Immunophenotyping and Transcriptional Profiling of Human Plasmablasts in Dengue.

Plasmablasts represent a specialized class of antibody-secreting effector B cells that transiently appear in blood circulation following infection or vaccination. The expansion of these cells generally tends to be massive in patients with systemic infections such as dengue or Ebola that cause hemorrhagic fever. To gain a detailed understanding of human plasmablast responses beyond antibody expression, here, we performed immunophenotyping and RNA sequencing (RNA-seq) analysis of the plasmablasts from dengue febrile children in India. We found that plasmablasts expressed several adhesion molecules and chemokines or chemokine receptors that are involved in endothelial interactions or homing to inflamed tissues, including skin, mucosa, and intestine, and upregulated the expression of several cytokine genes that are involved in leukocyte extravasation and angiogenesis. These plasmablasts also upregulated the expression of receptors for several B-cell prosurvival cytokines that are known to be induced robustly in systemic viral infections such as dengue, some of which generally tend to be relatively higher in patients manifesting hemorrhage and/or shock than in patients with mild febrile infection. These findings improve our understanding of human plasmablast responses during the acute febrile phase of systemic dengue infection. IMPORTANCE Dengue is globally spreading, with over 100 million clinical cases annually, with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody-dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the early cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the transcriptional profiles of human plasmablasts from dengue patients.

An HIV-1 Broadly Neutralizing Antibody from a Clade C-Infected Pediatric Elite Neutralizer Potently Neutralizes the Contemporaneous and Autologous Evolving Viruses.

Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite neutralizers are potential candidates for isolation of HIV-1 bNAbs. The coexistence of bNAbs such as BG18 with neutralization-susceptible autologous viruses in an HIV-1-infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1-infected children than in adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral-naive HIV-1 clade C chronically infected pediatric elite neutralizer, AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, a median inhibitory dilution (ID50) value of 1,246, and presence of N160 and N332 supersite-dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses, exhibited substantial indels despite limited somatic hypermutations, interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy, and demonstrated high binding affinity. In addition, AIIMS-P01 neutralized the coexisting and evolving autologous viruses, suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in the AIIMS_330 pediatric elite neutralizer. Such pediatric elite neutralizers can serve as potential candidates for isolation of novel HIV-1 pediatric bNAbs and for understanding the coevolution of virus and host immune response.IMPORTANCE More than 50% of the HIV-1 infections globally are caused by clade C viruses. To date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are under way to design immunogens that can elicit correlates of protection upon vaccination. Here, we report the isolation and characterization of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01, from a clade C chronically infected pediatric elite neutralizer. The N332 supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibited substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation and characterization of bNAbs contributing to the plasma breadth in HIV-1 chronically infected children may help provide a better understanding of their role in controlling HIV-1 infection.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India.

Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR- CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE: Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

Severe disease during both primary and secondary dengue virus infections in pediatric populations.

Dengue is a global epidemic causing over 100 million cases annually. The clinical symptoms range from mild fever to severe hemorrhage and shock, including some fatalities. The current paradigm is that these severe dengue cases occur mostly during secondary infections due to antibody-dependent enhancement after infection with a different dengue virus serotype. India has the highest dengue burden worldwide, but little is known about disease severity and its association with primary and secondary dengue infections. To address this issue, we examined 619 children with febrile dengue-confirmed infection from three hospitals in different regions of India. We classified primary and secondary infections based on IgM:IgG ratios using a dengue-specific enzyme-linked immunosorbent assay according to the World Health Organization guidelines. We found that primary dengue infections accounted for more than half of total clinical cases (344 of 619), severe dengue cases (112 of 202) and fatalities (5 of 7). Consistent with the classification based on binding antibody data, dengue neutralizing antibody titers were also significantly lower in primary infections compared to secondary infections (P ≤ 0.0001). Our findings question the currently widely held belief that severe dengue is associated predominantly with secondary infections and emphasizes the importance of developing vaccines or treatments to protect dengue-naive populations.

Antibody response patterns in chikungunya febrile phase predict protection versus progression to chronic arthritis.

Chikungunya virus (CHIKV) infection causes acute febrile illness in humans, and some of these individuals develop a debilitating chronic arthritis that can persist for months to years for reasons that remain poorly understood. In this study from India, we characterized antibody response patterns in febrile chikungunya patients and further assessed the association of these initial febrile-phase antibody response patterns with protection versus progression to developing chronic arthritis. We found 5 distinct patterns of the antibody responses in the febrile phase: no CHIKV binding or neutralizing (NT) antibodies but PCR positive, IgM alone with no NT activity, IgM alone with NT activity, IgM and IgG without NT activity, and IgM and IgG with NT activity. A 20-month follow-up showed that appearance of NT activity regardless of antibody isotype or appearance of IgG regardless of NT activity during the initial febrile phase was associated with a robust protection against developing chronic arthritis in the future. These findings, while providing potentially novel insights on correlates of protective immunity against chikungunya-induced chronic arthritis, suggest that qualitative differences in the antibody response patterns that have evolved during the febrile phase can serve as biomarkers that allow prediction of protection or progression to chronic arthritis in the future.

Amino acid Asp181 of 5'-flap endonuclease 1 is a useful target for chemotherapeutic development.

DNA alkylation-induced damage is one of the most efficacious anticancer therapeutic strategies. Enhanced DNA alkylation and weakened DNA repair capacity in cancer cells are responsible for the effectiveness of DNA-alkylating therapies. 5'-Flap endonuclease 1 (Fen1) is an important enzyme involved in base excision repair (BER), specifically in long-patch BER (LP-BER). Using the site-directed mutagenesis approach, we have identified an important role for amino acid Asp181 of Fen1 in its endonuclease activity. Asp181 is thought to be involved in Mg(2+) binding in the active site. Using structure-based molecular docking of Fen1 targeted to its metal binding pocket M2 (Mg(2+) site), we have identified a potent low-molecular weight inhibitor (LMI, NSC-281680) that efficiently blocks LP-BER. In this study, we have demonstrated that the interaction of this LMI with Fen1 blocked its endonuclease activity, thereby blocking LP-BER and enhancing the cytotoxic effect of DNA-alkylating agent Temozolomide (TMZ) in mismatch repair (MMR)-deficient and MMR-proficient colon cancer cells. The results further suggest that blockade of LP-BER by NSC-281680 may bypass other drug resistance mechanisms such as mismatch repair (MMR) defects. Therefore, our findings provide groundwork for the development of highly specific and safer structure-based small molecular inhibitors targeting the BER pathway, which can be used along with existing chemotherapeutic agents, like TMZ, as combination therapy for the treatment of colorectal cancer.

Analysis of dengue specific memory B cells, neutralizing antibodies and binding antibodies in healthy adults from India.

BACKGROUND: The Indian population is facing highest dengue burden worldwide supporting an urgent need for vaccines. For vaccine introduction, evaluation and interpretation it is important to gain a critical understanding of immune memory induced by natural exposure. However, immune memory to dengue remains poorly characterized in this region. METHODS: We enumerated levels of dengue specific memory B cells (MBC), neutralizing (NT) and binding antibodies in healthy adults (n=70) from New Delhi. RESULTS: NT-antibodies, binding antibodies and MBC were detectable in 86%, 86.56% and 81.63% of the subjects respectively. Among the neutralizing positive subjects, 58%, 27%, 5% and 10% neutralized all four, any three, any two and any one dengue serotypes respectively. The presence of the neutralizing antibodies was associated with the presence of the MBC and binding antibodies. However, a massive interindividual variation was observed in the levels of the neutralizing antibodies (range, <1:50-1:30,264), binding antibodies (range, 1:3,000-1:134,000,) as well as the MBC (range=0.006%-5.05%). CONCLUSION: These results indicate that a vast majority of the adults are immune to multiple dengue serotypes and show massive interindividual variation in neutralizing/binding antibodies and MBCs - emphasizing the importance of monitoring multiple parameters of immune memory in order to properly plan, evaluate and interpret dengue vaccines.

Cloning, expression, and functional analysis of rat liver cytosolic inorganic pyrophosphatase gene and characterization of its functional promoter.

Inorganic pyrophosphate (PPi) is formed in several metabolic processes and its hydrolysis by the ubiquitously expressed enzyme inorganic pyrophosphatase (iPPase) is essential for the reactions to proceed in the direction of biosynthesis. Recently, we have reported differential expression and activity of cytosolic iPPase in rat liver with aging. In this article we report the cloning of the coding region of rat liver cytosolic iPPase gene in a bacterial expression vector, its expression, purification, and functional analysis by in-gel enzyme assay. SDS-PAGE and Western blot analysis of this expressed protein revealed that its molecular weight (MW) is approximately 33 kDa, while in-gel assay showed that it is functionally active just as the liver cytosolic iPPase. We have determined the genomic organization of this gene by genome blast approach. We have also cloned and characterized its proximal approximate 1 kb functional promoter (-1009 to +82) by transient transfection and luciferase assay of different 5'-deleted iPPase promoter-luciferase constructs and also established its transcription start site by primer extension analysis, along with protein-DNA interaction studies for a few putative transcription factor binding sites.

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase ß and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase ß (Pol-ß) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-ß-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-ß and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-ß-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

MicroRNA-150 is a potential biomarker of HIV/AIDS disease progression and therapy.

BACKGROUND: The surrogate markers of HIV/AIDS progression include CD4 T cell count and plasma viral load. But, their reliability has been questioned in patients on anti-retroviral therapy (ART). Five microRNAs (miRNAs) - miR-16, miR-146b-5p, miR-150, miR-191 and miR-223 in peripheral blood mononuclear cells (PBMCs) were earlier found to assign HIV/AIDS patients into groups with varying CD4 T cell counts and viral loads. In this pilot study, we profiled the expression of these five miRNAs in PBMCs, and two of these miRNAs (miR-146b-5p and miR-150) in the plasma of HIV/AIDS patients, including those on ART and those who developed ART resistance, to evaluate if these are biomarkers of disease progression and therapy. RESULTS: We quantified miRNA levels by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RNA isolated from PBMCs and plasma of healthy persons or HIV-infected patients who were (1) asymptomatic; (2) symptomatic and ART naïve; (3) on ART; and (4) failing ART. Our results show miR-150 (p<0.01) and to a lesser extent miR-146b-5p (p<0.05) levels in PBMCs to reliably distinguish between ART-naïve AIDS patients, those on ART, and those developing drug resistance and failing ART. The plasma levels of these two miRNAs also varied significantly between patients in these groups and between patients and healthy controls (p values <0.05). CONCLUSIONS: We report for the first time that PBMC and plasma levels of miR-150 and miR-146b-5p are predictive of HIV/AIDS disease progression and therapy.

Adenomatous polyposis coli-mediated accumulation of abasic DNA lesions lead to cigarette smoke condensate-induced neoplastic transformation of normal breast epithelial cells.

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase ß and 5'-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model.

miR-200c is aberrantly expressed in leiomyomas in an ethnic-dependent manner and targets ZEBs, VEGFA, TIMP2, and FBLN5.

MicroRNA-200c (miR-200c) through repression of specific target genes has been associated with cellular transition, tumorigenesis, and tissue fibrosis. We explored the expression and functional aspects of miR-200c in genesis of leiomyomas (LYO), benign uterine tumors with fibrotic characteristic. Using LYO and matched myometrium (MYO; n=76) from untreated and from patients exposed to hormonal therapies (GNRH agonist (GNRHa), Depo-Provera, and oral contraceptives), we found that miR-200c was expressed at significantly lower levels (P<0.05) in LYO as compared with MYO. These levels were lower in LYO from African Americans as compared with Caucasians, patients experiencing abnormal uterine bleeding and those exposed to GNRHa therapy. Gain-of-function of miR-200c in isolated leiomyoma smooth muscle cells (LSMCs), myometrial smooth muscle cells (MSMCs), and leiomyosarcoma cell line (SKLM-S1) repressed ZEB1/ZEB2 mRNAs and proteins, with concurrent increase in E-cadherin (CDH1) and reduction in vimentin expression, phenotypic alteration, and inhibition of MSMC and LSMC proliferations. We further validated TIMP2, FBLN5, and VEGFA as direct targets of miR-200c through interaction with their respective 3' UTRs, and other genes as determined by microarray analysis. At tissue levels, LYO expressed lower levels of TIMP2 and FBLN5 mRNAs but increased protein expressions, which to some extent altered due to hormonal exposure. Given the regulatory functions of ZEBs, VEGFA, FBLN5, and TIMP2 on cellular activities that promote cellular transition, angiogenesis, and matrix remodeling, we concluded that altered expression of miR-200c may have a significant impact on the outcome of LYO growth, maintenance of their mesenchymal and fibrotic characteristics, and possibly their associated symptoms.

miR-93/106b and their host gene, MCM7, are differentially expressed in leiomyomas and functionally target F3 and IL-8.

miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3'-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.

Endometrial miR-181a and miR-98 expression is altered during transition from normal into cancerous state and target PGR, PGRMC1, CYP19A1, DDX3X, and TIMP3.

CONTEXT: Evidence suggests that a number of microRNA (miRNA) are aberrantly expressed in endometrial disorders with potential posttranscriptional regulation of their specific target genes, including ovarian steroid receptors. OBJECTIVES: Our objective was to assess the endometrial expression of miR-98 and miR-181a and their respective target genes, progesterone (P4) receptor membrane component 1 (PGRMC1) and P4 receptor (PGR). DESIGN, SETTING, AND PATIENTS: We evaluated tissue expression and in vitro regulation at an academic university medical center in endometrial biopsies and endometrial tissues from follicular and luteal phases with and without exposure to hormonal therapies and grade I-III endometrial cancer (n = 52). INTERVENTIONS: INTERVENTIONS included endometrial biopsies and in vitro transfection. MAIN OUTCOME MEASURES: We evaluated expression and function of miR-98 and miR-181a. RESULTS: Aberrant expression of miR-98 and miR-181a is associated with endometrial transition from normal into cancerous states, which to some extent is influenced by hormonal milieu, and exhibited an inverse relationship with PGMRC1 and PGR expression, respectively. Treatments of Ishikawa cells with 17ß-estradiol, P4, or medroxyprogesterone acetate had limited effects on miR-98, miR-181a, and PGRMC1 expression, whereas 17ß-estradiol treatment increased PGR expression. In Ishikawa cells, gain of function of miR-98 repressed PGRMC1 and CYP19A1, and miR-181a repressed PGR, DDX3X, and TIMP3 at mRNA and protein levels through direct interactions with their respective 3'-untranslated regions and CCNE1 through miR-181a-induced DDX3X repression, with miR-98 reducing the rate of cell proliferation as compared with controls. CONCLUSION: miR-98 and miR-181a through their regulatory functions on PGRMC1, PGR, CYP19A1, TIMP3, and DDX3X expression may influence a wide range of endometrial cellular activities during normal menstrual cycle and transition into disease states, including endometrial cancer.

Endometrial miR-200c is altered during transformation into cancerous states and targets the expression of ZEBs, VEGFA, FLT1, IKKß, KLF9, and FBLN5.

A number of microRNAs (miRNAs), including miR-200 family, are aberrantly expressed in endometriosis and endometrial cancer. Here we assessed the expression and functional aspects of miR-200c in endometrial tissues (N = 52) from normal endometrial biopsies (N = 15), endometrial tissues including those exposed to hormonal therapies (N = 20), and grade I-III endometrial cancer (N = 17). miR-200c expression was elevated in normal endometrial biopsies from mid- and late-luteal phase, and in endometrial tumors as compared to endometrial tissues from peri- and postmenopausal period (P < .05) and its pattern of temporal expression displayed an inverse relationship with the expression of ZEBs. The expression of E-cadherin (CDH1) varied, but expressed at low levels, specifically in endometrial tissues and endometrial tumors. The endometrial expression of ZEBs and CDH1 in patients who were exposed to Depo-Provera and gonadotropin-releasing hormone agonist GnRHa displayed a trend toward lower expression as compared to proliferative phase; however, treatment of Ishikawa cells with 17ß-estradiol, progesterone, and medroxy progesterone acetate had modest effects on the expression of miR-200c and ZEBs without affecting CDH1 expression. Gain of function of miR-200c in Ishikawa cells repressed ZEBs, as well as VEGFA, FLT1, IKKß, and KLF9 expression at transcriptional and translational levels through direct interaction with their respective 3'untranslated regions and increased the rate of their proliferation. These results indicated that endometrial miR-200c expression undergoes dynamic changes during transition from normal into cancerous states; possibly influenced by hormonal milieu and by targeting the expression of specific genes with key regulatory functions in cellular transformation, inflammation, and angiogenesis may influence these events during normal and disease progression.

A novel inhibitor of DNA polymerase beta enhances the ability of temozolomide to impair the growth of colon cancer cells.

The recent emerging concept to sensitize cancer cells to DNA-alkylating drugs is by inhibiting various proteins in the base excision repair (BER) pathway. In the present study, we used structure-based molecular docking of DNA polymerase beta (Pol-beta) and identified a potent small molecular weight inhibitor, NSC-666715. We determined the specificity of this small molecular weight inhibitor for Pol-beta by using in vitro activities of APE1, Fen1, DNA ligase I, and Pol-beta-directed single-nucleotide and long-patch BER. The binding specificity of NSC-666715 with Pol-beta was also determined by using fluorescence anisotropy. The effect of NSC-666715 on the cytotoxicity of the DNA-alkylating drug temozolomide (TMZ) to colon cancer cells was determined by in vitro clonogenic and in vivo xenograft assays. The reduction in tumor growth was higher in the combination treatment relative to untreated or monotherapy treatment. NSC-666715 showed a high specificity for blocking Pol-beta activity. It blocked Pol-beta-directed single-nucleotide and long-patch BER without affecting the activity of APE1, Fen1, and DNA ligase I. Fluorescence anisotropy data suggested that NSC-666715 directly and specifically interacts with Pol-beta and interferes with binding to damaged DNA. NSC-666715 drastically induces the sensitivity of TMZ to colon cancer cells both in in vitro and in vivo assays. The results further suggest that the disruption of BER by NSC-666715 negates its contribution to drug resistance and bypasses other resistance factors, such as mismatch repair defects. Our findings provide the "proof-of-concept" for the development of highly specific and thus safer structure-based inhibitors for the prevention of tumor progression and/or treatment of colorectal cancer.

Altered expression of trefoil factor 3 and cathepsin L gene in rat kidney during aging.

Alterations in a wide array of physiological functions are normal consequences of aging. It is likely that, decline in cellular and physiological functions that occur during aging are the net result of age related differential gene expression and their consequent down stream effects. In this report we demonstrate that in aged kidney there is a decrease in the expression of trefoil factor 3 gene and an age-related increase in the expression of cathepsin L gene as revealed by differential display PCR (DD-PCR) and northern blot analysis. Trefoil factor 3 is mainly expressed in the alimentary canal and protects it from the degradative effect of HCl by stimulating the goblet cells to synthesize mucin. Though the exact role of trefoil factor 3 in kidney is not known, we speculate that it has a protective role in kidney. Cathepsin L is a cysteine protease which degrades connective tissue proteins like collagen, elastin and fibronectin. Increase in the expression of cathepsin L in aged kidney leading to considerable loss of organ function in old age. Down regulation of trefoil factor 3 and up regulation of cathepsin L may contribute to lack of protection and increased age related tissue damage to kidney in aging.

Age-dependent differential expression and activity of rat liver cytosolic inorganic pyrophosphatase gene.

Besides epigenetic factors, the genetic make-up and differential gene expression not only determines aging and disease susceptibility but also the functional activity of cells in an individual. Analysis of a variety of mammalian tissues revealed that the age-associated differentially expressed genes mainly belong to inflammation, stress, and metabolism. Intracellular PPi is a by-product of multiple biosynthetic reactions and its hydrolysis by cytosolic inorganic pyrophosphatase (iPPase) has long been considered as an important homeostatic mechanism favoring biosynthesis. In this paper we report an age-associated increase ( approximately 2-fold) in the expression of rat liver cytosolic iPPase gene by differential display PCR and northern blot analysis. Expression profiling of iPPase by RNA slot blot analysis in several other tissues revealed no significant change with aging. A comparative spectrophotometric and in-gel analysis of iPPase activity in whole cell lysate (WCL) of liver, brain, skeletal muscle, heart, spleen and kidney exhibited that liver of old rats (24 months ) has approximately 2-fold more activity than the adult (4 months) ones and also its activity is highest among the tissues. The specificity of iPPase activity in the spectrophotometric assay and in-gel analysis was confirmed by specific iPPase inhibitors like CaCl(2) and NaF.

Increased phosphorylation and decreased level of IkappaBalpha during aging in rat liver.

Nuclear factor kappa B (NFkappaB) is an evolutionary conserved transcription factor, which coordinates various metabolic processes triggered by innate and adaptive immune responses. Supakar et al. (J. Biol. Chem. 270: 837-842, 1995) had reported a 10-fold increase in DNA binding activity of NFkappaB in liver of old rats. In this study, we have analyzed the changes in the level of NFkappaB, inhibitor of NFkappaB (IkappaBalpha), phosphorylated-IkappaBalpha (p-IkappaBalpha) and IkappaB kinase (IKK) in rat liver during aging by reverse transcription polymerase chain reaction and/or western blotting. Here we demonstrate that there is an age-dependent increase in the level of p-IkappaBalpha with concomitant decrease in the level of IkappaBalpha, which may be correlated with increased inflammation, oxidative stress and higher level of activated NFkappaB in rat liver in old age.

Decreased expression of the pancreatic secretory trypsin inhibitor II gene during aging of the rat liver.

The genetic constitution and differential gene expression of an organism play important roles in controlling the species-specific rate of aging and the maximum life span potential. We utilized a differential-display polymerase chain reaction technique to identify the age-dependent expression of genes in the rat liver. We demonstrate in this report, for the first time, that expression of the pancreatic secretory trypsin inhibitor II (PSTI-II) gene declines drastically during aging. We confirmed this decrease by Northern blot analysis. Low PSTI-II levels in aged animals might result in a lack of protection from prematurely activated trypsin-like proteases, which would thus enhance inflammation.