of 15 (66.7%) patients with borderline neoplasm, 4 of 15 (26.7%) patients with benign growths, and none of the normal group. The RRM2 mRNA levels were significantly over expressed in epithelial ovarian cancer (1.722 ± 0.639) and borderline ovarian neoplasms (1.365 ± 0.615), compared to the normal group (0.678 ± 0.446) and benign group (0.828 ± 0.545). Patients with ovarian caner in clinical FIGO-stages III-IV presented higher RRM2 gene expression than those in clinical FIGO-stages I-II. Furthermore, the survival of patients with low RRM2 mRNA level was significantly better than patients with high levels (P < 0.05). By Cox proportional risk model analysis, the risk of mortality of patients with high level expression of RRM2 mRNA was 2.553 times greater than those with low expression.

CONCLUSIONRRM2 expression closely correlates with the development of ovarian tumor and may serve as a novel predictive marker for diagnosis and prognosis of the disease.

Adolescent ; Adult ; Aged ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Ovarian Neoplasms ; enzymology ; genetics ; Real-Time Polymerase Chain Reaction ; Ribonucleoside Diphosphate Reductase ; genetics ; metabolism ; Young Adult

For cancer gene therapy, cancer-specific over-expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.
Breast Neoplasms/*genetics/therapy ; Cell Cycle Proteins/*genetics/metabolism ; Cell Line, Tumor ; Cloning, Molecular ; Cytomegalovirus ; Dependovirus ; Female ; *Gene Targeting ; Gene Therapy ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Promoter Regions, Genetic/*genetics ; Ribonucleoside Diphosphate Reductase/*genetics/metabolism ; *Transcriptional Activation

OBJECTIVETo study the relationship of changes in gene expression profiles of hydatidiform mole and choriocarcinoma with hyperplasia of trophoblasts.

METHODSThe differentially expressed genes were analyzed in two pairs of tissues of hydatidiform mole versus normal villi, and in two pairs of normal primary culture trophoblasts versus JAR cell line of chariocarcinoma, using cDNA microarray containing 4096 genes. To confirm the results of cDNA microarray analysis, expressions of some up-regulated genes related to DNA synthesis in normal villi, hydatidiform mole, and 2 choriocarcinoma cell lines (JAR and JEG-3) were examined by immunohistochemistry, immunoblotting and RT-PCR.

RESULTSA total of 89 genes were differentially expressed in all hydatidiform moles, accounting for 2.2% of the genes arrayed. Of the 89 genes, 24 were up-regulated and 65 were down-regulated. Compared with normal primary trophoblasts, there were 433 genes up-regulated and 380 genes down-regulated in JAR cell line. Forty six genes were up-regulated in both hydatidiform mole and choriocarcinoma, while 13 genes were down-regulated. Some genes associated with cell proliferative inhibition were significantly down-regulated, whereas those associated with cell proliferation, malignant transformation, metastasis and drug resistance were highly up-regulated. The expressions of thymidine kinase 1, the small subunit of ribonucleotide reductase (RRM2) were significantly increased in hydatidiform mole, JAR and JEG-3 cells.

CONCLUSIONAbnormal expression of genes exists in hydatidiform mole and choriocarcinoma. Hyperplasia of trophoblasts may be related to over-expression of genes coding for synthetic enzymes.

Adult ; Cell Line, Tumor ; Cell Proliferation ; Cell Transformation, Neoplastic ; Choriocarcinoma ; genetics ; metabolism ; pathology ; Drug Resistance, Neoplasm ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Hydatidiform Mole ; genetics ; metabolism ; Hyperplasia ; Neoplasm Metastasis ; Oligonucleotide Array Sequence Analysis ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Ribonucleoside Diphosphate Reductase ; metabolism ; Thymidine Kinase ; metabolism ; Trophoblasts ; pathology ; Uterine Neoplasms ; genetics ; metabolism ; pathology

OBJECTIVETo investigate the inhibitory effect of cinobufotalin (CBT) on the growth of xenograft endometrial carcinoma cell line ishikawa in nude mice, and its impact on the expression of ribonucleotide reductase subunit M2 (RRM2).

METHODSEleven nude mice with xenograft were randomly divided into two groups, the CBT group and the control group, which received intra-tumor injection of CBT and saline respectively for one week. The sizes of xenografts were measured before and after the treatment to calculate the inhibition ratio of tumor proliferation; the RRM2-mRNA and protein expressions in tumor tissue were measured by RT-PCR and Western blot respectively.

RESULTSAfter treatment, the size of xenografts in the CBT group was (0.1314 +/- 0.0304) cm3, which was significantly lower than that in the control group (0.360 0 +/- 0.1145) cm3, (P < 0.05), the tumor proliferation inhibition ratio being 43.46%. The differences of RRM2 mRNA and protein expression levels between the two group were significant (P = 0.019 and P = 0.001).

CONCLUSIONCBT significantly inhibits the growth of the xenografts of endometrial carcinoma Ishikawa in nude mice, and the action mechanism is possibly associated with the inhibition on RRM2 expression.

Animals ; Antineoplastic Agents ; pharmacology ; Bufanolides ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Endometrial Neoplasms ; genetics ; metabolism ; pathology ; Female ; Humans ; Materia Medica ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; RNA, Messenger ; genetics ; metabolism ; Ribonucleoside Diphosphate Reductase ; genetics ; metabolism ; Xenograft Model Antitumor Assays