Ashraful Islam

High expression of Survivin, mapped to 17q25, is significantly associated with poor prognostic factors and promotes cell survival in human neuroblastoma

Survivin (SVV) is a family member of inhibitor of apoptosis proteins (IAPs) and its expression is cell cycle regulated. The gene is mapped to chromosome 17q25, the region of which is frequently gained in advanced stages of neuroblastoma (NBL). However, the role of SVV in NBL is poorly understood. Here we studied the clinical and biological role of SVV in NBL. A 1.9 kb SVV transcript was expressed in all of 9 NBL cell lines at higher levels than those in adult cancer cell lines. In 34 primary NBLs, high levels of SVV expression was significantly associated with age greater than 12 months (two sample t-test: P=0.0003), advanced stages (P=0.0136), sporadic tumors (P=0.0027) and low levels of TrkA expression (P=0.0030). In NBL cell lines, SVV mRNA expression was dramatically down-regulated in CHP134 and IMR32 cells undergoing apoptosis after treatment with all-trans retinoic acid (RA) or serum deprivation. It was only moderately decreased in cells (SH-SY5Y and CHP901) undergoing RA-induced differentiation. On the other hand, in proliferating NBL cells or RA-treated SK-N-AS line which is refractory to RA, the SVV mRNA remained at steady state levels or rather up-regulated. Furthermore, transfection of SVV into CHP134 cells induced remarkable inhibition of the RA-induced apoptosis. Collectively, our results suggest that high expression of SVV is a strong prognostic indicator for the advanced stage neuroblastomas, and that it could be one of the candidate genes for the 17q gain.

c-Myc and Cancer Metabolism

The processes of cellular growth regulation and cellular metabolism are closely interrelated. The c-Myc oncogene is a “master regulator” which controls many aspects of both of these processes. The metabolic changes which occur in transformed cells, many of which are driven by c-Myc overexpression, are necessary to support the increased need for nucleic acids, proteins, and lipids necessary for rapid cellular proliferation. At the same time, c-Myc overexpression results in coordinated changes in level of expression of gene families which result in increased cellular proliferation. This interesting duality of c-Myc effects places it in the mainstream of transformational changes and gives it a very important role in regulating the “transformed phenotype.” The effects induced by c-Myc can occur either as a “primary oncogene” which is activated by amplification or translocation or as a downstream effect of other activated oncogenes. In either case, it appears that c-Myc plays a central role in sustaining the changes which occur with transformation. Although efforts to use c-Myc as a therapeutic target have been quite frustrating, it appears that this may change in the next few years. Clin Cancer Res; 18(20); 5546–53. ©2012 AACR.

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3½ years (HDNB1/UFD2, KIAA0591F/KIF1B-β, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-β, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes.

Genomic c-Myc Quadruplex DNA Selectively Kills Leukemia

c-Myc, a key regulator of cell cycle and proliferation, is commonly overexpressed in leukemia and associated with poor prognosis. Conventional antisense oligonucleotides targeting c-myc may attenuate leukemic cell growth, however, are poorly taken into cells, rapidly degraded, and have unwanted effects on normal cells. The c-myc promoter contains a guanine-rich sequence (PU27) capable of forming quadruplex (four-stranded) DNA, which may negatively regulate c-myc transcription. However, its biological significance is unknown. We show that treatment of leukemia with an oligonucleotide encoding the genomic PU27 sequence induces cell-cycle arrest and death by oncotic necrosis due to PU27-mediated suppression of c-myc mRNA/protein expression. Furthermore, PU27 is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on normal cells. Suppression of c-myc expression by PU27 caused significant DNA damage, cell and mitochondrial swelling, and membrane permeability characteristic of oncotic necrosis. Induction of oncosis caused mitochondrial dysfunction, depletion of cellular ATP levels, and enhanced oxidative stress. This novel antileukemic strategy addresses current concerns of oligonucleotide therapeutics including problems with uptake, stability, and unintentional effects on normal cells and is the first report of selective cancer cell killing by a genomic DNA sequence. Mol Cancer Ther; 11(1); 66–76. ©2011 AACR.

Identification of the homozygously deleted region at chromosome 1p36.2 in human neuroblastoma

BACKGROUND We have identified for the first time a homozygously deleted region within the smallest region of overlap at 1p36.2-3 in two neuroblastoma cell lines. PROCEDURE The 800-kb PAC contig covering the entire homozygously deleted region was made and sequenced. To date, approximately 70% of sequencing has been accomplished, and the estimated length of the deleted region was 500 kb. RESULTS Currently, we have found six genes within the region, which include three known genes as well as three other genes that have been reported during processing of our present project for the last 3(1/2) years. We report here the results of expression and mutation analyses of those genes. CONCLUSIONS Full sequencing for the region of homozygous deletion as well as further analyses of the genes mapped within the region may reveal whether or not there is a neuroblastoma suppressor gene as proposed by the Knudsons two-hit hypothesis.

Plagiochiline A Inhibits Cytokinetic Abscission and Induces Cell Death

We previously reported on the isolation and biological activities of plagiochiline A (1), a 2,3-secoaromadendrane-type sesquiterpenoid from the Peruvian medicinal plant, Plagiochila disticha. This compound was found to have antiproliferative effects on a variety of solid tumor cell lines, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from different plant species), but there are no prior reports regarding the mechanism for this bioactivity. Here, we have evaluated the effects of plagiochiline A on cell cycle progression in DU145 prostate cancer cells. A cell cycle analysis indicated that plagiochiline A caused a significant increase in the percentage of cells in the G2/M phase when compared with control cells. When cells were stained and observed by fluorescence microscopy to examine progress through the mitotic phase, we found a significant increase in the proportion of cells with features of late cytokinesis (cells connected by intercellular bridges) in the plagiochiline A-treated samples. These results suggest that plagiochiline A inhibits cell division by preventing completion of cytokinesis, particularly at the final abscission stage. We also determined that plagiochiline A reduces DU145 cell survival in clonogenic assays and that it induces substantial cell death in these cells.

Abstract 743: Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Throughout the genome as well as telomere region, there is presence of DNA sequences which forms quadruplex. Here we show Pu-27 triggers the damage of cells by interfering telomeric ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks (DSB). We found that Pu-27 frequently prompted chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal break (27%) by karyotyping of U937 metaphase cells and was associated with induction of DNA damage response sensor γ-H2AX. Analysis of DNA damage response regulators by RT-PCR array of Pu-27 treated U937 cells revealed downregulation of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), upstream kinase ATM, DNA damage response mediators (RAD17, RAD50 and 53BP1), and cell cycle arrest molecules (CHK1 and CHK2). Interestingly, there were no changes or low expression of DNA repair molecules (H2AX and BRCA1) and telomere maintenance gene TERT respectively. αB-U937 cell, where basic-domain of shelterin protein TRF2 was deleted became relatively resistance to Pu-27 and showed no changes of constitutively active form of γ-H2AX. RT-PCR array of Pu-27 treated αB-U937 cell appeared to be no changes of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), DNA damage response mediators (RAD17, RAD50 and 53BP1); down regulation of upstream kinase ATM; up regulation of cell cycle arrest molecules (SMC1, CHK1 and CHK2), DNA repair molecules (H2AX and BRCA1), and telomere maintenance gene TERT. Shelterin protein, TRF1 co-associated with γH2AX by inducing TIFs (Telomere-dysfunction Induced Foci) when U937 cell was treated with Pu-27. Sk-Lu-1, an Alt (Alternating Lengthening to Telomere) cell which does not use TERT for their growth and propagation is relatively resistance to Pu-27. ATM preserves the telomeric homeostasis by maintaining the integrity of ‘shelterin’ protein. To substantiate the specificity of Pu-27 to shelterin protein complex, we found ATM-deficient cell was more sensitive to Pu-27 than ATM-proficient cell. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these results suggest that Pu-27-treated sensitivity at least in part due to compromising telomeric shelterin complex and perhaps stabilizing telomeric quadruplex and not by directly inhibiting telomerase enzyme. U937 cell with chromosomal aberration and unstable telomere progress through cell cycle without arrest and repair leading to cell death whereas αB-U937 cells is continuously repaired by arresting and induction of repair machinery. Continuous exposure of Pu-27 results in refractory to repair of DNA damage leading to uncompensated genomic stability leading to cell death. Citation Format: Md Ashraful Islam, Shelia D. Thomas, Kara J. Sedoris, Donald M. Miller. Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2013-743 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Using Human Observations to Gain Biologic Insights and New Treatments; Discovery of a Quadruplex-Forming DNA Aptamer as an Anticancer Agent

The discovery and characterization of the primo vascular system represents a major new development in our understanding of human biology. The current understanding of this novel system is based on careful observations over an extended period of time. We have, in an analogous fashion, used clinical observations in patients with malignant diseases to develop new insights which have led to novel types of therapies. Beginning with the observation, in 1979, of in vivo differentiation of chronic myelogenous leukemia cells in a patient treated with the RNA synthesis inhibitor, mithramycin, we have characterized the ability of DNA binding compounds to inhibit gene expression. As a result of this work, we have discovered a quadruplex-forming, DNA aptamer, AS1411, which has significant anticancer activity with very little toxicity. AS1411 is currently in Phase IIb studies. We have recently shown that genomic quadruplex-forming sequences are selectively toxic to transformed cells and have therapeutic potential.

Abstract 2123: ATM mediated homeostasis of ‘shelterin’ at telomere is compromised by G-quadruplex leading to DNA damage

Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27 inhibits growth of malignant cells. Telomere integrity is maintained by the ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. ATM has been shown to play a role in the maintenance of telomere integrity. U937, histiocytic lymphoma cells were sensitive to Pu-27 as evident by MTT and colony formation assay and frequently showed chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal breaks (27%). RT-PCR array of Pu-27 treated U937 cells revealed downregulation of ‘shelterin’ proteins TRF2, TRF1, POT1 and TIN2; induction of DNA damage response sensor γ-H2AX; downregulation of upstream kinase ATM and other downstream DNA damage mediator RAD17, RAD50, BRCA1, 53BP1, CHK1, CHK2. To understand the specific role of ‘shelterin’ protein complex, we stably transfected U937 cells with a vector expressing a dominant negative mutant of a shelterin protein TRF2 (dn-U937). These dn-UP37 cells became relatively resistant when treated with Pu-27 and showed no changes of basal level expression of ‘shelterin’ protein complex but induction of γ-H2AX and DNA repair inducing molecules BRCA1 and CHK1. This suggests that intact TRF2 is required for Pu-27 mediated U937 cellular sensitivity and it may acted by altering the telomere-‘shelterin’ complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. To elucidate the specific role of ATM, we have shown by MTT and colony formation assay that ATM-deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM-proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and no alteration of basal level of ‘shelterin’ protein complex in Pu-27 treated ATM deficient cells (as determined by RT-PCR array). A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these data suggest that downregulation of ATM destabilizes telomere-shelterin homeostasis at telomere ends in response to Pu-27 of U937 cells. Continuous exposure of G-quadruplex results in refractory DNA damage repair leading to uncompensated genomic stability and cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2123. doi:1538-7445.AM2012-2123

Abstract 5193: The importance of primo vascular system (PVS) on cancer metastasis and therapeutic intervention: A new dimension of cancer stem cell

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The ability of cancer cells especially immature and multi-potent cancer stem cells to escape from conventional therapeutic intervention has been long investigated. The exact mechanisms and safe harboring of this population of cells have yet to be identified. The primo vascular system (PVS) which is composed of new types of micro-conduits named primo-vessels (PV) and primo-nodes (PN) has recently emerged as a third component of circulatory system. In this study, we have shown the existence of PVS and their networks in murine xenograft of human histiocytic lymphoma cells (U937) using trypan blue. These PVs are thread-like, elastic, multi-lumen structure and appears to radiate from PN, perpendicular of the conventional venuoles-arteriols and reside in close proximity to the tumor. PNs are about 500-600 μM membranous sac-like structures, containing numerous tiny cells evident by DAPI staining. Isolation of RNA from PVS signifies the presence of intact transcriptional machinery in the cells. Hematoxylin and Eosin (H&E) staining of PVS shows the presence of loose and abortive structure lined by fibroblast but filled with lightly dense fibers, cells, lacuna and nerve-like structures. To further characterize the origin and type of cells within the PVS, immunostaining with antibodies for CD68, CD45 and lysozyme has revealed the cells are same immunophenotype as of U937. RT-PCR arrays demonstrates a hundred to thousand fold up regulation of human cancer stem cell specific transcription factors most notably KLF4, an upstream regulator of NANOG which maintain the pluripotent and undifferentiated state of stem cells. These results suggest that PVS close to the tumor could be the safe haven for a selective population of cancer stem cell. Further understanding of the biological properties and responsiveness to therapy of these cells will shed light on new dimensions of anti-cancer interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5193. doi:1538-7445.AM2012-5193