Deepak Kaul

HIV-1 genome-encoded hiv1-mir-H1 impairs cellular responses to infection

The recent discovery of HIV-1 genome-encoded novel microRNA (miRNA; designated as hiv1-mir-H1) having ability to target selectively and specifically human cellular AATF gene, prompted us to explore the role of this miRNA in the regulation of genes involved in cellular apoptosis, proliferation and nucleic acid-based immune mechanism governed by miRNAs. Such a study revealed that this miRNA-induced knockdown of AATF gene, within normal human blood mononuclear cells, was responsible for the suppression of genes coding for Bcl-2, c-myc, Par-4 and Dicer. Further, hiv1-mir-H1 had the capacity to downregulate expression of cellular miR149 gene recognized to target Vpr gene encoded by HIV-1. Based upon these findings, we propose an “Epigenomic Pathway” through which hiv1-mir-H1 induced AATF gene knockdown within human mononuclear cells initiates their apoptosis.

Potential tumor suppressive function of miR-196b in B-cell lineage acute lymphoblastic leukemia.

Keeping in view the fact that genes coding microRNAs (miRNAs) have been found to be localized in chromosomal regions susceptible to genetic translocations, this study was addressed to identify and characterize the miRNAs that are present near/within the regions involved in genetic translocations characteristic of B-cell acute lymphoblastic leukemia (B-cell ALL). Out of six such identified miRNAs miR-196b was not only found to be significantly down-regulated in both EB-3 cell line as well as B-cell ALL patients as compared to that found in the corresponding controls, but also had the inherent capacity to down-regulate the highly expressed c-myc gene, a consequence of genetic translocation characteristic of EB-3 cells at both transcriptional and translational level. This phenomenon was in conformity with the observed reciprocal relationship between the expressed genes coding for miR-196b and c-myc in B-cells derived from ALL patients as well as c-myc gene was found to be a putative target of miR-196b as predicted by bioinformatic algorithms. Also down-regulation of c-myc gene was accompanied by decreased expressions of c-myc effector genes coding for hTERT, Bcl-2, and AATF. Based upon these results, we propose for the first time that miR-196b has the inherent capacity to down-regulate the overamplified c-myc gene recognized as a common pathognomonic feature leading to cancer in general and B-cell ALL in particular. Hence miR-196b can be assigned with the tumor suppressor function and can be of therapeutic importance in paving the way toward the treatment of B-cell ALL.

Regulation of cellular Cyclin D1 gene by arsenic is mediated through miR-2909

Arsenic through its ability to regulate genes that link cell cycle control with apoptosis has been widely recognized to play a crucial role in oncogenomics. However, the molecular event by which arsenic affects such genes is far from clear. Here we provide reasonably good evidence to support the view that arsenic exposure to human PBMCs (peripheral blood mononuclear cells) at low concentrations results in the over-expression of miR-2909 within these cells. This over-expressed miR-2909 was found to regulate CCND1 (Cyclin D1) gene expression, within these cells by inducing splice-switching of tumor suppresser CYLD (Cylindromatosis) gene as well as modulation of SP1 (Specificity Protein 1) activity through the repression of KLF4 (Kruppel-like factor4) expression at the translational level. Arsenic dependent regulation of AATF (Apoptosis Antagonizing Transcription factor) and BCL3 (B-cell Lymphoma 3) were also found to be modulated through its capacity to induce miR-2909 expression. Based upon these observations, a novel epigenomic pathway was proposed which may not only be useful in understanding the paradoxical role of arsenic in oncogenomics but also may even be useful in devising various strategies for the treatment/prevention of tumors induced by arsenic.

Functional characterization of AATF transcriptome in human leukemic cells

The study, addressed to explore the transcriptional expression and regulation of Apoptosis-antagonizing transcription factor (AATF) gene within various types of human leukemic cell lines, revealed that AATF gene was overexpressed ubiquitously in all the leukemic cell lines studied and this upregulation was accompanied by c-myc gene overamplification in these cells. Downregulation of AATF gene transcription within leukemic cells not only resulted in the downregulation of c-myc gene and vice-versa but also contributed to apoptosis leading to cell death. Further, the link between AATF expression and leukemic cellular apoptosis involved PI3K/Akt pathway. Based on these results we propose that AATF gene may be of crucial importance in maintaining the leukemic state of a cell compartment through its ability to initiate cell proliferation coupled with repression of cellular apoptosis.

Functional genomics of tumor suppressor miR-196b in T-cell acute lymphoblastic leukemia

Huge data accumulated in last few years have shown that differential expression of candidate miRNAs in normal versus transformed cell provides important insights into the pathogenesis of cancer including leukemias. In our previous report, we have revealed that miR-196b was significantly down-regulated in both EB-3 cells as well as B-cell ALL (acute lymphoblastic leukemia) patients as compared to their respective controls. We have unambiguously proven that miR-196b restoration in EB-3 cells leads to significant down-regulation of c-myc and its effector genes, i.e., human telomerase reverse transcriptase (hTERT), B-cell lymphoma/leukemia-2 (Bcl-2), apoptosis antagonizing transcription factor (AATF), and qualifies for tumor suppressor function in B-cell ALL. Keeping in view these results, the present study was aimed at dissecting the role of miR-196b and other miRNAs present near/within the genomic regions involved in genetic translocations characteristic of ALL in T-cell ALL cell lines and patient samples. We have demonstrated significant down-regulation in the expression of miR-196b in MOLT-4 and T-cell ALL patients with respect to the respective control cells. Transfection experiments revealed that none of the six identified miRNAs were able to knock down the expression of c-myc gene. Interestingly, it was found that miR-196b loses its ability to down-regulate c-myc gene expression in T-cell ALL as a consequence of mutations in target 3′-untranslated region (3′-UTR) of the c-myc gene. Results of the present study revealed that miR-196b becomes non-functional in T-cell ALL as a consequence of mutations in 3′-UTR of c-myc gene in T-cell ALL cellular models.

Inhibitory effects of Terminalia arjuna on platelet activation in vitro in healthy subjects and patients with coronary artery disease.

Terminalia arjuna (TA) is a medicinal plant used as a cardiotonic in ayurveda. Besides others, scientific evidence dictates its strong hypolipidemic and antioxidant properties. However, anti-inflammatory and antiplatelet aggregatory properties of TA are not known. The present study demonstrates in vitro effects of its ethanolic bark extract (TAE) on platelet function indices. Twenty patients of angiographically proven coronary artery disease (CAD) were included in Group I and 20 age and sex-matched controls were included in Group II. Platelet activation was monitored by determining P-selectin (CD62P) expression, intracellular free calcium (Ca2+) release and platelet aggregation. In vitro effect of TA on platelets function indices was determined by incubating the platelets with TAE in a time and dose-dependent manner in presence/absence of ADP. TAE was able to significantly inhibit platelet aggregation both in patient and control groups. Significant attenuation in Ca2+ release and expression of CD62P was also observed with TAE. Our data clearly demonstrates that the bark extract of TA decreases platelet activation and may possess antithrombotic properties. The possible mechanism of action could be by desensitizing platelets to the agonist by competing with platelet receptor or by interfering with signal transduction. Thus, TA can be exploited for its therapeutic potential in CAD and related cardiovascular disorders.

LXR-α selectively reprogrammes cancer cells to enter into apoptosis

There exists a general recognition of the fact that LXR-α, being a member of the nuclear receptor family, plays a crucial role in the biological process that connects inflammation, cholesterol homeostasis, and cellular decisions. In this context the present study was addressed to understand the role of LXR-α gene in the selective and specific reprogramming of cancer cells into a state of apoptosis leaving the normal cells unaffected. The results of this study revealed that LXR-α gene when activated in cancerous cells of diverse origin results in the regulation of genes coding for Bcl-2, AATF, and Par-4 in a fashion, forcing these cells to enter into the state of apoptosis leaving the normal cells unaffected. On the basis of this study we propose that in near future LXR-α agonist (Withaferin A) may definitely find its use in the therapeutic interventions directed towards the treatment of cancer.

Interleukin-18-induced atherosclerosis involves CD36 and NF-κB crosstalk in Apo E−/− mice

OBJECTIVE Interleukin (IL)-18 is a pleotropic cytokine involved in various inflammatory disorders. The transcription factor, nuclear factor kappa-B (NF-κB), is thought to play an important role in IL-18 signaling. The present study proposes a novel role for IL-18 in cholesterol efflux and plaque stability and demonstrates that pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor blocks IL-18 signaling in apolipoprotein (Apo) E-/- mice. METHODS Three groups of normal chow-diet-fed, male Apo E-/- mice, aged 12 weeks (n=6/group) were employed: Gp I, PBS (2mo); Gp II, recombinant (r)IL-18 (1mo) followed by PBS (1mo); Gp III, rIL-18 (1mo) followed by PDTC (1mo). RESULTS Significantly augmented expression of IL-18 receptor (R)α by fluorescence-activated cell sorting analysis and plasma IL-18 was observed in Gp II. There was a significant increase in total cholesterol and low-density lipoprotein cholesterol whereas high-density lipoprotein cholesterol was significantly decreased in Gp II. However, this pattern was reversed in Gp III. Significantly augmented mRNA expression of IL-18, CD36, matrix metalloproteinase (MMP)-9, and NF-κB was observed in Gp II but liver X receptor alpha (LXR-α) gene was significantly reduced. A significant increase in frequency of atherosclerotic lesions was observed in Gp II animals, whereas there was a significant decrease in the Gp III. CONCLUSION IL-18 administration initiates inflammatory cascade by binding with IL-18 Rα via NF-κB which is involved in progression and destabilization of atherosclerotic plaques in Apo E-/- mice. This study also reveals that NF-κB blockade with PDTC, blocks IL-18 signaling through down-regulation of IL-18, IL-18 Rα, CD36, and MMP-9, thus reducing inflammation and restoring plaque instability via upregulation of LXR-α.

Functional genomics of blood cellular LXR-α gene in human coronary heart disease

Recent studies on the liver X receptor-alpha (LXR-alpha) have recognized its crucial protective role in the initiation of a cross-talk between lipid metabolism and inflammation regarded as a prerequisite for the development of atherosclerotic lesions. The present study was directed to explore the functional genomics of LXR-alpha gene within blood mononuclear cells of subjects suffering from coronary heart disease (CHD), revealed a paradoxical relationship between blood cellular LXR-alpha mRNA expression and the severity of coronary occlusion. In order to resolve this apparent paradox, the ligand binding domain of LXR-alpha gene was analyzed. The results of such a study revealed that three critical mutations in the domain comprising of amino acids Asp324, Pro327 and Arg328, were responsible for inability of this domain to interact with its natural ligands leading thereby to deregulation of its effector genes that are known to play crucial role in the cross-talk between lipid peroxidation and inflammation. This phenomenon was in conformity with functional assay of LXR-alpha dependent transcriptional activity within cells derived from normal and CHD subjects. Based upon these results we propose that the mutations in the LXR-alpha gene reported here for the first time not only may be exploited for the diagnosis of CHD in human subjects but also could be used as a marker for exploring the predisposition of human subjects towards CHD.

Effect of herbal polyphenols on atherogenic transcriptome

The ancient Indian system of medicine supports the antiatherogenic properties of some herbs. The crosstalk amongst the genes coding for LDLR, LXRα, PPARs (α,γ), CD-36 and c-myc may be important in atherogenesis because these genes control lipid metabolism, cytokine production and cellular activity within the arterial wall. Hence, we attempted for the first time to explore whether or not the polyphenols extracted from medicinal herbs had any effect on the transcription of these genes. Normal human mononuclear cells were cultured in the presence of polyphenols (and their HPLC purified sub-fractions) extracted from Green tea (Camellia sinensis), Neem (Azadirachta indica) and Tulsi (Ocimum sanctum). Transcriptional expression of these genes was measured by using RT-PCR and SCION IMAGE analysis software. These polyphenolic extracts were found to have the inherent capacity to inhibit the transcriptional expression of genes having direct involvement in atherogenic process. On the basis of these results, we propose for the first time that HPLC purified polyphenolic fraction IV of Tulsi may have a profound antiatherogenic effect. (Mol Cell Biochem 278: 177–184, 2005)