Pharmacological inactivation of oncogenes is being investigated as a possible therapeutic strategy for cancer. One potential drawback is that cessation of such therapy may allow reactivation of the oncogene and tumor regrowth. We used a conditional transgenic mouse model for MYC-induced tumorigenesis to demonstrate that brief inactivation of MYC results in the sustained regression of tumors and the differentiation of osteogenic sarcoma cells into mature osteocytes. Subsequent reactivation of MYC did not restore the cells' malignant properties but instead induced apoptosis. Thus, brief MYC inactivation appears to cause epigenetic changes in tumor cells that render them insensitive to MYC-induced tumorigenesis. These results raise the possibility that transient inactivation of MYC may be an effective therapy for certain cancers.
Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.Agrobacterium tumefaciens causes crown gall tumors on many dicotyledonous plant species when the bacteria infect wounded tissue (18). The tumor-inducing (Ti) plasmid carries genes essential for tumorigenesis (63,72). The transferred DNA (T-DNA) portion of the Ti plasmid enters plant cells and integrates into nuclear DNA (8,9,74), in which expression of certain T-DNA genes leads to tumorous growth (2,5,32,46,59). Virulence (vir) genes necessary for T-DNA transmission (transfer and integration) lie elsewhere on the Ti plasmid (24, 55), and wounded plants release phenolic compounds that induce vir expression (54).Border sequences define the T-DNA ends (42,43,48,65). T-DNA transfer begins when the VirD2 endonuclease nicks the right-hand border sequence (66, 78) and attaches to the 5Ј end of the nicked DNA strand (22,27,30,67,79). Strand displacement continues leftward (to the nicked left-hand border sequence), generating linear VirD2-bound T strands (3,33,56), which the bacteria appear to export into plant cells (58, 80). VirE2 single-stranded DNA-binding (SSB) protein (11-13, 16, 26, 47) may also accompany T strands into plant cells. Both VirE2 and VirD2 contain plant nuclear localization signals and probably play important roles inside infected plant cells (14,28,31,51,62).Export of DNA and proteins from A. tumefaciens into plant cells depends on membrane-associated proteins encoded by the virB operon (10,23,34,49,50,60,61,(68)(69)(70)(71)) (for reviews, see references 29 and 81) and the virD4 gene (33, 38). The VirB proteins are similar to pertussis toxin liberation (Ptl) proteins of Bordetella pertussis that mediate export of pertussis toxin (15,73) and to proteins that facilitate conjugal transfer of IncP␣ plasmid RP4 (Trb proteins) (35) and IncN plasmid pKM101 (Tra proteins) (44). VirD4 protein has similarity to TraG, another protein required for conjugal transfer of RP4 (35). Thus, Agrobacterium proteins essential for tumorigenesis appear to facilitate export of both proteins and DNA into plant cells by using pathways that operate in other bacteria.VirE2 plays an important role inside plant cells but not inside Agrobacterium cells. T strands (56), which are widely accepted as intermediates of T-DNA transfer (45,75,81), accumulate to wild-type levels in virE2 mutants (57, 64), showing that VirE2 does not stabilize T strands inside bacterial cells. In addition, virE2 mutants can transfer T strands into plant cells (80), albeit with unknown efficiency, proving that VirE2 is not essential for export of T strands. Transgenic tobacco plants that produce VirE2 protein are susceptible to transformation by a virE mutant (14), indicating that VirE2 is necessary inside plant cells.
We previously identified HSP70 and HSC70 in complex with NS5A in a proteomic screen. Here, coimmunoprecipitation studies confirmed NS5A/HSC70 complex formation during infection, and immunofluorescence studies showed NS5A and HSC70 to colocalize. Unlike HSP70, HSC70 knockdown did not decrease viral protein levels. Rather, intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains directly bind NS5A whereas only the HSP70 nucleotide binding domain does. Knockdown of both HSC70 and HSP70 demonstrated an additive reduction in virus production. This data suggests that HSC70 and HSP70 play discrete roles in the viral life cycle. Investigation of these different functions may facilitate developing of novel strategies that target host proteins to treat HCV infection.
The human molecular chaperones heat shock protein 70 (Hsp70) and heat shock cognate protein 70 (Hsc70) bind to the hepatitis C viral nonstructural protein 5A (NS5A) and regulate its activity. Specifically, Hsp70 is involved in NS5A-augmented internal ribosomal entry site (IRES)-mediated translation of the viral genome, whilst Hsc70 appears to be primarily important for intracellular infectious virion assembly. To better understand the importance of these two chaperones in the viral life cycle, infected human cells were treated with allosteric Hsp70/Hsc70 inhibitors (AHIs). Treatment with AHIs significantly reduced the production of intracellular virus at concentrations that were non-toxic to human hepatoma Huh7.5 cells. The supernatant of treated cultures was then used to infect naïve cells, revealing that AHIs also lowered levels of secreted virus. In contrast to their effects on virion assembly, AHIs did not impact the stability of NS5A or viral protein translation in IRES assays. These results suggest that Hsc70 plays a particularly important and sensitive role in virion assembly. Indeed, it was found that combination of AHIs with a peptide-based viral translation inhibitor exhibited additive antiviral activity. Together these results suggest that the host Hsc70 is a new antiviral target and that its inhibitors utilise a new mechanism of action.
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