The potential of PCNA inhibition as a therapeutic strategy in cervical cancer.

Cervical cancers are the fourth most common and most deadly cancer in women worldwide. Despite being a tremendous public health burden, few novel approaches to improve care for these malignancies have been introduced. We discuss the potential for proliferating cell nuclear antigen (PCNA) inhibition to address this need as well as the advantages and disadvantages for compounds that can therapeutically inhibit PCNA with a specific focus on cervical cancer.

A Quantitative Analysis of Surgical Smoke Exposure as an Occupational Hazard.

OBJECTIVE: We hypothesized that OR airborne PM was different in quantity and mutagenic potential than office air and cigarette smoke. SUMMARY OF BACKGROUND DATA: Exposure to surgical smoke has been equated to cigarette smoking and thought to be hazardous to health care workers despite limited data. METHODS: PM was measured during 15 operations in ORs with 24.8 ±â€Š2.0 air changes/h, and in controls (cigarettes, office air with 1.9-2.9 air changes/h). Mutagenic potential was assessed by gamma Histone 2A family member X staining of DNA damage in small airway epithelial cells co-cultured with PM. RESULTS: Average PM concentration during surgery was 0.002 ±â€Š0.002 mg/m3 with maximum values at 1.08 ±â€Š1.30 mg/m3. Greater PM correlated with more diathermy (ρ = 0.69, P = 0.006). Values were most often near zero, resulting in OR average values similar to office air (0.002 ±â€Š0.001 mg/m3) (P = 0.32). Cigarette smoke average PM concentration was significantly higher, 4.8 ±â€Š5.6 mg/m3 (P < 0.001). PM collected from 14 days of OR air caused DNA damage to 1.6% ±â€Š2.7% of cultured cells, significantly less than that from office air (27.7% ±â€Š11.7%, P = 0.02), and cigarette smoke (61.3% ±â€Š14.3%, P < 0.001). CONCLUSIONS: The air we breathe during surgery has negligible quantities of PM and mutagenic potential, likely due to low frequency of diathermy use coupled with high airflow. This suggests that exposure to surgical smoke is associated with minimal occupational risk.

How Does the Proliferating Cell Nuclear Antigen Modulate Binding Specificity to Multiple Partner Proteins?

Proliferating cell nuclear antigen (PCNA) is a member of the family of sliding clamp proteins that serves as a clamp during DNA repair, DNA replication, cell cycle control, and multiple forms of chromatin modification. PCNA functions as a homotrimer and complexes with multiple proteins in order to carry out each of these varied functions. PCNA binds to different partner proteins in the same region of its structure, called the " interdomain connecting loop", but with different affinities. This interdomain connecting loop is an intrinsically disordered region that takes different conformations when binding to different partner proteins. In this work, we performed all-atom molecular dynamics simulations on PCNA trimer unbound to any partner protein, PCNA bound to peptides from different partner proteins, and PCNA bound to the full Fen 1 protein in two different conformations. Using this massive amount of simulation results, we analyzed whether PCNA in its free trimeric form samples conformations that are similar to those when it is bound to different partner proteins. We observed that PCNA samples many of these peptide-bound conformations even when not bound to the peptides and selects specific conformations when binding to partner proteins. We also identified PCNA-peptide interactions formed in the peptide bound simulation that play a crucial role in complex formation. The calculated binding energies correlate well with the measured binding affinities of various peptides to PCNA. Lastly, we studied the internal dynamics of PCNA and propose a mechanism through which PCNA recruits binding partners. This work highlights the functional role of intrinsically disordered regions in multifunctional proteins such as PCNA.

A Peptide mimicking a region in proliferating cell nuclear antigen specific to key protein interactions is cytotoxic to breast cancer.

Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has implications for targeted breast cancer therapy. In the present communication, we characterize a novel peptide (caPeptide) that has been synthesized to mimic the sequence identified as critical to the cancer-associated isoform of PCNA. This peptide is delivered into cells using a nine-arginine linking mechanism, and the resulting peptide (R9-cc-caPeptide) exhibits cytotoxicity in a triple-negative breast cancer cell line, MDA-MB-436, while having less of an effect on the normal counterparts (MCF10A and primary breast epithelial cells). The novel peptide was then evaluated for cytotoxicity using various in vivo techniques, including ATP activity assays, flow cytometry, and clonogenetic assays. This cytotoxicity has been observed in other breast cancer cell lines (MCF7 and HCC1937) and other forms of cancer (pancreatic and lymphoma). R9-cc-caPeptide has also been shown to block the association of PCNA with chromatin. Alanine scanning of the peptide sequence, combined with preliminary in silico modeling, gives insight to the disruptive ability and the molecular mechanism of action of the therapeutic peptide in vivo.

The Novel Ribonucleotide Reductase Inhibitor COH29 Inhibits DNA Repair In Vitro.

COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1-defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 + BRCA1 cells. By EJ5- and DR-green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 + BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering drug-induced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent.

Error-promoting DNA synthesis in ovarian cancer cells.

OBJECTIVE: The objective of this study is to determine whether an altered DNA replication process is responsible for some of genetic damage observed in ovarian cancer. METHODS: The replication fidelity of the DNA synthetic process was evaluated in both malignant and non-malignant human ovarian cells. The types of replication errors produced were identified. In addition, kinetic analyses of the efficiency of ovarian cancer DNA polymerases for misincorporating nucleotides were performed. RESULTS: We report for the first time that ovarian cancer cells harbor an error promoting DNA replication apparatus which contributes to the decrease in DNA synthetic fidelity exhibited by these cells. Our study also shows that the decrease in DNA replication fidelity was not a result of an increased DNA replication activity. In addition, it was observed that the higher rate of DNA replication errors does not result in significant differences in the type of DNA replication-errors made during the DNA replication process; just the relative abundance. A detailed kinetic analysis of the efficiency of misincorporating nucleotides demonstrated that the DNA polymerases within the ovarian cancer cells exhibited a significant propensity for creating purine-pyrimidine nucleotide mismatches relative to non-malignant ovarian cells, while being only slightly more efficient at incorrectly pairing a purine nucleotide with a purine nucleotide. CONCLUSIONS: All together, these data suggest that the systematic analysis of the DNA replication process in ovarian cancer could uncover information on some of the molecular mechanisms that drive the accumulation of genetic damage, and probably contribute to the pathogenesis of the disease.

Therapeutic Targeting of DNA Replication Stress in Cancer.

This article reviews the currently used therapeutic strategies to target DNA replication stress for cancer treatment in the clinic, highlighting their effectiveness and limitations due to toxicity and drug resistance. Cancer cells experience enhanced spontaneous DNA damage due to compromised DNA replication machinery, elevated levels of reactive oxygen species, loss of tumor suppressor genes, and/or constitutive activation of oncogenes. Consequently, these cells are addicted to DNA damage response signaling pathways and repair machinery to maintain genome stability and support survival and proliferation. Chemotherapeutic drugs exploit this genetic instability by inducing additional DNA damage to overwhelm the repair system in cancer cells. However, the clinical use of DNA-damaging agents is limited by their toxicity and drug resistance often arises. To address these issues, the article discusses a potential strategy to target the cancer-associated isoform of proliferating cell nuclear antigen (caPCNA), which plays a central role in the DNA replication and damage response network. Small molecule and peptide agents that specifically target caPCNA can selectively target cancer cells without significant toxicity to normal cells or experimental animals.

Arginine shortage induces replication stress and confers genotoxic resistance by inhibiting histone H4 translation and promoting PCNA polyubiquitination.

The unique arginine dependencies of cancer cell proliferation and survival creates metabolic vulnerability. Here, we investigate the impact of extracellular arginine availability on DNA replication and genotoxic resistance. Using DNA combing assays, we find that when extracellular arginine is limited, cancer cells are arrested at S-phase and DNA replication forks slow or stall instantly until arginine is re-supplied. The translation of new histone H4 is arginine-dependent and impacts DNA replication and the expression of newly synthesized histone H4 is reduced in the avascular nutrient-poor breast cancer xenograft tumor cores. Furthermore, we demonstrate that increased PCNA occupancy and HLTF-catalyzed PCNA K63-linked polyubiquitination protects arginine-starved cells from hydroxyurea-induced, DNA2-catalyzed nascent strand degradation. Finally, arginine-deprived cancer cells are tolerant to genotoxic insults in a PCNA K63-linked polyubiquitination-dependent manner. Together, these findings reveal that extracellular arginine is the "linchpin" for nutrient-regulated DNA replication. Such information could be leveraged to expand current modalities or design new drug targets against cancer.

Arginine shortage induces replication stress and confers genotoxic resistance by inhibiting histone H4 translation and promoting PCNA ubiquitination.

The arginine dependency of cancer cells creates metabolic vulnerability. In this study, we examine the impact of arginine availability on DNA replication and genotoxicity resistance. Using DNA combing assays, we find that limiting extracellular arginine results in the arrest of cancer cells at S phase and a slowing or stalling of DNA replication. The translation of new histone H4 is arginine dependent and influences DNA replication. Increased proliferating cell nuclear antigen (PCNA) occupancy and helicase-like transcription factor (HLTF)-catalyzed PCNA K63-linked polyubiquitination protect arginine-starved cells from DNA damage. Arginine-deprived cancer cells display tolerance to genotoxicity in a PCNA K63-linked polyubiquitination-dependent manner. Our findings highlight the crucial role of extracellular arginine in nutrient-regulated DNA replication and provide potential avenues for the development of cancer treatments.

Small molecule targeting of transcription-replication conflict for selective chemotherapy.

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.

Small-molecule targeting of proliferating cell nuclear antigen chromatin association inhibits tumor cell growth.

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 µM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 µM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 µM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.

Clinical significance of serum biomarkers in pediatric solid mediastinal and abdominal tumors.

Childhood cancer is the leading cause of death by disease among U.S. children between infancy and age 15. Despite successes in treating solid tumors such as Wilms tumor, disappointments in the outcomes of high-risk solid tumors like neuroblastoma have precipitated efforts towards the early and accurate detection of these malignancies. This review summarizes available solid tumor serum biomarkers with a special focus on mediastinal and abdominal cancers in children.

Nuclear protein isoforms: implications for cancer diagnosis and therapy.

Post-translational modifications (PTMs) of nuclear proteins play essential roles in the regulation of gene transcription and signal transduction pathways. Numerous studies have demonstrated a correlation between specific nuclear protein isoforms and cellular malignant process. This communication reviews the impact of major PTM events such as phosphorylation, acetylation, methylation, ubiquitination, and sumoylation on several important nuclear proteins including p53, histones, proliferating cellular nuclear antigen (PCNA), and retinoblastoma protein (Rb) in the process. In addition, the implications of the PTMs as cancer biomarkers and therapeutic targets are considered.

Elevated expression of cancer-associated proliferating cell nuclear antigen in high-grade prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) plays an important role in DNA replication and repair. The expression and potential utility of this marker in prostatic neoplasia is uncertain. With the development of this new caPCNA selective antibody, we explored the potential utility of this marker in prostate cancer. METHODS: Using a traditional primary Fab2' rabbit anti-caPCNA antibody-HRP conjugated secondary anti-Fab2' antibody format, the expression of the caPCNA was analyzed in prostate tissue from 89 radical prostatectomy specimens. The caPCNA expression was correlated with clinicopathologic characteristics. RESULTS: The fraction of cells staining positively with caPCNA antibody in prostatic adenocarcinoma (mean, 23%) was significantly higher than that in benign prostatic epithelium (mean, 2%; P < 0.001) or high-grade prostatic intraepithelial neoplasia (PIN) (mean, 6%; P < 0.05). Moreover, the intensity of caPCNA expression in prostatic adenocarcinoma (mean, 2.9) was significantly higher than that in benign prostatic tissue (mean, 0.7; P < 0.001) or high-grade PIN (mean, 2.0; P < 0.001). Benign prostatic epithelium showed only minimal or negative reactivity. There was significant correlation between the percentage of caPCNA expression and primary Gleason grade (P = 0.01), and with Gleason score (P = 0.02). Adenocarcinomas with positive vascular invasion had a significantly higher percentage of cells staining with caPCNA antibody (P < 0.0001) and a higher intensity of caPCNA expression (P = 0.04). CONCLUSIONS: Our data indicate that increased expression of the cancer-associated isoform of PCNA is common in prostatic adenocarcinoma and its precursor and may be a useful biomarker.

Novel Peptide Therapeutic Approaches for Cancer Treatment.

Peptides are increasingly being developed for use as therapeutics to treat many ailments, including cancer. Therapeutic peptides have the advantages of target specificity and low toxicity. The anticancer effects of a peptide can be the direct result of the peptide binding its intended target, or the peptide may be conjugated to a chemotherapy drug or radionuclide and used to target the agent to cancer cells. Peptides can be targeted to proteins on the cell surface, where the peptide-protein interaction can initiate internalization of the complex, or the peptide can be designed to directly cross the cell membrane. Peptides can induce cell death by numerous mechanisms including membrane disruption and subsequent necrosis, apoptosis, tumor angiogenesis inhibition, immune regulation, disruption of cell signaling pathways, cell cycle regulation, DNA repair pathways, or cell death pathways. Although using peptides as therapeutics has many advantages, peptides have the disadvantage of being easily degraded by proteases once administered and, depending on the mode of administration, often have difficulty being adsorbed into the blood stream. In this review, we discuss strategies recently developed to overcome these obstacles of peptide delivery and bioavailability. In addition, we present many examples of peptides developed to fight cancer.

Molecular Targeting of Cancer-Associated PCNA Interactions in Pancreatic Ductal Adenocarcinoma Using a Cell-Penetrating Peptide.

Pancreatic ductal adenocarcinoma is a particularly difficult cancer to treat due to a lack of effective screening or treatment. Pancreatic cancer cells exhibit high proliferating cell nuclear antigen (PCNA) expression, which is associated with poor prognosis. PCNA, an important nuclear DNA replication and repair protein, regulates a myriad of proteins via the interdomain connector loop. Within this region, amino acids 126-133 are critical for PCNA interactions in cancer cells. Here, we investigate the ability of a decoy cell-penetrating peptide, R9-caPeptide, that mimics the interdomain connector loop region of PCNA to disrupt PCNA-protein interactions in pancreatic cancer cells. Our data suggest that R9-caPeptide causes dose-dependent toxicity in a panel of pancreatic cancer cell lines by inhibiting DNA replication fork progression and PCNA-regulated DNA repair, ultimately causing lethal DNA damage. Overall, these studies lay the foundation for novel therapeutic strategies that target PCNA in pancreatic cancer.

Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome.

We previously reported on the purification and characterization of a functional multi-protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry-out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30-fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7-10 ribonucleotides long and DNA primers that are 20-40 deoxyribonucleotides long using a poly(dT) template of exogenous single-stranded DNA. The synthesome-catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double-stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10-20 nt in length and the primers are covalently link to DNA to form RNA-primed nascent DNA of 100-200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins.

Proteomic approaches in neuroblastoma: a complementary clinical platform for the future.

Neuroblastoma (NB) is one of the most common solid tumors of childhood and displays a remarkable diversity in both biologic characteristics and clinical outcomes. Availability of high-throughput 'omics technologies and their subsequent application towards oncology has provided insight into the complex pathways of tumor formation and progression. Investigation of NB 'omics profiles may better define tumor behavior and provide targeted therapy with the goal of improving outcomes in patients with high-risk disease. Utilization of these technologies in NB has already led to advances in classification and risk stratification. The gradual emergence of NB-directed proteomics adds a layer of intricacy to the analysis of biologic organization but may ultimately provide a better comprehension of this complex disease. In this review, we cite specific examples of how NB-directed proteomics has provided information regarding novel biomarkers and possible therapeutic targets. We finish by examining the impact of high-throughput 'omics in the field of NB and speculate on how these emerging technologies may further be incorporated into the discipline.

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome.

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC(50) of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases alpha, delta and epsilon is 15, 45 and 125 muM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC(50)=88 muM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.

Proteomic analysis of neuroblastoma microenvironment: effect of the host-tumor interaction on disease progression.

BACKGROUND: Children with advanced-stage neuroblastoma (NB) traditionally experience poor outcomes. Because early detection of advanced-stage disease may impact survival, finding new targets for early diagnosis is crucial. Evidence suggests the tumor microenvironment may have profound effects on cancer progression. METHODS: As little is known concerning the NB-host microenvironment, this study applied proteomic techniques, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to determine protein differences between cell cultured NB and tumors grown in mice for 2, 4, and 5 wk. RESULTS: We found an increase in proteins in cultured NB compared with implanted mouse tumors during tumor progression. Additionally, analyzing in vivo tumors to cultured NB, we observed less expressed proteins. However, 16 out of 19 proteins were of mouse origin, thus inferring host-derived factors contributing to tumor growth. CONCLUSION: We show that the dynamic relationship between NB and host microenvironment is important for tumor growth and better understanding of this milieu maybe relevant towards finding unique approaches for identifying advanced-stage disease.