Jochen Heinrich

A Short Hairpin DNA Analogous to miR-125b Inhibits C-Raf Expression, Proliferation, and Survival of Breast Cancer Cells

The noncoding RNA miR-125b has been described to reduce ErbB2 protein expression as well as proliferation and migration of cancer cell lines. As additional target of miR-125b, we identified the c-raf-1 mRNA by sequence analysis. We designed a short hairpin-looped oligodeoxynucleotide (ODN) targeted to the same 3′ untranslated region of c-raf-1 mRNA as miR-125b. The fully complementary ODN antisense strand is linked to a second strand constituting a partially double-stranded structure of the ODN. Transfection of the c-raf-1–specific ODN (ODN-Raf) in a breast cancer cell line reduced the protein levels of C-Raf, ErbB2, and their downstream effector cyclin D1 similar to miR-125b. MiR-125b as well as ODN-Raf showed no effect on the c-raf-1 mRNA level in contrast to small interfering RNA. Unlike miR-125b, ODN-Raf induced a cytopathic effect. This may be explained by the structural properties of ODN-Raf, which can form G-tetrads. Thus, the short hairpin-looped ODN-Raf, targeting the same region of c-raf-1 as miR-125b, is a multifunctional molecule reducing the expression of oncoproteins and stimulating cell death. Both features may be useful to interfere with tumor growth. (Mol Cancer Res 2009;7(10):1635–44)

RNase H-mediated retrovirus destruction in vivo triggered by oligodeoxynucleotides

The HIV-1 RNase H can be prematurely activated by oligodeoxynucleotides targeting the highly conserved polypurine tract required for second strand DNA synthesis. This inhibits retroviral replication in cell-free HIV particles and newly infected cells. Here we extend these studies to an in vivo model of retroviral replication. Mice that are chronically infected with the spleen focus-forming virus and treated with oligodeoxynucleotides that target the polypurine tract, exhibit either transient or long-term reductions in plasma virus titer, depending on the therapeutic regimen. Treatment prior to, during or shortly after infection can delay disease progression, increase survival rates and prevent viral infection. This strategy destroys viral RNA template in virus particles in serum as well as early retroviral replication intermediates in infected cells. As it targets events common to the replication cycle of all retroviruses, this approach may be broadly applicable to retroviruses of medical and agricultural importance.

Silencing of HIV by hairpin‐loop‐structured DNA oligonucleotide

We describe inhibition of HIV replication by a partially double‐stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple‐helix formation. Upon treatment of HIV‐infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN‐mediated effect was highly sequence‐specific. The ODN against HIV‐IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV‐BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.

Regulation of c-Src by binding to the PDZ domain of AF-6

c‐Src is a tightly regulated non‐receptor tyrosine kinase. We describe the C‐terminus of c‐Src as a ligand for a PDZ (postsynaptic density 95, PSD‐95; discs large, Dlg; zonula occludens‐1, ZO‐1) domain. The C‐terminal residue Leu of c‐Src is essential for binding to a PDZ domain. Mutation of this residue does not affect the intrinsic kinase activity in vitro, but interferes with c‐Src regulation in cells. As a candidate PDZ protein, we analysed AF‐6, a junctional adhesion protein. The AF‐6 PDZ domain restricts the number of c‐Src substrates, whereas knockdown of AF‐6 has the opposite effect. Binding of c‐Src to the AF‐6 PDZ domain interferes with phosphorylation of c‐Src at Tyr527 by the C‐terminal kinase, and reduces c‐Src autophosphorylation at Tyr416, resulting in a moderately activated c‐Src kinase. Unphosphorylated Tyr527 allows binding of c‐Src to AF‐6. This can be overcome by overexpression of CSK or strong activation of c‐Src. c‐Src is recruited by AF‐6 to cell–cell contact sites, suggesting that c‐Src is regulated by a PDZ protein in special cellular locations. We identified a novel type of c‐Src regulation by interaction with a PDZ protein.

A WD-FYVE protein binds to the kinases Akt and PKCζ/λ

WD (tryptophan-aspartic acid dipeptide)-repeat proteins play a central role in signal transduction cascades by co-ordinating the interaction of key signalling molecules. We identified a novel propeller-FYVE [domain identified in Fab1p, YOTB, Vac1p and EEA1 (early endosome antigen 1)] protein, ProF, which is expressed in various cell lines and tissues and consists of seven WD-repeats and a FYVE domain. WD-repeat proteins offer a platform for protein-protein interactions by folding into a seven-bladed propeller-like structure, while the FYVE domain binds to phosphatidylinositol 3-phosphate present mainly on intracellular membranes. The ProF protein partially co-localizes with EEA1 on vesicular structures and binds to the protein kinases Akt and PKCzeta/lambda (protein kinase Czeta/lambda) via its WD-repeat propeller. ProF interacts more strongly with the kinases after hormonal stimulation. Endogenously expressed ProF and the two kinases interact in brain and in the preadipocyte cell line 3T3-L1, suggesting a role in secretory vesicular processes. In summary, we describe a new binding partner for kinases, located on vesicular structures in specialized cells, which may play a role for the spatial organization of signalling cascades.

Oligonucleotide-mediated retroviral Rnase H activation leads to reduced Hiv-1 titer in patient-derived plasma

Background:The retroviral RNase H is essential for viral replication. This component has not yet been extensively studied for antiviral therapy. It can be activated by an oligodeoxynucleotide (ODN) resulting in self-destruction of the virions. Objective:To examine antiviral potential of ODN in clinical samples using plasma of HIV-1-infected patients. Design:Plasma of 19 HIV-1-infected patients from Zurich and 10 HIV-1 isolates from Africa and drug-resistant strains were processed for ex-vivo treatment. Methods:Cell-free virions were treated with ODN in the plasma and HIV RNA was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, infectivity of the treated virions was tested on primary human peripheral blood mononuclear cells. Results:Cell-free virions in plasma contained significantly less intact HIV RNA upon treatment with ODN (P = 0.0004), and their infectivity was decreased 52-fold (P = 0.0004). In 39% of the Zurich samples, infectivity was reduced more than 10-fold, in 33% more than 100-fold, and in 28% more than 1000-fold. Also, the isolates from Africa exhibited a 63-fold reduction in infectivity (P = 0.0069) with 80% of the isolates responding more than 10-fold, 40% more than 100-fold, and 10% more than 1000-fold. Conclusion:Significant reduction of plasma HIV RNA levels and infectivity of treated virions was achieved on the basis of induced self-destruction of HIV observed with clinical samples. Reduction of viral load ex vivo was designed as model for potential effects in vivo. Premature activation rather than inhibition of a viral enzyme could be a model strategy for future antiretroviral control.

Inhibition of influenza A virus replication by short double-stranded oligodeoxynucleotides.

Influenza A virus causes prevalent respiratory tract infections in humans. Small interfering RNA (siRNA) and antisense oligonucleotides (asODNs) have been used previously for silencing the RNA genome of influenza virus. Here, we explored the use of partially double-stranded oligodeoxynucleotides (dsODNs) to suppress the production of influenza A virus in cell cultures and animal models. We were able to inhibit influenza A virus replication in cultured human lung cells as well as in the lungs of infected C57BL/6 mice by treatment with dsODN 3-h post-infection. In about 20% of the cases (15/77) the titer was reduced by 10- to 100-fold and in 10% up to 1,000-fold. The antiviral effects of dsODNs were dose-dependent, sequence-dependent and comparable to those of its antisense and siRNA analogues. Thus, dsODNs may be developed as an additional class of nucleic acids for the inhibition of influenza virus replication.

The PDZ protein MPP2 interacts with c-Src in epithelial cells.

c-Src is a non-receptor tyrosine kinase involved in regulating cell proliferation, cell migration and cell invasion and is tightly controlled by reversible phosphorylation on regulatory sites and through protein-protein interactions. The interaction of c-Src with PDZ proteins was recently identified as novel mechanism to restrict c-Src function. The objective of this study was to identify and characterise PDZ proteins that interact with c-Src to control its activity. By PDZ domain array screen, we identified the interaction of c-Src with the PDZ protein Membrane Protein Palmitoylated 2 (MPP2), a member of the Membrane-Associated Guanylate Kinase (MAGUK) family, to which also the Discs large (Dlg) tumour suppressor protein belongs. The function of MPP2 has not been established and the functional significance of the MPP2 c-Src interaction is not known. We found that in non-transformed breast epithelial MCF-10A cells, endogenous MPP2 associated with the cytoskeleton in filamentous structures, which partially co-localised with microtubules and c-Src. MPP2 and c-Src interacted in cells, where c-Src kinase activity promoted increased interaction of c-Src with MPP2. We furthermore found that MPP2 was able to negatively regulate c-Src kinase activity in cells, suggesting that the functional significance of the MPP2-c-Src interaction is to restrict Src activity. Consequently, the c-Src-dependent disorganisation of the cortical actin cytoskeleton of epithelial cells expressing c-Src was suppressed by MPP2. In conclusion we demonstrate here that MPP2 interacts with c-Src in cells to control c-Src activity and morphological function.

Induction of putative tumor-suppressing genes in Rat-1 fibroblasts by oncogenic Raf-1 as evidenced by robot-assisted complex hybridization

The growth factor receptor-dependent protein kinase Raf-1 is activated by GTP-bound Ras, thereby activating the mitogen-activated protein kinase pathway. To study the role of Raf in transformation we transduced Rat-1 cells with a tetracycline-regulatable retroviral vector encoding the constitutively active oncogenic C-terminal fragment of the human Raf-1 protein. Using subtractive hybridization of mRNAs from induced and noninduced cells and robot-assisted screening by complex hybridization, Raf-induced genes with various different characteristics of induction were investigated. Among the strongly induced genes were those involved in carcinogenesis such as metalloproteinases 3, 10 and 13, cathepsin L, ornithine decarboxylase, and putative tumor-suppressing genes such as monocyte chemoattracting protein 1, interferon-induced protein 10, a recently identified 2′-5′ oligoadenylate synthetase-like protein, and plasminogen activator inhibitor type 2. Other components of the plasminogen activator system were not induced. Plasminogen activator inhibitor type 2 is a downregulator of the proteolytic cascade consisting of various metalloproteinases, some of which are induced by a carboxy-terminal Raf mutant (RafCT). In conclusion, RafCT induces factors which act in a conflicting manner in respect of carcinogenesis, especially within the proteolytic system of the extracellular matrix.

Antitumor activity of small double-stranded oligodeoxynucleotides targeting telomerase RNA in malignant melanoma cells.

Human telomerase RNA (hTR) is an intrinsic component of telomerase enzyme. Small interfering RNAs (siRNAs) and single-stranded antisense oligonucleotides have been used previously for silencing of the hTR. The objective of this study was to investigate the effect of partially double-stranded oligodeoxynucleotides (ODNs), in vitro and in vivo in comparison to single-stranded antisense ODNs and siRNAs. ODNs were designed on the basis of structural properties of an ODN from previous studies on HIV, to target the hTR in the human cervical carcinoma HeLa cell line and mouse telomerase RNA (mTR) in the murine metastatic melanoma B16-F10 cell line, respectively. Our results indicate that ODNs were able to inhibit the hTR by 68% and the mTR by 81% in the respective cell lines. This correlated with ODN-mediated rapid inhibition of cell proliferation and induction of apoptosis excluding slow effects on telomerase function. The inhibition of the hTR was decreased by knock-down of the cellular RNases H suggesting their contribution. Furthermore, we showed a reduction in numbers of metastases by 70% after intravenous administration of ODN-transfected B16-F10 cells in C57BL/6 mice. Our study demonstrates the potential utility of these hairpin-loop-structured ODNs as a different group of nucleic acids for telomerase-based antiproliferative strategies.