Mitochondrial DNA (mtDNA) depletion syndromes are disorders characterized by infantile-onset, severe progression, and the drastic loss of mtDNA content in affected tissues. In a patient who showed severe hypotonia, proximal tubulopathy and sensorineural hearing loss after birth, we observed severe mtDNA depletion and impaired respiratory chain activity in muscle due to heterozygous variants c.686G > T and c.551-2A > G in RRM2B, encoding the p53R2 subunit of the ribonucleotide reductase.

Ribonucleotide reductase (RR)M2B is an enzyme belonging to the ribonucleotide reductase enzyme family, which is essential for DNA synthesis and repair. RRM2B plays an important role in tumor progression and metastasis; however, little is known about the expression and underlying molecular mechanisms of RRM2B in hepatocellular carcinoma (HCC). In the present study, we report that down-regulation of RRM2B in HCC is negatively associated with intrahepatic metastasis, regardless of p53 status. Moreover, the ectopic overexpression of RRM2B decreased HCC cell migration and invasion in vitro, whereas silencing RRM2B expression resulted in increased migration and invasion in vitro and intrahepatic and lung metastasis in vivo. Additionally, knockdown of RRM2B by short hairpin RNA (shRNA) in HCC cells was associated with epithelial-mesenchymal transition (EMT), including the down-regulation of E-cadherin, and the concomitant up-regulation of N-cadherin and slug. A further experiment showed that RRM2B inhibited cell migration and spreading through regulation of the early growth response protein 1 (Egr-1)/phosphatase and tensin homolog (PTEN)/Akt1 pathway. Consistently, we also detected a significant correlation between RRM2B and E-cadherin protein expression in HCC tissues. Furthermore, Egr-1 also directly bound to the RRM2B promoter and repressed RRM2B transcription, thereby establishing a negative regulatory feedback loop.

These findings indicate that RRM2B suppresses cell migration and spreading by way of modulation of the Egr-1/PTEN/Akt1 pathway.

Several studies suggest estrogen to be protective against the development of colon cancer. Estrogen receptor β (ERβ) is the predominant estrogen receptor expressed in colorectal epithelium and is the main candidate to mediate the protective effects. We have previously shown that expression of ERβ reduces growth of colorectal cancer in xenografts. Little is known of the actions of ERβ and its effect on gene transcription in colon cancers. To dissect the processes that ERβ mediates and to investigate cell-specific mechanisms, we reexpressed ERβ in three colorectal cancer cell lines (SW480, HT29, and HCT-116) and conducted genome-wide expression studies in combination with gene-pathway analyses and cross-correlation to ERβ-chromatin-binding sites. Although induced gene regulation was cell specific, overrepresentation analysis of functional classes indicated that the same biological themes, including apoptosis, cell differentiation, and regulation of the cell cycle, were affected in all three cell lines. Novel findings include a strong ERβ-mediated down-regulation of IL-6 and downstream networks with significant implications for inflammatory mechanisms involved in colon carcinogenesis. We also discovered cross talk between the suggested nuclear receptor coregulator PROX1 and ERβ, demonstrating that ERβ both regulates and shares target genes with PROX1. The influence of ERβ on apoptosis was further explored using functional studies, which suggested an increased DNA-repair capacity. We conclude that reexpression of ERβ induces transcriptome changes that, through several parallel pathways, converge into antitumorigenic capabilities in all three cell lines. We propose that enhancing ERβ action has potential as a novel therapeutic approach for prevention and/or treatment of colon cancer.

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In the present study, we examined changes in gene expression profiles associated with p53R2 in cancer cells, using the analysis of cDNA microarray. Gene set enrichment analysis identified that the gene set regulating cell-cycle progression was significantly enriched in p53R2-silencing human oropharyngeal carcinoma KB cells. Attenuation of p53R2 expression significantly reduced p21 expression and moderately increased cyclin D1 expression in both wild-type p53 cancer cells (KB and MCF-7) and mutant p53 cancer cells (PC3 and MDA-MB-231). Conversely, overexpression of p53R2-GFP resulted in an increase in the expression of p21 and decrease in the expression of cyclin D1, which correlated with reduced cell population in S-phase in vitro and suppressed growth in vivo. Furthermore, the MAP/ERK kinase inhibitor PD98059 partially abolished modulation of p21 and cyclin D1 expression by p53R2. Moreover, under the conditions of nonstress and adriamycin-induced genotoxic stress, attenuation of p53R2 in KB cells significantly increased phosphorylated H2AX, which indicates that attenuation of p53R2 may enhance DNA damage induced by adriamycin. Overall, our study shows that p53R2 may suppress cancer cell proliferation partially by upregulation of p21 and downregulation of cyclin D1; p53R2 plays critical roles not only in DNA damage repair but also in proliferation of cancer cells.

Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C-->T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.

The p53-inducible p53R2 gene has been isolated and shown to play a crucial role in DNA repair and synthesis after DNA damage. Moreover, the expression and activity of p53R2 has been reported to be associated with the anticancer agent resistance of human cancer cells. Previously, we reported that the presence of p53R2 expression was a predictive factor for regional lymph node metastasis in oral squamous cell carcinoma; however, the mechanism of cancer metastasis by p53R2 expression is still unclear. In the present study, we analyzed the correlation of p53R2 expression with cancer invasion in vitro. Three human oral cancer cell lines (SAS, HSC-3 and Ca9-22) were cultured, and the invasive potential of these cancer cells was evaluated using Matrigel invasion assay. To investigate the effect of p53R2 on cancer invasion, the down-regulation of p53R2 was examined by small interfering RNA (siRNA). Moreover, we examined the intracellular localization of cell adhesion molecules (E-cadherin and beta-catenin) in subcellular extractions of cancer cells by immunoblotting. The proteolytic activity of matrix metalloproteinases (MMPs) was assessed by gelatin zymography. Down-regulation of p53R2 significantly enhanced the invasion potential (p<0.01), and enhanced nuclear translocation of beta-catenin with loss of total cellular E-cadherin expression in p53 mutant cancer cells, but not in p53 wild-type cancer cells. These changes in the invasion index by p53R2 siRNA transfection were not accompanied by alterations in MMP activity and expression. These results suggested that the expression of p53R2 could be associated with the invasion of cancer cells, and indicated that p53R2 might promote cancer invasion via the E-cadherin/beta-catenin pathway without the alteration of MMP activity.

p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying deoxynucleotide triphosphate pools in response to DNA damage. In this study, we found that p53R2 was overexpressed in prostate tumor cell lines compared with immortalized prostatic epithelial cells and that the protein was induced upon DNA damage. We investigated the effects of p53R2 silencing on DNA damage in LNCaP cells (wild-type p53). Silencing p53R2 potentiated the apoptotic effects of ionizing radiation and doxorubicin treatment as shown by increased sub-G(1) content and decreased colony formation. This sensitizing effect was specific to DNA-damaging agents. Comet assay and gamma-H2AX phosphorylation status showed that the decreased p53R2 levels inhibited DNA repair. Silencing p53R2 also reduced the levels of p21(WAF1/CIP1) at the posttranscriptional level, suggesting links between the p53-dependent DNA repair and cell cycle arrest pathways. Using LNCaP sublines stably expressing dominant-negative mutant p53, we found that the sensitizing effect of p53R2 silencing is mediated by p53-dependent apoptosis pathways. In the LNCaP sublines (R273H, R248W, and G245S) that have defects in inducing p53-dependent apoptosis, p53R2 silencing did not potentiate DNA damage-induced apoptosis, whereas p53R2 silencing was effective in a LNCaP subline (P151S) which retains the ability to induce p53-dependent apoptosis. This study shows that p53R2 is a potential therapeutic target that could be used to enhance the effectiveness of ionizing radiation or DNA-damaging chemotherapy in a subset of patients with prostate cancer.

Cancer contributes to 50% of deaths worldwide and new anti-tumour therapeutics with novel mechanisms of actions are essential to develop. Metabolic inhibitors represent an important class of anti-tumour agents and for many years, agents targeting the nutrient folate were developed for the treatment of cancer. This is because of the critical need of this factor for DNA synthesis. Similarly to folate, Fe is an essential cellular nutrient that is critical for DNA synthesis. However, in contrast to folate, there has been limited effort applied to specifically design and develop Fe chelators for the treatment of cancer. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) group of ligands that demonstrate marked and selective anti-tumour activity in vitro and also in vivo against a wide spectrum of tumours. Indeed, administration of these compounds to mice did not induce whole body Fe-depletion or disturbances in haematological or biochemical indices due to the very low doses required. The mechanism of action of these ligands includes alterations in expression of molecules involved in cell cycle control and metastasis suppression, as well as the generation of redox-active Fe complexes. This review examines the alterations in Fe metabolism in tumour cells and the systematic development of novel aroylhydrazone and thiosemicarbazone Fe chelators for cancer treatment.

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.

The p53 family regulates cell-cycle arrest, triggers apoptosis or is involved in repair of DNA damage. In the present study, we analysed the expression of some p53 family proteins and their responses to chemoradiation therapy (CRT) in cases of oesophageal squamous cell carcinoma (ESCC). We immunohistochemically investigated the relationship between p53, p53R2, and p21 expression in biopsy specimens of untreated primary tumours and their clinical and histological responses to CRT in 62 patients with ESCC. Chemoradiation therapy consisted of 5-fluorouracil plus cisplatin and 40 Gy of radiation. The rates of clinical and histological responses (complete or partial) to CRT were 71.0% (clinical) and 52.8% (histological). The rate of positive expression was 43.5% for p53, 37.1% for p53R2, and 54.8% for p21 expression. Statistically significant correlations were found between p53 or p53R2 expression and favourable response to CRT (P=0.0001 or 0.041 clinical, P=0.016 or 0.0018 histological, respectively). Furthermore, in p53-negative tumours, CRT was more effective in tumours with p53R2 negative expression than those with p53R2 positive expression (P=0.0014). We demonstrated that the negative expression of p53 and p53R2 expression was closely related to the effect of CRT and should predict the CRT outcome in patients with ESCC.

Mammalian ribonucleotide reductase (mRR) is a chemotherapeutic target. In common with other class Ia RRs, the enzyme is composed of two subunits (mR1 and mR2), with mR1 containing both the active site and allosteric effector sites and mR2 containing a stable tyrosyl radical that is essential for enzymatic activity. mRR is inhibited by Ac-FTLDADF (denoted P7), corresponding to the C-terminus of mR2, which competes with mR2 for binding to mR1. The enzyme has two physiologically important active forms, mR12mR22 and mR16(mR22)j (j=1-3), with high ATP concentrations favoring the latter. Here, we report on our progress in using structural and functional studies in conjunction with library screening to identify derivatives of tri-, tetra- and hexapeptides, and cyclic heptapeptides, having equal or significantly higher activities than P7 toward inhibition of one or both active forms. These identifications were made by screening candidate peptides both for their abilities to bind to mR1 competitively with P7 and to inhibit ribonucleotide reductase activity. A significant feature of both P7 and the newly identified derivatives is that they are stronger inhibitors of mR12mR22 than of mR16(mR22)j. For the tetrapeptides, this is due in part to their preferential binding to mR1 monomer. The possible application of these peptide derivatives in cancer chemotherapy, exploiting their preferential inhibition of mR12mR22, is considered.

The p53R2 gene encodes the ribonucleotide reductase (RR) small subunit 2 homologue, and is induced by several stress signals activating p53, such as DNA-damaging agents. The p53R2 gene product causes an increase in the deoxynucleotide triphosphate (dNTP) pool in the nucleus, which facilitates DNA repair and synthesis. We hypothesized that p53R2 would be a good molecular target for cancer gene therapy. In this study, three human oral cancer cell lines (SAS, HSC-4 and Ca9-22), a human breast cancer cell line MCF-7, and a normal human fibroblast cell line NHDF were tested. We silenced the expression of p53R2 with the highly specific post-transcriptional suppression of RNA interference (RNAi). We investigated p53R2 expression with the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The sensitivity to anticancer agents was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of p53R2 showed no association with the mutational status of p53. The cancer cell lines with higher p53R2 expression were more resistant to 5-FU. RNAi-mediated p53R2 reduction selectivity inhibited growth and enhanced chemosensitivity in cancer cell lines but not in normal fibroblasts. These results suggest that basal transcription of p53R2 could be associated with the sensitivity to anticancer agents. Moreover, we assessed the possibility that p53R2 would be a good molecular target, and report that RNAi targeting of p53R2 could be useful for oral cancer gene therapy.

Functional defects in DNA repair have been shown to be associated with genomic instability followed by cancer. Recently, p53R2 [p53-inducible ribonucleotide reductase (RR) small subunit 2 homologous] was identified as a novel RR gene which is directly regulated by p53 protein in the G1 and G2 phases of the cell cycle supplying nucleotides to repair damaged DNA.

We performed genetic analyses of p53R2 to determine whether p53R2 alterations play significant roles in urothelial tumorigenesis. Genomic DNA from 108 primary transitional cell carcinomas (TCCs; 81 of the urinary bladder and 27 of the renal pelvis or ureter) was analyzed for mutation in the p53R2 gene by direct sequencing. We focused on three domains of the p53R2 gene: one RR small subunit signature involving codons 120-146 (region 1) and two putative nuclear localization signal sequences, involving codons 149-155 (region 2) and codons 163-169 (region 3). In addition, a p53-binding site of 20 nucleotides in intron 1 of p53R2 was also analyzed.

One renal pelvic TCC (0.9%: 1/108) had a single-base substitution in p53R2 with a G to T transversion resulting in the amino acid substitution Glu136 --> Asp. This base substitution was localized within the domain of exon 4 encoding the RR small subunit signature, and causes an amino acid substitution in one of the most highly conserved regions of p53R2, in which human R2 and yeast RNR2 and RNR4 proteins are highly homologous.

This finding provides the in vivo evidence for the infrequent involvement of alterations in p53R2 inhuman urothelial TCCs.

Class I ribonucleotide reductases (RRs), which are well-recognized targets for cancer chemotherapeutic and antiviral agents, are composed of two different subunits, R1 and R2, and are inhibited by oligopeptides corresponding to the C-terminus of R2, which compete with R2 for binding to R1. These peptides specifically inhibit the RRs from which they are derived, and closely homologous RRs, but do not inhibit less homologous RRs. Here we review results obtained for oligopeptide inhibition of RRs from several sources, including related x-ray, NMR, and modeling results. The most extensive studies have been performed on herpes simplex virus-RR (HSV-RR) and mammalian-RR (mRR). A common model fits the data obtained for both enzymes, in which the C-terminal residue of the oligopeptide (Leu for HSV-RR, Phe for mRR) binds with high specificity to a narrow and deep hydrophobic subsite, and two or more hydrophobic groups at the N-terminal portion of the peptide bind to a broad and shallow second hydrophobic subsite. The studies have led to the development of highly potent and specific inhibitors of HSV-RR and promising inhibitors of mRR, and indicate possible directions for the development of inhibitors of bacterial and fungal RRs.

The current systems of risk grouping in pediatric acute lymphoblastic leukemia (ALL) fail to predict therapeutic success in 10-35% of patients. To identify better predictive markers of clinical behavior in ALL, we have developed an integrated approach for gene expression profiling that couples suppression subtractive hybridization, concatenated cDNA sequencing, and reverse transcriptase real-time quantitative PCR. Using this approach, a total of 600 differentially expressed genes were identified between t(4;11) ALL and pre-B ALL with no determinant chromosomal translocation. The expression of 67 genes was analyzed in different cytogenetic ALL subgroups and B lymphocytes isolated from healthy donors. Three genes, BACH1, TP53BPL, and H2B/S, were consistently expressed as a significant cluster associated with the low-risk ALL subgroups. A total of 42 genes were differentially expressed in ALL vs normal B lymphocytes, with no specific association with any particular ALL subgroups. The remaining 22 genes were part of a specific expression profile associated with the hyperdiploid, t(12;21), or t(4;11) subgroups. Using an unsupervised hierarchical cluster analysis, the discriminating power of these specific expression profiles allowed the clustering of patients according to their subgroups. These genes could help to understand the difference in treatment response and become therapeutical targets to improve ALL clinical outcomes.

Recently, the p53R2 gene has been isolated and shown to play a crucial role in DNA repair after DNA damage. The p53R2 gene encodes the p53 inducible ribonucleotide reductase small subunit 2 homologue, which is part of the p53 pathway. However, the function of p53R2 in human cancer is still unclear. We investigated p53R2 mRNA expression in human oral normal epithelium, epithelial dysplasias and squamous cell carcinomas (SCCs). Surgical or biopsy-proven specimens of 10 normal epithelium, 48 epithelial dysplasias and 63 SCCs were collected in our department. Then, p53R2 was identified by in situ hybridization to visualize and localize the expression of specific mRNAs. The authors examined the p53 gene mutation by polymerase chain reaction-single strand conformation polymorphism analysis. p53, mdm2, p21(WAF1/CIP1) and Ki-67 expression was detected by immunohistochemistry. p53R2 expression was detected in none of ten normal epithelium (0%), ten of 48 dysplasias (20.8%) and 33 of 63 SCCs (52.4%). In oral SCC, the expression of p53R2 was significantly associated with tumor size, lymph node metastasis and histological differentiation (P=0.014, 0.046 and 0.022, respectively). p53R2 expression was significantly associated with p53 abnormality in epithelial dysplasia and SCC (P=0.034 and 0.009, respectively). Of 63 patients, 37 received preoperative radiochemotherapy. p53R2 mRNA expression was significantly associated with the pathologic response to radiochemotherapy (P=0.031). This study suggested that p53R2 expression could be associated with oral carcinogenesis. The presence of p53R2 mRNA expression would be a predictive factor for tumor development, tumor cell differentiation and the sensitivity to radiochemotherapy in oral SCC.