Mechanism of replication blocking and bypass of Y-family polymerase {eta} by bulky acetylaminofluorene DNA adducts

Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA and RNA synthesis. These lesions are consequently strong replication blocks. In addition bulky adducts give rise to point and frameshift mutations. The translesion synthesis (TLS) DNA polymerase η is able to bypass slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol η depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.     

miR-93 suppresses proliferation and colony formation of human colon cancer stem cells

AIM:To identify differentially expressed microRNAs(miRNAs) in human colon cancer stem cells(SW1116csc) and study their function in SW1116csc proliferation.METHODS:SW1116csc were isolated from the humancolon cancer cell line,SW1116 and cultured in serumfree medium. A miRNA microarray was used to detect differential expression profiles of miRNAs in SW1116cscand SW1116 cells. Real-time quantitative polymer asechain reaction(PCR) was performed to verify the differential expression of candidate miRNAs obtained f...     

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O(8) atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.     

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very low-fidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.Replicative DNA polymerases (DNAPs) from A and B families, collectively termed replicases, exhibit a “tight fit” for their DNA and dNTP substrates and are wondrously adapted to form correct Watson–Crick base pairs, resulting in very pronounced fidelity (1, 2). This strict preference to produce A:T and G:C base pairs is also the Achilles heel of faithful DNA polymerases, however, because they are strongly inhibited by modified nucleotides present at sites of DNA damage, leading to the stalling of replication fork and eventually to replicative stress and cell death (3). At the stalled replication fork, the DNA polymerase may be exchanged by a translesion synthesis (TLS) polymerase, generally belonging to the Y family. These enzymes possess looser solvent-exposed active sites, which allows them to deal with aberrant DNA features much better, although with a very low polymerization accuracy with the risk of the accumulation of mutations and genetic instability (4, 5). Alternatively, nonbulky modified bases, such as uracil and 8-oxo-deoxyguanosine (8oxoG), can be bypassed by replicases, with faithful or mutagenic outcomes that can be modified by the sequence context and dNTP availability (6, 7).Abasic or apurinic/apyrimidinic (AP) sites are the most common DNA lesions arising in cells when the N-glycosydic bond between the sugar moiety and the nucleobase is broken, either spontaneously or by a DNA glycosylase reaction product in the base excision repair pathway (8, 9). Unrepaired abasic sites are highly blocking lesions for replicative DNA polymerases (10), although mutant polymerases with impaired proofreading activity or with mutations in the polymerization active site residues that affect the incoming nucleotide selection have been shown to have enhanced AP site bypass capacity (11–15).Among family B DNAPs, protein-primed polymerases constitute a heterogeneous group with an apparent monophyletic origin that can be found in various prokaryotic and eukaryotic viruses, mitochondrial plasmids, and eukaryotic mobile elements (16–19). This designation refers to their capacity, apparent for adenoviruses as well as different bacteriophage enzymes (20, 21), to perform genome replication primed by a specific protein, termed terminal protein (TP), that becomes covalently linked to the 5′ DNA ends. Identification and annotation of these polymerases in databases is based on the presence of two specific amino acid insertions, TPR1 and TPR2, involved in the interaction with TP and in processivity and strand displacement capacity, respectively (22, 23).Bacillus subtilis bacteriophage Φ29 DNA polymerase (Φ29DNAP) is the paradigm of protein-primed DNAPs, with well-understood biochemical and structural properties (reviewed in refs. 18, 20, 24). Φ29DNAP is a highly faithful enzyme (25, 26) able to generate very long DNA molecules (27), coupling DNA synthesis and strand displacement. Φ29, along with other protein-primed genome replication phages, such as PRD1 or Cp-1 (28, 29), can undergo a lytic cycle only after infection of the host cell occurs. In contrast, phage Bam35, which infects Bacillus thuringiensis and related tectiviruses infecting Bacillus cereus sensu lato group (30, 31), are temperate viruses that can self-replicate as linear episomes within lysogenic cells.In this work, we describe the biochemical properties of Bam35 DNA polymerase (B35DNAP) as a faithful, processive DNAP endowed with intrinsic strand displacement activity. Surprisingly, we also found that, despite its high fidelity, it can elude to some extent the tight quality check of proofreading activity, allowing the enzyme to processively bypass abasic sites in DNA. Furthermore, deletion of the TPR2 subdomain does not substantially reduce the insertion of nucleotides opposite the abasic site, but does impair its further extension. We discuss the potential implications of these findings for the bacteriophage replication cycle and possible applications.     

Direct visualization of translesion DNA synthesis polymerase IV at the replisome

In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these translesion DNA synthesis DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the β sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses, we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the β clamp but not its catalytic activity, and is reinforced by the presence of the γ subunit of the β clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential.

Replication fork progression in Escherichia coli is catalyzed by a replisome composed of the DNA polymerase III holoenzyme (Pol III HE), which provides both the leading- and lagging-strand DNA polymerases, the hexameric replicative DNA helicase, DnaB, and the Okazaki fragment primase, DnaG (1). The Pol III HE itself contains 10 subunits: two copies of the core DNA polymerase (αεθ, where α is the catalytic DNA polymerase and ε is the proofreading 3′→5′ exonuclease), the dimeric sliding processivity clamp β, and the DnaX complex, τ₂γδδ′χψ, which loads β to the primer template. Pol III* possess all the subunits except β (1). The core polymerases are bound to the DnaX complex via interaction with the τ subunit (2, 3). An alternative form of the DnaX complex, τ₃δδ′χψ, allows the assembly in vitro of a Pol III HE with three core polymerases (4), the existence of which in vivo is supported by imaging of fluorescently tagged polymerase subunits (5, 6). However, cells that do not produce the γ subunit are ultraviolet (UV) sensitive and have reduced mutagenic break repair (7), an activity that requires DNA polymerase IV (Pol IV) (8).DNA synthesis catalyzed by the Pol III HE is highly accurate, with an error rate of roughly 10⁻⁷ (9). In general, Pol III cannot bypass bulky template lesions, although we have shown that it can bypass a cis-syn thymidine dimer (10). Bypass of most template lesions in the cell is ascribed to the action of translesion DNA synthesis (TLS) polymerases, of which E. coli has three: DNA polymerases II, IV, and V (11). These polymerases demonstrate different activities with various template lesions. Pol V Mut, a RecA-activated form of Pol V (12, 13) [UmuD′₂UmuC (14, 15)], is the major activity under conditions of high replication stress when the SOS response has been activated (16, 17). Pol IV, which is encoded by dinB (18), is a Y family DNA polymerase that is well conserved from bacteria to eukaryotic cells. Pol IV has long been thought to be present at the highest concentration of all DNA polymerases in E. coli under unstressed conditions, about 250 copies per cell, with this concentration increasing about 10-fold upon induction of the SOS response (19). However, a recent study that measured the signal generated by fluorescently tagged Pol IV molecules in live cells argued that these high values were inaccurate, with the basal level of Pol IV being about 20 copies/cell and the SOS-induced level about 280 copies/cell (20).Unlike Pol III HE, which is a rapid and highly processive DNA polymerase (21–23), Pol IV is distributive, incorporating only one nucleotide per primer binding event (24). However, like all E. coli DNA polymerases, it can interact with the β processivity clamp, which increases its processivity to 300–400 nucleotides (24). Overproduction of Pol IV in the absence of replication stress slows DNA replication (25, 26) and it has been demonstrated that it is the main factor contributing to slowing replication fork progression under conditions of stress (27). Studies in vitro have shown that Pol II and Pol IV can generate slow-moving replication forks in the presence of DnaB and DnaG and that very high concentrations of Pol IV can slow the canonical Pol III–DnaB–DnaG replisome (28).The ability of all E. coli TLS polymerases to bind to β led to the formulation of the “tool belt” model to account for rapid localization of TLS polymerases to the site of replisome stalling (29). The conceptual basis of the model was that because β is a dimer, the TLS polymerase could ride along with the replisome on the same sliding clamp that was bound to the α subunit of the Pol III HE and switch places with a stalled Pol III to catalyze lesion bypass. How the polymerase switch occurs is still unclear. It has been suggested that switching occurs only at a stalled polymerase and that the stalled Pol III dissociates from β and is replaced by Pol IV (28, 30, 31). It has also been a common view that Pol IV association with the replisome is concentration dependent (32, 33), accounting for association only when it is needed (i.e., when the SOS response is induced). We have shown that Pol IV–dependent bypass at a thymidine dimer in the leading-strand template in the presence of an active replisome competes with lesion skipping (34), when the stalled leading-strand polymerase cycles ahead to a new primer made on the leading-strand template to continue replication downstream (35), suggesting that polymerase switching had occurred.Here we have addressed association of Pol IV with the replisome by using single-molecule DNA replication (23). We show that in the absence of template damage Pol IV can associate in a concentration-dependent manner with the replisome and proceeds along with it during replication fork progression. Association of Pol IV with the replisome requires its β binding motif, is stabilized by the presence of the γ subunit of the DnaX complex, and generates two classes of replisomes: those without Pol IV bound that progress rapidly and those with Pol IV bound that proceed slowly. Slowing of the replisome by Pol IV does not require its catalytic activity, suggesting that the decrease in rapid replication fork progression is a result of Pol IV binding directly to one of the two polymerase binding clefts on β. The constant presence of Pol IV in the replisome may act as a template damage surveillance mechanism.     

Regulation of polymerase exchange between Poleta and Poldelta by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme

To ensure efficient and timely replication of genomic DNA, organisms in all three kingdoms of life possess specialized translesion DNA synthesis (TLS) polymerases (Pols) that tolerate various types of DNA lesions. It has been proposed that an exchange between the replicative DNA Pol and the TLS Pol at the site of DNA damage enables lesion bypass to occur. However, to date the molecular mechanism underlying this process is not fully understood. In this study, we demonstrated in a reconstituted system that the exchange of Saccharomyces cerevisiae Poldelta with Poleta requires both the stalling of the holoenzyme and the monoubiquitination of proliferating cell nuclear antigen (PCNA). A moving Poldelta holoenzyme is refractory to the incoming Poleta. Furthermore, we showed that the Poleta C-terminal PCNA-interacting protein motif is required for the exchange process. We also demonstrated that the second exchange step to bring back Poldelta is prohibited when Lys-164 of PCNA is monoubiquitinated. Thus the removal of the ubiquitin moiety from PCNA is likely required for the reverse exchange step after the lesion bypass synthesis by Poleta.     

Gene expression profiling of the response of esophageal carcinoma cells to cisplatin

SUMMARY.  Cisplatin is the most common chemotherapeutic agent used in esophageal cancer. However, sensitivity to cisplatin varies greatly between patients. It is important to identify the gene(s) that are related to the sensitivity to cisplatin in esophageal cancer patients. The IC50 for cisplatin was measured for 15 esophageal cancer cell lines (TE1–5, TE8–15, KYSE140, and KYSE150). RNA was extracted from each of these cell lines and a normal esophageal epithelial cell line, namely, Het1A, and gene expression profiles were analyzed using an oligonucleotide microarray consisting of 34 594 genes. TE4 was highly resistant and TE12, 14, and 15 were sensitive to cisplatin. Thirty-seven genes were differentially expressed in the cisplatin-resistant esophageal cancer cell line. Our investigation provides a list of candidate genes that may be associated with resistance to cisplatin in esophageal cancer cells, which may serve as a basis for additional functional studies.     

DNA修复相关蛋白与肺癌顺铂耐药的关系

目的研究DNA修复相关蛋白ERCC1、BRCA1和hMLH1与肺癌顺铂耐药的关系。方法应用免疫组化链霉素抗生物素蛋白——过氧化物酶连接法检测60例肺癌组织中ERCC1、BRCA1和hMLH1蛋白的表达,分析其与临床病理特征和顺铂耐药的关系及3种DNA修复蛋白的相关性。结果60例肺癌组织ERCC1、BRCA1、hMLH1蛋白的阳性表达率分别为30％、25％、68．3％。顺铂化疗耐药组ERCC1、BRCA1蛋白阳性表达率明显高于化疗敏感组（P〈0．05）。BRCA1和hMLH1蛋白的表达具有相关性（P〈0．05）。结论ERCC1、BRCA1蛋白的表达与肺癌顺铂耐药有关,可能成为预测肺癌顺铂敏感性的指标。     

Higher expression of SIRT1 induced resistance of esophageal squamous cell carcinoma cells to cisplatin

Background

High expression of Sirtuin type 1 (SIRT1) exists in some cancer cells. However, it is still unclear whether SIRT1 affects the sensitivity of esophageal cancer cells to cisplatin. This study was designed to explore the relationship between SIRT1 expression and resistance of esophageal squamous cell carcinoma (ESCC) cells to cisplatin and reveal the underlying mechanism.

Methods

The tissue samples of 68 ESCC patients were collected from Nanjing Drum Tower Hospital, China. All the patients had undergone cisplatin based combination chemotherapy. The expression of SIRT1and Noxa in tissue samples were analyzed by quantitative real-time reverse PCR (qRT-PCR) and Western blot. Human ESCC cell line (ECa9706 cells) was cultured and a cisplatin-resistant subline (ECa9706-CisR cells) was established by continuous exposure to cisplatin at different concentrations. The expression of SIRT1 and Noxa in both cell lines was analyzed by qRT-PCR and Western blot. siRNA technology was utilized to down-regulate the SIRT1 expression in ECa9706-CisR cells. The influence of SIRT1 silence on sensitivity of ECa9706-CisR cells to cisplatin was confirmed using CCK-8 assay and flow cytometry. Furthermore, the level change of Noxa after SIRT1 silence in ECa9706-CisR cells was determined by qRT-PCR and Western blot.

Result

SIRT1 and Noxa expression in chemo-resistant patients was significantly increased and decreased respectively, compared with chemo-sensitive patients. SIRT1 expression in ECa9706-CisR cells was significantly increased with a lower Noxa level, compared with normal ECa9706 cells. Cisplatin 5 µM could cause proliferation inhibition, G2/M phase arrest and apoptosis in ECa9706-CisR cells and these effects could be enhanced dramatically by SIRT1 silencing. Moreover, Noxa expression was increased after treated with SIRT1 siRNA.

Conclusions

Over-expression of SIRT1 may cause resistance of ESCC cells to cisplatin through the mechanism involved with Noxa expression.     

Efficient and accurate bypass of N2-(1-carboxyethyl)-2'-deoxyguanosine by DinB DNA polymerase in vitro and in vivo

DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N(2)-dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) as the major stable DNA adduct. Here, we found that N(2)-CEdG could be detected at a frequency of one lesion per 10(7) nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N(2)-CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N(2)-CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N(2)-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase kappa), opposite the N(2)-CEdG is both accurate and efficient. Taken together, our data support that N(2)-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.     

Novel therapeutic target for cancer stem cells in hepatocellular carcinoma

Cancer is a disease of genetic and epigenetic alterations, which are emphasized as the central mechanisms of tumor progression in the multi-stepwise model. Discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. The heterogeneity of tumors can be explained with the help of CSCs supported by anti-apoptotic signaling. CSCs mimic normal adult stem cells by demonstrating unique characteristics of self-renewal and pluripotency, and the critical role for tumor growth and resistance to anti-cancer therapy. We found that CD13 was a surface marker for CSCs in human liver cancer cell lines and clinical samples, and that CD13+ CSCs were associated with a hypoxic marker in clinical hepatocellular carcinoma (HCC) sample, suggesting that CD13+ CSCs have the critical role in tumor growth and resistance to anti-cancer therapy in liver cancers. In this review article, we update recent findings regarding the involvement of CSCs, especially in HCC.     

A model for DNA polymerase switching involving a single cleft and the rim of the sliding clamp

The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagenesis are managed by its interaction with the β sliding clamp. In the structure reported by Bunting et al. [EMBO J (2003) 22:5883–5892], the C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while residues V303–P305 reach over the dimer interface to contact the rim of the adjacent clamp protomer. Using mutant forms of these proteins impaired for either the rim or the cleft contacts, we determined that the rim contact was dispensable for Pol IV replication in vitro, while the cleft contact was absolutely required. Using an in vitro assay to monitor Pol III*-Pol IV switching, we determined that a single cleft on the clamp was sufficient to support the switch, and that both the rim and cleft contacts were required. Results from genetic experiments support a role for the cleft and rim contacts in Pol IV function in vivo. Taken together, our findings challenge the toolbelt model and suggest instead that Pol IV contacts the rim of the clamp adjacent to the cleft that is bound by Pol III* before gaining control of the same cleft that is bound by Pol III*.     

Yeast Rev1 protein promotes complex formation of DNA polymerase ζ with Pol32 subunit of DNA polymerase δ

Yeast DNA polymerase (Pol) δ, essential for DNA replication, is comprised of 3 subunits, Pol3, Pol31, and Pol32. Of these, the catalytic subunit Pol3 and the second subunit Pol31 are essential, whereas the Pol32 subunit is not essential for DNA replication. Although Pol32 is an integral component of Polδ, it is also required for translesion synthesis (TLS) by Polζ. To begin to decipher the bases of Pol32 involvement in Polζ-mediated TLS, here we examine whether Pol32 physically interacts with Polζ or its associated proteins and provide evidence for the physical interaction of Pol32 with Rev1. Rev1 plays an indispensable structural role in Polζ-mediated TLS and it binds the Rev3 catalytic subunit of Polζ. Here, we show that although Pol32 does not directly bind Polζ, Pol32 can bind the Rev1–Polζ complex through its interaction with Rev1. We find that Pol32 binding has no stimulatory effect on DNA synthesis either by Rev1 in the Rev1–Pol32 complex or by Polζ in the Polζ–Rev1–Pol32 complex, irrespective of whether proliferating cell nuclear antigen has been loaded onto DNA or not. We discuss evidence for the biological significance of Rev1 binding to Pol32 for Polζ function in TLS and suggest a structural role for Rev1 in modulating the binding of Polζ with Pol32 in Polδ stalled at a lesion site.     

Cancer stem cells in lung cancer: Evidence and controversies

The cancer stem cell (CSC) model is based on a myriad of experimental and clinical observations suggesting that the malignant phenotype is sustained by a subset of cells characterized by the capacity for self‐renewal, differentiation and innate resistance to chemotherapy and radiation. CSC may be responsible for disease recurrence after definitive therapy and may therefore be functionally synonymous with minimal residual disease. Similar to other solid tumours, several putative surface markers for lung CSC have been identified, including CD133 and CD44. In addition, expression and/or activity of the cytoplasmic enzyme aldehyde dehydrogenase ALDH and capacity of cells to exclude membrane permeable dyes (known as the ‘side population’) correlate with stem‐like function in vitro and in vivo. Embryonic stem cell pathways such as Hedgehog, Notch and WNT may also be active in lung cancers stem cells and therefore may be therapeutically targetable for maintenance therapy in patients achieving a complete response to surgery, radiotherapy or chemotherapy. This paper will review the evidence regarding the existence and function of lung CSC in the context of the experimental and clinical evidence and discuss some ongoing controversies regarding this model.     

Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells

A new paradigm in oncology establishes a spectrum of tumorigenic potential across the heterogeneous phenotypes within a tumor. The cancer stem cell hypothesis postulates that a minute fraction of cells within a tumor, termed cancer stem cells (CSCs), have a tumor-initiating capacity that propels tumor growth. An application of this discovery is to target this critical cell population using chemotherapy; however, the process of isolating these cells is arduous, and the rarity of CSCs makes it difficult to test potential drug candidates in a robust fashion, particularly for individual patients. To address the challenge of screening drug libraries on patient-derived populations of rare cells, such as CSCs, we have developed a drug-eluting microarray, a miniaturized platform onto which a minimal quantity of cells can adhere and be exposed to unique treatment conditions. Hundreds of drug-loaded polymer islands acting as drug depots colocalized with adherent cells are surrounded by a nonfouling background, creating isolated culture environments on a solid substrate. Significant results can be obtained by testing <6% of the cells required for a typical 96-well plate. Reliability was demonstrated by an average coefficient of variation of 14% between all of the microarrays and 13% between identical conditions within a single microarray. Using the drug-eluting array, colorectal CSCs isolated from two patients exhibited unique responses to drug combinations when cultured on the drug-eluting microarray, highlighting the potential as a prognostic tool to identify personalized chemotherapeutic regimens targeting CSCs.Tumor-initiating cancer stem cells (CSCs) are being investigated as a promising therapeutic target (1). The rarity of CSCs, which constitute ∼1% of tumor cells (1, 2), limits their availability for testing, and traditional screening methods require substantial cell quantities. Industrial pharmaceutical capabilities have successfully reduced cell requirements in drug screening, but such capital-intensive facilities are typically unavailable to clinicians and pathology laboratories. The past decade has witnessed the emergence of multiple cell-based microarray platforms that address availability and cell source limitations (3–5), although these systems have inherent shortcomings. Many rely on immobilizing target molecules (6–9), limiting applicability to small molecule drug libraries, whereas others rely on robotically spotting cells (10), a technique not amenable to widespread adoption. Array platforms capable of capturing single cells have been established (11, 12), but determination of chemotherapeutic efficacy is better investigated through methods using greater cell numbers, which better capture variability in cellular responses. Furthermore, arrays of drug-loaded polymer films with an overlying cell monolayer have been developed (13), but monolayers of cells are susceptible to juxtacrine and paracrine signaling, which are particularly important for multipotent cells. In the present work, the provision of differential cell adhesion to promote seeding onto spotted drug-loaded films against a surrounding nonfouling background (i.e., a surface that resists protein adsorption and thus cell adhesion) can separate drug-eluting polymer films to create isolated culture environments. The use of programmable arraying techniques can then enable fabrication of uniquely formulated drug-eluting spots that provide prescribed drug doses and drug combinations to overlying cells for simultaneous testing on a single device.It is becoming increasingly evident in cancer treatment that simultaneously targeting multiple critical pathways using combinations of chemotherapeutic drugs can enhance outcomes (14–17). Conventional screening of chemotherapeutics uses an established panel of cancer cell lines (18) that have been derived from bulk tumors. A recently developed clinical approach involves performing in vitro chemosensitivity testing of tumor biopsy specimens to individualize treatment (19, 20). Unfortunately, benefits have been limited, with poor correlations between bulk tumor cell sensitivity and clinical efficacy. This lack of efficacy has been attributed to patient to patient variability, owing in part to intratumor heterogeneity (21–23).Tumors consist of multiple cell phenotypes. In the CSC model, a rare cell population of tumor-initiating cells perpetually self-renew and are responsible for tumor heterogeneity, metastasis, and disease recurrence (1, 24). Recent identification of unique cell surface markers that enrich tumor cell isolates for CSCs have led to novel techniques for isolating enriched colorectal CSC (CCSC) populations from patient tumor samples (25–28). For example, xenotransplantation of a single CCSC identified by high Wnt/Β-catenin signaling activity generates tumors that recapitulate the diverse phenotypic heterogeneity of the original tumor (29). Thus, identifying and isolating CCSCs out of the tumor bulk from an individual cancer patient and determining sensitivity to chemotherapeutic drugs in vitro is possible (30, 31). An approach such as the drug-eluting microarray, enabling use of low cell numbers, could potentiate personalized combination drug treatment screens for efficacy against patient-specific CSCs.     

Pancreatic cancer stem cells: regulatory networks in the tumor microenvironment and targeted therapy

Recent evidence has demonstrated that the existence of a cancer stem cell (CSC) subset in a solid tumor is responsible for the progression and relapse of cancer as well as its resistance to current therapies. Over the past decade, CSC research on pancreatic cancer has progressed. A fundamental understanding of pancreatic CSCs may improve therapies and deepen insight into the role of cell–cell interactions within a tumor microenvironment in pancreatic cancer progression. This review focuses on the impact of pancreatic CSCs on the regulatory networks in the tumor microenvironment, and the implications of targeting CSCs to treat pancreatic cancer.