Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

Leukocyte recruitment from blood to tissue is a process that occurs at the level of capillary vessels during both physiological and pathological conditions. This process is also relevant for evaluating novel adoptive cell therapies, in which the trafficking of therapeutic cells such as chimeric antigen receptor (CAR)-T cells throughout the capillaries of solid tumors is important. Local variations in blood flow, mural cell concentration, and tissue stiffness contribute to the regulation of capillary vascular permeability and leukocyte trafficking throughout the capillary microvasculature. We developed a platform to mimic a biologically functional human arteriole-venule microcirculation system consisting of pericytes (PCs) and arterial and venous primary endothelial cells (ECs) embedded within a hydrogel, which self-assembles into a perfusable, heterogeneous microvasculature. Our device shows a preferential association of PCs with arterial ECs that drives the flow-dependent formation of microvasculature networks. We show that PCs stimulate basement membrane matrix synthesis, which affects both vessel diameter and permeability in a manner correlating with the ratio of ECs to PCs. Moreover, we demonstrate that hydrogel concentration can affect capillary morphology but has no observed effect on vascular permeability. The biological function of our capillary network was demonstrated using an inflammation model, where significantly higher expression of cytokines, chemokines, and adhesion molecules was observed after tumor necrosis factor-alpha (TNF-α) treatment. Accordingly, T cell adherence and transendothelial migration were significantly increased in the immune-activated state. Taken together, our platform allows the generation of a perfusable microvasculature that recapitulates the structure and function of an in vivo capillary bed that can be used as a model for developing potential immunotherapies.

T-cadherin is a novel regulator of pericyte function during angiogenesis.

Pericytes are mural cells that play an important role in regulation of angiogenesis and endothelial function. Cadherins are a superfamily of adhesion molecules mediating Ca2+-dependent homophilic cell-cell interactions that control morphogenesis and tissue remodeling. To date, classical N-cadherin is the only cadherin described on pericytes. Here, we demonstrate that pericytes also express T-cadherin (H-cadherin, CDH13), an atypical glycosyl-phosphatidylinositol (GPI)-anchored member of the superfamily that has previously been implicated in regulation of neurite guidance, endothelial angiogenic behavior, and smooth muscle cell differentiation and progression of cardiovascular disease. The aim of the study was to investigate T-cadherin function in pericytes. Expression of T-cadherin in pericytes from different tissues was performed by immunofluorescence analysis. Using lentivirus-mediated gain-of-function and loss-of-function in cultured human pericytes, we demonstrate that T-cadherin regulates pericyte proliferation, migration, invasion, and interactions with endothelial cells during angiogenesis in vitro and in vivo. T-cadherin effects are associated with the reorganization of the cytoskeleton, modulation of cyclin D1, α-smooth muscle actin (αSMA), integrin ß3, metalloprotease MMP1, and collagen expression levels, and involve Akt/GSK3ß and ROCK intracellular signaling pathways. We also report the development of a novel multiwell 3-D microchannel slide for easy analysis of sprouting angiogenesis from a bioengineered microvessel in vitro. In conclusion, our data identify T-cadherin as a novel regulator of pericyte function and support that it is required for pericyte proliferation and invasion during active phase of angiogenesis, while T-cadherin loss shifts pericytes toward the myofibroblast state rendering them unable to control endothelial angiogenic behavior.

Oxidative stress markers in patient-derived non-cancerous cervical tissues and cells.

High-risk human papillomaviruses (HPV) are the causative agents of cervical cancer. However, not all infected women develop cervical cancer. Cervical tumorigenesis is characterized by a multifactorial etiology, with oxidative stress (OS) likely playing a major role. In addition to exogenous sources, metabolic processes also contribute to OS. In principle, variability in levels of cervical OS has the potential to influence the likelihood of conversion to cervical cancer. To ask whether such variability indeed existed, we assessed the levels of ROS and the oxidative DNA damage biomarker 8-oxodG in normal non-cancerous cervical tissues and cells obtained from women with uterovaginal pelvic organ prolapse following vaginal hysterectomy. We demonstrated five and ten-fold variability between tissues isolated from the transformation zone (TZ) and ectocervix (EC) of different women, respectively. Despite the greater variability (likely due to differences in tissue composition), the overall pattern of ROS levels in EC tissues mirrored those obtained in their corresponding TZ tissues. Our results also show that the levels of ROS in TZ tissues were always higher than or equal to those found in the respective EC tissues, providing a possible explanation for TZ tissue being the primary target for HPV infection and cervical carcinogenesis. Interestingly, primary keratinocytes isolated and cultured from these cervical specimens also displayed high variability in ROS levels, with some strongly mirroring the levels of ROS observed in their corresponding tissues, while others were less closely associated. Finally, we demonstrated that the levels of DNA damage mirrored the levels of ROS in the cultured primary cells. Understanding the factors and mechanisms that dispose certain individuals to develop cervical cancer has the potential to enable the development of approaches that make the conversion of HPV infection to cancer development even more rare.

Approaches and Methods to Measure Oxidative Stress in Clinical Samples: Research Applications in the Cancer Field.

Reactive oxygen species (ROS) are common by-products of normal aerobic cellular metabolism and play important physiological roles in intracellular cell signaling and homeostasis. The human body is equipped with antioxidant systems to regulate the levels of these free radicals and maintain proper physiological function. However, a condition known as oxidative stress (OS) occurs, when ROS overwhelm the body's ability to readily detoxify them. Excessive amounts of free radicals generated under OS conditions cause oxidative damage to proteins, lipids, and nucleic acids, severely compromising cell health and contributing to disease development, including cancer. Biomarkers of OS can therefore be exploited as important tools in the assessment of disease status in humans. In the present review, we discuss different approaches used for the evaluation of OS in clinical samples. The described methods are limited in their ability to reflect on OS only partially, revealing the need of more integrative approaches examining both pro- and antioxidant reactions with higher sensitivity to physiological/pathological alternations. We also provide an overview of recent findings of OS in patients with different types of cancer. Identification of OS biomarkers in clinical samples of cancer patients and defining their roles in carcinogenesis hold great promise in promoting the development of targeted therapeutic approaches and diagnostic strategies assessing disease status. However, considerable data variability across laboratories makes it difficult to draw general conclusions on the significance of these OS biomarkers. To our knowledge, no adequate comparison has yet been performed between different biomarkers and the methodologies used to measure them, making it difficult to conduct a meta-analysis of findings from different groups. A critical evaluation and adaptation of proposed methodologies available in the literature should therefore be undertaken, to enable the investigators to choose the most suitable procedure for each chosen biomarker.

EphrinB2/EphB4 signaling regulates non-sprouting angiogenesis by VEGF.

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best-understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine-tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF-R2 activation by VEGF nor its internalization, but it modulates VEGF-R2 downstream signaling through phospho-ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF-induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.

Overexpression of HPV16 E6* Alters ß-Integrin and Mitochondrial Dysfunction Pathways in Cervical Cancer Cells.

BACKGROUND: High-risk human papillomaviruses (HPV) cause nearly all cases of cervical cancer, as well as many types of oral and anogenital cancer. Alternative splicing increases the capacity of the HPV genome to encode the proteins necessary for successful completion of its infectious life cycle. However, the roles of these splice variants, including E6*, the smaller splice isoform of the E6 oncogene, in carcinogenesis are not clear. MATERIALS AND METHODS: SiHa (HPV16(+)) and C33A (HPV(-)) cells were transfected with the E6* plasmid, and tandem mass tag-labeled protein levels were quantified by mass spectrometry. Proteomic analyses identified pathways affected by E6* in both HPV(+) and HPV(-) cells, and pathways were validated using in vitro methods. RESULTS: A total of 4,300 proteins were identified and quantified in lysates of SiHa and C33A cells with and without HPV16 E6* expression. SiHa and C33A cells expressing E6* underwent changes in protein expression affecting integrin signaling and mitochondrial dysfunction pathways, respectively. Subsequent experiments were performed to validate selected E6*-mediated alterations in protein levels. CONCLUSION: E6* modifies the expression of proteins involved in mitochondrial dysfunction and oxidative phosphorylation in C33A cells, and ß-integrin signaling in SiHa cells.

Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes.

Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis.

Recent Progress in Therapeutic Treatments and Screening Strategies for the Prevention and Treatment of HPV-Associated Head and Neck Cancer.

The rise in human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has elicited significant interest in the role of high-risk HPV in tumorigenesis. Because patients with HPV-positive HNSCC have better prognoses than do their HPV-negative counterparts, current therapeutic strategies for HPV⁺ HNSCC are increasingly considered to be overly aggressive, highlighting a need for customized treatment guidelines for this cohort. Additional issues include the unmet need for a reliable screening strategy for HNSCC, as well as the ongoing assessment of the efficacy of prophylactic vaccines for the prevention of HPV infections in the head and neck regions. This review also outlines a number of emerging prospects for therapeutic vaccines, as well as for targeted, molecular-based therapies for HPV-associated head and neck cancers. Overall, the future for developing novel and effective therapeutic agents for HPV-associated head and neck tumors is promising; continued progress is critical in order to meet the challenges posed by the growing epidemic.

Combined ultrasound-curcumin treatment of human cervical cancer cells.

OBJECTIVES: Human papillomavirus (HPV) is associated with cervical cancer. Studies showed curcumin inhibits HPV oncogenes expression but curcumin has low bioavailability. The objectives were: (1) to study ultrasound enhancement of curcumin effects on HeLa, SiHa and C33A, (2) to compare two frequencies for sonoporation and (3) to detect cell-free DNA released by the treatment. STUDY DESIGN: HeLa, SiHa and C33A cells (non-HPV control) were processed and exposed to either: (1) 10µM curcumin only, (2) 10µM curcumin with 8s of 7.5MHz ultrasound, (3) 10µM curcumin with 8s of 5.0MHz ultrasound, (4) control medium, or (5) 8s of 7.5MHz ultrasound. The five treated groups were incubated (48h) and analyzed by dual fluorescence apoptosis/necrosis assay. DNA in spent media was analyzed by capillary analysis. RESULTS: Combined curcumin ultrasound resulted in 9-, 12- and 16-fold higher necrosis in HeLa, SiHa and C33A cells respectively. Increased necrosis correlated with higher ultrasound frequencies. There was increased apoptosis in HeLa or SiHa cells with the combined treatment. Curcumin alone resulted in a lesser 2-4-fold increase in necrosis in the groups. Cell-free DNA was detected in the spent media of HeLa and SiHa but not C33A cultures. CONCLUSIONS: The results showed enhanced necrosis in cervical carcinoma cell lines after combined treatment and confirmed the ultrasound capacity to increase effectiveness of curcumin. Cancer cells were smaller post-treatment suggesting microtubule structural disruption. Cell-free DNA was low molecular weight consistent with lysed host cell.

Cellular levels of oxidative stress affect the response of cervical cancer cells to chemotherapeutic agents.

Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT-PCR array. The analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress affect responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy.

Viral carcinogenesis: factors inducing DNA damage and virus integration.

Viruses are the causative agents of 10%-15% of human cancers worldwide. The most common outcome for virus-induced reprogramming is genomic instability, including accumulation of mutations, aberrations and DNA damage. Although each virus has its own specific mechanism for promoting carcinogenesis, the majority of DNA oncogenic viruses encode oncogenes that transform infected cells, frequently by targeting p53 and pRB. In addition, integration of viral DNA into the human genome can also play an important role in promoting tumor development for several viruses, including HBV and HPV. Because viral integration requires the breakage of both the viral and the host DNA, the integration rate is believed to be linked to the levels of DNA damage. DNA damage can be caused by both endogenous and exogenous factors, including inflammation induced by either the virus itself or by co-infections with other agents, environmental agents and other factors. Typically, cancer develops years to decades following the initial infection. A better understanding of virus-mediated carcinogenesis, the networking of pathways involved in transformation and the relevant risk factors, particularly in those cases where tumorigenesis proceeds by way of virus integration, will help to suggest prophylactic and therapeutic strategies to reduce the risk of virus-mediated cancer.

Human papillomavirus type 16 E6* induces oxidative stress and DNA damage.

UNLABELLED: High-risk types of human papillomavirus (HPV) are the causative agents of virtually all cases of cervical cancer and a significant proportion of other anogenital cancers, as well as both oral and pharyngeal cancers. The high-risk types encode two viral oncogenes, E6 and E7, which work together to initiate cell transformation. Multiple steps involving the activities and interactions of both viral and cellular proteins are involved in the progression from HPV infection to cell transformation to cancer. The E6 oncoprotein is expressed as several isoforms: a full-length variant referred to as E6 and a few shorter isoforms collectively referred to as E6*. In this study, we found that expression of E6* increased the level of reactive oxygen species (ROS) in both HPV-positive and HPV-negative cells. This increased oxidative stress led to higher levels of DNA damage, as assessed by the comet assay, quantification of 8-oxoguanine, and poly(ADP-ribose) polymerase 1. The observed increase in ROS may be due to a decrease in cellular antioxidant activity, as we found that E6* expression also led to decreased expression of superoxide dismutase isoform 2 and glutathione peroxidase. These studies indicate that E6* may play an important role in virus-induced mutagenesis by increasing oxidative stress and DNA damage. IMPORTANCE: Our findings demonstrate for the first time that an HPV gene product, E6*, can increase ROS levels in host cells. This ability may play a significant role both in the viral life cycle and in cancer development, because an increase in oxidative DNA damage may both facilitate HPV genome amplification and increase the probability of HPV16 DNA integration. Integration, in turn, is thought to be an important step in HPV-mediated carcinogenesis.

The small splice variant of HPV16 E6, E6, reduces tumor formation in cervical carcinoma xenografts.

High-risk types of human papillomavirus (HPV) cause nearly all cases of cervical cancer. The E6 oncoprotein is produced as a full-length variant (E6) as well as several shorter isoforms (E6). E6 inhibits certain oncogenic activities of E6, suggesting that it might play an anti-oncogenic role in vivo. To test this, we created E6-expressing SiHa (HPV(+)) and C33A (HPV(-)) cells, then examined the ability of both the parental and E6-expressing cells to form tumors in nude mice. We found that over-expression of E6 indeed decreased the growth of tumors derived from both SiHa and C33A cells, with the reduction greatest in tumors derived from E6-expressing SiHa cells. These findings point to multiple anti-oncogenic characteristics of E6, some of which likely involve down-regulation of the full-length isoform, and others that are independent of HPV. These data represent the first demonstration of biologically-relevant E6 activities distinct from those of the full-length isoform in vivo.

Modulation of apoptotic pathways by human papillomaviruses (HPV): mechanisms and implications for therapy.

The ability of the host to trigger apoptosis in infected cells is perhaps the most powerful tool by which viruses can be cleared from the host organism. To avoid elimination by this mechanism, human papillomaviruses (HPV) have developed several mechanisms that enable the cells they infect to elude both extrinsic and intrinsic apoptosis. In this manuscript, we review the current literature regarding how HPV-infected cells avoid apoptosis and the molecular mechanisms involved in these events. In particular, we will discuss the modifications in intrinsic and extrinsic apoptotic pathways caused by proteins encoded by HPV early genes. Many of the current efforts regarding anti-cancer drug development are focused on directing tumor cells to undergo apoptosis. However, the ability of HPV-infected cells to resist apoptotic signals renders such therapies ineffective. Possible mechanisms for overcoming the resistance of HPV-infected tumor cells to anticancer drugs will be discussed.

DNA damage induces down-regulation of UDP-glucose ceramide glucosyltransferase, increases ceramide levels and triggers apoptosis in p53-deficient cancer cells.

DNA damaging agents typically induce an apoptotic cascade in which p53 plays a central role. However, absence of a p53-mediated response does not necessarily abrogate programmed cell death, due to the existence of p53-independent apoptotic pathways, such as those mediated by the pro-apoptotic molecule ceramide. We compared ceramide levels before and after DNA damage in human osteosarcoma (U2OS) and colon cancer (HCT116) cells that were either expressing or deficient in p53. When treated with mitomycin C, p53-deficient cells, but not p53-expressing cells, showed a marked increase in ceramide levels. Microarray analysis of genes involved in ceramide metabolism identified acid ceramidase (ASAH1, up-regulated), ceramide glucosyltransferase (UGCG, down-regulated), and galactosylceramidase (GALC, up-regulated) as the three genes most affected. Experiments employing pharmacological and siRNA agents revealed that inhibition of UGCG is sufficient to increase ceramide levels and induce cell death. When inhibition of UGCG and treatment with mitomycin C were combined, p53-deficient, but not p53-expressing cells, showed a significant increase in cell death, suggesting that the regulation of sphingolipid metabolism could be used to sensitize cells to chemotherapeutic drugs.

Small molecule inhibitors of the HPV16-E6 interaction with caspase 8.

High-risk strains of human papillomaviruses (HPVs) cause nearly all cases of cervical cancer as well as a growing number of head and neck cancers. The oncogenicity of these viruses can be attributed to the activities of their two primary oncoproteins, E6 and E7. The E6 protein has among its functions the ability to prevent apoptosis of infected cells through its binding to FADD and caspase 8. A small molecule library was screened for candidates that could inhibit E6 binding to FADD and caspase 8. Flavonols were found to possess this activity with the rank order of myricetin>morin>quercetin>kaempferol=galangin≫(apigenin, 7-hydroxyflavonol, rhamnetin, isorhamnetin, geraldol, datiscetin, fisetin, 6-hydroxyflavonol). Counter screening, where the ability of these chosen flavonols to inhibit caspase 8 binding to itself was assessed, demonstrated that myricetin, morin and quercetin inhibited GST-E6 and His-caspase 8 binding in a specific manner. The structure-activity relationships suggested by these data are unique and do not match prior reports on flavonols in the literature for a variety of anticancer assays.

The stress oncoprotein LEDGF/p75 interacts with the methyl CpG binding protein MeCP2 and influences its transcriptional activity.

The lens epithelium-derived growth factor p75 (LEDGF/p75) is a transcription coactivator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kDa (DFS70) and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull-down and AlphaScreen assays, coimmunoprecipitation, and nuclear colocalization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS osteosarcoma cells, whereas this effect was more pronounced in PC3 prostate cancer cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75-MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes.

Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells.

Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment.

Mass spectrometric studies on epigenetic interaction networks in cell differentiation.

Arrest of cell differentiation is one of the leading causes of leukemia and other cancers. Induction of cell differentiation using pharmaceutical agents has been clinically attempted for the treatment of these cancers. Epigenetic regulation may be one of the underlying molecular mechanisms controlling cell proliferation or differentiation. Here, we report on the use of proteomics-based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation. During phorbol-12-myristate 13-acetate-induced differentiation of U937 cells, fatty acid synthesis and its metabolic processing, the clathrin-coated pit endocytosis pathway, and the ubiquitin/26 S proteasome degradation pathways were up-regulated. In addition, global histone H3/H4 acetylation and H2B ubiquitination were down-regulated concomitantly with impaired chromatin remodeling machinery, RNA polymerase II complexes, and DNA replication. Differential protein expression analysis established the networks linking histone hypoacetylation to the down-regulated expression/activity of p300 and linking histone H2B ubiquitination to the RNA polymerase II-associated FACT-RTF1-PAF1 complex. Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation.

HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress.

HPV-DNA integration into cellular chromatin is usually a necessary event in the pathogenesis of HPV-related cancer; however, the mechanism of integration has not been clearly defined. Breaks must be created in both the host DNA and in the circular viral episome for integration to occur, and studies have shown that viral integration is indeed increased by the induction of DNA double strand breaks. Inflammation generates reactive oxygen species, which in turn have the potential to create such DNA strand breaks. It is plausible that these breaks enable a greater frequency of HPV-DNA integration, and in this way contribute to carcinogenesis. Consistent with this idea, co-infections with certain sexually transmitted diseases cause cervical inflammation, and have also been identified as cofactors in the progression to cervical cancer. This article examines the idea that inflammation facilitates HPV-DNA integration into cellular chromatin through the generation of reactive oxygen species, thereby contributing to carcinogenesis.