Identification of a Novel Gammaretrovirus in Prostate Tumors of Patients Homozygous for R462Q RNASEL Variant

Ribonuclease L (RNase L) is an important effector of the innate antiviral response. Mutations or variants that impair function of RNase L, particularly R462Q, have been proposed as susceptibility factors for prostate cancer. Given the role of this gene in viral defense, we sought to explore the possibility that a viral infection might contribute to prostate cancer in individuals harboring the R462Q variant. A viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses identified the presence of gammaretroviral sequences in cDNA samples from seven of 11 R462Q-homozygous (QQ) cases, and in one of eight heterozygous (RQ) and homozygous wild-type (RR) cases. An expanded survey of 86 tumors by specific RT-PCR detected the virus in eight of 20 QQ cases (40%), compared with only one sample (1.5%) among 66 RQ and RR cases. The full-length viral genome was cloned and sequenced independently from three positive QQ cases. The virus, named XMRV, is closely related to xenotropic murine leukemia viruses (MuLVs), but its sequence is clearly distinct from all known members of this group. Comparison of gag and pol sequences from different tumor isolates suggested infection with the same virus in all cases, yet sequence variation was consistent with the infections being independently acquired. Analysis of prostate tissues from XMRV-positive cases by in situ hybridization and immunohistochemistry showed that XMRV nucleic acid and protein can be detected in about 1% of stromal cells, predominantly fibroblasts and hematopoietic elements in regions adjacent to the carcinoma. These data provide to our knowledge the first demonstration that xenotropic MuLV-related viruses can produce an authentic human infection, and strongly implicate RNase L activity in the prevention or clearance of infection in vivo. These findings also raise questions about the possible relationship between exogenous infection and cancer development in genetically susceptible individuals

Biological Stoichiometry in Human Cancer

A growing tumor in the body can be considered a complex ecological and evolutionary system. A new eco-evolutionary hypothesis (the "Growth Rate Hypothesis", GRH) proposes that tumors have elevated phosphorus (P) demands due to increased allocation to P-rich nucleic acids, especially ribosomal RNA, to meet the protein synthesis demands of accelerated proliferation.We determined the elemental (C, N, P) and nucleic acid contents of paired malignant and normal tissues from colon, lung, liver, or kidney for 121 patients. Consistent with the GRH, lung and colon tumors were significantly higher (by approximately two-fold) in P content (fraction of dry weight) and RNA content and lower in nitrogen (N):P ratio than paired normal tissue, and P in RNA contributed a significantly larger fraction of total biomass P in malignant relative to normal tissues. Furthermore, patient-specific differences for %P between malignant and normal tissues were positively correlated with such differences for %RNA, both for the overall data and within three of the four organ sites. However, significant differences in %P and %RNA between malignant and normal tissues were not seen in liver and kidney and, overall, RNA contributed only approximately 11% of total tissue P content.Data for lung and colon tumors provide support for the GRH in human cancer. The two-fold amplification of P content in colon and lung tumors may set the stage for potential P-limitation of their proliferation, as such differences often do for rapidly growing biota in ecosystems. However, data for kidney and liver do not support the GRH. To account for these conflicting observations, we suggest that local environments in some organs select for neoplastic cells bearing mutations increasing cell division rate ("r-selected," as in colon and lung) while conditions elsewhere may select for reduced mortality rate ("K-selected," as in liver and kidney)

Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

The c-Myc Target Glycoprotein1bα Links Cytokinesis Failure to Oncogenic Signal Transduction Pathways in Cultured Human Cells

An increase in chromosome number, or polyploidization, is associated with a variety of biological changes including breeding of cereal crops and flowers, terminal differentiation of specialized cells such as megakaryocytes, cellular stress and oncogenic transformation. Yet it remains unclear how cells tolerate the major changes in gene expression, chromatin organization and chromosome segregation that invariably accompany polyploidization. We show here that cancer cells can initiate increases in chromosome number by inhibiting cell division through activation of glycoprotein1b alpha (GpIbα), a component of the c-Myc signaling pathway. We are able to recapitulate cytokinesis failure in primary cells by overexpression of GpIbα in a p53-deficient background. GpIbα was found to localize to the cleavage furrow by microscopy analysis and, when overexpressed, to interfere with assembly of the cellular cortical contraction apparatus and normal division. These results indicate that cytokinesis failure and tetraploidy in cancer cells are directly linked to cellular hyperproliferation via c-Myc induced overexpression of GpIbα

Progressive Telomere Dysfunction Causes Cytokinesis Failure and Leads to the Accumulation of Polyploid Cells

Most cancer cells accumulate genomic abnormalities at a remarkably rapid rate, as they are unable to maintain their chromosome structure and number. Excessively short telomeres, a known source of chromosome instability, are observed in early human-cancer lesions. Besides telomere dysfunction, it has been suggested that a transient phase of polyploidization, in most cases tetraploidization, has a causative role in cancer. Proliferation of tetraploids can gradually generate subtetraploid lineages of unstable cells that might fire the carcinogenic process by promoting further aneuploidy and genomic instability. Given the significance of telomere dysfunction and tetraploidy in the early stages of carcinogenesis, we investigated whether there is a connection between these two important promoters of chromosomal instability. We report that human mammary epithelial cells exhibiting progressive telomere dysfunction, in a pRb deficient and wild-type p53 background, fail to complete the cytoplasmatic cell division due to the persistence of chromatin bridges in the midzone. Flow cytometry together with fluorescence in situ hybridization demonstrated an accumulation of binucleated polyploid cells upon serial passaging cells. Restoration of telomere function through hTERT transduction, which lessens the formation of anaphase bridges by recapping the chromosome ends, rescued the polyploid phenotype. Live-cell imaging revealed that these polyploid cells emerged after abortive cytokinesis due to the persistence of anaphase bridges with large intervening chromatin in the cleavage plane. In agreement with a primary role of anaphase bridge intermediates in the polyploidization process, treatment of HMEC-hTERT cells with bleomycin, which produces chromatin bridges through illegimitate repair, resulted in tetraploid binucleated cells. Taken together, we demonstrate that human epithelial cells exhibiting physiological telomere dysfunction engender tetraploid cells through interference of anaphase bridges with the completion of cytokinesis. These observations shed light on the mechanisms operating during the initial stages of human carcinogenesis, as they provide a link between progressive telomere dysfunction and tetraploidy

Synthesis and Biological Evaluation of 2-Methyl-4,5-Disubstituted Oxazoles as a Novel Class of Highly Potent Antitubulin Agents

Antimitotic agents that interfere with microtubule formation are one of the major classes of cytotoxic drugs for cancer treatment. Multiple 2-methyl-4-(3′,4′,5′-trimethoxyphenyl)-5-substituted oxazoles and their related 4-substituted-5-(3′,4′,5′-trimethoxyphenyl) regioisomeric derivatives designed as cis-constrained combretastatin A-4 (CA-4) analogues were synthesized and evaluated for their antiproliferative activity in vitro against a panel of cancer cell lines and, for selected highly active compounds, interaction with tubulin, cell cycle effects and in vivo potency. Both these series of compounds were characterized by the presence of a common 3′,4′,5′-trimethoxyphenyl ring at either the C-4 or C-5 position of the 2-methyloxazole ring. Compounds 4g and 4i, bearing a m-fluoro-p-methoxyphenyl or p-ethoxyphenyl moiety at the 5-position of 2-methyloxazole nucleus, respectively, exhibited the greatest antiproliferative activity, with IC50 values of 0.35-4.6 nM (4g) and 0.5–20.2 nM (4i), which are similar to those obtained with CA-4. These compounds bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. Furthermore, 4i strongly induced apoptosis that follows the mitochondrial pathway. In vivo, 4i in a mouse syngeneic model demonstrated high antitumor activity which significantly reduced the tumor mass at doses ten times lower than that required for CA-4P, suggesting that 4i warrants further evaluation as a potential anticancer drug

Heterologous Replacement of the Supposed Host Determining Region of Avihepadnaviruses: High In Vivo Infectivity Despite Low Infectivity for Hepatocytes

Hepadnaviruses, including hepatitis B virus (HBV), a highly relevant human pathogen, are small enveloped DNA viruses that replicate via reverse transcription. All hepadnaviruses display a narrow tissue and host tropism. For HBV, this restricts efficient experimental in vivo infection to chimpanzees. While the cellular factors mediating infection are largely unknown, the large viral envelope protein (L) plays a pivotal role for infectivity. Furthermore, certain segments of the PreS domain of L from duck HBV (DHBV) enhanced infectivity for cultured duck hepatocytes of pseudotyped heron HBV (HHBV), a virus unable to infect ducks in vivo. This implied a crucial role for the PreS sequence from amino acid 22 to 90 in the duck tropism of DHBV. Reasoning that reciprocal replacements would reduce infectivity for ducks, we generated spreading-competent chimeric DHBVs with L proteins in which segments 22–90 (Du-He4) or its subsegments 22–37 and 37–90 (Du-He2, Du-He3) are derived from HHBV. Infectivity for duck hepatocytes of Du-He4 and Du-He3, though not Du-He2, was indeed clearly reduced compared to wild-type DHBV. Surprisingly, however, in ducks even Du-He4 caused high-titered, persistent, horizontally and vertically transmissable infections, with kinetics of viral spread similar to those of DHBV when inoculated at doses of 108 viral genome equivalents (vge) per animal. Low-dose infections down to 300 vge per duck did not reveal a significant reduction in specific infectivity of the chimera. Hence, sequence alterations in PreS that limited infectivity in vitro did not do so in vivo. These data reveal a much more complex correlation between PreS sequence and host specificity than might have been anticipated; more generally, they question the value of cultured hepatocytes for reliably predicting in vivo infectivity of avian and, by inference, mammalian hepadnaviruses, with potential implications for the risk assessment of vaccine and drug resistant HBV variants