Characterization of an HIV-targeted transcriptional gene-silencing RNA in primary cells.

Small antisense RNAs targeted to the HIV-1 promoter have been shown to remodel the surrounding chromatin to a state unfavorable for transcriptional activation, yet transcriptional gene silencing (TGS) of HIV-1 has, to date, not been shown in primary human cells. We demonstrate here that TGS can reduce viral transcription in primary human CD4(+) T cells; however, increasing viral burden results in the loss of this antiviral effect. This observation suggests a critical level at which viral RNA can dilute out effective targeting by TGS-based RNAs. Furthermore, studies into off-target effects have identified a potential interaction between the small nucleolar RNA pathway and the TGS-based antisense RNA, resulting in activation of p53. Although not overtly toxic to primary cells, this represents a novel interaction between antisense RNAs and a cellular pathway that should be considered when pursuing small antisense RNA-based therapeutics.

Local anesthetics inhibit kinesin motility and microtentacle protrusions in human epithelial and breast tumor cells.

Detached breast tumor cells produce dynamic microtubule protrusions that promote reattachment of cells and are termed tubulin microtentacles (McTNs) due to their mechanistic distinctions from actin-based filopodia/invadopodia and tubulin-based cilia. McTNs are enriched with vimentin and detyrosinated α-tubulin, (Glu-tubulin). Evidence suggests that vimentin and Glu-tubulin are cross-linked by kinesin motor proteins. Using known kinesin inhibitors, Lidocaine and Tetracaine, the roles of kinesins in McTN formation and function were tested. Live-cell McTN counts, adhesion assays, immunofluorescence, and video microscopy were performed to visualize inhibitor effects on McTNs. Viability and apoptosis assays were used to confirm the non-toxicity of the inhibitors. Treatments of human non-tumorigenic mammary epithelial and breast tumor cells with Lidocaine or Tetracaine caused rapid collapse of vimentin filaments. Live-cell video microscopy demonstrated that Tetracaine reduces motility of intracellular GFP-kinesin and causes centripetal collapse of McTNs. Treatment with Tetracaine inhibited the extension of McTNs and their ability to promote tumor cell aggregation and reattachment. Lidocaine showed similar effects but to a lesser degree. Our current data support a model in which the inhibition of kinesin motor proteins by Tetracaine leads to the reductions in McTNs, and provides a novel mechanism for the ability of this anesthetic to decrease metastatic progression.

Epithelial-to-mesenchymal transition promotes tubulin detyrosination and microtentacles that enhance endothelial engagement.

Epithelial-to-mesenchymal transition (EMT) is associated with increased breast tumor metastasis; however, the specific mechanisms by which EMT promotes metastasis remain somewhat unclear. Despite the importance of cytoskeletal dynamics during both EMT and metastasis, very few current studies examine the cytoskeleton of detached and circulating tumor cells. Specific posttranslational α-tubulin modifications are critical for adherent cell motility and implicated in numerous pathologies, but also remain understudied in detached cells. We report here that EMT induced through ectopic expression of Twist or Snail promotes α-tubulin detyrosination and the formation of tubulin-based microtentacles in detached HMLEs. Mechanistically, EMT downregulates the tubulin tyrosine ligase enzyme, resulting in an accumulation of detyrosinated α-tubulin (Glu-tubulin), and increases microtentacles that penetrate endothelial layers to facilitate tumor cell reattachment. Confocal microscopy shows that microtentacles are capable of penetrating the junctions between endothelial cells. Suppression of endogenous Twist in metastatic human breast tumor cells is capable of reducing both tubulin detyrosination and microtentacles. Clinical breast tumor samples display high concordance between Glu-tubulin and Twist expression levels, emphasizing the coupling between EMT and tubulin detyrosination in vivo. Coordinated elevation of Twist and Glu-tubulin at invasive tumor fronts, particularly within ductal carcinoma in situ samples, establishes that EMT-induced tubulin detyrosination occurs at the earliest stages of tumor invasion. These data support a novel model where the EMT that occurs during tumor invasion downregulates tubulin tyrosine ligase, increasing α-tubulin detyrosination and promoting microtentacles that could enhance the reattachment of circulating tumor cells to the vascular endothelium during metastasis.

Microtentacles tip the balance of cytoskeletal forces in circulating tumor cells.

Detection of circulating tumor cells (CTC) is advancing as an effective predictor of patient outcome and therapeutic response. Unfortunately, our knowledge of CTC biology remains limited, and the impact of drug treatments on CTC metastatic potential is currently unclear. Improved CTC imaging in vivo and analysis of free-floating tumor cells now show that cytoskeletal regulation in CTCs contrasts starkly with tumor cells attached to extracellular matrix. In this review, we examine how persistent microtubule stabilization promotes the formation of microtentacles on the surface of detached breast tumor cells and enhances metastatic potential.

Antimitotic chemotherapeutics promote adhesive responses in detached and circulating tumor cells.

In the clinical treatment of breast cancer, antimitotic cytotoxic agents are one of the most commonly employed chemotherapies, owing largely to their antiproliferative effects on the growth and survival of adherent cells in studies that model primary tumor growth. Importantly, the manner in which these chemotherapeutics impact the metastatic process remains unclear. Furthermore, since dissemination of tumor cells through the systemic circulation and lymphatics necessitates periods of detached survival, it is equally important to consider how circulating tumor cells respond to such compounds. To address this question, we exposed both nontumorigenic and tumor-derived epithelial cell lines to two antitumor compounds, jasplakinolide and paclitaxel (Taxol), in a series of attached and detached states. We report here that jasplakinolide promoted the extension of microtubule-based projections and microtentacle protrusions in adherent and suspended cells, respectively. These protrusions were specifically enriched by upregulation of a stable post-translationally modified form of alpha-tubulin, and this occurred prior to, and independently of any reductions in cellular viability. Microtubule stabilization with Taxol significantly enhanced these effects. Additionally, Taxol promoted the attachment and spreading of suspended tumor cell populations on extracellular matrix. While the antiproliferative effects of these compounds are well recognized and clinically valuable, our findings that microfilament and microtubule binding chemotherapeutics rapidly increase the mechanisms that promote endothelial adhesion of circulating tumor cells warrant caution to avoid inadvertently enhancing metastatic potential, while targeting cell division.

Delocalization of gamma-tubulin due to increased solubility in human breast cancer cell lines.

The centrosome is the major organelle responsible for the nucleation and organization of microtubules into arrays. Recent studies demonstrate that microtubules can nucleate outside the centrosome. The molecular mechanisms controlling acentrosomal microtubule nucleation are currently poorly defined, and the function of this type of microtubule regulation in tumor cell biology is particularly unclear. Since microtubule nucleation is initiated by the gamma-tubulin protein, we examined the regulation of gamma-tubulin in a panel of human breast tumor cell lines, ranging from non-tumorigenic to highly aggressive. We have identified a more dispersive subcellular localization of gamma-tubulin in aggressive breast cancer cell lines, while gamma-tubulin localization remains largely centrosomal in non-aggressive cell lines. Delocalization of gamma-tubulin occurs independently from changes in protein expression and is therefore regulated at the post-translational level. Subcellular fractionation revealed that tumor cell lines show an aberrantly increased release of gamma-tubulin into a soluble cytoplasmic fraction, with the most dramatic changes observed in tumor cell lines of greater aggressiveness. Extraction of soluble gamma-tubulin revealed acentrosomal incorporation of gamma-tubulin in cytoplasmic microtubules and along cell junctions. Moreover, acentrosomal delocalization of gamma-tubulin yielded resistance to colchicine-mediated microtubule collapse. These findings support a model where the solubility of gamma-tubulin can be altered through post-translational modification and provides a new mechanism for microtubule dysregulation in breast cancer. Gamma-tubulin that is delocalized from the centrosome can still clearly be incorporated into filaments, and defines a novel mechanism for tumor cells to develop resistance to microtubule-targeted chemotherapies.

Vimentin filaments support extension of tubulin-based microtentacles in detached breast tumor cells.

Solid tumor metastasis often involves detachment of epithelial carcinoma cells into the vasculature or lymphatics. However, most studies of cytoskeletal rearrangement in solid tumors focus on attached cells. In this study, we report for the first time that human breast tumor cells produce unique tubulin-based protrusions when detached from extracellular matrix. Tumor cell lines of high metastatic potential show significantly increased extension and frequency of microtubule protrusions, which we have termed tubulin microtentacles. Our previous studies in nontumorigenic mammary epithelial cells showed that such detachment-induced microtentacles are enriched in detyrosinated alpha-tubulin. However, amounts of detyrosinated tubulin were similar in breast tumor cell lines despite varying microtentacle levels. Because detyrosinated alpha-tubulin associates strongly with intermediate filament proteins, we examined the contribution of cytokeratin and vimentin filaments to tumor cell microtentacles. Increased microtentacle frequency and extension correlated strongly with loss of cytokeratin expression and up-regulation of vimentin, as is often observed during tumor progression. Moreover, vimentin filaments coaligned with microtentacles, whereas cytokeratin did not. Disruption of vimentin with PP1/PP2A-specific inhibitors significantly reduced microtentacles and inhibited cell reattachment to extracellular matrix. Furthermore, expression of a dominant-negative vimentin mutant disrupted endogenous vimentin filaments and significantly reduced microtentacles, providing specific genetic evidence that vimentin supports microtentacles. Our results define a novel model in which coordination of vimentin and detyrosinated microtubules provides structural support for the extensive microtentacles observed in detached tumor cells and a possible mechanism to promote successful metastatic spread.

The effect of aerial parts of Echinacea on the circulating white cell levels and selected immune functions of the aging male Sprague-Dawley rat.

Echinacea is one of the most widely used over-the-counter herbal preparations that purport to "improve immune system function", especially when taken as a short course of therapy (6-8 weeks). Since many purchasers are older individuals, a double-blind, placebo-controlled study was performed to investigate whether Echinacea could affect total and differential white cell counts, phagocytic activity and interleukin (IL-2) levels in 12-month-old, healthy, male Sprague-Dawley rats when administered over an 8-week period. Echinacea (50 mg/kg of aerial parts) mixed with peanut butter or peanut butter alone was fed to 16 rats, which were receiving regular food and water ad libitum. Cell counts and immune functions were determined on rat tail vein blood on a weekly basis. Echinacea significantly increased circulating total white cell counts during the first 2 weeks of administration, and IL-2 levels during the final 5 weeks of the study period (p<0.05). Differential counts were altered during the entire 8-week study, with mononuclear cells significantly increased to the detriment of granulocytes (p<0.05). No such changes were observed in animals given peanut butter alone. No difference was observed in phagocytic function between animals given Echinacea or peanut butter alone. These studies suggest that aerial components of Echinacea affect both mononuclear cell levels and circulating IL-2 levels in older animals.