Viatcheslav A. Soldatenkov

Regulation of Poly(ADP-ribose) Polymerase-1 by DNA Structure-specific Binding

Poly(ADP-ribose) polymerase-1 (PARP-1) is an intracellular sensor of DNA strand breaks and plays a critical role in cellular responses to DNA damage. In normally functioning cells, PARP-1 enzymatic activity has been linked to the alterations in chromatin structure associated with gene expression. However, the molecular determinants for PARP-1 recruitment to specific sites in chromatin in the absence of DNA strand breaks remain obscure. Using gel shift and enzymatic footprinting assays and atomic force microscopy, we show that PARP-1 recognizes distortions in the DNA helical backbone and that it binds to three- and four-way junctions as well as to stably unpaired regions in double-stranded DNA. PARP-1 interactions with non-B DNA structures are functional and lead to its catalytic activation. DNA hairpins, cruciforms, and stably unpaired regions are all effective co-activators of PARP-1 auto-modification and poly(ADP-ribosyl)ation of histone H1 in the absence of free DNA ends. Enzyme kinetic analyses revealed that the structural features of non-B form DNA co-factors are important for PARP-1 catalysis activated by undamaged DNA. K0.5 constants for DNA co-factors, which are structurally different in the degree of base pairing and spatial DNA organization, follow the order: cruciform ≤ hairpin « loop. DNA structure also influenced the reaction rate; when a hairpin was substituted with a stably unpaired region, the maximum reaction velocity decreased almost 2-fold. These data suggest a link between PARP-1 binding to non-B DNA structures in genome and its function in the dynamics of local modulation of chromatin structure in the normal physiology of the cell.

Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor.

Ewings sarcoma (EWS) cells accumulate elevated steady-state levels of poly (ADP-ribose) polymerase (PARP) mRNA and protein. To understand the molecular mechanisms underlying PARP upregulation, we cloned and analysed the 5′-flanking region of the PARP gene from EWS cells. Nucleotide sequence analysis demonstrated no variations in the PARP promoter region in EWS cells. The PARP promoter encompasses multiple binding motifs for the ETS transcription factor. We have also observed that there is a coordinated up-regulation of the expression of both PARP and ETS1, relative to cells of other human tumor types expressing lower levels of PARP. Transient co-expression of ETS1 in EWS cells resulted in a strong enhancement of PARP-promoter activity. The participation of ETS in the regulation of PARP gene expression was further demonstrated in EWS cells stably transfected with Ets1 antisense cDNA constructs. Antisense-mediated down-regulation of endogenous ETS1 resulted in the inhibition of PARP expression in EWS cells, and sensitized these cells to ionizing radiation. These data provide support for ETS regulation of PARP expression levels, and implicate ETS transcription factors in the radiation response of EWS cells.

Progressive Loss of Syk and Abnormal Proliferation in Breast Cancer Cells

The tumor suppressor gene Syk tyrosine kinase is absent or reduced in invasive breast cancer tissues and cell lines; its loss in breast tissues is linked to poor prognosis and metastasis. Also, evidence shows that in vitro Syk is involved in regulating proliferation. Here, we show by in situ hybridization on breast tissue sections that the loss of Syk expression is progressive during tumor development. Strikingly, Syk is already partially lost in normal epithelial tissue adjacent to the cancer lesion. In vivo, cell proliferation (as measured by the proliferative index Ki67) increased from normal to ductal carcinoma in situ to invasive, whereas Syk in situ staining in the same tissues decreased. In vitro, the presence of Syk was associated with reduced cell proliferation in an epidermal growth factor receptor-overexpressing breast cancer cell line, BT549, whereas changes in apoptosis were undetected. Concomitantly, the kinase activity of the proto-oncogene Src was reduced by ∼30%. A 5-fold increase in abnormal mitoses was observed in the Syk-transfected cells compared with vector control. We propose that Syk is involved in the regulation of cell proliferation, possibly by controlling mechanisms of mitosis and cytokinesis via Src signal transduction pathway(s). Because of its progressive and early loss during tumor onset and development, monitoring of Syk loss in breast epithelial cells by noninvasive techniques such as ductal lavage may be a powerful tool for screening purposes.

Poly(ADP‐ribose) polymerase in DNA damage‐response pathway: Implications for radiation oncology

Poly(ADP‐ribose) polymerase (PARP) catalyzes the transfer of successive units of ADP‐ribose moiety from NAD+ covalently to itself and other nuclear acceptor proteins. PARP is a zinc finger‐containing protein, allowing the enzyme to bind to either double‐ or single‐strand DNA breaks without any apparent sequence preference. The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification. Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with the breaking and rejoining of DNA strands, and plays a pivotal role in DNA damage repair. Recent advances in apoptosis research identified PARP as one of the intracellular “death substrates” and demonstrated the involvement of polymerase in the execution of programmed cell death. This review summarizes the biological effects of PARP function that may have a potential for targeted sensitization of tumor cells to genotoxic agents and radiotherapy. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 59–67 (2000).

Nonviral Nanoscale-Based Delivery of Antisense Oligonucleotides Targeted to Hypoxia-Inducible Factor 1α Enhances the Efficacy of Chemotherapy in Drug-Resistant Tumor

Purpose: To enhance the efficacy of cancer treatment, we propose a complex approach: simultaneous delivery to the tumor of a chemotherapeutic agent and a suppressor of hypoxia-inducible factor 1α (HIF1A). Experimental Design: The novel complex liposomal drug delivery system was developed and evaluated in vitro and in vivo on nude mice bearing xenografts of multidrug-resistant human ovarian carcinoma. The proposed novel complex drug delivery system consists of liposomes as a nanocarrier, a traditional anticancer drug (doxorubicin) as a cell death inducer, and antisense oligonucleotides targeted to HIF1A mRNA as a suppressor of cellular resistance and angiogenesis. Results: The system effectively delivers active ingredients into tumor cells, multiplies the cell death signal initiated by doxorubicin, and inhibits cellular defensive mechanisms and angiogenesis by down-regulating BCL2, HSP90, and vascular endothelial growth factor proteins. This, in turn, activates caspases, promotes apoptosis, necrosis, and tumor shrinkage. The proposed novel complex multipronged approach enhances the efficiency of chemotherapy. Conclusions: The proposed combination therapy prevents the development of resistance in cancer cells, and thus, increases the efficacy of chemotherapy to an extent that cannot be achieved by individual components applied separately. It could form the foundation for a novel type of cancer therapy based on simultaneous delivery of an anticancer drug and a suppressor of HIF1A.

End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes

Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine–homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA–DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.

Combined transcriptional and translational targeting of EWS/FLI-1 in Ewing's sarcoma

Purpose: To show the efficacy of targeting EWS/FLI-1 expression with a combination of specific antisense oligonucleotides and rapamycin for the control of Ewings sarcoma (EWS) cell proliferation in vitro and the treatment of mouse tumor xenografts in vivo. Experimental Design: EWS cells were simultaneously exposed to EWS/FLI-1–specific antisense oligonucleotides and rapamycin for various time periods. After treatment, the following end points were monitored and evaluated: expression levels of the EWS/FLI-1 protein, cell proliferation, cell cycle distribution, apoptotic cell death, caspase activation, and tumor growth in EWS xenografts implanted in nude mice. Results: Simultaneous exposure of EWS cells in culture to an EWS/FLI-1–targeted suppression therapy using specific antisense oligonucleotides and rapamycin resulted in the activation of a caspase-dependent apoptotic process that involved the restoration of the transforming growth factor-β–induced proapoptotic pathway. In vivo, individual administration of either antisense oligonucleotides or rapamycin significantly delayed tumor development, and the combined treatment with antisense oligonucleotides and rapamycin caused a considerably stronger inhibition of tumor growth. Conclusions: Concurrent administration of EWS/FLI-1 antisense oligonucleotides and rapamycin efficiently induced the apoptotic death of EWS cells in culture through a process involving transforming growth factor-β. In vivo experiments conclusively showed that the combined treatment with antisense oligonucleotides and rapamycin caused a significant inhibition of tumor growth in mice. These results provide proof of principle for further exploration of the potential of this combined therapeutic modality as a novel strategy for the treatment of tumors of the Ewings sarcoma family.

Protein changes associated with ionizing radiation‐induced apoptosis in human prostate epithelial tumor cells

Ionizing radiation (IR) is an important component in the therapy of localized prostate cancer. Identification of protein alterations during IR‐induced apoptosis prostate cancer cells is an important step toward understanding the new metabolic status of the dying cell. In the present study, we report changes in protein profile that define the execution phase of the apoptotic response in the in vitro model of tumorigenic radiation‐transformed SV40‐immortalized human prostate epithelial cells (267B1‐XR), induced to undergo programmed cell death by IR. We employed an approach that involves use of analytical two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) coupled with Western blotting with specific antisera. Our results point out that apoptotic cells experience significant reduction in the levels of the intermediate filament proteins, keratins‐18, 19, vimentin and the associated 14‐3‐3 adapter proteins. At the same time, molecular chaperones such as glucose‐regulated protein 94, calreticulin, calnexin, and protein disulfide isomerase exhibit marked accumulation in these dying cells. The present data indicate that apoptosis‐associated processes in prostate epithelial cells include solubilization of the rigid intermediate filament network by specific proteolysis as well as increased levels of endoplasmic reticulum (ER) proteins with chaperone functions.

Sodium butyrate induces apoptosis and accumulation of ubiquitinated proteins in human breast carcinoma cells

To investigate the possible relationship between apoptosis and the ubiquitin pathway we examined the patterns of ubiquitinated proteins in the human breast carcinoma MCF-7 cell line following induction of apoptotic death by sodium butyrate. Apoptosis in these cells was associated with internucleosomal DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase. By dual in situ antiubiquitin immunofluorescence and chromatin DNA staining, we demonstrated that ubiquitin fluorescence was increased specifically in cells that underwent sodium butyrate-mediated apoptosis. The extent of ubiquitin incorporation into protein conjugates was examined in both adherent (not yet apoptotic) and floating (apoptotic) cell populations. We found that apoptotic cells exhibited enhanced intensity of ubiquitin-immunoreactive conjugates, whereas adherent cells did not. In addition, two-dimensional immunoblot analysis of proteins from apoptotic cells identified a set of isomeric ubiquitinated conjugates located at a pI range of 4.2–4.6 and a Mr approximately of 30 kDa. These data indicate that the ubiquitin pathway may play a role in the sodium butyrate-induced apoptotic program in breast carcinoma cells.

Isolation and characterization of SpTRK, a gene from schizosaccharomyces pombe predicted to encode a K+ transporter protein

A novel gene, SpTRK, has been isolated from DNA of the fission yeast Schizosaccharomyces pombe (Sp) by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a sequence fully conserved between the potassium transporter genes TRK1 and TRK2 of Saccharomyces cerevisiae (Sc). SpTRK is a single-copy gene located on Sp chromosome I. Nucleotide sequence analysis of the cloned gene identified an open reading frame (ORF) with coding capacity for a protein of 833 amino acids (aa). The predicted SpTRK aa sequence showed a high level of conservation relative to the potassium transporters of Sc and Saccharomyces uvarum (Su), particularly within their transmembrane (TM) domains and in aa required for their ion transport functions. A single SpTRK transcript of about 2.7 kb is expressed at high levels in exponentially growing Sp cells, but it is downregulated in cells from stationary cultures.